Sensitive analysis of anti-HIV drugs, efavirenz, lopinavir and ritonavir, in human hair by liquid chromatography coupled with tandem mass spectrometry

A highly sensitive and selective method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed and validated for the measurement of three antiretroviral agents, efavirenz, lopinavir and ritonavir, in human hair. Hair samples from adherent HIV-infected patients on antiretroviral therapies were cut into about 1 mm length segments and drugs were extracted by first shaking the samples with methanol in a 37 degrees C water bath overnight (>14 h), followed by methyl tert-butyl ether/ethyl acetate (1:1) extraction under weak alkaline conditions. The extracted lopinavir and ritonavir were separated by reversed-phase chromatography and detected by tandem mass spectrometry in electrospray positive ionization mode with multiple reaction monitoring (MRM), while efavirenz; was monitored in negative ionization MRM mode. This method was validated from 0.01 to 4.0 ng/mg hair for ritonavir and 0.05-20 ng/mg hair for lopinavir and efavirenz by using 2 mg of a human hair sample. The interday and intraday assay precision (coefficients of variation, CV) for spiked quality control (QC) samples at low, medium and high concentrations were within 15% and accuracy ranged from 89% to 110%. Assay reproducibility was also demonstrated by analysis of incurred hair QC samples (CV <14%). No significant matrix ionization suppression was observed. This developed method allowed for the monitoring of these target medications in the hair samples of HIV-infected women on antiretroviral therapy in an observational study using small amounts of hair. Copyright (C) 2008 John Wiley & Sons, Ltd.