Validation of an electrospray ionization LC-MS/MS method for quantitative analysis of raltegravir, etravirine, and 9 other antiretroviral agents in human plasma samples.
BACKGROUND
Raltegravir is the first human immunodeficiency virus-1 (HIV-1) integrase inhibitor used in treatment-experienced patients who have evidence of viral replication and HIV-1 strains resistance to multiple antiretroviral regimens. Etravirine is a novel NNRTI, active against HIV-1 strains harboring multiple NNRTI mutations. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of raltegravir, etravirine, and 9 other antiretroviral agents (amprenavir, atazanavir, darunavir, efavirenz, indinavir, lopinavir, ritonavir, saquinavir, and tipranavir) in plasma at the concentrations associated with therapy.


METHODS
The ritonavir analog, methyl indinavir, and lopinavir-d8 were used as internal standards, added to 100 microL of plasma sample prior to a protein precipitation using methanol. Chromatographic separation was achieved on a C18 HPLC column (Waters Sunfire 100 x 2.1 mm, 3.5 microm) with a mobile phase gradient at a flow rate of 0.3 mL/min. Five microL of sample were injected into the LC-MS/MS system (Waters Quattro Premier XE) to determine concentrations of raltegravir, etravirine, and other antiretroviral agents.


CONCLUSIONS
This method showed an excellent linearity for all calibration curves (r2 > 0.998). The lower limit of quantification was established at 5 ng/mL for raltegravir and 40 ng/mL for etravirine, with precision and accuracy within +/-20% and 80% to 120% for all analytes. Intraassay and interassay precision and inaccuracy ranged from -9.2% to 6.9% for raltegravir and from -14.3% to 12.3% for etravirine and were less than 15% for all other compounds. No matrix effect was observed for any of the antiretrovirals studied.


CONCLUSIONS
A rapid, specific, and sensitive LC-MS/MS method for quantification of raltegravir, etravirine, and 9 other antiretrovirals in human plasma was developed and was successfully applied for routine therapeutic drug monitoring.
Laboratoire de Pharmacocinétique et Toxicologie, Hôpital de la Timone, Marseille, France. sylvie.quaranta@ap-hm.fr
Sylvie
S
Quaranta
Christian
C
Woloch
Alexandre
A
Paccou
Madeleine
M
Giocanti
Caroline
C
Solas
Bruno
B
Lacarelle
D053000:Analytic Sample Preparation Methods; D019380:Anti-HIV Agents / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods; D017320:HIV Protease Inhibitors / Q000097:blood; D006801:Humans; D057230:Limit of Detection; D008832:Microchemistry / Q000379:methods; D009570:Nitriles; D011724:Pyridazines / Q000097:blood*; D011743:Pyrimidines; D011760:Pyrrolidinones / Q000097:blood*; D000068898:Raltegravir Potassium; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000097:blood; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D019380:Anti-HIV Agents; D017320:HIV Protease Inhibitors; D009570:Nitriles; D011724:Pyridazines; D011743:Pyrimidines; D011760:Pyrrolidinones; D018894:Reverse Transcriptase Inhibitors; C451734:etravirine; D000068898:Raltegravir Potassium