Biomedical subjects

G Blaschke

Publications and source records attributed to G Blaschke.

At least 73 records · Page 4Linked to original sources

PubMed1998

Capillary electrophoresis with laser-induced fluorescence: a routine method to determine moxifloxacin in human body fluids in very small sample volumes.

The feasibility of capillary electrophoresis with HeCd laser-induced fluorescence detection as a validated routine method for bioanalytical analysis is reported. Method evaluation, validation and results of the determination of moxifloxacin (BAY 12-8039), a new antimicrobially active 8-methoxy-quinolone, in plasma and microdialysate are described. After a one step sample preparation the samples can be injected directly into the capillary. The volume of microdialysate and plasma, respectively, needed for more than 50 injections is only 10 microl and 20 microl. Total run time is less than 7 min using a 27 cm capillary on commercial instrumentation. An analysis time of less than 1 min was shown to be possible, however it could not be used routinely since appropriate instrumentation was not available. Evaluation is based on the relative corrected peak area (analyte/I.S.). The method's dynamic range comprises three orders of magnitude (plasma: 2.5-5000 microg/l; microdialysate: 5-5000 microg/l). Validation according to international guidelines yielded data on accuracy and precision of the method throughout the entire working range of inter-day precision: plasma <6%, microdialysate <5% and inter-day accuracy: plasma <2%, microdialysate <4%. The crossvalidation with an existing HPLC method utilizing clinical study samples shows linear correlation. In view of its adequate sensitivity and high selectivity capillary electrophoresis with laser-induced fluorescence is a very versatile tool in pharmacokinetic studies of quinolones, especially in situations with limited sample volumes: e. g. pediatrics, patients at risk, animal-, microdialysis- and tissue-kinetic studies. Validation parameters and other features, like high sample throughput and robustness, are comparable to or even better than HPLC. Further necessary improvements of the capillary electrophoresis with laser-induced fluorescence instrumentation (autosampler, vials, parallel capillaries) and its use in bioanalytical routine analysis are discussed.

Anti-Infective Agents↗

PubMed1998

Stereoselective determination of clenbuterol in human urine by capillary electrophoresis.

The effectiveness of capillary electrophoresis (CE) in the field of stereoselective determination of drugs in biological matrices is demonstrated by analyzing clenbuterol in human urine. Due to the very low therapeutical doses of 20-40 microg per day the total concentrations in urine are 1-10 ng/ml. The sample was extracted with hexane-tert.-butyl methyl ether (99.5:0.5). The reconstituted sample was injected electrokinetically (50 s, 10 kV). Using phosphate buffer, pH 3.3 and hydroxyethyl-beta-cyclodextrin as chiral selector the total analysis time was below 15 min. The limit of determination was estimated as 0.5 ng/ml per enantiomer. S-(-)-Bupranolol was used as internal standard. Both precision and accuracy of the method were within the limits for biological samples. The application to human urine from patients having received therapeutical doses showed a slightly predominant excretion of the (+)-enantiomer to the (-)-enantiomer.

Adult↗

PubMed1998

Separation of ergot alkaloids and their epimers and determination in sclerotia by capillary electrophoresis.

Capillary electrophoresis has been shown to be a very useful analysis technique for secondary metabolites of plants. In the present study a method is described for the qualitative and quantitative determination of ergot alkaloids and their epimers. The extraction from the biological matrix yields recoveries of 50-97%, depending on the individual alkaloid. Using a mixture of 20 mM beta-cyclodextrin (CD), 8 mM gamma-CD, 2 M urea and 0.3% (w/w) poly(vinyl alcohol) to phosphate buffer at pH 2.5 the simultaneous separation of all analytes was achieved. A 37 cm (30 cm) fused-silica capillary, at a voltage of 25 kV and a temperature of 20 degrees C, was used for the analysis. Overall analysis time for the separation was 12 min. The limit of detection of the alkaloids using UV detection at 214 nm can be improved 30-fold to about 9.10(-8) M when laser-induced fluorescence detection is applied.

Buffers↗

PubMed1998

Investigation of the stereoselective metabolism of praziquantel after incubation with rat liver microsomes by capillary electrophoresis and liquid chromatography-mass spectrometry.

Two different separation methods for the antischistosomal drug praziquantel and its metabolites by capillary electrophoresis are described. Achiral separation was obtained by micellar electrokinetic capillary chromatography using sodium dodecyl sulfate as micelle-forming surfactant. On the other hand, the negatively charged sulfobutylether-beta-cyclodextrin as a chiral selector enabled the separation of the drug and its metabolites as well as their enantioseparation. Based on this separation, the enantioselectivity of the metabolism of praziquantel was studied by incubation of the drug with rat liver microsomes. Whereas trans- and cis-4-hydroxypraziquantel were mainly formed from the R-(-)-enantiomer, another, different monohydroxylated metabolite was only formed from the S-(+)-enantiomer. Information about the structure of these metabolites was obtained, using LC-MS.

Animals↗

PubMed1998

Kinetics of racemization of (+)- and (-)-diethylpropion: studies in aqueous solution, with and without the addition of cyclodextrins, in organic solvents and in human plasma.

The configurational stability of (+)- and (-)-diethylpropion [(+)- and (-)-2-(diethyl)-1-phenyl-1-propanone or (+)- and (-)-DEP] was investigated systematically from chemical, pharmaceutical, and pharmacological aspects. The enantiomeric ratio was monitored directly with a recently developed stability-indicating enantioselective HPLC method. In aqueous solutions, the rate of racemization increased non-linearly with increasing pH and with increasing phosphate buffer concentration. The racemization rate showed a positive slope with increasing temperature and decreasing ionic strength. The racemization rates of (+)- and (-)-DEP in the presence of cyclodextrins (CDs) did not differ significantly. CDs that were added to (+)- and (-)-DEP in a molar ratio 5:1 showed the following effects after dissolution in 10 mM phosphate buffer (final pH 6.7): sulfobutyl ether-beta-CD (SBE-beta-CD) and methylated-beta-CD (Me-beta-CD) retarded racemization; whereas hydroxypropyl-beta-CD (HP-beta-CD), acetyl-gamma-CD (Ac-gamma-CD), acetyl-beta-CD (Ac-beta-CD), gamma-CD, and beta-CD showed a weak destabilising effect. In contrast to the described CDs, alpha-CD distinctly accelerated the rate of racemization. The configurational stability of (+)- and (-)-DEP was also studied under physiological conditions. The half-life of racemization in heparinised human plasma was for both enantiomers determined to be approximately 23-25 min. In phosphate buffer (10 mM, pH 7.4), rac-DEP showed a high, but unselective affinity towards human alpha 1-acid glycoprotein (orosomucoid) immobilised on silica (Chiral AGP). The rate of racemization of the free base of (-)-DEP dissolved in organic solutions generally increases with the polarity of the solvating agent.

Appetite Depressants↗

PubMed1998

Capillary electrophoresis, nuclear magnetic resonance and mass spectrometry studies of opposite chiral recognition of chlorpheniramine enantiomers with various cyclodextrins.

Markedly different chiral separation abilities were observed for native beta-cyclodextrin (beta-CD), carboxymethyl-beta-CD (CM-beta-CD) and heptakis (2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD) towards the enantiomers of (+/-)-chlorpheniramine ((+/-)-CHL) in capillary electrophoresis (CE). Native beta-CD afforded almost baseline enantioseparation at a concentration of 18 mg/mL, whereas only 1 mg/mL solution of CM-beta-CD was required for adequate enantioseparation. TM-beta-CD allowed the nearly baseline enantioseparation only at a concentration as high as 80 mg/mL. Moreover, the migration order of (+/-)-CHL in the presence of TM-beta-CD was opposite to that with beta-CD and CM-beta-CD. 1H and 13C-NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) have been used in order to obtain preliminary information about the stoichiometry and the binding constants in the intermolecular diastereomeric complexes of (+/-)-CHL with these CDs.

Chlorpheniramine↗

PubMed1998

Enantiomeric ratio of shikonin derivatives as a possible key for the determination of the origin of lithospermi radix.

An HPLC method was developed to resolve the enantiomers of shikonin derivatives of the Lithospermi Radix. The optimum mobile phase on a Chiracel AD column was 5% isopropanol in n-hexane with flow rate of 1 ml/min. Establishment of this method made possible to determine the ratios of shikonin/acetylshikonin or alkanin/acetylalkanin in the same root. The correlation of the ratios of these substance pairs appeared characteristic for the country where they were originated from. All of the Korean species showed significantly higher ratios of shikonin/acetylshikonin and alkanin/acetylalkanin than the Chinese ones. This method would be useful to determine the origin of Lithospermi Radix.

2-Propanol↗

PubMed1998

A specific immunoassay for the determination of morphine and its glucuronides in human blood.

The development of specific antisera for immunochemical determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide is described. Morphine was N-demethylated to normorphine and N-alkylated to give N-aminopropyl-normorphine as hapten for antisera against morphine. As haptens for antisera against morphine-3-glucuronide and morphine-6-glucuronide, N-aminopropyl-nor-morphine was glucuronidated in position 3 or 6 respectively. Each of these three haptens were coupled to BSA employing the glutaraldehyde method to obtain three different immunogens. Immunisation of rabbits with these conjugates gave anti-morphine, anti-morphine-3-glucuronide and anti-morphine-6-glucuronide antisera, which were tested in a competitive, heterogeneous radioimmunoassay. Tracers for this radioimmunoassay procedure were synthesised by substitution of morphine and morphine-6-glucuronide in position 2 with 125I and indirect iodination of the morphine-3-glucuronide hapten according to the method of Bolton and Hunter. The resulting antisera show very specific reactions with morphine, morphine-3-glucuronide and morphine-6-glucuronide. Cross reactivities of each antiserum with structurally related opiates and opioides are very low. The cross reactivities of the anti-morphine antiserum against morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine were less than 0.3%, the anti-morphine-3-glucuronide antiserum against morphine, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine less than 0.1% and the anti-morphine-6-glucuronide antiserum against morphine, morphine-3-glucuronide, codeine or dihydrocodeine less than 0.1%, against codeine-6-glucuronide less than 2.3%. The determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in blood samples (limit of detection= 3, 1, 0.5 ng/g) of nine cases of fatal heroin overdose with this radioimmunoassay method and the comparison with a GC/MS method is described.

Animals↗

PubMed1998

Chemically induced isomerization and differential uptake modulate retinoic acid disposition in HL-60 cells.

The successful introduction of 13-cis-retinoic acid (13-cis-RA) and all-trans-retinoic acid (all-trans-RA) in the chemoprevention and treatment of cancer along with the discovery of different retinoic acid receptors transactivated by different retinoic acid isomers resulted in a number of in vitro studies of the antitumor effects of single retinoic acid isomers. Since the formation of retinoic acid isomers with different receptor affinities might modulate retinoic acid response in vitro, we determined retinoic acid disposition in HL-60 cells and cell culture medium during incubation with 13-cis-, 9-cis-, and all-trans-RA. In medium, retinoic acids underwent a thiol-radical mediated isomerization resulting in a mixture of 13-cis-, 9-cis-, 9,13-di-cis-, and all-trans-RA. Except for the 9, 13-di-cis-RA, all isomers generated in medium were also detected in HL-60 cells. Whereas 9-cis-RA and 13-cis-RA showed similar cellular pharmacokinetics, all-trans-RA reached about fourfold higher concentrations in HL-60 cells compared to 9-cis-RA and 13-cis-RA. Due to its better uptake, all-trans-RA became the main isomer within cells as it was formed in the medium when incubated with 13-cis-RA and 9-cis-RA. Thus, due to the simple chemically induced isomerization and its profound influence on cellular retinoic acid concentrations, studies of the efficacy of single retinoic acid isomers in vitro should be interpreted with caution.

Biological Transport↗

PubMed1998

Excretion kinetics of ifosfamide side-chain metabolites in children on continuous and short-term infusion.

Ifosfamide (IFO) requires metabolic activation by hydroxylation of the ring system to exert cytotoxic activity. A second metabolic pathway produces the cytostatically inactive metabolites 2-dechloroethyl-ifosfamide (2-D-IFO) and 3-dechloroethyl-ifosfamide (3-D-IFO) under release of chloroacetaldehyde. This side-chain metabolism has been suggested to be involved in CNS- and renal toxicity. The total urinary excretion of ifosfamide and its metabolites was investigated during 23 cycles in 22 children at doses ranging from 400 mg/m2 to 3 g/m2. The kinetics of the excretion were compared following short-term and continuous ifosfamide infusion at a dosage of 3 g/m2. IFO and side-chain metabolites were analyzed by gas chromatography, the active metabolites by indirect determination of acrolein (ACR) and IFO mustard (IFO-M) with the NBP test. 59+/-15% of the applied dose could be recovered in the urine, 23+/-9% as unmetabolized IFO. The main metabolite was 3-D-IFO (14+/-4%) followed by isophosphoramide mustard (IFO-M) (13+/-4%) and 2-D-IFO (8+/-3%). Neither the total amount recovered nor the excretion kinetics of ifosfamide and side-chain metabolites showed obvious schedule dependency. The excretion kinetics of side-chain metabolites as well as unmetabolized IFO were nearly superimposable on short-term and continuous infusion. Even after 1-hour infusion there was a lag of 3 - 6 hours until dechloroethylation became relevant. Therefore, differences in toxicity and efficacy cannot be explained by an influence of the application time on the metabolic profile of ifosfamide.

Acetaldehyde↗

PubMed1997

Analysis of the antidiabetic drug acarbose by capillary electrophoresis.

This study describes the derivatization of the pseudooligosaccharide acarbose and its main metabolite, component 2, with 7-aminonaphthalene-1,3-disulfonic acid (ANDS) in human urine. Their efficient separation was possible by means of capillary zone electrophoresis, using a capillary tube of fused-silica containing 100 mM triethylammonium phosphate buffer, pH 1.5. On column laser-induced fluorescence allowed the detection of the pseudooligosaccharides in human urine in the nanomolar range. With this method, acarbose and component 2 were quantified in human urine after application of 300 mg of acarbose.

Acarbose↗

PubMed1997

Determination of idarubicin and idarubicinol in plasma by capillary electrophoresis.

A rapid and sensitive capillary electrophoretic method for the determination of idarubicin and its metabolite idarubicinol in plasma has been developed and validated. Plasma is extracted by liquid-liquid extraction using chloroform. Idarubicin, idarubicinol and the internal standard daunorubicin can be separated in less than 5 min using a phosphate buffer of pH 5 with 70% acetonitrile. Laser-induced fluorescence detection with an Ar ion laser operated at 488 nm provides a sensitive and selective detection method without interferences from biological fluids. The small sample volume of 100 microl is of particular advantage for studies in pediatric oncology. The reproducibility of the method has been shown to be sufficient for drug monitoring or pharmacokinetic studies. The limit of quantification for idarubicin in plasma is 0.5 ng/ml.

Antibiotics, Antineoplastic↗

PubMed1997

Determination of LSD in blood by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis (CE) with HeCd laser-induced fluorescence (LIF) detection and its application in forensic toxicology is demonstrated by the determination of D-lysergic acid diethylamide (LSD) in blood. Following precipitation of proteins, washing of the evaporated supernatant and extraction, the residue was reconstituted in methanol and injected electrokinetically (10 s, 10 kV). The total analysis time for quantification of LSD was 8 min using a citrate-methanol buffer, pH 4.0. With this buffer system it is possible to separate LSD, nor-LSD, iso-LSD and iso-nor-LSD. Using a specific sample preparation, electrokinetic injection, extended light path (bubble cell) capillaries and especially LIF detection (lambda(ex) 325 nm, lambda(em) 435 nm), a limit of detection of 0.1-0.2 ng LSD per ml blood could be obtained. The limit of quantitation was about 0.4-0.5 ng/ml. The quantitative evaluation for LSD was carried out using methylergometrine as internal standard. The precision expressed as coefficient of variation (C.V.) and accuracy of the method were <20% and 86-110%, respectively. The application of the method to human blood samples from two forensic cases and a comparison with radioimmunoassay demonstrated that the results were consistent.

Electrophoresis, Capillary↗

PubMed1997

Capillary electrophoresis with laser-induced fluorescence detection, an adequate alternative to high-performance liquid chromatography, for the determination of ciprofloxacin and its metabolite desethyleneciprofloxacin in human plasma.

A method to determine plasma concentrations of ciprofloxacin and its metabolite desethyleneciprofloxacin (M1) by CE with HeCd laser-induced fluorescence detection is described. Following precipitation of proteins and centrifugation supernatant is injected hydrodynamically (10 s, 0.5 p.s.i.) into the capillary. Overall analysis time for the quantification of both analytes was 7 min. The total amount of plasma needed for multiple injections (n>5) was 10-20 microl. Data on accuracy and precision are presented. The assay performance is compared to the specifications of a validated HPLC method, which is routinely used for the quantification of ciprofloxacin and M1 in body fluids. Both methods showed comparable accuracy and precision for both analytes throughout the whole working range (inter-day precision <9%; inter-day accuracy 96-110%). The limit of quantification (LOQ) of 20 microg/l (M1 10 microg/l) for the CE procedure was slightly higher than for the HPLC method, where 10 microg/l (M1 2.5 microg/l) was determined. However, application of the methods to human plasma samples derived from a clinical study proved that comparable results are obtained and that the sensitivity of the HPCE method was sufficient to fully describe typical plasma concentration time profiles of ciprofloxacin and its metabolite M1. Both the adequate sensitivity and the required smaller sample volume compared to HPLC indicate that the method is feasible for clinical studies where sample amounts are limited, e.g., studies to investigate pharmacokinetics in pediatric patients. Preclinical studies form another possible application of this technique.

Anti-Infective Agents↗

PubMed1997

Investigation of the stereoselective in vitro metabolism of the chiral antiasthmatic/antiallergic drug flezelastine by high-performance liquid chromatography and capillary zone electrophoresis.

An achiral HPLC method using a silica gel column as well as two independent chiral analytical methods by HPLC and capillary zone electrophoresis (CZE) were developed in order to investigate the in vitro metabolism of the racemic antiasthmatic/antiallergic drug flezelastine. The chiral HPLC analysis was performed on a Chiralpak AD column, which also allowed the simultaneous separation of the N-dephenethyl metabolite. The chiral separation by CZE was achieved by the addition of beta-cyclodextrin to the run buffer. The stereoselectivity of the in vitro biotransformation of flezelastine was investigated using liver homogenates of different species. Depending on the species, diverse stereoselective aspects were demonstrated. The determination of the enantiomeric ratios of flezelastine and of N-dephenethylflezelastine after incubations of racemic flezelastine with liver microsomes revealed that porcine liver microsomes showed the greatest enantioselectivity of the biotransformation. (-)-Flezelastine was preferentially metabolized. After incubations with bovine liver microsomes the enantiomer of N-dephenethylflezelastine formed from (+)-flezelastine dominated. Incubations of the pure enantiomers of flezelastine with induced rat liver microsomes resulted in the stereoselective formation of a hitherto unknown metabolite, which was only detected in samples of (+)-flezelastine. Initial structure elucidation of the compound indicated that the new metabolite was most likely an aromatically hydroxylated derivative of the N-dephenethylflezelastine.

Animals↗

PubMed1997

Investigation of the stereoselective in vitro biotransformation of glutethimide by high-performance liquid chromatography and capillary electrophoresis.

Due to our interest in drugs with a glutarimide structure, we reinvestigated the stereoselectivity of the in vitro biotransformation of the chiral hypnotic-sedative drug glutethimide. Glutethimide enantiomers were separated on a preparative scale by HPLC on cellulose tris(4-methylbenzoate) as chiral stationary phase. The enantiomeric purity was higher than 99%. A reversed-phase HPLC method was developed to determine the metabolites of glutethimide. After incubations with rat liver microsomes both enantiomers formed 5-hydroxyglutethimide as the main metabolite, as well as additional metabolites, of which some were formed stereoselectively. Mass spectrometry of the unknown metabolites indicated a hydroxylation in the ethyl side chain for two of the metabolites. A third metabolite was tentatively identified as desethylglutethimide.

Animals↗

PubMed1997

Studies of the metabolism of the antihistaminic drug dimethindene by high-performance liquid chromatography and capillary electrophoresis including enantioselective aspects.

A reversed-phase high-performance liquid chromatography method for the determination of dimethindene and its main metabolites N-demethyldimethindene, 6-hydroxydimethindene and 6-hydroxy-N-demethyldimethindene in human urine was developed. The assay was also applied to the quantification of dimethindene-N-oxide in rat urine. Conjugates of the hydroxylated metabolites were determined after enzymatic deconjugation. Moreover the direct determination of dimethindene and its metabolites without prior extraction from urine was performed by capillary electrophoresis. The direct simultaneous determination of the enantiomers of dimethindene and N-demethyldimethindene was achieved on a Chiralcel OD column. Urinary data after oral administration of dimethindene are presented. The assays were used to study dimethindene and it metabolites in urine upon oral administration of the drug to rats and human volunteers.

Adult↗

PubMed1997

Enantioselective determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in serum, urine and hair by capillary electrophoresis.

A capillary electrophoresis method has been developed for the enantioselective determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in serum and urine. After addition of the internal standard diphenhydramine, the serum and urine samples were extracted at pH 9-10 with n-hexane. Different cyclodextrins were tested for use as chiral selectors. The best results for simultaneous separation of methadone and EDDP were obtained using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB). Data on the accuracy and precision of the method are presented. The method was applied to serum, urine, and hair samples, from individuals undergoing methadone therapy, under consideration of multi-drug abuse.

Electrophoresis, Capillary↗