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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 a method for the determination of tetramethylpyrazine (TMP) and its metabolite 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) in plasma of rats by high performance liquid chromatography. Methods The plasma was alkalified and extracted with chloroform-1-chloro-n-butane (3: 1) and detected by HPLC. The mobile phase was Hypersil BDS C18 (4.6 mm × 250 mm, 5 m) Potassium dihydrogen phosphate buffer (pH 5.6) -methanol (72:28), 2-methylpyrazine as internal standard, flow rate: 1.0 mL.min 1. The detection wavelength was 285 nm and the column temperature was room temperature. Results The plasma concentration of TMP was linear within 0.031 25 ~ 50 g · mL 1 (r = 0.999 5). The linear range of HTMP was 0.031 25 ~ 5 g.mL 1 (r = 0.999 8). The lower limit of quantification of TMP and HTMP were 0.031 25 g.mL 1, the recoveries were 86.2% -93.0% (TMP), 73.8% -95.1% (HTMP), and the relative recoveries were 96.9% -117.7% (TMP) 97.5% to 104.9% (HTMP), both intra-and inter-assay RSD <9% (n = 5). Conclusion The method is simple, accurate, reproducible and sensitive. It is suitable for the determination of TMP and its major metabolite HTMP and its pharmacokinetics.