Liquid chromatography-tandem mass spectrometry in chiral study of amlodipine biotransformation in rat hepatocytes.
A high proportion of drugs are chiral compounds used as racemic mixtures in a clinical practice. Very often only one of two enantiomers exhibits a desired pharmacological effect. Amlodipine, 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine, is a chiral calcium channel blocker, currently used as a racemate in clinical practice. Racemic mixture is used even though it is known that R- and S-amlodipine do not have the same biological activity and only S-amlodipine possesses vasodilating properties. In this work a novel reversed phase liquid chromatography (RP-LC) separation method for amlodipine and its metabolites was developed. Based on this separation chiral aspects of amlodipine biotransformation were studied by incubation of amlodipine and its two individual enantiomers with primary culture of rat hepatocytes. Structure of the metabolites was elucidated using a liquid chromatography (LC) separation with ultraviolet (UV) and mass spectrometry (MS) detection. An LC-tandem MS (MS/MS) method was used to establish fragmentation pattern of amlodipine and its metabolites. Eight metabolites presented in the highest amount were identified and semiquantified by employing an LC separation. Basically two types of metabolites were detected, reduced type--dihydropyridine metabolites and oxidized type--pyridine metabolites. Other metabolic modification included changes of functional groups, e.g., methylester hydrolysis or acetylation of amino group. The results exhibited that R-amlodipine was stereoselectively metabolized by the respective biotransformation enzymes in rat liver hepatocytes and it is also demonstrated by greater extent of R-amlodipine conversion into metabolites where the values for R-amlodipine are for the most metabolites higher than those for metabolites of S-amlodipine.
Suchanova
Bohumila
B
Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, CZ-500 05 Hradec Kralove, Czech Republic.
Sispera
Ludek
L
Wsol
Vladimir
V