Simple High-Performance Liquid Chromatographic Method for the Determination of Tetramethylpyrazine Phosphate in Very Small Volumes of Dog Plasma: Application to a Pharmacokinetic Study

A rapid and sensitive high-performance liquid chromatographic (HPLC) method is developed for the determination of tetramethylpyrazine phosphate, an antiplatelet aggregation agent, in 100 µL of dog plasma. Sample preparations are carried out by deproteinization with an internal standard (carbamazepine) solution in acetonitrile. An aliquot of the supernatant (20 µL) is directly injected into an HPLC apparatus with methanol‐phosphate buffer (0.01M, pH 3.0) (62:38, v/v) as the mobile phase at a flow rate of 1.0 mL/min. Separation is performed with a C 18 column at 30°C. The peak is detected using a UV detector set at 279 nm. The capacity factors are 1.48 for tetramethylpyrazine phosphate and 2.09 for carbamazepine, with a total run time of 10 min. The calibration curve is linear in the 0.2‐50-µg/mL range. The limit of detection is 0.05 µg/mL. Mean recoveries are 92.6‐98.1%. The within- and between-day variation coefficients are less than 4.9% and 7.5%, respectively. The present method has been successfully used to provide pharmacokinetic data after oral administration of tetramethylpyrazine phosphate pulsincap capsules and immediaterelease tablets to dogs.