Establishment and performance evaluation of a candidate reference method for 4 anti-tuberculosis drugs in human plasma

doi:10.3969/j.issn.1006-7795.2026.03.007

Abstract

Abstract: Objective To establish a candidate reference method for the simultaneous, accurate and sensitive determination of four first-line anti-tuberculosis drugs (rifampicin, isoniazid, pyrazinamide and ethambutol) in human plasma, and to provide technical support for individualized and precise clinical diagnosis and treatment. Methods An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed. Rifampicin-d8, isoniazid-d4, ethambutol-d4 and pyrazinamide-d3 were used as internal standards, and acetonitrile was selected as the extraction solvent for the quantitative determination of the four drugs in human plasma. The blood samples were centrifuged, and the supernatant was directly injected for analysis. The mobile phase was composed of 0.1% formic acid aqueous solution (v/v) (A) and methanol (B). The flow rate was set as 0.3 mL/min, the column temperature was set at 40℃ and the injection volume was 1 μL. Methodological validation was performed according to General Chapter 9012 of the Chinese Pharmacopoeia, including specificity, linearity, precision, accuracy, matrix effect and stability. Results All four drugs showed good linearity in their respective concentration ranges, with all the correlation coefficients (r2) of the calibration curves greater than 0.995. The intra-batch recoveries were in the range of 94.4% to 108.2% with the relative standard deviations (RSD) less than 6.34%, and the inter-batch recoveries were 92.4% to 104.8% with the RSD less than 4.44%. All the validation parameters met the requirements of methodological verification. Conclusion The established UPLC-MS/MS method for the determination of four first-line anti-tuberculosis drugs in human plasma is accurate, rapid, simple, and highly sensitive, with a wide linear range. It provides a reliable technical basis for therapeutic drug monitoring (TDM) and individualized dosing regimens for anti-tuberculosis treatment.

Key words: anti-tuberculosis drugs, therapeutic drug monitoring, rifampicin, isoniazid, pyrazinamide, ethambutol, ultra performance liquid chromatography-tandem mass spectrometry

CLC Number:

R969

Miao Wenxuan , Chen Qi , Mei Shenghui. Establishment and performance evaluation of a candidate reference method for 4 anti-tuberculosis drugs in human plasma[J]. Journal of Capital Medical University, 2026, 47(3): 457-469.

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