Determination of Coumarin, 7-Hydroxy-Coumarin, 7-Hydroxycoumarin-Glucuronide, and 3-Hydroxycoumarin by High-Performance Liquid Chromatography

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Abstract
A selective and sensitive method for the determination of coumarin and its main metabolites 7-hydroxycoumarin, 7-hydroxycoumarin-glucuronide and 3-hydroxycoumarin in human plasma and/or urine is described. Coumarin and 7-hydroxycoumarin were extracted from plasma with n-hexane/chloroform and with chloroform. After evaporation under vacuum, the residue was redissolved in methanol/water and injected onto the HPLC column (LiChroCART 250-4, RP 8e 5 μm; Merck, Darmstadt, Germany). The mobile phase consisted of methanol/water/tetrahydrofuran/acetic acid (45:A0:10:5). 7-Hydroxycoumarin-qlucuronide in plasma was enzymatically converted to 7-hydroxycoumarin by incubation with a mixture of β-glucuronidase and sulphatase in citrate-hydrochloric acid-buffer. Analysis of 7-hydroxycoumarin and its glucuronide in Correspondence Address: Dr. med. Sheida Sharifi. University of Gottingen, Robert-Koch-Strasse 40, D-3400 Göttingen, Germany urine was performed without extraction using a linear gradient system of methanol/water/tetrahydrofuran/acetic acid as mobile phase. 3-Hydroxycoumarin was determined in urine by extraction and HPLC separation procedures as described for coumarin or 7-hydroxycoumarin in plasma samples. The assay was linear (mean: r=0.9993) in the concentration ranges studied; coumarin: 500 25,000 nmol/1, 7-hydroxycoumarin: 500-25.000 nmol/1; (plasma), 250 - 10,000 nmol/1 (urine), 7-hydroxycoumarin-glucuronide: 1000 -50,000 nmol/1 (plasma), 250 - 10,000 nmol/1 (urine) and 3-hydroxycoumarin: 500 - 25,000 nmol/1); with a mean accuracy of 4.6%. The lower limits of detection ranged from 60 nmol/1 (7-hydroxy-metabolites in plasma) up to 300 nmol/1 (coumarin). The interday precision data were determined using spiked plasma samples (range 4000-8500 nmol/1); with a mean coefficient of variation of 4.3% The assay was used for the determination of coumarin and its 7- or 3-hydroxy-metaboiites in healthy volunteers; examples of chromatograms and concentration vs. time curves are shown.

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