2).11 Since sufficient analytical methods have not been reported for the quantitative estimation of pyrazinamide, there is a necessity for investigation of selective and sensitive new analytical methods for quantitative estimation of pyrazinamide in human plasma. Additionally, pyrazinamide has a strong chromophore showing reddish brown color at wavelength of 268 nm. This chromophore not only allows for successful determination in human plasma by UV detection but also offers acceptable sensitivity as offered by LC-MS/MS detection. Although LC-MS/MS is a
versatile tool, the development of HPLC based separation methods makes it more economical and simpler both in terms of maintenance and data interpretation. The present article describes find more a simple and sensitive RP-HPLC method with a low LLOQ for UV detection of PZA using metronidazole (Fig. 2) as an internal standard (IS) eluted under isocratic mode which can be directly applied to the successful estimation
of rifampicin in a bioequivalence study and to validate the developed method according to FDA guidelines.12 Pyrazinamide (purity 98.00% w/w) was used as received from Lupin Laboratories Ltd. Metronidazole (MTZ) (used as internal standard, purity 99.0% w/w) is purchased from Sigma Aldrich Inc. HPLC grade methanol and potassium dihydrogen phosphate (purified grade) were purchased from Merck Ltd (Mumbai, India). Deionized water was processed through a Milli-Q water purification system (Millipore, USA). All other chemicals and reagents were of analytical grade. The chromatographic system Selleckchem Caspase inhibitor consisted of a Shimadzu Class VP Binary pump LC 10ATvp, SIL-10ADvp Auto sampler, CTO-10Avp Column Temperature Oven, SPD-10Avp UV–Visible Detector. All the components of the system were controlled using SCL-10Avp System Controller. Data acquisition was done using LC Solutions SB-3CT software. The detector is set at a wavelength of 268 nm. Chromatographic separations were accomplished using a Phenomenex C18, 5 μm, 150 mm × 4.6 mm column. The mobile phase was composed of a mixture of 15 parts of methanol and 85 parts of 10 mM potassium dihydrogen phosphate (pH 7.4), adjusted with potassium hydroxide. The mixture was
filtered through 0.22 μm membrane (Millipore, Bedford, MA, USA) under vacuum, and then degassed by flushing with nitrogen for 5 min. The mobile phase was pumped isocratically at a flow rate of 1.0 ml/min during analysis, at ambient temperature. The rinsing solution consisted of a mixture of 50: 50% v/v of methanol: HPLC grade water. A stock solution of pyrazinamide was prepared in diluent solution (mixture of 50:50% v/v of methanol: HPLC grade water) such that the final concentration was approximately 10 mg/ml. Stock solution of metronidazole (approx 5 mg/ml) is prepared in HPLC grade methanol. The solutions were stored at 4 °C and they were stable for two weeks. Aqueous stock dilutions were prepared initially. Aqueous stock dilution, 0.