| Mucuna pruriens, the seed of Mucuna pruriens var. utilis Burck containing levodopa(4-7%), is an ancient Indian herbal medicine widely used for the treatment of Parkinson's disease. Comparing with classic levodopa products, Mucuna pruriens extracts(MPE) would not cause side effects, such as dyskinesia during the long-term treatment of Parkinson's disease. MPE consists of not only just levodopa, but also the trace derivatives with structure of isoquinoline amino acids including 1,1-dimethyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline(MD01). The present study developed a reliable and sensitive LC-MS/MS method for simultaneous determination of levodopa and MD01, which was successfully applied to a pharmacokinetic study in rats after a single and multiple oral administrations of levodopa, MPE and levodopa/MD01, respectively. The present study provided scientific data for the impact of the trace constituents in MPE on the pharmacokinetic characteristics of levodopa.As for the LC-MS/MS method development, the systematic optimization of mass spectral and chromatographic conditions together with sample treatment procedure was achieved. The mass spectrometric analysis was conducted in positive ionization mode with electrospray ionization(ESI) and multiple reaction monitoring(MRM) transitions at m/z 198.1 ? 135.0 for levodopa, m/z 238.3 ? 175.2 for MD01 and m/z 212.1 ? 139.0 for methyldopa, respectively. The chromatographic conditions and sample preparation procedures were optimized for the purpose of eliminating the ion suppression caused by matrix effect and improving the extraction recovery. Finally, separation of analytes was performed on a Thermo Aquasil C18 column(50 × 2.1 mm, 3 ?m), and the mobile phase consisted of 0.2% formic acid and acetonitrile with a gradient elution at a flow rate of 0.6 m L/min. The impact of matrix effect was minimized on this condition. The recovery of analytes was very low when acetonitrile was used for precipitation due to protein binding of analytes, thus acetonitrile containing 2% formic acid was used for the protein precipitation in order to improve the extract recovery. Since levodopa and MD01 were easily oxidized, the sodium metabisulfite was used as a stabilizer in neat solutions and matrix samples. The dynamic range of the present method was 20.0-10000 ng/m L. The analysis time was 3.5 min per injection with acceptable accuracy(-9.7%-10.8% relative error) and intra- and interbatch precision(RSD?12.3%).The validated method was applied for determination of levodopa and MD01 in rat plasma after a single and multiple oral administrations of levodopa, MPE and levodopa/MD01, respectively. The pharmacokinetic characteristics of these analytes indicated that no significant differences were observed among groups except for the increase of mean retention time(MRT) in MPE and levodopa/MD01 groups compared with levodopa group. However, the AUC(0-?) value in MPE group(641405 ± 81451 ng?min/mL) was significant increased compared with that of levodopa group after multiple oral administrations for seven consecutive days. These results revealed that the main bioactive component levodopa in MPE possessed higher bioavailability after multiple oral administration of MPE, which made MPE more advantageous than levodopa for the long-term treatment of Parkinson's disease. |
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