Lack of specificity for the analysis of raltegravir using online sample clean-up liquid chromatography–electrospray tandem mass spectrometry

BackgroundRaltegravir is the first antiretroviral agent to target the human immunodeficiency virus-1 (HIV-1) integrase. It is indicated, in association with other antiretrovirals, in the treatment of acquired immunodeficiency syndrome (AIDS) in antiretroviral treatment-experienced adult patients with viral resistance. To evaluate the feasibility of raltegravir therapeutic drug monitoring, we developed a rapid and specific analytical method for the quantification of raltegravir in human plasma by online sample clean-up liquid chromatography–tandem mass spectrometry (LC–MS/MS).MethodsAfter protein precipitation (with 100 μL of acetonitrile/methanol (50/50)) of 25 μL of plasma, fast online matrix-clean-up was performed using a column switching program. The chromatographic step was optimized to separate raltegravir and its glucuronide metabolite (G-raltegravir). Multiple reaction monitoring (MRM) was used for detection of raltegravir and G-raltegravir. In the absence of G-raltegravir standard, G-raltegravir identification was confirmed by β-glucuronidase pre-treatment.ResultsA total analysis of 3.8 min was needed to separate raltegravir to G-raltegravir. The method was linear between 10 and 3000 ng/mL for raltegravir. Analytical recovery was 94 ± 1%. Variation coefficients ranged between 5% and 8.4%. Pre-treatment of plasma from a patient under raltegravir treatment with β-glucuronidase suppressed G-raltegravir peak.ConclusionWe describe a fast online LC–MS/MS assay that is valid and reliable for the quantification of raltegravir, despite the lack of specificity that could occur in MRM scanning mode experiments.