INTRODUCTION:

Cobicistat^1-5 is a chemically known as (1,3-thiazol-5-yl)methyl N-[(2R,5R)-5-[(2S)-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}-4-(morpholin-4-yl)butanamido]-1,6-diphenylhexan-2-yl] Cobicistat, trade name Tybost (formerly GS-9350), is a licensed drug for use in the treatment of infection with human immunodeficiency virus (HIV). Although it does not have any anti-HIV activity, Cobicistat acts as a pharmacokinetic enhancer by inhibiting cytochrome P450 3A isoforms (CYP3A) and therefore increases the systemic exposure of co administered agents that are metabolized by CYP3A enzymes.

More specifically, Cobicistat is indicated to increase systemic exposure of atazanavir or darunavir (once daily dosing regimen) in combination with other antiretroviral agents in the treatment of HIV-1 infection. Increasing systemic exposure of anti-retrovirals (ARVs) without increasing dosage allows for better treatment outcomes and a decreased side effect profile.

Following oral administration of Cobicistat with food in HIV-1 infected subjects, peak plasma concentrations were observed 4 hours post-dose for Cobicistat. The steady-state mean Cmax, AUCtau, and Ctrough (mean ± SD) following multiple doses of Cobicistat in HIV-1 infected subjects (n = 68), respectively, were 1.2 ± 0.3 μg/ml, 10.9 ± 3.8 μg/ml, and 0.07 ± 0.07 μg/ml. Cobicistat is 97-98% bound to human plasma proteins and the mean plasma to blood drug Cobicistat is metabolized via CYP3A (major)- and CYP2D6 (minor)-mediated oxidation and does not undergo glucuronidation. Following oral administration of [14C] Cobicistat, 99% of circulating radioactivity in plasma was unchanged cobicistat. Low levels of metabolites are observed in urine and faeces and do not contribute to the CYP3A inhibitory activity of cobicistat. Following oral administration of [14C] Cobicistat, 86% and 8.2% of the dose were recovered in faeces and urine, respectively. The median terminal plasma half-life of Cobicistat following administration of Tybost is approximately 3-4 hours.

Based on the literature available, Cobicistat is quantitative determined by high performance liquid chromatography (RP–HPLC), gas chromatography (GC) with mass spectrometric (MS) detection ¹¹ and radioimmunoassay (RIA). The usual HPLC methods have the limitation in sensitivity and selectivity and GC methods are requires more time for the sample preparation and longer run time for analysis. A RIA method requires handling of radioactive materials, prolonged incubation and is more prone to cross reactivity. Hence there is a necessity to develop sensitive, selective and rapid analytical methods such as LC-MS/MS.

Two liquid chromatography (LC) mass spectrometry (MS) methods have been reported for the determination of Cobicistat with other steroids in serum and water effluents. But there were no reports available for the determination of Cobicistat alone in human plasma. Many formulations available for Cobicistat alone in the market and should have a proper analytical method to quantify the drug concentrations in plasma. Hence I made an attempt to develop and validate a simple HPLC method for the estimation of Cobicistat in human plasma with better sensitivity and short runtime.

In the present work the author has been developed and fully validated a HPLC method for quantifying Cobicistat in human plasma. The sample preparation is simple and rapid with no derivatization process. This is the first validated assay to determine Cobicistat alone in human plasma.

Fig. 1: Cobicistat structure

Fig. 2: Febuxostat (IS) structure

MATERIALS AND METHODS:

Instrumentation:

Chromatographic separation was performed on WATERS HPLC model: 2695, separation module, photodiode array detector model 2996, automatic injection, sampling vial 120. The instrument was operated using computer via Empower-2 software. The mobile phase was filtered through a 0.45µm membrane filter and degassed for 30 min. Analysis was performed at 30⁰c temperature.

Preparation of stock and intermediate working solutions of Cobicistat:

Stock solution of Cobicistat was prepared in HPLC grade methanol at a concentration of 1 mg/mL. The prepared stock concentrations were corrected based on its potency and actual amount weighed. Two separate stock solutions were prepared for the preparation of calibration standards (CC) and quality control samples (QC). The working solutions required for plotting calibration curve were also prepared in methanol. Likewise, quality controls (QCs) for the determination of precision and accuracy were prepared by appropriate dilution of the second stock solution prepared above using the same diluents. The QC samples were prepared in five different concentration levels covering the entire CC range.

Preparation of stock solution and working solution of IS:

Take 10 mg of Febuxostat in 10 ml volumetric flask and make up the volume with diluent. From that stock solution take 0.1 ml of solution into 10 ml volumetric flask and make up the volume with diluent to produce 10µg/ml solution. The working concentration of Take 100 mg of Cobicistat in 100 ml volumetric flask and make the volume with diluent to produce 1 mg/ml

Calibration curve standards and quality control samples preparation in human plasma:

Screened K₂EDTA human plasma batches (n=6) including one hemolytic and lipemic plasmas were pooled and used to prepare calibration standards and quality control samples. CC standards were prepared by spiking (50µl of working standard in to 950 µl of blank plasma) respective CC working solutions in plasma. Similarly, QC samples were also prepared in a similar way to produce QC samples in plasma. These samples were prepared in a bulk and an aliquot of 500 µl of CCs and QCs were pipetted in to prelabelled micro centrifuge tubes and stored at –70±10 °C in a deep freezer.

Calibration samples were prepared at a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 5.0 and 10.0µg/ml. Similarly, QC samples were at concentrations of 0.01 (QC-LLOQ), 0.1 (low; QC-L), 1.0 (middle; QC–M1),) and 5.0 µg/ml (high; QC-H).

Extraction protocol:

Take 250µl of plasma and 50µl of internal standard ,10µl of Cobicstat from the spiking solutions of both into a centrifuging tube and add 2 ml of acetonitrile go for cyclomixer for 15 sec. Then vertex for 2 min and finally centrifuge for 3 min at 3200 rpm speed. After the centrifugation collect the organic layer and directly inject 10 µl into HPLC(liquid-liquid extraction).

Validation ^6-15:

System suitability test:

System suitability^6,7(SST) experiment was performed by injecting 6 consecutive injections using aqueous sample equivalent to QC-M2 concentration. SST was performed at the start of the method validation and on each day as a first experiment.

Specificity:

The selectivity of the HPLC-UV method was established by screening the standard blanks of different lots of human plasma. For selectivity experiment ten different lots of plasma including one hemolytic and lipemic were screened for the specificity experiment.

Sensitivity:

The sensitivity^8,9of the method was evaluated by analyzing 6 LLOQ samples (0.01 µg/ml).

Linearity:

The linearity of the method was determined by using a 1/ x² weighted least square regression analysis of standard plots associated with eight-point standard curve. All the 3 calibration curves analyzed were found to be linear for the standards concentration ranging from 0.01 to 10.0 µg/ml.

Precision and Accuracy:

The precision (% CV) of the RP-HPLC method was evaluated by analyzing 6 replicates at different concentration levels corresponding to QC-LLOQ, QC-L, QC-M1, QC-M2 and QC-H during the course of validation. The accuracy of the assay was defined as the absolute value of the ratio of the calculated mean values of the LOQ, low, middle and high quality control samples to their respective nominal value, expressed as percentage.

Recovery:

The % mean recoveries were determined by measuring the responses of the extracted plasma quality control samples against un-extracted quality control samples at QC-L, QC-M1, QC-M2 and QC-H levels.

Ruggedness precision and accuracy:

Ruggedness was performed by analyzing one P&A batch (PA Batch-III) using different column and different analyst.

Stabilities studies:

Short term stock solution stability:

Short term stock solution stability of the Cobicistat and the IS was determined by using aqueous standard equivalent to QC-M2 concentration for the Cobicistat and working concentration for the IS after storage of stock solution over a period of 6 hours at room temperature.

Long term stock solution stability:

Long term stock solution stability for the Cobicistat and the IS was determined at a concentration of QC-M2 level for the analyte and working concentration for the IS using aqueous standard solution after a storage period of 39 days at 2-8 °C in refrigerator.

Reinjection reproducibility:

Reinjection reproducibility was performed by re injecting the P&A-01 after a period of 70 hours which were remain kept in auto sampler at 2-8 °C.

RESULTS:

Optimised chromatographic conditions

Column:	BDS 250, 4.6µ, 5mm
Mobile phase composition:	0.1% OPA Buffer : Acetonitrile 45:55
Flow rate :	1 ml/min
Injection volume:	50 µl
Run time:	10 min
Detection wavelength:	210nm
Column temperature:	30 ºC

System suitability test:

The % CV for system suitability test was in the range of 0.00–5.00% for retention time and 4.00–5.48% for the area ratio (analytes area/IS area).

Selectivity:

All the investigated human plasma lots were found to be free of significant interferences at the retention time of Cobicistat and the IS. Six plasma lots were pooled and used as a blank matrix to prepare calibration curve standards and quality control samples. Representative chromatograms of standard blank and blank with IS sample using pooled plasma are shown in Fig.3 and 4, respectively.

Sensitivity:

The % CV and % mean accuracy for analyte at LLOQ level were found to be 11.50% and 101.67% respectively. The sensitivity results Cobicistat are summarized in the Table 1.

Linearity:

Linearity and range were estimated by using calibration curve. By using calibration standards prepared by spiking drug (Cobicistat) and internal standard (febuxostat) in plasma at different concentrations 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 5.0 and 10.0µg/ml, the calibration graph was plotted taking concentration of spiked plasma on x-axis and peak area on y-axis. All the 3 calibration curves analyzed were found to be linear for the standards concentration ranging from 0.01 to 10.0µg/ml. A representative calibration curve is shown in Figure.10, which is obtained during the first precision and accuracy batch. The back calculated concentration data obtained for 3 calibration curves are summarized in the Table.2. .

Precision and Accuracy:

Intra batch precision (intra day):

The % CV of back calculated concentrations of all quality control samples analyzed in a single day at QC-L, QC-LLOQ, QC-M1, and QC-H concentration levels were ranged from 5.42% to 8.17%.

The % mean accuracy of back calculated concentrations for all quality control samples analyzed in a single day at QC-L, QC-M1, LLOQ and QC-H concentration levels were ranged from 98.33% to 100.71%.

Recovery:

The % mean recovery of Cobicistat at QC-L, QC-M1, and QC-H levels was found to be 47.15%, 63.81%, and 51.20%, respectively. Overall % mean recovery of analyte and % CV at all QC levels was found to be 54.055% and 16.08%, respectively. The recovery of Cobicistat at four different QC levels is summarized in the Table.4.

The overall % mean recovery for the internal standard was found to be 64.91%. The mean recovery of Febuxostat d6 is summarized in Table.5.

Ruggedness precision and accuracy:

Ruggedness was performed by analyzing one P&A batch (PA Batch-III) using different column and different analyst. The results were summarized in Table.6.

Stabilities:

Stability was assessed by comparing the stability stock solution(short term stock solution stability at room temperature for 6 hours) against the freshly prepared stock solution. The % mean stability of LQC and HQC Cobicistat was found to be 100.50% and 101.73% respectively. The results were summarized in Table.7.

The % mean stability of LQC and HQC Cobicistat long term stock solution stability at 2-8⁰c for 39 days was found to be 96.57 and 99.17% respectively.

Reinjection reproducibility:

The % CV of back calculated concentrations for HQC, MQC1, LQC, LLOQ QC was in between 5.12% to 10.79% respectively.

The % mean accuracy of back calculated concentrations for HQC, MQC1, LQC, LLOQ QC was in between 99.74% to 108.33 % respectively.

Table 2: Linearity data for cobicistat

Acquisition Batch ID	Std1			Std2	Std3	Std4	Std5	Std6	Std7	Std8
	Nominal Concentration (µg/ml)
	0.010	0.050			0.100	0.500	1.000	2.000	5.000	10.000
	Nominal Concentration Range(µg/ml)
	0.008-0.012		0.043-0.058		0.085-0.115	0.425-0.575	0.850-1.150	1.700-2.300	4.250-5.750	8.500-11.500
	Back Calculated Concentration(µg/ml)
P AND A1 P AND A2 P AND A3 n mean SD %CV %Mean Accuracy	0.009 0.011 0.010 3 0.010 0.001 10.00 100.0	0.049 0.052 0.050 3 0.0503 0.0015 3.03 100.67			0.089 0.099 0.110 3 0.0993 0.0105 10.57 99.33	0.510 0.469 0.512 3 0.4970 0.0242 4.88 99.40	0.895 0.965 1.005 3 0.9550 0.0556 5.83 95.50	1.986 2.003 2.125 3 2.0380 0.0758 3.72 101.90	4.695 5.412 5.025 3 5.0440 0.3588 7.11 100.88	8.965 10.965 11.025 3 10.3183 1.17240 11.36 103.18

Table 3: Accuracy and precision data for each individual run of cobicistat

Acquisition Batch ID	HQC	MQC1	LQC		LLOQ QC
	Nominal Concentration (µg/ml)
	5.000	1.000	0.100		0.010
	Nominal Concentration Range(µg/ml)
	4.250-5.750	0.850-1.150		0.0850-0.115	0.008-0.012
n Mean SD %CV %Mean Accuracy n Mean SD %CV %Mean Accuracy n Mean SD %CV %Mean Accuracy	4.986 5.215 5.362 4.912 4.856 4.901 6 5.0387 0.20336 4.04 100.77 4.669 4.900 5.024 5.028 5.009 5.698 6 5.0547 0.34374 6.80 101.09 5.363 5.258 4.696 4.715 5.237 4.811 6 5.0132 0.30400 6.06 100.26	0.965 0.935 1.102 1.095 1.125 1.120 6 1.0570 0.08415 7.96 105.70 0.998 0.915 0.927 1.025 0.985 1.085 6 0.9892 0.06313 6.38 98.92 1.106 1.125 1.105 1.102 1.008 1.139 6 1.0975 0.04612 4.20 109.75		0.098 0.102 0.115 0.093 0.096 0.093 6 0.0995 0.00835 8.40 99.47 0.101 0.112 0.095 0.097 0.090 0.092 6 0.0980 0.00785 8.01 98.02 0.096 0.105 0.108 0.110 0.097 0.091 6 0.1012 0.00757 7.49 101.17	0.009 0.010 0.010 0.011 0.010 0.009 6 0.0098 0.00075 7.64 98.00 0.010 0.009 0.010 0.009 0.011 0.010 6 0.0097 0.00088 9.08 97.00 0.010 0.009 0.011 0.009 0.010 0.011 6 0.0100 0.00089 8.94 100.00
Between Batch Precision and Accuracy
n Mean SD %CV %Mean Accuracy	18 5.0355 0.27277 5.42 100.71	18 1.0479 0.07743 7.39 104.79	18 0.0996 0.00757 7.60 99.55		18 0.0098 0.00080 8.17 98.33

Table 4: Recovery data of cobicistat

Replicate no.	HQC		MQC1		LQC
	Un extracted response	extracted response	Un extracted response	extracted response	Un extracted response	extracted response
1 2 3 4 5 6 n Mean SD %CV	522548 523258 523328 521748 523365 528569 6 523803 2417.74 0.46	263269 266987 266178 272203 271230 269325 6 268199 3356.94 1.25	91562 94558 94158 91456 94569 94518 6 93470 1527.08 1.63	59569 59985 59226 59593 59985 59523 6 59647 293.29 0.49	30268 30265 30698 30885 30222 30269 6 30435 283.33 0.93	14632 14547 14021 14398 14479 14023 6 14350 265.50 1.85
%Mean Recovery	51.20		63.81		47.15
overall% Mean Recovery overall SD overall %CV	54.055 8.6904 16.08

Table 5: Recovery- febuxostat-D6 (IS)

S.NO.

Un extracted Area Ratio

Extracted Area Ratio

Mean

%CV

405689

412568

395684

402535

395624

389586

400281.0

8276.48

2.07

264854

265487

241154

256147

265894

265335

259811.8

9865.85

3.80

%Mean Recovery

64.91

Table 6: Ruggedness data

	HQC	MQC1		LQC	LLOQ QC
	Nominal Concentration (µg/ml)
	5.000	5.000		5.000	5.000
	Nominal Concentration Range(µg/ml)
	4.250-5.750	4.250-5.750		4.250-5.750	4.250-5.750
	Calculated Concentration(µg/ml)
Different column n Mean SD %CV %Mean Accuracy	4.598 4.985 5.236 5.145 5.412 5.365 6 5.1235 0.30000 5.86 102.47		0.896 0.914 0.966 1.145 1.025 1.005 6 0.9918 0.09016 9.09 99.18	0.097 0.114 0.106 0.110 0.092 0.086 6 0.1008 0.01093 10.84 100.83	0.009 0.010 0.012 0.011 0.010 0.009 6 0.0102 0.00117 11.50 101.67
Different analyst n Mean SD %CV %Mean Accuracy	5.124 4.689 4.985 4.478 4.966 4.856 6 4.8496 0.23283 4.80 96.99		1.110 1.089 0.938 0.987 0.954 0.896 6 0.9957 0.08585 8.62 99.57	0.098 0.106 0.108 0.093 0.097 0.113 6 0.1026 0.00761 7.42 102.57	0.008 0.009 0.010 0.011 0.011 0.009 6 0.0097 0.00121 12.53 96.67

Table 7: Short term stock solution stability at room temperature (6 hours)

Replicate no.	HQC	LQC
	Nominal Concentration (µg/ml)
	5.000	0.100
	Nominal Concentration Range(µg/ml)
	4.250-5.750	0.085-0.115
	Calculated Concentration(µg/ml)
1 2 3 4 5 6 n Mean SD %CV %Mean Accuracy	4.365 4.968 4.991 5.256 5.612 5.326 6 5.0863 0.42567 8.37 101.73	0.096 0.112 0.103 0.091 0.092 0.109 6 0.1005 0.00887 8.83 100.50

Table 8: Long term stock solution stability (at 2 – 8 °C for 39 days)

Replicate no.	HQC			LQC
	Nominal Concentration (µg/ml)
	5.000			0.100
	Nominal Concentration Range(µg/ml)
	4.250-5.750		0.085-0.115
	Comparision sample	Stability sample	Comparision sample		Stability sample
1 2 3 4 5 6 n Mean SD %CV %Mean Accuracy	5.321 4.626 5.126 5.695 5.015 4.985 6 5.1279 0.35906 7.00 102.56	4.398 4.698 5.698 5.125 5.326 5.267 6 5.0853 0.46690 9.18 101.71	0.099 0.102 0.112 0.114 0.109 0.109 6 0.1075 0.00582 5.42 107.50		0.097 0.100 0.111 0.103 0.113 0.100 6 0.1038 0.00666 6.42 103.81
%Mean stability	99.17		96.57

Table 9: Reinjection reproducibility results - Cobicistat for 70 hours

Replicate no.	HQC	MQC1	LQC	LLOQ QC
	Nominal Concentration (µg/ml)
	5.000	1.000	0.100	0.010
	Nominal Concentration Range(µg/ml)
	4.250-5.750	0.850-1.150	0.0850-0.115	0.008-0.012
1 2 3 4 5 6 n Mean SD %CV %Mean Accuracy	4.695 5.214 5.363 4.966 4.895 4.789 6 4.9869 0.25529 5.12 99.74	0.895 0.984 1.125 1.112 1.025 0935 6 1.0127 0.09310 9.19 101.27	0.097 0.100 0.112 0.110 0.113 0.093 6 0.041 0.00862 8.28 104.10	0.009 0.012 0.011 0.010 0.011 0.012 6 0.0108 0.00117 10.79 108.33

DISCUSSION AND CONCLUSION:

RP-HPLC methods have been reported for the determination of Cobicistat with other steroids in serum and water effluents. But there were no reports available for the determination of Cobicistat alone in human plasma. Many formulations available for Cobicistat alone in the market and should have a proper analytical method to quantify the drug concentrations in plasma. Hence a simple and rapid RP-HPLC has been proposed for the determination of Cobicistat in human plasma. This method employed commercially available Febuxostat as an internal standard. Chromatography was performed on inertsil BDS (250mm×4.6mm, 5µm), Orthophosphoric acid: Acetonitrile (45:55 v/v) delivered at a flow rate of 1.0 ml/min. Precision and accuracy results over 3 analytical batches are meeting acceptance criteria. Stock solutions were stable for 6 hours at room temperature and 39 days at 2-8°C in refrigerator. Cobicistat was stable at variety of stability studies namely long term stock solution stability (at 2-8°C for 39 days), auto sampler stability (73 hours), bench top stability (13 hours), re-injection stability (70 hours). Real time sample concentrations above the calibration curve range can be quantified by half and quarter dilution with blank plasma.

A rapid, sensitive and highly selective method for the determination of Cobicistat in human plasma was developed, using RP-HPLC technique. This method can be used for analysis of large number of samples with greater precision during pharmacokinetic and bioequivalence studies. The assay method is more selective than previously reported HPLC, RIA and allows for a much higher sample throughput due to the short chromatographic time and simple sample preparation. As robust RP-HPLC instrument performance was observed and hence this method is an excellent analytical option for rapid quantification of Cobicistat in human plasma. The method was used successfully for the determination of plasma drug concentrations in human plasma samples under fasting conditions.

ACKNOWLEDGEMENT:

Authors are thankful to University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Nagarjuna nagar, Guntur, Andhra Pradesh, India.for providing necessary analytical facilities

REFERENCES:

1. Chandni Saha, Md. Nazeeruddin Ahmed, development and validation of a simple UV spectrophotometric method for the determination of cobicistat in its bulk form, Indo American Journal of Pharmaceutical Research, 2014. ISSN NO: 2231-6876, pg no 5792-5796.

2. Shiny Ganji, D. Satyavati, Development and validation of RP-HPLC method for the analysis of Cobicistat and related impurities in bulk and pharmaceutical dosage forms, Asian J. Pharm. Ana. 2015; Vol. 5: Issue 1, Pg 1-8.

3. D. Sindu Priya and D. Gowri Sankar, Stability indicating RP-HPLC Method Development and Validation for Simultaneous Determination of Atazanavir and Cobicistat in Bulk and Pharmaceutical Formulation, Am. J. Pharm Tech Res. 2015; 6(1).

4. Raghu Ram Jampala, V. Kiran Kumar, Development and Application of Liquid Chromatographic Method for Simultaneous Determination of Elvitegravir, Tenofovir Disoproxil Fumarate, Emtricitabine, and Cobicistat in Fixed Dosage Form, Pharmaceutical Methods Vol 5, Issue 1, Jan-Jun 2014.

5. Swati S. Pardhi, Kiran B.A her, development and validation of uv spectrophotometric method for the estimation of elvitegravir (evg) in bulk and pharmaceutical formulations. IJPSR, ISSN: 0975-9492 Vol 6 No 01 Jan 2015(213-218).

6. N. Tamilselvi, Dona Sara Kurian. Bioanalytical method development and validation of pirfenidone by RPHPLC method and its application to the determination of drug food interaction study in wister rats., IJPBR, ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012(132-142).

7. A. Kirthi1, R. Shanmugam, M. Shanti Prathyusha1, D. Jamal Basha, a review on bioanalytical method development and validation by RP–HPLC, Journal of Global Trends in Pharmaceutical Sciences, 5(4) -(2014) 2265-2271

8. A. B. Pharne, B. Santhakumari, A. S. Ghemud, H. K. Jain, M. J. Kulkarni, Bioanalytical method development and validation of vildagliptin a novel dipeptidyl peptidase iv inhibitor by RP-HPLC method, International journal of Pharmacy and Pharmaceutical Sciences, Issue 3 - 0975-1491,

9. Senthil Rajan Dharmalingam, Srinivasan Ramamurthy, A Simple HPLC Bioanalytical Method for the Determination of Doxorubicin Hydrochloride in Rat Plasma: Application to Pharmacokinetic Studies, Tropical Journal of Pharmaceutical Research, March 2014; 13 (3): 409-415.

10. S. Jayaseelan, S. Suresh, G. Sathishkumar, V. Sekar, P. Perumal, bioanalytical method development and validation of lamivudine by rp-hplc method, International Journal of ChemTech Research, ISSN : 0974-4290, Vol.2, No.1, pp 163-167, Jan-Mar 2010.

11. Surajpal Verma, Pooja Mullick, Md. Sleem Bhatt, Nadeem Siddiqui, Ozair Alam, Indu Bala and Suroor A. Khan, Bioanalytical method development and validation for the simultaneous estimation of lamivudine and stavudine in human plasma by hplc, Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 67 No. 4 pp. 429-437, 2010 ISSN 0001-6837.

12. Pranay Wal, Brijesh Kumar, Anil Bhandari, A. K. Rai, Ankita Wal, Bioanalytical Method Development –Determination of Drugs in Biological Fluids, Journal of Pharmaceutical Science and Technology Vol. 2 (10), 2010,333-347.

13. Vishal. P. Awari, Subramania Nainar Meyyanathan, Yamjala. Karthik, Natarajan Jawahar, HPLC Method Development and Validation of Rizatriptan in Rabbit Plasma, J. Pharm. Sci. & Res. Vol. 6(1), 2014, 24 – 26.

14. T. Manish K development and validation of HPLCUV method for the estimation of levofloxacin in human plasmamara, gurrala Srikant HB, J. Venkateshwar Rao, CKRS. Sambasiva Rao, International Journal of Pharmacy and Pharmaceutical Sciences, ISSN- 0975-1491,Vol 3, Issue 2, 2011.

15. Santosh Kumar Bhardwaj, K. Dwivedia and D. D. Agarwala, A Review: HPLC Method Development and Validation, International Journal of Analytical and Bioanalytical Chemistry, ISSN-2231-5012, 2015; 5(4): 76-81.

Received on 17.07.2016 Accepted on 20.10.2016

Asian J. Pharm. Ana. 2016; 6(4): 216-226.

DOI: 10.5958/2231-5675.2016.00033.8

Replicate no.	LLOQ(nominal concentration) 0.010µg/ml
	nominal concentration range (0.008-0.012µg/ml)
	Calculated concentration (µg/ml)
1 2 3 4 5 6 n Mean SD %CV %Mean Accuracy	0.009 0.010 0.012 0.011 0.009 0.010 6 0.0102 0.00117 11.50 101.67