MSACL 2025 Abstract

Evaluation of an Assay for Seven Nontuberculosis Mycobacterial Pulmonary Disease Drugs Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

Kyunghoon Lee (1,2), Joon Hee Lee (1,2), Sun-Hee Jun (2), Sang Hoon Song (1,3), Junghan Song (1,2)
(1) Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; (2) Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea;(3) Department of Laboratory Medicine, Seoul National University Hospital, Seoul, Republic of Korea

Kyunghoon Lee, Ph.D. Candidate (Presenter) Seoul National University Bundang Hospital

Relevant Financial Disclosures (within past 24 months, reported on Jul 06, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

BACKGROUND:
Nontuberculous mycobacteria (NTM), with over 200 identified species, are ubiquitous in natural environments such as soil and water. Recently, the global prevalence of nontuberculous mycobacterial pulmonary disease (NTM-PD), caused by lung tissue damage and inflammation, has risen rapidly, with a reported rate of 56.7 cases per 100,000 population in Korea as of 2021. Despite the growing burden of NTM-PD, treatment remains empirical, typically involving two or three antibiotics administered over 18 to 24 months. Therefore, there is am increasing need for therapeutic drug monitoring (TDM) to enable optimized treatment. The aim of this study was to develop and evaluate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous quantification of seven antibiotics commonly used in NTM-PD treatment.

METHODS:
Seven antibiotics – azithromycin, clarithromycin, clofazimine, ethambutol, linezolid, moxifloxacin, and rifampicin – were selected for assay development. Each serum sample was analyzed using a UPLC-MS/MS system. Analytical performance was evaluated in terms of precision, linearity, lower limit of quantification (LLOQ), carryover, and method comparison.

RESULTS:
All analytes and internal standards were clearly separated on the UPLC-MS/MS system without ion suppression. Within-run and between-day precision at three concentration levels for each drug ranged from 2.2% to 9.2% and from 5.8% to 16.7%, respectively. Calibration curves were linear over the following range: 0 – 10 μg/mL for azithromycin, clarithromycin, ethambutol, moxifloxacin, and rifampicin; 0 – 5 μg/mL for clofazimine; and 0 – 20 μg/mL for linezolid. The LLOQ was 0.0625 μg/mL for clofazimine; 0.125 μg/mL for azithromycin, clarithromycin, and rifampicin; and 0.25 μg/mL for ethambutol, linezolid, and moxifloxacin. No carryover was observed. Method comparison using four serum samples showed a difference ranging from -0.5% to 12.3% between the new and existing methods for ethambutol and rifampicin.

CONCLUSIONS:
The newly developed antibiotics assay demonstrated acceptable analytical performance including precision, linearity, LLOQ, carryover and method comparability. This method may provide a valuable foundation for implementing TDM and supporting personalized therapy for NTM-PD in clinical practice.