普萘洛尔在人肝细胞色素P450转基因细胞中的立体选择性测定

目的：建立一种手性色谱法，用于普萘洛尔经人肝细胞色素P450特基因细胞S9代谢的立体选择性研究。方法：以S—( )—普罗帕酮为内标，用柱前GITC手性衍生化反相高效液相色谱法测定普萘洛尔对映体在S9孵育液中的浓度。结果：5—(-)—普萘洛尔、内标及R—( )—普萘洛尔获基线分离。在5—500μmol/L／L范围内线性良好。定量限5μmol/L／L(22＝5，RSD＜10％)。S—(-)—和R—( )—普萘洛尔的平均回收率分别为98．7％和98．1％。日内、日间精密度均小于10％。经时孵育试验表明，普萘洛尔经CYP2Cl8有S—(-)—体优先代谢的立体选择性，而经CYP2C9有R—( )—体优先代谢的立体选择性。结论：该法简便、准确、重现性好、专属性强，可以用于普萘洛尔体外代谢的立体选择性研究。

Stereoselective determination of propranolol enantiomer in transgenic cell lines expressing human cytochrome P450

OBJECTIVE: To establish a chiro chromatography for studying the stereoselective metabolism of propranolol (PL) in S(9) incubates prepared from transgenic cell lines expressing human cytochrome P450. METHODS: The concentration of each enantiomer in S(9) incubates was determined through precolumn derivatization with GITC, followed by RP-HPLC assay using S-(+)-propafenone as internal standard. RESULTS: Baseline separations among the diastereomers of S(-)-P, internal standard and R(+)-PL were achieved on Shimpack CLC C(18)ODS column, with UV detection and methanol:water:glacial acetic acid (67/33/0.05,v/v/v) as mobile phase. The assay was simple, accurate, precise and specific. The linear range was from 5 to 500 micromol/L for each enantiomer. The limit of quantitation (LOQ) for the method was 5 micromol/L for the S(-)-and R(+)-PL, respectively (n=5, RSD<10%). The analytical method afforded average recoveries of 98.7 and 98.1% for S(-)- and R(+)-PL, respectively. The reproducibility of the assay was good (RSD<10%). The time-dependent studies showed that PL had the stereoselectivity of S-(-)-isomer in metabolism via CYP2C18 and the stereoselectivity of R-(+)-isomer in metabolism via CYP2C9. CONCLUSION: The method allows to study of stereoselective metabolism of PL in vitro.