Analytical Sciences

Simultaneous Determination of Isoniazid, Pyrazinamide, Rifampicin and Acetylisoniazid in Human Plasma by High-Performance Liquid Chromatography

Zhifeng ZHOU,* Lingyun CHEN,* Peng LIU,** Mei SHEN,* and Fei ZOU***

*Hygiene Detection Center, School of Public Health and Tropical Medicine, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, P. R. China
**Department of Orthopedic and Spinal Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, P. R. China
***Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, P. R. China

A sensitive and accurate high-performance liquid-chromatography method was developed and validated for the simultaneous determination of pyrazinamide (PZA), isoniazid (INH), rifampicin (RFP) and acetylisoniazid (AcINH) in human plasma. Separation was performed on a Max-RP C₁₂ column using gradient elution and a flow-rate program. The mobile phase was methanol–acetonitrile–buffer (20 mM of heptanesulfonic acid sodium, pH 2.5) with a ratio of 10:8:82 (v/v/v) at the initial phase. All calibration curves had good linearity (r² > 0.99) between the test ranges. The intra- and inter-day precision was less than 8.8% in good accuracy (<15%). The limit of detection with a signal-to-noise (S/N) of 3 was 0.014, 0.009, 0.023 and 0.054 μg mL⁻¹ for PZA, AcINH, INH and RFP, respectively. The method was selective, sensitive and reliable, and is a good alternative for routine therapeutic drug monitoring of the four compounds during the treatment of tuberculosis patients.

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