Analysis of Antiretrovirals in Single Hair Strands for Evaluation of Drug Adherence with Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging

Abstract

Adherence to a drug regimen can be a strong predictor of health outcomes, and validated measures of adherence are necessary at all stages of therapy from drug development to prescription. Many of the existing metrics of drug adherence (e.g., self-report, pill counts, blood monitoring) have limitations, and analysis of hair strands has recently emerged as an objective alternative. Traditional methods of hair analysis based on LC-MS/MS (segmenting strands at ≥1 cm length) are not capable of preserving a temporal record of drug intake at higher resolution than approximately 1 month. Here, we evaluated the detectability of HIV antiretrovirals (ARVs) in hair from a range of drug classes using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) with 100 μm resolution. Infrared laser desorption of hair strands was shown to penetrate into the strand cortex, allowing direct measurement by MSI without analyte extraction. Using optimized desorption conditions, a linear correlation between IR-MALDESI ion abundance and LC-MS/MS response was observed for six common ARVs with estimated limits of detection less than or equal to 1.6 ng/mg hair. The distribution of efavirenz (EFV) was then monitored in a series of hair strands collected from HIV infected, virologically suppressed patients. Because of the role hair melanin plays in accumulation of basic drugs (like most ARVs), an MSI method to quantify the melanin biomarker pyrrole-2,3,5-tricarboxylic acid (PTCA) was evaluated as a means of normalizing drug response between patients to develop broadly applicable adherence criteria.

Figure 1

Optimization of IR-MALDESI analysis of hair strands. (A) Optical image of hair strand indicating laser desorption of material in region analyzed by IR-MALDESI. (B) Ion maps of an endogenous lipid, cholesterol, and incubated ARV efavirenz collected with varying numbers of laser pulses (1–5) at each voxel.

Figure 2

Calibration of IR-MALDESI MSI response to ARV-incubated hair strands. (A) IR-MALDESI ion maps of EFV response, representative of other investigated ARVs, from strands incubated in stock solution concentrations of 1 µg/mL, 10 µg/mL, and 100 µg/mL as noted in each panel. (B) Log–log plot summarizing +ESI IRMALDESI response to all six ARVs relative to LC–MS/MS measurements of ARV accumulation from incubated hair strands. Linear plots comparing IR-MALDESI and LC–MS/MS results for each individual analyte are shown in the Supporting Information, Figure S-1.

Figure 3

Analysis of EFV in hair strands from three dosed patients. (A) IR-MALDESI MSI shows uniform longitudinal distribution of EFV in hair strands collected from virally suppressed dosed patients, with high intrapatient repeatability of response. (B) Longitudinal profiling of EFV response from proximal end of dosed hair strands using MSiReader.

Figure 4

(A) Scheme of melanin degradation to analytical target PTCA based on hair oxidation by 15% H₂O₂ and 1 M NH₄OH. (B) −ESI IR-MALDESI MS²I ion maps of [PTCA − H]⁻ (m/z 198.0039) and fragments associated with the loss of one (m/z 154.01330) and two (m/z 110.02320) carboxylic acid units from oxidized pigmented and unpigmented hair strands. Unpigmented, gray strands (denoted by white asterisks) show no response to PTCA. (C) Ion map of PTCA response from hair strands varying in color (dyed, blonde, brown, and black). D) Average [PTCA − H]⁻ ion abundance from colored hair strands indicating increased abundance as hair color darkens.

Figure 5

(A) IR-MALDESI response to [EFV + H]⁺ from strands (n = 4) of three dosed patients before (left) and after (right) oxidation of melanin by H₂O₂ indicating no significant degradation in response to EFV. (B) Top panel: ion map of [EFV + H]⁺ (left) and average longitudinal profile (right) for each of three patients, indicating a 4-fold difference in response to accumulated EFV in hair. Middle panel: Normalization of [EFV + H]⁺ response by [PTCA − H]⁻ results in similar longitudinal profiles for each of three patients to fixed-dose intake of EFV. Bottom panel: Comparative normalization approach for EFV, matching ionization mechanisms ([EFV − H]⁻/PTCA − H]⁻).