Jan X. De Vries

CHARACTERIZATION OF HEPARINS BY HIGH-PERFORMANCE SIZE EXCLUSION LIQUID CHROMATOGRAPHY

Abstract A method for the determination of the molecular weight distribution of heparins using heparin oligosaccharides as calibration standards and high-performance size exclusion liquid chromatography has been developed. An organic microparticulate stationary phase (TSK 3000), an aqueous mobile phase and UV detection at 206 nm were adopted. Commercial heparins, low-molecular-weight heparins and polysulphated mucopolysaccharides with heparin-like activities were characterized by this procedure. Average molecular weights [weight average (Mw), number average (Mn), z average (Mz)] and molecular weight distribution (cumulative weight fraction and differential weight fraction) were calculated from the chromatographic data.

Direct column liquid chromatographic enantiomer separation of the coumarin anticoagulants phenprocoumon, warfarin, acenocoumarol and metabolites on an α1-acid glycoprotein chiral stationary phase

Abstract The enantiomers of the racemic coumarin anticoagulants phenprocoumon (PH) and metabolites (4′-, 6-, 7- and 8-hydroxy-PH), warfarin (WA) and metabolites (6-, 7-hydroxy-WA and the two diastereomeric WA alcohols) and acenocoumarol were resolved by column liquid chromatography using an immobilized α1-acid glycoprotein stationary phase; elution was performed using a phosphate buffer and isopropanol gradient with and without dimethyloctylamine as modifier, and detection by ultraviolet or fluorescence. The advantages of this method are: the procedure is simple and fast and does not require pre-column derivatization; the configuration of the enantiomers can be assigned by comparison with a reference sample with already known absolute configuration; the optical purities of these compounds can be analysed with high sensitivity; the method can be applied to the determination of the enantiomers in biological samples.

Thermospray and particle beam liquid chromatographic-mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography-thermospray mass spectrometry (LC-TSP-MS) and liquid chromatography-electron impact mass spectrometry (LC-EI-MS) to assess the use of LC-MS methods for the determination of these compounds in biological materials. LC-TSP mass spectra showed a single [M + 1]+ ion with no fragmentation; LC-EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC-MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC-TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.

Analysis of heparins by size-exclusion and reversed-phase high-performance liquid chromatography with photo-diode-array detection

Abstract High-performance liquid chromatography (HPLC) of heparins was carried out with on-line photodiode-array detection. Average molecular weights and molecu

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.

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.

Determination of benzarone in human plasma and urine by high-performance liquid chromatography and gas chromatography—mass spectrometry : Identification of the conjugates

Benzarone (the debrominated metabolite of the uricosuric drug benzbromarone) has been proposed for treatment of vascular disorders. An assay was developed for the quantitation of total benzarone (conjugated and unconjugated) in plasma and urine, following oral intake of benzarone. Enzymatic hydrolysis of the samples with beta-glucuronidase/arylsulphatase, extraction, gradient elution high-performance liquid chromatography with reversed-phase columns and UV detection were used for the assay. The concentration ranges, precision and sensitivities were: 0.01-2 micrograms/ml, 3-5% and 0.01 microgram/ml, respectively, for both plasma and urine. These results were validated by gas chromatography-mass spectrometry after methylated derivatives were prepared. Enzymatic hydrolysis of plasma with pure beta-glucuronidase or arylsulphatase showed that the relative amounts of unconjugated, glucuronidated, and sulphated benzarone were 6, 12 and 82% respectively, for both plasma and urine.