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°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.