Per Olof Edlund

Determination of coenzyme Q10, α-tocopherol and cholesterol in biological samples by coupled-column liquid chromatography with coulometric and ultraviolet detection

Coenzyme (Co) Q10, Co Q10H2, alpha-tocopherol and cholesterol were dissociated from lipoproteins in plasma by treatment with 1-propanol. The supernatant obtained was injected directly for determination of Co Q10 and Co Q10H2. Precolumn reduction with borohydride was used for determination of total Co Q10 simultaneously with alpha-tocopherol and cholesterol. Total Co Q10 in freeze-dried myocardial biopsies was determined after extraction with 1-propanol and oxidation of Co Q10H2 with ferric chloride. The chromatographic system comprised two reversed-phase columns and a three-electrode coulometric detector and a UV detector coupled in series. A pre-fractionation on the first column protected the coulometric detector from contamination and reduced the time for analysis by eliminating strongly retained solutes. The coulometric electrodes were operated in the oxidation-reduction-oxidation mode, and the last electrode was used for detection of alpha-tocopherol, Co Q10 and Co Q10H2, while cholesterol was detected by UV at 215 nm. The fast isolation procedure made it possible to determine the reduced and oxidized forms of Co Q10 in plasma. Quantitative recoveries were obtained for all the analytes studied and normal levels were determined with a coefficient of variation of 2-3%.

Determination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry.

Monitoring steroid use requires an understanding of the metabolism in the species in question and development of sensitive methods for screening of the steroid or its metabolites in urine. Qualitative information for confirmation of methandrostenolone and identification of its metabolites was primarily obtained by coupled-column high-performance liquid chromatography-tandem mass spectrometry. The steroids and a sulphuric acid conjugate were isolated and identified by their daughter ion mass spectra in the urine of both man and the horse following administration of methandrostenolone. Spontaneous hydrolysis of methandrostenolone sulphate gave 17-epimethandrostenolone and several dehydration products. This reaction had a half-life of 16 min in equine urine at 27 degrees C. Mono- and dihydroxylated metabolites were also identified. Several screening methods were evaluated for detection and confirmation of methandrostenolone use including thin-layer chromatography and high-performance liquid chromatography. Coupled-column liquid chromatography was used for automated clean-up of analytes difficult to isolate by manual methods. The recovery of methandrostenolone was 101 +/- 3.3% (mean +/- S.D.) at 6.5 ng/ml and both methandrostenolone and 17-epimethandrostenolone were quantified in urine by ultraviolet detection up to six days after a 250-mg intramuscular dose to a horse. The utility of on-line tandem mass spectrometry for confirmation of suspected metabolites is also shown.

Determination of ergot alkaloids in plasma by high-performance liquid chromatography and fluorescence detection

Liquid chromatographic methods for the determination of ergotamine and methylergometrine in plasma have been developed. The samples are extracted with an organic solvent at pH 9.0 cleaned by extractions and finally injected on an ODS-Hypersil reversed-phase column with acetonitrile-ammonium carbonate buffer as the mobile phase. THe polarity of solvents used for extraction and the mobile phase are varied with the compounds of interest. Ergocristine is used as internal standard for ergotamine, and methysergide for the determination of methylergometrine. The stability of samples and standard solutions for calibration are discussed. Conditions for high selectivity and sensitivity of detection are given. Concentrations down to 100 pg/ml of plasma can be detected with a 3-ml sample.

Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent coorelation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS. (Journal of Biomolecular Screening 2003:247-256)

Rapid determination of methandrostenolone in equine urine by isotope dilution liquid chromatography-tandem mass spectrometry

Urine samples were spiked with [17-methyl-2H3]methandrostenolone as internal standard and extracted with a mixture of dichloromethane and cyclohexane. The organic phase was concentrated and injected onto a short octyl-silica column (30 mm x 4.6 mm I.D.) for separation of methandrostenolone and 17-epimethandrostenolone. The effluent from the column was connected to a Sciex TAGA 6000E triple quadrupole mass spectrometer equipped with an atmospheric pressure ion source for sampling of ions generated by a heated pneumatic nebulizer with corona discharge ionization. This ion source produced abundant [M + H]+ ions and a weak fragment ion due to loss of water. The protonated molecular ions at m/z 301 and 304 for methandrostenolone, 17-epimethandrostenolone and the internal standard were transmitted to the second quadrupole for collision-induced dissociation. Quantification was obtained by selected reaction monitoring of three daughter ions. Methandrostenolone and 17-epimethandrostenolone were separated by liquid chromatography, but gave identical mass spectra. The method detection limit by injection of a urine extract corresponding to 2.8 ml urine was 180 pg/ml at the 99% confidence level. The precision (relative standard deviation) was 3% at the 16 ng/ml level and the linear dynamic range was at least 3 orders of magnitude. Screening for unknown metabolites in urine after administration of methandrostenolone to horses and humans was accomplished by a parent ion scan of m/z 121, a fragment corresponding to the intact A-ring of the steroids.

Determination of melengestrol acetate in bovine tissues by automated coupled-column normal-phase high-performance liquid chromatography

A method has been developed for the determination of melengestrol acetate in bovine tissues at lower levels than previously reported. Liquid-liquid extraction of tissue homogenates provided crude clean-up while final isolation, screening, and quantification was done on-line with an automated, normal-phase, coupled-column high-performance liquid chromatographic system. The chromatographic system included phenyl and silica analytical columns for the purposes of isolation and final separation, respectively. These columns provided a large difference in selectivity when operated under normal-phase conditions which allowed for the efficient isolation of melengestrol acetate from the complex tissue extracts. Mobile phases were composed of hexane and dichloromethane modified with methanol and water. Transfer and enrichment of the analyte from the primary phenyl column to the silica column was via a short (12 mm x 4 mm I.D.) silica column. Regeneration and equilibration of the phenyl column was performed after the injection of each tissue extract and was accomplished simultaneously while analytical separation occurred on the final silica column. Routing of the mobile phases and regeneration solvent was performed with automated switching valves. The total time required for each analysis was 12 min. Quantification is demonstrated using external standards with UV detection at 287 nm. The overall recovery of the method was 86% with a coefficient of variation of 9.84% at the 10 ppb [the American billion (10(9] is used in this article] level in bovine liver extracts.