Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

Determination of piribedil and its basic metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of piribedil and its p-hydroxylated, catechol and N-oxide metabolites in plasma is described. After addition of an internal standard (buspirone), the plasma samples were subjected to a three-step extraction procedure. The final extracts were evaporated to dryness under nitrogen, and the residues were reconstituted in 100 microliters of mobile phase (0.01 M phosphate buffer-acetonitrile, 50:50, v/v) and chromatographed by acetonitrile gradient elution on a C18 reversed-phase column coupled to an ultraviolet detector set at 240 nm. The method was selective for piribedil and its metabolites, and sufficiently sensitive and precise for studies aimed at elucidating the role of the metabolites in the parent drug's pharmacological effects.     

Determination of free phenytoin in plasma by ultrafiltration and high-performance liquid chromatography

A process for determining the free concentration of the highly protein-bound anti-convulsant drug phenytoin (5,5-diphenylhydantoin) is described. The procedure involves rapid isolation of the unbound drug from the drug/protein complex by ultrafiltration followed by high-performance liquid chromatography. Total time for a free phenytoin determination is about 15 min. The procedure is used to examine the protein-binding of phenytoin, as well as the effects of a displacer, salicylic acid. Commonly prescribed anticonvulsant drugs are resolved under the selected chromatographic conditions.     

Determination of diazepam and its metabolites by high-performance liquid chromatography and thin-layer chromatography

A sensitive, simple high-performance liquid chromatographic assay, capable of simultaneously measuring diazepam, its active metabolites oxazepam, temazepam and N-desmethyldiazepam and two phenyl hydroxylated metabolites, 4'-hydroxy-N-desmethyldiazepam and 4'-hydroxydiazepam, is described. The assay is easily modified to include separation of additional metabolite(s), e.g. oxazepam glucuronide(s). A thin-layer chromatographic assay, which resolves diazepam, the active metabolites and the two phenyl hydroxylated derivatives in one solvent system, is also reported. Application of these procedures to the quantitation of diazepam and its metabolites was shown, after delivery of diazepam (5 micrograms/ml or 16 microM) at a constant flow-rate (10 ml/min per liver) through the single-pass perfused rat liver preparation. Blood perfusion medium and bile were analysed for parent drug and metabolites before and after enzyme hydrolysis. These assay methods are found to be particularly pertinent and useful in providing a more comprehensive metabolic profile of diazepam metabolism, especially when aromatic hydroxylation pathways predominate.     

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 alpidem and its metabolites in human plasma by high-performance liquid chromatography and fluorimetric detection

A high-performance liquid chromatographic method has been developed for the simultaneous determination of alpidem and its metabolites in human plasma. The method involved a single extraction of the parent drug and metabolites into diethyl ether from alkalinized plasma, evaporation of the organic solution and chromatography of the extracts on a C18 column coupled to a fluorimetric detector. An internal standard was used for the quantitative determination of the compounds. The method was selective for alpidem and three of its metabolites and has a limit of detection of less than 1 ng ml-1 for all the compounds. Since the chromatographic run took more than 20 min, the chromatographic process was fully automated and performed overnight.     

Determination of bromazepam in plasma and of its main metabolites in urine by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bromazepam in plasma and of its main metabolites in urine is described. The unchanged drug is extracted from plasma with dichloromethane, using Extrelut 1 extraction tubes. The residue from this extract is subsequently analysed by reversed-phase high-performance liquid chromatography with ultraviolet detection (230 nm). The limit of detection is 6 ng/ml of plasma, using a 1-ml specimen. For the determination of the metabolites, the urine samples are incubated to effect enzymatic deconjugation and are then extracted with dichloromethane. Following two clean-up steps (back extractions), the final residue is analysed on the same reversed-phase system as the plasma samples. The limit of detection for the two metabolites is 200 ng/ml.     

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.     

Determination of lansoprazole and its metabolites in plasma by high-performance liquid chromatography using a loop column.

A high-performance liquid chromatographic method for the simultaneous determination of lansoprazole, a new proton pump inhibitor, and its metabolites in human plasma is described. Lansoprazole, its metabolites and an internal standard were extracted with tert.-butyl methyl ether. Samples were injected using an automatic injector via a loop column, and separation was obtained using a reversed-phase column under isocratic conditions. The absorbance was monitored at 285 and 303 nm. The quantification limit was 2 ng/ml for lansoprazole and 3 or 5 ng/ml for the metabolites. No endogenous compounds were found to interfere. The mean overall recovery was between 75 and 95% for lansoprazole and its metabolites. This method is suitable for pharmacokinetic studies.     

Determination of the serum concentration of spironolactone and its metabolites by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic assay is described for the simultaneous determination in serum of the aldosterone antagonist spironolactone and its metabolites 7 alpha-thiomethylspirolactone, 6 beta-hydroxy-7 alpha-thiomethylspirolactone and canrenone. Ultraviolet absorption at 240 nm was used to detect the different compounds after elution on a normal-phase column. Endogenous serum substances did not interfere with the assay. This method provides a convenient tool in pharmacokinetic studies of spironolactone, in contrast to previously reported aspecific fluorimetric assays or time-consuming thin-layer chromatographic analyses of radioactive biological material.     

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.     

Determination of piroxicam and its major metabolites in the plasma, urine and bile of humans by high-performance liquid chromatography.

A simple and sensitive liquid chromatographic method with ultraviolet detection is described for the determination of the nonsteroidal anti-inflammatory drug piroxicam and its major metabolites in human plasma, urine and bile. Separation of these components occurs on a reversed phase C10CN column with a mobile phase consisting of acetonitrile-water-sodium dihydrogenphosphate solution. The detection limit of the assay was 50 ng/ml with intra- and inter-assay coefficients of variation for piroxicam of the order of 2 and 5%, respectively. The assay linearity was good (typically r = 0.9999). This method can be readily utilised for clinical pharmacokinetic and mass-balance studies.     

Determination of diclofenac and its metabolites in plasma and cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic method with electrochemical detection for the quantitation of diclofenac and metabolites in plasma and cerebrospinal fluid has been developed. Pirprofen is employed as internal standard. Samples are extracted with C18 solid-phase extraction columns and eluted with methanol. Oxidation potentials for detection were established by constructing voltammograms for each compound. In the concentration range found in human studies, the intra-day coefficients of variation were always less than 6%. The procedure allows the simultaneous determination of diclofenac and its four major metabolites with very low detection limits (less than 1 ng/ml), which were sufficient even for kinetic studies in cerebrospinal fluid.