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

Background: Raltegravir 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). Methods: After protein precipitation (with 100 mu L of acetonitrile/methanol (50/50)) of 25 mu L 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 beta-glucuronidase pre-treatment. Results: A 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 beta-glucuronidase suppressed G-raltegravir peak. Conclusion: We 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. (C) 2009 Elsevier B.V. All rights reserved.