HPLC法同时测定Beagle犬血浆中烟酸和烟尿酸的浓度

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

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：Diamonsil C18柱（4．6mm×200mm，5μm），流动相：甲醇-0．04mol·L^-1四丁基溴化铵溶液（体积比为30：70），流速：1．0mL·min^-1，柱温：20℃，检测波长：262nm。结果烟酸在0．20～80．0mg·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0．10～5．0mg·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。

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, 5μm）, 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℃2. 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.