The present study describes the optimization of a simple and reliable method for the determination of four first line antitubercular drugs in human plasma. The studied analytes were isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E) in fixed dose combination recommended to patients under the Revised National Tuberculosis Control Programme (RNTCP, India). The analytes were extracted from the human plasma (150 μL) using the single step liquid-liquid extraction approach and were analyzed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The method was fully validated, according to USFDA guidelines. A linear range of 0.05-10 μg mL-1, 0.1-20 μg mL-1, 0.5-100 μg mL-1 and 0.05-10 μg mL-1 for H, R, Z and E respectively was established, presenting determination coefficients above 0.99. Concerning imprecision, the CV was lower than 15% for all analytes. All tested analytes were found to be stable in the samples. Although the values obtained for recovery were above 85%, the method proved to be sensitive, since low detection limits of 0.05 μg mL-1 for H and E, 0.1 μg mL-1 for R and 0.5 μg mL-1 for Z were obtained. The intra-day and inter-day accuracy and imprecision were within CV ±15%. The use of the conventional silica column in the extraction of these compounds through a single step protein precipitation method simplifies the analytical process. In addition due to its simplicity and sensitivity, it can be applied to carry out therapeutic drug monitoring and drug level assessment in human plasma samples. The results of analyte levels are comparable to other reported methods. The method had been successfully applied for simultaneous determination of first line anti-tubercular drugs in pulmonary tubercular patients. The method requires 150 μL of patient plasma and offers low volume for injection (10 μL) and blood sample collection (3 mL) which will be an added advantage for pediatric anti-TB drug level assessment and monitoring.

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