RP-HPLC荧光检测法测定大鼠血浆中甲磺酸二氢麦角毒碱浓度

RP-HPLC determination of dihydroergotoxine mesylate in rats plasma with fluorescence detection

目的:建立测定大鼠血浆中甲磺酸二氢麦角毒碱的反相高效液相荧光检测方法。方法:血浆样品预处理采用乙腈直接沉淀法去除蛋白质。采用 L-7000型高效液相色谱仪,Phenomenex C_(18)柱(250 mm×4.6 mm,5μm);流动相为水-乙腈-三乙胺(32:18:1),流速为1.1 mL·min^(-1);荧光检测波长:λ_(Ex)=290 nm,λ_(Ex)=348 nm;柱温:30℃;进样量:50μL。外标法峰面积定量。结果:血浆中甲磺酸二氢麦角毒碱能得到较好的分离,无明显的干扰峰。测定方法在1.0～200.0 ng·mL^(-1)范围内具有良好的线性关系,方法回收率为96.2%～100%,日内、日间 RSD 均小于15%,最低检测浓度为1.0 ng·mL^(-1),最低检测限为50 pg。结论:本法灵敏、准确、快速,适用于甲磺酸二氢麦角毒碱鼻腔喷雾剂的药物动力学和生物利用度的研究。

Objective： To establish a simple, sensitive and rapid BP - HPLC with fluorescence detector method for the determination of dihydroergotoxine mesylate in rats plasma. Method：The plasma samples were precipitated by adding acetonitrile. The HPLC was L- 7000 and the chromatographic column was a Phenomenex C18 column （250 mm ×4. 6 mm,5 μm）. The mobile phase,water - acetonitrile - triethylamine （32： 18：1 ） ,was used at a flow rate of 1.1 mL· min^-1. The fluorimetric excitation and emission wavelengths were set at 290 nm and 348 nm,respectively. The column temperature was 30 ℃ and injection volume was 50 μL. The peak area was quantitated with external reference method. Results： Under the condition of test, dihydroergotoxine mesylate was well separated and there were not obvious interactive peak in plasma. A good linearity was obtained in the range of 1.0 - 200. 0 ng · mL^-1 （r =0. 9997）. The method recovery was between 96. 2% - 100%. The relative standard deviations of within - day and between - day were less than 15% （ n = 5 ）. The lowest concentration of detection was 1.0 ng · mL^-1, the lowest limit of detection was 50 pg. Conclusion：The established method is shown to be sensifive, accurate and simple for the determination of dihydroergotoxine mesylate levels in rats plasma. It is suitable for the pharmacokinetics and bioavailability study of dihydroergotoxine mesylate nasal spray.