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Simultaneous quantification of four first line antitubercular drugs and metabolites in human plasma by hydrophilic interaction chromatography and tandem mass spectrometry

Journal article
Authors Jesper Sundell
E. Bienvenu
Sofia Birgersson
Angela Äbelö
Michael Ashton
Kurt-Jürgen Hoffmann
Published in Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume 1105
Pages 129-135
ISSN 1570-0232
Publication year 2019
Published at Institute of Neuroscience and Physiology, Department of Pharmacology
Pages 129-135
Language en
Keywords Biomolecules, Diseases, Extraction, Formic acid, Hydrazine, Hydrophilicity, Liquid chromatography, Liquids, Mass spectrometry, Metabolites, Plasma (human), Chromatographic separations, Hydrophilic interaction chromatography, Liquid chromatography-tandem mass spectrometry, Lower limit of quantifications, Mass spectrometric detection, Multiple-reaction monitoring modes, Tandem mass spectrometry, US Food and Drug Administration, Drug interactions
Subject categories Pharmacology, Analytical Chemistry


Co-infection of tuberculosis in HIV-patients is a major health concern worldwide and especially so in Sub-Saharan Africa. To enhance the study of potential drug-drug interactions when simultaneously treating the two infections, a liquid chromatography tandem mass spectrometry method was developed for the quantitation of the four first line anti-tuberculosis drugs isoniazid, rifampicin, pyrazinamide, ethambutol and four of their major metabolites in human plasma. Analytes were extracted from 200 μL of plasma using a sequential liquid-liquid extraction with ethyl acetate at neutral and acidic pH. The combined extracts were analyzed by liquid chromatography with mass spectrometric detection in a multiple reaction monitoring mode. The chromatographic separation was performed on a hydrophilic interaction column using a stepwise gradient with two mobile phases consisting of water with 0.3% formic acid and methanol with 0.3% formic acid, respectively. The total run time of each analysis was 4 min. The lower limit of quantification applied was 40 ng/mL for ethambutol, acetylisoniazid and 25-desacetylrifampicin, 60 ng/mL for 5-hydroxypyrazinamide, 80 ng/mL for isoniazid and isonicotinic acid, 200 ng/mL for rifampicin and 320 ng/mL for pyrazinamide. The method was validated according to US Food and Drug Administration guidance. The method exhibited adequate accuracy (87.1–114.9%), precision (CV < 12.8%) and specificity. Recovery and matrix effect were consistent (CV < 11.9%). The extracted samples were stable in the autosampler at 8 °C for up to 24 h as well as after three freeze-thaw cycles (recovery > 86.3%). The method has been shown to be robust for the analysis of the stated drugs and metabolites in human plasma obtained from 73 patients receiving these four first line anti-tuberculosis drugs. © 2018 Elsevier B.V.

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