Determination of Tetramethylpyrazine and its Active Metabolite in Rat Plasma by HPLC

OBJECTIVE To establish an HPLC method for the determination of concentration of tetramethylpyrazine (TMP) and its active metabolite, 2-hydroxymethyl-3,5,6-trimethylpyrarzine (HTMP) in rat plasma and apply this method to the pharmacokinetic study of TMP. METHODS 2-Methylpyrazine was used as internal standard. The alkalified serum samples were extracted with a chloroform-1-chloro-butane (3∶1) induced liquid-liquid extraction. The target analytes were quantitatively determined by HPLC. Hypersil BDS C₁₈ column(4.6 mm×250 mm, 5 mm) was used. The mobile phase consisted of 20 mmol·L^-1 potassium dihydrogen phosphate buffer(pH 5.6)-methanol(72∶28). The flow rate was 1.0 mL·min^-1, 15 mL sample was injected and detected by the ultra-violet detector at 285 nm. RESULTS The linearity was obtained over the concentration ranges of 0.031 25-50 μg·mL^-1 for TMP(r=0.999 5) and 0.031 25-5 μg·mL^-1 for HTMP(r=0.999 8). The lower limit of quantitation (LLOQ) was 0.031 25 μg·mL^-1 for TMP and HTMP. The inter- and intra-batch precisions (RSD%) were less than 9% for both analyses. The accuracies and extracted recoveries were 86.2%-93.0% for TMP and 73.8%-95.1% for HTMP. The relative recoveries were 96.9%-117.7% for TMP and 97.5%-104.9% for HTMP. CONCLUSION The method has good selectivity, acceptable accuracy, precision and sensitivity. It can be applied to pharmacokinetic study of TMP in rats.