High-performance liquid chromatographic determination of ursodeoxycholic acid after solid phase extraction of blood serum and detection-oriented derivatization.
Ursodeoxycholic acid (3 alpha,7 beta-dihydroxy-5 beta-cholanoic acid, UDCA) is a therapeutically applicable bile acid widely used for the dissolution of cholesterol-rich gallstones and in the treatment of chronic liver diseases associated with cholestasis. UDCA is more hydrophilic and less toxic than another therapeutically valuable bile acid, chenodeoxycholic acid (CDCA), the 7 alpha-epimer of UDCA. Procedures for sample preparation and HPLC determination of UDCA in blood serum were developed and validated. A higher homologue of UDCA containing an additional methylene group in the side chain was synthetized and used as an internal standard (IS). Serum samples with IS were diluted with a buffer (pH=7). The bile acids and IS were captured using solid phase extraction (C18 cartridges). The carboxylic group of the analytes was derivatized using 2-bromo-2'-acetonaphthone (a detection-oriented derivatization), and reaction mixtures were analyzed (HPLC with UV 245 nm detection; a 125--4 mm column containing Lichrospher 100 C18, 5 microm; mobile phase: acetonitrile--water, 6:4 (v/v)). Following validation, this method was used for pharmacokinetic studies of UDCA in humans.
Institute of Experimental Biopharmaceutics, Joint Research Centre of PRO.MED.CS Praha a.s. and Academy of Sciences of the Czech Republic, Heyrovského 1207, CZ-500 02 Hradec Králové, Czech Republic. nobilis@uebf.cas.cz
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Nobilis
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Pour
J
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Kunes
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Kopecký
J
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Kvĕtina
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Svoboda
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Sládková
J
J
Vortel
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D010865:Pilot Projects; D012015:Reference Standards; D012016:Reference Values; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D014580:Ursodeoxycholic Acid / Q000097:blood* / Q000493:pharmacokinetics
D014580:Ursodeoxycholic Acid