Zakir Ahmad et al. Int. Res. J. Pharm. 2016, 7 (1)

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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

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Research Article DEVELOPMENT OF A VALIDATED RP-HPLC METHOD FOR ESTIMATION OF DARUNAVIR IN SPIKED HUMAN PLASMA WITH UV DETECTION Zakir Ahmad, Sandeep S. Sonawane *, Atharva S. Bhalerao, Sanjay J. Kshirsagar MET’s Institute of Pharmacy, MET League of Colleges, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra, India *Corresponding Author Email: sandeeps.iop@gmail.com Article Received on: 11/10/15 Revised on: 13/11/15 Approved for publication: 28/12/15 DOI: 10.7897/2230-8407.0714 ABSTRACT A simple, rapid and accurate RP-HPLC method was developed and validated for the quantification of Darunavir in spiked human plasma using liquid-liquid extraction. Sufficient recovery was obtained when drug and internal standard (Efavirenz) were extracted using diethyl ether. The chromatographic separation was performed on C18 (250 X 4.6 mm, 5 µm) column with mobile phase acetonitrile: water (60: 40 %, v/v). The flow rate was kept constant at 1 mL/min and detection was performed at 267 nm. The calibration curve was found linear in the range of 600-19200 ng/mL. During calibration experiments, it was found that heteroscedasticity can be minimized using weighted regression calibration model with weighing factor of 1/x². Selectivity was evaluated at lower limit of quantification of Darunavir at 600 ng/mL. During accuracy and precision studies, the intra-day and inter-day % RE was found between ±15 % and % RSD was less than 15%. Stability study experiments indicates that the drug remain stable after three freeze-thaw cycles. Keywords: Bioanalytical; weighted-regression; liquid-liquid extraction; Darunavir INTRODUCTION Chemically, Darunavir is, [(1S,2R)-3-[[(4-amino-phenyl)sulfonyl](2-methyl propyl)amino]-2-hydroxy-1-(phenyl-methyl)propyl] carbamic acid 1. A HIV peptidic protease inhibitor, with high levels of antiviral activity against wide type virus and stains with phenotypic resistance to other protease inhibitors2. Literature survey revealed few quantitative analytical methods for Darunavir in pharmaceutical formulations using RP-HPLC3, 4, 5 and also in biological fluids, like LC-MS6, 7, LC-tandem mass spectrometry8, UPLC-MS/MS9, direct injection HILIC-MS/MS in rat plasma10 and determination of intracellular Darunavir by HPLC with Fluorescence detection11. The objective of the present work was to develop a simple and rapid RP-HPLC-UV based bioanalytical method using liquid-liquid extraction technique for Darunavir. Also the study suggests an approach in selection of optimum calibration model and to validate the method according to US-FDA guidelines13. MATERIALS AND METHODS Chemicals and Reagents Pharmaceutical grade Darunavir and Efavirenz were used as drug and internal standard respectively and were kindly supplied as gift samples by Indoco Remedies Ltd., Mumbai, India. Blank human plasma was procured as a gift sample from Arpan Blood Bank, Nashik, India. Plasma from six different sources was mixed thoroughly to get pooled blank plasma. Acetonitrile and Methanol used in analysis were of HPLC grade and all other chemicals were of analytical reagent grade, purchased from SD Fine Chemicals, Mumbai, India. The DURAPORE 0.45 µ ´ 47 mm membrane filter papers were purchased from Millipore (India) Pvt. Ltd., Bangalore, India. Freshly prepared double distilled water used in analysis was prepared from Borosil All Glass Double Distillation Assembly (Borosil, Mumbai, India) and filtered through 0.45 µ membrane filters paper.

Instrumentation Chromatographic analysis was carried out using a HPLC system consisting of pump PU-2080 Plus (JASCO Corporation, Tokyo, Japan) equipped with 100 µL Rheodyne loop injector (7725i) and detection was carried out on UV-2075 detector (JASCO Corporation, Tokyo, Japan) using Borwin Chromatography software (Version 1.50). Liquid-liquid Extraction (LLE) Experiments An aliquot of pooled human plasma (1 mL) was taken in a 20 mL stoppered glass tube and spiked with 3000 ng of Darunavir by addition of 30 µL of 100 mL methanolic solution. This was vortex mixed for 1 min, to this, 5 mL of diethyl ether was added and the tube was shaken in an inclined position on a reciprocating shaker at 100 strokes/min for 30 min. The tube was centrifuged at 3000 rpm for 10 min. From this, the organic layer was transferred to a separate tube and evaporated to dryness under a stream of nitrogen. The residue obtained upon evaporation to dryness was reconstituted with 250 µL of mobile phase and 100 µL were injected into HPLC system under appropriate chromatographic conditions. Preparation of Standard stock solution and working standard solution of Internal Standard Quantity equivalent to 25 mg of the Darunavir was accurately weighed transferred to 25 mL volumetric flask; volume was made up to the mark with methanol to get 1000 µg/mL standard stock solution for Darunavir. Similarly, quantity equivalent to 15 mg of the Efavirenz was accurately weighed and transferred to 10 mL volumetric flask; volume was made up to the mark with methanol to get 1500 µg/mL standard stock solution for Efavirenz.

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Preparation of Calibration curve (CC) standards and Quality Control (QC) samples From above prepared standard stock solution of Darunavir, appropriate dilutions were made to get six different working standard solutions of concentrations; 20, 40, 80, 160, 320, 640 µg/mL. Aliquots of 1 mL of pooled blank plasma were taken in stoppered glass tubes of capacity 20 mL. To these tubes 30 µL of appropriate working standard solutions of Darunavir was added and vortex mixed for 1 min get CC standards containing 600, 1200, 2400, 4800, 9600, and 19200 ng/mL of Darunavir, respectively. The QC samples were similarly prepared to contain three concentrations [1800 ng/mL Low Quality Control (LQC), 9600 ng/ml Middle Quality Control (MQC) and 19200 ng/ml High Quality Control (HQC)]. Chromatographic conditions Chromatographic analysis was carried out on a C18 Phenomenex Hyperclone column (250 × 4.6 mm, 5 µm) with mobile phase consisting of acetonitrile: water (60:40 %, v/v) at flow rate of 1 mL/min. The detection was carried out at 267 nm. Calibration runs In the calibration runs, 1 mL aliquots of all CC standards were analyzed in six replicates using optimized LLE and chromatographic conditions. All calibration standards (CC) were analyzed in six replicates. Prior to analysis, each CC standard was mixed with 30 µL of 1500 µg/mL of methanolic solution of Efavirenz (as an internal standard). At the end of the calibration runs, the chromatograms of CC standards were processed to get the peak areas for Darunavir and Efavirenz. For each CC standard the area ratio of Danuravir to Efavirenz was calculated. Selection of Calibration model and range The purpose of calibration experiments is to select and evaluate the calibration range and model. As the calibration data obtained is prone to heteroscedasticity that can be overcome by applying appropriate weights12. Thus, data obtained from the calibration run experiments were subjected to unweighted and weighted least square regression analysis to generate the respective calibration equations. In weighted regression, weighing factors (w) of 1/x and 1/x2 was used, where x is the concentration of the CC standards of Darunavir. In order to select best calibration model, each calibration model and equation was evaluated with respect to % relative error (% RE), residual plot and homogeneity of variance (homoscedasticity) in the linear range. The area ratios for the CC standards were referred to the calibration equations to get the back calculated concentrations (interpolated concentrations) for the each CC standards. % RE was calculated for each CC standard as, % RE = (Interpolated concentration – Nominal concentration) / Nominal concentration × 100 The total % RE was calculated as sum of the % RE for all CC standards. The predicted area ratios for the CC standards were calculated by entering the nominal concentration of each CC standard into the calibration equations. A plot of residuals was constructed by plotting the differences between measured and predicted area ratios against log nominal concentration and the scatter of residual was evaluated. To evaluate the homoscedasticity in the linear range, the variance of the residuals at the highest CC standard to the lowest CC standard was evaluated by means of one- way ANOVA.

The calibration model with, minimum % RE, random scatter of points in the plot of residuals and no significant difference in one- way ANOVA was selected. Validation Studies The developed method was validated as per US-FDA Guidance for Industry: Bioanalytical methods Validation13. Selectivity was studied at the Lower Limit of Quantification (LLOQ) at 600 ng/mL by comparing blank responses of plasma from six different sources with peak areas afforded by the LLOQ samples. The Calibration curve standards were evaluated by preparing and analyzing CC standard solutions spiked with internal standard for five days. The concentrations of each CC standard was back calculated using suggested calibration model and the deviation of the back calculated concentrations from the nominal values was studied and expressed as % nominal. Precision and accuracy were studied by analyzing five bioanaytical batches over five days. Each batch consists of one blank, all CC standards and five replicates of LQC, MQC and HQC samples. The calibration equation was determined for each batch from the analysis of CC standards and was used to calculate the concentration of Darunavir in LQC, MQC and HQC samples. The within batch and between batch accuracy and precision were determined in terms of % RE and % RSD, respectively. The recovery of the extraction procedure was calculated as, % Mean recovery = Mean area for processed samples / Mean peak area for standard dilution × 100 Stability of Darunavir in plasma was evaluated under various conditions viz. freeze-thaw cycles, stability at – 20° C for 30 days and stability at room temperature for 6 h. The amount of Darunavir in the stability samples was found out and the % nominal and % RSD of the determinations were calculated. RESULTS AND DISCUSSION When Darunavir and Efavirenz were subjected to chromatographic analysis in mobile phases of different strengths and compositions, it was found that mobile phase comprising of acetonitrile: water (60:40 %, v/v) gave adequate retention and resolution at flow rate of 1 mL/min. The detection was carried out at 267 nm. The retention for Darunavir and Efavirenz obtained at 4.07 min and 8.44 min respectively. When liquid-liquid extraction experiments were performed using different solvents like ethyl acetate, n-hexane and diethyl ether, it was found that both Darunavir and Efavirenz were comparably extracted with diethyl ether. Also, when aliquots of blank plasma were extracted with diethyl ether and chromatographed, it was found that there were no significant interfering peaks at the retention times of Darunavir and Efavirenz. Thus, concluded that diethyl ether can be used as liquid-liquid extraction solvent for Darunavir and Efavirenz. The chromatogram of blank plasma extracted in diethyl ether is shown in FIGURE 1 and the representative chromatogram of Darunavir and Efavirenz extracted in diethyl ether is shown in FIGURE 2. The extraction recovery for Darunavir and Efavirenz was 67.41 % and 81.05 %, respectively. When the highest CC standard containing 19200 ng/ mL of Darunavir in plasma and plasma samples spiked with varying amounts of Efavirenz concentration were analyzed, it was found that 45 µg of Efavirenz was required to get an internal standard peak area that was between 30-60 % of the peak area afforded by the highest CC standard of Darunavir. It was thus decided to add 30 µL of 1500 µg/mL methanolic solution of Efavirenz to each CC standard prior to analysis.

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Table 1: Area ratios from calibration experiments

CC No. Amount of Drug ng/mL Area Ratios (mean ± SD, n = 6) CC-1 600 0.1170± 0.0114 CC-2 1200 0.2337± 0.0083 CC-3 2400 0.4276± 0.0295 CC-4 4800 0.8047± 0.0206 CC-5 9600 1.6401± 0.1359 CC-6 19200 3.4255± 0.1178

Table 2: Results of evaluation of various calibration models

Unweighted regression Weighted regression (1/X) Weighted regression (1/X2)

∑% RE Nature of residuals plot

F5,5* value ∑% RE Nature of residuals plot

F5,5** value ∑% RE Nature of residuals plot

F5,5** value

163.03 Random scatter 106.742 -3.79E-13 Random scatter 0.1042 -8E-13 Random scatter 0.0001 * F value calculated as ratio of variances at the extremes of calibration range , ** F value calculated as ratio of variances of the weighted residuals.

Table 3: Blank responses and LLOQ peak areas for darunavir

Sr. No. Blank response (µV.sec) Peak areas at LLOQ (µV.sec)

1 11471 251801 2 27247 349581 3 22174 286879 4 12742 319938 5 17960 332526 6 25939 302156

Table 4: Standard curve parameters for darunavir

CC No.

Nominal Conc.

(µg/mL)

Back Calculated concentrations (µg/mL) (Days)

Mean ±SD % RSD

% Accuracy

1 2 3 4 5 6 1 600 597.12 587.5 587.5 587.52 597.12 580.17 583.48 4.03 0.69 97.24 2 1200 1296.72 1252.50 1252.50 1265.69 1296.71 1299.07 1277.19 20.78 1.62 106.43 3 2400 2412.35 2397.5 2397.5 2391.88 2412.35 2416.76 2404.72 9.40 0.39 100.19 4 4800 4622.94 4451.38 4451.38 4582.88 4622.94 4631.39 4560.85 78.67 1.72 95.01 5 9600 9263.84 9900.27 9900.27 9436.43 9263.64 9280.72 9507.57 283.93 2.98 99.03 6 19200 19643.64 19199.72 19199.72 19808.7 19643.64 19679.57 19529.17 239.45 1.22 101.71

Table 5: Results of accuracy and precision studies

Level Concentration

added (ng/mL) Intra – day (n = 5) Inter – day (n = 5) Recovery

(n = 5) Mean concentrations found (ng/mL)

% RE % RSD Mean concentrations found (ng/mL)

% RE % RSD

LQC 1800 1816.15 0.89 3.99 1898.17 5.45 4.87 65.35 HQC 9600 9600.27 0.0029 2.56 9779.06 3.94 4.18 67.10 HQC 19200 19113.01 -0.45 7.63 20340.53 5.94 3.91 80.52

IS -- -- -- -- -- -- -- 81.05

Table 6: Results of one-way ANOVA at each QC level

Level Source Sum of squares df Mean squares Total Variance ±SD F value LQC Within run 200123 20 10006 16683.49 129.16 4.509

Between run 200281 4 50070 MQC Within run 4591165 20 229558 391739 625.89 5.239

Between run 4810606 4 1202651 HQC Within run 2.208E+07 20 1103874 1749401 1322.64 5.004

Between run 1.991E+07 4 4977034

Table 7: Results of stability studies for darunavir (n = 5)

QC level

Stability at room temperature Stability at -200C Freeze thaw (FT) stability

% Nominal % RSD % Nominal % RSD % Nominal % RSD 2h 4h 6h 2h 4h 6h 10

days 20

days 30

days 10

days 20

days 30

days FT 1

FT 2

FT 3

FT1 FT2 FT3

LQC 99.83 100.66 101.4 2.31 1.16 1.39 101.32 103.05 98.51 3.23 5.78 4.12 103.64 99.27 97.34 3.30 4.17 8.73 HQC 95.85 97.35 99.09 2.48 3.50 4.11 101.89 98.35 98.35 3.37 7.70 4.71 98.98 93.46 100.93 3.43 5.81 6.24

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Figure 1: Chromatogram of blank plasma extracted in diethyl ether and detected at 267 nm

Figure 2: Representative chromatogram of Darunavir and Efavirenz extracted in diethyl ether and detected at 267 nm

During calibration experiments, when data obtained from Table 1 subjected to unweighted and weighted linear regression with a weighing factor 1/x and 1/x2, unweighted regression resulted in the equation, Y = 0.0002x + 0.008 and with 1/x and 1/x2 weights, resulted in equation, Y = 0.0002x + 0.0127 and Y = 0.0002x + 0.0159 respectively. For selection of calibration model, each of these obtained linear regression equations evaluated for the % RE, random scatter and homoscedasticity for selection of calibration model (Table 2). From this, it was concluded that although all calibration equations gave a random scatter residuals, the total % RE was minimal when weighted regression with weighing factor 1/x2 was applied. Further, when the Fcalculated values were compared with Ftabulated (a = 0.05), it became evident that a weighing factor of 1/x2 was suitable to homogenize the variance of the residuals. Thus, it was decided to adopt calibration model of weighted linear regression with weighing factor 1/x2 in the calibration range of 600 to 19200 ng/mL of drug. The range was acceptable considering that Darunavir results in a Cmax of 6000 ng/mL upon administration of an oral dose of 600 mg. During validation studies it was found that the peak areas for the Lower Limit of Quantification (LLOQ) samples were more than five times the blank responses obtained using six different plasma sources as seen in Table 3, which concludes that the method was deemed to be selective for an LLOQ of 600 ng/mL. From Table 4, it was concluded that the % nominal values of the back calculated concentrations of CC standards were between 95-106 %, which was in acceptable limit. The evaluation of accuracy and precision showed that the intra- day % RE was between ± 15 %, while the % RSD was less than 15. The US- FDA Guidance requires that the % RE be between ± 15 %, while the % RSD should be less than 15. The results of assay precision and accuracy as well as extraction recovery for Darunavir at LQC, MQC and HQC and for Efavirenz are presented in Table 5. Further, the intermediate precision of the method was determined by using a one-way ANOVA. For each QC level, within mean square and between mean square values were determined. The total variance was taken as a sum of within and between mean squares and standard deviation was determined as a square root of total variance and F value was determined. The Fcalculated was found less than Ftabulated (a = 0.05), Table 6, which indicates that there is no significant difference between intra- day and inter- day precision. The results of stability evaluation of Darunavir are presented in Table 7. Analysis cycles viz. three freeze-thaw cycles, stability at -20°C for 30 days and stability at room temperature for 6 h indicated that Darunavir was stable in human plasma under these conditions. CONCLUSION In this report, a simple, rapid, selective and accurate RP-HPLC-UV method is described for the quantification of Darunavir in spiked

human plasma using liquid-liquid extraction. The developed bioanalytical method is capable of quantifying Darunavir from spiked human plasma in the concentration range of 600 – 19200 ng/mL. The method meets the requirements of the US- FDA guidance and can be applied to BA/BE studies. Also, the developed method described does not require expensive chemicals and solvents and does not involve complex instrumentation or complicated sample preparation. ACKNOWLEDGEMENT Authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities, Indoco Remedies Ltd., Mumbai for providing gift samples of drugs and to Arpan Blood Bank, Nashik for providing human plasma samples. REFERENCES 1. O’Meil M, Heckelman P, Koch C, Roman K, Kenny C,

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Cite this article as: Zakir Ahmad, Sandeep S. Sonawane, Atharva S. Bhalerao, Sanjay J. Kshirsagar. Development of a validated RP-HPLC method for estimation of Darunavir in spiked human plasma with UV detection. Int. Res. J. Pharm. 2016;7(1):24-28 http://dx.doi.org/10.7897/2230-8407.0714

Source of support: Nil, Conflict of interest: None Declared

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