Determination of coumarin, 7-hydroxycoumarin, 7-hydroxycoumarin-glucuronide, and 3-hydroxycoumarin by high-performance liquid chromatography

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 mu m; Merck, Darmstadt, Germany). The mobile phase consisted of methanol/water/tetrahydrofuran/acetic acid (45:40:10:5). 7-Hydroxycoumarin-glucuronide in plasma was enzymatically converted to 7-hydroxycoumarin by incubation with a mixture of B-glucuronidase and sulphatase in citrate-hydrochloric acid-buffer. Analysis of 7-hydroxycoumarin and its glucuronide in 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/l; (plasma). 250 - 10,000 nmol/l (urine), 7-hydroxycoumarin-glucuronide: 1000 - 50,000 nmol/l (plasma). 250 - 10,000 nmol/l (urine) and 3-hydroxycoumarin: 500 - 25,000 nmol/l); with a mean accuracy of 4.6%. The lower limits of detection ranged from 60 nmol/l (7-hydroxy-metabolites in plasma) up to 300 nmol/l (coumarin). The interday precision data were determined using spiked plasma samples (range 4000-8500 nmol/l); with a mean coefficient of variation of 4.3%. The assay was used for the determination of coumarin and its 7- or 3-hydroxy-metabolites in healthy volunteers; examples of chromatograms and concentration vs. time curves are shown.