Evaluation of a versatile reversed-phase high-performance liquid chromatographic system using cethexonium bromide as ion-pairing reagent for the analysis of glucuronic acid conjugates.
An ion-pair high-performance liquid chromatographic technique has been developed to reduce the elution times of both glucuronic acid conjugates and their free aglycones. The chromatographic system combines the use of a reversed-phase column (LiChrospher CH-18; 5 microns) and a mobile phase of methanol (70-80%)-0.01 M phosphate buffer (pH 6.0) containing 2.5 mM cethexonium bromide as counter-ion at a flow-rate of 1 ml min-1. The hydrophobicity of this quaternary ammonium ion-pairing reagent and the high content of the organic modifier in the mobile phase provide close and short elution times for a wide structural variety of compounds (i.e. alcohols, phenols, steroids, carboxylic acids) and their conjugates with glucuronic acid (capacity factors lower than 7.5), without compromising the selectivity with respect to endogenous compounds of the microsomal incubation medium and urine. Advantages of cethexonium bromide over conventional tetrabutylammonium salts are clearly demonstrated, and the described system was applied to the simultaneous quantitation of clofibric acid and its acylglucuronide in human urine and validated for a pharmacogenetics purpose.
Laboratoire de Chimie Analytique, URA CNRS 597, Faculté des Scieces Pharmaceutiques et Biologiques, Nancy, France.
H F
HF
Liu
P
P
Leroy
A
A
Nicolas
J
J
Magdalou
G
G
Siest
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002995:Clofibric Acid / Q000378:metabolism / Q000652:urine; D005965:Glucuronates / Q000378:metabolism* / Q000652:urine; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D000644:Quaternary Ammonium Compounds; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet
D005965:Glucuronates; D007202:Indicators and Reagents; D000644:Quaternary Ammonium Compounds; C013871:cethexonium; D002995:Clofibric Acid