Simultaneous Quantification of Doravirine, Lamivudine, and Tenofovir Disoproxil Fumarate in Human Plasma by UPLC-MS/MS: Method Development and Validation.

Objectives: A novel, high-throughput liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique has been developed that uses Etravirine (ETR) as the internal standard (IS) to simultaneously quantify Doravirine (DOR), Lamivudine (LAM), and Tenofovir Disoproxil Fumarate (TDF) in human plasma. The procedure employs a precipitation extraction technique to analyze analytes from human plasma. This study aims to develop and validate a novel and reliable stability-indicating UPLC-MS/MS method for the simultaneous determination of DOR, LAM, and TDF in human plasma, using ETR as the IS. Materials and Methods: ETR, based on its stable-isotopic nature and structural and physicochemical similarity to the analytes of interest, was used as an IS. Precipitation extraction was the technique used to prepare samples. An agilent zorbax XDB C18 analytical column (2.1 × 50 mm, 3.5 μm) was used for chromatographic separation, and its isocratic mobile phase consists of acetonitrile and buffer (5 mM of ammonium formate with 0.1 % formic acid) in the ratio 80:20, Results: The parent-to-product ion transitions for the drugs were as follows : LAM: m/z 231.08 amu → 112.00 amu, TDF: m/z 288.33 amu → 176.17 amu, DOR: m/z 426.38 amu → 112.02 amu, and IS ETR: m/z 437.36 amu → 164.97 amu. These transitions were observed using a triple quadrupole mass spectrometer in the multiple reaction monitoring (MRM) positive ion mode. The compound's basic group content determined which positive mode to choose. For DOR, LAM and TDF, the method was validated throughout concentration ranges of 2.5-1000 ng/mL with correlation coefficients (r Conclusion: A triple quadrupole mass spectrometer running in the MRM positive ion mode was used to track these transitions. The compounds' functional group content served as the basis for choosing the positive mode. The mean recovery values were obtained for three APIs from spiked plasma samples. The run times were found to be both reliable and quick. The method was proven to produce precise and specific results for the determination of selected drugs through the current study. The method is stable when exposed to various stress conditions, demonstrating minimal degradation. The current method was validated as per the ICH M10 guidelines and was found to meet the desired acceptance criteria. The developed bioanalytical method, validated in accordance with ICH M10 guidelines, demonstrated high accuracy, precision, and reproducibility for the simultaneous quantification of DOR, LAM, and TDF. Its streamlined design and reliable performance make it a valuable tool for routine analysis.