Research Article | Volume: 8, Issue: 9, September, 2018

Simultaneous Determination of Pyrazinamide, Rifampicin, Ethambutol, Isoniazid and Acetyl Isoniazid in Human Plasma by LC-MS/MS Method

Le Thi Luyen Ta Manh Hung Le Thu Huyen Le Anh Tuan Duong Thi Ly Huong Hoang Van Duc Bui Thanh Tung   

Open Access   

Published:  Sep 30, 2018

DOI: 10.7324/JAPS.2018.8910

Monitoring of anti-tuberculosis drug concentrations in human plasma is very important in the treatment of TB disease. In this study, we described a method that can rapidly and simultaneously measure the plasma concentrations of four anti-tuberculosis drugs (isoniazid, rifampicin, pyrazinamide, and ethambutol) and one major metabolite (acetylisoniazid) using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). To adjust for degradation and losses during sample preparation, Diltiazem hydrochloride was used as an internal standard. Samples were prepared by using protein precipitation with methanol. Four drugs and one metabolite were simultaneously separated by using the high-performance liquid chromatography system with Gemini C18 column and a mobile phase consisting of MeOH and Ammonium acetate 5mM, pH 3.5. The method was found to have high selectivity. Precision estimated by the coefficient of variation was <15% for all drugs. The linear range of the calibration curve for PZA 1.0–100 μg/mL; RIF 0.2–20 μg/mL; INH 0.1–10 μg/mL; AcINH 0.1–10 μg/mL; and EMB 20–5000 ng/mL. The lower limit of quantification was 1 μg/mL; 0.2 μg/mL; 0.1 μg/mL; 0.1 μg/mL and 20 ng/mL, respectively. The results of validation for specificity/selectivity, precision, accuracy, linearity, limit of quantification, recovery, and stability show that the method meets the requirements of a bioanalytical method. Our method may serve well for routine therapeutic monitoring of the first-line anti-TB drugs in patient plasma.

Keyword:     Pyrazinamide Rifampicin Isoniazid Acetyl Isoniazid Ethambutol LC-MS/MS Tuberculosis.


Luyen LT, Hung TM, Huyen LT, Tuan LA, Huong DTL, Duc HV, Tung BT. Simultaneous determination of Pyrazinamide, Rifampicin, Ethambutol, Isoniazid and Acetyl Isoniazid in human plasma by LC-MS/MS method. J App Pharm Sci, 2018; 8(09): 061-073.

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

HTML Full Text


Agency EM. Guideline on bioanalytical method validation. Committee for Medicinal Products for Human Use (CHMP). 2011.

Alsultan A, Peloquin CA. Therapeutic drug monitoring in the treatment of tuberculosis: an update. Drugs, 2014; 74(8):839-854.

Health UDo, Services H, Food, Administration D (2017). Analytical procedures and methods validation for drugs and biologics-guidance for industry. 2015.

Hoa NB, Sy DN, Nhung NV, Tiemersma EW, Borgdorff MW, Cobelens FG. National survey of tuberculosis prevalence in Viet Nam. Bulletin of the World Health Organization, 2010; 88(4):273-280.

Mirsaeidi M, Sadikot RT. Patients at high risk of tuberculosis recurrence. International journal of mycobacteriology, 2018; 7(1):1.

Molden E, Bøe GH, Christensen H, Reubsaet L. High-performance liquid chromatography-mass spectrometry analysis of diltiazem and 11 of its phase I metabolites in human plasma. Journal of pharmaceutical and biomedical analysis, 2003; 33(2):275-285.

Mukherjee A, Velpandian T, Singla M, Kanhiya K, Kabra SK, Lodha R. Pharmacokinetics of isoniazid, rifampicin, pyrazinamide and ethambutol in Indian children. BMC infectious diseases, 2015; 15(1):126.

Mukherjee JS, Rich ML, Socci AR, Joseph JK, Virú FA, Shin SS, Furin JJ, Becerra MC, Barry DJ, Kim JY. Programmes and principles in treatment of multidrug-resistant tuberculosis. The Lancet, 2004; 363(9407):474-481.

Prahl JB, Lundqvist M, Bahl J, Johansen IS, Andersen ÅB, Frimodt-Møller N, Cohen AS. Simultaneous quantification of isoniazid, rifampicin, ethambutol and pyrazinamide by liquid chromatography/ tandem mass spectrometry. Apmis, 2016; 124(11):1004-1015.

Song SH, Jun SH, Park KU, Yoon Y, Lee JH, Kim JQ, Song J. Simultaneous determination of first-line anti-tuberculosis drugs and their major metabolic ratios by liquid chromatography/tandem mass spectrometry. Rapid communications in mass spectrometry, 2007; 21(7):1331-1338.

Sturkenboom M, van der Lijke H, Jongedijk E, Kok WT, Greijdanus B, Uges D, Alffenaar J-WC. Quantification of isoniazid, pyrazinamide and ethambutol in serum using liquid chromatography-tandem mass spectrometry. Journal of Applied Bionalysis, 2015; 1(3):89.

Um S, Lee S, Kwon S, Yoon H, Park K, Song J, Lee C, Lee J. Low serum concentrations of anti-tuberculosis drugs and determinants of their serum levels. The International Journal of Tuberculosis and Lung Disease, 2007; 11(9): 972-978.

Zhou Z, Wu X, Wei Q, Liu Y, Liu P, Ma A, Zou F. Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the simultaneous determination of five first-line antituberculosis drugs in plasma. Analytical and bioanalytical chemistry, 2013; 405(19):6323-6335.

Article Metrics

514 Absract views 33 PDF Downloads 547 Total views

   Abstract      Pdf Download

Related Search

By author names

Citiaion Alert By Google Scholar

Name Required
Email Required Invalid Email Address

Comment required