Simultaneous Estimation of Levodopa and Carbidopa in Mice Plasma by LC-MS/MS

A simple and sensitive liquid chromatographic method along with tandem mass detection has been developed for the determination of levodopa and carbidopa in mice plasma. Owing the hydrophilic nature of both the analytes, a hydrophilic interaction liquid chromatography (HILIC) setup was used for their separation by a Merck (Germany) HILIC column (4.6 × 250 mm, 5 μm; 200 Å). The mobile phase composed equal proportion of water and acetonitrile both containing 0.1% formic acid at a flow rate of 1.4 mL/min to achieve rapid separation of the compounds. The column was coupled with a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source using multi reaction monitoring (MRM) analysis. Since, recovery of both analytes in mice plasma posed significant challenge, a customized extraction procedure based on protein precipitation was adopted with best recovery. The optimized HILIC-MS/MS condition led to yield lower limit of quantification (LLOQ) of 9.9 ng/mL and 2.47 ng/mL for levodopa and carbidopa, respectively. The method was validated with suitable determination of correlation coefficient (R2: 0.997), precision (1.6-17.2%), accuracy (84.7-120%). Successful application of this validated method was accomplished for analytes in biological samples.