Quantification of Pyrazinamide in Human Plasma by Validated High Performance Liquid Chromatography Method

Abstract

Objective: To be able to accurately determine the quantity of Pyrazinamide (PZA) in different tablet preparations and human plasma using an Ultra violet detector equipped high performance liquid chromatography (HPLC). Study Design: Experimental study Place and Duration of Study: Department of Bioequivalence Studies, University of Veterinary and Animal Sciences Lahore and the Department of Pharmacology, University of Health Sciences, Lahore the from 1st April 2017 to 31st March 2018. Methodology: Two mobile phases were used, the first compromised of disodium hydrogen phosphate buffer having a pH of 6.8 and acetonitrile in the proportion of (95:5) and the second was a combination of aforesaid substances in equivalent proportion (50:50 v/v). The gradient for the first 5 min was exclusively Mobile phase “a” after which 5-6 min Mobile phase “b” was raised from 0 to 100% and was kept at 100% till the completion of the cycle. The flow of mobile phase was kept at 1000 µl/min. Determination of PZA was done using a ultraviolet detector at a wavelength of 238 nm. Amount of sample injected was 40 μl. Procedure was done by using Shizmadu Chromatographic System, Japan equipped with a SIL-20AC HT auto-sampler, SPD-M20A, CTO 20 AC, a LC-20AT VP pump, and CBM 20A controller unit. A C18 column was used as well. Results: Retention time of PZA was 6.1±2%. Precision was 0.46 to 2.20% relative standard deviation for intra assay and for inter assay we obtained 0.29 to 34.45% RSD for all quality control levels. The overall recovery of PZA was 96.75%. Conclusion: High selectivity for PZA was seen and no other spikes from drugs present in FDC regimen were observed at the time when PZA is detected in blank plasma samples Key words: Chromatography, High pressure liquid. Pyrazinamide. Tuberculosis