Combined determination of tenofovir disoproxil and emtricitabine in tablet dosage forms by hydrophilic interaction high performance liquid chromatography method

Determination of Tenofovir Disoproxil and Emtricitabine in Tablet Dosage Forms by HILIC-HPLC Method", Trakya University Institute of Health Sciences, Department of Pharmaceutical Nanotechnology, Master's Degree, Edirne, 2023. Tenofovir disoproxil fumarate and emtricitabine are antiviral active ingredients used against human immunodeficiency virus and hepatitis B viruses in adults. These drugs, which are often used together in treatment, are prescribed to patients in combined tablet dosage form. High performance liquid chromatography is a widely used technique for the quantification of analytes in complex samples. Hydrophilic interaction chromatography has been successfully applied to analyze small molecules with polar properties. In this study, a method for the quantification of the active substances tenofovir disoproxil fumarate and emtricitabine in the combined tablet dosage form using hydrophilic interaction column with high performance liquid chromatography technique was developed and method validation was provided. The developed chromatographic method was applied on a commercially available product and chromatographic analysis of tenofovir diproxyl fumarate and emtricitabine in the product content was performed. The analysis was performed using a high-performance liquid chromatography-diode array detector system. Suitable mobile phase composition, flow rate and analysis temperature were determined to ensure chromatographic separation of active substances. Method validation was performed with specificity, linearity, limit of observability, limit of detection, linear range, accuracy, precision and robustness studies. Chromatographic analysis was performed using acetonitrile:ammonium acetate buffer (100 mM) 85:15 (h:h) and HILIC amide column as mobile phase. Separation was achieved at a flow rate of 0.4 ml.min.-1 , injection volume of 10 µL, temperature 25 ⁰C. Detection wavelengths were 260 nm for tenofovir diproxyl fumarate and 284 nm for emtricitabine. The retention times were 4.34 minutes for tenofovir diproxyl fumarate and 7.58 minutes for emtricitabine. The detection limit for tenofovir diproxil fumarate was 0.149 µg.ml-1 and the limit of detection was 0.352 µg.ml-1 ; the detection limit for emtricitabine was 0.108 µg.ml-1 and the limit of detection was 0.327 µg.ml-1 . Within the scope of the developed method validation, it was determined that the active substances met the regulatory criteria for specificity (Rs>1.5), linearity (R2 ≥0.999), accuracy (% recovery = 98-102%), precision (% BSS<2%),and robustness studies. Tenofovir diproxil fumarate and emtricitabine in the commercial product were quantified with 99.17% and 101.58% analysis percentages, respectively. Keywords: HILIC, HPLC, Antiviral, Tenofovir Disoproxil Fumarate, Emtricitabine, Supporting Institution: This study was supported by Trakya University Scientific Research Projects Unit. Project No: 2021/138