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Free Content Determination of rifampicin, isoniazid and pyrazinamide by high performance liquid chromatography after their simultaneous extraction from plasma

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SETTING: Isoniazid (INH), rifampicin (RIF), and pyrazinamide (PZA) are first-line antituberculosis drugs. Due to difficulties in producing effective combined formulations of these three drugs, the bioavailability of new combination formulations needs to be assessed prior to registration.

OBJECTIVE: To develop a rapid, simple and sensitive high performance liquid chromatography (HPLC) assay method suitable for assaying RIF, INH and PZA in large numbers of plasma samples generated in bioavailability studies.

METHOD: RIF, desacetyl-rifampicin (DRIF), INH and PZA were extracted simultaneously from plasma using a solid phase extraction column. RIF and DRIF were quantitated by HPLC using an 80% acetonitrile/0.1% trifluoroacetic acid (TFA) mobile phase and a C8 reversed phase column. INH and PZA were also quantitated on a C8 reversed phase column, but a 3% acetonitrile/0.6% TFA mobile phase was used.

RESULTS: Mean recovery of RIF, DRIF and PZA from plasma was well over 90%, and over 70% for INH. Calibration graphs were linear for all the drugs in their therapeutic range. Correlation coefficients were all above 0.9995, and between- and within-run coefficients of variation below 10%.

CONCLUSION: A simple, accurate, sensitive and cost effective assay for first-line antituberculosis drugs has been developed.
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Keywords: HPLC; isoniazid; pyrazinamide; rifampicin

Document Type: Regular Paper

Affiliations: Department of Pharmacology, University of Cape Town Medical School, Observatory, Western Cape, South Africa

Publication date: 01 November 1999

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  • The International Journal of Tuberculosis and Lung Disease publishes articles on all aspects of lung health, including public health-related issues such as training programmes, cost-benefit analysis, legislation, epidemiology, intervention studies and health systems research. The IJTLD is dedicated to the continuing education of physicians and health personnel and the dissemination of information on tuberculosis and lung health world-wide.

    Certain IJTLD articles are selected for translation into French, Spanish, Chinese or Russian. They are available on the Union website

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