Research ArticleStability-Indicating HPLC Determination of Gemcitabine inPharmaceutical Formulations

Rahul Singh1 Ashok K Shakya2 Rajashri Naik2 and Naeem Shalan2

1Faculty of Pharmacy Integral University Kursi Road Lucknow 226-026 India2Faculty of Pharmacy and Medical Sciences Al-Ahliyya Amman University PO Box 263 Amman 19328 Jordan

Correspondence should be addressed to Ashok K Shakya ashokkumar2811gmailcom

Received 28 September 2014 Revised 23 January 2015 Accepted 26 January 2015

Academic Editor David M Lubman

Copyright copy 2015 Rahul Singh et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A simple sensitive inexpensive and rapid stability indicating high performance liquid chromatographic method has beendeveloped for determination of gemcitabine in injectable dosage forms using theophylline as internal standard Chromatographicseparation was achieved on a Phenomenex Luna C-18 column (250mm times 46mm 5120583) with a mobile phase consisting of 90 waterand 10 acetonitrile (pH 700plusmn005)The signals of gemcitabine and theophylline were recorded at 275 nm Calibration curves werelinear in the concentration range of 05ndash50 120583gmLThe correlation coefficient was 0999 or higher The limit of detection and limitof quantitation were 01498 and 04541 120583gmL respectively The inter- and intraday precision were less than 2 Accuracy of themethod ranged from 1002 to 1004 Stability studies indicate that the drug was stable to sunlight and UV lightThe drug gives 6different hydrolytic products under alkaline stress and 3 in acidic condition Aqueous and oxidative stress conditions also degradethe drug Degradation was higher in the alkaline condition compared to other stress conditionsThe robustness of the methods wasevaluated using design of experiments Validation reveals that the proposed method is specific accurate precise reliable robustreproducible and suitable for the quantitative analysis

1 Introduction

Gemcitabine hydrochloride (Figure 1) (4-amino-1-[(2R4R 5R)-33-difluoro-4-hydroxy-5-(hydroxymethyl) oxolan-2-yl] pyrimidin-2-one) is a 120573-difluoronucleoside purineantimetabolite The drug is an antitumor agent employedextensively against several human malignancies like ovarianlung pancreatic bladder urothelial and breast cancer It iscurrently marketed as a lyophilized powder The drug is alsoextensively employed as antiviral agent enzyme inhibitorimmunosuppressive agent and radiation-sensitizing agentsGemcitabine is a prodrug that enters the cell by means ofnucleoside transporters and becomes active through an intra-cellular transformation catalyzed by deoxycytidine kinase toits diphosphate and triphosphate derivatives The triphos-phate derivative is incorporated into theDNA strand inhibit-ing thymidylate synthetase which inhibits DNA synthesis andchain elongation contributing to the antineoplastic activity of

the drug The diphosphate derivative inhibits ribonucleotidereductase the enzyme responsible for catalyzing synthesis ofdeoxynucleoside-triphosphate required for DNA synthesisGemcitabine triphosphate competes with endogenous nucle-oside triphosphate for incorporation into DNA [1ndash3]

A literature survey reveals that only a few methods basedon ultraviolet spectroscopy [4] HPTLC [5] and HPLC [6ndash13] are available for determination of drug in formulationAlthough several HPLC [14ndash22] and LC-MSMS [23ndash28]methods have been reported for estimation of drug andits metabolites in biological fluids these methods [23ndash28]are complicated costly and time consuming in comparisonto a simple HPLC-UV method A few stability indicatingthat HPLC methods [3 11 12] have been reported whichprovides variable level of degradation of gemcitabine Jansenet al [3] reported the separation and identification ofdegraded product of gemcitabine in acidic stress conditionMastanamma et al [11] and Kudikala et al [12] have reported

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2015 Article ID 862592 12 pageshttpdxdoiorg1011552015862592

2 International Journal of Analytical Chemistry

N O

N

O

OH F

FHO

(1)

HN

NNO

N

O

(2)

NH2

CH3

H3C

Figure 1 Chemical structures of gemcitabine (1) and theophylline (2)

the validated stability indicating method which can separatethe hydrolytic degraded product of gemcitabine Howeverto the best of our knowledge none of the HPLC methodreported the oxidative degraded product of gemcitabinePreviously published methods for formulation are less robustand need more investigations for method development andvalidation Stability-indicating methods have to demonstratethat they are specific which involves evaluating the drug inthe presence of its degradation products [29] The presentinvestigation describes a simple rapid accurate preciserobust stability indicating RP-HPLC method for the deter-mination of gemcitabine for dosage forms The robustness ofthemethodwas studied using 24 factorial designThemethodwas validated as per the ICH guidelines

2 Experimental

21 Chemicals and Reagents The reference sample of gem-citabine was supplied by Ms Shilpa Medicare LimitedRaichur India Theophylline (2) was received as gift samplefrom Hetero Pharmaceutical Ltd Hyderabad India Themarketed formulation of drug (Cytogem Dr Reddys Mum-bai India) was purchased from the local market All reagentswere of analytical grade unless stated otherwise Reverseosmosis quality water (purified with a Milli-RO plus Milli-Qstation Millipore Corp USA) and HPLC quality water wereused throughout Acetonitrile andmethanol were supplied byPanreac (Barcelona Spain)

22 HPLC Instrumentation and Conditions A ShimadzuProminence high pressure liquid chromatographic instru-ment provided with a Luna C-18 column (250mm times 46mm5 120583) an LC 20AT-VP solvent delivery system a universalloop injector (Rheodyne 7725 i) of injection capacity of20120583L and an SPD 20A UV-visible detector (120582max 275 nm)was employed in the study Data acquisition was carriedout using LC-Solution software Chromatographic analyseswere carried out using the mobile phase of acetonitrile-water (10 90 pH adjusted to 70 using trietylamine andorthophosphoric acid) The mobile phase was prepared dailyand filtered through a 045120583m membrane filter (MilliporeCorp USA) The temperature of column was maintained at25 plusmn 1∘C Robustness cross-validation and stability studies

were carried out on Shimadzu Prominence Liquid Chro-matographic system consists of quaternary gradient pumpShimadzu-20-AD UFLC Degasser DGU-20A3 ProminenceDegasser Autosampler and Injector SIL-20A ProminenceAutosampler Detector Diode Array Detector (SPD-M20A)Communication Bus module CBM-20A and Column LunaC-18 column (250mm times 46mm 5120583) The signals were cap-tured using Windows based LC-Solution software (version125)

23 Preparation of Stock and Standards Solutions

231 Gemcitabine Standard and Working Solutions Anaccurately weighed amount (100mg) of gemcitabine wastransferred into 100mL calibrated flask and dissolved inappropriate volume of methanol Then the void volumewas completed with methanol to produce a stock solutionof 1000 120583gmL The stock solution was further diluted withmobile phase to obtain working solutions (25 100 and200120583gmL)

232 Preparation of the Internal Standard (IS) Solution Anaccurately weighed amount (100mg) of theophylline wastransferred into 100mL volumetric flask and dissolved in25mL of methanol The resultant solution was thoroughlysonicated till complete dissolution of the drug has occurredVolume was made up to the mark with water

233 Calibration Standards Calibration standards were pre-pared freshly using either stock or intermediate workingsolution of gemcitabine and internal standard Standardsolution of concentrations 05 10 20 50 10 15 25 40 and50 120583gmL was prepared All these solutions were containing20120583gmL of theophylline as standards These solutions wereanalyzed immediately to avoid degradation

234 Quality Control Samples Similarly quality controlsamples of concentration 1 5 20 30 and 45120583gmL contain-ing IS (20 120583gmL) were prepared freshly and analyzed

24 Preparation of Sample for Assay Twelve injection vialscontaining the drug in the lyophilized powder form of two

International Journal of Analytical Chemistry 3

Table 1 Linearity data of the proposed method

Conc (120583gmL) Mean peak area (gemcitabine) (119899 = 6) Mean peak area (IS) Mean area ratio Conc found (120583gmL) assay05 115562 4901386 002358 050 99010 221962 5272420 004210 103 102620 401196 5162028 007772 205 102350 992068 5492566 018062 499 999100 1977022 5469878 036144 1018 1018150 2980964 5516108 054041 1530 1020250 4960840 5603166 088536 2519 1008400 7917220 5666864 139711 3985 996500 9875806 5534780 178432 5095 1019

119910 = 00353119909 + 00063 1199032 = 09998

Table 2 Accuracy of the method

Amount taken (120583gmL) Amount added Amount recovered (mean plusmn SD) (119899 = 6) recovery (mean plusmn SD) RSD 120583gmL

20 25 5 2509 plusmn 010 10037 plusmn 040 04020 50 10 3006 plusmn 009 10019 plusmn 031 03120 80 16 3615 plusmn 021 10042 plusmn 059 05920 100 20 4014 plusmn 013 10034 plusmn 033 03320 120 24 4407 plusmn 006 10015 plusmn 013 013

different batches were studiedTheir aluminum closures wereremoved The vials were weighed with the drug and afterremoving the content in empty stateWith the help of the dataavailable weight of the lyophilized powder was calculated Anaccurately weighed portion from this powder equivalent to10mg of drugwas transferred to 100mL calibrated volumetricflask and 10mL of internal standard solution was also trans-ferred to it quantitatively 50mL of methanol was added tothe flask and the contents of the flask were swirled sonicatedfor 5min and then completed to volume with water Theprepared solutions were diluted quantitatively with mobilephase to obtain a solution of 20120583gmL drug and internalstandard for the analysis

25 Analytical Method Validation

251 Linearity Limit of Detection (LOD) and Limit of Quan-titation (LOQ) Appropriate volumes of gemcitabine stockstandard solution (1000mgmL) were diluted with mobilephase to produce concentrations of 05 10 20 50 1015 25 40 and 50 120583gmL Replicates of each concentrationwere independently prepared and injected into the chromato-graph Linearity was evaluated by the linear least-squaresregression model using weighting factor 119909 Microsoft officeexcel 2007 was used for statistical analysis The method wasvalidated according to ICH guidelines of the validation ofanalytical methods [29] A 5 significance level was usedfor evaluation The method was evaluated by determinationof the correlation coefficient and intercept values LOD andLOQ were determined from the best fitted calibration curveLOD and LOQwere calculated as 33times120590

119899minus1119878 and 10times120590

119899minus1119878

where 120590119899minus1

is the standard deviation of the intercept and 119878 isthe slope of the calibration curve

252 Precision Precision was measured using triplicatedetermination of quality control samples of 1 5 20 30and 45 120583gmL of gemcitabine on different occasions (0 3and 6 h) and different days The precision (RSD) of themethod was determined as intraday precision (repeatability)and intermediate precision The intermediate precision wasestimated from the RSD of the analysis of the samplesprepared at the same concentration but on 3 different daysat different concentration levels while intraday precision wascalculated by analyzing the same concentration during thesame day at different time

253 Accuracy Accuracy of method was determined byaddition of known amounts of gemcitabine (119899 = 3 ateach level of 25 50 80 100 and 120 levels) to a samplesolution of known concentration (formulation) From thesesolutions appropriate solution was prepared and analyzedand the total amount recovered was calculated In this workthe mean recovery of the target concentration was 100 plusmn 2for acceptance

254 Robustness It is a measure of reproducibility of testresults under normal expected operational condition fromanalyst to analyst The robustness of the method was evalu-ated on the basis of precision as measured by percent coef-ficient of variation (RSD) determined as each concentrationlevel was required not to exceed 2 Design of experiments

4 International Journal of Analytical Chemistry

Table 3 Precision study of the proposed method

Concentration (120583gmL)Intraday precision Interday precision

Conc found RSD Conc found RSDMean plusmn SD Mean plusmn SD

1 0994 plusmn 0015 152 0985 plusmn 0018 1815 5030 plusmn 0074 148 5041 plusmn 0092 18320 19920 plusmn 0243 122 19840 plusmn 0280 14130 29886 plusmn 0292 098 29861 plusmn 0233 07845 44759 plusmn 0255 057 44742 plusmn 0326 073

Table 4 System suitability

Parameters Mean RSDTheoretical plates (Drug) 7716 125Platesmeter 30864 125HETP 3110 013Tailing factor 110 011LOD (120583gmL) 01498 105LOQ (120583gmL) 04541 105Resolution (Rs) 110 045Retention time of drug 395min 150Retention time of IS 780min 150

was used to study robustness of the method A 24 factorialdesign was used to test the robustness of chromatographicseparation The experimental design is useful for this kind ofstudy as it facilitates the investigation of several parametersby reducing the number of experiments Acetonitrile contentof the mobile phase pH column oven temperature and flowrate was investigated Upper and lower limits are shown inTable 5 The experiments were run randomly with samplecontaining gemcitabine and internal standard (20 120583gmL ofeach) The selected responses were resolution (119877

119904) tailing

factor of drug (119879119891-D) and tailing factor of IS (119879

119891-I)

255 Stability Studies Stress study like oxidative alkalineand acidic stress exposure to sunlight andUV light (254 nm)was carried out using raw material Chromatograms wererecorded in order to study the specificity of the methodThe chromatograms of the samples were compared withthose of control samples that were freshly prepared from thestock standard solution and without stress All samples wereanalyzed in triplicate The peak purity was checked using thetools of the LC-Solution software This assessment was basedon the comparison of spectra recorded during the elution ofthe peak UV spectra and peak purity were used to assesspurity of analytes

(1) Oxidative Stress Gemcitabine (5mg) was weighed accu-rately and transferred to 100mL flask for evaluation ofoxidative stress 10mL of 5 hydrogen peroxide was added toit It was shaken for one hour at 60∘C and then contents werecooled to room temperature Internal standard (2mL) was

(nm)20000 25000 30000 32500

Abs

2000

1500

1000

0500

0000minus0150

Figure 2 UV spectra of the gemcitabine in mobile phase

(min)

(mAU

)

00 25 50 75 100

0

25

50

1 PDA multi 1275nm 4nm

3997

gem

cita

bine

7777

theo

phyl

line

Figure 3 Representative chromatogram showing signals of gemc-itabine and theophylline in the selected mobile phase

added the contents were transferred quantitatively to 100mLvolumetric flask and diluted to the mark with mobile phase

(2) Effect of Acidic Alkaline and Aqueous Media Similarlyfor evaluation of acidic alkaline or hydrolytic stress gem-citabine (5mg) was weighed accurately and transferred to100mL flask These samples were shaken (at 60∘C 1 h) witheither hydrochloric acid (5mL 1NHCl) or sodiumhydroxide(5mL 1NNaOHor 20mLwater) After one hour the contentwas cooled and processed as described above (in oxidativestress)

(3) Effect of UV Light or Sunlight Gemcitabine (500mg)was placed in an open watch glass and exposed to either

International Journal of Analytical Chemistry 5

Table 5 Factorial design data for robustness of chromatographic separation

(a)

Selected parameters and their variations minus1 (lower limit) +1 (upper limit)Acetonitrile in mobile phase () (119860) 8 12Final pH of the mobile phase (119861) 68 72Column oven temperature (∘C) (119862) 20 30Flow rate (mLmin) (119863) 08 12

(b)

Exp number Run order 119860 119861 119862 119863 119877119904

119879119891-D 119879

119891-I

1 6 8 68 20 08 1401 112 122 11 12 68 20 08 131 111 1193 5 8 72 20 08 141 109 1184 13 12 72 20 08 132 108 115 1 8 68 30 08 143 109 1116 7 12 68 30 08 102 109 1087 10 8 72 30 08 145 111 1158 15 12 72 30 08 1185 11 1089 12 8 68 20 12 1351 113 11110 14 12 68 20 12 1021 105 10711 9 8 72 20 12 1441 109 11212 2 12 72 20 12 1005 106 10713 8 8 68 30 12 1325 112 1114 4 12 68 30 12 1015 104 10715 3 8 72 30 12 1412 11 11116 16 12 72 30 12 1015 105 105119877119904 resolution factor 119879119891-D tailing factor for drug and 119879119891-I tailing factor for IS

UV-irradiation (sim100Wm2) or direct sunlight for one hourwith occasionally shifting of the content using stainless steelspatula After exposure 5mg of sample was weighed andtransferred to 100mL volumetric flask internal standard(2mL) was added to it and further processed as describedabove

Chromatograms of these sample solutions were recordedand compared with the chromatograms of unexposed API

(4) Stock StabilityThestability of stock solutionwas evaluatedat zero time and stored in the refrigerator (2ndash8∘C) Sampleswere prepared and analyzed at days 0 7 14 and 21

3 Results and Discussion

31 Analytical Method Development In order to achieveoptimum separation various parameters like solvent solventstrength detection wavelength flow rate elution time asym-metry and plate numbers were considered During optimiza-tion gemcitabine hydrochloride and internal standard wereinjected into various mobile phases of water methanol orwater acetonitrile (90 10 80 20 70 30 60 40 and 50 50pH 5 6 or 7) and the retention time tailing factor along withresolution factor was recorded In certain mobile phases thepeak was distorted while in others the compound eluted outquickly indicating the lesser retention time and thus lesser

separation on the column As the pKa of gemcitabine is 35and unstable in an acidic pH [3] different mobile phases ofpH 70 were used Mobile phase of acetonitrile-water (10 90vv pH adjusted to 70) was used as suitable mobile phaseas it was able to separate the analytes Using the C-18 ODSanalytical columnwith an isocraticmobile phase at a flow rateof 1 mLmin the drug and IS were eluted at sim40 and 78minrespectively Although temperature was found not to be acritical parameter for this analysis it was set at 25∘C to avoidshifting of signals The absorption maximum of the drug at275 nm was selected for detection (Figure 2) as there wasno interference from excipients present in drug or baselinedisturbance The resolution factor was sim11 Figure 3 depictsthe representative chromatogram obtained with the presentmethod

32 Method Validation The method was validated withrespect to parameters including linearity limit of quantitation(LOQ) limit of detection (LOD) precision accuracy speci-ficity robustness system suitability and stability

321 Linearity Different calibration curves (119899 = 6) that wereconstructed for gemcitabine were linear over the concentra-tion range of 05ndash50120583gmL Peak area ratios of gemcitabineto IS were plotted versus gemcitabine concentration and

6 International Journal of Analytical Chemistry

0

05

1

A B C D

minus05

minus1

minus2

minus15

AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(a)

0

0005

001

0015

minus0005

minus001

minus002

minus0015

minus0025

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(b)

0

001

002

minus001

minus002

minus003

minus004

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(c)

Figure 4 Scaled and centered coefficient of variation () of (a) resolution factor (b) tailing factor of drug and (c) tailing factor of IS duringrobustness studies

Table 6 Stability data under different stressed conditions

Stress conditions Percent gemcitabine remained Retention time of degraded products

Alkaline stress (1 N NaOH 60∘C 1 h) 161 plusmn 02 3023 (d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944(d-5) and 5375 (d-6)

Acidic stress (1 N HCl 60∘C 1 h) 950 plusmn 02 4953 (d-5) 6082 (d-7) and 7131 (d-8)Oxidative stress (5 60∘C 1 h) 971 plusmn 01 3772 (d-3)Aqueous hydrolytic stress (60∘C 1 h) 992 plusmn 01 4952 (d-5)Ultraviolet light (100Wm2 1 h) 1000 00Direct sunlight (1 h) 1000 00Aqueous stability (after 21 days) 999 plusmn 01 00

Table 7 Assay of formulations

Sample Label claim (mgvial) (119899 = 6) Amount found assay RSDMean plusmn SD

Batch 1 200 19935 plusmn 046 997 023Batch 2 200 19927 plusmn 052 996 026

linear regressionwas performed using Spinchrome-Clarity orLC-solution software Different calibration curves (119899 = 6)were prepared on three different days The mean regressionequation for gemcitabine was found to be 119910 = 00353119909 +00063 with 09998 correlation coefficient using weighting

factor-119909 (Table 1) The linearity range reported in othermethods ranged between 0020 and 300 120583gmL [3 5ndash28]

322 LOD and LOQ The LOD and LOQ values were 01498and 04541120583gmL calculated using calibration curve as per

International Journal of Analytical Chemistry 7

0

00

10

20

(min)

(mAU

)

1 PDA multi1275nm4nm3023d-1

3645d-3

3422

5375d-6

4944d-5

4342d-4

3202d-2

7589

theo

phyl

line

3965

gem

cita

bine

10025 50 75

(a)

0 2 4 6 8 10340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Peak purity index 1000000Impurity not detected

Single point threshold 0998065Minimum peak purity index 1934

39 40 41 42000

025

050

075

0

10

20

Retention time 3964Compound name gemcitabine

(min)

ID2

(c)

05

1015202530354045

200 300

267822363222395 25075

33965

(mAU

)(nm)

Retention time 3965Compound name gemcitabine

ID2

(d)

102030405060

7080

90100

200 300

27635

24183

(mAU

)

(nm)

Retention time 3023Compound name d-1

ID6

minus0

(e)

05

1015202530354045

200 300

25830

23260

(mAU

)

(nm)

Retention time 3202Compound name d-2

ID7

(f)

05

10

15

20

25

200 300

25862

22758

(mAU

)

(nm)

Retention time 4342Compound name d-4

ID9

minus00

(g)

0

5

10

15

20

200 300

25728

22795

34558

(mAU

)

(nm)

Retention time 4944Compound name d-5

ID3

(h)

Figure 5 Continued

8 International Journal of Analytical Chemistry

102030405060708090

100110

200 300

20214

27131

2421833250

(mAU

)

(nm)

Retention time 7589Compound name theophylline

ID 1

minus0

(i)

Figure 5 Typical HPLC chromatogram of (a) gemcitabine exposed to alkaline stress (1 N NaOH 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectra of (d) gemcitabine (e) degraded product d-1 (f) d-2 (g) d-4 (h) d-5 and (i) theophylline

ICH guideline The LOD and LOQ reported by Lanz et al[14] were 10 and 20 ngmL based on signal to noise ratiomethod while Xu et al [7] reported 12 and 375 ngmL basedon calibration curve (external standard method)

323 Accuracy and Precision The accuracy and precisionof the analytical method were established across its linearrange as indicated in the guideline As shown from the datain Table 2 excellent recoveries were made at different addedconcentration level The results obtained for the intraday andinterday precision of themethod expressed as RSD values Asshown in the table the intraday and interday RSDwerelt20for all concentrations tested in different situations studied(Tables 2 and 3)

324 Specificity Specificity of the method was assessed bycomparing the chromatograms obtained from lyophilizedpowder from internal standard and from the drug standardsThe retention times of drug from standard solutions andfrom lyophilized powder were identical and no coelutingpeaks from the diluents were observed indicating specificmethod for quantitative estimation of drug in the commercialformulation

325 System Suitability System suitability parameters werestudied with six replicates standard solution of the drug andthe calculated parameters are within the acceptance criteriaThe tailing factor the number of theoretical plates andHETPwere in the acceptable limits (RSD less than 2) The systemsuitability results are shown in Table 4

326 Robustness Robustness of the methods was illustratedby getting the resolution factor and tailing factor whenmobile phase flow rate (plusmn02mLmin) acetonitrile content(plusmn2) pH (plusmn02 units) and column temperature (plusmn5∘C)were deliberately varied It was studied using factorial design

experiment The deliberate changes in the method do notaffected the resolution tailing factor of drug and IS signifi-cantly The scaled and centered coefficient plots for the aboveresponses revealed that different parameters did not affectresponses so that the developed method was consideredrugged and robust Results are presented in Figure 4 andTable 5

327 Stability Studies The prepared stock and workingsolutions were stable up to 21 days when stored in refrigerator(2ndash8∘C) and did not produce degraded compounds duringexperimental conditions The peak purity was 0985 ormore during the validation studies Gemcitabine producessix different degradation products on alkaline stress withretention time (min) 3023 (as d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944 (d-5) and 5375 (d-6) The percentageof gemcitabine remained was 161 Mastanamma et al [11]and Kudikala et al [12] have reported two degraded productof gemcitabine Figure 5 represents the chromatogram con-tour plot peak purity and UV spectra of gemcitabine anddegraded products The extracted UV spectra indicate thatthe entire degradation products are derived fromgemcitabineor its intermediates In case of acidic stress the degradationproducts were observed at 4953 (as d-5) 6082 (d-7) and7131min (d-8) (Figure 6) Jansen et al [3] have reported thepresence of a coeluted degraded product with gemcitabinewhich does not possess UV absorption at 275 nm Thehydrolytic product d-5 (495min) was observed in alkalineas well as in aqueous stress condition On exposure tohydrogen peroxide (5 60∘C 1 h) gemcitabine producesonly one minor degradation products having retention time3772min (Table 6 Figure 7) The percentage of unoxi-dized gemcitabine was 971 Gemcitabine was completelydegraded on exposure to drastic oxidative condition (5060∘C 1 h) However these degraded compounds have noultraviolet (UV) absorbance at 275 nm the wavelength usedto monitor the gemcitabine concentrations Borisagar et al

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

2 International Journal of Analytical Chemistry

N O

N

O

OH F

FHO

(1)

HN

NNO

N

O

(2)

NH2

CH3

H3C

Figure 1 Chemical structures of gemcitabine (1) and theophylline (2)

the validated stability indicating method which can separatethe hydrolytic degraded product of gemcitabine Howeverto the best of our knowledge none of the HPLC methodreported the oxidative degraded product of gemcitabinePreviously published methods for formulation are less robustand need more investigations for method development andvalidation Stability-indicating methods have to demonstratethat they are specific which involves evaluating the drug inthe presence of its degradation products [29] The presentinvestigation describes a simple rapid accurate preciserobust stability indicating RP-HPLC method for the deter-mination of gemcitabine for dosage forms The robustness ofthemethodwas studied using 24 factorial designThemethodwas validated as per the ICH guidelines

2 Experimental

21 Chemicals and Reagents The reference sample of gem-citabine was supplied by Ms Shilpa Medicare LimitedRaichur India Theophylline (2) was received as gift samplefrom Hetero Pharmaceutical Ltd Hyderabad India Themarketed formulation of drug (Cytogem Dr Reddys Mum-bai India) was purchased from the local market All reagentswere of analytical grade unless stated otherwise Reverseosmosis quality water (purified with a Milli-RO plus Milli-Qstation Millipore Corp USA) and HPLC quality water wereused throughout Acetonitrile andmethanol were supplied byPanreac (Barcelona Spain)

22 HPLC Instrumentation and Conditions A ShimadzuProminence high pressure liquid chromatographic instru-ment provided with a Luna C-18 column (250mm times 46mm5 120583) an LC 20AT-VP solvent delivery system a universalloop injector (Rheodyne 7725 i) of injection capacity of20120583L and an SPD 20A UV-visible detector (120582max 275 nm)was employed in the study Data acquisition was carriedout using LC-Solution software Chromatographic analyseswere carried out using the mobile phase of acetonitrile-water (10 90 pH adjusted to 70 using trietylamine andorthophosphoric acid) The mobile phase was prepared dailyand filtered through a 045120583m membrane filter (MilliporeCorp USA) The temperature of column was maintained at25 plusmn 1∘C Robustness cross-validation and stability studies

were carried out on Shimadzu Prominence Liquid Chro-matographic system consists of quaternary gradient pumpShimadzu-20-AD UFLC Degasser DGU-20A3 ProminenceDegasser Autosampler and Injector SIL-20A ProminenceAutosampler Detector Diode Array Detector (SPD-M20A)Communication Bus module CBM-20A and Column LunaC-18 column (250mm times 46mm 5120583) The signals were cap-tured using Windows based LC-Solution software (version125)

23 Preparation of Stock and Standards Solutions

231 Gemcitabine Standard and Working Solutions Anaccurately weighed amount (100mg) of gemcitabine wastransferred into 100mL calibrated flask and dissolved inappropriate volume of methanol Then the void volumewas completed with methanol to produce a stock solutionof 1000 120583gmL The stock solution was further diluted withmobile phase to obtain working solutions (25 100 and200120583gmL)

232 Preparation of the Internal Standard (IS) Solution Anaccurately weighed amount (100mg) of theophylline wastransferred into 100mL volumetric flask and dissolved in25mL of methanol The resultant solution was thoroughlysonicated till complete dissolution of the drug has occurredVolume was made up to the mark with water

233 Calibration Standards Calibration standards were pre-pared freshly using either stock or intermediate workingsolution of gemcitabine and internal standard Standardsolution of concentrations 05 10 20 50 10 15 25 40 and50 120583gmL was prepared All these solutions were containing20120583gmL of theophylline as standards These solutions wereanalyzed immediately to avoid degradation

234 Quality Control Samples Similarly quality controlsamples of concentration 1 5 20 30 and 45120583gmL contain-ing IS (20 120583gmL) were prepared freshly and analyzed

24 Preparation of Sample for Assay Twelve injection vialscontaining the drug in the lyophilized powder form of two

International Journal of Analytical Chemistry 3

Table 1 Linearity data of the proposed method

Conc (120583gmL) Mean peak area (gemcitabine) (119899 = 6) Mean peak area (IS) Mean area ratio Conc found (120583gmL) assay05 115562 4901386 002358 050 99010 221962 5272420 004210 103 102620 401196 5162028 007772 205 102350 992068 5492566 018062 499 999100 1977022 5469878 036144 1018 1018150 2980964 5516108 054041 1530 1020250 4960840 5603166 088536 2519 1008400 7917220 5666864 139711 3985 996500 9875806 5534780 178432 5095 1019

119910 = 00353119909 + 00063 1199032 = 09998

Table 2 Accuracy of the method

Amount taken (120583gmL) Amount added Amount recovered (mean plusmn SD) (119899 = 6) recovery (mean plusmn SD) RSD 120583gmL

20 25 5 2509 plusmn 010 10037 plusmn 040 04020 50 10 3006 plusmn 009 10019 plusmn 031 03120 80 16 3615 plusmn 021 10042 plusmn 059 05920 100 20 4014 plusmn 013 10034 plusmn 033 03320 120 24 4407 plusmn 006 10015 plusmn 013 013

different batches were studiedTheir aluminum closures wereremoved The vials were weighed with the drug and afterremoving the content in empty stateWith the help of the dataavailable weight of the lyophilized powder was calculated Anaccurately weighed portion from this powder equivalent to10mg of drugwas transferred to 100mL calibrated volumetricflask and 10mL of internal standard solution was also trans-ferred to it quantitatively 50mL of methanol was added tothe flask and the contents of the flask were swirled sonicatedfor 5min and then completed to volume with water Theprepared solutions were diluted quantitatively with mobilephase to obtain a solution of 20120583gmL drug and internalstandard for the analysis

25 Analytical Method Validation

251 Linearity Limit of Detection (LOD) and Limit of Quan-titation (LOQ) Appropriate volumes of gemcitabine stockstandard solution (1000mgmL) were diluted with mobilephase to produce concentrations of 05 10 20 50 1015 25 40 and 50 120583gmL Replicates of each concentrationwere independently prepared and injected into the chromato-graph Linearity was evaluated by the linear least-squaresregression model using weighting factor 119909 Microsoft officeexcel 2007 was used for statistical analysis The method wasvalidated according to ICH guidelines of the validation ofanalytical methods [29] A 5 significance level was usedfor evaluation The method was evaluated by determinationof the correlation coefficient and intercept values LOD andLOQ were determined from the best fitted calibration curveLOD and LOQwere calculated as 33times120590

119899minus1119878 and 10times120590

119899minus1119878

where 120590119899minus1

is the standard deviation of the intercept and 119878 isthe slope of the calibration curve

252 Precision Precision was measured using triplicatedetermination of quality control samples of 1 5 20 30and 45 120583gmL of gemcitabine on different occasions (0 3and 6 h) and different days The precision (RSD) of themethod was determined as intraday precision (repeatability)and intermediate precision The intermediate precision wasestimated from the RSD of the analysis of the samplesprepared at the same concentration but on 3 different daysat different concentration levels while intraday precision wascalculated by analyzing the same concentration during thesame day at different time

253 Accuracy Accuracy of method was determined byaddition of known amounts of gemcitabine (119899 = 3 ateach level of 25 50 80 100 and 120 levels) to a samplesolution of known concentration (formulation) From thesesolutions appropriate solution was prepared and analyzedand the total amount recovered was calculated In this workthe mean recovery of the target concentration was 100 plusmn 2for acceptance

254 Robustness It is a measure of reproducibility of testresults under normal expected operational condition fromanalyst to analyst The robustness of the method was evalu-ated on the basis of precision as measured by percent coef-ficient of variation (RSD) determined as each concentrationlevel was required not to exceed 2 Design of experiments

4 International Journal of Analytical Chemistry

Table 3 Precision study of the proposed method

Concentration (120583gmL)Intraday precision Interday precision

Conc found RSD Conc found RSDMean plusmn SD Mean plusmn SD

1 0994 plusmn 0015 152 0985 plusmn 0018 1815 5030 plusmn 0074 148 5041 plusmn 0092 18320 19920 plusmn 0243 122 19840 plusmn 0280 14130 29886 plusmn 0292 098 29861 plusmn 0233 07845 44759 plusmn 0255 057 44742 plusmn 0326 073

Table 4 System suitability

Parameters Mean RSDTheoretical plates (Drug) 7716 125Platesmeter 30864 125HETP 3110 013Tailing factor 110 011LOD (120583gmL) 01498 105LOQ (120583gmL) 04541 105Resolution (Rs) 110 045Retention time of drug 395min 150Retention time of IS 780min 150

was used to study robustness of the method A 24 factorialdesign was used to test the robustness of chromatographicseparation The experimental design is useful for this kind ofstudy as it facilitates the investigation of several parametersby reducing the number of experiments Acetonitrile contentof the mobile phase pH column oven temperature and flowrate was investigated Upper and lower limits are shown inTable 5 The experiments were run randomly with samplecontaining gemcitabine and internal standard (20 120583gmL ofeach) The selected responses were resolution (119877

119904) tailing

factor of drug (119879119891-D) and tailing factor of IS (119879

119891-I)

255 Stability Studies Stress study like oxidative alkalineand acidic stress exposure to sunlight andUV light (254 nm)was carried out using raw material Chromatograms wererecorded in order to study the specificity of the methodThe chromatograms of the samples were compared withthose of control samples that were freshly prepared from thestock standard solution and without stress All samples wereanalyzed in triplicate The peak purity was checked using thetools of the LC-Solution software This assessment was basedon the comparison of spectra recorded during the elution ofthe peak UV spectra and peak purity were used to assesspurity of analytes

(1) Oxidative Stress Gemcitabine (5mg) was weighed accu-rately and transferred to 100mL flask for evaluation ofoxidative stress 10mL of 5 hydrogen peroxide was added toit It was shaken for one hour at 60∘C and then contents werecooled to room temperature Internal standard (2mL) was

(nm)20000 25000 30000 32500

Abs

2000

1500

1000

0500

0000minus0150

Figure 2 UV spectra of the gemcitabine in mobile phase

(min)

(mAU

)

00 25 50 75 100

0

25

50

1 PDA multi 1275nm 4nm

3997

gem

cita

bine

7777

theo

phyl

line

Figure 3 Representative chromatogram showing signals of gemc-itabine and theophylline in the selected mobile phase

added the contents were transferred quantitatively to 100mLvolumetric flask and diluted to the mark with mobile phase

(2) Effect of Acidic Alkaline and Aqueous Media Similarlyfor evaluation of acidic alkaline or hydrolytic stress gem-citabine (5mg) was weighed accurately and transferred to100mL flask These samples were shaken (at 60∘C 1 h) witheither hydrochloric acid (5mL 1NHCl) or sodiumhydroxide(5mL 1NNaOHor 20mLwater) After one hour the contentwas cooled and processed as described above (in oxidativestress)

(3) Effect of UV Light or Sunlight Gemcitabine (500mg)was placed in an open watch glass and exposed to either

International Journal of Analytical Chemistry 5

Table 5 Factorial design data for robustness of chromatographic separation

(a)

Selected parameters and their variations minus1 (lower limit) +1 (upper limit)Acetonitrile in mobile phase () (119860) 8 12Final pH of the mobile phase (119861) 68 72Column oven temperature (∘C) (119862) 20 30Flow rate (mLmin) (119863) 08 12

(b)

Exp number Run order 119860 119861 119862 119863 119877119904

119879119891-D 119879

119891-I

1 6 8 68 20 08 1401 112 122 11 12 68 20 08 131 111 1193 5 8 72 20 08 141 109 1184 13 12 72 20 08 132 108 115 1 8 68 30 08 143 109 1116 7 12 68 30 08 102 109 1087 10 8 72 30 08 145 111 1158 15 12 72 30 08 1185 11 1089 12 8 68 20 12 1351 113 11110 14 12 68 20 12 1021 105 10711 9 8 72 20 12 1441 109 11212 2 12 72 20 12 1005 106 10713 8 8 68 30 12 1325 112 1114 4 12 68 30 12 1015 104 10715 3 8 72 30 12 1412 11 11116 16 12 72 30 12 1015 105 105119877119904 resolution factor 119879119891-D tailing factor for drug and 119879119891-I tailing factor for IS

UV-irradiation (sim100Wm2) or direct sunlight for one hourwith occasionally shifting of the content using stainless steelspatula After exposure 5mg of sample was weighed andtransferred to 100mL volumetric flask internal standard(2mL) was added to it and further processed as describedabove

Chromatograms of these sample solutions were recordedand compared with the chromatograms of unexposed API

(4) Stock StabilityThestability of stock solutionwas evaluatedat zero time and stored in the refrigerator (2ndash8∘C) Sampleswere prepared and analyzed at days 0 7 14 and 21

3 Results and Discussion

31 Analytical Method Development In order to achieveoptimum separation various parameters like solvent solventstrength detection wavelength flow rate elution time asym-metry and plate numbers were considered During optimiza-tion gemcitabine hydrochloride and internal standard wereinjected into various mobile phases of water methanol orwater acetonitrile (90 10 80 20 70 30 60 40 and 50 50pH 5 6 or 7) and the retention time tailing factor along withresolution factor was recorded In certain mobile phases thepeak was distorted while in others the compound eluted outquickly indicating the lesser retention time and thus lesser

separation on the column As the pKa of gemcitabine is 35and unstable in an acidic pH [3] different mobile phases ofpH 70 were used Mobile phase of acetonitrile-water (10 90vv pH adjusted to 70) was used as suitable mobile phaseas it was able to separate the analytes Using the C-18 ODSanalytical columnwith an isocraticmobile phase at a flow rateof 1 mLmin the drug and IS were eluted at sim40 and 78minrespectively Although temperature was found not to be acritical parameter for this analysis it was set at 25∘C to avoidshifting of signals The absorption maximum of the drug at275 nm was selected for detection (Figure 2) as there wasno interference from excipients present in drug or baselinedisturbance The resolution factor was sim11 Figure 3 depictsthe representative chromatogram obtained with the presentmethod

32 Method Validation The method was validated withrespect to parameters including linearity limit of quantitation(LOQ) limit of detection (LOD) precision accuracy speci-ficity robustness system suitability and stability

321 Linearity Different calibration curves (119899 = 6) that wereconstructed for gemcitabine were linear over the concentra-tion range of 05ndash50120583gmL Peak area ratios of gemcitabineto IS were plotted versus gemcitabine concentration and

6 International Journal of Analytical Chemistry

0

05

1

A B C D

minus05

minus1

minus2

minus15

AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(a)

0

0005

001

0015

minus0005

minus001

minus002

minus0015

minus0025

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(b)

0

001

002

minus001

minus002

minus003

minus004

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(c)

Figure 4 Scaled and centered coefficient of variation () of (a) resolution factor (b) tailing factor of drug and (c) tailing factor of IS duringrobustness studies

Table 6 Stability data under different stressed conditions

Stress conditions Percent gemcitabine remained Retention time of degraded products

Alkaline stress (1 N NaOH 60∘C 1 h) 161 plusmn 02 3023 (d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944(d-5) and 5375 (d-6)

Acidic stress (1 N HCl 60∘C 1 h) 950 plusmn 02 4953 (d-5) 6082 (d-7) and 7131 (d-8)Oxidative stress (5 60∘C 1 h) 971 plusmn 01 3772 (d-3)Aqueous hydrolytic stress (60∘C 1 h) 992 plusmn 01 4952 (d-5)Ultraviolet light (100Wm2 1 h) 1000 00Direct sunlight (1 h) 1000 00Aqueous stability (after 21 days) 999 plusmn 01 00

Table 7 Assay of formulations

Sample Label claim (mgvial) (119899 = 6) Amount found assay RSDMean plusmn SD

Batch 1 200 19935 plusmn 046 997 023Batch 2 200 19927 plusmn 052 996 026

linear regressionwas performed using Spinchrome-Clarity orLC-solution software Different calibration curves (119899 = 6)were prepared on three different days The mean regressionequation for gemcitabine was found to be 119910 = 00353119909 +00063 with 09998 correlation coefficient using weighting

factor-119909 (Table 1) The linearity range reported in othermethods ranged between 0020 and 300 120583gmL [3 5ndash28]

322 LOD and LOQ The LOD and LOQ values were 01498and 04541120583gmL calculated using calibration curve as per

International Journal of Analytical Chemistry 7

0

00

10

20

(min)

(mAU

)

1 PDA multi1275nm4nm3023d-1

3645d-3

3422

5375d-6

4944d-5

4342d-4

3202d-2

7589

theo

phyl

line

3965

gem

cita

bine

10025 50 75

(a)

0 2 4 6 8 10340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Peak purity index 1000000Impurity not detected

Single point threshold 0998065Minimum peak purity index 1934

39 40 41 42000

025

050

075

0

10

20

Retention time 3964Compound name gemcitabine

(min)

ID2

(c)

05

1015202530354045

200 300

267822363222395 25075

33965

(mAU

)(nm)

Retention time 3965Compound name gemcitabine

ID2

(d)

102030405060

7080

90100

200 300

27635

24183

(mAU

)

(nm)

Retention time 3023Compound name d-1

ID6

minus0

(e)

05

1015202530354045

200 300

25830

23260

(mAU

)

(nm)

Retention time 3202Compound name d-2

ID7

(f)

05

10

15

20

25

200 300

25862

22758

(mAU

)

(nm)

Retention time 4342Compound name d-4

ID9

minus00

(g)

0

5

10

15

20

200 300

25728

22795

34558

(mAU

)

(nm)

Retention time 4944Compound name d-5

ID3

(h)

Figure 5 Continued

8 International Journal of Analytical Chemistry

102030405060708090

100110

200 300

20214

27131

2421833250

(mAU

)

(nm)

Retention time 7589Compound name theophylline

ID 1

minus0

(i)

Figure 5 Typical HPLC chromatogram of (a) gemcitabine exposed to alkaline stress (1 N NaOH 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectra of (d) gemcitabine (e) degraded product d-1 (f) d-2 (g) d-4 (h) d-5 and (i) theophylline

ICH guideline The LOD and LOQ reported by Lanz et al[14] were 10 and 20 ngmL based on signal to noise ratiomethod while Xu et al [7] reported 12 and 375 ngmL basedon calibration curve (external standard method)

323 Accuracy and Precision The accuracy and precisionof the analytical method were established across its linearrange as indicated in the guideline As shown from the datain Table 2 excellent recoveries were made at different addedconcentration level The results obtained for the intraday andinterday precision of themethod expressed as RSD values Asshown in the table the intraday and interday RSDwerelt20for all concentrations tested in different situations studied(Tables 2 and 3)

324 Specificity Specificity of the method was assessed bycomparing the chromatograms obtained from lyophilizedpowder from internal standard and from the drug standardsThe retention times of drug from standard solutions andfrom lyophilized powder were identical and no coelutingpeaks from the diluents were observed indicating specificmethod for quantitative estimation of drug in the commercialformulation

325 System Suitability System suitability parameters werestudied with six replicates standard solution of the drug andthe calculated parameters are within the acceptance criteriaThe tailing factor the number of theoretical plates andHETPwere in the acceptable limits (RSD less than 2) The systemsuitability results are shown in Table 4

326 Robustness Robustness of the methods was illustratedby getting the resolution factor and tailing factor whenmobile phase flow rate (plusmn02mLmin) acetonitrile content(plusmn2) pH (plusmn02 units) and column temperature (plusmn5∘C)were deliberately varied It was studied using factorial design

experiment The deliberate changes in the method do notaffected the resolution tailing factor of drug and IS signifi-cantly The scaled and centered coefficient plots for the aboveresponses revealed that different parameters did not affectresponses so that the developed method was consideredrugged and robust Results are presented in Figure 4 andTable 5

327 Stability Studies The prepared stock and workingsolutions were stable up to 21 days when stored in refrigerator(2ndash8∘C) and did not produce degraded compounds duringexperimental conditions The peak purity was 0985 ormore during the validation studies Gemcitabine producessix different degradation products on alkaline stress withretention time (min) 3023 (as d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944 (d-5) and 5375 (d-6) The percentageof gemcitabine remained was 161 Mastanamma et al [11]and Kudikala et al [12] have reported two degraded productof gemcitabine Figure 5 represents the chromatogram con-tour plot peak purity and UV spectra of gemcitabine anddegraded products The extracted UV spectra indicate thatthe entire degradation products are derived fromgemcitabineor its intermediates In case of acidic stress the degradationproducts were observed at 4953 (as d-5) 6082 (d-7) and7131min (d-8) (Figure 6) Jansen et al [3] have reported thepresence of a coeluted degraded product with gemcitabinewhich does not possess UV absorption at 275 nm Thehydrolytic product d-5 (495min) was observed in alkalineas well as in aqueous stress condition On exposure tohydrogen peroxide (5 60∘C 1 h) gemcitabine producesonly one minor degradation products having retention time3772min (Table 6 Figure 7) The percentage of unoxi-dized gemcitabine was 971 Gemcitabine was completelydegraded on exposure to drastic oxidative condition (5060∘C 1 h) However these degraded compounds have noultraviolet (UV) absorbance at 275 nm the wavelength usedto monitor the gemcitabine concentrations Borisagar et al

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

International Journal of Analytical Chemistry 3

Table 1 Linearity data of the proposed method

Conc (120583gmL) Mean peak area (gemcitabine) (119899 = 6) Mean peak area (IS) Mean area ratio Conc found (120583gmL) assay05 115562 4901386 002358 050 99010 221962 5272420 004210 103 102620 401196 5162028 007772 205 102350 992068 5492566 018062 499 999100 1977022 5469878 036144 1018 1018150 2980964 5516108 054041 1530 1020250 4960840 5603166 088536 2519 1008400 7917220 5666864 139711 3985 996500 9875806 5534780 178432 5095 1019

119910 = 00353119909 + 00063 1199032 = 09998

Table 2 Accuracy of the method

Amount taken (120583gmL) Amount added Amount recovered (mean plusmn SD) (119899 = 6) recovery (mean plusmn SD) RSD 120583gmL

20 25 5 2509 plusmn 010 10037 plusmn 040 04020 50 10 3006 plusmn 009 10019 plusmn 031 03120 80 16 3615 plusmn 021 10042 plusmn 059 05920 100 20 4014 plusmn 013 10034 plusmn 033 03320 120 24 4407 plusmn 006 10015 plusmn 013 013

different batches were studiedTheir aluminum closures wereremoved The vials were weighed with the drug and afterremoving the content in empty stateWith the help of the dataavailable weight of the lyophilized powder was calculated Anaccurately weighed portion from this powder equivalent to10mg of drugwas transferred to 100mL calibrated volumetricflask and 10mL of internal standard solution was also trans-ferred to it quantitatively 50mL of methanol was added tothe flask and the contents of the flask were swirled sonicatedfor 5min and then completed to volume with water Theprepared solutions were diluted quantitatively with mobilephase to obtain a solution of 20120583gmL drug and internalstandard for the analysis

25 Analytical Method Validation

251 Linearity Limit of Detection (LOD) and Limit of Quan-titation (LOQ) Appropriate volumes of gemcitabine stockstandard solution (1000mgmL) were diluted with mobilephase to produce concentrations of 05 10 20 50 1015 25 40 and 50 120583gmL Replicates of each concentrationwere independently prepared and injected into the chromato-graph Linearity was evaluated by the linear least-squaresregression model using weighting factor 119909 Microsoft officeexcel 2007 was used for statistical analysis The method wasvalidated according to ICH guidelines of the validation ofanalytical methods [29] A 5 significance level was usedfor evaluation The method was evaluated by determinationof the correlation coefficient and intercept values LOD andLOQ were determined from the best fitted calibration curveLOD and LOQwere calculated as 33times120590

119899minus1119878 and 10times120590

119899minus1119878

where 120590119899minus1

is the standard deviation of the intercept and 119878 isthe slope of the calibration curve

252 Precision Precision was measured using triplicatedetermination of quality control samples of 1 5 20 30and 45 120583gmL of gemcitabine on different occasions (0 3and 6 h) and different days The precision (RSD) of themethod was determined as intraday precision (repeatability)and intermediate precision The intermediate precision wasestimated from the RSD of the analysis of the samplesprepared at the same concentration but on 3 different daysat different concentration levels while intraday precision wascalculated by analyzing the same concentration during thesame day at different time

253 Accuracy Accuracy of method was determined byaddition of known amounts of gemcitabine (119899 = 3 ateach level of 25 50 80 100 and 120 levels) to a samplesolution of known concentration (formulation) From thesesolutions appropriate solution was prepared and analyzedand the total amount recovered was calculated In this workthe mean recovery of the target concentration was 100 plusmn 2for acceptance

254 Robustness It is a measure of reproducibility of testresults under normal expected operational condition fromanalyst to analyst The robustness of the method was evalu-ated on the basis of precision as measured by percent coef-ficient of variation (RSD) determined as each concentrationlevel was required not to exceed 2 Design of experiments

4 International Journal of Analytical Chemistry

Table 3 Precision study of the proposed method

Concentration (120583gmL)Intraday precision Interday precision

Conc found RSD Conc found RSDMean plusmn SD Mean plusmn SD

1 0994 plusmn 0015 152 0985 plusmn 0018 1815 5030 plusmn 0074 148 5041 plusmn 0092 18320 19920 plusmn 0243 122 19840 plusmn 0280 14130 29886 plusmn 0292 098 29861 plusmn 0233 07845 44759 plusmn 0255 057 44742 plusmn 0326 073

Table 4 System suitability

Parameters Mean RSDTheoretical plates (Drug) 7716 125Platesmeter 30864 125HETP 3110 013Tailing factor 110 011LOD (120583gmL) 01498 105LOQ (120583gmL) 04541 105Resolution (Rs) 110 045Retention time of drug 395min 150Retention time of IS 780min 150

was used to study robustness of the method A 24 factorialdesign was used to test the robustness of chromatographicseparation The experimental design is useful for this kind ofstudy as it facilitates the investigation of several parametersby reducing the number of experiments Acetonitrile contentof the mobile phase pH column oven temperature and flowrate was investigated Upper and lower limits are shown inTable 5 The experiments were run randomly with samplecontaining gemcitabine and internal standard (20 120583gmL ofeach) The selected responses were resolution (119877

119904) tailing

factor of drug (119879119891-D) and tailing factor of IS (119879

119891-I)

255 Stability Studies Stress study like oxidative alkalineand acidic stress exposure to sunlight andUV light (254 nm)was carried out using raw material Chromatograms wererecorded in order to study the specificity of the methodThe chromatograms of the samples were compared withthose of control samples that were freshly prepared from thestock standard solution and without stress All samples wereanalyzed in triplicate The peak purity was checked using thetools of the LC-Solution software This assessment was basedon the comparison of spectra recorded during the elution ofthe peak UV spectra and peak purity were used to assesspurity of analytes

(1) Oxidative Stress Gemcitabine (5mg) was weighed accu-rately and transferred to 100mL flask for evaluation ofoxidative stress 10mL of 5 hydrogen peroxide was added toit It was shaken for one hour at 60∘C and then contents werecooled to room temperature Internal standard (2mL) was

(nm)20000 25000 30000 32500

Abs

2000

1500

1000

0500

0000minus0150

Figure 2 UV spectra of the gemcitabine in mobile phase

(min)

(mAU

)

00 25 50 75 100

0

25

50

1 PDA multi 1275nm 4nm

3997

gem

cita

bine

7777

theo

phyl

line

Figure 3 Representative chromatogram showing signals of gemc-itabine and theophylline in the selected mobile phase

added the contents were transferred quantitatively to 100mLvolumetric flask and diluted to the mark with mobile phase

(2) Effect of Acidic Alkaline and Aqueous Media Similarlyfor evaluation of acidic alkaline or hydrolytic stress gem-citabine (5mg) was weighed accurately and transferred to100mL flask These samples were shaken (at 60∘C 1 h) witheither hydrochloric acid (5mL 1NHCl) or sodiumhydroxide(5mL 1NNaOHor 20mLwater) After one hour the contentwas cooled and processed as described above (in oxidativestress)

(3) Effect of UV Light or Sunlight Gemcitabine (500mg)was placed in an open watch glass and exposed to either

International Journal of Analytical Chemistry 5

Table 5 Factorial design data for robustness of chromatographic separation

(a)

Selected parameters and their variations minus1 (lower limit) +1 (upper limit)Acetonitrile in mobile phase () (119860) 8 12Final pH of the mobile phase (119861) 68 72Column oven temperature (∘C) (119862) 20 30Flow rate (mLmin) (119863) 08 12

(b)

Exp number Run order 119860 119861 119862 119863 119877119904

119879119891-D 119879

119891-I

1 6 8 68 20 08 1401 112 122 11 12 68 20 08 131 111 1193 5 8 72 20 08 141 109 1184 13 12 72 20 08 132 108 115 1 8 68 30 08 143 109 1116 7 12 68 30 08 102 109 1087 10 8 72 30 08 145 111 1158 15 12 72 30 08 1185 11 1089 12 8 68 20 12 1351 113 11110 14 12 68 20 12 1021 105 10711 9 8 72 20 12 1441 109 11212 2 12 72 20 12 1005 106 10713 8 8 68 30 12 1325 112 1114 4 12 68 30 12 1015 104 10715 3 8 72 30 12 1412 11 11116 16 12 72 30 12 1015 105 105119877119904 resolution factor 119879119891-D tailing factor for drug and 119879119891-I tailing factor for IS

UV-irradiation (sim100Wm2) or direct sunlight for one hourwith occasionally shifting of the content using stainless steelspatula After exposure 5mg of sample was weighed andtransferred to 100mL volumetric flask internal standard(2mL) was added to it and further processed as describedabove

Chromatograms of these sample solutions were recordedand compared with the chromatograms of unexposed API

(4) Stock StabilityThestability of stock solutionwas evaluatedat zero time and stored in the refrigerator (2ndash8∘C) Sampleswere prepared and analyzed at days 0 7 14 and 21

3 Results and Discussion

31 Analytical Method Development In order to achieveoptimum separation various parameters like solvent solventstrength detection wavelength flow rate elution time asym-metry and plate numbers were considered During optimiza-tion gemcitabine hydrochloride and internal standard wereinjected into various mobile phases of water methanol orwater acetonitrile (90 10 80 20 70 30 60 40 and 50 50pH 5 6 or 7) and the retention time tailing factor along withresolution factor was recorded In certain mobile phases thepeak was distorted while in others the compound eluted outquickly indicating the lesser retention time and thus lesser

separation on the column As the pKa of gemcitabine is 35and unstable in an acidic pH [3] different mobile phases ofpH 70 were used Mobile phase of acetonitrile-water (10 90vv pH adjusted to 70) was used as suitable mobile phaseas it was able to separate the analytes Using the C-18 ODSanalytical columnwith an isocraticmobile phase at a flow rateof 1 mLmin the drug and IS were eluted at sim40 and 78minrespectively Although temperature was found not to be acritical parameter for this analysis it was set at 25∘C to avoidshifting of signals The absorption maximum of the drug at275 nm was selected for detection (Figure 2) as there wasno interference from excipients present in drug or baselinedisturbance The resolution factor was sim11 Figure 3 depictsthe representative chromatogram obtained with the presentmethod

32 Method Validation The method was validated withrespect to parameters including linearity limit of quantitation(LOQ) limit of detection (LOD) precision accuracy speci-ficity robustness system suitability and stability

321 Linearity Different calibration curves (119899 = 6) that wereconstructed for gemcitabine were linear over the concentra-tion range of 05ndash50120583gmL Peak area ratios of gemcitabineto IS were plotted versus gemcitabine concentration and

6 International Journal of Analytical Chemistry

0

05

1

A B C D

minus05

minus1

minus2

minus15

AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(a)

0

0005

001

0015

minus0005

minus001

minus002

minus0015

minus0025

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(b)

0

001

002

minus001

minus002

minus003

minus004

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(c)

Figure 4 Scaled and centered coefficient of variation () of (a) resolution factor (b) tailing factor of drug and (c) tailing factor of IS duringrobustness studies

Table 6 Stability data under different stressed conditions

Stress conditions Percent gemcitabine remained Retention time of degraded products

Alkaline stress (1 N NaOH 60∘C 1 h) 161 plusmn 02 3023 (d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944(d-5) and 5375 (d-6)

Acidic stress (1 N HCl 60∘C 1 h) 950 plusmn 02 4953 (d-5) 6082 (d-7) and 7131 (d-8)Oxidative stress (5 60∘C 1 h) 971 plusmn 01 3772 (d-3)Aqueous hydrolytic stress (60∘C 1 h) 992 plusmn 01 4952 (d-5)Ultraviolet light (100Wm2 1 h) 1000 00Direct sunlight (1 h) 1000 00Aqueous stability (after 21 days) 999 plusmn 01 00

Table 7 Assay of formulations

Sample Label claim (mgvial) (119899 = 6) Amount found assay RSDMean plusmn SD

Batch 1 200 19935 plusmn 046 997 023Batch 2 200 19927 plusmn 052 996 026

linear regressionwas performed using Spinchrome-Clarity orLC-solution software Different calibration curves (119899 = 6)were prepared on three different days The mean regressionequation for gemcitabine was found to be 119910 = 00353119909 +00063 with 09998 correlation coefficient using weighting

factor-119909 (Table 1) The linearity range reported in othermethods ranged between 0020 and 300 120583gmL [3 5ndash28]

322 LOD and LOQ The LOD and LOQ values were 01498and 04541120583gmL calculated using calibration curve as per

International Journal of Analytical Chemistry 7

0

00

10

20

(min)

(mAU

)

1 PDA multi1275nm4nm3023d-1

3645d-3

3422

5375d-6

4944d-5

4342d-4

3202d-2

7589

theo

phyl

line

3965

gem

cita

bine

10025 50 75

(a)

0 2 4 6 8 10340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Peak purity index 1000000Impurity not detected

Single point threshold 0998065Minimum peak purity index 1934

39 40 41 42000

025

050

075

0

10

20

Retention time 3964Compound name gemcitabine

(min)

ID2

(c)

05

1015202530354045

200 300

267822363222395 25075

33965

(mAU

)(nm)

Retention time 3965Compound name gemcitabine

ID2

(d)

102030405060

7080

90100

200 300

27635

24183

(mAU

)

(nm)

Retention time 3023Compound name d-1

ID6

minus0

(e)

05

1015202530354045

200 300

25830

23260

(mAU

)

(nm)

Retention time 3202Compound name d-2

ID7

(f)

05

10

15

20

25

200 300

25862

22758

(mAU

)

(nm)

Retention time 4342Compound name d-4

ID9

minus00

(g)

0

5

10

15

20

200 300

25728

22795

34558

(mAU

)

(nm)

Retention time 4944Compound name d-5

ID3

(h)

Figure 5 Continued

8 International Journal of Analytical Chemistry

102030405060708090

100110

200 300

20214

27131

2421833250

(mAU

)

(nm)

Retention time 7589Compound name theophylline

ID 1

minus0

(i)

Figure 5 Typical HPLC chromatogram of (a) gemcitabine exposed to alkaline stress (1 N NaOH 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectra of (d) gemcitabine (e) degraded product d-1 (f) d-2 (g) d-4 (h) d-5 and (i) theophylline

ICH guideline The LOD and LOQ reported by Lanz et al[14] were 10 and 20 ngmL based on signal to noise ratiomethod while Xu et al [7] reported 12 and 375 ngmL basedon calibration curve (external standard method)

323 Accuracy and Precision The accuracy and precisionof the analytical method were established across its linearrange as indicated in the guideline As shown from the datain Table 2 excellent recoveries were made at different addedconcentration level The results obtained for the intraday andinterday precision of themethod expressed as RSD values Asshown in the table the intraday and interday RSDwerelt20for all concentrations tested in different situations studied(Tables 2 and 3)

324 Specificity Specificity of the method was assessed bycomparing the chromatograms obtained from lyophilizedpowder from internal standard and from the drug standardsThe retention times of drug from standard solutions andfrom lyophilized powder were identical and no coelutingpeaks from the diluents were observed indicating specificmethod for quantitative estimation of drug in the commercialformulation

325 System Suitability System suitability parameters werestudied with six replicates standard solution of the drug andthe calculated parameters are within the acceptance criteriaThe tailing factor the number of theoretical plates andHETPwere in the acceptable limits (RSD less than 2) The systemsuitability results are shown in Table 4

326 Robustness Robustness of the methods was illustratedby getting the resolution factor and tailing factor whenmobile phase flow rate (plusmn02mLmin) acetonitrile content(plusmn2) pH (plusmn02 units) and column temperature (plusmn5∘C)were deliberately varied It was studied using factorial design

experiment The deliberate changes in the method do notaffected the resolution tailing factor of drug and IS signifi-cantly The scaled and centered coefficient plots for the aboveresponses revealed that different parameters did not affectresponses so that the developed method was consideredrugged and robust Results are presented in Figure 4 andTable 5

327 Stability Studies The prepared stock and workingsolutions were stable up to 21 days when stored in refrigerator(2ndash8∘C) and did not produce degraded compounds duringexperimental conditions The peak purity was 0985 ormore during the validation studies Gemcitabine producessix different degradation products on alkaline stress withretention time (min) 3023 (as d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944 (d-5) and 5375 (d-6) The percentageof gemcitabine remained was 161 Mastanamma et al [11]and Kudikala et al [12] have reported two degraded productof gemcitabine Figure 5 represents the chromatogram con-tour plot peak purity and UV spectra of gemcitabine anddegraded products The extracted UV spectra indicate thatthe entire degradation products are derived fromgemcitabineor its intermediates In case of acidic stress the degradationproducts were observed at 4953 (as d-5) 6082 (d-7) and7131min (d-8) (Figure 6) Jansen et al [3] have reported thepresence of a coeluted degraded product with gemcitabinewhich does not possess UV absorption at 275 nm Thehydrolytic product d-5 (495min) was observed in alkalineas well as in aqueous stress condition On exposure tohydrogen peroxide (5 60∘C 1 h) gemcitabine producesonly one minor degradation products having retention time3772min (Table 6 Figure 7) The percentage of unoxi-dized gemcitabine was 971 Gemcitabine was completelydegraded on exposure to drastic oxidative condition (5060∘C 1 h) However these degraded compounds have noultraviolet (UV) absorbance at 275 nm the wavelength usedto monitor the gemcitabine concentrations Borisagar et al

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 International Journal of Analytical Chemistry

Table 3 Precision study of the proposed method

Concentration (120583gmL)Intraday precision Interday precision

Conc found RSD Conc found RSDMean plusmn SD Mean plusmn SD

1 0994 plusmn 0015 152 0985 plusmn 0018 1815 5030 plusmn 0074 148 5041 plusmn 0092 18320 19920 plusmn 0243 122 19840 plusmn 0280 14130 29886 plusmn 0292 098 29861 plusmn 0233 07845 44759 plusmn 0255 057 44742 plusmn 0326 073

Table 4 System suitability

Parameters Mean RSDTheoretical plates (Drug) 7716 125Platesmeter 30864 125HETP 3110 013Tailing factor 110 011LOD (120583gmL) 01498 105LOQ (120583gmL) 04541 105Resolution (Rs) 110 045Retention time of drug 395min 150Retention time of IS 780min 150

was used to study robustness of the method A 24 factorialdesign was used to test the robustness of chromatographicseparation The experimental design is useful for this kind ofstudy as it facilitates the investigation of several parametersby reducing the number of experiments Acetonitrile contentof the mobile phase pH column oven temperature and flowrate was investigated Upper and lower limits are shown inTable 5 The experiments were run randomly with samplecontaining gemcitabine and internal standard (20 120583gmL ofeach) The selected responses were resolution (119877

119904) tailing

factor of drug (119879119891-D) and tailing factor of IS (119879

119891-I)

255 Stability Studies Stress study like oxidative alkalineand acidic stress exposure to sunlight andUV light (254 nm)was carried out using raw material Chromatograms wererecorded in order to study the specificity of the methodThe chromatograms of the samples were compared withthose of control samples that were freshly prepared from thestock standard solution and without stress All samples wereanalyzed in triplicate The peak purity was checked using thetools of the LC-Solution software This assessment was basedon the comparison of spectra recorded during the elution ofthe peak UV spectra and peak purity were used to assesspurity of analytes

(1) Oxidative Stress Gemcitabine (5mg) was weighed accu-rately and transferred to 100mL flask for evaluation ofoxidative stress 10mL of 5 hydrogen peroxide was added toit It was shaken for one hour at 60∘C and then contents werecooled to room temperature Internal standard (2mL) was

(nm)20000 25000 30000 32500

Abs

2000

1500

1000

0500

0000minus0150

Figure 2 UV spectra of the gemcitabine in mobile phase

(min)

(mAU

)

00 25 50 75 100

0

25

50

1 PDA multi 1275nm 4nm

3997

gem

cita

bine

7777

theo

phyl

line

Figure 3 Representative chromatogram showing signals of gemc-itabine and theophylline in the selected mobile phase

added the contents were transferred quantitatively to 100mLvolumetric flask and diluted to the mark with mobile phase

(2) Effect of Acidic Alkaline and Aqueous Media Similarlyfor evaluation of acidic alkaline or hydrolytic stress gem-citabine (5mg) was weighed accurately and transferred to100mL flask These samples were shaken (at 60∘C 1 h) witheither hydrochloric acid (5mL 1NHCl) or sodiumhydroxide(5mL 1NNaOHor 20mLwater) After one hour the contentwas cooled and processed as described above (in oxidativestress)

(3) Effect of UV Light or Sunlight Gemcitabine (500mg)was placed in an open watch glass and exposed to either

International Journal of Analytical Chemistry 5

Table 5 Factorial design data for robustness of chromatographic separation

(a)

Selected parameters and their variations minus1 (lower limit) +1 (upper limit)Acetonitrile in mobile phase () (119860) 8 12Final pH of the mobile phase (119861) 68 72Column oven temperature (∘C) (119862) 20 30Flow rate (mLmin) (119863) 08 12

(b)

Exp number Run order 119860 119861 119862 119863 119877119904

119879119891-D 119879

119891-I

1 6 8 68 20 08 1401 112 122 11 12 68 20 08 131 111 1193 5 8 72 20 08 141 109 1184 13 12 72 20 08 132 108 115 1 8 68 30 08 143 109 1116 7 12 68 30 08 102 109 1087 10 8 72 30 08 145 111 1158 15 12 72 30 08 1185 11 1089 12 8 68 20 12 1351 113 11110 14 12 68 20 12 1021 105 10711 9 8 72 20 12 1441 109 11212 2 12 72 20 12 1005 106 10713 8 8 68 30 12 1325 112 1114 4 12 68 30 12 1015 104 10715 3 8 72 30 12 1412 11 11116 16 12 72 30 12 1015 105 105119877119904 resolution factor 119879119891-D tailing factor for drug and 119879119891-I tailing factor for IS

UV-irradiation (sim100Wm2) or direct sunlight for one hourwith occasionally shifting of the content using stainless steelspatula After exposure 5mg of sample was weighed andtransferred to 100mL volumetric flask internal standard(2mL) was added to it and further processed as describedabove

Chromatograms of these sample solutions were recordedand compared with the chromatograms of unexposed API

(4) Stock StabilityThestability of stock solutionwas evaluatedat zero time and stored in the refrigerator (2ndash8∘C) Sampleswere prepared and analyzed at days 0 7 14 and 21

3 Results and Discussion

31 Analytical Method Development In order to achieveoptimum separation various parameters like solvent solventstrength detection wavelength flow rate elution time asym-metry and plate numbers were considered During optimiza-tion gemcitabine hydrochloride and internal standard wereinjected into various mobile phases of water methanol orwater acetonitrile (90 10 80 20 70 30 60 40 and 50 50pH 5 6 or 7) and the retention time tailing factor along withresolution factor was recorded In certain mobile phases thepeak was distorted while in others the compound eluted outquickly indicating the lesser retention time and thus lesser

separation on the column As the pKa of gemcitabine is 35and unstable in an acidic pH [3] different mobile phases ofpH 70 were used Mobile phase of acetonitrile-water (10 90vv pH adjusted to 70) was used as suitable mobile phaseas it was able to separate the analytes Using the C-18 ODSanalytical columnwith an isocraticmobile phase at a flow rateof 1 mLmin the drug and IS were eluted at sim40 and 78minrespectively Although temperature was found not to be acritical parameter for this analysis it was set at 25∘C to avoidshifting of signals The absorption maximum of the drug at275 nm was selected for detection (Figure 2) as there wasno interference from excipients present in drug or baselinedisturbance The resolution factor was sim11 Figure 3 depictsthe representative chromatogram obtained with the presentmethod

32 Method Validation The method was validated withrespect to parameters including linearity limit of quantitation(LOQ) limit of detection (LOD) precision accuracy speci-ficity robustness system suitability and stability

321 Linearity Different calibration curves (119899 = 6) that wereconstructed for gemcitabine were linear over the concentra-tion range of 05ndash50120583gmL Peak area ratios of gemcitabineto IS were plotted versus gemcitabine concentration and

6 International Journal of Analytical Chemistry

0

05

1

A B C D

minus05

minus1

minus2

minus15

AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(a)

0

0005

001

0015

minus0005

minus001

minus002

minus0015

minus0025

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(b)

0

001

002

minus001

minus002

minus003

minus004

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(c)

Figure 4 Scaled and centered coefficient of variation () of (a) resolution factor (b) tailing factor of drug and (c) tailing factor of IS duringrobustness studies

Table 6 Stability data under different stressed conditions

Stress conditions Percent gemcitabine remained Retention time of degraded products

Alkaline stress (1 N NaOH 60∘C 1 h) 161 plusmn 02 3023 (d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944(d-5) and 5375 (d-6)

Acidic stress (1 N HCl 60∘C 1 h) 950 plusmn 02 4953 (d-5) 6082 (d-7) and 7131 (d-8)Oxidative stress (5 60∘C 1 h) 971 plusmn 01 3772 (d-3)Aqueous hydrolytic stress (60∘C 1 h) 992 plusmn 01 4952 (d-5)Ultraviolet light (100Wm2 1 h) 1000 00Direct sunlight (1 h) 1000 00Aqueous stability (after 21 days) 999 plusmn 01 00

Table 7 Assay of formulations

Sample Label claim (mgvial) (119899 = 6) Amount found assay RSDMean plusmn SD

Batch 1 200 19935 plusmn 046 997 023Batch 2 200 19927 plusmn 052 996 026

linear regressionwas performed using Spinchrome-Clarity orLC-solution software Different calibration curves (119899 = 6)were prepared on three different days The mean regressionequation for gemcitabine was found to be 119910 = 00353119909 +00063 with 09998 correlation coefficient using weighting

factor-119909 (Table 1) The linearity range reported in othermethods ranged between 0020 and 300 120583gmL [3 5ndash28]

322 LOD and LOQ The LOD and LOQ values were 01498and 04541120583gmL calculated using calibration curve as per

International Journal of Analytical Chemistry 7

0

00

10

20

(min)

(mAU

)

1 PDA multi1275nm4nm3023d-1

3645d-3

3422

5375d-6

4944d-5

4342d-4

3202d-2

7589

theo

phyl

line

3965

gem

cita

bine

10025 50 75

(a)

0 2 4 6 8 10340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Peak purity index 1000000Impurity not detected

Single point threshold 0998065Minimum peak purity index 1934

39 40 41 42000

025

050

075

0

10

20

Retention time 3964Compound name gemcitabine

(min)

ID2

(c)

05

1015202530354045

200 300

267822363222395 25075

33965

(mAU

)(nm)

Retention time 3965Compound name gemcitabine

ID2

(d)

102030405060

7080

90100

200 300

27635

24183

(mAU

)

(nm)

Retention time 3023Compound name d-1

ID6

minus0

(e)

05

1015202530354045

200 300

25830

23260

(mAU

)

(nm)

Retention time 3202Compound name d-2

ID7

(f)

05

10

15

20

25

200 300

25862

22758

(mAU

)

(nm)

Retention time 4342Compound name d-4

ID9

minus00

(g)

0

5

10

15

20

200 300

25728

22795

34558

(mAU

)

(nm)

Retention time 4944Compound name d-5

ID3

(h)

Figure 5 Continued

8 International Journal of Analytical Chemistry

102030405060708090

100110

200 300

20214

27131

2421833250

(mAU

)

(nm)

Retention time 7589Compound name theophylline

ID 1

minus0

(i)

Figure 5 Typical HPLC chromatogram of (a) gemcitabine exposed to alkaline stress (1 N NaOH 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectra of (d) gemcitabine (e) degraded product d-1 (f) d-2 (g) d-4 (h) d-5 and (i) theophylline

ICH guideline The LOD and LOQ reported by Lanz et al[14] were 10 and 20 ngmL based on signal to noise ratiomethod while Xu et al [7] reported 12 and 375 ngmL basedon calibration curve (external standard method)

323 Accuracy and Precision The accuracy and precisionof the analytical method were established across its linearrange as indicated in the guideline As shown from the datain Table 2 excellent recoveries were made at different addedconcentration level The results obtained for the intraday andinterday precision of themethod expressed as RSD values Asshown in the table the intraday and interday RSDwerelt20for all concentrations tested in different situations studied(Tables 2 and 3)

324 Specificity Specificity of the method was assessed bycomparing the chromatograms obtained from lyophilizedpowder from internal standard and from the drug standardsThe retention times of drug from standard solutions andfrom lyophilized powder were identical and no coelutingpeaks from the diluents were observed indicating specificmethod for quantitative estimation of drug in the commercialformulation

325 System Suitability System suitability parameters werestudied with six replicates standard solution of the drug andthe calculated parameters are within the acceptance criteriaThe tailing factor the number of theoretical plates andHETPwere in the acceptable limits (RSD less than 2) The systemsuitability results are shown in Table 4

326 Robustness Robustness of the methods was illustratedby getting the resolution factor and tailing factor whenmobile phase flow rate (plusmn02mLmin) acetonitrile content(plusmn2) pH (plusmn02 units) and column temperature (plusmn5∘C)were deliberately varied It was studied using factorial design

experiment The deliberate changes in the method do notaffected the resolution tailing factor of drug and IS signifi-cantly The scaled and centered coefficient plots for the aboveresponses revealed that different parameters did not affectresponses so that the developed method was consideredrugged and robust Results are presented in Figure 4 andTable 5

327 Stability Studies The prepared stock and workingsolutions were stable up to 21 days when stored in refrigerator(2ndash8∘C) and did not produce degraded compounds duringexperimental conditions The peak purity was 0985 ormore during the validation studies Gemcitabine producessix different degradation products on alkaline stress withretention time (min) 3023 (as d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944 (d-5) and 5375 (d-6) The percentageof gemcitabine remained was 161 Mastanamma et al [11]and Kudikala et al [12] have reported two degraded productof gemcitabine Figure 5 represents the chromatogram con-tour plot peak purity and UV spectra of gemcitabine anddegraded products The extracted UV spectra indicate thatthe entire degradation products are derived fromgemcitabineor its intermediates In case of acidic stress the degradationproducts were observed at 4953 (as d-5) 6082 (d-7) and7131min (d-8) (Figure 6) Jansen et al [3] have reported thepresence of a coeluted degraded product with gemcitabinewhich does not possess UV absorption at 275 nm Thehydrolytic product d-5 (495min) was observed in alkalineas well as in aqueous stress condition On exposure tohydrogen peroxide (5 60∘C 1 h) gemcitabine producesonly one minor degradation products having retention time3772min (Table 6 Figure 7) The percentage of unoxi-dized gemcitabine was 971 Gemcitabine was completelydegraded on exposure to drastic oxidative condition (5060∘C 1 h) However these degraded compounds have noultraviolet (UV) absorbance at 275 nm the wavelength usedto monitor the gemcitabine concentrations Borisagar et al

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

International Journal of Analytical Chemistry 5

Table 5 Factorial design data for robustness of chromatographic separation

(a)

Selected parameters and their variations minus1 (lower limit) +1 (upper limit)Acetonitrile in mobile phase () (119860) 8 12Final pH of the mobile phase (119861) 68 72Column oven temperature (∘C) (119862) 20 30Flow rate (mLmin) (119863) 08 12

(b)

Exp number Run order 119860 119861 119862 119863 119877119904

119879119891-D 119879

119891-I

1 6 8 68 20 08 1401 112 122 11 12 68 20 08 131 111 1193 5 8 72 20 08 141 109 1184 13 12 72 20 08 132 108 115 1 8 68 30 08 143 109 1116 7 12 68 30 08 102 109 1087 10 8 72 30 08 145 111 1158 15 12 72 30 08 1185 11 1089 12 8 68 20 12 1351 113 11110 14 12 68 20 12 1021 105 10711 9 8 72 20 12 1441 109 11212 2 12 72 20 12 1005 106 10713 8 8 68 30 12 1325 112 1114 4 12 68 30 12 1015 104 10715 3 8 72 30 12 1412 11 11116 16 12 72 30 12 1015 105 105119877119904 resolution factor 119879119891-D tailing factor for drug and 119879119891-I tailing factor for IS

UV-irradiation (sim100Wm2) or direct sunlight for one hourwith occasionally shifting of the content using stainless steelspatula After exposure 5mg of sample was weighed andtransferred to 100mL volumetric flask internal standard(2mL) was added to it and further processed as describedabove

Chromatograms of these sample solutions were recordedand compared with the chromatograms of unexposed API

(4) Stock StabilityThestability of stock solutionwas evaluatedat zero time and stored in the refrigerator (2ndash8∘C) Sampleswere prepared and analyzed at days 0 7 14 and 21

3 Results and Discussion

31 Analytical Method Development In order to achieveoptimum separation various parameters like solvent solventstrength detection wavelength flow rate elution time asym-metry and plate numbers were considered During optimiza-tion gemcitabine hydrochloride and internal standard wereinjected into various mobile phases of water methanol orwater acetonitrile (90 10 80 20 70 30 60 40 and 50 50pH 5 6 or 7) and the retention time tailing factor along withresolution factor was recorded In certain mobile phases thepeak was distorted while in others the compound eluted outquickly indicating the lesser retention time and thus lesser

separation on the column As the pKa of gemcitabine is 35and unstable in an acidic pH [3] different mobile phases ofpH 70 were used Mobile phase of acetonitrile-water (10 90vv pH adjusted to 70) was used as suitable mobile phaseas it was able to separate the analytes Using the C-18 ODSanalytical columnwith an isocraticmobile phase at a flow rateof 1 mLmin the drug and IS were eluted at sim40 and 78minrespectively Although temperature was found not to be acritical parameter for this analysis it was set at 25∘C to avoidshifting of signals The absorption maximum of the drug at275 nm was selected for detection (Figure 2) as there wasno interference from excipients present in drug or baselinedisturbance The resolution factor was sim11 Figure 3 depictsthe representative chromatogram obtained with the presentmethod

32 Method Validation The method was validated withrespect to parameters including linearity limit of quantitation(LOQ) limit of detection (LOD) precision accuracy speci-ficity robustness system suitability and stability

321 Linearity Different calibration curves (119899 = 6) that wereconstructed for gemcitabine were linear over the concentra-tion range of 05ndash50120583gmL Peak area ratios of gemcitabineto IS were plotted versus gemcitabine concentration and

6 International Journal of Analytical Chemistry

0

05

1

A B C D

minus05

minus1

minus2

minus15

AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(a)

0

0005

001

0015

minus0005

minus001

minus002

minus0015

minus0025

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(b)

0

001

002

minus001

minus002

minus003

minus004

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(c)

Figure 4 Scaled and centered coefficient of variation () of (a) resolution factor (b) tailing factor of drug and (c) tailing factor of IS duringrobustness studies

Table 6 Stability data under different stressed conditions

Stress conditions Percent gemcitabine remained Retention time of degraded products

Alkaline stress (1 N NaOH 60∘C 1 h) 161 plusmn 02 3023 (d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944(d-5) and 5375 (d-6)

Acidic stress (1 N HCl 60∘C 1 h) 950 plusmn 02 4953 (d-5) 6082 (d-7) and 7131 (d-8)Oxidative stress (5 60∘C 1 h) 971 plusmn 01 3772 (d-3)Aqueous hydrolytic stress (60∘C 1 h) 992 plusmn 01 4952 (d-5)Ultraviolet light (100Wm2 1 h) 1000 00Direct sunlight (1 h) 1000 00Aqueous stability (after 21 days) 999 plusmn 01 00

Table 7 Assay of formulations

Sample Label claim (mgvial) (119899 = 6) Amount found assay RSDMean plusmn SD

Batch 1 200 19935 plusmn 046 997 023Batch 2 200 19927 plusmn 052 996 026

linear regressionwas performed using Spinchrome-Clarity orLC-solution software Different calibration curves (119899 = 6)were prepared on three different days The mean regressionequation for gemcitabine was found to be 119910 = 00353119909 +00063 with 09998 correlation coefficient using weighting

factor-119909 (Table 1) The linearity range reported in othermethods ranged between 0020 and 300 120583gmL [3 5ndash28]

322 LOD and LOQ The LOD and LOQ values were 01498and 04541120583gmL calculated using calibration curve as per

International Journal of Analytical Chemistry 7

0

00

10

20

(min)

(mAU

)

1 PDA multi1275nm4nm3023d-1

3645d-3

3422

5375d-6

4944d-5

4342d-4

3202d-2

7589

theo

phyl

line

3965

gem

cita

bine

10025 50 75

(a)

0 2 4 6 8 10340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Peak purity index 1000000Impurity not detected

Single point threshold 0998065Minimum peak purity index 1934

39 40 41 42000

025

050

075

0

10

20

Retention time 3964Compound name gemcitabine

(min)

ID2

(c)

05

1015202530354045

200 300

267822363222395 25075

33965

(mAU

)(nm)

Retention time 3965Compound name gemcitabine

ID2

(d)

102030405060

7080

90100

200 300

27635

24183

(mAU

)

(nm)

Retention time 3023Compound name d-1

ID6

minus0

(e)

05

1015202530354045

200 300

25830

23260

(mAU

)

(nm)

Retention time 3202Compound name d-2

ID7

(f)

05

10

15

20

25

200 300

25862

22758

(mAU

)

(nm)

Retention time 4342Compound name d-4

ID9

minus00

(g)

0

5

10

15

20

200 300

25728

22795

34558

(mAU

)

(nm)

Retention time 4944Compound name d-5

ID3

(h)

Figure 5 Continued

8 International Journal of Analytical Chemistry

102030405060708090

100110

200 300

20214

27131

2421833250

(mAU

)

(nm)

Retention time 7589Compound name theophylline

ID 1

minus0

(i)

Figure 5 Typical HPLC chromatogram of (a) gemcitabine exposed to alkaline stress (1 N NaOH 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectra of (d) gemcitabine (e) degraded product d-1 (f) d-2 (g) d-4 (h) d-5 and (i) theophylline

ICH guideline The LOD and LOQ reported by Lanz et al[14] were 10 and 20 ngmL based on signal to noise ratiomethod while Xu et al [7] reported 12 and 375 ngmL basedon calibration curve (external standard method)

323 Accuracy and Precision The accuracy and precisionof the analytical method were established across its linearrange as indicated in the guideline As shown from the datain Table 2 excellent recoveries were made at different addedconcentration level The results obtained for the intraday andinterday precision of themethod expressed as RSD values Asshown in the table the intraday and interday RSDwerelt20for all concentrations tested in different situations studied(Tables 2 and 3)

324 Specificity Specificity of the method was assessed bycomparing the chromatograms obtained from lyophilizedpowder from internal standard and from the drug standardsThe retention times of drug from standard solutions andfrom lyophilized powder were identical and no coelutingpeaks from the diluents were observed indicating specificmethod for quantitative estimation of drug in the commercialformulation

325 System Suitability System suitability parameters werestudied with six replicates standard solution of the drug andthe calculated parameters are within the acceptance criteriaThe tailing factor the number of theoretical plates andHETPwere in the acceptable limits (RSD less than 2) The systemsuitability results are shown in Table 4

326 Robustness Robustness of the methods was illustratedby getting the resolution factor and tailing factor whenmobile phase flow rate (plusmn02mLmin) acetonitrile content(plusmn2) pH (plusmn02 units) and column temperature (plusmn5∘C)were deliberately varied It was studied using factorial design

experiment The deliberate changes in the method do notaffected the resolution tailing factor of drug and IS signifi-cantly The scaled and centered coefficient plots for the aboveresponses revealed that different parameters did not affectresponses so that the developed method was consideredrugged and robust Results are presented in Figure 4 andTable 5

327 Stability Studies The prepared stock and workingsolutions were stable up to 21 days when stored in refrigerator(2ndash8∘C) and did not produce degraded compounds duringexperimental conditions The peak purity was 0985 ormore during the validation studies Gemcitabine producessix different degradation products on alkaline stress withretention time (min) 3023 (as d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944 (d-5) and 5375 (d-6) The percentageof gemcitabine remained was 161 Mastanamma et al [11]and Kudikala et al [12] have reported two degraded productof gemcitabine Figure 5 represents the chromatogram con-tour plot peak purity and UV spectra of gemcitabine anddegraded products The extracted UV spectra indicate thatthe entire degradation products are derived fromgemcitabineor its intermediates In case of acidic stress the degradationproducts were observed at 4953 (as d-5) 6082 (d-7) and7131min (d-8) (Figure 6) Jansen et al [3] have reported thepresence of a coeluted degraded product with gemcitabinewhich does not possess UV absorption at 275 nm Thehydrolytic product d-5 (495min) was observed in alkalineas well as in aqueous stress condition On exposure tohydrogen peroxide (5 60∘C 1 h) gemcitabine producesonly one minor degradation products having retention time3772min (Table 6 Figure 7) The percentage of unoxi-dized gemcitabine was 971 Gemcitabine was completelydegraded on exposure to drastic oxidative condition (5060∘C 1 h) However these degraded compounds have noultraviolet (UV) absorbance at 275 nm the wavelength usedto monitor the gemcitabine concentrations Borisagar et al

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 International Journal of Analytical Chemistry

0

05

1

A B C D

minus05

minus1

minus2

minus15

AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(a)

0

0005

001

0015

minus0005

minus001

minus002

minus0015

minus0025

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(b)

0

001

002

minus001

minus002

minus003

minus004

A B C D AlowastB

AlowastC

AlowastD

BlowastC

BlowastD

ClowastD

(c)

Figure 4 Scaled and centered coefficient of variation () of (a) resolution factor (b) tailing factor of drug and (c) tailing factor of IS duringrobustness studies

Table 6 Stability data under different stressed conditions

Stress conditions Percent gemcitabine remained Retention time of degraded products

Alkaline stress (1 N NaOH 60∘C 1 h) 161 plusmn 02 3023 (d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944(d-5) and 5375 (d-6)

Acidic stress (1 N HCl 60∘C 1 h) 950 plusmn 02 4953 (d-5) 6082 (d-7) and 7131 (d-8)Oxidative stress (5 60∘C 1 h) 971 plusmn 01 3772 (d-3)Aqueous hydrolytic stress (60∘C 1 h) 992 plusmn 01 4952 (d-5)Ultraviolet light (100Wm2 1 h) 1000 00Direct sunlight (1 h) 1000 00Aqueous stability (after 21 days) 999 plusmn 01 00

Table 7 Assay of formulations

Sample Label claim (mgvial) (119899 = 6) Amount found assay RSDMean plusmn SD

Batch 1 200 19935 plusmn 046 997 023Batch 2 200 19927 plusmn 052 996 026

linear regressionwas performed using Spinchrome-Clarity orLC-solution software Different calibration curves (119899 = 6)were prepared on three different days The mean regressionequation for gemcitabine was found to be 119910 = 00353119909 +00063 with 09998 correlation coefficient using weighting

factor-119909 (Table 1) The linearity range reported in othermethods ranged between 0020 and 300 120583gmL [3 5ndash28]

322 LOD and LOQ The LOD and LOQ values were 01498and 04541120583gmL calculated using calibration curve as per

International Journal of Analytical Chemistry 7

0

00

10

20

(min)

(mAU

)

1 PDA multi1275nm4nm3023d-1

3645d-3

3422

5375d-6

4944d-5

4342d-4

3202d-2

7589

theo

phyl

line

3965

gem

cita

bine

10025 50 75

(a)

0 2 4 6 8 10340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Peak purity index 1000000Impurity not detected

Single point threshold 0998065Minimum peak purity index 1934

39 40 41 42000

025

050

075

0

10

20

Retention time 3964Compound name gemcitabine

(min)

ID2

(c)

05

1015202530354045

200 300

267822363222395 25075

33965

(mAU

)(nm)

Retention time 3965Compound name gemcitabine

ID2

(d)

102030405060

7080

90100

200 300

27635

24183

(mAU

)

(nm)

Retention time 3023Compound name d-1

ID6

minus0

(e)

05

1015202530354045

200 300

25830

23260

(mAU

)

(nm)

Retention time 3202Compound name d-2

ID7

(f)

05

10

15

20

25

200 300

25862

22758

(mAU

)

(nm)

Retention time 4342Compound name d-4

ID9

minus00

(g)

0

5

10

15

20

200 300

25728

22795

34558

(mAU

)

(nm)

Retention time 4944Compound name d-5

ID3

(h)

Figure 5 Continued

8 International Journal of Analytical Chemistry

102030405060708090

100110

200 300

20214

27131

2421833250

(mAU

)

(nm)

Retention time 7589Compound name theophylline

ID 1

minus0

(i)

Figure 5 Typical HPLC chromatogram of (a) gemcitabine exposed to alkaline stress (1 N NaOH 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectra of (d) gemcitabine (e) degraded product d-1 (f) d-2 (g) d-4 (h) d-5 and (i) theophylline

ICH guideline The LOD and LOQ reported by Lanz et al[14] were 10 and 20 ngmL based on signal to noise ratiomethod while Xu et al [7] reported 12 and 375 ngmL basedon calibration curve (external standard method)

323 Accuracy and Precision The accuracy and precisionof the analytical method were established across its linearrange as indicated in the guideline As shown from the datain Table 2 excellent recoveries were made at different addedconcentration level The results obtained for the intraday andinterday precision of themethod expressed as RSD values Asshown in the table the intraday and interday RSDwerelt20for all concentrations tested in different situations studied(Tables 2 and 3)

324 Specificity Specificity of the method was assessed bycomparing the chromatograms obtained from lyophilizedpowder from internal standard and from the drug standardsThe retention times of drug from standard solutions andfrom lyophilized powder were identical and no coelutingpeaks from the diluents were observed indicating specificmethod for quantitative estimation of drug in the commercialformulation

325 System Suitability System suitability parameters werestudied with six replicates standard solution of the drug andthe calculated parameters are within the acceptance criteriaThe tailing factor the number of theoretical plates andHETPwere in the acceptable limits (RSD less than 2) The systemsuitability results are shown in Table 4

326 Robustness Robustness of the methods was illustratedby getting the resolution factor and tailing factor whenmobile phase flow rate (plusmn02mLmin) acetonitrile content(plusmn2) pH (plusmn02 units) and column temperature (plusmn5∘C)were deliberately varied It was studied using factorial design

experiment The deliberate changes in the method do notaffected the resolution tailing factor of drug and IS signifi-cantly The scaled and centered coefficient plots for the aboveresponses revealed that different parameters did not affectresponses so that the developed method was consideredrugged and robust Results are presented in Figure 4 andTable 5

327 Stability Studies The prepared stock and workingsolutions were stable up to 21 days when stored in refrigerator(2ndash8∘C) and did not produce degraded compounds duringexperimental conditions The peak purity was 0985 ormore during the validation studies Gemcitabine producessix different degradation products on alkaline stress withretention time (min) 3023 (as d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944 (d-5) and 5375 (d-6) The percentageof gemcitabine remained was 161 Mastanamma et al [11]and Kudikala et al [12] have reported two degraded productof gemcitabine Figure 5 represents the chromatogram con-tour plot peak purity and UV spectra of gemcitabine anddegraded products The extracted UV spectra indicate thatthe entire degradation products are derived fromgemcitabineor its intermediates In case of acidic stress the degradationproducts were observed at 4953 (as d-5) 6082 (d-7) and7131min (d-8) (Figure 6) Jansen et al [3] have reported thepresence of a coeluted degraded product with gemcitabinewhich does not possess UV absorption at 275 nm Thehydrolytic product d-5 (495min) was observed in alkalineas well as in aqueous stress condition On exposure tohydrogen peroxide (5 60∘C 1 h) gemcitabine producesonly one minor degradation products having retention time3772min (Table 6 Figure 7) The percentage of unoxi-dized gemcitabine was 971 Gemcitabine was completelydegraded on exposure to drastic oxidative condition (5060∘C 1 h) However these degraded compounds have noultraviolet (UV) absorbance at 275 nm the wavelength usedto monitor the gemcitabine concentrations Borisagar et al

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

International Journal of Analytical Chemistry 7

0

00

10

20

(min)

(mAU

)

1 PDA multi1275nm4nm3023d-1

3645d-3

3422

5375d-6

4944d-5

4342d-4

3202d-2

7589

theo

phyl

line

3965

gem

cita

bine

10025 50 75

(a)

0 2 4 6 8 10340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Peak purity index 1000000Impurity not detected

Single point threshold 0998065Minimum peak purity index 1934

39 40 41 42000

025

050

075

0

10

20

Retention time 3964Compound name gemcitabine

(min)

ID2

(c)

05

1015202530354045

200 300

267822363222395 25075

33965

(mAU

)(nm)

Retention time 3965Compound name gemcitabine

ID2

(d)

102030405060

7080

90100

200 300

27635

24183

(mAU

)

(nm)

Retention time 3023Compound name d-1

ID6

minus0

(e)

05

1015202530354045

200 300

25830

23260

(mAU

)

(nm)

Retention time 3202Compound name d-2

ID7

(f)

05

10

15

20

25

200 300

25862

22758

(mAU

)

(nm)

Retention time 4342Compound name d-4

ID9

minus00

(g)

0

5

10

15

20

200 300

25728

22795

34558

(mAU

)

(nm)

Retention time 4944Compound name d-5

ID3

(h)

Figure 5 Continued

8 International Journal of Analytical Chemistry

102030405060708090

100110

200 300

20214

27131

2421833250

(mAU

)

(nm)

Retention time 7589Compound name theophylline

ID 1

minus0

(i)

Figure 5 Typical HPLC chromatogram of (a) gemcitabine exposed to alkaline stress (1 N NaOH 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectra of (d) gemcitabine (e) degraded product d-1 (f) d-2 (g) d-4 (h) d-5 and (i) theophylline

ICH guideline The LOD and LOQ reported by Lanz et al[14] were 10 and 20 ngmL based on signal to noise ratiomethod while Xu et al [7] reported 12 and 375 ngmL basedon calibration curve (external standard method)

323 Accuracy and Precision The accuracy and precisionof the analytical method were established across its linearrange as indicated in the guideline As shown from the datain Table 2 excellent recoveries were made at different addedconcentration level The results obtained for the intraday andinterday precision of themethod expressed as RSD values Asshown in the table the intraday and interday RSDwerelt20for all concentrations tested in different situations studied(Tables 2 and 3)

324 Specificity Specificity of the method was assessed bycomparing the chromatograms obtained from lyophilizedpowder from internal standard and from the drug standardsThe retention times of drug from standard solutions andfrom lyophilized powder were identical and no coelutingpeaks from the diluents were observed indicating specificmethod for quantitative estimation of drug in the commercialformulation

325 System Suitability System suitability parameters werestudied with six replicates standard solution of the drug andthe calculated parameters are within the acceptance criteriaThe tailing factor the number of theoretical plates andHETPwere in the acceptable limits (RSD less than 2) The systemsuitability results are shown in Table 4

326 Robustness Robustness of the methods was illustratedby getting the resolution factor and tailing factor whenmobile phase flow rate (plusmn02mLmin) acetonitrile content(plusmn2) pH (plusmn02 units) and column temperature (plusmn5∘C)were deliberately varied It was studied using factorial design

experiment The deliberate changes in the method do notaffected the resolution tailing factor of drug and IS signifi-cantly The scaled and centered coefficient plots for the aboveresponses revealed that different parameters did not affectresponses so that the developed method was consideredrugged and robust Results are presented in Figure 4 andTable 5

327 Stability Studies The prepared stock and workingsolutions were stable up to 21 days when stored in refrigerator(2ndash8∘C) and did not produce degraded compounds duringexperimental conditions The peak purity was 0985 ormore during the validation studies Gemcitabine producessix different degradation products on alkaline stress withretention time (min) 3023 (as d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944 (d-5) and 5375 (d-6) The percentageof gemcitabine remained was 161 Mastanamma et al [11]and Kudikala et al [12] have reported two degraded productof gemcitabine Figure 5 represents the chromatogram con-tour plot peak purity and UV spectra of gemcitabine anddegraded products The extracted UV spectra indicate thatthe entire degradation products are derived fromgemcitabineor its intermediates In case of acidic stress the degradationproducts were observed at 4953 (as d-5) 6082 (d-7) and7131min (d-8) (Figure 6) Jansen et al [3] have reported thepresence of a coeluted degraded product with gemcitabinewhich does not possess UV absorption at 275 nm Thehydrolytic product d-5 (495min) was observed in alkalineas well as in aqueous stress condition On exposure tohydrogen peroxide (5 60∘C 1 h) gemcitabine producesonly one minor degradation products having retention time3772min (Table 6 Figure 7) The percentage of unoxi-dized gemcitabine was 971 Gemcitabine was completelydegraded on exposure to drastic oxidative condition (5060∘C 1 h) However these degraded compounds have noultraviolet (UV) absorbance at 275 nm the wavelength usedto monitor the gemcitabine concentrations Borisagar et al

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 International Journal of Analytical Chemistry

102030405060708090

100110

200 300

20214

27131

2421833250

(mAU

)

(nm)

Retention time 7589Compound name theophylline

ID 1

minus0

(i)

Figure 5 Typical HPLC chromatogram of (a) gemcitabine exposed to alkaline stress (1 N NaOH 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectra of (d) gemcitabine (e) degraded product d-1 (f) d-2 (g) d-4 (h) d-5 and (i) theophylline

ICH guideline The LOD and LOQ reported by Lanz et al[14] were 10 and 20 ngmL based on signal to noise ratiomethod while Xu et al [7] reported 12 and 375 ngmL basedon calibration curve (external standard method)

323 Accuracy and Precision The accuracy and precisionof the analytical method were established across its linearrange as indicated in the guideline As shown from the datain Table 2 excellent recoveries were made at different addedconcentration level The results obtained for the intraday andinterday precision of themethod expressed as RSD values Asshown in the table the intraday and interday RSDwerelt20for all concentrations tested in different situations studied(Tables 2 and 3)

324 Specificity Specificity of the method was assessed bycomparing the chromatograms obtained from lyophilizedpowder from internal standard and from the drug standardsThe retention times of drug from standard solutions andfrom lyophilized powder were identical and no coelutingpeaks from the diluents were observed indicating specificmethod for quantitative estimation of drug in the commercialformulation

325 System Suitability System suitability parameters werestudied with six replicates standard solution of the drug andthe calculated parameters are within the acceptance criteriaThe tailing factor the number of theoretical plates andHETPwere in the acceptable limits (RSD less than 2) The systemsuitability results are shown in Table 4

326 Robustness Robustness of the methods was illustratedby getting the resolution factor and tailing factor whenmobile phase flow rate (plusmn02mLmin) acetonitrile content(plusmn2) pH (plusmn02 units) and column temperature (plusmn5∘C)were deliberately varied It was studied using factorial design

experiment The deliberate changes in the method do notaffected the resolution tailing factor of drug and IS signifi-cantly The scaled and centered coefficient plots for the aboveresponses revealed that different parameters did not affectresponses so that the developed method was consideredrugged and robust Results are presented in Figure 4 andTable 5

327 Stability Studies The prepared stock and workingsolutions were stable up to 21 days when stored in refrigerator(2ndash8∘C) and did not produce degraded compounds duringexperimental conditions The peak purity was 0985 ormore during the validation studies Gemcitabine producessix different degradation products on alkaline stress withretention time (min) 3023 (as d-1) 3202 (d-2) 3645 (d-3) 4342 (d-4) 4944 (d-5) and 5375 (d-6) The percentageof gemcitabine remained was 161 Mastanamma et al [11]and Kudikala et al [12] have reported two degraded productof gemcitabine Figure 5 represents the chromatogram con-tour plot peak purity and UV spectra of gemcitabine anddegraded products The extracted UV spectra indicate thatthe entire degradation products are derived fromgemcitabineor its intermediates In case of acidic stress the degradationproducts were observed at 4953 (as d-5) 6082 (d-7) and7131min (d-8) (Figure 6) Jansen et al [3] have reported thepresence of a coeluted degraded product with gemcitabinewhich does not possess UV absorption at 275 nm Thehydrolytic product d-5 (495min) was observed in alkalineas well as in aqueous stress condition On exposure tohydrogen peroxide (5 60∘C 1 h) gemcitabine producesonly one minor degradation products having retention time3772min (Table 6 Figure 7) The percentage of unoxi-dized gemcitabine was 971 Gemcitabine was completelydegraded on exposure to drastic oxidative condition (5060∘C 1 h) However these degraded compounds have noultraviolet (UV) absorbance at 275 nm the wavelength usedto monitor the gemcitabine concentrations Borisagar et al

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

International Journal of Analytical Chemistry 9

Table8Com

paris

onbetweenanalyticalmetho

ds

Snu

mber

Analytic

almetho

d(reference)

Drugs

Colum

nDetectio

n(120582

max)

Silent

features

andadvantages

Disa

dvantage

1HPL

C-PD

A(Ja

nsen

etal

2000)[3]

Gem

citabine

Zorbax

C-8(5120583m46times

250m

m)

275n

mSensitivesim

plem

etho

dapplicableforseparationof

drug

anddegraded

prod

ucts

Degradatedprod

uctswere

identifi

edusingspectro

scop

y

Gradientelutio

nOnlythed

egradatedprod

uctsof

acidic

stressstudies

weres

eparatedR

untim

e20

min

2HPL

C(Rao

etal2007)[6]

Gem

citabine

ODScolumn(5120583m46times

250m

m)

234n

mLinearity

rang

e1ndash300120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notp

erform

ed

3HPL

C(Xuetal2014)[7]

Gem

citabine

and

curcum

inPh

enom

enex

C-18

(5120583m

46times250m

m)

270n

m(G

em)

420n

m(curcumin)

LOD0012120583gmLLO

Q0038120583gmL

Linearity

range0038ndash15120583gmL

Sensitiveaccurateandprecise

metho

ddevelopedusing

desig

nof

experim

ent

Gradientelutio

nnarrow

rangeo

flinearityruntim

e15minN

onsta

bilityindicatin

gmetho

d(effectof

pHand

oxidation

expo

sure

tosunlight

orUVlightw

asno

tstu

died)

4HPL

C(M

astanammae

tal

2010)[11]

Gem

citabine

C-18

(5120583m46times250m

m)

270n

mLinearity

range10ndash6

0120583gmLSimplea

ndrapidsta

bility

indicatin

gmetho

dOrganicwaste(40

methano

l)andlowsensitivity

5HPL

C(Kud

ikalae

tal2014)

[12]

Gem

citabine

EnableC1

8Gcolumn(5120583m

46times250m

m)

285n

mLO

Q1120583gmLLinearity

rang

e1ndash45120583gmLSimple

stabilityindicatin

gmetho

dHydrolytic

(aq)a

ndoxidatived

egradedprod

uctsno

tstu

diedG

emcitabine

show

shightailing

factor

6HPL

C(D

evanaboyinae

tal

2014)[13]

Gem

citabine

Krom

asil(5120583m46times

150m

m)

247n

mLinearity

rang

e50ndash300120583gmL

Highorganicw

aste(30

aceton

itrile)Stabilitystu

dies

notp

erform

ed

7HPL

C(Rajeshetal2011)

[10]

Gem

citabine

Capecitabine

Intersil3C-

18column

(5120583m46times150m

m)

260n

mLinearity

rang

e10ndash50120583

gmL

Highorganicw

aste(70

aceton

itrile)Stabilitystu

dies

notstudied

8HPL

C(Lanze

tal2007)

[14]

Gem

citabine

C18(3120583m

particlesiz

e46m

mtimes100m

m)

276n

m

LOQ002120583

gmL

Linearity

range002ndash20120583

gmL

Sensitive

metho

dfora

nalysis

ofdrug

inplasmaa

ndserum

Gradientelutio

nruntim

e17minA

pplicableon

lyfor

serum

andplasmas

amplesE

ffectof

pHoxidatio

nor

light

onsta

bilityof

rawmaterialformulationisno

tstu

died

9LC

-MS-MS(N

ussbaumer

etal2010)[28]

Gem

citabine

and

othera

nticancer

drugs

mdashMassspectrometry

(MS-MS)

LOQ025

Linearity

range025ndash2

ngm

LSensitivesim

plem

etho

dapplicableford

etectio

nof

severald

rugs

Lower

accuracy

(85ndash110

)a

ndprecision

(15

)Ap

plicableforb

ioequivalen

ceandph

armacok

inetic

studies

where

15precision

isperm

ittedG

radient

elutio

nruntim

e21min

10HPT

LC(Borisa

gare

tal

2012)[5]

Gem

citabine

mdash268n

mLinearity

rang

e500ndash

3000

ngspo

tRa

pidHPT

LCmetho

dfora

nalysisStabilityindicatin

gmetho

dmdash

11Prop

osed

HPL

Cmetho

dGem

citabine

Phenom

enex

Luna

C-18

column(5120583m46times

250m

m)

275n

m

LOD015120583gmLLinearity

rang

esfro

m05to

50120583gmL

Isocratic

econo

mical(le

ssorganicw

aste)effi

cient

stabilityindicatin

gmetho

dCa

pableo

fseparating

different

hydrolyticandoxidativep

rodu

ctso

fdrugwhich

canbe

estim

ated

separately

Symmetric

alpeak

shape

Degradedprod

uctsares

eparated

butn

otqu

antized

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

10 International Journal of Analytical Chemistry

25 50 75 100

0

00

10

20

(min)

(mAU

)

1 PDA multi 1275nm 4nm3964

gem

cita

bine

4953d-5

7131d-8

6082d-7

7615

theo

phyl

line

(a)

0 2 4 6 8340320300280260240220200

(min)

(nm

)

minus5

34

(b)

Retention time 3964Compound name gemcitabine

39 40 41 42 43

000

025

050

0

50

100

150

Peak purity index 1000000Impurity not detected

Single point threshold 0999923Minimum peak purity index 76

(min)

ID 2

(c)

(nm)

00102030405060

200 300

25655

22764

(mAU

)

Retention time 4953Compound name d-5

ID 3

(d)

(nm)

010020030040050060070

080090

100

200 300

21213

26987

20705

24760

(mAU

)

Retention time 6082Compound name d-7

ID 10

minus000

(e)

(nm)

005015025035045055065075085

200 300

21208

27031

20534

24518

31355

(mAU

)

Retention time 7131Compound name d-8

ID 5

(f)

Figure 6 Typical HPLC chromatogram of (a) gemcitabine exposed to acidic stress (1 N HCl 60∘C 1 h) (b) contour plot (c) peak purityindex and extracted UV spectrum of (d) degraded product d-5 (e) d-7 and (f) d-8

[5] reported the oxidative degradation [138] of gemcitabineutilizing HPTLC The present results indicate that usingappropriate chromatographic conditions the structurallyrelated degraded products of gemcitabine can be separatedwhich were not studied earlier (Table 8)

328 Assay The proposed method was applied to the deter-mination gemcitabine in injectable formulations The resultsof these assay yielded 996 (RSD 026) of labeled claimedLow value of precision indicates that the method can be usedprecisely for the estimation of drug in formulations (Table 7)

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

International Journal of Analytical Chemistry 11

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

3965

gem

cita

bine

7621

theo

phyl

line

4952

d-5

(a)

(min)

(mAU

)

00 25 50 75 100

0

10

20

1 PDA multi 1275nm 4nm

416

0ge

mci

tabi

ne

7531

theo

phyl

line

3772

d-3

(b)

Figure 7 Typical HPLC chromatogram of gemcitabine exposed to (a) hydrolytic (H2O 60∘C 1 h) and (b) oxidative stress (5 60∘C 1 h)

4 Conclusion

A validated HPLC method has been developed for determi-nation of gemcitabine in formulationsThe proposed stabilityindicating method is simple economical accurate precisespecific and robust Method is capable of separating differentdegraded products of drugwhich can be estimated separatelyThe experimental design was found to be very useful intesting the robustness of chromatographic separation duringthe validation step Hence this method can be easily andconveniently adopted for the routine analysis of gemcitabinein pharmaceutical dosage form

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the Head of Faculty of PharmacyIntegral University Lucknow India and the Dean of Facultyof Pharmacy andMedical Sciences Al-Ahliyya AmmanUni-versity Amman Jordan for providing necessary facilities

References

[1] Q Xu Y Zhang and L A Trissel ldquoPhysical and chemicalstability of gemcitabine hydrochloride solutionsrdquo Journal of theAmerican Pharmaceutical Association (Washington) vol 39 no4 pp 509ndash513 1999

[2] S L Anliker M S McClure T C Britton E A StephanS R Maple and G G Cooke ldquoDegradation chemistry ofgemcitabine hydrochloride a new antitumor agentrdquo Journal ofPharmaceutical Sciences vol 83 no 5 pp 716ndash719 1994

[3] P J Jansen M J Akers R M Amos et al ldquoThe degradationof the antitumor agent gemcitabine hydrochloride in an acidicaqueous solution at pH 32 and identification of degradationproductsrdquo Journal of Pharmaceutical Sciences vol 89 no 7 pp885ndash891 2000

[4] D G Sankar P V M Latha B A Kumar and P J BabuldquoUV spectrophotometric determination of temozolamide and

gemcitabinerdquoAsian Journal of Chemistry vol 19 no 2 pp 1605ndash1607 2007

[5] S L Borisagar H U Patel and C Patel ldquoA validated stability-indicating HPTLC method for the estimation of gemcitabineHCl in its dosage formrdquo Journal of Planar Chromatography vol25 no 1 pp 77ndash80 2012

[6] J V L N S Rao M M Krishna P B Prakash and P RKumar ldquoRP-HPLC analysis of gemcitabine in pure form and inpharmaceutical dosage formsrdquo Asian Journal of Chemistry vol19 no 5 pp 3399ndash3402 2007

[7] H Xu J Paxton J Lim Y Li and Z Wu ldquoDevelopment ofa gradient high performance liquid chromatography assay forsimultaneous analysis of hydrophilic gemcitabine and lipophiliccurcumin using a central composite design and its applica-tion in liposome developmentrdquo Journal of Pharmaceutical andBiomedical Analysis vol 98 pp 371ndash378 2014

[8] S S Bansal C Celia S Ferrati et al ldquoValidated RP-HPLCmethod for the simultaneous analysis of gemcitabine and LY-364947 in liposomal formulationsrdquo Current Drug Targets vol14 no 9 pp 1061ndash1069 2013

[9] Q Zhou L Liu D Zhang and X Fan ldquoAnalysis of gemcitabineliposome injection by HPLC with evaporative light scatteringdetectionrdquo Journal of Liposome Research vol 22 no 4 pp 263ndash269 2012

[10] V Rajesh B Anupama V Jagathi and P Sai PraveenldquoSimultaneous estimation of gemcitabine hydrochloride andcapecitabine hydrochloride in combined tablet dosage form byRP-HPLC methodrdquo E-Journal of Chemistry vol 8 no 3 pp1212ndash1217 2011

[11] S Mastanamma G Ramkumar D A Kumar and J V L N SRao ldquoA stability indicating RP-HPLCmethod for the estimationof gemcitabine HCl in injectable dosage formsrdquo E-Journal ofChemistry vol 7 no S1 pp S239ndashS244 2010

[12] S Kudikala S RMalladi SThota andV R Kumar ldquoRP-HPLCmethod for the estimation gemcitabine in API and parenteraldosage formrdquo Journal of Scientific Research in Pharmacy vol 3pp 16ndash18 2014

[13] N Devanaboyina S Sushma B Sekhar E Asha KMutyalamma and N Trimurthulu ldquoA novel RP-HPLCmethoddevelopment and validation for analysis of gemcitabine inbulk and pharmaceutical dosage formrdquo International Journal ofPharma Sciences vol 4 pp 522ndash525 2014

[14] C LanzM FruhWThormann TCerny andBH LauterburgldquoRapid determination of gemcitabine in plasma and serum

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

12 International Journal of Analytical Chemistry

using reversed-phase HPLCrdquo Journal of Separation Science vol30 no 12 pp 1811ndash1820 2007

[15] M N Kirstein I Hassan D E Guire et al ldquoHigh-performanceliquid chromatographic method for the determination of gem-citabine and 2101584021015840-difluorodeoxyuridine in plasma and tissueculture mediardquo Journal of Chromatography B vol 835 no 1-2pp 136ndash142 2006

[16] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[17] R Losa M I Sierra C Guardado et al ldquoDevelopmentand validation of an ion pair HPLC method for gemcitabineand 2101584021015840-difluoro-21015840-deoxyuridine determinationrdquo AnalyticaChimica Acta vol 528 no 2 pp 255ndash260 2005

[18] B Yılmaz and Y Kadıoglu ldquoComparison of zero- and second-order derivative spectrophotometric andHPLCmethods for thedetermination of gemcitabine in human plasmardquo Il-Farmacovol 59 no 5 pp 425ndash429 2004

[19] B Keith Y Xu and J L Grem ldquoMeasurement of the anti-canceragent gemcitabine in human plasma by high-performanceliquid chromatographyrdquo Journal of Chromatography B vol 785no 1 pp 65ndash72 2003

[20] B Yilmaz Y Kadıoglu and Y Aksoy ldquoSimultaneous deter-mination of gemcitabine and its metabolite in human plasmaby high-performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 791 no 1-2 pp 103ndash109 2003

[21] K B Freeman S Anliker M Hamilton et al ldquoValidatedassays for the determination of gemcitabine in human plasmaand urine using high-performance liquid chromatography withultraviolet detectionrdquo Journal of Chromatography B BiomedicalApplications vol 665 no 1 pp 171ndash181 1995

[22] N-M Lin S Zeng S-L Ma Y Fan H-J Zhong and L FangldquoDetermination of gemcitabine and its metabolite in humanplasma using high-pressure liquid chromatography coupledwith a diode array detectorrdquo Acta Pharmacologica Sinica vol25 no 12 pp 1584ndash1589 2004

[23] Y Xu B Keith and J L Grem ldquoMeasurement of the anticanceragent gemcitabine and its deaminated metabolite at low con-centrations in human plasma by liquid chromatography-massspectrometryrdquo Journal of Chromatography B vol 802 no 2 pp263ndash270 2004

[24] E Marangon F Sala O Caffo E Galligioni M DrsquoIncalciandM Zucchetti ldquoSimultaneous determination of gemcitabineand its main metabolite dFdU in plasma of patients withadvanced non-small-cell lung cancer by high-performanceliquid chromatography-tandem mass spectrometryrdquo Journal ofMass Spectrometry vol 43 no 2 pp 216ndash223 2008

[25] H Khoury A Deroussent L H Reddy P Couvreur G Vassaland A Paci ldquoSimultaneous determination of gemcitabine andgemcitabine-squalene by liquid chromatography-tandem massspectrometry in human plasmardquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol858 no 1-2 pp 71ndash78 2007

[26] L D Vainchtein H Rosing B Thijssen J H M Schellensand J H Beijnen ldquoValidated assay for the simultaneousdetermination of the anti-cancer agent gemcitabine and itsmetabolite 2101584021015840-difluorodeoxyuridine in human plasma byhigh-performance liquid chromatography with tandem mass

spectrometryrdquo Rapid Communications in Mass Spectrometryvol 21 no 14 pp 2312ndash2322 2007

[27] R Honeywell A C Laan C J van Groeningen et al ldquoThedetermination of gemcitabine and 21015840-deoxycytidine in humanplasma and tissue by APCI tandemmass spectrometryrdquo Journalof Chromatography B vol 847 no 2 pp 142ndash152 2007

[28] S Nussbaumer S Fleury-Souverain P Antinori et al ldquoSimul-taneous quantification of ten cytotoxic drugs by a validated LC-ESI-MSMS methodrdquo Analytical and Bioanalytical Chemistryvol 398 no 7-8 pp 3033ndash3042 2010

[29] IFPMA ldquoICH validation of analytical procedures text andmethodology Q2 (R1)rdquo in Proceedings of the International Con-ference on Harmonization pp 1ndash13 IFPMA Geneva Switzer-land 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of