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Simple, Selective and High throughput Estimation of Pyrazinamide in Human Plasma using LC-MS/MS

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Author(s): Gaurang Shah | Vivek Upadhyay | Vikas Trivedi | Jaysukh Rathod | Sujal Shah, | Manish Yadav | Dr. Deepak Sawhney

Journal: Journal of Pharmaceutical and Bioanalytical Science
ISSN 2278-828X

Volume: 1;
Issue: 2;
Start page: 36;
Date: 2012;
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Keywords: Pyrazinamide | Pyrazinamide-D3 as ISTD | HPLC-MS/MS | Protein Precipitation | high throughput | human

ABSTRACT
A simple, sensitive and fast throughput high performance liquid chromatography tandem mass spectrometry (HPLCMS/MS) method has been developed for the determination of pyrazinamide. The method involved protein precipitation ofpyrazinamide and pyrazinamide-D3 as the internal standard (ISTD) in Acetic Acid in water 1%v/v: Acetonitrile (15:85v/v)from 100μL human plasma. The chromatographic separation is achieved on a Waters Symmetry shield TM RP8 3.5 μm,4.6x100mm analytical column using isocratic mobile phase, consisting of Acetic acid in water (1.0%v/v) : Acetonitrile(45:55v/v), at a flow-rate of 0.8mL/min. The precursor → product ion transition for pyrazinamide (m/z 124.1/78.90), and IS(m/z 126.90/81.90) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring(MRM) and positive ion mode. The method is validated over a wide dynamic concentration range of 0.500-50.000μg/mL.Matrix effect is assessed by post-column analyte infusion experiment for pyrazinamide. The method is simple, rugged andrapid with a total run time of 2.8 minutes and the same applied to a bioequivalence study 400mg tablet formulation in 36healthy Indian male subjects under fasting condition.

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