ZALTOPROFEN Bioanalytical method development and ...shodhganga.inflibnet.ac.in/bitstream/10603/30958/15/15_chapter 6.pdf · ZALTOPROFEN Bioanalytical method development and Validation

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ZALTOPROFEN

Bioanalytical method development and Validation using HPLC

6.1 DRUG PROFILE

Chemical name : 2-(10, 11 dihydro-10-oxodibenzo [b, f ] thiepin -2-yl)

Propionic acid.

Molecular formula : C17H14O3S

Molecular weight : 298.4

Melting point : 131-1330C

Physical Appearance: Off-White to Pale Yellow Crystalline Solid

Solubility : It is freely soluble in acetone, chloroform and

methanol; slightly soluble in ethanol and benzene; practically insoluble in water and

cyclohexane. 1, 2

Mechanism of action:

Zaltoprofen, a propionic acid derivative of non-steroidal anti-inflammatory

drugs (NSAIDs), which as more powerful inhibitory effects to bradykini (BK) than

other NSAIDs. However, the molecular mechanisms underlying this potent

analgesia are not yet fully understood. It has been used clinically for treatment of

post-operative pain and low back pain. Zaltoprofen is a unique compound that also

has anti-bradykinin activity. Its analgesic effects may be a result of inhibition of

bradykinin B receptor-mediated bradykinin responses not only of cyclo-oxygenases

but also of bradykinin-induced 12-lipoxygenase inhibitors.3

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6.2. LITERATURE REVIEW- ZALTOPROFEN

Wang H et al (2011)4 reported a simple and rapid RP-HPLC analysis

method for determination of Zaltoprofen glucuronide in rat hepatic microsomes.

The calibration curves of (+) - and (−) - Zaltoprofen glucuronide showed a good

linearity in the given concentration range from 0.15 to 31.13µM. Kinetic analysis

found that (−) Zaltoprofen had a greater glucuronidation rate in rat liver microsome

than that of (+) Zaltoprofen.

Kiran BA et al (2011)5 reported a simple, rapid and precise LC method for

determination of Zaltoprofen in pharmaceutical bulk dosage forms.

Chromatographic separation for Zaltoprofen and its degradation products was

carried on a C18 analytical column with mobile phase ratio, 80:20(v/v) of 0.01 M

potassium dihydrogen phosphate-acetonitrile. The flow rate was 1.0 ml/min with the

column temperature 25°C, and detection was by UV absorption at 233 nm using a

photodiode array detector and UV detector. The linearity of the method was good

over the range 10–140 µg/ml.

Lee XP et al (2010)6 reported a LC/MS method using electron spray

ionization tandem mass spectrometry (MS-MS) for the estimation of six

non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma using a

hydrophilic polymer column (MSpak GF-310 4B). Gradient elution was employed

for separation of the six NSAIDs, Alminoprofen, Flurbiprofen, Ibuprofen,

Pranoprofen, Tiaprofenic acid and Zaltoprofen, with 10 mM ammonium

acetate/acetonitrile as mobile phase. All NSAIDs spiked into plasma showed

recoveries of 77.0% - 88.2%.

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Yang HK et al (2009)7 reported a column-switching High-performance

liquid chromatographic method for a comparative study on pharmacokinetic

parameters of Zaltoprofen and those of its sodium salt in rats. Zaltoprofen has a poor

solubility in water, but its sodium salt had excellent water solubility. To study the

effect of aqueous solubility microcapsules containing the drug powders were

administrated orally to rats and blood samples were collected via the common

carotid artery.

Nirogi RVS et al (2006)8 reported a simple reversed-phase chiral HPLC method

for direct separation of the enantiomers of Zaltoprofen. Separation was carried on a

Chiralcel OJ-RH stationary phase. Retention times for S and R enantiomers were

approximately 14 and 16 min. Mobile phase composition was ammonium buffer and

organic modifier used to find the optimum chromatographic conditions .

Lee HW et al (2006)9 reported a RP-HPLC method coupling with liquid-

liquid extraction using positive ion electrospray ionization tandem mass

spectrometry for estimation of Zaltoprofen in human plasma. Enalapril was

employed as internal standard, ion acquisition for Zaltoprofen and Enalapril was

done on MRM (multiple reaction monitoring mode). Linearity found to be 0.05 to

20.0µg/ml.

Choi SO et al (2006)10 reported a direct injection column-switching High-

performance liquid chromatography (HPLC) method for quantitation of Zaltoprofen

in rat plasma. In this method two mobile phases are used, mobile phase A,

acetonitrile-10mM potassium phosphate buffer (pH-6.8) (12:88 v/v) in which

samples were directly injected to the pre-column without sample pre-purification

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step, when all endogenous plasma components were eluted to waste, the system was

switched and the analyte was eluted to the trap column and into the second mobile

phase B, acetonitrile-10mM potassium phosphate buffer (pH-6.8) (35:65 v/v)

Zaltoprofen was then back-flushed to the analytical column for separation with an

ultraviolet detector at 230 nm. Concentration range was 40-5000 ng/ml.

Okamoto T et al (2001)11 studied the effect of Zaltoprofen on the loss of

body weight by using rodent sickness behavior models and reported the sickness

behaviors such as anorexia, decrease in body weight and loss of locomotor activity

in concanavalin A (Con A) - induced mouse hepatitis and carbon tetrachloride-

induced rat hepatitis. Zaltoprofen is a non-steroidal anti-inflammatory drug

(NSAID) that causes potent inhibition of cyclooxygenase-2 with fewer side effects

on the gastrointestinal tract.

Davies NM (1997)12 reported a review which gives the information about the

general principles which allows the separation of chiral NSAID enantiomers and

gives both advantages and disadvantages of various analytical methods which

involve chromatographic separations.

Yamamoto M et al (1990)13 reported the purity of enatiomers by using chiral

cellulose column (CHIRALCEL OJ), a separation factor (alpha) of 1.73 was

obtained. X-ray crystallographic analysis of the (+) -isomer [salt of (-) -1-(4-

bromophenyl) ethylamine] showed that this enantiomer has S-configuration, which

had anti-inflammatory activity.

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Oshima J et al (1987)14 reported a reversed phase High performance liquid

chromatographic method for the estimation of a new anti-inflammatory agent,

Zaltoprofen (CN-100; I). The method consists in the extraction of samples

containing Zaltoprofen and Mefenamic acid which is used as internal standard,

under acidic conditions using ultraviolet detection at 330nm.

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6.3. EXPERIMENTAL: METHOD DEVELOPMENT

6.3.1. MATERIALS AND METHODS

Materials

Zaltoprofen and Nevirapine were obtained from CIPLA Pharmaceuticals,

Mumbai. HPLC grade acetonitrile, ethylacetate and methanol were purchased from

SD fine chemicals, Mumbai, India. Analytical Grade Potassium dihydrogen

phosphate was purchased from SD fine chemicals, Mumbai, India. Pooled drug free

expired human plasma was donated by Red Cross Society, Warangal.

Instrumentation

The HPLC system consisted of Alliance Waters 2695 with dual λ Absorbance UV

detector. Separation was carried out on Inertsil C18 column (4.6x250mmx5µm). A

vortex-mixer (Remi), ultrasonic bath (Bransonic), a model C-30 centrifuge (Remi)

and a model cool safe 110-4/scan speed 32 were used for sample preparation.

Preparation of Stock solutions

Stock solutions of Zaltoprofen (500 µg/ml) and Nevirapine (1000 µg/ml)

internal standard were prepared in Methanol. Further dilutions were carried out in

Methanol.

Preparation of calibration standards

Calibration standards were prepared freshly by spiking drug free plasma with

Zaltoprofen stock solution to give the concentrations of 0.15, 0.3, 0.6, 1.2, 2.5, 5.0,

10.0 and 20.0µg/ml they were stored at -200c till the time of analysis.

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Preparation of quality control standards

Quality control standards were prepared at three levels namely LQC, MQC

and HQC. These standards were prepared freshly by spiking drug free plasma with

Zaltoprofen stock solution to give the concentrations of 0.5, 9.0 and 18.0 µg/ml

respectively. They were stored at -200c till the time of analysis. Detailed procedure

for the preparation is shown in Table 66.

Sample preparation method

Drug free human plasma (500 µl) was spiked with appropriate volume of

drug stock. To the above prepared sample, 50µl of Nevirapine (60µg/ml) was added

as an ISTD. The sample was vortex mixed for 4 min to assure complete mixing.

Analytes were extracted by adding with 3.0ml of ethyl acetate followed by vortex

mixing for 4 min and centrifugation at 2000 rpm/min in a cooling centrifuge for

15min at 40C. The organic phase containing analytes were separated and analytes

were obtained as dried residues after drying in lyophilizer. The analytes residue

obtained was reconstituted with 300µl of mobile phase and analysed using HPLC

system according to parameters optimized. Detailed procedure for preparation is

shown in Table 61, calibration data is shown in Table 62, calculated concentrations

of calibration standards and mean concentrations of calibration standards data were

shown in Tables 63 and 64.

Chromatographic conditions

The HPLC system consisted of Alliance waters 2695 with dual λ Absorbance

UV detector. The wavelength of detection was set at 254nm. Separation was carried

out on Inertsil C18 column (4.6x250mmx5µm) using 40:60 v/v phosphate buffer pH-

3.0 and acetonitrile as mobile phase at a flow rate of 1.0 ml/min. The mobile phase

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was filtered through nylon milli pore (0.2µm) membrane filter, purchased from pall

life sciences, Mumbai and degassed with Ultrasonicator prior to use.

Chromatography was carried out at room temperature 250c.

6.4. EXPERIMENTAL: METHOD VALIDATION

6.4.1. VALIDATION PARAMETERS

Specificity

A solution containing 0.15µg/ml Zaltoprofen was injected onto the column

under optimized chromatographic conditions to show the separation of Zaltoprofen

from the impurities from the plasma. The specificity of the method was checked for

the interference from plasma.

Linearity and Range

Six samples of each calibration standard were analysed. The spiked

concentration and their respective peak area ratios with respective to internal

standards were subjected to least squares regression. After examining the percent

deviation, a proper model was chosen. The slope, Y-intercept and coefficient of

determination (r2) were obtained from linear regression equation.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The limit of quantitation refers to the lowest amount of an analyte in a

sample that can be quantitatively determined with suitable precision and accuracy.

There are different approaches to determine the LOD and LOQ. Typically the

concentration level that generates a signal-to-noise (S/N) of 3.3 is regarded as the

LOD and the concentration level that generates S/N = 10 is regarded as the LOQ.

Here LOD was calculated using the formula 3.3 times σ/s, similarly LOQ was

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calculated using the formula 10 times σ/s where “σ” is the standard deviation of the

intercept obtained for calibration curve and “s” is the slope of the calibration curve.

Accuracy and precision

Intraday precision and accuracy was determined by analyzing quality control

standards (0.5, 9.0 and 18.0 µg/ml) and LLOQC standards (0.15µg/ml) five times a

day randomly, interday precision and accuracy were determined from the analysis of

each quality control standards (0.5, 9.0 and 18.0 µg/ml) and LLOQC standards

(0.15µg/ml) once on each of five different days.

Ruggedness

The ruggedness of the method was determined by analysing spiked control

samples (n=6) of medium concentration, i.e. 9.0 µg/ml using two different columns.

Recovery studies

The percent mean recoveries were determined by measuring the responses of

the extracted plasma Quality Control samples at HQC, MQC and LQC against

unextracted Quality Control samples at HQC, MQC and LQC. For calculation of

recovery of the compound, spiked control samples were prepared at low, medium

and high concentrations i.e. 0.5, 9.0 and 18.0 µg/ml concentrations. The samples

were processed as mentioned above and the concentration of the compound was

determined from the regression of the analytical standard calibration curve.

Recovery was calculated by comparing the observed concentrations in spiked

samples to that of respective unextracted samples.

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Stability studies

The stability of Zaltoprofen was determined by measuring concentration

change in control samples overtime under set conditions.

Freeze-thaw stability study of Zaltoprofen was carried out by subjecting

samples to three freeze and thaw cycles. Samples before study and after study were

analysed by developed method. Similarly Stock solution stability study of

Zaltoprofen (Stability after ‘0’ hrs), Stock solution stability study (Stability after ‘8’

hrs), Bench top stability study of Zaltoprofen (Stability after ‘10’ hrs) and Inter

injection stability study of Zaltoprofen were carried out by subjecting samples to

study conditions.

6.4.2 DATA ANALYSIS 15

Data analysis was done using MS-Excel package for calculating mean,

standard deviation and % relative standard deviation.

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6.5. RESULTS AND DISCUSSION

Under the chromatographic conditions employed, the sample showed sharp

peaks for Zaltoprofen and Nevirapine (IS) with good resolution. The retention time

for the Zaltoprofen was found to be 10.710±0.05 minutes and the retention time for

Nevirapine (IS) was 4.074±0.03 minutes (Figure 44) and the method developed was

validated for specificity, accuracy, precision, linearity, range and stability as per

USFDA guidelines. The results of validation parameters are given below.

The specificity of the method was proven by the absence of peaks near the

retention time of the Zaltoprofen as well as the Nevirapine (IS) (Figure 45).

The calibration function was developed for Peak area ratio Vs Concentration

(in µg/ml) and it was linear over the concentration range of 0.15 to 20.0µg/ml.

The regression line equation for the analysis was y= 0.394x + 0.025 with a

coefficient of correlation (r2) = 0.999. The chromatograms of calibration standard

and calibration curve were shown in Figures 46 to 54.

The LOD is calculated using the formula 3.3 times σ/s, similarly LOQ is

calculated using the formula 10 times σ/s where “σ” is standard deviation of the

intercept obtained for calibration curve and “s” is the slope of the calibration curve.

The calculated LOD and LOQ are shown in Table 65.

A system suitability test was carried out by injecting six aqueous mixtures of

Zaltoprofen and Nevirapine (IS). The following parameters were observed for

repeated injections (n=6) of both Zaltoprofen and Nevirapine (IS) (Table 67).

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The intraday and interday precision and accuracy of the method was found to

be 1.36 to 1.64% and 100.01 to 100.02% respectively for the quality control

samples. This is within the acceptance limits of precision is 15% and accuracy is 85

to 115% (Table 68). The limit of quantitation was found to be 0.15µg/ml, at such

concentration the interday precision was found to be 1.35 to 2.52% and the accuracy

was found to be 100.01 to 100.05% which are within the acceptance limits of

precision is 20% and accuracy is 80 to 120% (Table 69). The results for ruggedness

on different columns were shown in Table 71.

The percent mean recovery for Zaltoprofen in LQC, MQC and HQC was

50.2%, 49.99% and 51.67% respectively (Tables 72 to 74).

Stability was assessed by comparing against the freshly thawed quality

control samples. The percent mean stability for HQC and LQC were 100.01% and

100.57% respectively, which is within the acceptance limits of 85 to 115%. Plasma

Quality Control samples of Zaltoprofen were found to be stable for at least one

month and results were shown in the Table 75.The results for Stock solution

stability study (Stability after ‘0’ hrs), Stock solution stability (Stability after ‘8’

hrs), Bench top stability study (Stability after ‘10’ hrs) and Inter injection stability

study data were shown in Tables 76 to 79.

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Table 61: Preparation of Zaltoprofen calibration standards in Plasma

Zaltoprofen Concentration (µg/ml)

Drug stock solution (µl)*

Blank plasma (µl)

ZLP CS1 0.15 15 985

ZLP CS2 0.3 30 970

ZLP CS3 0.6 60 940

ZLP CS4 1.2 120 880

ZLP CS5 2.5 250 750

ZLP CS6 5.0 500 500

ZLP CS7 10.0 1000 0

ZLP CS8 20.0 40 Δ 960

* indicates drug stock solution from 10 µg/ml

Δ indicates drug stock solution from 500 µg/ml

Table 62: Calibration data of Zaltoprofen

Concentration (µg/ml) Peak Area Ratio of Peak

Area Zaltoprofen Internal Standard

0.15 23911 413071 0.057

0.3 52441 418492 0.125

0.6 110580 421175 0.262

1.2 214279 409897 0.522

2.5 416266 395978 1.051

5.0 823758 411692 2.000

10.0 1660076 415925 3.991

20.0 3229705 409099 7.894

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Table 63: Calculated concentrations of Zaltoprofen calibration standards

Zaltoprofen Actual

Concentration (µg/ml)

calculated concentrations

1 2 3

ZLP CS1 0.156 0.163 0.162 0.161

ZLP CS2 0.312 0.289 0.288 0.289

ZLP CS3 0.625 0.611 0.613 0.615

ZLP CS4 1.25 1.255 1.254 1.259

ZLP CS5 2.5 2.458 2.431 2.521

ZLP CS6 5.0 4.959 4.948 5.015

ZLP CS7 10.0 10.087 10.099 10.202

ZLP CS8 20.0 20.483 20.012 20.266

Table 64: Mean concentrations of Zaltoprofen calibration standards

Zaltoprofen Concentration (µg/ml)

Mean of calculated

concentrationsSD % RSD

ZLP CS1 0.156 0.162 0.001 0.62

ZLP CS2 0.312 0.288 0.001 0.20

ZLP CS3 0.625 0.611 0.003 0.47

ZLP CS4 1.25 1.256 0.003 0.21

ZLP CS5 2.5 2.483 0.033 1.32

ZLP CS6 5.0 4.974 0.036 0.72

ZLP CS7 10.0 10.129 0.063 0.62

ZLP CS8 20.0 20.108 0.137 0.68

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Table 65: Calibration curve parameters for Zaltoprofen

1 2 3 Mean SD

Slope 0.00343 0.00349 0.00313 0.00335 0.000193

Intercept 0.0796 0.0796 0.0789 0.0793 0.0004

r2 0.998 0.998 0.998 - -

LOD 0.4µg/ml - - - -

LOQ 1.2µg/ml - - - -

Table 66: Preparation of Zaltoprofen quality control standards in Plasma Zaltoprofen Concentration

(µg/ml) Drug stock solution

(µl) * Blank plasma (µl)

ZLP LQC 0.5 50 950

ZLP MQC 9.0 18 982.0

ZLP HQC 18.0 36 Δ 964.0

* indicates drug stock solution from 10 µg/ml

Δ indicates drug stock solution from 500 µg/ml

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Table 67: HPLC System suitability parameter (Precision) for Zaltoprofen and Nevirapine (ISTD)

Name Replicates RT Peak area

Zaltoprofen (n=6)

1 10.71 722350

2 10.713 742435

3 10.715 727623

4 10.711 749878

5 10.718 739874

6 10.710 722658

Mean 10.71 734136

SD 0.0031885 11512.45

% RSD 0.029 1.56

Internal Standard (n=6)

1 4.074 525098

2 4.057 522984

3 4.0351 535724

4 4.071 543785

5 4.067 523957

6 4.055 530217

Mean 4.059 530294

SD 0.014282 8149.72

% RSD 0.351 1.53

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Table 68: Intra-day accuracy and precision for Zaltoprofen

Zaltoprofen QC ID LQC MQC HQC

Actual conc.( µg/ml) 0.5 9 18

Intraday

1 0.446 9.699 20.675

2 0.45 9.379 20.156

3 0.459 9.639 20.182

4 0.459 9.7 19.825

5 0.459 9.606 20.522

Mean 0.454 9.604 20.272

± SD 0.0061 0.1323 0.3340

% RSD 1.3613 1.3782 1.6476

% Accuracy 100.01 100.01 100.02

Table 69: Inter-day accuracy and precision for Zaltoprofen

Zaltoprofen

QC ID LQC MQC HQC

Actual conc. (µg/ml) 0.5 9 18

Interday

1 0.446 9.714 20.225

2 0.448 9.393 19.846

3 0.464 9.637 20.135

4 0.474 9.707 19.308

5 0.458 9.621 20.099

Mean 0.458 9.614 19.922

± SD 0.0115 0.1304 0.3712

% RSD 2.5274 1.3568 1.8634

% Accuracy 100.05 100.01 100.02

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Table 70: Precision & Accuracy of LLOQC Standard

QC ID LLOQC

Actual conc.(µg/ml)

0.156

1 0.154

2 0.151

3 0.158

4 0.154

5 0.153

Mean 0.154

±SD 0.0025

%RSD 1.6555

% Accuracy 100.02

Table 71: Ruggedness of the method developed for Zaltoprofen (on different columns)

MQC

Replicates

Column 1

(area ratio)

Column 2

(area ratio)

1 1.784 1.716

2 1.814 1.81

3 1.807 1.797

4 1.741 1.736

5 1.789 1.779

6 1.809 1.794

Mean 1.791 1.772

± SD 0.027 0.038

% RSD 1.51 2.12

% Accuracy 101.05

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Table 72: Recovery study for Zaltoprofen from human plasma

Zaltoprofen

Replicates

LQC

Unextracted

(area ratio)

Extracted

(area ratio)

%Recovery

1 0.123 0.059 47.97

2 0.122 0.065 53.28

3 0.124 0.062 50.00

4 0.124 0.063 50.81

5 0.124 0.061 49.19

6 0.124 0.062 50.00

Mean 0.124 0.062 50.208

±SD 0.001 0.002 1.785

%RSD 0.68 3.23 3.55


Zaltoprofen

Replicates

MQC

Unextracted

(area ratio)

Extracted

(area ratio)

%Recovery

1 2.377 1.125 47.33

2 2.375 1.169 49.22

3 2.371 1.242 52.38

4 2.368 1.179 49.79

5 2.366 1.266 53.51

6 2.37 1.131 47.72

Mean 2.371 1.185 49.992

±SD 0.004 0.058 2.489

%RSD 0.18 4.86 4.98

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Zaltoprofen

Replicates

HQC

Unextracted

(area ratio)

Extracted

(area ratio)

%Recovery

1 4.356 2.612 59.96

2 4.262 2.136 50.12

3 4.356 2.106 48.35

4 4.356 2.227 51.12

5 4.345 2.255 51.90

6 4.355 2.116 48.59

Mean 4.338 2.242 51.673

±SD 0.038 0.191 4.291

%RSD 0.87 8.53 8.30

Table 75: Freeze-thaw Stability of Quality Control Standards

Stability after 3 freeze thaw cycles

Zaltoprofen LQC HQC

Actual conc.(µg/ml) 0.5 18

1 0.535 18.796

2 0.431 18.292

3 0.478 18.762

4 0.49 18.824

5 0.433 18.323

6 0.451 18.887

Mean 0.469 18.647

± SD 0.0398 0.2665

% RSD 8.4843 1.4296

% Accuracy 100.57 100.01

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Table 76: Stock solution stability study of Zaltoprofen (Stability at ‘0’ hrs)

Replicates SSS(0hrs) LQC SSS(0hrs) HQC

1 0.126 4.792

2 0.127 4.773

3 0.126 4.773

4 0.127 4.778

5 0.126 4.783

6 0.126 4.778

Mean 0.126 4.780

± SD 0.001 0.007

% RSD 0.41 0.15

Table 77: Stock solution stability study of Zaltoprofen (Stability after ‘8’ hrs)

Replicates SSS(8hrs) LQC SSS(8hrs) HQC

1 0.127 4.793

2 0.127 4.809

3 0.121 4.762

4 0.126 4.81

5 0.127 4.721

6 0.125 4.802

Mean 0.126 4.783

± SD 0.002 0.035

% RSD 1.87 0.73

%Accuracy 99.34 100.07

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Table 78: Bench top stability study of Zaltoprofen (Stability after 10 Hours)

Time (h) 10.00(h)BTS

Zaltoprofen LQC HQC

Actual conc.(µg/ml) 0.5 18

1 0.47 19.091

2 0.48 17.507

3 0.483 18.876

4 0.475 18.464

5 0.462 18.156

6 0.516 18.614

Mean 0.481 18.451

± SD 0.019 0.565

% RSD 3.89 3.06

% Accuracy 96.20 102.51

Table 79: Inter injection stability study of Zaltoprofen

Zaltoprofen LQC HQC Actual

conc.(µg/ml) 0.5 18

1 0.491 18.856

2 0.497 17.823

3 0.439 18.515

4 0.446 18.224

5 0.473 17.873

6 0.488 18.478

Mean 0.472 18.295

± SD 0.025 0.401

% RSD 5.19 2.19

% Accuracy 94.47 101.64

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CHROMATOGRAMS OF DEVELOPED METHOD

Figure 44: Chromatogram showing Retention times of Aqueous Mixture consists of Zaltoprofen (5µg/ml) and Nevirapine (5µg/ml)

Figure 45: Chromatogram of Blank plasma sample showing no interference at the RT of Zaltoprofen and Nevirapine.

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Figure 46: Chromatogram of Zaltoprofen calibration standard-1(ZLP CS1)


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Figure 54: Calibration curve of spiked concentrations (ZLP CS1-CS8)

Figure 55: Chromatogram of Zaltoprofen Low Quality Control (ZLP LQC)

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Figure 56: Chromatogram of Zaltoprofen Middle Quality Control (ZLP MQC)

Figure 57: Chromatogram of Zaltoprofen High Quality Control (ZLP HQC)

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6.6. SUMMARY

A Simple, rapid, selective and sensitive HPLC method was developed and

validated for the determination of Zaltoprofen from human plasma. The drug was

extracted with ethyl acetate.Zaltoprofen was measured in plasma using a validated

HPLC method with UV detector at 254nm chromatographic peaks were separated

on 5µm Intensil, C18 column (4.6x250mmx5µm) using 40:60 v/v Phosphate buffer

pH-3.0 and Acetonitrile as mobile phase at a flow rate of 1.0 ml/min.

The chromatograms showed good resolution and no interference from plasma.

The retention time of Zaltoprofen and Nevirapine (IS) were approximately 4.0±0.05

min and 10.7±0.03 min respectively. The mean recovery from human plasma was

found to be above 50%. The method was linear over the concentration range of 0.15

to 20.0 µg/ml with a coefficient of correlation (r2) 0.999. Both intraday and interday

accuracy and precision data showed good reproducibility. This method was

successfully applied to pharmacokinetic studies.

6.7. CONCLUSION

Various methods reported in literature were studied. In the present study a

simple, rapid, specific, rugged, accurate, precise and stable method was developed

for estimation of Zaltoprofen in human plasma. The regression equation for the

analysis was y= 0.394x + 0.025 with a coefficient of correlation (r2) = 0.999. The

percent mean recovery for Zaltoprofen in LQC, MQC and HQC was 50.2, 49.99 and

51.6% respectively. The method is accurate, precise and rugged with % RSD < 15%

and 20% when tested at MQC, HQC and LQC levels respectively. The stability was

assessed at different levels. The results of the Freeze-thaw stability, Bench top

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stability, Inter injection stability studies showed that the compound under analysis

was stable under test conditions. Hence the method developed can be used for

estimation of Zaltoprofen present in human plasma.

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LIST OF PUBLICATIONS

1. Manish Thimmaraju, Srikanth Gurrala, J.Venkateshwar Rao, K.R.S. Sambasiva Rao,

Development and validation of HPLC-UV method for the estimation of Eprosartan in

human plasma, International Journal of Pharmacy and Pharmaceutical Sciences,

2011;3,(2).191-194.

2. Manish Thimmaraju, Srikanth Gurrala, Vandana Pamulaparthy, J.Venkateshwar Rao,

K.R.S. Sambasiva Rao, Development and validation of HPLC-UV method for the

estimation of 0xaprozin in human plasma, Journal of Pharmacy Research

2011,4(11),4202-4204.



estimation of Fenofibrate in human plasma, Journal of Pharmacy Research 2011,4(10),

3735-373.



estimation of Zaltoprofen in human plasma, Journal of Pharmacy Research 2011,4(10),

3753-375.

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