Quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in peripheral blood mononuclear cell lysate using liquid chromatography coupled with tandem mass spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2009-02, Vol.877 (5), p.575-580
• Main Authors: ter Heine, R., Davids, M., Rosing, H., van Gorp, E.C.M., Mulder, J.W., van der Heide, Y.T., Beijnen, J.H., Huitema, A.D.R.
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Antiretroviral; Antiviral Agents - blood; Biological and medical sciences; Blood Cell Count; Cell Extracts - chemistry; Chromatography, Liquid; Drug Stability; Fundamental and applied biological sciences. Psychology; General pharmacology; HIV; HIV Protease Inhibitors - blood; HPLC; Humans; Intracellular; LC-MS; LC-MS/MS; Leukocytes, Mononuclear - chemistry; Medical sciences; Nucleosides - blood; Pharmacology; Pharmacology. Drug treatments; Reproducibility of Results; Reverse Transcriptase Inhibitors - blood; Tandem Mass Spectrometry - methods; TDM
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.01.011

For pharmacokinetic monitoring, measurement of antiretroviral agents in plasma is the gold standard. However, human immunodeficiency virus protease inhibitors (PIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs) exert their action within the infected cell. Cell-associated concentrations may therefore more adequately reflect therapy outcome. Therefore, for the quantification of nine PIs (amprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), 1 active PI metabolite (nelfinavir M8) and 2 NNRTIs (efavirenz and nevirapine) in lysate of peripheral blood mononuclear cells (PBMCs) an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Analytes were extracted from a PBMC pellet by means of a one-step extraction with 50% methanol containing the internal standards D6-indinavir, D5-saquinavir, 13C6-efavirenz and dibenzepine. Chromatographic separation was performed on a reversed phase C18 column (150 mm × 2.0 mm, particle size 5 μm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. The analytical run time was 10 min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 1–500 ng/mL in PBMC lysate for all analytes. The method was proven to be specific, accurate, precise and robust. The mean precision and accuracy was less than ±12% at all concentration levels. Using the developed assay and a previously developed assay for these analytes in plasma, the relationship between plasma and intracellular pharmacokinetics and their relationship with therapy outcome can now be determined.