Detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography-mass spectrometry after extractive methylation.

A gas chromatography-mass spectrometry (GC-MS) procedure was developed for the detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 291, 294, 295, 309, 313, 322, 324, 336, 343 and 354, the possible presence of 4-hydroxycoumarin anticoagulants and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of phenprocoumon and warfarin in human urine samples. In absence of human urine, acenocoumarol, coumachlor, coumatetrayl, pyranocoumarin (cyclocumarol) could be detected only in rat urine.

Identification of phenprocoumon metabolites in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

The oral anticoagulant phenprocoumon is eliminated in urine mainly as the glucuronide conjugate to an extent of 20% of the dose. The urine from patients undergoing phenprocoumon therapy was investigated and the following metabolites were isolated and identified: 7-hydroxyphenprocoumon as the main component, and 4'-hydroxyphenprocoumon and 6-hydroxyphenprocoumon as conjugates. They were characterized by high-performance liquid chromatography and, after methylation, by gas chromatography-mass spectrometry.

The excretion of coumarin and hydroxycoumarins by the avian kidney in vivo.

The mechanics involved in the renal excretion of C14-labeled coumarin (C), 7-hydroxycoumarin (7HC) and 4-hydroxycoumarin (4HC) were studied in unanesthetized chickens by use of the Sperber technique. Analysis of urine collected during unilateral portal infusion of C, 4HC or 7HC indicated that the chicken excreted these coumarins almost entirely in conjugated form. Both 7HC and 4HC appeared in the urine only as the corresponding glucuronide. During C infusion, 84% of excreted radioactivity was identified as 7HC-glucuronide with only 8% appearing as a conjugate of open-ring metabolite o-hydroxyphenylacetic acid (HPAA). This demonstrated that, unlike other commonly-used laboratory animals, the chicken excretes C in a manner similar to man and suggests that this species is the most appropriate model for C pharmacodynamic studies applicable to humans. Comparative analysis of urines from the infused and control side kidneys indicated that neither C or 4HC undergoes net tubular secretion. In contrast 7HC was excreted by an active process which was significantly inhibited by simultaneous probenecid infusion. In addition it was demonstrated that the renal tubule cells conjugate 7HC as it crosses from blood to lumen suggesting that the kidney may contribute to the metabolism of C.

Determination of the rodenticide difenacoum in biological materials by high-pressure liquid chromatography with confirmation of identity by mass spectrometry.

A method for determining difenacoum in liver, plasma, urine and feedingstuffs by high-pressure liquid chromatography is described. Samples are cleaned up by molecular exclusion chromatography on porous glass. In some cases this also serves for determination; if not, the separated difenacoum is determined on an adsorption column. Identity is confirmed by chemical ionisation mass spectrometry. Recoveries at levels of 0.025-5 ppm from plasma were 101-113% by exclusion chromatography alone and 93-101% after adsorption chromatography. Recoveries from liver after both chromatographic steps were 62-86%. Reasons for the lower recoveries from liver are suggested.