Jan Bosman

The Effect of Preanalytical Factors on Stability of the Proteome and Selected Metabolites in Cerebrospinal Fluid (CSF)

To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples. The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.

Multiresidue analysis of β2-agonists in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection

Abstract Various β2-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four β-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The β-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the β-agonists were eluted with ammoniated ethanol–hexane. The extract was analysed with an HPLC method with electrochemical detection. The β-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90–105% vs. 65–75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that β2-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.

A study of the separation of enantiomers of some aromatic carboxylic acids by high-performance liquid chromatography on a β-cyclodextrin-bonded stationary phase

Abstract The enantiomers of some aromatic carboxylic acids of biomedical interest were separated by high-performance liquid chromatography on a β-cyclodextin-bonded stationary phase. Evaluation of the system shows that the structure of the acids plays an important role in the possibility for separation of the enantiomers. Moreover, the pH as well as the buffer components of the mobile phase have a pronounced effect on the retention times and the selectivity of the system. In order to reduce tailing, the column temperature was increased; this resulted in better peak resolution. Anomalous behaviour was seen in the relation between retention time and sample concentration. At low amounts of injected sample the retention times increased, but this could not be ascribed to adsorption processes. This phenomenon caused great problems in the quantitative analysis of the two enantiomers with this system.

Optimization of the direct enantiomeric separation by high-performance liquid chromatography of the D-2 dopamine agonist N-0437 using response surface methodology and the multi-criteria decision-making method

Abstract The applicability of a Chiralcel OD column for the separation of the enantiomers of the racemic dopamine agonist 2- (N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437) was studied. The resolution between the enantiomers was described by means of the response surface methodology and optimum chromatographic conditions were found using Smildes multi-criteria decision-making technique. The variables studied were eluent composition, flow-rate and temperature. The effects of these variables on the retention time of the last-eluting enantiomer and the resolution between the enantiomers were examined. The optimum result was considered to be the highest resolution possible within a short retention time for the last-eluting enantiomer. It appeared that a decrease in temperature gave higher resolutions. With hexane-ethanol (95:5, v/v) as eluent at 10°C and a flow-rate of 0.5 ml/min, a resolution of 2.5 and a retention time of the last-eluting enantiomer of 23 min were obtained. Under these conditions calibration graphs for the (+)- and (−)-enantiomers were prepared using racemic N-0437.

INFLUENCE OF CHEMICAL-STRUCTURE OF TRICYCLIC TERTIARY DIMETHYLAMINES ON CHIRAL SEPARATION BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AFTER DERIVATIZATION WITH (-)-MENTHYLCHLOROFORMATE

Abstract In the present study (−)-menthylchloroformate was used as a chiral derivatizing agent for promethazine, trimeprazine, trimipramine and N-(2-dimethylaminopropyl)iminodibenzyl. (−)-Menthylchloroformate reacted with the tertiary dimethylamine moiety in these tricyclic antihistamines and antidepressants, resulting in the formation of diastereoisomers. Owing to the reaction conditions, during the derivatization with (−)-menthylchloroformate, the possibility of racemization had to be established. For this purpose different ratios of (+) and (−)-promethazine were prepared. Enantiomeric separation of these mixtures took place on a Chiral α AGP column or, after derivatization with (−)-menthylchloroformate, on a C18 column. The results from these two independent separation systems were compared with trace racemization. No racemization was found during the experiments. To study the effects of changes in the molecular structures of the tertiary dimethylamines on the chromatographic behavior of the derivatization products, four tertiary dimethylamines [promethazine, trimipramine, trimeprazine and N-(2-dimethylaminopropyl) iminodibenzyl] were derivatized and analyzed. With these amines the effects on resolution and capacity factor of replacing a phenothiazine ring by an iminodibenzyl ring or insertion of a carbon molecule between the chiral centre in the chain and the place where (−)-menthylchloroformate reacts were studied. Not only the distance between the chiral centres in the diastereoisomers (a longer distance caused less resolution and higher capacity factors) but also the kind of ring influenced resolution and capacity factor. Finally, the influence of eluent composition on resolution and capacity factor was studied. Three different mixtures of methanol and acetic acid were tested. More acetic acid in the eluent caused a better resolution and higher capacity factor. The higher capacity factor, however, resulted in unacceptable retention times.

DIRECT DETERMINATION OF THE ENANTIOMERIC PURITY OF OXYPHENONIUM USING CHIRAL HPLC WITH POST-COLUMN EXTRACTION DETECTION

SummaryA method is described for the determination of the enantiomeric purity (enantiomeric excess) of the anticholinergic drug oxyphenonium. The method for this quaternary ammonium compound is based on the direct HPLC analysis with a chiral stationary phase. Two kinds of α1-acid glycoprotein-bonded phases were used.For the detection a post-column extraction with fluorescence detection of the ion-pair counter ion dimethoxyantracene sulphonate was used.

Attempts to Obtain Separations of Chiral Anticholinergic Drugs

Where a drug has enantiomeric forms, each should be measurable in the same sample, as now investigated with oxyphenonium and some quaternary atropine analogues. Resolution was attempted with a chiral-HPLC system, containing d-camphorsulphonic acid as a mobile phase component or (cf. W.U. Pirkle’s work) with chiral stationary phases. Neither approach was successful, due to the relatively long distance between the chiral centre and the quaternized nitrogen.