Method for simultaneous analysis of nine second-line anti-tuberculosis drugs using UPLC-MS/MS

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Therapeutic drug monitoring (TDM) of anti-tuberculosis (TB) drugs is beneficial for patients responding slowly to treatment and those with multidrug-resistant TB. We used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to develop a rapid method for simultaneously measuring the blood concentrations of nine second-line anti-TB drugs: streptomycin, kanamycin, clarithromycin, cycloserine, moxifloxacin, levofloxacin, para-aminosalicylic acid, prothionamide and linezolid.


Serum samples were extracted with acidified methanol and neutralized with NaOH. A Waters Acquity HSS T3 column and gradients of ammonium formate and acetonitrile in 0.1% formic acid were used for UPLC separation. Drug concentrations were determined by multiple reaction monitoring in positive ion mode, and assay performance was evaluated. We applied this method to TDM, analysing random serum samples from 85 patients treated with second-line drugs.


Sample preparation using acidified methanol extraction followed by neutralization yielded good recovery and ionization efficiency, with chromatographic separation achieved within 3 min per sample. Within-run and between-run precisions were 1.7%–7.5% and 1.7%–12.4%, respectively, at concentrations representing low and high levels for the nine drugs. Lower limits of detection and quantification were 0.025–0.5 and 0.25–5.0 μg/mL, respectively. Linearity was acceptable at five concentrations for each drug. No ion suppression was observed at the retention time for most compounds, except for streptomycin, kanamycin and cycloserine, which were eluted close to the void volume of the column. In a limited pilot study, all quantifiable human samples had values within the validated assay ranges.


The performance of our MS/MS detection technique was generally acceptable. The method provided rapid, sensitive and reproducible quantification of nine second-line anti-TB drugs and should facilitate drug monitoring during treatment.

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