Simultaneous Quantitation of Rosiglitazone and Glibenclamide in Human Plasma by LC-MS/MS; Method Development and Validation

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated according to the FDA guidelines, for the simultaneous quantitation of rosiglitazone and glibenclamide in human plasma using glipizide (is an internal standard (IS). The bioanalytical method consists in the liquid-liquid extraction with a mixture of chloroform and isoamylalcohol (9:1) and quantitation by triple quadrupole mass spectrometer (API- 2000) using electrospray ionization technique, operating in multiple reaction monitoring (MRM) and positive ion mode. The compounds were eluted isocratically injecting 20 mu L aliquots of the processed samples on a C18 column with an mobile phase consisting of a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and methanol. The ion transitions monitored were m/z 358 -> 135 for rosiglitazone, m/z 494 -> 369 for glibenclamide, and m/z 446 -> 321 for glipizide (internal standard, IS). The method demonstrated linearity from 5 to 800 ng/mL (r(2) = 0.998) for both the analytes with desired accuracy and precision. Intra- and inter-day precision ranged from 4 to 6 and 5 to 9% (RSD), respectively. The accuracy determined at quality control levels was within 90-105%. The validated method is very robust, accurate and requires a single IS for determination of both the analytes. This developed method proved to be reproducible and sensitive and can be used successfully to support a pharmacokinetic and/or bioequivalence study.