HPLC法同时测定Beagle犬血浆中烟酸和烟尿酸的浓度

目的建立比格犬血浆中烟酸及其代谢物烟尿酸的RP-HPLC测定方法。方法血浆样品用甲醇沉淀蛋白,上清液氮气流吹干,残留物用流动相复溶后分析,以富马酸为内标。色谱柱:Diamon-sil C18柱(4.6 mm×200 mm,5μm),流动相:甲醇-0.04 mol.L-1四丁基溴化铵溶液(体积比为30∶70),流速:1.0 mL.min-1,柱温:20℃,检测波长:262 nm。结果烟酸在0.20~80.0 mg.L-1质量浓度内线性关系良好,高、中、低3种质量浓度QC样品的日内、日间RSD均小于8.0%,提取回收率在90.0%~97.8%之间;烟尿酸在0.10~5.0 mg.L-1浓度范围内线性关系良好,高、中、低3种浓度QC样品的日内、日间RSD均小于9.1%,提取回收率在95.9%~99.5%之间。结论该方法适用于血浆中烟酸和烟尿酸浓度的测定和药动学研究。

Development and validation of an RP-HPLC method for simultaneous determination of nicotinic acid and nicotinuric acid in Beagle dog plasma

Objective To establish a method for the determination of nicotinic acid and its metabolite, nicotinuric acid in Beagle dog plasma by RP-HPLC. Methods Plasma protein was precipitated by the addition of methanol. The supernatant solution was evaporated to dryness under nitrogen. The residue was reconstituted in mobile phase and analyzed by RP-HPLC. Fumaric acid was used as the internal standard. The following chromatographic conditions were used: The column was Diamonsil C₁₈ (4.6 mm × 200 mm, 5 mm), mobile phase was methanol-0.04 mol·L^-1 tetrabutylammonium bromide (V:V=30:70). The flow rate was set at 1.0 mL·min^-1. The column temperature was 20 ˚C. Detection wavelength was set at 262 nm. Results Nicotinic acid showed a good linearity in the range of 0.2-80.0 mg·L^-1, The intra-day RSD and inter-day RSD were less than 8.0%, The extraction recovery was within 90.0-97.8%; nicotinuric acid showed a good linearity in the range of 0.1-5.0 mg·L^-1. The intra-day RSD and inter-day RSD were less than 9.1%. The extraction recovery was within 95.9-99.5%. Conclusions The method is proved to be sensitive, specificity and accurate. It is suitable for plasma drug concentration and pharmacokinetic study of nicotinic acid in Beagle dogs.