W. D. van Dongen

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples I. Determination of clenbuterol in urine

The potential of the direct coupling of solid-phase extraction (SPE) with mass spectrometry (MS) for the analysis of biological samples is demonstrated. For SPE a cartridge exchanger is used and the eluate is directly introduced into the mass spectrometer. This system has been investigated for the determination of clenbuterol in urine. With mixed-mode cartridges, a considerable ion suppression has been obtained. The mass spectrum at the elution time of clenbuterol is dominated by that of creatinine and adduct formation of clenbuterol and creatinine has been observed. The whole procedure including injection of 1 ml urine, washing and desorption has been developed with cartridges containing 8-microm C18-bonded silica. If only a single MS step is used, the selectivity and, therefore, the sensitivity are insufficient. The detection limit is about 100 ng/ml. However, with atmospheric pressure chemical ionisation and the tandem MS mode the detection limit has been decreased to about 2 ng/ml and the ion suppression is only about 10%. For the electrospray ionisation the detection limit is about 10-times higher and the ion suppression is less favourable. The repeatability for the SPE-MS-MS procedure was 6.5% at 10 ng/ml (n=5) and the difference between the response factors at 10 ng/ml and 100 ng/ml was only 2.5%. The MS behaviour of clenbuterol and the matrix under the present conditions is discussed.

Comparison between collision induced dissociation of electrosprayed protonated peptides in the up-front source region and in a low-energy collision cell

A systematic comparison between the up-front Collision induced dissociation (CID) mass spectra and low-energy CID tandem mass spectra from twenty-one singly and/or doubly charged peptides has been made. CID spectra of the peptides were recorded at different electrode voltages in the up-front source region of a single quadrupole instrument and different collision energies in the collision cell of a tandem quadrupole instrument. It was observed that up-front CID and low-energy CID yielded comparable product ion spectra from protonated peptides, and that the instrumental settings necessary for obtaining comparable CID mass spectra from the two methods are correlated.

The B1-fragment Ion from Protonated Glycine is an Electrostatically-bound Ion/Molecule Complex of CH2=NH+2 and CO

The stability of the B1-ion from glycylglycine dipeptides was studied using collision-induced dissociation experiments and ab initio calculations. This study reveals that the B1-type ion from glycine should be considered as an electrostatically bound ion/moecule complex, which dissociates into a glycine immonium ion (A1) and a carbon monoxide molecule. The stability of this complex has been calculated at various levels of the theory. At the highest level of theory (#RHF/6-311G**) it is found that the energy requirement, including zero-point vibrational energy correction, for dissociation into a glycine immonium A1 ion and a neutral carbon monoxide CO molecule is −4.0 kJ mol−1. Furthermore it could be concluded that B1-ions from glycine are stable if they are present as N-acylated species. The formation of stable B ions requires at least two carbonyl groups in the peptide backbone moiety.

Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine κ-casein

The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine κ-casein was studied. A 4-h digest (pH 6.2, 15°C) of κ-casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1–160, 1–151, 1–95 and 1–79 of κ-casein, whereas the main LMM products found were the 161–169 and 152–160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160–161 and 151–152 peptide bonds. Two minor LMM products were identified as the fragments 96–104 and 103–106, indicating additional cleavage at positions 102–103, 104–105 and 106–107 of the sequence. Also several peptide bonds within the 161–169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococaal CEPI, CEPIII and several mixed-type proteinases all act on the peptide bonds at positions 79–80 and 95–96. However, the C-terminal region of the κ-casein sequence is the exclusive target of the CEPIII-aand, to variable extents, of the mixed-type enzymes.

The use of mobile phase additives in the determination of 55 (polar) pesticides by column liquid chromatography-thermospray-mass spectrometry

Abstract The effect of various additives (ammonium acetate, ammonium formate, triethyl-and tripropylammonium formate and chloroacetonitrile) on the sensitivity and selectivity in liquid chromatography-thermospray-mass spectrometry (positive and negative ion mode) is studied for a group of 55 pesticides including phenoxy and carboxylic acids, hydroxy-keto-lactones, substituted amides, triazines, (thio)carbamates, phenylureas, substituted phenols, organo-phosphorus compounds, thiocyanates and anilines. In the positive ion mode the base peaks correspond to [M + H]+ and [M + NH4]+ for the majority of the test compounds. Fragmentation is observed in some isolated cases only. In the negative ion mode processes like (dissociative) electron capture and ion-molecule reactions take place. Fragment ions such as [M-H-CONCH3]− for the carbamates, [M-C3H6O]− for the hydroxy-keto-lactones and [M-H]− for the chlorinated phenoxy acids are observed. Depending on the additive used, adduct ions such as [M + CH3COO]−, [M + HC...

Binding of selected carbohydrates to apo-concanavalin A studied by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry was used to investigate the binding of selected carbohydrates to different multimeric forms of apo-concanavalin A (apo-Con A). Information on the stoichiometry of the different quaternary Con A complexes, bound to high-affinity carbohydrate ligands, could be derived from the mass spectra. It was observed that specific carbohydrate binding takes place with metal-free apo-Con A. The data revealed that the apo-dimer and -tetramer (which are both present in solution) have similar affinities for the studied carbohydrates.

Rapid analysis of enzymatic digests of a bacterial protease of the subtilisin type and a “bio-engineered” variant by high-performance liquid chromatography—frit fast atom bombardment mass spectrometry

Amino acid sequencing of a subtilisin-type bacterial protease and a bio-engineered variant was carried out by investigating various enzymatic digests using HPLC-frit fast atom bombardment MS methods. The fast atom bombardment mass spectral data allowed rapid identification of the enzymatically generated peptides and differentiation between both proteins. The feasibility of determining the positions and nature of mutations in the amino acid sequence depends mainly on the size of the peptides containing the modifications.