Research Article High-Performance Liquid Chromatographic ...downloads.hindawi.com/archive/2013/329072.pdf · RP-HPLC method for a combination of tenofovir diso-proxil fumarate, emtricitabine,

Hindawi Publishing CorporationISRN ChromatographyVolume 2013 Article ID 329072 6 pageshttpdxdoiorg1011552013329072

Research ArticleHigh-Performance Liquid Chromatographic Method forAnalysis of Emtricitabine in Rat Plasma Method DevelopmentValidation and Application to a Pharmacokinetic Study

Gurinder Singh and Roopa S Pai

Department of Pharmaceutics Faculty of Pharmacy Al-Ameen College of Pharmacy Near Lal Bagh Main gate Hosur RoadBangalore Karnataka 560027 India

Correspondence should be addressed to Roopa S Pai roopaspaiyahoocom

Received 16 August 2013 Accepted 8 October 2013

Academic Editors F Couderc C Fanali J Millership and F J Senorans

Copyright copy 2013 G Singh and R S Pai This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A new reverse phase liquid chromatographic method for the investigation of emtricitabine in rat plasma was developed afteroral administration to Wistar rats The desired chromatographic separation was achieved on Phenomenex C

18column (250mm

times 46mm ID 5 120583m) column under isocratic conditions using UV detection at 280 nm The optimized mobile phase consistedof a mixture of 10mM potassium dihydrogen phosphate buffer- (adjusted to pH 68) methanol-2 acetic acid in a ratio of(73 25 2 vvv) at a flow rate of 1mLminminus1 The system was found to produce sharp and well-resolved peaks for emtricitabinewith retention time of 578min The linear regression analysis for the calibration curves showed a good linear correlation overthe concentration range 0050ndash30 120583gmLminus1 with determination coefficients R2 exceeding 09970 The limits of detection (LOD)and quantitation (LOQ) were found to be 0016120583gmLminus1 and 0049 120583gmLminus1 respectively The method was successfully appliedfor the pharmacokinetic in rats Emtricitabine concentration in plasma reached (119862max) was 1357 120583gmLminus1 about 2 h after oraladministration of 15mgkgrat The AUC

0minus24was 12175 120583gmLminus1lowast h and the apparent elimination half-life (t

12) was 8153 h This

method was found to be suitable for examining emtricitabine concentration in rats after oral administration of emtricitabine in asingle dose

1 Introduction

Emtricitabine (5-fluoro-1-(2R 5S)-[2-(hydroxymethyl)-13-oxathiolan-5-yl]cytosine) (Figure 1(a)) is a potent deoxy-cytidine nucleoside reverse transcriptase inhibitor for thetreatment of human immunodeficiency virus (HIV) infection[1] In adults emtricitabine recommended dose is 200mgonce a day (QD) [2] Both in vitro [3] and in vivo [4] testingdemonstrated that emtricitabine presents enough potentialto be tested in the prevention of HIV-1 either alone or incombination [5]

A few HPLC and a brief reference to one UPLC methodfor simultaneous determination of emtricitabine in com-bination with other antiretroviral drugs in human plasmaand rats have been described in the literature mainly withthe objective of method development for application to abioequivalence study [6ndash8] A simultaneous determination of

emtricitabine and tenofovir in human plasma was described[9] HPLC-UV detection method was developed for simul-taneous determination of emtricitabine and tenofovir intablet dosage form with LOQ of 0091120583gmLminus1 [10] A rapidRP-HPLC method for a combination of tenofovir diso-proxil fumarate emtricitabine and efavirenz was developedand subjected to forced degradation studies with LOQ ofemtricitabine that was 119 120583gmLminus1 [11] A validated RP-HPLC method for the estimation of emtricitabine was notappropriate for detection of low emtricitabine concentrationin capsules In this method the LOQ value was found to be16786 120583gmLminus1 and retention time was more than 9341min[12] In the above reported HPLC methods acetonitrile wasemployed as the organic phase It is a toxic chemical as it cancause pollution and health hazards to humans and animals[13]

2 ISRN Chromatography

N

N

F

S

O

OH

ONH2

(a)

N

N

O

S

O OH

NH2

(b)

Figure 1 Chemical structure of emtricitabine (a) and lamivudine (b)

The purpose of the present study was to develop andvalidate a simple and time-saving RP-HPLC method withUV detection for the investigation of emtricitabine after oraladministration to Wistar rats The method was validatedaccording to Food and Drug Administration (FDA) andInternational Conference on Harmonization (ICH) guide-lines with respect to linearity precision accuracy and speci-ficity and stability studies [14 15] Lamivudine (08 120583gmLminus1)was used as an internal standard (IS)

2 Experimental

21 Materials and Reagents Emtricitabine and lamivudine(Figure 1(b)) (998 ww and 987 ww HPLC) were pro-vided ex-gratis by MS Cipla Laboratories Mumbai IndiaHPLC grade methanol was purchased from SD fine-chemlimited (Mumbai India) Deionized water used in all theexperiments was passed through a Milli-Q water purifica-tion system (182MΩcm) Millipore (Bangalore KarnatakaIndia)

22 Instrumentation The HPLC (Shimadzu Kyoto Japan)instrument was equipped with binary pump and SPD-20AVPUVdetector Sample injection was done by Rheodyneinjector with a 50 120583L loop and a computer running Varianworkstation version 642 software for data acquisition andprocessingThe chromatographic analysis was carried out onPhenomenex C

18column (250mm times 46mm ID 5 120583m)

23 Chromatographic Conditions Chromatographic sepa-ration was achieved using Phenomenex C

18column The

mobile phase was composed of 10mMpotassium dihydrogenphosphate buffer (adjusted to pH 68)-methanol-2 aceticacid in a ratio of 73 25 2 (vvv) run under isocratic elutionand pumped at a flow rate of 1mL minminus1 The column wasthermostated at 30∘C Under these conditions the run timewas less than 8min

24 Optimization and Chromatographic Method Develop-ment Trial experiments were carried out initially in order tochoose most appropriate solvent system for precise analysisand to achieve good resolution Column chemistry solventtype solvent ratio (volume of organic solvents in the mobile

phase) flow rate detection and wavelength were varied toestablish the chromatographic conditions giving the goodseparation On the basis of time required for analysis sensi-tivity of the assay resolution time and use of readily availablecost-effective solvents the aptness of the mobile phase andthe flow rate was decided These included water-methanol(50 50 vv) 10mM pH 68 PBS-methanol (50 50 vv)10mM pH 68 PBS-methanol (70 30 vv) 10mM pH 68PBS-methanol-2 acetic acid (70 25 5 vvv) and 10mMpH 68 PBS-methanol-2 acetic acid (73 25 2 vvv) Amobile phase system comprised of 10mM potassium dihy-drogen phosphate buffer (pH 68)-methanol-2 acetic acid(73 25 2 vvv) at a flow rate of 1mL minminus1 was found to beoptimum The experimental work was performed in an air-conditioned room maintained at 20ndash25∘C

25 Preparation of Calibration Curve (CC) and Quality Con-trol Samples (QC) Eight-point calibration curve (CC) wasprepared by serial dilution of emtricitabine stock solution(100 120583gmLminus1) in the range of 0050 01 05 10 15 2025 and 3 120583gmLminus1 that were obtained by measuring therequired amount of 100 120583gmLminus1 working standard solu-tion mixed with a sufficient quantity of mobile phase andmaking up to 10mL Similarly six standard solutions wereprepared by serial dilution of emtricitabine stock solution(10 120583gmLminus1) in the range of 0005 0010 0020 0030 0040and 0050 120583gmLminus1 Six standard solutions were obtainedfrom the 10 120583g mLminus1 working standard solution in order todetermine the LOD and LOQ of the method

Calibration standards were prepared daily by spiking100 120583L of blank plasma with 10 120583L of the appropriate workingsolution resulting in concentrations of 0050 01 05 1015 20 25 and 3120583gmLminus1 and 0005 0010 0020 00300040 and 0050 120583gmLminus1 respectively of emtricitabineStock solution (08120583gmLminus1) of lamivudine (IS) was preparedin methanol and stored at minus20∘C The solutions were stablefor one day when stored at room temperature (20ndash25∘C)Thestock and standard solutions) were prepared on a daily basisand stored in the dark at about 5∘C All solutions were usedon the day they were prepared

Quality control (QC) samples (low quality control (LQC)01 120583gmLminus1 medium quality control (MQC) 10 120583gmLminus1

ISRN Chromatography 3

high quality control (HQC) 25 120583gmLminus1 limit of quantifica-tion (LOQ) 0049 120583gmLminus1 were prepared by spiking 01mLaliquot of blank plasma with 10 120583L of spiking solution of drugas well as the IS All solutions were stored in the refrigeratorat 40 plusmn 20∘C The bulk spiked CC and QC samples werestored at minus20∘C and brought to room temperature beforeuse

26 Sample Preparation To a 50 120583L of rat plasma 10 120583L of ISand 100 120583L of emtricitabine were added and the mixture wasincubated at 37∘C for 1 h Emtricitabine was then extractedusing 30120583L of acetonitrile followed by vortexing for 2minAfter vortexing the samples were subjected to centrifugeat 12000timesg for 10min The supernatant was decanted intoa china dish and evaporated to dryness at room tempera-ture This was further reconstituted with 100 120583L of mobilephase and vortexed for 30 s and 20120583L was injected into anHPLC system Emtricitabine was detected at a wavelength of280 nm

27 Method Validation Study The developed method wasvalidated as per ICH guidelines using emtricitabine withrespect to the following parameters accuracy precisionLOD LOQ specificity stability and system suitability

271 Linearity For testing linearity seven calibration stan-dards were prepared in the concentration range of 005ndash30 120583gmLminus1 (0050 01 05 15 10 20 25 and 30 120583gmLminus1)Standard curve was achieved by plotting peak area againstconcentration and the evaluation of linearity was completedby linear regression analysis using least square method

272 Limit of Detection and Limit of Quantitation Normallylimit of detection (LOD) and limit of quantitation (LOQ)are estimated at a signal to noise ratio of 3 1 and 10 1respectively LOD and LOQ were determined based on theresponse and slope of a specific calibration curve obtainedfrom six standard solutions (0005 0010 0020 0030 0040and 0050 120583gmLminus1) that were in proximity of these limitconcentration values

273 Selectivity Specificity and Linearity Selectivity wasverified by analyzing the blank plasma from rats to testinterference at the analyte retention times By employing theproposed extraction procedure each blank plasma samplewas tested and then compared with the results of plasmasamples spiked with emtricitabine (119899 = 6) in calibrationstandard to ensure no interference of emtricitabine fromplasma

Spiked plasma samples that contained increasing con-centrations of emtricitabine from 0050 to 30 120583gmLminus1 wereanalyzed according to the procedure described above Thelinearity was detected by calculating the correlation coef-ficient (119903) of the curves by means of least-squared linearregression method All calibration curves of emtricitabinewere constructed prior to the experiments with correlationcoefficient of (1199032 gt 09987)

274 Accuracy The accuracy of the assay method was evalu-ated in triplicate at three different concentration levels (0110 and 25 120583gmLminus1) and the percentage recoveries werecalculated

275 Precision The precision is usually reported as thepercent relative standard deviation (RSD) of a set ofresponses Precision was represented into two categoriesnamely repeatability (intraday precision) and intermediateprecision (interday precision)

276 Repeatability or Intraday Precision Repeatability wastested by analyzing six determinations at three differentconcentrations namely low medium and high within thelinearity range

277 Intermediate or Interday Precision The inter-day vari-ability of this method was assessed over three days atthree low medium and high concentrations of emtricitabinestandard in replicates of six

278 Pharmacokinetic Study in Rats The pharmacokineticstudies were carried out in healthy male Wistar rats (200ndash250 g) and the animals were fasted overnight before dosingwith free access to water The animals were acclimatized tolaboratory conditions over the week before experiments andfed with standard rat diet under controlled conditions of a12 12 h light dark cycle with a temperature of 22plusmn3∘C and arelative humidity of 50 plusmn 5 RH The experimental protocolwas approved by the Institutional Animal Ethical Committee(AACPIAECJun-2012-02)

Twelve rats were randomly separated into two groups (sixanimals each group)The grouping of animals was as follows

Group I control normal rats (received saline solu-tion)Group II administered with pure drug (as solution)(15mgkgrat) [16]

At regular time intervals 0 025 05 1 2 3 6 10 12 20and 24 h samples of blood were withdrawn (100120583L) fromthe retro-orbital plexus by microcapillary technique underlight ether anesthesia into heparinized microcentrifuge tubes(50 units heparinmL of blood) Plasma was separated bycentrifugation at 12000timesg for 15min and analyzed by thefollowing method Plasma samples were deproteinated with1mL of acetonitrile vortexed for 2min and centrifuged at12000timesg for 10min The supernatant was decanted into aChina dish and evaporated to dryness at room tempera-ture This was further reconstituted with 100 120583L of mobilephase and vortexed for 30 s and 20120583L was injected into anHPLC system Emtricitabine was detected at a wavelength of280 nm

3 Results and Discussion

31 Method Development and Optimization of HPLC-UVConditions A liquid chromatographic method for the esti-mation of emtricitabine in rat plasma has been developed


and validated according to the principles of Good LaboratoryPractices An appropriate wavelength was important forgood sensitivity It is shown in Figure 1(a) that emtricitabinehas a special conjugation structure which leads to strongUV absorption at the wavelength of 280 nm Therefore thedetection wavelength was set at 280 nm It was necessaryto use an IS in extraction techniques and HPLC method tocompensate for extraction variation efficiency and analyticalerrors Lamivudine was adopted as the IS in this study forthe reasons that it is structurally similar to emtricitabine andits behavioural characteristics and properties conform to thechemical requirement for IS in HPLC In addition lamivu-dine is commercially available in high purity and it is stableand nonreactive with sample or mobile phase Meanwhile italso has good response at the detectionwavelength of 280 nm

To acquire short run time and good resolution for bothanalyte and IS several trials were carried out to optimizedthe mobile phase The feasibility of various combinationsof solvents such as acetonitrile and methanol with alteredflowrates (in the range 1ndash12mLminminus1) was investigated forcomplete chromatographic resolution of the emtricitabinewith best sensitivity efficiency and peak shape Methanolwas selected as organic phase because of its least viscosityand has strong eluting power Therefore according to thechemical characteristics of emtricitabine and IS appropriateconcentration of acidic modifier acetic acid added intomobile phase and an increase in the methanol content couldimprove peak shape An increase in the water content notonly broadened the peak but also resulted in extremely rapiddesorption and elution of emtricitabine

Finally it was found that a mixture of 10mM potassiumdihydrogen phosphate buffer- (adjusted to pH 68)methanol-2 acetic acid in a ratio of (73 25 2 vvv) with pH adjustedwith orthophosphoric acid to 35 at a flowrate of 1mL minminus1could achieve the above purpose that was found to beoptimum and provided adequate peak separation with lesstailing and resulted in good resolution among all the othercombinations tested which was finally adopted as the mobilephase

32 Limit of Detection and Limit of Quantitation Concen-trations of LOD and LOQ were found to be 0016 and0049 120583gmLminus1 respectively

33 Specificity Specificity is expressed as the capability of amethod to distinguish the analyte from all potentially intru-sive substance The specificity of the method was scrutinizedby blank plasma detection peak purity and spiking blankplasma with pure standard compounds Blank plasma hadno interference when emtricitabine and the IS were elutedAt optimized conditions the separation of emtricitabine andlamivudine was completed within 8min (Figure 2)

34 Linearity Each sample was analyzed in replicates of sixto verify the reproducibility of detector response at eachconcentration level The detector responses were found to belinear over the concentration range from0050 to 30 120583gmLminus1as portrays in Figure 3The regression equation for the graph

EmtricitabineLamivudine

Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100

Figure 2 At optimized conditions (a) chromatographic profile ofthe plasma spiked with emtricitabine (2 120583gmLminus1) in the presence ofthe IS (08 120583gmLminus1)

0200400600800

10001200140016001800

0 05 1 15 2 25 3 35

Are

a (m

Vmiddots)

Conc (120583gmL)

y = 5339926x + 357234

R2 = 09970

Figure 3 Linearity graph of emtricitabine at 0050 01 05 15 1020 25 and 30 120583gmLminus1

is 119910 = 53399119909 + 3572 and the correlation coefficient 1198772is 09970 showing excellent correlation between the area andthe concentration

35 Precision The percentage relative standard deviation(RSD) of the area of emtricitabine during intraday studywas found to be less than 5 and for interday study was foundto be less than 65 which indicated a good precision ofthe method (Table 1) Intra-day and inter-day precision (RSD) of the methods were lower than 7 and were withinthe acceptable limits to meet the guidelines for bioanalyticalmethod validation which is considered to be le15 [13 15]

36 Accuracy The quantitative recovery of emtricitabineachieved ranged from 920 to 9820 with a low RSDvalue The results of the recovery experiments done at threeconcentration levels and the RSD values are given inTable 1

37 Stability Bench-top stability was investigated to ensurethat emtricitabine was not degraded in plasma samples atroom temperature for a time period to cover the sample


Table 1 Intra-day and inter-day precision and accuracy of emtric-itabine in rat plasma (119899 = 6)

Concentration(120583gmLminus1)

Observedconcentration(120583gmLminus1)

Precision Accuracy

Intraday01 0097 plusmn 0003 309 97010 0982 plusmn 0044 448 982025 2465 plusmn 0078 316 980

Interday01 0092 plusmn 0005 545 92010 0975 plusmn 0063 646 975025 2427 plusmn 0105 432 9708

Table 2 Stability of emtricitabine in rat plasma (119899 = 6)

Samplecondition

Spikedconcentration

(120583gmL)

Mean determinedconcentration

(120583gmL)Accuracy ()

Bench-topstabilityclubs

01 0094 940010 098 980025 242 9680

Freeze-thawstability998835

01 0099 990010 103 1030025 246 9840

One-weekstabilityQ

01 0092 920010 089 890025 238 9520

clubsExposed at ambient temperature (25∘C) for 4 h998835After three freeze-thaw cyclesQStored at minus16∘C

preparation It was measured by divulging the QC samplesto ambient laboratory conditions for 10 h Freeze-thaw sta-bility was measured over three cycles Because of the needfor occasional delayed injection of extraction samples thestability of reconstituted samples was assessed at ambienttemperature for 24 h The freezer storage stability of emtric-itabine rat plasma at minus20∘C was evaluated by assaying QCsamples at the beginning and one week later All stability QCsamples were analyzed in six replicates The results indicatedthat emtricitabine had an acceptable stability under thoseconditions (Table 2)

38 Application of the Assay The validated method was suc-cessfully applied to investigate the content of emtricitabine inin vivo after administered orally to rats Oral administrationof emtricitabine in the present study resulted in a sharp119862max of 1357 120583gmLminus1 within 2 h after which the plasmaconcentration declined rapidly indicating a rapid absorptionof emtricitabine The areas under the concentration versustime curve were 12175 120583gmLminus1lowast h The representative chro-matogram of a plasma sample which was collected fromWistar rats 2 h following oral administration of emtricitabineas portrays in Figure 4 The plasma profile of emtricitabine


Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100

Figure 4 Chromatogram of plasma sample collected from rats 2 hafter oral administration of emtricitabine

0

04

08

12

16

2

0 6 12 18 24Time (h)

Plas

ma c

once

ntra

tion

(120583g

mL)

Figure 5 Plasma concentration-time curve of emtricitabine afterbeing orally administered male Wistar rats at dose of 15mgkgrat(119899 = 6 mean plusmn SD)

Table 3 Pharmacokinetic parameters of emtricitabine at a dose of15mgkgrat

Pharmacokinetic parameters Emtricitabine119862max (120583gmLminus1) 1357 plusmn 0029119905max (h) 20 plusmn 000711990512

(h) 8153 plusmn 0670AUC0ndash24 (120583gmLminus1 lowast h) 12175 plusmn 0326

AUMC0ndash24 (120583gmLminus1 lowast h) 16351 plusmn 24038

119870119890(1h) 00681 plusmn 0007

MRT 156 plusmn 0743Data presented as mean plusmn standard deviation (119899 = 6)

is shown in Figure 5 The results substantiate the suitabilityof the developed method for determining emtricitabineconcentration in plasma after oral administration The phar-macokinetic data of emtricitabine after oral administration inrats is shown in Table 3


4 Conclusion

A specific linear accurate reliable and reproducible newmethod of emtricitabine in rat plasma was developed andfully validated over the range 0050ndash30120583gmLminus1 with LOQof 0049 120583gmLminus1 The method was successfully appliedto measure the drug concentration in plasma after oraladministration to rats Such low LOQ is very important forpharmacokinetic study of sustained release oral formulationswhere a drug concentration in blood is very low at severaltime points Reproducible high recovery of emtricitabinewas achieved Because of its highly satisfactory sensitivityaccuracy linearity and specificity this HPLC methodologycould thus be an appropriate tool for further determinationof emtricitabine in plasma samples in the pharmacokineticstudies

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors gratefully acknowledge financial support andgranting research fellowship (45382011Nan-BMS) fromIndian Council of Medical Research Govt of India NewDelhi (ICMR) The authors are also grateful to Cipla Lab-oratories Mumbai India for providing the gift samples ofemtricitabine and lamivudine

References

[1] L H Wang A A Wiznia M H Rathore et al ldquoPharma-cokinetics and safety of single oral doses of emtricitabine inhuman immunodeficiency virus-infected childrenrdquo Antimicro-bial Agents and Chemotherapy vol 48 no 1 pp 183ndash191 2004

[2] P D Hamarapurkar and A N Parate ldquoHPLC method forthe determination of emtricitabine and related degradationsubstancesrdquoThe Journal of Chromatographic Science vol 51 no5 pp 419ndash424 2013

[3] Y van Herrewege J Michiels J van Roey et al ldquoIn vitro eval-uation of nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 as human immunodeficiency virusmicrobicidesrdquo Antimicrobial Agents and Chemotherapy vol 48no 1 pp 337ndash339 2004

[4] K Terrazas-Aranda Y van Herrewege P J Lewi J van Roeyand G Vanham ldquoIn vitro pre- and post-exposure prophy-laxis using HIV inhibitors as microbicides against cell-freeor cell-associated HIV-1 infectionrdquo Antiviral Chemistry andChemotherapy vol 18 no 3 pp 141ndash151 2007

[5] S di Fabio J van Roey G Giannini et al ldquoInhibition of vaginaltransmission of HIV-1 in hu-SCID mice by the non-nucleosidereverse transcriptase inhibitor TMC120 in a gel formulationrdquoAIDS vol 17 no 11 pp 1597ndash1604 2003

[6] A Darque G Valette F Rousseau L H Wang J-P Somma-dossi and X-J Zhou ldquoQuantitation of intracellular triphos-phate of emtricitabine in peripheral blood mononuclearcells from human immunodeficiency virus-infected patientsrdquoAntimicrobial Agents andChemotherapy vol 43 no 9 pp 2245ndash2250 1999

[7] N L Rezk R D Crutchley and A D M Kashuba ldquoSimulta-neous quantification of emtricitabine and tenofovir in humanplasma using high-performance liquid chromatography aftersolid phase extractionrdquo Journal of Chromatography B vol 822no 1-2 pp 201ndash208 2005

[8] A K Peepliwal and C G Bonde ldquoDetermination of emtric-itabine in human plasma by RP-HPLC with UV-detectionrdquoJournal of Pharmacy Research vol 3 no 8 pp 1712ndash1715 2010

[9] N A Gomes V V Vaidya A Pudage S S Joshi and S AParekh ldquoLiquid chromatography-tandem mass spectrometry(LC-MSMS) method for simultaneous determination of teno-fovir and emtricitabine in human plasma and its applicationto a bioequivalence studyrdquo Journal of Pharmaceutical andBiomedical Analysis vol 48 no 3 pp 918ndash926 2008

[10] A Karunakaran K Kamarajan andVThangarasu ldquoA validatedRP-HPLC method for simulataneous estimation of emtric-itabine and tenofovir disoproxil fumarate in pure and in tabletdosage formrdquo Der Pharmacia Sinica vol 1 no 2 pp 52ndash562010

[11] P S Devrukhakar R Borkar N Shastri and K V H Suren-dranat ldquoA validated stability-indicating rp-hplc method for thesimultaneous determination of tenofovir emtricitabine and anefavirenz and statistical approach to determine the effect ofvariablesrdquo ISRN Chromatography vol 2013 Article ID 8782958 pages 2013

[12] P Kumar S C Dwivedi and A kushnoor ldquoA validatedstability indicating RP-HPLC method for the determination ofemtricitabine in bulk and capsulesrdquo Farmacia vol 60 no 3 pp402ndash410 2012

[13] C Mathew M Ajitha and P R S Babu ldquoCefpodoximeproxetil a new stability indicating RP-HPLC methodrdquo ISRNChromatography vol 2013 Article ID 328157 8 pages 2013

[14] Food and Drug Administration Guidance for Industry Bio-analytical Method Validation US Department of Health andHuman Services FDA Center for Drug Evaluation andResearch Rockville Md USA 2001

[15] ICH Harmonised Tripartite Guideline Validation of AnalyticalProcedures Methodology Q2 (R1) International Conference onHarmonisation of Technical Requirements for Registrations ofPharmaceuticals for Human Use ICH Geneva Switzerland2005

[16] R Nirogi G Bhyrapuneni V Kandikere et al ldquoPharmacoki-netic profiling of efavirenz-emtricitabine-tenofovir fixed dosecombination in pregnant and non-pregnant ratsrdquo Biopharma-ceutics amp Drug Disposition vol 33 no 5 pp 265ndash277 2012

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N

N

F

S

O

OH

ONH2

(a)

N

N

O

S

O OH

NH2

(b)

Figure 1 Chemical structure of emtricitabine (a) and lamivudine (b)

The purpose of the present study was to develop andvalidate a simple and time-saving RP-HPLC method withUV detection for the investigation of emtricitabine after oraladministration to Wistar rats The method was validatedaccording to Food and Drug Administration (FDA) andInternational Conference on Harmonization (ICH) guide-lines with respect to linearity precision accuracy and speci-ficity and stability studies [14 15] Lamivudine (08 120583gmLminus1)was used as an internal standard (IS)

2 Experimental

21 Materials and Reagents Emtricitabine and lamivudine(Figure 1(b)) (998 ww and 987 ww HPLC) were pro-vided ex-gratis by MS Cipla Laboratories Mumbai IndiaHPLC grade methanol was purchased from SD fine-chemlimited (Mumbai India) Deionized water used in all theexperiments was passed through a Milli-Q water purifica-tion system (182MΩcm) Millipore (Bangalore KarnatakaIndia)

22 Instrumentation The HPLC (Shimadzu Kyoto Japan)instrument was equipped with binary pump and SPD-20AVPUVdetector Sample injection was done by Rheodyneinjector with a 50 120583L loop and a computer running Varianworkstation version 642 software for data acquisition andprocessingThe chromatographic analysis was carried out onPhenomenex C

18column (250mm times 46mm ID 5 120583m)

23 Chromatographic Conditions Chromatographic sepa-ration was achieved using Phenomenex C

18column The

mobile phase was composed of 10mMpotassium dihydrogenphosphate buffer (adjusted to pH 68)-methanol-2 aceticacid in a ratio of 73 25 2 (vvv) run under isocratic elutionand pumped at a flow rate of 1mL minminus1 The column wasthermostated at 30∘C Under these conditions the run timewas less than 8min

24 Optimization and Chromatographic Method Develop-ment Trial experiments were carried out initially in order tochoose most appropriate solvent system for precise analysisand to achieve good resolution Column chemistry solventtype solvent ratio (volume of organic solvents in the mobile

phase) flow rate detection and wavelength were varied toestablish the chromatographic conditions giving the goodseparation On the basis of time required for analysis sensi-tivity of the assay resolution time and use of readily availablecost-effective solvents the aptness of the mobile phase andthe flow rate was decided These included water-methanol(50 50 vv) 10mM pH 68 PBS-methanol (50 50 vv)10mM pH 68 PBS-methanol (70 30 vv) 10mM pH 68PBS-methanol-2 acetic acid (70 25 5 vvv) and 10mMpH 68 PBS-methanol-2 acetic acid (73 25 2 vvv) Amobile phase system comprised of 10mM potassium dihy-drogen phosphate buffer (pH 68)-methanol-2 acetic acid(73 25 2 vvv) at a flow rate of 1mL minminus1 was found to beoptimum The experimental work was performed in an air-conditioned room maintained at 20ndash25∘C

25 Preparation of Calibration Curve (CC) and Quality Con-trol Samples (QC) Eight-point calibration curve (CC) wasprepared by serial dilution of emtricitabine stock solution(100 120583gmLminus1) in the range of 0050 01 05 10 15 2025 and 3 120583gmLminus1 that were obtained by measuring therequired amount of 100 120583gmLminus1 working standard solu-tion mixed with a sufficient quantity of mobile phase andmaking up to 10mL Similarly six standard solutions wereprepared by serial dilution of emtricitabine stock solution(10 120583gmLminus1) in the range of 0005 0010 0020 0030 0040and 0050 120583gmLminus1 Six standard solutions were obtainedfrom the 10 120583g mLminus1 working standard solution in order todetermine the LOD and LOQ of the method

Calibration standards were prepared daily by spiking100 120583L of blank plasma with 10 120583L of the appropriate workingsolution resulting in concentrations of 0050 01 05 1015 20 25 and 3120583gmLminus1 and 0005 0010 0020 00300040 and 0050 120583gmLminus1 respectively of emtricitabineStock solution (08120583gmLminus1) of lamivudine (IS) was preparedin methanol and stored at minus20∘C The solutions were stablefor one day when stored at room temperature (20ndash25∘C)Thestock and standard solutions) were prepared on a daily basisand stored in the dark at about 5∘C All solutions were usedon the day they were prepared

Quality control (QC) samples (low quality control (LQC)01 120583gmLminus1 medium quality control (MQC) 10 120583gmLminus1



























Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100


0200400600800

10001200140016001800

0 05 1 15 2 25 3 35

Are

a (m

Vmiddots)

Conc (120583gmL)

y = 5339926x + 357234

R2 = 09970










Precision Accuracy




Samplecondition

Spikedconcentration

(120583gmL)




01 0094 940010 098 980025 242 9680


01 0099 990010 103 1030025 246 9840

One-weekstabilityQ

01 0092 920010 089 890025 238 9520





Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100


0

04

08

12

16

2

0 6 12 18 24Time (h)

Plas

ma c

once

ntra

tion

(120583g

mL)






119870119890(1h) 00681 plusmn 0007




4 Conclusion




Acknowledgments


References


























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



























Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100


0200400600800

10001200140016001800

0 05 1 15 2 25 3 35

Are

a (m

Vmiddots)

Conc (120583gmL)

y = 5339926x + 357234

R2 = 09970










Precision Accuracy




Samplecondition

Spikedconcentration

(120583gmL)




01 0094 940010 098 980025 242 9680


01 0099 990010 103 1030025 246 9840

One-weekstabilityQ

01 0092 920010 089 890025 238 9520





Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100


0

04

08

12

16

2

0 6 12 18 24Time (h)

Plas

ma c

once

ntra

tion

(120583g

mL)






119870119890(1h) 00681 plusmn 0007




4 Conclusion




Acknowledgments


References


























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100


0200400600800

10001200140016001800

0 05 1 15 2 25 3 35

Are

a (m

Vmiddots)

Conc (120583gmL)

y = 5339926x + 357234

R2 = 09970










Precision Accuracy




Samplecondition

Spikedconcentration

(120583gmL)




01 0094 940010 098 980025 242 9680


01 0099 990010 103 1030025 246 9840

One-weekstabilityQ

01 0092 920010 089 890025 238 9520





Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100


0

04

08

12

16

2

0 6 12 18 24Time (h)

Plas

ma c

once

ntra

tion

(120583g

mL)






119870119890(1h) 00681 plusmn 0007




4 Conclusion




Acknowledgments


References


























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





Precision Accuracy




Samplecondition

Spikedconcentration

(120583gmL)




01 0094 940010 098 980025 242 9680


01 0099 990010 103 1030025 246 9840

One-weekstabilityQ

01 0092 920010 089 890025 238 9520





Time (min)

Volta

ge (m

V)

1 2 4 6 8 10

0

25

50

75

100


0

04

08

12

16

2

0 6 12 18 24Time (h)

Plas

ma c

once

ntra

tion

(120583g

mL)






119870119890(1h) 00681 plusmn 0007




4 Conclusion




Acknowledgments


References


























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


4 Conclusion




Acknowledgments


References


























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article High-Performance Liquid Chromatographic ...downloads.hindawi.com/archive/2013/329072.pdf · RP-HPLC method for a combination of tenofovir diso-proxil fumarate, emtricitabine,