Wolfgang Blum

Preparation on inert glass capillary columns for gas chromatography : A revised, comprehensive description

Abstract A critical comparison of commercial with laboratory-mad columns has revealed important new facts. Whereas commercial columns are now preferentially made of fused silica, there are strong arguments for retaining glass for laboratory- mad capillary columns. In parallel with the intense development of commercial columns there has been a considerable increase in interest in making individual columns. A primary reason may be that the study and optimization of new analytical applications require rapid and flexible column tailoring, which is not available from commercial manufacturers. The laboratory preparation on inert columns has recently been facilitated. Simpler and safer preparation techniques have become available, which permit non- specialized laboratories to include column preparation in their regular analytical activities. To support such laboratories, a detailed and comprehensive description of recent column preparation techniques is presented.

In vivo metabolism of epothilone B in tumor-bearing nude mice: identification of three new epothilone B metabolites by capillary high-pressure liquid chromatography/mass spectrometry/tandem mass spectrometry

Epothilone B is a 16-membered macrolide produced by the myxobacterium Sorangium cellulosum and is currently under clinical investigation. Experimentally, epothilone B demonstrates potent antiproliferative activity at the nanomolar level in vitro, and potent regression-producing antitumor activity in vivo, similar to paclitaxel (Taxol). In order to foster the design of improved derivatives, the potential biotransformation products of epothilone B formed in liver of tumor-bearing mice after intravenous administration of 10 mg/kg were characterized in an early stage of compound development. Solely on the basis of capillary high-performance liquid chromatography, combined either with electrospray tandem mass spectrometry (in precursor and product ion scan mode) and single analyzing time-of-flight mass spectrometry (H/D exchange and accurate mass measurement), three main metabolites could be detected. The three metabolites, formed by the liver, have in common that the epoxide ring was hydrolyzed and that the macrocyclic lactone ring was opened to the acid. In two cases it is assumed that open-chain intermediates re-cycled either to a lactone or, after conjugation with taurine, to the respective lactam. The proposed structures were additionally supported by the determination of the number of the exchangeable hydrogen atoms and by confirmation of the proposed elemental composition by exact mass measurement.

Parallel flame ionization detection-total ion current recording in capillary gas chromatography-chemical-ionization mass spectrometry

Abstract The need for independent dual detection in the analysis of complex mixtures by gas chromatography-chemical-ionization mass spectrometry is illustrated. Monitoring of the gas chromatographic effluent by means of universal detection and unaltered uniform response by flame ionization detection is a desirable supplement to the customary, highly selective, monitoring of the total ion current, especially in consecutive analyses of the same sample, where the chemical-ionization reagent gas is varied. Since the gas chromatograms obtained by flame ionization detection are unaffected by changes in chemical-ionization conditions, they provide a safer common basis for interrelating spectral data from successive runs than do the total ion current records. For the special case of reconstructed gas chromatograms generated by computers from digitized total ion current values, direct correlations are conveniently achieved by calibrating both the flame ionization detection and total ion current records in spectrum index numbers through the use of an event marker, triggered by the data system.

Characterization of a novel diclofenac metabolite in human urine by capillary gas chromatography-negative chemical ionization mass spectrometry.

A sensitive analytical method was developed to characterize diclofenac metabolites in small amounts of body fluids. Desalted and lyophilized urine samples were extracted with supercritical carbon dioxide directly or after acidic hydrolysis. The extracts were derivatized with N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide. The derivatives were separated by capillary gas chromatography and identified by negative chemical ionization mass spectrometry. Full mass spectra were obtained at a level of 1.10(-9) g/ml. With direct extraction, the metabolites could be analysed in one step as open-chained acids and as (cyclic) oxindoles. By acidic hydrolysis the conjugates were transformed to the oxindoles. With both methods, a new main metabolite, [2-[2,6-dichloro-4-hydroxy-3-methoxyphenyl)amino]phenyl]acetic acid, was identified The mechanism of its formation is discussed.

Pharmacokinetics and metabolism of formestane in breast cancer patients

Formestane (Lentaron(R), 4-hydroxyandrostenedione) is a steroidal aromatase inhibitor used for treatment of advanced breast cancer. Clinically, it is administered as a depot form once fortnightly by intramuscular (i.m.) injection. To investigate the pharmacokinetics, bioavailability and metabolism of the drug, seven patients received single 250 mg i.m. doses of commercial formestane on Days 0, 21, 35, 49 and 63 of this trial. On Day 63, three of the patients received an additional single intravenous (i.v.) pulse dose of 1 mg of 14C-labelled formestane. The plasma kinetics after i.m. dosing confirmed a sustained release of formestane from the site of injection. Within 24-48 h of the first dose, the circulating drug reached a C(max) of 48.0+/-20.9 nmol/l (mean+/-S.D.; N=7). At the end of the dosing interval, after 14 days, the plasma concentration was still at 2.3+/-1.8 nmol/l. The kinetic variables did not significantly change during prolonged treatment. Intramuscular doses appear to be fully bioavailable. Following i.v. injection of 14C-formestane, the unchanged drug disappeared rapidly from plasma, the terminal elimination half-life being 18+/-2 min (N=3). Plasma clearance, CL was 4.2+/-1.3 l/(h kg) and the terminal distribution volume V(z) was 1.8+/-0.5 l/kg. The drug is mainly eliminated by metabolism, renal excretion of metabolites accounting for 95% of dose. The excretory balance of 14C-compounds in urine and faeces totals up to 98.9+/-0.8% of the i.v. dose after 168 h. The 14C-compounds in plasma and urine were separated by HPLC, and three major metabolites were submitted to structural analysis by MS, NMR and UV spectroscopy. One of the metabolites is the direct 4-O-glucuronide of formestane. The other two represent 3-O-sulfates of the exocons 3beta,4beta-dihydroxy-5alpha-androstane-17-one and 3alpha,4beta-dihydroxy-5alpha-androstane-17-one, their ratio being 7:3. These exocons are formed by stereoselective 3-keto reduction, accompanied by reduction of the 4,5-enol function. The exocons do not inhibit human placental aromatase activity in vitro.

Determination of the GABAB receptor agonist CGP 44532 (3-amino-2-hydroxypropylmethylphosphinic acid) in rat plasma after pre-column derivatization by micro-high-performance liquid chromatography combined with negative electrospray tandem mass spectrometry

Abstract An assay, based on pre-column derivatization and micro-high-performance liquid chromatography–tandem mass spectrometry, was developed for the determination of the GABA B agonist CGP 44532 in rat plasma. CGP 44532, a highly polar 3-amino-2( S )-hydroxypropylmethylphosphinic acid, presented difficulties in developing a chromatographic method for the analysis of the compound in rat plasma. Instead of analyzing the target compound directly, it was derivatized prior to separation to a 4-nitrobenzylcarbamate isopropyliden derivative. In order to reach the required quantitation limit, on-line solid-phase extraction was utilized for sample clean-up and reversed-phase micro-column high-performance liquid chromatography, for separation of the plasma samples. The separated compounds were detected by negative electrospray tandem mass spectrometry in selected reaction monitoring mode. The derivatives show good chromatographic and mass spectrometric properties and both the target compound and the internal standard, could be eluted as symmetrical peaks with good signal/noise ratio. The MS–MS detection was selective and sensitive due to the straight fragmentation pattern. After injection of 200-μl sample aliquots, the limit of quantification was 10 ng ml −1 . The analytical assay is useable in the range of 10–500 ng ml −1 .

Special ergolines are highly selective, potent antagonists of the chemokine receptor CXCR3: discovery, characterization and preliminary SAR of a promising lead.

The special ergoline 1 is a highly potent, selective antagonist of the chemokine receptor CXCR3. The surprising selectivity of this LSD-related compound can be explained by different electronic and steric properties of the ergoline core structure caused by the urea portion of the molecule. Discovery, biopharmaceutical properties and first derivatives of this promising lead compound are discussed.

Rapid characterization of artemether and its in vitro metabolites on incubation with bovine hemoglobin, rat blood and dog blood by capillary gas chromatography–chemical ionization mass spectrometry

A fast and sensitive analytical method was developed to characterize artemether and its metabolites in small amounts in body fluids. The extracts were derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide, separated on an optimized capillary gas chromatographic system and identified by chemical ionization mass spectrometry by using ammonia as reagent gas. The analytical assay is demonstrated on samples extracted from bovine hemoglobin, rat blood and dog blood. Full mass spectra of artemether and three metabolites were obtained at a level of 1 x 10(-6) g/ml.

Preparation of glass and fused-silica capillary columns coated with immobilized XE-60

Revetement statique par une solution de XE-60 et de peroxyde de cumyle, puis immobilisation par traitement thermique. La stabilite de la phase est ainsi augmentee

GC/MS/MS studies of unusual chemical ionization (CI) processes with a triple stage quadrupole (TSQ) mass spectrometer

Abstract For 2,2,6,6-tetramethylcyclohexanone (I), the CI(CH4)-induced fragmentation of MH+ into Me2C=O N + H and [MH - H2O]+ ions is shown to proceed through common intermediates via two interconnected paths. Route A (ca. 75% of either fragment ion current/ involves sequential [1,2]-methyl migration, ring contraction and double [1.2]-methyl migration, route B (ca. 25%) may proceed via [1,2]-methyl migration, [1,21-hydroxyl migration, ring contraction and double [1,2]-methyl migration. A study of the effect of methyl substitution on the relative ease of these routes by GC/MS/MS analysis of mixtures of the lower methyl homologues of I sheds additional light on the mechanisms.