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

Abstract

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.

Figures

Figure 1

Comparison of the extraction methods for assaying lopinavir/ritonavir and efavirenz in samples incurred from human hair. The methanol and enzyme digestion were carried out at 37°C with shaking in a water bath for 14 h. Data are represented by mean ± standard deviation (SD) (n = 4). *p <0.01, compared with cut hair/MeOH by t-test.

Figure 2

Time course for extraction of ritonavir, lopinavir and efavirenz from samples incurred from cut hair (1 mm length segments) in methanol at 37°C with shaking in a water bath. After the methanol extraction, the samples were then subjected to MTBE/EA (1:1) extraction and analyzed by LC/MS/MS. Each point represents mean ± SD (n = 4).

Figure 3

LC/MS/MS chromatograms for measuring ritonavir and lopinavir in positive ionization MRM mode: (A) blank hair; (B) blank hair spiked with ritonavir/lopinavir at low QC concentration (lopinavir, 0.15 ng/mg, ritonavir, 0.03 ng/mg); and (C) authentic hair sample from a patient on HAART (lopinavir, 3.0 ng/mg, ritonavir, 0.70 ng/mg). The detailed LC/MS/MS conditions are described in the text.

Figure 4

LC/MS/MS chromatograms for measuring efavirenz in negative ionization MRM mode: (A) blank hair; (B) blank hair spiked with efavirenz at low QC concentration (efavirenz, 0.15 ng/mg); and (C) authentic hair sample from a patient on HAART (efavirenz, 5.3 ng/mg). The detailed LC/MS/MS conditions are described in the text.