Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Andrew Reckers; Anita Wen; David Aguilar; Peter Bacchetti; Monica Gandhi; John Metcalfe; Roy Gerona

doi:10.3791/60861

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

7.3K views

⸱

DOI:

10.3791/60861

⸱

May 19th, 2020

Andrew Reckers¹ , Anita Wen¹ , David Aguilar¹ , Peter Bacchetti² , Monica Gandhi³ , John Metcalfe⁴ , Roy Gerona¹

¹Department of Obstetrics, Gynecology and Reproductive Sciences, TB Hair Analysis Laboratory, University of California San Francisco, ²Department of Epidemiology and Biostatistics, University of California San Francisco, ³Department of Medicine, Division of HIV, Infectious Diseases, and Global Medicine, University of California San Francisco, ⁴Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California San Francisco

Summary

Current methods of analyzing patients’ adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Abstract

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, ¹⁵N-, or ¹³C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide’s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Introduction

In the twenty-first century, drug-resistant TB (DR-TB) is an evolving catastrophe for already weak national TB control programs, with confirmed cases doubling in the past 5 years alone, accounting for nearly one-third of all deaths related to antimicrobial resistance globally¹^,². Successful treatment of DR-TB has conventionally required longer and more toxic second-line regimens than treatment for drug-sensitive TB. Moreover, patients with DR-TB often have significant pre-existing challenges to adherence, which contributed to the emergence of resistance initially³.