Chiral reversed phase high-performance liquid chromatography for determining propranolol enantiomers in transgenic Chinese hamster CHL cell lines expressing human cytochrome P450.
An enantioselective assay for S-(-)- and R-(+)-propranolol in transgenic Chinese hamster CHL cell lines, expressing human cytochrome P450 (CYP), was developed. The method involves extraction of propranolol from the S(9) incubates, using S-(+)-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-beta-D-glucopranosyl isothiocyanate and quantitation by reversed phase high-performance liquid chromatography system with UV detection (lambda=220 nm). A baseline separation of propranolol enantiomers was achieved on a 5-microm reverse-phase ODS column, with a mixture of methanol/water/glacial acetic acid (67:33:0.05, v/v) as mobile phase. The assay is linear from 5 to 500 microM for each enantiomer. The analytical method affords average recoveries of 99.2% and 98.8% for S-(-)- and R-(+)-propranolol, respectively. The limit of quantitation for the method is 5 microM for both S-(-)- and R-(+)-propranolol. The reproducibility of the assay is satisfactory (RSD < 10%). The method allowed study of the depletion of S-(-)- and R-(+)-propranolol in transgenic Chinese hamster CHL cell lines expressing CYP3A4, CYP2C18 and CYP2C9.
Zhou
Q
Q
Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Zhejiang 310031, Hangzhou, People's Republic of China.
Yao
T W
TW
Zeng
S
S
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D016466:CHO Cells / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D011433:Propranolol / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013237:Stereoisomerism
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D011433:Propranolol