Validation of a UPLC-MS/MS method for the simultaneous measurement of the antiretroviral drugs atazanavir, darunavir, efavirenz, etravirine, lopinavir, raltegravir, ritonavir from human plasma
MetadataShow full item record
Background: The current standard of treatment for HIV infection is a combination of antiretroviral drugs, including at least one protease inhibitor (PI), a non-nucleoside transcriptase inhibitor (NNRTI) and/or one or more nucleoside or nucleotide transcriptase inhibitor (NRTIs-NtRTI) and/or a fusion inhibitor (FI). A mutation in HIV genome conferring resistance to different compounds is not an infrequent occurrence and there is a need for new therapeutic regimens able to overcome the extensive class resistances observed in multi treated patients. Accurate measurement of blood plasma level of antiretroviral drugs is crucial for these studies, as well as the study of side effects, optimization of efficacy, metabolic impairment and therapeutic drug monitoring (TDM). The aim of this study is to validate an UPLC-MS/MS analytical tool for the simultaneous quantification of multiple antiretroviral drugs in human plasma. Methods and Materials: Plasma samples (250 uL) were spiked with four stable isotope labeled internal standards (Etravirine-d6, Atazanavir-d5, DPQ and L-737,345), precipitated with 4% formic acid solution and extracted by solid phase extraction (SPE) on Oasis HMB cartridges. The SPE elutent was evaporated and reconstituted with water: acetonitrile (70:30 v/v) containing 0.5% formic acid. Samples were injected onto a Waters Acquity BEH C18 2.1 X 100mm column and eluted with a binary gradient of Waters Acquity UPLC system. ARV drug analytes were detected using ESI- tandem mass spectrometry by multiple reaction monitoring. Results: All of the drugs were separated within 9 minutes. Out of five runs, three runs were considered for accuracy, precision and linearity results. Sensitivity was achieved optimal for all of the drugs at lowest concentrations. Accuracy and precision were compromised for all the drugs. Etravirine and efavirenz failed to pass re-injection stability after 24 hours. Conclusion: The current analytical accuracy and precision of this assay is unacceptable for routine diagnostic used in human patient samples and further method development is required.