Analysis of clomiphene isomers in human plasma and detection of metabolites using reversed-phase chromatography and fluorescence detection.
A method is described for the quantitative clinical analysis of plasma concentrations of the E and Z isomers of clomiphene, which is used in the induction of ovulation. The isomers of clomiphene, in addition to metabolites, are extracted from plasma with tert-butyl methyl ether (MTB). The MTB layer is dried, reconstituted and an aliquot subjected to chromatography. The drug and metabolites are separated by reversed-phase high-performance liquid chromatography. The eluent is fed into a knitted or braided, cylindrical reaction coil made of Teflon, into which is inserted a low-energy mercury lamp. This results in a photoinduced stilbene-to-phenanthrene oxidation yielding highly fluorescent analytes; this provides excellent sensitivity for the quantitation of the intact drug isomers and the detection of presently uncharacterized metabolites. Use of the reversed-phase chromatographic mode results in elution of the polar metabolites prior to the intact drug isomers. A combination of reversed-phase chromatography and an in-line post-column reaction coil results in a sensitive method that is more reliable and rapid than those previously reported and is applicable to the routine analysis of clinical samples. The method has been applied to individual isomers of clomiphene in plasma at concentrations of 0.06-600 ng/ml.
Baustian
C L
CL
Pharmaceutical Chemistry Department, University of Kansas, Lawrence, KS 66045, USA.
Mikkelson
T J
TJ