Pharmacokinetic Study on Levodopa and Benserazide Hydrochloride Tablets in Beagle Dogs by HPLC-ESI-MS/MS

The simultaneous determination of levodopa and benserazide in Beagle dog plasma was presented by HPLC-ESI-MS/MS method applied to actual pharmacokinetic study using Beagle dogs. Effective chromatographic separation was achieved by using a C18 analytical column with an ODS guard column maintained at 25 degrees C. The mobile phase was methanol : 0.5 % formic acid aqueous solution (8/92, v/v) system in the gradient elution profile at a flow- rate of 0.8 mL/min, methyldopa was used as the internal standard (IS). The retention times of levodopa, benserazide and methyldopa were 3.9, 2.2 and 7.1 min respectively. The tandem MS optimal conditions were analyzed, and hypothetical electrospray ionization ESI(+)- MS/MS fragmentation pattern for levodopa, benserazide, dopamine and methyldopa was illustrated. Quantification was achieved using a positive electrospray ionization (ESI+) interface under multiple reaction monitoring (MRM) condition. No endogenous interferences were found for levodopa, benserazide and methyldopa, the matrix effects were negligible. The limit of detection (LOD) for levodopa and benserazide were 0.32 and 0.20 ng/mL in plasma, respectively. The calibration curve for levodopa showed linearity in the range 1.28- 1280 ng/mL with a regression coefficient of 0.9997 and the calibration curve for benserazide showed linearity in the range 0.80-800 ng/mL with a regression coefficient of 0.9999. The pharmacokinetics of levodopa and benserazide was studied in Beagle dogs following intragastric administration. The t1/2(beta) were 1.78 +/- 0.24 and 2.28 +/- 0.57 h, the T-max were 0.67 +/- 0.14 and 0.75 +/- 0.00 h, the Cmax were 55.21 +/- 13.92 and 0.02 +/- 0.01 mu g/mL, the AUC(0)-(infinity) were 74.60 +/- 12.16 and 0.04 +/- 0.01 h mu g/mL, the MRT were 1.78 +/- 0.06 and 2.61 +/- 0.39 h for levodopa and benserazide, respectively. The novel method was validated and suitably applied to measuring of levodopa and benserazide in plasma samples and its oral single dose pharmacokinetics to Beagle dog. Compared with the methods reported in literatures before, the result showed excellent accuracy, specificity and sensitivity, suggesting its applicability to preclinical and clinical pharmacological research.