Nanofluid of magnetic-activated charcoal and hydrophobic deep eutectic solvent: Application in dispersive magnetic solid-phase extraction for the determination and preconcentration of warfarin in biological samples by high-performance liquid chromatography.

In this study, for the first time, nanofluid of magnetic-activated charcoal and hydrophobic deep eutectic solvent (AC@Fe3 O4 -DES) based dispersive magnetic solid-phase extraction was successfully applied for the determination and preconcentration of warfarin in plasma and urine samples. The hydrophobic DES was prepared by mixing tetramethylammonium chloride (as hydrogen bond acceptor) and thymol (as hydrogen bond donor) and acted simultaneously as both carrier and stabilizer for magnetic nanoparticles. In this method, the nanofluid as a new extraction solvent was rapidly injected into the aqueous sample, which led to improvement of the mass transfer of the analytes into the sorbent and reduction of the extraction time. In the screening step, the fractional factorial design was applied for selecting some important parameters which significantly affected the extraction procedure. The effective parameters were then optimized by Box-Behnken design. Under the optimal conditions, the limits of detection were in the range of 0.3-1.6 ng/ml. A good linear range was observed in the range of 1.0-500.0 ng/ml for water and 5.0-500.0 ng/ml for urine and plasma. The intra- and inter-day relative standard deviations were 2.7-3.2 and 1.9-4.5% for five replications, respectively. Based on the results, the proposed method was successfully applied for the determination of warfarin in biological samples, using high-performance liquid chromatography.

Novel revelation of warfarin resistant mechanism in roof rats (Rattus rattus) using pharmacokinetic/pharmacodynamic analysis.

Roof rats (Rattus rattus) live mainly in human habitats. Heavy use of rodenticides, such as warfarin, has led to the development of drug resistance, making pest control difficult. There have been many reports regarding mutations of vitamin K epoxide reductase (VKOR), the target enzyme of warfarin, in resistant rats. However, it has been suggested there are other mechanisms of warfarin resistance. To confirm these possibilities, closed colonies of warfarin-susceptible roof rats (S) and resistant rats from Tokyo (R) were established, and the pharmacokinetics/pharmacodynamics of warfarin in rats from both colonies was investigated. R rats had low levels of warfarin in serum and high clearance activity. These rats can rapidly metabolize warfarin by hydroxylation. The levels of accumulation in the organs were lower than those of S rats. R rats administered warfarin showed high expression levels of CYP2B, 2C, and 3A, which play roles in warfarin hydroxylation, and may explain the high clearance ability of R rats. The mechanism of warfarin resistance in roof rats from Tokyo involved not only mutation of VKOR but also high clearance ability due to high levels of CYP2B, 2C and 3A expression possibly induced by warfarin.

Synthesis of molecularly imprinted polypyrrole as an adsorbent for solid-phase extraction of warfarin from human plasma and urine.

The aim of this work was to develop a method for the clean-up and preconcentration of warfarin from biological sample employing a new molecularly imprinted polymer (MIP) as a selective adsorbent for solid-phase extraction (SPE). This MIP was synthesized using warfarin as a template, pyrrole as a functional monomer and vinyl triethoxysilane as a cross-linker. The molar ratio of 1:4:20 (template-functional monomer-cross-linker) showed the best results. Nonimprinted polymers (NIPs) were prepared and treated with the same method, but in the absence of warfarin. The prepared polymer was characterized by Fourier transmission infrared spectrometry and scanning electron microscopy. An adsorption process (SPE) for the removal of warfarin using the fabricated MIPs and NIPs was evaluated under various conditions. Effective parameters on warfarin extraction, for example, type and volume of elution solvent, pH of sample solution, breakthrough volume and maximum loading capacity, were studied. The limits of detection were in the range of 0.0035-0.0050 µg mL(-1). Linearity of the method was determined in the range of 0.0165-10.0000 µg mL(-1) for plasma and 0.0115-10.0000 µg mL(-1) for urine with coefficients of determination (R(2)) ranging from 0.9975 to 0.9985. The recoveries for plasma and urine samples were >95%.

Clinical factors associated with negative urinary antigen tests implemented for the diagnosis of community-acquired pneumococcal pneumonia in adult patients.

OBJECTIVE: This study investigated clinical factors associated with negative urinary antigen tests (UAT) implemented for the diagnosis of pneumococcal community-acquired pneumonia (CAP) in adult patients. SUBJECTS AND METHODS: We reviewed the medical records of 755 adult patients who completed the UAT in our hospital between 2009 and 2012. Of these, we evaluated 63 patients with bacteriologically confirmed definite pneumococcal CAP (33 were UAT-positive, and 30 were UAT-negative). RESULTS: There was no significant difference between the UAT-positive and the UAT-negative patients regarding age, dehydration, respiratory failure, orientation, blood pressure (ADROP) score (the CAP severity score proposed by the Japanese Respiratory Society), gender, white blood cell counts, liver/kidney function tests, or urinalysis. However, serum C-reactive protein (CRP) concentrations were 31% lower in the UAT-negative patients than in the UAT-positive patients (p = 0.02). Furthermore, the prothrombin time-international normalized ratio was 50% higher in the UAT-negative patients than in the UAT-positive patients, although the difference did not reach statistical significance (p = 0.06). The prevalence of comorbidities was similar in both UAT-positive and UAT-negative patients. However, warfarin had been prescribed in 8 (27%) of the UAT-negative patients compared to only 1 (3%) of the UAT-positive patients (odds ratio = 11.6; p = 0.01). CONCLUSIONS: These results suggested that low serum CRP concentrations and the use of warfarin increased the possibility with which false-negative UAT results occurred in these patients with pneumococcal CAP.

Hydrophobic borneol-based natural deep eutectic solvents as a green extraction media for air-assisted liquid-liquid micro-extraction of warfarin in biological samples.

In the present study, a new generation of water-immiscible natural deep eutectic solvents (DESs) was synthesized using borneol as a hydrogen-bonding acceptor and decanoic acid, oleic acid, and thymol as a hydrogen-bonding donor in different molar ratios. These green hydrophobic solvents which are chemically stable in aqueous solutions were used as extraction solvents for isolation and pre-concentration of warfarin in biological samples. In this method, fine droplets of DESs were dispersed into the sample solution by using the air-assisted liquid-liquid micro-extraction method to accelerate the cloudy emulsion system formation and increase the mass transfer of the analyte to the DES-rich phase. The borneol based deep eutectic solvent is a worthy generation of the extraction solvents in the ALLME method due to low-cost and less toxicity. A Plackett-Burman design was utilized for screening the experimental parameters. The effective parameters were then optimized by Box-Behnken design (BBD). Optimized extraction conditions were pH of sample solution of 3.9, number of aspiration/dispersion cycles of 15, the volume of DES of 60 µL, and rate and time of centrifuge of 6000 rpm and 10 min, respectively. Under the optimized conditions, the developed NADES-ALLME method exhibited a wide linear range of 5-500 µg L - 1 for plasma and urine samples with satisfactory recoveries above 88.80%. Limit of detections (LODs) and Limit of quantifications (LOQs) of warfarin were in the ranges of 0.5-2.7 and 1.65-8.91, respectively. The enrichment factors were obtained in the range of 148-164 and precisions were lower than 5.87%. Finally, the proposed method was successfully employed for the analysis of warfarin in human urine and plasma samples.

Assessing cytochrome P450 and UDP-glucuronosyltransferase contributions to warfarin metabolism in humans.

As a step toward exploring a targeted metabolomics approach to personalized warfarin (Coumadin) therapy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying specific enantiomeric (R and S) contributions of warfarin (WAR) and the corresponding hydroxywarfarins (OH-WAR) and glucuronides (-GLUC) generated by cytochrome P450s (CYP) and UDP-glucuronosyltransferases (UGTs), respectively. Evaluation of quality control samples and three commercially available human samples showed that our analytical approach has the ability to measure 24 unique WAR metabolites in human urine. Evaluation of the human data also provides new insights for evaluating WAR toxicity and begins characterizing important UGT metabolic pathways responsible for WAR detoxification. Data revealed the significance of specific metabolites among patients and the corresponding enzymatic capacity to generate these compounds, including the first report of direct WAR glucuronidation. On the basis of total OH-WAR levels, (S)-7-OH-WAR was the predominant metabolite indicating the significance of CYP2C9 in WAR metabolism, although other CYP2C enzymes also contributed to clearance of this isomer. (R)-WAR hydroxylation to OH-WARs was more diverse among the patients as reflected in varying contributions of CYP1A2 and multiple CYP2C enzymes. There was wide variation in the glucuronidation of WAR and the OH-WARs with respect to the compounds and patients. 6- and 7-OH-WAR were primarily (>70%) excreted as glucuronides unlike 4'-OH-WAR and 8-OH-WAR. For all patients, UGT1A1 is likely responsible for 6-O-GLUC production, although UGT1A10 may also contribute in one patient. 7-O-GLUC levels reflected contributions from potentially five different UGT1A enzymes. In all cases, WAR, 4'-OH-WAR, 8-OH-WAR, and the corresponding glucuronides were minor metabolites with respect to the others. Taken together, these data suggest that both P450 and UGT reactions contribute to the generation of excretable products in human urine, thereby generating complex metabolic networks.

[Patient with warfarin poisoning caused by coumarin rodenticide].

We report a patient who attempted suicide by taking warfarin as a coumarin rodenticide twice, and showed oral bleeding and an abnormally prolonged bleeding tendency after 10 days. He was conscious. His body temperature was 36.6 degrees C, the respiratory rate was 23/min, and blood pressure was 142/87 mmHg. A bite wound showing continuous bleeding was observed on the right side of the oral cavity. CT of the head and upper gastrointestinal endoscopy showed no intracranial and gastrointestinal bleeding,respectively. The PT and INR were extremely high, and complete blood count revealed anemia. Blood chemistry showed hypoproteinemia. Occult blood was strongly positive. Routine urine examination and electrocardiography showed neither abnormal nor characteristic findings. In drug/ poison screening tests, GC/MS analysis revealed warfarin artifacts in serum and urine samples. Warfarin was isolated from each sample, converted to a TMS derivative, and quantified by SIM analysis of GC/MS. During the clinical course, KaytwoN (vitamin K) was intravenously administered at a dose of 20 mg since the INR at the first consultation was 20.89. After admission to the ICU, 20 mg Kaywan (vitamin K) was orally administered, and the INR after 5 hours improved to 2.32. Kaywan administration (20 mg/day) was initiated, and he improved and was discharged 5 days after admission. The definite cause could be clarified by drug/poison analysis, and effective treatment could be performed.

Simultaneous electrochemical sensing of warfarin and maycophenolic acid in biological samples.

In this study, for the first time a rapid, selective and highly sensitive method was developed for simultaneous determination of warfarin and mycophenolic acid using carbon paste electrode modified by ß-cyclodextrin/multi-walled carbon nanotubes/cobalt oxide nanoparticles (ß-CD/MWCNTs/Co3O4NPs/CPE). The oxidation peaks of desired drugs was separated enough using the constructed electrode. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were utilized for study the electrochemical response of the fabricated electrode and its surface modification was investigated by electrochemical impedance spectroscopy (EIS). Under optimal conditions, the adsorptive stripping voltammetric responses were linear in the concentration ranges 0.05-150 µM and 0.5-200 µM for WAR and MPA, respectively. The correlation coefficients were greater than 0.99. The limits of detection for WAR and MPA were 0.02 and 0.03 µM. The fabricated electrode was applied for the simultaneous determination of WAR and MPA in urine and human serum samples with satisfactory results.

Warfarin metabolism in man: identification of metabolites in urine.

After administration of the coumarin anticoagulant racemic warfarin to normal humans, seven fluorescent compounds were chromatographically separated from extracts of their urine. Four of these were identified using mass spectrometry, thin-layer chromatography, and ultraviolet absorption spectroscopy. One metabolic pathway, reduction of the acetonyl side chain of warfarin, resulted in the formation of a second asymmetric carbon atom, and two diastereoisomer alcohols were identified. These warfarin alcohols are structurally similar to pharmacologically active coumarin derivatives. They have not been reported in animal studies. In addition, 6- and 7-hydroxywarfarin were identified. These are the first studies to document the metabolic fate of warfarin in the normal human.

Warfarin. Stereochemical aspects of its metabolism and the interaction with phenylbutazone.

An examination of the metabolic fate of the R and the S isomers of warfarin revealed that the two isomers were metabolized by different routes. R warfarin was oxidized to 6-hydroxywarfarin and was reduced to the (R,S) warfarin alcohol. In contrast, S warfarin was oxidized to 7-hydroxywarfarin and was reduced to the (S,S) warfarin alcohol. S warfarin was also oxidized to 6-hydroxywarfarin. These observations suggested that interactions between warfarin and other drugs might be manifest stereo-specifically, i.e., have a different effect on the isomers of warfarin, so a series of experiments were conducted with each isomer of warfarin, before and after phenylbutazone. The plasma clearance of S warfarin was slowed from 3.1 to 1.1% per h in one subject and from 2.3 to 1.6% per h in another. In contrast, the clearance of R warfarin was increased from 1.5 to 3.0% per h and from 0.9 to 1.6% per h in two subjects after phenylbutazone. The rate of clearance of racemic warfarin was unaffected by phenylbutazone; the depression of the rate of clearance of the S isomer masked the stimulation of the clearance of the R isomer. Since S warfarin is five times more potent an anticoagulant than R warfarin, it is concluded that inhibition of the metabolism of S warfarin provides one mechanism for the augmented anticoagulation which follows phenylbutazone.

Simultaneous Determination of Warfarin and Aspirin Contents in Biological Fluids Using Excitation-Emission Matrix Fluorescence Coupled with a Second-order Calibration Method.

In the present work, a practical method that combines excitation-emission matrix fluorescence with a second-order calibration method based on an alternating trilinear decomposition (ATLD) algorithm was developed in order to simultaneously and directly determine the contents of warfarin (WAR) and aspirin (ASA) in human plasma and urine samples, even in the presence of unknown interferences. With the pre-estimated component number of 4, the obtained average spiked recoveries were 105.4 ± 7.8 and 104.2 ± 8.3% for WAR, 96.5 ± 2.8 and 91.2 ± 2.3% for ASA in human plasma and urine samples, respectively. Furthermore, the figures of merit were calculated and also inter- and intra-day experiments were performed that proved the proposed method is of great significance to the monitoring of clinical administration and also being a simple sample pretreatment at low-cost.

CYP2C9 Genotype-Dependent Warfarin Pharmacokinetics: Impact of CYP2C9 Genotype on R- and S-Warfarin and Their Oxidative Metabolites.

Multiple factors can impact warfarin therapy, including genetic variations in the drug-metabolizing enzyme cytochrome P450 2C9 (CYP2C9). Compared with individuals with the wild-type allele, CYP2C9*1, carriers of the common *3 variant have significantly impaired CYP2C9 metabolism. Genetic variations in CYP2C9, the primary enzyme governing the metabolic clearance of the more potent S-enantiomer of the racemic anticoagulant warfarin, may impact warfarin-drug interactions. To establish a baseline for such studies, plasma and urine concentrations of R- and S-warfarin and 10 warfarin metabolites were monitored for up to 360 hours following a 10-mg warfarin dose in healthy subjects with 4 different CYP2C9 genotypes: CYP2C9*1/*1 (n = 8), CYP2C9*1/*3 (n = 9), CYP2C9*2/*3 (n = 3), and CYP2C9*3/*3 (n = 4). Plasma clearance of S-warfarin, but not R-warfarin, decreased multiexponentially and in a CYP2C9 gene-dependent manner: 56%, 70%, and 75% for CYP2C9*1/*3, CYP2C9*2/*3, and CYP2C9*3/*3 genotypes, respectively, compared with CYP2C9*1/*1, resulting in pronounced differences in the S:R ratio that identified warfarin-sensitive genotypes. CYP2C9 was the primary P450 enzyme contributing to S-warfarin metabolism and a minor contributor to R-warfarin metabolism. In the presence of a defective CYP2C9 allele, switching of warfarin metabolism to other oxidative pathways and P450 enzymes for the metabolic elimination of S-warfarin was not observed. The 10-hydroxywarfarin metabolites, whose detailed pharmacokinetics are reported for the first time, exhibited a prolonged half-life with no evidence of renal excretion and displayed elimination rate-limited kinetics. Understanding the impact of CYP2C9 genetics on warfarin pharmacokinetics lays the foundation for future genotype-dependent warfarin-drug interaction studies.

Validation and Application of a Simple UHPLC-MS-MS Method for the Enantiospecific Determination of Warfarin in Human Urine.

A simple and rapid liquid chromatographic-tandem mass spectrometric method has been developed and validated for the enantiospecific determination of R- and S-warfarin in human urine. Warfarin enantiomers were extracted from urine using methyl tert-butyl ether. Chromatographic separation of warfarin enantiomers and the internal standard d5-warfarin was achieved using a Astec Chirobiotic V column with gradient mobile phase at a flow rate of 400 µL/min over 10 min. Detection was performed on a TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with a heated electrospray ionization source. Analytes were detected in negative ionization mode using selected reaction monitoring. Calibration curves were linear with a correlation coefficient of ≥0.996 for both enantiomers over a concentration range of 5-500 ng/mL. The intra- and interday accuracy and precision for both analytes were within ±9.0%. Excellent extraction efficiency and negligible matrix effects were observed. The applicability of the method was demonstrated by successful measurement of warfarin enantiomers in urine of patients with kidney disease. The method is simple, accurate and reproducible and is currently being used to support warfarin pharmacokinetic studies.

Reduction in the plasma clearance rate of warfarin induced by cimetidine.

The interaction between the histamine H-2 receptor antagonist, cimetidine, and warfarin sodium was prospectively studied in 14 patients who were previously anticoagulated for five years. The patients received warfarin and cimetidine concomitantly for a minimum of ten days. Seven of the patients experienced increases in plasma warfarin concentrations. This correspondingly resulted in abnormal prolongation of their prothrombin times. In these patients the elevation in the steady-state warfarin concentration demonstrates that coadministration of cimetidine significantly reduces the plasma clearance rate of warfarin. The serum and urine metabolite levels of warfarin were not qualitatively different in the absence in contrast to the presence of cimetidine. It is apparent that cimetidine can act as an inhibitory influence on the catabolic degradation of warfarin.

Electrooxidation behavior of warfarin in Fe3O4 nanoparticles modified carbon paste electrode and its determination in real samples.

In the present work, a simple and sensitive electrochemical sensor based on magnetic Fe3O4 nanoparticles modified carbon paste electrode (CPE) was introduced for detection of low level with 0.21µM of warfarin. Under the optimum experimental conditions the oxidation peak current of warfarin was used for its monitoring for the first time. The analytical curve was linear for warfarin concentrations from 0.5 to 1000µM with a limit of detection of 0.21µM by square wave anodic stripping voltammetry (SWASV). The proposed sensor showed excellent stability and was used for the determination of warfarin in tablet, human serum and urine with satisfactory results.

The warfarin-sulfinpyrazone interaction: stereochemical considerations.

To allow the simultaneous evaluation of the interaction between sulfinpyrazone and each enantiomer of racemic warfarin, pseudoracemic warfarin (1:1 12C-R(+) and 13C-S(-)warfarin) was given to six normal subjects both before and during oral sulfinpyrazone dosing. Serial blood and urine samples were analyzed for unchanged warfarin and its metabolic products by GC/MS. A mass balance of an oral dose of pseudoracemic warfarin, containing a tracer quantity of 14C-warfarin, was carried out in one of the subjects by monitoring 14C levels in urine and feces for 15 days. Concomitant sulfinpyrazone dosing markedly increased hypoprothrombinemia, decreased clearance of (S)-warfarin, and increased clearance of (R)-warfarin. Sulfinpyrazone also decreased the urinary excretion of warfarin-related products but increased their fecal excretion by an equivalent amount. Virtually all of the administered warfarin dose could be accounted for either as unchanged drug or known metabolites. Pharmacokinetic analysis of the data suggests the following: At least four distinct enzymes (two oxidases and two reductases) are involved in the metabolism of warfarin. Sulfinpyrazone increases the hypoprothrombinemia caused by warfarin primarily by inhibition of the cytochrome P-450-mediated oxidation of (S)-warfarin, the biologically more potent enantiomer. The increased clearance of (R)-warfarin results not from induction, but from its selective displacement from plasma protein binding sites.

Population differences in S-warfarin metabolism between CYP2C9 genotype-matched Caucasian and Japanese patients.

OBJECTIVE: Our objective was to investigate population differences in the metabolic activity of cytochrome P450 (CYP) 2C9 between genotypically matched Caucasian and Japanese patients by using the unbound oral clearance of S-warfarin as an in vivo phenotypic trait measure. METHODS: Ninety Japanese and 47 Caucasian patients receiving maintenance warfarin therapy were studied. Steady-state plasma unbound concentrations of S-warfarin were measured by a chiral HPLC method coupled with an ultrafiltration technique, and unbound oral clearance for S-warfarin was estimated. By combining plasma unbound concentrations of S-warfarin with the urinary excretion rates of S-7-hydroxywarfarin, the formation clearance of S-7-hydroxywarfarin was also determined. Genotyping of CYP2C9 was performed for 6 distinct alleles (CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C9*4, CYP2C9*5, and a T/C transition in intron 2). RESULTS: The frequency distribution of unbound oral clearance for S-warfarin obtained from Japanese patients was shifted toward higher values as compared with that in Caucasian patients. Japanese patients had lower allelic frequencies for the 5 variants than Caucasian patients. When interpopulation comparisons of CYP2C9 activity were made for genotype-matched subjects, Japanese patients with the homozygous CYP2C9*1 (wild-type) genotype (n = 85) had significantly (P <.01) greater median values for unbound oral clearance and formation clearance than Caucasian patients with the corresponding genotype (n = 26), 10.4 mL x min(-1) x kg(-1) versus 4.25 mL x min(-1) x kg(-1) and 0.015 mL x min(-1) x kg(-1) versus 0.010 mL x min(-1) x kg(-1), respectively. In addition, Japanese patients heterozygous for the CYP2C9*3 genotype (n = 4) showed a significantly (P <.05) reduced unbound oral clearance for S-warfarin, by 63%, as compared with Japanese patients possessing the homozygous CYP2C9*1 genotype. By contrast, in Caucasian patients, no significant differences were observed in this parameter between CYP2C9(*)1 homozygous subjects and those with heterozygous CYP2C9(*)2 or CYP2C9(*)3 genotypes. CONCLUSIONS: These findings indicate that population differences in the frequencies of known variant CYP2C9 alleles account only in part for the variability observed in in vivo CYP2C9 activity in different populations. In addition, a gene-dose effect of defective CYP2C9 alleles on the in vivo CYP2C9 activity is evident in Japanese patients but not in Caucasian patients. Further studies are required to identify currently unknown factor(s) (eg, transcriptional regulation) responsible for the large intrapopulation and interpopulation variability in CYP2C9 activity.

Potentiation of anticoagulant effect of warfarin caused by enantioselective metabolic inhibition by the uricosuric agent benzbromarone.

OBJECTIVE: To clarify the mechanism(s) for the interaction between warfarin and benzbromarone, a uricosuric agent, and to predict changes in the in vivo pharmacokinetics of (S)-warfarin from in vitro data. METHODS: Warfarin enantiomers and benzbromarone in serum, 7-hydroxywarfarin in urine, and serum unbound fractions of warfarin enantiomers were measured in patients with heart disease given warfarin with (n = 13) or without (n = 18) oral benzbromarone (50 mg/d). In vitro inhibition constants (K(i)) of benzbromarone for (S)-warfarin 7-hydroxylation were determined with use of human CYP2C9 and liver microsomes. The magnitude of changes in the formation clearance for 7-hydroxylation (CLf), the unbound oral clearance (CL(oral,u)), and the oral clearance (CL(oral)) for (S)-warfarin were predicted by equations incorporating the in vitro Ki, the theoretical maximum unbound hepatic benzbromarone concentration, and the fractions of warfarin eliminated through metabolism and of CYP2C9-mediated metabolic reaction susceptible to inhibition by benzbromarone. RESULTS: The patients given warfarin with benzbromarone required a 36% less (P < .01) warfarin dose than those given warfarin alone (2.5 versus 3.9 mg/d) to attain similar international normalized ratios (2.1 and 2.2, respectively), and the former had 65%, 53%, and 54% lower (P < .05 or P < .01) CLf, CL(oral),u, and CL(oral) for (S)-warfarin than the latter, respectively. In contrast, no significant differences were observed for (R)-warfarin kinetics between the groups. Benzbromarone was found to be a potent competitive inhibitor (Ki < 0.01 micromol/L) for (S)-warfarin 7-hydroxylation mediated by CYP2C9. The average changes in the in vivo CLf, CL(oral),u, and CL(oral)values for (S)-warfarin induced by benzbromarone were largely predictable by the proposed equations. CONCLUSION: Benzbromarone would intensify anticoagulant response of warfarin through an enantioselective inhibition of CYP2C9-mediated metabolism of pharmacologically more potent (S)-warfarin. The magnitude of changes in the in vivo warfarin kinetics may be predicted by in vitro data.

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.

Stereospecific fluorescence high-performance liquid chromatographic analysis of warfarin and its metabolites in plasma and urine.

A stereospecific assay for the simultaneous determination of the enantiomers of warfarin and its major metabolites, 6- and 7-hydroxywarfarin and warfarin alcohols, in plasma and urine was developed. Involved in this determination was the formation of diastereoisomeric esters with carbobenzyloxy-L-proline, separation by normal-phase high-performance liquid chromatography, and detection by fluorescence after postcolumn aminolysis with n-butylamine. The determination limit for any enantiomer is in the order of 50-100 ng. The method was applied to the analysis of the enantiomers of warfarin and metabolites in plasma and urine of human subjects receiving racemic drug. The results for warfarin enantiomers are comparable with those obtained by an MS method, involving administration of a synthetic pseudoracemate [12C(R), 13C(S)]warfarin. In addition to all known metabolites, the detection of 7-R-hydroxywarfarin indicates that 7-hydroxylation is stereoselective rather than stereospecific.