Quantitative Analysis of Bictegravir, Emtricitabine, Tenofovir Alafenamide in Human Plasma by HPLC-MS/MS and its Application to Bioequivalence Study in Healthy Subjects

Kiran Kumar.A^1*, Komal Lata Nagpal² and Jaswanth Kumar.I^3
1Research Scholar, Department of Life Sciences, OPJS University, Churu, Rajasthan -313301, India.
²Associate Professor, Department of Life Sciences, OPJS University, Churu, Rajasthan -313301, India.
³Jaswanth Kumar.I, Head-Operations, Clinse labs private limited, Hyderabad, TG-500018, India.

A B S T R A C T
Combination antiretroviral (cARV) treatment is more common in human immunodeficiency virus (HIV) infection. In many instances, treatment regimen includes two or more combination of drugs from six different classes. Some of the antiretroviral combination medications are under study at preclinical and clinical stages. A precise method is required to quantify the drug concentration in biological matrices to study pharmacokinetic behavior and tissue distribution profile in animals and/or humans. We have developed and validated a sensitive and precise liquid chromatography-tandem mass spectrometry method for simultaneous quantification of Bictegravir (BG), Emtricitabine (EC) & Tenofovir Alafenamide (TA) in human plasma using  Bictegravir-D2 (BGIS), Emtricitabine 15ND2 (ECIS) and Tenofovir Alafenamide D5 Fumarate (TAIS) as an internal standards (IS). Chromatographic separation was performed on a ZODIAC CN column using an isocratic mobile phase composed of methanol and 5Mm ammonium acetate (75:25, v/v) and delivered at a flow rate of 1.0 mL/min. Analytes were extracted from plasma by a solid phase extraction technique. BG, EC and TA and BGIS, ECIS and TAIS were detected with protonated adducts at m/z 450.30→289.20, 248.30→130.00, 477.40→470.00 and 452.30→289.10, 251.10→130.80, 482.20→470.00 in multiple reaction monitoring (MRM) using AB Sciex API 4500 triple quard mass spectrometer. The method was validated over a linear concentration range of 20.131 to 10015.534 ng/mL, 12.086 to 3006.433 ng/mL and 6.015 to 1496.289 ng/mL for BG, EC and TA, respectively. This method demonstrated acceptable intra and inter-day precision within 3.55 to 5.71 for BG, 0.47 to 7.58 for EC and 1.90 to 6.61 for TA and 3.28 to 5.37 for BG,1.04 to 8.20 fot EC and 1.74 to 7.11 for TA intraday precision, and 5.37 to 3.28 for BG , 1.04 to 8.20 for EC and 1.74 to 7.11 inter-day precision, This method demonstrated acceptable intra and inter-day accuracy within 89.58 to 98.18 for BG,91.25 to 98.04 for EC and 88.16 to100.09 for TA intraday accuracy and  91.04 to 95.68 for BG,93.90 to 98.27 for EC and 92.80 to 99.97 for TA, respectively. The obtained results are according to current U.S Food and Drug Administration (U.S. FDA) Bioanalytical method validation guidelines. This method was successfully applied to a comparative bioavailability/bioequivalence study in 12 healthy human subjects under fed conditions.
Keywords: Bictegravir, Emtricitabine, Tenofovir Alafenamide, LC-MS/MS, Bioequivalence, Human Plasma.