DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF TENOFOVIR DISOPROXIL FUMERATE AND EFAVIRENZ IN BULK AND FORMULATION

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF TENOFOVIR DISOPROXIL FUMERATE AND EFAVIRENZ IN BULK AND FORMULATION

D. Sri Vidya*¹, Y.A. Chowdary ³ and T.E.G.K. Murthy ²

Department of Pharmaceutical Analysis & Quality Assurance ¹, Department of Pharmaceutics ², Bapatla College of Pharmacy, Bapatla-522101, Andhra Pradesh, India

Department of Pharmaceutics, NRI College of Pharmacy ³, Agiripalli, Vijayawada-521137, Andhra Pradesh, India

ABSTRACT: The objective of the present work was to develop a simple, rapid, accurate RP-HPLC method for the simultaneous estimation of Tenofovir disoproxil fumerate (TDF), Efavirenz in bulk and formulation. The chromatographic separation was achieved by isocratic mode of elution by using Agilent Zorbax Eclipse XDB C₁₈ (150 x 4.6mm, particle size 5µm) analytical column. The mobile phase consisting of Acetonitrile and phosphate buffer (0.03M KH₂PO₄, pH 2.5) the ratio of 70:30v/v with 0.6 ml/min flow rate and the eluents are monitored at 255nm.The retention times were 2.44min for TDF and 5.52 min for Efavirenz. The detector response was linear for TDF between 3-18µg/ml with R² =0.9987 and 6-36µg/ml for Efavirenz with R²=0.9982 respectively. The % relative standard deviation (%R.S.D) values were found to be <2. The method was validated by determining accuracy, precision and linearity range. The results  of the proposed RP-HPLC method is precise, rapid and accurate which is useful for quantitative determination and routine analysis of Tenofovir disoproxil fumerate and Efavirenz in bulk and  in formulation.

Tenofovir disoproxil fumerate, Efavirenz, RP-HPLC, Agilent Zorbax column

INTRODUCTION:A novel antiretroviral formulation with combining doses of the non-nucleoside reverse transcriptase inhibitor Efavirenz (600mg) and nucleoside reverse transcriptase inhibitor Tenofovir disoproxil fumerate (300mg) are more effective. Tenofovir disoproxil fumerate ^{1, 2} is fumaric acid salt of bis isopropoxycarbonyloxy o-methyl ester derivative of Tenofovir is 9-[(R)-2-[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adeninefumerate.

It is   chemically the prodrug of Tenofovir. Efavirenz chemically (S)-6-chloro-4-(cyclopropylethynyl)-1, 4-dihydro4-(trifluoro methyl)-2H-3, 1-benzoxazin-2-one. Literature review ^3-10 reveals a few chromatographic methods are developed for the determination of Tenofovir disoproxil fumerate Emtricitabine and Efavirenz, but there are no analytical methods for Tenofovir disoproxil fumerate and Efavirenz in biological fluids and pharmaceutical dosage forms along with other antiretroviral drugs.

The chemical structures of the drugs are furnished below. During formulation of a tablet dosage form, there is a possibility of formulation and process derived impurities generation and interference of such impurities in the analytical method.

Further there is a need to identify and control such impurities in the formulation as per the regulatory requirement. So studies were conducted to develop a validated analytical method for quantitative estimation of Tenofovir disoproxil fumerate and Efavirenz simultaneously from the pharmaceutically acceptable tablet dosage form and the results are reported here.

A

B

FIGURE 1: CHEMICAL STRUCTURE OF TENOFOVIR DISOPROXIL FUMERATE AND EFAVIRENZ

MATERIALS AND METHOD: Tenofovir disoproxil fumerate, Efavirenz were obtained as a gift samples from NATCO Pharmaceuticals, Hyderabad.KH₂PO₄ was analytical grade  supplied  by S.D. Fine chemicals, Mumbai, Ortho phosphoric acid(OPA),  Acetonitrile(Merck HPLC grade) and Water for HPLC method( in House).

Instrument: Quantitative HPLC was performed on Agilent technologies1200 series, PDA detector module equipped with auto injector with Ezchrome elite software. A reverse phase Agilent Zorbax Eclipse XDB C₁₈ (150 x 4.6mm, particle size 5µm) analytical column was used.

HPLC Conditions: Preliminary studies were conducted andtrails are made for the method development. The optimized mobile phase consists of Acetonitrile and phosphate buffer (0.03M KH₂PO₄, pH adjusted to 2.5 with orthophosphoric acid) in the ratio of 70:30 v/v. They were filtered through 0.45µm membrane filter using vaccum pump.

Flow rate was maintained at 0.6ml/min and run time for 10 min. prior to sample injection, column was saturated with mobile phase for 40 min and injection volume was 10µl injected by auto sampler. The detection response was measured at 255nm and maintained at ambient temperature.

Buffer preparation 0.03M: 10.08g of KH₂PO₄ was dissolved in 1000ml of double distilled water, pH adjusted to 2.5 with orthophosphoric acid.

Preparation of standard stock solution: Accurately 3mg of Tenofovir disoproxil fumerate and 6mg of Efavirenz standard drugs were weighed and transferred into 10ml volumetric flask and made up to the mark with Acetonitrile diluent-1(3000µg/ml and 6000 µg/ml).

Diluent1- Acetonitrile

Diluent-2 Mobile phase (Acetonitrile: Phosphate buffer, 70:30v/v)

Working standard solution: From above standard stock solution, working standard solution was prepared by pippeting 0.1ml into 10ml volumetric flask and made up to the mark with diluent-2. Final concentration of 3µg/ml and 6µg/ml of Tenofovir disoproxil fumerate and Efavirenzworking standard solution were injected.

Preparation of in house tablet: Tenofovir disoproxil fumerate (300mg), Efavirenz (600mg), Starch paste(15mg), starch(60mg), Magnesium stearate (6mg), talc(6mg) were weighed.

Procedure: Tenofovir disoproxil fumerate (300mg), Efavirenz (600mg) were blended in mortar. The mixture was moistened with the binder starch paste. The damp mass was subjected to wet sieving (sieve no.12). The granules were dried at 60⁰ for 4 hr and re-sieved.

The granules having a size of 44/60 were collected and compressed to form a tablet with 12mm compression tool by using Cad mach rotary tablet machine.

Preparation of sample solution for Assay: Ten tablets (in house) were accurately weighed and crushed into fine powder. The powder equivalent to Tenofovir disoproxil fumerate (300mg) and Efavirenz (600mg) was transferred to 10ml volumetric flask then sonicated for 10 min, filtered through whatmann filter paper no.1 and made up to the mark with diluent-1.From the above stock solution 0.1 ml solution was pippeted into 10 ml volumetric flask and makeup to mark with diluents-2. Final concentration of 3µg/ml and 6µg/ml of Tenofovir disoproxil fumerate and Efavirenz respectively was injected.

Validation of Analytical method: ¹¹

1. Linearity and Range: Appropriate aliquots of Tenofovir disoproxil fumerate and Efavirenz standard stock solution of 3000µg/ml and 6000µg/ml was transferred to series of 10 ml volumetric flasks (Each 0.1 ml contains 3µg/ml of Tenofovir disoproxil fumerate and 6µg/ml Efavirenz respectively.) 0.1-0.6ml of standard stock was pippeted and diluted to final volume with diluent-2.
2. Accuracy studies: Accuracy was determined by adding the known amount of standard drug to the pre fixed concentrations of assay samples by standard addition method. The percentage recovery studies were carried out in triplicate of three different levels 50%, 100%, 150% by spiking standard drug solution to the placebo.
3. Precision Studies and System suitability: The method precision of the proposed method is ascertained by injecting 6 replicates of test sample and % recovery, %RSD were calculated.
4. LOD and LOQ: The sensitivity of HPLC was determined from LOD and LOQ which were calculated from the calibration curve using the following equations

LOD = 3.3σ/S and

LOQ = 10 σ/S, where

σ= Standard deviation of y intercept of regression line

S = Slope of the calibration curve

1. Robustness and Ruggedness: Robustness is the measure of the ability of an analytical method to remain unaffected by small but deliberate variations in method parameters, pH change (±0.5), mobile phase composition (±0.2), flow rate (±1ml/min), wave length (2nm) were considered. The developed method is robust with deliberate changes with variation of analyst to analyst, column to column Intraday and Inter day precision and provides an indication of its reliability.

RESULTS AND DISCUSSION: The present study describes a new RP- HPLC method for the estimation of Tenofovir disoproxil fumerate and Efavirenzin bulk and formulation (in house). Preliminary studies were conducted, in first trail by using Acetonitrile: pH 4.0 buffer in 90:10 ratio split peak were observed. In the second trail using Acetonitrile: pH 6.8 buffer peak shape is not good. In the third trail by using

Acetonitrile: pH 2.5 buffer in 90:10 ratio peak shapes is good but poor resolution. In the fourth   trail Acetonitrile: pH 2.5 buffer were used in 80:20 ratio good peaks were observed with low resolution. In the fourth trail by using Acetonitrile: pH 2.5 buffer in 70:30 ratio good peak shape with high resolution were observed and the corresponding chromatograms were shown in figure 2 (working standard) and figure 3 (sample extracted from tablet).

FIGURE 2: TYPICAL CHROMATROGRAM OF STANDARD CONTAINING TENOFOVIR DISOPROXIL FUMERATE AND EFAVIRENZ

 FIGURE 3: TYPICAL CHROMATROGRAM OF SAMPLE CONTAINING TENOFOVIR DISOPROXIL FUMERATE AND EFAVIRENZ

The developed method was validated in terms of Accuracy, Precision, Specificity, Detection limit Quantitation limit, Linearity Range Ruggedness, Robustness and System suitability parameters as per the recommendations of ICH guidelines.

From Table 1 and 2, the recovery studies demonstrated the better accuracy. The mean% recovery studies are within the assay limit100±1% with deliberate relative standard deviation (%RSD).

TABLE 1:  ACCURACY STUDIES OF THE TENOFOVIR DISOPROXIL FUMERATE

TABLE 2: ACCURACY STUDIES OF THE EFAVIRENZ

From Table 3, the %RSD of method and system precision parameter was found to be less than 2 for both repeatability and intermediate precision as per ICH guidelines. The mean recovery studies from the system and method precision were found to be 100.71 and 100.74 for Tenofovir disoproxil fumerate and Efavirenzrespectively are within the assay limit100±1% with deliberate relative standard deviation (%RSD).

TABLE 3: PRECISION TABLE

To determine specificity during the validation of blanks, sample matrix (placebo) and known related impurities are analyzed to determine whether interferences occur. It was observed that there were no analytical peaks observed in the blank indicating the method is specific. Moreover, the method is highly sensitive as evidenced by the LOD and LOQ values of Tenofovir disoproxil fumerate and Efavirenz are 0.0850 and 0.259, 0.199 and 0.605 respectively.

From figure 4 and 5, the standard calibration curve was linear over the concentration range of 3-18µg/ml and 6-36µg/ml for Tenofovir disoproxil fumerate and Efavirenzrespectively. The linear regression equation was found to be Y=47873x+12581 for Tenofovir disoproxil fumerate with R² =0.9987 and Y=86782x+52813 for Efavirenz with R² =0.9982 and respectively.

Standard calibration curve was linear and best fit line with in the linear range.

FIGURE 4: CALIBRATION CURVE OF TENOFOVIR DISOPROXIL FUMERATE

FIGURE 5: CALIBRATION CURVE OF EFAVIRENZ

As there are no significant change in the chromatographic parameters when variations in the optimized conditions indicates that the method is robust, less deviations were observed between analyst to analyst, column to column so the proposed method is rugged. The system suitability parameters shows that the method is highly selective and specific, as the retention time of Tenofovir disoproxil fumerate and Efavirenz were found to be 2.440 and 5.527min respectively so the method was rapid.

The theoretical plates were found to be >3000, thus demonstrating good column efficiency with significant resolution and better separation within accepted limits. As the asymmetry values are found to <1.5 it indicates the good peak shape. From the above results it was found that the optimized method is simple rapid, accurate, precise and within the limit of validation parameters.

CONCLUSION: The developed and validated HPLC method was found to be rapid, accurate, precise and reliable which is useful for routine quantification purpose in quality control. The system suitability parameters indicate good sensitivity, more ruggedness and robustness of the method. Therefore, the proposed method has proven simple, selective, specific which meets the ICH guidelines for analytical method validation.

ACKNOWLEDGMENTS: The author grateful to Management, Bapatla college of Pharmacy, Bapatla for encouragement and providing the necessary facilities during the course of investigation, authors are also gratified to NATCO Pharmaceuticals, Hyderabad for providing the gift sample.

REFERENCES:

1. Indian Pharmacopoeia 6^th edition. New Delhi: The Indian Pharmacopoeial Commission; 2010 Volume 2, 1272.
2. Indian Pharmacopoeia 6^th edition. New Delhi: The Indian Pharmacopoeial Commission; 2010 Volume 2, 1274.
3. N. Appala raju, J. Venkateswara Rao, K. Vanitha prakash, K. Mukkanti and K. Srinivasu Simultaneous estimation of Tenofovir disoproxil, emtricitabine and efavirenz in tablet dosage form by RP-HPLC, Oriental Journal of Chemistry, 2008 Volume.24(2), 645-650.
4. Raju N Appala, Begum Shabana: Simultaneous RP-HPLC Method for the “Estimation of the emtricitabine, Tenofovir disoproxil fumerate and Efavirenz in Tablet Dosage Forms”Indian Journals 2008;Volume 1(4);522-525
5. 5. Rao J, Gondkar S, Yadav S. Simultaneous HPTLC–Densitometric analysis of Tenofovir and Emtricitabine in Tablet dosage form. International Journal Pharmaceutical and Technology Research: 2011; Volume 3:1430–1434.
6. Sharma R, Mehta K. Simultaneous Spectrophotometric estimation of tenofovir disoproxil fumarate and lamivudine in three component tablet formulation containing efavirenz. Indian Journal of Pharmaceutical Science.2010; volume 72:527–30.
7. N. A. Gomes, V. V. Vaidya, A. Pudage, S. S. Joshi, and S. A. Parekh, “Liquid chromatography-Tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir and emtricitabine in human plasma and its application to a bioequivalence study,” Journal of Pharmaceutical and Biomedical Analysis:2008,volume48(3),  918–926.
8. Sethi P.D. Quantitative analysis of Pharmaceutical Formulation.3^rd edition New Delhi: CBS publishers and distributers 2010, Volume 4:434,435.
9. Ter Heine, Rob; Alderden-Los, Carolien G.;Rosing, Hilde; Hillebrand, Michel J. X.; vanGorp, Eric C. M.; Huitema, Alwin D. R.;Beijnen, Jos H. Rapid Communications in Mass Spectrometry, 2007 volume 21(15): 2505-2514.
10. Anogaokar.K and Desai. A.SimultaneousRP-HPLC Method for the “Estimation of the emtricitabine, Tenofovir disoproxil fumerate and Efavirenz in Tablet Dosage Forms Indian Drugs 2008, Volume 45(3), 188-192.
11. Part-2 Validation of analytical procedure: Methodology Q2B, ICH Harmonized Tripartite Guidelines, 1996; 6-12.
    

    ---

How to cite this article:

Vidya DS, Chowdary YA and Murthy TEGK: Development and validation of RP-HPLC method for simultaneous estimation of Tenofovir disoproxil fumerate and Efavirenz in bulk and formulation. Int J Pharm Sci Res 2013; 4(12): 4619-24. doi: 10.13040/IJPSR. 0975-8232.4(12).4619-24

All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.