A LC–tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir, and etravirine

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 877; no. 11; pp. 1057 - 1069
• Main Authors: Fayet, A., Béguin, A., Zanolari, B., Cruchon, S., Guignard, N., Telenti, A., Cavassini, M., Günthard, H.F., Buclin, T., Biollaz, J., Rochat, B., Decosterd, L.A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15-04-2009; Elsevier
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Anti-HIV Agents - analysis; Anti-HIV Agents - blood; Anti-HIV Agents - pharmacokinetics; Antiretroviral Therapy, Highly Active; Biological and medical sciences; Calibration; CCR5 co-receptor antagonists; Chromatography, High Pressure Liquid; Darunavir; Drug Stability; Etravirine; Fundamental and applied biological sciences. Psychology; General pharmacology; Glucuronosyltransferase - metabolism; HPLC–MS/MS; Human immunodeficiency virus; Humans; Integrase inhibitors; Maraviroc; Medical sciences; Non-nucleoside reverse transcriptase inhibitors; Pharmacology. Drug treatments; Protease inhibitors; Quality Control; Raltegravir; Reference Standards; Ritonavir; Tandem Mass Spectrometry; Non-nucleoside reverse transcriptase inhibitors; HPLC–MS/MS; Protease inhibitors; Maraviroc; Darunavir; Ritonavir; Etravirine; CCR5 co-receptor antagonists; Integrase inhibitors; Raltegravir; inhibitors; Antiretroviral agent; RNA-directed DNA polymerase; Non nucleoside compound; Non-nucleoside reverse transcriptase; Non peptide compound; Integrase inhibitor; Reverse transcriptase inhibitor; Antiviral; Antagonist; Sulfonamide; Enzyme; Transferases; Enzyme inhibitor; Retroviridae; Lentivirus; Virus; CCR5 chemokine receptor; Peptidases; Nucleotidyltransferases; HPLC-MS/MS; Simultaneous measurement; Hydrolases; Coreceptor; Human immunodeficiency virus; Protease inhibitor
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.02.057

Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100
μl) is performed by protein precipitation using 600
μl of acetonitrile, after the addition of 100
μl darunavir-d
9 (DRV-d
9) at 1000
ng/ml in MeOH/H
2O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10
min, vortex-mixed again and centrifuged. An aliquot of supernatant (150
μl) is diluted 1:1 with a mixture of 20
mM ammonium acetate/MeOH 40/60 and 10
μl is injected onto a 2.1
×
50
mm Waters Atlantis™-dC18 3
μm analytical column. Chromatographic separations are performed using a gradient program with 2
mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000
ng/ml, 2.5 to 1000
ng/ml, 25 to 10,000
ng/ml, 10 to 4000
ng/ml, and 5 to 2000
ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1–9.8%, and accurate (range of inter-day deviation from nominal values −3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir–glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.