Development and validation of high performance liquid chromatographic method for the determination of pyrazinamide in human plasma

Abstract

BackgroundPyrazinamide is one of the key APIs used in the combination treatment of tuberculosis recommended by WHO. Since sufficient analytical methods have not been reported officially for the quantitative estimation of pyrazinamide, there is necessity for investigation of new analytical methods for quantitative estimation of pyrazinamide in human plasma. ObjectiveA rapid, sensitive, simple and cost-effective high performance liquid chromatographic method for the determination of pyrazinamide by UV detection in human plasma is to be developed and validated. Materials and methodsThe extraction process involved a liquid–liquid extraction using a 70:30% v/v mixture of t-butyl methyl ether and dichloromethane. Both pyrazinamide and the internal standard were eluted under isocratic mode using a 150 x 4.6 mm i.d, 5 μm Phenomenex ODS 2 C18 column. The mobile phase was composed of a mixture of 15:85 % v/v methanol and 0 mM potassium dihydrogen phosphate (pH adjusted to 7.4) at a flow rate of 1.0 ml/min. The wavelength of detection was 268 nm. The injection volume was 20 μl. The runtime of the method was 8 min. ResultsThe method showed good linearity in the range of 1.02–50.23 μg/ml. The overall recovery of pyrazinamide was 27.21% with a CV of 2.71% and recovery of internal standard was 83.34% with a CV of 4.38%. ConclusionA rapid, sensitive, simple and cost effective method for the estimation of pyrazinamide in human plasma using metronidazole as internal standard was developed and validated according to FDA guidelines.