Original Article LIQUID CHROMATOGRAPHIC QUANTIFICATION OF TERNARY MIXTURE OF ANTI-VIRAL DRUGS AND APPLICATION TO ASSESSMENT OF THEIR TABLET DOSAGE FORM ANUMOLU P. D., ANUSHA K., MRUDULA KIRAN A., MONIKA P., SOWNDARYA NSKR, SUNITHA G.* Gokaraju Rangaraju College of Pharmacy, Department of Pharmaceutical Analysis and Quality Assurance, Osmania University, Hyderabad 500090, Telangana state, India Email: g.sunitha88@gmail.com Received: 17 Apr 2015 Revised and Accepted: 25 Nov 2015 ABSTRACT Objective: To establish a validated liquid chromatographic method for the quantification of Tenfovir disoproxil fumarate (TENO), Lamivudine (LAM) and Nevirapine (NEV) in ternary combination and in its tablet dosage form. Methods: The three drugs were well resolved using ODS C18 Results: The response was a linear function of analyte concentration over the concentration range of 45-105 µg/ml for tenofovir disoproxil fumarate, lamivudine and between 30-70 µg/ml for nevirapine with a correlation coefficient>0.9997. The % RSD values of precision and accuracy studies were found to be less than 2. column (250 x 4.6 mm, 5μm) with a mobile phase consisting of phosphate buffer, pH 3.0- acetonitrile (60:40, v/v) with a flow rate of 1.0 ml/min at UV detection wavelength 252 nm. The developed method was validated as per ICH guidelines. Conclusion: The proposed method was validated as per standard guidelines. The results obtained from assay values were in good agreement with the labeled amount of the marketed tablet dosage form RICOVIR-LN. The method holds promise for routine quality control of this ternary combination in bulk and pharmaceutical formulations. Keywords: Column liquid chromatography, Tenofovir Disoproxil Fumarate, Lamivudine, Nevirapine. © 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/ ) INTRODUCTION Tenofovir chemically designated as {[(2R)-1-(6-amino-9H-purin-9-yl) propan-2-yl) oxy] methyl} phosphonic acid, is an anti-retroviral agent currently used in the treatment of HIV. Its mechanism of action involves the inhibition of HIV reverse transcriptase activity by competing with natural substrate deoxyadenosine5’-triphosphate by DNA chain termination. Nevirapine is chemically designated as 2-cyclopropyl-7- methyl- 2,4,9,15-tetra aza tricyclo [9.4.0.0^{3,8}] pentadeca- 1(15)3,5,7,11,13-hexane-10-one, is an antiretroviral agent used in HIV treatment. Its mechanism of action involves binding of reverse transcriptase directly and blocks RNA-dependent and DNA–dependent polymerase activities by causing disruption of enzyme catalytic site. Lamivudine is also an anti-retroviral agent which is chemically designated as 4-amino-1-[(2R, 5S)-2-(hydromethyl)-1,3-oxathiolan-5- yl]-1,2-dihydropyrimidin-2-one, used in the treatment of HIV. Chemical structures of these drugs were shown in fig.-1. The combination of three drugs used for the treatment of HIV as a ternary combination [1-2]. Fig. 1: Chemical structures of tenofovir disoproxil fumarate (A), Nevirapine (B) and Lamivudine (C) An all-embracing literature search revealed that only one analytical method using high-performance liquid chromatography has been previously reported for the determination of this combination by gradient elution [3]. Few analytical methods based on HPLC, HPTLC, UV spectroscopy are available for the determination of these drugs individually and in combination with other drugs in different dosage forms, but there is no isocratic HPLC method for simultaneous quantification of three drugs in combined dosage form [4-14]. Keeping this point into consideration, present investigation has been undertaken with the objective to develop and validate simple isocratic RP-HPLC method for evaluation of this tertiary combination of drugs in bulk and tablet dosage form. MATERIALS AND METHODS Instrument and chromatographic conditions The ternary combination of three drugs were well separated by using a Shimadzu model LC-20AD HPLC system connected to LC solutions software, equipped with an SPD-20A prominence UV detector and ODS, C18 Chemicals and reagents column (250x4.6 mm, 5 µm) maintained at 25ºC. Isocratic elution was performed using acetonitrile and phosphate buffer, pH 3.0 (40:60, v/v) with flow rate 1.0 ml/min at UV detection wavelength 252 nm. 20 µl of sample was injected into the HPLC system. Tenofovir disoproxil fumarate, nevirapine and lamivudine standards and tablet dosage forms were obtained from Mylan laboratories (Hyderabad, India). Milli-Q-water was used throughout the experiment. Acetonitrile and methanol (HPLC grade), sodium hydroxide, potassium dihydrogen phosphate, orthophosphoric acid were purchased from Merck (Mumbai, India). Preparation of standard stock solution The standard stock solution was prepared by dissolving 75 mg of tenofovir diisopropyl fumarate, 50 mg of nevirapine and 75 mg of lamivudine in 100 ml of mobile phase to get concentrations of 750 µg/ml, 500 µg/ml and 750 µg/ml respectively. This stock solution was further diluted with mobile phase to get the required concentration of drugs. Solutions were filtered through 0.22 µm membrane filter prior to injection into HPLC system. Assay of commercial tablets Twenty tablets of RICOVIR-LN were purchased, weighed and crushed into fine powder. An accurately weighed portion of tablet powder equivalent to 75 mg, 50 mg and 75 mg of tenofovir International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 8, Issue 1, 2016