A Validated Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Tenofovir, Emtricitabine, and a Efavirenz and Statistical Approach to Determine the Effect of Variables
A simple, rapid, and stability-indicating RP-HPLC method for a combination of tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) was developed and validated with the help of a suitable statistical software as an application tool for the quality by design. The drugs individually, and in combination, were subjected to forced degradation (thermal, photolytic, hydrolytic, and oxidative stress conditions) and accelerated stability studies (40？±？1°C/75？±？3% RH for three months). Successful separation of combined drugs from degradation products was achieved by gradient elution on a reverse-phase C18 column, using a mobile phase containing phosphate buffer (pH 3.5): acetonitrile at 1.5？mL min？1 flow rate, detection wavelength 256？nm, column oven temperature 25°C, and injection volume 10 μL. Linearity was established in the range of 20–300 μg mL？1, 24.5–367.5 μg mL？1 and 60–900 μg mL？1 for FTC, TDF, and EFV, respectively. The method was successfully applied for quantifying the drugs in marketed dosage forms and on stability samples. 1. Introduction Around 33.4 million people were living with HIV in year 2008 and around 2 million people have died in the same year [1]. Atripla, a fixed dose combination of tenofovir, emtricitabine, and efavirenz, was approved by the Food and Drug Administration (FDA) on July 12, 2006, for the treatment of this disease. Atripla was the first fixed dose formulation available in the United States to combine two different classes of antiviral drugs in a single pill. Many generic products of Atripla are also available, such as Viraday from Cipla Ltd. and Vonavir from Emcure Ltd. Tenofovir is chemically [(2R)-1-(6-aminopurin-9-yl) propan-2-yl]oxymethylphosphonic acid, a nucleotide analog of adenosine monophosphate. Tenofovir disoproxil fumarate (TDF) is an oral prodrug of tenofovir. TDF, a nucleotide reverse-transcriptase inhibitor (NRTI) blocks the enzyme reverse transcriptase, an essential enzyme that is required for the replication of viral DNA [2]. Emtricitabine (FTC) is chemically 4-amino-5-fluoro-1-[(2R, 5S)-2-(hydroxymethyl)-1, 3-oxathiolan-5-yl]pyrimidin-2-one. FTC is fluorinated NRTI. TDF requires two phosphorylation steps while FTC undergoes one step [3, 4]. Efavirenz (EFV) is chemically (4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-1H-3,1-benzoxazin-2-one. EFV is a non-nucleoside reverse-transcriptase inhibitor (NNRTI). NNRTIs block HIV replication by inhibiting HIV reverse transcriptase [4, 5]. Several HPLC methods are available in the literature for individual drugs and for a
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