A new and simple HPLC-UV method for measuring seven anti-tuberculosis drugs simultaneously in human plasma for drug-resistant tuberculosis

Abstract Therapeutic drug monitoring (TDM) of anti-tuberculosis (anti-TB) drugs is important for optimizing drug dosages to improve treatment efficacy for drug-resistant tuberculosis (DR-TB). However, existing high-pressure liquid chromatography-ultraviolet (HPLC-UV) detection methods are limited either by the number of anti-TB drugs that can be measured simultaneously or by laborious sample preparation procedures. Although liquid chromatography–mass spectrometry (LC-MS) offers advantages over HPLC-UV methods, its availability is limited, particularly in resource-constrained settings, for routine TDM. Therefore, the study aimed to develop a simple HPLC-UV method for the simultaneous quantification of anti-TB drugs, namely, levofloxacin, moxifloxacin, linezolid, pretomanid, clofazimine, bedaquiline, and delamanid, and to evaluate its applicability to clinical samples. Plasma samples (100 µL) were deproteinized with acetonitrile (100 µL), and 25 µL of the supernatant was injected into the HPLC system. Chromatographic analysis was performed using an Atlantis T3 column (4.6 × 150 mm, 3 μm) with gradient elution using 10 mM ammonium formate buffer containing 0.5% formic acid (pH 2.6) and acetonitrile at a flow rate of 0.8 mL/min for 30 min. The method demonstrated linearity (R² ≥ 0.999) over concentration ranges of 1.00–20.00 µg/mL for levofloxacin and moxifloxacin (305 nm), 3.00–60.00 µg/mL for linezolid (254 nm), and 0.50–10.00 µg/mL for clofazimine (305 nm), pretomanid, bedaquiline, and delamanid (330 nm), with acceptable intra-day and inter-day accuracy and precision (< 15%). Furthermore, the method was found suitable for measuring these anti-TB drugs in clinical samples. The newly developed HPLC-UV method offers simple sample preparation for simultaneously analyzing seven anti-TB drugs in a single run and it is easily adoptable in resource-limited settings for routine TDM.