Direct analysis of drugs by continuous-flow fast-atom bombardment and tandem mass spectrometry

The techniques of continuous-flow fast-atom bombardment (CF-FAB) and tandem mass spectrometry (MS/MS) are combined and applied to the analysis of small molecular mass drugs (mol.wt less than 500 Da). The approach involves the interfacing of a CF-FAB inlet with a triple-stage quadrupole mass spectrometer, enabling the acquisition of collision-activated decomposition mass spectra of the drugs after FAB ionization. The relationship between a stable sample surface on the CF-FAB probe tip and the quality of the mass spectrum is discussed, as are practical methods for obtaining and maintaining surface stability. CF-FAB MS/MS spectra for several drugs are presented, including penicillin G, phentolamine, cocaine and benzoylecgonine. Minimum detection limits range from 50-500 pg injected, depending on the compound. The reproducibility of the integrated areas of peaks from repetitive injections is approximately five per cent. Data are also presented for the direct CF-FAB MS/MS analysis of cocaine and benzoylecgonine in spiked urine samples.     

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%.     

Analysis of bovine beta-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 arachidonic-acid-containing molecular species in glycerophospholipid classes from rat kidney by fast-atom bombardment mass spectrometry

The qualitative analysis of the molecular species of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine from a rat kidney, by fast-atom bombardment mass spectrometry after separation and purification by liquid chromatography is described. The present results indicate that a high content of arachidonic-acid-containing molecular species within major glycerophospholipid classes is present in this biological sample.     

Capillary-zone electrophoresis/fast-atom bombardment mass spectrometry: design of an on-line coaxial continuous-flow interface

An on-line coaxial continuous-flow capillary-zone electrophoresis/fast-atom bombardment mass spectrometry (CZE/FAB-MS) interface is described. This interface is shown to be capable of acquiring mass spectra in an on-line fashion from low femtomole amounts of peptides while maintaining high (hundreds of thousands of plates) electrophoretic separation efficiencies. Active electrophoretic transport of the analytes directly to the FAB probe tip obviates the need for a transfer line from the end of the CZE capillary to this point, and thereby precludes the zone broadening that would otherwise occur both within such a transfer line and in the connections between the CZE column and the transfer line. The capability of acquiring an on-line tandem mass spectrometry (MS/MS) spectrum of an electrophoretically separated analyte using this interface is also demonstrated.     

Application of continuous-flow liquid chromatography/fast-atom bombardment mass spectrometry to the analysis of diagnostic acylcarnitines in human urine

Acylcarnitine profiles of diagnostic value were generated from the equivalent of 0.1 microL of raw urine using the continuous-flow liquid chromatography/fast-atom bombardment mass spectrometry (LC/FAB-MS) interface for sample introduction. Further analysis was accomplished by gradient LC/MS using a commercially available packed fused-silica microbore column.     

Characterization of phosphorylated amino acids by fast-atom bombardment mass spectrometry

Positive- and negative-ion fast-atom bombardment (FAB) mass spectrometry and linked-field scan techniques at constant B/E are used to characterize phosphorylated serine, threonine, and tyrosine amino acids. Abundant molecular ions are formed for all three amino acids in both modes of ionization. The dominant fragmentation is cleavage of the phosphate ester bond with charge retention in positive-ion FAB by the amino acid backbone and in the negative-ion mode by the phosphate group. The unique feature of positive-ion FAB mass spectra of phosphoserine and -threonine is the loss, from the ion [M + H]+, of a molecule of phosphoric acid (98 Da), whereas the corresponding tyrosine expels a HPO4 (96 Da) moiety to yield a stable phenylalanine ion.     

Multisample probe for fast-atom bombardment / mass spectrometry

An inexpensive multisample fast-atom bombardment (FAB) probe assembly was designed for high-throughput analysis of samples on a VG ZAB-SE mass spectrometer. The system consists of a vacuum lock system and a FAB probe whose tip contains five or more sample wells. The probe enters the mass spectrometer source region perpendicular to the secondary ion beam axis, The probe is maintained at high voltage on contact with a spring clip attached to the screen plate of the source block. Sample throughput with the multisample probe is twice that of a coaxial probe, with about twice the sensitivity and no sample cross contamination.     

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 αA-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.     

Structural determination of cyclitol derivatives by fast-atom bombardment tandem mass spectrometry

Three cyclitol derivatives were isolated from the marine sponge Sarcotragus sp. by reversed-phase high-performance liquid chromatography and analyzed by fast-atom bombardment mass spectrometry (FAB-MS). Their structural elucidation was carried out with FAB tandem mass spectrometry (FAB-MS/MS). FAB-MS spectra produced a significant abundance of the sodium adducts [M+Na]+ and [M+2Na-H]+ from a mixture of m-NBA and NaI. In addition, trifluoroacetylation of the cyclitol derivatives was used for confirmation of the presence of the cyclitol ring. High abundance [M-5H+5CF3CO+Na]+ ions were observed in the FAB-MS spectra of the trifluoroacetyl-cyclitol derivatives. Collision-induced dissociation (CID) of the [M+Na]+ ions produced diverse product ions via a series of dissociative processes. Charge-remote fragmentation (CRF) patterns of [M+Na]+ ions were very useful for the identification of product ions which are characteristic for the cyclitol ring and long hydrocarbon chains substituted at the glycerol backbone. Moreover, the CID-MS/MS spectra of the [M+Na]+ ions yielded characteristic product ions at m/z 53, 83, 113, 155 and 171 for the cyclitol moiety, and at m/z 213, 229 and 245 for the glycerol backbone attached to the cyclitol ring.     

Rapid analysis of a peptide by fast-atom bombardment mass spectrometry after polyacrylamide gel electrophoresis

Gel electrophoresis has been a powerful technique for the separation of peptides and proteins for many years. After electrophoresis separation on a polyacrylamide gel, the peptide bradykinin was localized using Coomassie Blue as a staining dye. Excess dye was removed by washing the gel with water. For mass spectrometric analysis, bands containing the peptide were crushed, extracted with acetic acid and the eluent applied to the fast-atom bombardment probe. Under these conditions the protonated molecule of bradykinin was clearly observed. Also apparent were sequence ions at about the same intensity observed from authentic bradykinin.     

Study of adduct ions of meso-phenyl-substituted tetrabenzoporphyrins by fast-atom bombardment mass spectrometry

Mass spectra of meso-phenyl-substituted tetrabenzoporphyrins were investigated by fast-atom bombardment mass spectrometry and tandem mass spectrometry. A cluster of adduct ions with mass-to-charge ratio values higher than the corresponding molecular ions of the porphyrins has been observed. The mass number differences among the series of cluster ions are constant depending on the para-phenyl substituents. Under certain conditions, dimers or trimers of molecular ions with low abundances have been detected. To trace the origin of the adduct ions, a series of experiments based on mass spectrometry have been carried out. The mass spectrum of tetrabenzoporphyrin showed no adduct ions with mass number differences of 90 even with the addition of phenylacetic acid. The mass spectrum of meso-tetraphenylte-trabenzoporphyrin ¹³C-labeled at the meso carbons showed adduct ions with mass number differences of 91. Product spectra of [2M + H]⁺ or [3M + H]⁺ of porphyrins exhibited adduct ions. All these results suggest that fragmentations of [2M + H]⁺ or [3M + H]⁺ may be one of the many possible routes to form the adduct ions, and the mass number differences among the series of these cluster ions should correspond to the benzyl group from the meso positions of meso-phenyl-substituted tetrabenzoporphyrins.     

Kinetic analysis of cyclic CMP-specific and multifunctional phosphodiesterases by quantitative positive-ion fast-atom bombardment mass spectrometry

Two enzymes, cyclic CMP-specific phosphodiesterase and multifunctional phosphodiesterase, are responsible for the hydrolysis of cytidine 3',5'-cyclic monophosphate in living cells. Quantitation of both enzymes has been carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubates after termination of the reaction. The kinetic data obtained are in close agreement with parallel data obtained by the conventional radiometric assay. The extra facility of the mass spectrometry based assay to monitor several incubation components simultaneously has been exploited to study the concurrent hydrolysis of alternate cyclic nucleotide substrates and provides kinetic parameters of significance in interpreting substrate-enzyme interactions. This is extended by the use of collisionally-induced dissociation of the protonated molecules of the liberated products to identify the mononucleotide isomers resulting from the cyclic nucleotide hydrolysis.     

A coordinated approach towards the molecular weight determination of peptides by gel electrophoresis and fast-atom bombardment mass spectrometry

The molecular weight of peptides can be determined by fast-atom bombardment mass spectrometry (FAB-MS) following polyacrylamide gel electrophoresis (PAGE). Initial results combining the fast and efficient separation of peptides by PAGE and the unambiguous determination of molecular weights by FAB-MS have been demonstrated for the three peptides bradykinin, neurotensin and gramicidin S. This method has also been applied to the determination of the molecular weights of two fragments from the tryptic digest of horse-heart cytochrome c.     

Analysis of some eicosanoids by continuous-flow fast atom bombardment mass spectrometry

Continuous-flow fast atom bombardment has been used to analyze eicosanoids by selected-ion monitoring on a sector-field mass spectrometer operating in the negative-ion mode. The method has been optimized with respect to solvent composition and flow-rates. Detection limits were below 50 pg, and under optimal conditions a linear relationship between response and amount of substance was achieved. The method was successfully applied to the analysis of two spiked urine samples.     

Electron ionisation and fast-atom bombardment mass spectrometry study of diaryl carbonates

Electron ionisation mass spectrometry studies were performed previously for p-diphenyl carbonate and some monosubstituted diphenyl carbonates. In this work, p-diphenyl carbonate and p-methoxyphenylphenyl carbonate are re-examined, and p-chlorophenyl phenyl carbonate and two disubstituted diphenyl carbonates, bis (p-chlorophenyl) carbonate and p-methoxyphenyl-p-fluorophenyl carbonate, are studied for the first time. The previously established fragmentation routes were observed for all compounds investigated. Some other different sequences were observed, and a fragmentation path, other than decarboxylation, of the molecular ion is proposed. In the fast-atom bombardment study it was observed that the M(+*)/[MH](+) ion abundance ratio increased from 0.44 for compound 1 to 2.95 for compound 5. [MH](+) is not a dominant ion in most of compounds studied, in spite of the presence of a carbonyl group, a strong proton acceptor. The presence of two oxygen atoms bonded to the carbonyl group appears to induce delocalisation of the electron pairs, thus deactivating the carbonyl site for protonation. In addition, m-nitrobenzyl alcohol (NBA) being a relatively aprotic/hydrophobic matrix reinforces the deactivation for protonation. Because the carbonate group and NBA are common features to the study, the contributions of the substituents were taken into account to explain the different behaviour of the five compounds with respect to protonation.     

Characterization of beam-induced reactions occurring in liquid secondary ion mass spectrometry/fast-atom bombardment by tandem mass spectrometry

Department of General and Physical Chemistry, University of Liege, Liege, Belgium A specific beam-induced secondary reaction involving the condensation of hydroxylic matrices with some organic groups (aldehydes, ketones, etc.) accompanied by the loss of a water molecule was investigated by liquid secondary ion mass spectrometry/fast-atom bombardment (LSIMS/FAB). A mechanistic scheme and a structure of the induced product are proposed. The features of this secondary reaction were studied and the influence of the types of solutes, acidic additives, and matrices analyzed. Rather than a drawback, LSIMS/FAB mass spectrometry can take advantage of this matrix effect to infer analytical information through tandem mass spectrometry experiments. Specific neutral loss scans can be conducted to highlight beam-induced reactive molecules, even when the detection of these species is prevented in normal scan spectra by other surface-active components.