Continuous-flow Fast-atom Bombardment Mass Spectrometry Research Articles

Chromatographic behaviour of the chromium(III) complex of 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone

The separation of the two geometrical isomers of the chromium(III) complex of 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone was carried out using acetonitrile–water (90:10, v/v) and n-hexane–tetrahydrofuran (70:30, v/v) as the mobile phases for an ODS column and a silica gel column, respectively. Identification of the isomers was performed using a high-performance liquid chromatography–continuous-flow fast atom bombardment–mass spectrometry system. It was found that the fac complex eluted first, followed by the mer complex on the ODS column.

HPLC-continuous-flow fast atom bombardment mass spectrometry (HPLC-CFFAB) — a convenient method for the analysis of bile acids in bile and serum

In order to analyse bile acids in biological matrices, e.g. rat bile and human serum, high performance liquid chromatography (HPLC) was coupled to continuous-flow fast atom bombardment mass spectrometry (CFFAB-MS). A gradient elution system which had already proved to be well suited for the quantitative determination of conjugated bile acids in bile was modified to allow HPLC-CFFAB-MS-coupling. Due to the sensitivity of this coupling method it is possible to obtain more information about the biliary bile acid pattern and species-specific secondary bile acids. Furthermore, we were able to identify obviously unknown bile acid species in rat bile which most likely classify as mono-oxo and di-oxo-taurocholates (MW 513 Da, 511 Da) and mono-oxo-glycocholates (MW 463 Da). In the present study we show that using this system it is possible to determine both conjugated and unconjugated as well as sulfated bile acids, without time consuming group separation and derivatization, from rat bile and human serum. In addition, it is suggested that the method presented here should be considered for use in routine analysis.

Investigation of mammalian lignan precursors in flax seed: First evidence of secoisolariciresinol diglucoside in two isomeric forms by liquid chromatography/mass spectrometry

A strong research effort has been devoted in recent years to the mammalian lignans enterolactone and enterodiol because of their claimed protective effects against breast and colon cancer. Some plant lignans appear to be precursors of these molecules. This paper reports a novel identification procedure for one of these precursors, the secoisolariciresinol diglucoside, in the extracts of defatted flax-seed meal. The procedure is based on high-performance liquid chromatography coupled with either ionspray or continuous-flow fast-atom bombardment mass spectrometry. The application of these techniques allowed the identification in flax seed of two isomeric forms of this precursor for the first time. The isomers are reasonable the two diastereoisomers, due to the two equivalent stereogenic centers present in the molecule.

Identification and Measurement of Food and Cosmetic Dyes in a Municipal Wastewater Treatment Plant

Acid Blue 9, Acid Violet 17, Quinoline Yellow, Acid Red 51, Acid Red 87, and Acid Red 92 along with N-benzyl-N-ethylaniline sulfonic acid (BEASA), a synthetic precursor, were identified and measured in colored wastewater samples from a municipal treatment plant. Continuous-flow fast-atom bombardment mass spectrometry was used to analyze BEASA. Liquid chromatography with ultraviolet detection was used to analyze the other dyes, but its lack of selectivity required prior isolation of the analytes from interfering compounds by solid-phase extraction onto C[sub 18] extraction disks and onto cartridges packed with strong anion-exchange resins. The xanthene dyes (Acid Red 51, 87, and 92) were found in low parts per billion (ppb) concentrations in the plant influent and were rapidly removed by adsorption to sludge. Acid Red 92 was found to be over 35 times more concentrated on secondary sludge than in the corresponding liquid samples, indicating high levels of accumulation. The other dyes and BEASA were found in hundred ppb concentrations in both the influent and the effluent of the plant, indicating a resistance to both degradation and removal by sorption. 32 refs., 4 figs., 3 tabs.

Probing high order structure of proteins by fast-atom bombardment mass spectrometry

During the past decade, numerous investigations have demonstrated that the rate at which amide hydrogens located at peptide linkages undergo isotopic exchange is a sensitive probe of the high order structure and dynamics of proteins. The present investigation demonstrates that microbore high-performance liquid chromatography (HPLC) continuous-flow fast-atom bombardment mass spectrometry (FABMS) can be used to accurately quantify deuterium located at peptide linkages in short segments of large proteins. This result is important because it demonstrates the feasibility of using mass spectrometry as a tool for studying the high order structure and dynamics of large proteins. Following a period of deuterium exchange-in, a protein was placed into slow-exchange conditions and fragmented into peptides with pepsin. The digest was analyzed by continuous-flow HPLC FABMS to determine the molecular weights of the peptides, from which the number of deuterons located at the peptide linkages could be deduced. The HPLC step was used both to fractionate the peptides according to their hydrophobicities and to remove through back-exchange all deuterium except that located at peptide amide linkages. This approach has been applied to α-crystallin, a lens protein composed of two gene products with monomer molecular weights of 20 kDa and an aggregate molecular weight approaching 1000 kDa. Results from this study show that some of the peptide amide hydrogens in α A-crystallin exchange very rapidly (k > 10 h −1) while others exchange very slowly (k < 10 −3 h −1). The ability not only to detect that a conformational change has occurred, but also to identify the specific regions within the protein where the change occurred, was demonstrated by measuring changes in the exchange rates within these regions as the deuterium exchange-in temperature was increased from 10 to 80 °C.

Analysis of cyclic nucleotide-related enzymes by continuous-flow fast-atom bombardment mass spectrometry.

Continuous-flow fast-atom bombardment mass spectrometry has been developed to directly monitor cyclic nucleotide (substrate) and its product levels from an on-going phosphodiesterase reaction. Analysis of cAMP and cCMP phosphodiesterase incubates have been performed where the temporal evolution of the enzymic reaction is monitored and the effect of enzyme concentration upon the rate of reaction determined. Quantitative data on the enzyme kinetics have been obtained, in the form of Lineweaver-Burke plots, that are shown to correlate well with well-established radiometric methods.

Fungal metabolites. Part 5. Rapid structure elucidation of antibiotic peptides, minor components of Trichosporin Bs from Trichoderma polysporum. Application of linked-scan and continuous-flow fast-atom bombardment mass spectrometry

From the minor-components mixture of Trichosporin Bs (TS-Bs) produced by Trichoderma polysporum, ten components could be elucidated by means of linked-scan and continuous-flow fastatom bombardment. The combination of their complementary results made the rapid structure elucidation of the minor components possible without isolation and purification. The identified compounds are antibiotic eicosapeptides and have very similar structures with relative molecular masses of ∼2000. Four new peptide groups were found. In their structures, the first group has Gly at the amino acid position 3 in contrast to the known TS-Bs, the second one has Vxx (valine or isovaline) at position 12, and the third and the fourth ones have Ala at position 13 or 17, respectively.

Determination of the coumarin derivative cloricromene acid in rabbit plasma and platelets

Two methods for the determination of cloricromene acid in biological samples are described. Cloricromene acid is a catabolite of cloricromene, a coumarin derivative which is active in the cardiovascular system. After oral administration of cloricromene to a rabbit, plasma and platelets were taken at different times and cloricromene acid was then isolated by solid-phase extraction with Sep-Pak C 18 cartridges using acetonitrile—tetrahydrofuran—20% aqueous acetic acid (15:11:74, v/v/v) as eluent. The analyses were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with fluorescence detection with excitation at 310 nm and emission at 390 nm. The limit of quantification by RP-HPLC was about 50 pg. The catabolite in the plasma was identified by continuous-flow fast atom bombardment mass spectrometry (CF-FAB-MS), also used as a complementary means of RP-HPLC determination. The results obtained by RP-HPLC and CF-FAB-MS showed good agreement.

Analysis of saxitoxin in urine by continuous-flow fast-atom bombardment mass spectrometry.

An improved method of saxitoxin analysis in urine using continuous-flow fast-atom bombardment mass spectrometry was developed. Parameters studied were matrix composition, matrix flow, temperature of probe tip, probe-tip design and sample extraction. Optimal detection was obtained using the following matrix composition: 5% glycerol, 0.5% acetic acid, 0.025% sodium dodecylsulfate, 0.1% polyethylene glycol (PEG) 400 and 0.5% PEG 300; probe-tip temperature: (approximately 55 degrees C); flow rate: 5 or 8 microL per min.; probe tip: Olson-Hogge design. The STX standard was detected at 200 pg with signal-to-noise ratio of 11. The percent recovery of saxitoxin from human urine after clean-up on a weak cation exchange column was 75%.

Quantitative analysis of the molecular species of monosialogangliosides by continuous-flow fast-atom bombardment mass spectrometry

Negative-ion continuous-flow fast-atom bombardment mass spectrometry was evaluated as a means for the quantitative analysis of N-acetylneuraminyl-galactosyl-glucosyl-ceramide (NeuAc-GM3) and N-acetylgalactosaminyl-(N-acetylneuraminyl)galactosyl-glucosyl-ceramide (NeuAc-GM2). This study was carried out on a 7070-EQ mass spectrometer (VG Analytical, Manchester, UK) using a home-made continuous-flow fast-atom bombardment probe with a mixture of methanol + water + triethanolamine (70:27:3, v/v/v) as the mobile phase. Utilizing 100 ng of acetyl-lysogalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)g alactosyl-glucosyl-ceramide (acetyl-lysoGM1) as an internal standard, standard curves for NeuAc-GM3 d18:1-16:0, NeuAc-GM3 d18:1-18:0 and Neuac-GM2 d18:1-18:0 were found to be linear over the range 5-250 ng, with associated correlation coefficients of 0.990-0.997. The lower limit of detection was found to be 2.5 ng. Satisfactory results could also be obtained when the calibration curves were derived from the deprotonated molecular ions of a mixture of the NeuAc-GM2 and NeuAc-GM3 classes. Using this approach, quantitative determination of NeuAc-GM3 d18:1-16:0 from rat adrenal gland was performed using N-acetylneuraminic acid assay as a test control. We found 278 +/- 36 ng of this species in 1 mg of tissue (three replicate experiments). The procedure represents a sensitive method for the quantitation of monosialogangliosides and its capability to give molecular species information.

Approach to studying proteinase specificity by continuous-flow fast atom bombardment mass spectrometry and high-performance liquid chromatography combined with photodiode-array ultraviolet detection

Fast atom bombardment mass spectrometry (FAB-MS) and high-performance liquid chromatography using a photodiode-array ultraviolet detector were applied to study a dynorphin-converting endopeptidase from the human pituitary gland. The specificity of the enzyme was tested towards various opioid peptides derived from the prodynorphin precursor, i.e. dynorphin A, dynorphin B and α-neoendorphin. Peptide fragments were analysed directly by continuous-flow FAB-MS and those containing aromatic amino acids were detected independently by the photodiode-array ultraviolet detector. The results obtained suggest a similar processing of these structure-related substrates and it appears that the enzyme recognizes the dibasic stretch in their sequence. It is also clear from this study that the combination of the above techniques provides a powerful tool for studies of enzymatic conversion among the prodynorphin-derived peptides and it should be applicable to studies of similar mechanisms in other peptide systems.

Analysis of bovine β-casein tryptic digest by continuous-flow fast-atom bombardment mass spectrometry

Liquid chromatography/fast-atom bombardment mass spectrometry was used to partially confirm the amino-acid sequence of the protein, beta-casein. The study demonstrates that the technique is capable of the rapid and accurate identification of peptide fragments from tryptic digests and that chromatographic integrity is maintained during the analysis. The power of the technique derives from the ability to determine both the retention time and the molecular weight of the eluting components. Each of the components yields a prominent pseudo-molecular ion (MH+), the majority exhibiting sufficient fragmentation to confirm their structure unambiguously.

Analysis of tyrosine- and methionine-containing neuropeptides by fast atom bombardment mass spectrometry

A simple and unambiguous method for the detection of the amino acids tyrosine and methionine in peptide structures has been developed. The procedure, which was applied in studies of opioid peptides, is based on continuous-flow fast atom bombardment mass spectrometry (CF-FAB-MS) following chemical modification of the residue to be analyzed. Thus, for the detection of tyrosine, modification reactions such as acetylation or non-radioactive iodination were performed prior to analysis by CF-FAB-MS. O-Acetylation of the tyrosine residue with N-acetylimidazole was accompanied by a shift of 42 Da in the molecular mass of the peptide under investigation. This modification was reversed by treatment with hydroxylamine hydrochloride. Incorporation of iodine resulted in a molecular weight shift of 126 Da per iodine atom. Methionine residues were detected in methionine-enkephalin-containing peptides following S-oxidation with hydrogen peroxide. The procedures described may have a wide application in peptide chemistry, particularly for the identification of peptide fragments containing the above residues, e.g. in studies of processing or degradation of the enkephalins or other neuropeptides ( e.g. endorphins and tachykinins).

Phase-system switching in liquid chromatography/continuous-flow fast-atom bombardment mass spectrometry

A new, attractive application of the phase-system switching (PSS) technique is demonstrated. The PSS system is used in combination with liquid chromatography coupled to continuous-flow fast-atom bombardment mass spectrometry (LC/CF-FABMS) to achieve flow-rate reduction from 1.0 mL/min in the high-performace liquid chromatography (HPLC) system to 15μL/min going into the mass spectrometer. In PSS, a compound of interest, in this case erythromycin 2′-ethylsuccinate, is enriched after the analytical separation, on a short trapping column, from which it is desorbed subsequently to the LC/MS interface. Optimal mobile phases and flow-rates can be used for the HPLC separation as well as for the mass spectrometric detection. Desorption of the analyte is achieved either by using a flow-rate of 15 μL/min without a splitter or a flow-rate of 230 μL/min with a splitter. Compared to the LC/CF-FAB MS methods using a conventional bore HPLC column and a splitter, improved detection limits for the model compound were achieved using the PSS method.

Optimisation of continuous flow fast atom bombardment mass spectrometry for bionalysis

Fast atom bombardment mass spectrometry (FAB-MS) has been employed very successfully over the last years for the analysis of polar and thermally- labile compounds which are frequently encountered in bioanalysis. The recently developed method of continuous flow FAB-MS (CF-FAB) as a liquid chromatography-MS interface and ionization method has opened up a new area of research in the analysis of biomolecules such as peptides and proteins. Ion suppression effects observed in standard FAB analysis seem to be reduced, and increased signal-to-noise gives better sensitivity for qualification. The optimisation of CF-FAB on the MAT90 magnetic sector spectrometr will be descussed. Application in bioanaysis, e.g., peptide analysis in conjunction with linked scan techniques, will also be reported.

Microbore high-performance liquid chromatography—mass spectrometry for the analysis of proteolytic digests by continuous-flow fast-atom bombardment mass spectrometry

Microbore high-performance liquid chromatographic (HPLC) techniques have been combined with fast atom bombardment mass spectrometry (FAB-MS) for the mass-specific detection of mixtures of peptides produced by proteolytic hydrolysis of proteins. The continuous-flow FAB interface has been utilized for direct coupling of the microbore HPLC system and the mass spectrometer. Conditions are reported for the effective separation of 100 pmol of peptides at flow-rates of 5 μl/min with acetonitrile gradients and 1-mm I.D. C 8 columns. A comparison is also made between columns of 5 and 25 cm lengths for the separation of peptide mixtures. Data are presented for the separation of peptides on a slurry-packed C 18 fused-silica capillary column with the continuous-flow HPLC—FAB-MS interface at flow-rates of 3 μl/min.

Continuous-flow fast atom bombardment-mass spectrometry of permethylated oligosaccharides: A comparative study of direct mixture analysis with packed capillary column liquid chromatography-fast atom bombardment-mass spectrometry

Conventional positive fast atom bombardment (FAB) and continuous-flow FAB analysis were carried out with permethylated lacto- N-tetraose. This latter method, a new approach, has been used to analyze a mixture of permethylated oligosaccharides by liquid chromatography—mass spectrometry (LC-MS) with a packed capillary fused-silica column and the continuous-flow probe as interface. Under these conditions, we found the continuous-flow probe to be superior to the conventional probe because its low matrix level increased the signal-to-noise ratio. The analysis of the mixture of permethylated oligosaccharide alditols obtained from hen ovomucoid by LC-MS using the continous-flow probe as interface is described.