Determination of phenprocoumon, warfarin and their monohydroxylated metabolites in human plasma and urine by liquid chromatography-mass spectrometry after solid-phase extraction.

A high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the quantification of phenprocoumon, warfarin, and their known monohydroxylated metabolites in human plasma and urine was developed using a simple, selective solid-phase extraction scheme. Chromatographic separation was achieved on a reversed-phase Luna C18 column and step gradient elution resulted in a total run time of about 13 min. Limits of quantification (LOQ) were < or = 40 nM for the parent compounds and < or = 25 nM for the metabolites and the limit of detection (LOD) was < or = 2.5 nM for all analytes. Average recovery was 84% (+/- 3.7) and 74% (+/- 13.2) in plasma and urine, respectively. Intra- and inter-day coefficients of variation were < or = 8.6 and < or = 10.6% in plasma and urine, respectively. The method was successfully applied to the analysis of phenprocoumon samples from four healthy volunteers and should prove useful for future comparative studies of warfarin and phenprocoumon pharmacokinetics.

Effects of CYP2C9 polymorphisms on the pharmacokinetics of R- and S-phenprocoumon in healthy volunteers.

CYP2C9 catalyses the biotransformation of the oral anticoagulants S-warfarin and R- and S-acenocoumarol. According to data obtained in vitro, phenprocoumon is also metabolized by CYP2C9 but the impact of the CYP2C9 polymorphism on phenprocoumon pharmacokinetics has not been studied. Twenty-six healthy heterozygous and homozygous carriers of the CYP2C9 alleles *1 (wild-type), *2 (Arg144Cys), and *3 (Ile359Leu) received a single oral dose of 12 mg of racemic phenprocoumon. Plasma and 12 h urine concentrations of both enantiomers and their monohydroxylated metabolites were measured by high-performance liquid chromatography with mass spectrometry detection. No significant effect of the CYP2C9 variants *2 and *3 on R-phenprocoumon pharmacokinetic parameters was detected, but S-phenprocoumon clearance tended to decrease with increasing number of CYP2C9*2 and *3 alleles. The ratios of S- to R-phenprocoumon plasma clearances were higher with a median of 0.95 in carriers of *1/*1 versus 0.65 in *3/*3 (P < 0.001 for trend). Plasma and urine concentrations of 4'-, 6- and 7-hydroxyphenprocoumon were significantly lower in homozygous carriers of the CYP2C9*2 and *3 variants compared to CYP2C9*1/*1. Carriers of CYP2C9*3/*3 had a median AUC of (R,S) 7-OH-phenprocoumon of only approximately 25% compared to the wild-type genotype. The AUC of (R,S) 6-OH-phenprocoumon was only approximately 50% in CYP2C9*3/*3 compared to the homozygous wild-type genotype. In conclusion, carriers of CYP2C9*2 and *3 alleles had a lower metabolic capacity regarding phenprocoumon hydroxylation than those with CYP2C9*1/*1. However, regarding phenprocoumon hydroxylation CYP2C9 genotypes had only marginal effects on S- and R-phenprocoumon total clearance in healthy volunteers.

Enantioseparation of the anticoagulant drug phenprocoumon in capillary electrophoresis with UV and laser-induced fluorescence detection and application of the method to urine samples.

The enantioseparation of phenprocoumon (PhC) in capillary electrophoresis (CE) has been studied using various cyclodextrins (CDs) such as native alpha, beta and gamma-CD and several neutral and randomly, as well as selectively substituted charged CD derivatives. Reversal of the enantiomer migration order was observed when using heptakis(2,3,6-tri-O-methyl (TM)-beta-CD as a chiral selector compared to all other CDs used. The detection of PhC was performed using either UV or laser-induced fluorescence (LIF) detection. The limit of detection (LOD) observed with LIF detection was ca. 20 times lower compared to UV. The method has been applied to the analysis of urine samples of the patient under treatment with PhC in combination with other drugs such as ramipril, hydrochlorothiazide, and nifedipine.

Determination of phenprocoumon in plasma and urine using at-line solid-phase extraction-capillary electrophoresis.

The use of capillary electrophoresis (CE) for the analysis of biological samples is rather problematic because of the large number of interferences present in the matrix. One of the possibilities to solve such problems is to couple solid-phase extraction (SPE) at-line with CE, a technique developed in our laboratory. In this study at-line SPE-CE is performed for the determination of the anticoagulant phenprocoumon in biological fluids. Plasma samples are injected after the addition of 1 vol.% of formic acid to release the drug from binding proteins, while urine samples can be directly injected. The procedure is linear between 0.2 and 30 microg ml(-1) with a correlation coefficient, r2, of 0.9996. The detection limit in plasma is 0.1 microg ml(-1), which is fully adequate in view of the concentrations, that have to be dealt with in practice. The phenprocoumon concentration in a plasma sample of a patient treated with the anticoagulant was 3.8 microg ml(-1).

Determination of the coumarin anticoagulant phenprocoumon and metabolites in human plasma, urine and breast milk by high-performance liquid chromatography after solid-phase extraction.

The coumarin anticoagulant phenprocoumon (PH) and metabolites (6-, 7- and 4'-hydroxyphenprocoumon) were analysed in plasma and urine samples from anticoagulated patients using solid-phase extraction and high-performance liquid chromatography with reversed-phase columns and ultraviolet and fluorescence detection; a simpler handling of samples, higher selectivity, precision, accuracy and analytical recovery were obtained compared to analysis using liquid-liquid extraction. Similarly, a method for the analysis of PH in human breast milk was developed to assess the passage of anticoagulants into breast milk in anticoagulated lactating women.

Analysis of phenprocoumon and its hydroxylated and conjugated metabolites in human urine by high-performance liquid chromatography after solid-phase extraction.

The anticoagulant phenprocoumon is mainly metabolized in humans to hydroxylated metabolites and their glucuronides. A method is described for the determination of phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon, 7-hydroxyphenprocoumon, and their glucuronide and sulphate conjugates in human urine. Reversed-phase high-performance liquid chromatography is performed after selective extraction with disposable quaternary amine columns of untreated, and beta-glucuronidase- or sulphatase-treated urine samples. Urinary excretion data are presented for total, glucuronidated, sulphated and free phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon and 7-hydroxyphenprocoumon in twelve patients after an average daily dosage of 1.3-4.2 mg phenprocoumon.

The effects of frusemide and probenecid on the pharmacokinetics of phenprocoumon.

We have studied the pharmacokinetics of phenprocoumon with and without co-administration of frusemide and probenecid in two groups of 17 healthy volunteers. Frusemide 40 mg b.i.d. for 7 days did not interact with phenprocoumon to a significant extent. Probenecid 500 mg q.i.d. for 7 days significantly accelerated the overall elimination of phenprocoumon, as indicated by a decrease in AUC from 295 to 157 micrograms.h.ml-1, and a reduction in the fraction of the dose excreted by the kidneys. The data are consistent with inhibition of the glucuronidation of phenprocoumon by probenecid. Its accelerated elimination may be a consequence of the increased formation of hydroxylated metabolites.

Separation of the enantiomers of phenprocoumon and warfarin by high-performance liquid chromatography using a chiral stationary phase. Determination of the enantiomeric ratio of phenprocoumon in human plasma and urine.

The enantiomers of the chiral coumarin-type anticoagulants phenprocoumon, warfarin and p-chlorophenprocoumon were separated by high-performance liquid chromatography on a chiral stationary phase (Nucleosil-Chiral 2) and normal-phase conditions. Chromatographic peak identification was performed with authentic reference compounds of the enantiomers and on-line UV spectra comparison. This method was applied to the determination of the enantiomeric ratio of phenprocoumon in plasma and urine extracts from patients under racemic drug therapy. The limit of detection (50 and 80 ng/ml) and precision (less than 5%) of the method are adequate for pharmacokinetic and enantioselective disposition studies, respectively, of phenprocoumon. No racemization was detected during the extraction procedures.

Biliary excretion of phenprocoumon and metabolites.

To evaluate phenprocoumon elimination its possible biliary excretion was evaluated in addition to the known pathway of renal elimination. Bile samples were obtained during diagnostic endoscopy in patients receiving chronic phenprocoumon therapy and were analyzed for phenprocoumon and its metabolites by HPLC and GC-MS. The following substances were detected, mainly in conjugated form: unchanged phenprocoumon and the metabolites 7-hydroxy-, 4'-hydroxy-, and 6-hydroxy-phenprocoumon. The data provide direct evidence of the biliary elimination of unchanged phenprocoumon and its metabolites in humans.

Metabolic fate of phenprocoumon in humans.

Samples of urine and feces were collected daily from a normal human volunteer who had received a dose of pseudoracemic phenprocoumon [an equimolar mixture of (R)-[12C]- and (S)-[2-13C]phenprocoumon] containing a tracer dose of 10 microCi of [14C]phenprocoumon and analyzed by TLC, HPLC, and GC-MS. After 25 days, 96% of the radiolabeled material was recovered (62.8% in urine and 33.3% in feces). By isotopic dilution and comparison to the Rf values, retention times, and mass fragmentograms of synthetic standards, the metabolites of the drug were identified as the 4'-, 6-, and 7-hydroxy analogues of phenprocoumon. Virtually all of the recovered radioactivity could be accounted for by the parent drug (approximately 40%) and the three metabolites (approximately 60%). The formation of both 4'-(8.1% of administered dose) and 7- (33.4% of administered dose) hydroxyphenprocoumon was highly stereoselective, giving S/R ratios of 2.86 and 1.69, respectively. The formation of 6- (15.5% of administered dose) hydroxyphenprocoumon showed little stereoselectivity (S/R ratio equal to 0.85). The urinary excretion pattern was also confirmed in four additional healthy male subjects who received a single oral dose of pseudoracemic phenprocoumon and whose urine was analyzed by GC-MS. All the drug-related materials (both hydroxylated metabolites and parent compound) that were excreted into the urine were extensively conjugated.

The effect of wheat bran on the pharmacokinetics of phenprocoumon in normal volunteers.

Observations of a variable anticoagulatory response in patients being treated by phenprocoumon and wheat bran resulted in a study of seven healthy volunteers. Up to 168 h after the administration of phenprocoumon, both in the fasting state and following the ingestion of 35 g wheat bran in a crossover design, blood samples were taken and urine was collected. The usual pharmacokinetic parameters were calculated from the concentrations measured. Following the ingestion of wheat bran, a decreased absorption rate for phenprocoumon but no decrease in overall bioavailability was observed. In addition, a decrease in total body clearance of phenprocoumon and an increase in the free plasma fraction of phenprocoumon, resulting in decreased free drug clearance, was seen after wheat bran administration. No differences were observed in the urinary excretion of either phenprocoumon or its metabolite. These findings cannot be completely explained by our present knowledge and need further investigation.

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.

Interaction between phenprocoumon and phytomenadion: a pharmacokinetic investigation in healthy volunteers.

In 23 female and male healthy volunteers the pharmacokinetic behaviour of [3H]phenprocoumon and [3H]phytomenadion was investigated. The time-dependent changes in plasma radioactivity and the amount of urinary excretion were measured. Under simultaneous administration of phytomenadion (PHY) in daily doses of 10 mg, the elimination of phenprocoumon (PPC) was not altered (t0.5 beta 141.4 h, CUE50 20.4% of administered dose) as compared with data of previous investigations, while its anticoagulant effect was inhibited. PHY in doses of 10 mg was absorbed quickly and eliminated with a mean half-life of 60.6 h at a total body clearance (Cltot) of 9.3 ml/min and a renal clearance (Clren) of 1.9 ml/min. Under the co-administration of PPC the vitamin was eliminated with a half-life time of 23.1 h (Cltot 16.7 ml/min) and the amount of excreted radioactivity was enhanced (26.3% of administered dose; Clren 4.5 ml/min) as compared with the data of PHY without co-medication. From the experiments it is concluded that (1) PHY inhibits the action of PPC without influencing its pharmacokinetics, and (2) that PPC distinctly changes the pharmacokinetics of PHY and diminishes its pharmacodynamic action as measured by changes in the prothrombin complex activity.

The effect of liver cirrhosis on the pharmacokinetics of phenprocoumon.

Phenprocoumon was given orally to 9 patients with biopsy proven liver cirrhosis (dose range 0.12-0.25 mg/kg) and to 7 healthy volunteers (0.23 mg/kg). Concentrations of phenprocoumon were determined using HPLC in plasma and urine samples obtained for 6-7 days after drug administration. The binding of [3H]-phenprocoumon in plasma from all subjects was determined by equilibrium dialysis. Antipyrine plasma concentrations were determined spectrophotometrically following oral administration of antipyrine (1200 mg). The total body clearance of phenprocoumon was higher in the cirrhotic patients (1.64 +/- 0.16 ml/h/kg mean +/- SEM) than in the healthy volunteers (0.90 +/- 0.07 ml/h/kg), however the free drug clearance was not significantly different in the patients (144 +/- 14 ml/h/kg) compared with normal (113 +/- 11 ml/h/kg). In contrast the clearance of antipyrine was much reduced in the cirrhotic group (17.5 +/- 2.9 ml/h/kg) compared with normal (35.6 +/- 3.9 ml/h/kg). The metabolic clearance of phenprocoumon via glucuronidation, is relatively unaffected during cirrhosis compared with antipyrine clearance via oxidation.

Determination of the anticoagulant phenprocoumon in human plasma and urine by high-performance liquid chromatography.

The determination of the anticoagulant phenprocoumon in plasma, after acidification and extraction with 1,2-dichloroethane was effected through isocratic high-performance liquid chromatography; a C18 reversed-phase column was used as stationary phase using aqueous acetonitrile as eluent and UV detection at 313 nm; p-chlorophenprocoumon was used as internal standard. A high proportion of phenprocoumon in urine is eliminated as the glucuronide and must be hydrolyzed enzymatically before extraction; the same column and detector as for plasma were used, but with gradient elution. The method was used in the range 0.1-5 mg/1, the sensitivity was 0.1 mg/1 for plasma and 0.02 mg/1 for urine, the precision was in the range 3-5% and the absence of interference due to other anticoagulants, drugs or endogenous compounds allows the specific determination of phenprocoumon in plasma and urine from patients and volunteers in clinical relevant cases, drug interaction, compliance, toxicological and pharmacokinetic studies.

[Study on possible interactions between tiaprofenic acid and phenprocoumon (author's transl)]. / Untersuchung zur Frage einer Interaktion von Tiaprofensüre and Phenprocoumon.

The study deals with the clinical and pharmacokinetic interaction of tiaprofenic acid (Surgam) and phenprocoumon. 6 healthy volunteers received phenprocoumon p.o. with a view to decreasing Quick's value. After the first administration a pharmacokinetic profile of phenprocoumon was taken. As Quick's value dropped to 37% at the 4th day of the study, the subjects received tiaprofenic acid additionally. Afterwards the pharmacokinetic profiles of phenprocoumon and tiaprofenic acid were taken again. The study led to the following results: 1. The clotting parameters are not changed on synchronous administration of tiaprofenic acid and phenprocoumon. 2. The pharmacokinetic profile of tiaprofenic acid is not changed by pretreatment with phenprocoumon. 3. The parmacokinetic profile of phenprocoumon is not changed by tiaprofenic acid.