HPLC测定大鼠血浆中盐酸川芎嗪及其代谢物

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

关键词:  川芎嗪  血药浓度  高效液相色谱法

Key words:  tetramethylpyrazine  serum concentration of drug  HPLC

摘要:

目的 建立川芎嗪(TMP)及其代谢物2-羟甲基-3，5，6-三甲基吡嗪(HTMP)大鼠血浆中的高效液相色谱测定方法。方法 血浆碱化后用氯仿-1-氯正丁烷(3∶1)萃取，采用HPLC检测，色谱柱为Hypersil BDS C₁₈(4.6 mm×250 mm，5 mm)，流动相为20 mmol·L^-1磷酸二氢钾缓冲液(pH 5.6)-甲醇(72∶28)，2-甲基吡嗪为内标，流速：1.0 mL·min^-1。检测波长为285 nm，柱温为室温。结果 TMP大鼠血浆浓度测定方法在0.031 25~50 μg·mL^-1内线性良好(r=0.999 5)；HTMP线性范围为0.031 25~5 μg·mL^-1(r=0.999 8)。TMP、HTMP的定量下限均为0.031 25 μg·mL^-1，提取回收率为86.2%~93.0%(TMP)、73.8%~95.1%(HTMP)，相对回收率为96.9%~117.7%(TMP)、97.5%~104.9%(HTMP)，批内和批间RSD均<9%(n=5)。结论 该方法简便、准确，重复性好，灵敏度高，适合于TMP及其主要代谢产物HTMP血药浓度测定及药动学研究。

Abstract:

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.