Determination of glibenclamide and its two major metabolites in human serum and urine by column liquid chromatography

doi:10.1016/0378-4347(91)80084-P

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 564, Issue 1, 8 March 1991, Pages 223-233

https://doi.org/10.1016/0378-4347(91)80084-P Get rights and content

Abstract

A simple reversed-phase liquid chromatographic method for the measurement of low concentrations of glibenclamide (glyburide) and its two major metabolites, 4-trans- and 3-cis-hydroxyglibenclamide, in human serum and urine has been developed. The compounds were extracted with n-hexane—dichloromethane (1:1). The UV detection wavelength was 203 nm. The minimum detectable serum level of glibenclamide was 1 ng/ml (2 nM), and the relative standard deviation was 8.9% (n = 9). When maximum sensitivity was desired the metabolites were chromatographed separately. Metabolites in urine were measured by the same method after five-fold sample dilution. The utility of the method was tested on a healthy volunteer who ingested 3.5 mg of glibenclamide. The parent drug was present in the serum for at least 18 h, and the metabolites in the urine for at least 24 h.

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Recently, charged aerosol detection (CAD), a new kind of universal detection method, has been widely employed in the HPLC system. In the present study, four kinds of anti-diabetic drug standards, glipizide, gliclazide, glibenclamide and glimepiride were determined by ultraviolet (UV) detection, evaporative light scattering detection (ELSD) and the aforementioned CAD. The results were compared with reference to linearity, accuracy, precision and limit of detection (LOD). All of the experiments were performed on a reverse phase column with water and acetonitrile as the mobile phase. Separations were achieved under the same chromatographic conditions for each detection method. As a result, CAD generated nearly uniform responses compared with UV detection and ELSD. It showed the best accuracy and LOD among 3 detectors and had similar precision with UV detection at higher concentrations while UV detection showed a better precision at lower concentrations than did CAD or ELSD. The LOD of CAD, in fact, can be up to two times higher than that of ELSD. The UV and ELSD linearity was satisfactory at R² > 0.99, though in the case of CAD, a log–log transformation was needed. The proposed methods were also applied to the real anti-diabetic drugs and diabetes-related dietary supplements.
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In this investigation, human hepatic and placental CYP isozyme(s) responsible for the formation of each metabolite of glyburide were identified. The data revealed that CYP3A4 is responsible for the formation of three metabolites, namely, M3 (3-trans-), M4 (2-trans-cyclohexyl glyburide), and M5 (ethylene-hydroxylated glyburide), while its contribution to the formation of the previously known metabolites M1 (4-trans-) and M2b (3-cis-) [8–11], as well as M2a (4-cis-), was relatively small. Therefore, human hepatic CYP3A4 introduces a hydroxyl group into all positions of the cyclohexyl ring [6,7,20] as well as in one of the two positions [6,14] in the ethylene bridge of glyburide (M5).
One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans-(M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.
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The quantification of M1 in this assay was 10-fold lower than previously reported [26].
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Determination of glibenclamide and its two major metabolites in human serum and urine by column liquid chromatography

Abstract

J. Chromatogr.

J. Pharm. Sci.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Drugs

Drugs

Clin. Pharmacokin.