HPLC测定犬血浆中的视黄醇、视黄醇棕榈酸酯和视黄醇硬脂酸酯

Determination and pharmacokinetics study of Vitamin A in beagle dogs plasma by HPLC

目的采用HPLC测定比格犬血浆中的视黄醇（RL）、视黄醇棕榈酸酯（RP）、视黄醇硬脂酸酯（RS），并研究RP在比格犬体内的药动学。方法采用Agilent Extend—C18色谱柱（250mm×4．6mm，5μm），乙腈-甲醇-1％乙酸铵溶液-四氢呋喃为流动相梯度洗脱，检测波长325nm，以视黄醇醋酸酯为内标；血浆样本以无水乙醇沉淀蛋白后，用正己烷提取，N2吹干后复溶测定。结果RL、RP、RS的线性范围分别为：0．5620～56．20、0．4698～46．98、0．5215～52．15μg·mL^-1，r分别为0．9977、0．9971、0．9977（n=7），日内、日间RSD均≤10．2％，RL、RP、RS的提取回收率分别为78．0％～93．0％、79．4％～101．7％、78．8％-101．8％。比格犬单次口服视RP微囊后，RP的Tmax为7．333±1．033h，Cmax为2．342±0．337mg·L^-1，AUC（0-t）为44．913±12．963mg·L^-1·h。结论所用方法简便准确，适于比格犬血浆中RL、RP、RS的同时测定及药动学研究。

OBJECTIVE To establish a method for determination of vitamin A in beagle dogs plasma,including retinol（ RL）, retinol palmitate（RP） and retinol stearate（ RS）, and study on the pharmacokinetics of RP in beagle dogs. METHODS HPLC method was developed with a Agilent Extend - C18 column. A mobile phase consisting of acetonitrile - methnol - 1% ammonium acetate - tetrahydrofuran was used with gradient elution, and the detection wavelength was 325 nm. Proteins were precipitated with anhydrous ethanol. RL, RP and RS in beagle dogs plasma were extracted with n - hexane and determined with HPLC. RESULT The linear ranges of RL, RP and RS were 0. 5620 -56.20μg·mL^-1 （r =0. 9997 ,n =7） ,0. 4698 -46.98μg·mL^-1 （ r =0. 9971 ,n = 7） and 0. 5215 -52.15μg·mL^-1 （ r = 0. 9977, n = 7 ） , respectively. The RSD for RL, RP and RS were less than 10.2%. The proposed method recovered 78.0% - 93.0% RL,79.4% - 101.7% RP and 78.8% - 101.8% RS in dog plasma. The method was applied for the pharmacokineties study of RP 250CWS by oral administration. The AUC（0-t） ,tmax and Cmax for RP were 44. 913 ± 12. 963 mg·L^-1·h^-1 ,7. 333 ± 1. 033 h, 2. 342 ± 0. 337 mg·L^-1, respectively. CONCLUSION The method was simple, accurate and applicable to analysis and pharmacokinetic study of RL,RP and RS.