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

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 m),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 g.mL 1内线性良好(r=0.999 5);HTMP线性范围为0.031 25～5 g.mL 1(r=0.999 8)。TMP、HTMP的定量下限均为0.031 25 g.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。

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-l-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 μm） 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 μL 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-l 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.