A Single-Run HPLC–MS Multiplex Assay for Therapeutic Drug Monitoring of Relevant First- and Second-Line Antibiotics in the Treatment of Drug-Resistant Tuberculosis

Abstract

The treatment of drug-resistant Mycobacterium tuberculosis relies on complex antibiotic therapy. Inadequate antibiotic exposure can lead to treatment failure, acquired drug resistance, and an increased risk of adverse events. Therapeutic drug monitoring (TDM) can be used to optimize the antibiotic exposure. Therefore, we aimed to develop a single-run multiplex assay using high-performance liquid chromatography–mass spectrometry (HPLC–MS) for TDM of patients with multidrug-resistant, pre-extensively drug-resistant and extensively drug-resistant tuberculosis. A target profile for sufficient performance, based on the intended clinical application, was established and the assay was developed accordingly. Antibiotics were analyzed on a zwitterionic hydrophilic interaction liquid chromatography column and a triple quadrupole mass spectrometer using stable isotope-labeled internal standards. The assay was sufficiently sensitive to monitor drug concentrations over five half-lives for rifampicin, rifabutin, levofloxacin, moxifloxacin, bedaquiline, clofazimine, terizidone/cycloserine, ethambutol, delamanid, pyrazinamide, meropenem, prothionamide, and para-amino salicylic acid (PAS). Accuracy and precision were sufficient to support clinical decision making (≤±15% in clinical samples and ±20–25% in spiked samples, with 80% of future measured concentrations predicted to fall within ±40% of nominal concentrations). The method was applied in the TDM of two patients with complex drug-resistant tuberculosis. All relevant antibiotics from their regimens could be quantified and high-dose therapy was initiated, followed by microbiological conversion. In conclusion, we developed a multiplex assay that enables TDM of the relevant first- and second-line anti-tuberculosis medicines in a single run and was able to show its applicability in TDM of two drug-resistant tuberculosis patients.

Identification of Mycobacterium tuberculosis Resistance to Common Antibiotics: An Overview of Current Methods and Techniques

“…High-performance liquid chromatography mass was used to monitor the first- and second-line antibiotic concentrations in plasma or serum and was successfully applied in two clinical patients. 45 The drug resistance of MTB is mainly about the peripheral lipids of the MTB cell envelope and the increased amounts of nonpolar lipids. 46 The classic laboratory examination method for MTB includes an E-test, nitrate reductase assay, bacterial culture method, and molecular diagnostic technique, with different features and limitations.…”

“…Highperformance liquid chromatography mass was used to monitor the first-and second-line antibiotic concentrations in plasma or serum and was successfully applied in two clinical patients. 45 The drug resistance of MTB is mainly about the peripheral lipids of the MTB cell envelope and the increased amounts of nonpolar lipids. 46 inhA Encodes NADH-enoyl-dependent acetyl carrier protein reductase [4] Streptomycin rpsL Encodes S12 [5] rrs Encodes16S RNA [5] gidB Encodes 7-methylguanosine-methyltransferase [6] Ethambutol embB Encodes arabinofuranosyltransferase, related to arabinogalactan synthesis [7][8][9] embC Encodes arabinofuranosyltransferase, related to arabinoglycan lipid synthesis [7][8][9] embA Encodes arabinofuranosyltransferase, related to arabinogalactan synthesis.…”