Development, validation and clinical implementation of a HPLC-MS/MS method for the simultaneous quantification of bictegravir, emtricitabine, doravirine, cabotegravir, lenacapavir, fostemsavir, tenofovir alafenamide and the corresponding metabolites temsavir and tenofovir, in human plasma

A highly sensitive and specific liquid chromatography-tandem mass spectrometry method was developed, fully validated, and successfully implemented for routine analysis to simultaneously quantify Bictegravir, Emtricitabine, Doravirine, Cabotegravir, Lenacapavir, Fostemsavir, Tenofovir alafenamide, and their metabolites, Temsavir and Tenofovir, in human plasma. The sample preparation employed a commercial liquid-liquid extraction kit optimized for low plasma volumes (50 mu L), which also included the Internal Standard. The method demonstrated excellent precision, accuracy, and robustness, making it suitable for pharmacokinetic and therapeutic drug monitoring applications. Analyte separation was carried out using a gradient elution program over a total run time of seven minutes, with a flow rate of 0.35 mL/min. The mobile phase consisted of solvent A (water containing 0.1 % formic acid) and solvent B (acetonitrile containing 0.1 % formic acid). Detection was performed using a QTRAP (R) 5500 triple quadrupole mass spectrometer (SCIEX) equipped with an electrospray ionization source operating in positive ion mode. Ion monitoring was performed in multiple reaction monitoring (MRM) mode for all analytes. The method was validated in accordance with European Medicines Agency (EMA) guidelines across clinically relevant concentration ranges. The proposed method was successfully implemented in routine analysis. Following the initial months of application, biological samples from 165 patients were analyzed primarily to assess therapy adherence and confirm that drug blood concentrations reached the minimum threshold. Additionally, data on drug pharmacokinetics were obtained. Our findings indicate that the proposed method is a reliable and accurate tool for high-throughput screening that could be readily used by the clinicians to optimize therapeutic treatments, verify patients' adherence and reduce drug-related toxicities.