高效液相色谱法分离测定大鼠肝微粒体中普萘洛尔葡醛酸化代谢产物

目的　建立分离测定外消旋普萘洛尔两种对映体的葡醛酸化代谢产物的反相高效液相色谱法。方法用生物合成法合成R-(+)-普萘洛尔葡醛酸苷,并经C₁₈固液萃取柱纯化、浓集,得到标准液储备液,以此标准液进行普萘洛尔葡醛酸化代谢物的分析测定。结果　R-(+)-普萘洛尔葡醛酸苷在0.24-73.17μmol·L^-1 浓度范围内线性良好,方法专属性高,平均回收率为99.4%±1.0% ,日内精密度RSD小于3.3% ,日间精密度小于4.5%。结论　此法可用于外消旋普萘洛尔两种对映体体外葡醛酸化代谢研究

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY SEPARATION AND QUANTITATION OF PROPRANOLOL GLUCURONIDE IN RAT MICROSOMES

AIM To establish an HPLC assay to determine directly, the propranolol glucuronides in microsomal incubate without hydrolysis to their parent enantiomers. METHODS The standard for this direct assay was prepared by incubation of liver microsomes with propranolol. The glucuronide obtained was purified and concentrated with solid-phase extraction and the concentration was measured by an indirect method, i.e. HPLC assay of the propranolol after enzymatic hydrolysis with β-glucuronidase. The direct assay involved separation by HPLC using a C₁₈ reversed phase column, with UV detection at 290 nm. RESULTS The recovery of the assay was 99.4%±1.0% (n=15), the reproducibility of the assay was less than 3% (RSD) for inter-day and 5% (RSD) for intra-day. The standard curves showed excellent linearity over the range 0.24-73.17 μmol·L^-1. Followed Michaelis Menten kinetics, the K_m and C_max for the two enantiomers were very different. The R-enantiomer was glucuronidated at a more efficient rate than its enantiomorph, and was a better substrate. CONCLUSION The sensitive, reproducible and accurate HPLC method was developed to measure directly the propranolol glucuronides and has been applied to determine the stereoselectivity of glucuronidation metabolism of racemic propranolol in rat liver microsomes.