Combined high-performance liquid chromatographic-continuous-flow fast atom bombardment mass spectrometric analysis of acylcoenzyme A compounds

A high-performance liquid chromatographic method for the analysis of coenzyme A thioesters which employs continuous-flow fast atom bombardment mass spectrometric detection is presented. The chromatographic system utilizes gradient elution with reversed-phase conditions using ammonium acetate-acetonitrile from both standard analytical (3.9 mm I.D.) and microbore (1 mm I.D.) columns. Applications to coenzyme A thioesters of various acyl group chain length (C2-C18) and functionality (-COOH, -OH, -C = C-) are described. The system is also applied to an in vitro enzyme reaction (crotonase) to directly follow the disappearance of substrate and appearance of product. The mass spectrometry of coenzyme A thioesters, their chromatographic behavior, system stability, and sensitivity of detection are discussed.     

High-performance liquid chromatographic separation and immunological characterization of soluble bovine viral diarrhea virus antigen

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis of the protein extracts from bovine viral diarrhea virus (BVDV, Singer strain) infected primary calf testicle cells (soluble antigen) showed the presence of four virus specific polypeptides of 105, 90, 84 and 67 kiloDaltons (kD) the 84-kD being the most abundant. Anion-exchange high-performance liquid chromatography (HPLC) of soluble antigen separated the virus specific polypeptides in individual peaks while the gel permeation HPLC collected all of them in a single protein aggregate peak of 290 kD. Except for the 84-kD polypeptide peak in anion-exchange HPLC, all peak fractions were found to be heterogeneous in nature having more than one polypeptide. Analysis of the antisera raised against the peaks having antigen activity showed that antisera against the 84-kD polypeptide peak did not neutralise BVDV while those against the fractions containing the 90- and 105-kD polypeptides neutralised the virus.     

High-performance liquid chromatographic mass spectrometric identification of the photoproducts of cymoxanil

The photochemical degradation of the fungicide cymoxanil {2-cyano-N-[(ethylamino)carbonyl]-2-(methoxyimino) acetamide} was studied in aqueous buffer solution (pH 5.9+/- 0.1) under UV light and in laboratory conditions. The degradation followed a pseudo- first-order kinetic with significant correlation coefficient. The main photoproducts were separated and tentatively identified by HPLC/UV and HPLC/MS data, as 3-ethyl-4-(methoxyamino)-2,5-dioxo-4-imidazolidinecarbonitrile, 1-ethyl-5-(methoxyimino)-2,4-imidazolidin-2,4-dione, ethylimidazolidinetrione and {[(ethylamino)carbonyl]-amino}oxoacetic acid. A photolysis pathway of cymoxanil is proposed.     

High-performance liquid chromatographic separation and indirect fluorescence detection of thiols

A fluorescent post-column reaction detection scheme has been devised for selective determination of thiols. The post-column reagent is 40 microM Cd2+ and 100 microM 8-hydroxyquinoline-5-sulfonic acid (HQS) in non-complexing buffer at pH 10. HQS complexes Cd2+ to form a fluorescent product. Thiols in the HPLC effluent compete for complexation of the Cd2+, resulting in a decrease in the fluorescence response. Detection limits of 0.2 microM (0.04 ppm) are achieved for cysteine, homocysteine and glutathione in a 5 min separation. Recoveries from spiked synthetic urine samples are 87-120%.     

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 alpha-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.     

A fast atom bombardment mass spectrometric analysis of crown ether complexes [1]

Analyses of crown ether complexes of alkali metal ions and characterization of the complexes formed inm-nitrobenzyl alcohol have been carried out by fast atom bombardment (FAB) mass spectrometry. By using m-nitrobenzyl alcohol as a matrix for measurements, the stoichiometry of the complexes was assessed on the basis of the observed FAB peaks. In addition, the formation of crown ether-alkali metal complexes at a 2 : 1 molar ratio was enhanced by increasing the ionic radius of the metal ion in agreement with previous observations. On these grounds, FAB mass spectrometry may provide a rapid means for investigation of the complexation behavior of crown ethers and the stoichiometry of the complexes.     

High-performance liquid chromatographic separation of cobalamins

Physiological cobalamins were separated by means of high-performance liquid chromatography (HPLC). Optimal conditions for elution of methylcobalamin, adenosylcobalamin, hydroxycobalamin and cyanocobalamin were determined. Excellent separation and resolution of these physiological cobalamins by HPLC were achieved. In addition, several cobalamin analogues were also studied and shown to be separable from the physiological forms. HPLC provides a rapid, sensitive, reproducible means of characterizing physiological cobalamins.     

Negative-ion fast atom bombardment mass spectrometry in the characterization of deoxyfluorinated sugars

Negative-ion fast atom bombardment mass spectrometry and metastable ion studies were applied to the characterization of three isomeric fluorine-containing sugars. Peculiar fragmentation processes were evidenced for each isomer, enabling their differentiation. Comparison with the behaviour of a non-fluorinated analogue allowed investigation of the role of fluorine in the decomposition processes of these compounds.     

High-performance liquid chromatographic separation and detection methods for anabolic compounds.

The role of high-performance liquid chromatography (HPLC) in methods of analysis for anabolic compounds in biological samples is reviewed. Special attention is given to both the separation and detection of anabolic compounds. A distinction is made between on-line detection systems, such as ultraviolet detection and diode-array detection, and off-line detection methods with special emphasis on immunochemical detection methods using non-isotopic labels. A number of applications are given to elucidate the possibilities of HPLC in the analysis of anabolic compounds.     

Fast atom bombardment combined with mass spectrometry for characterization of polycyclic aromatic hydrocarbons

A new approach using fast atom bombardment combined with mass spectrometry to characterize polycyclic aromatic hydrocarbons (PAHs) in the range of 128-252 u molecular weight is described. Sulfolane was employed as a liquid matrix for these π-conjugated hydrocarbons. Bombardment of sulfolane solution of certain PAHs with an atom beam produces both radical cation (M^+.) and protonated molecule [(M + H)⁺], with no evidence of fragmentation. Collisional activation of the fast atom bombardment-desorbed M^+. ions, however, results in several structure-specific fragment ions. Structural differences in a few isomeric hydrocarbons can be detected using the [(M + H)⁺]/[M^+.] abundance ratio and in the pyrene-fluoranthene pair by the B/E linked-field-collision-activated dissociation data. The [(M + H)⁺]/[M^+.] was found to be compound-specific and correlated well with certain properties (resonance energy, proton affinity, and ionizing energy) of PAHs.     

Evaluation of fast atom bombardment mass spectrometric and low-energy collisional activation tandem mass spectrometric data for the structural elucidation of bisbenzylisoquinoline alkaloids

The fast atom bombardment (FAB) mass spectra and low-energy collisional activation mass spectra of ions generated under FAB were investigated for twelve bisbenzylisoquinoline (BBI) alkaloids. The relative molecular mass of the free base and diquaternary BBI alkaloids can be obtained from FAB data. However, monoquaternary ammonium salts produce only an [M — X]⁺ ion and the relative molecular mass cannot be determined. For Type A (single ether linkage) BBI alkaloids, fragmentation occurs primarily from benzylic and ether cleavages. Thus, the total number of aromatic substituents (OH, OCH₃ or OCH₂O) can be determined for rings A-B, C-D, E and F. For Type B (two either linkages) BBI alkaloids, fragmentation occurs primarily from double benzylic cleavages. Hence only the total number of aromatic substituents can be determined for the upper half of the Type B BBI alkaloids, i.e. rings A-B and C-D. An unknown alkaloid was examined to illustrate the utility of the fragmentation schemes.     

Continuous-flow fast atom bombardment mass spectrometry of oligonucleotides

Although frit-fast atom bombardment (frit-FAB) and continuous-flow FAB mass spectrometry have become standard methods for the analysis of peptides and peptide mixtures, these techniques have not been applied previously to the analysis of oligonucleotides. Mobilephase composition, flow rate, and sample size were optimized for the analysis of oligonucleotides by negative ion frit-FAB mass spectrometry (a type of continuous-flow FAB mass spectrometry). With a mobile phase consisting of methanol/water/triethanolamine (80:20:0.5, v/v/w), flow injection frit-FAB analysis of oligonucleotides showed lower limits of detection compared to standard probe FAB mass spectrometry. For example, in order to obtain a signal-to-noise ratio of 3:1, 38 prnol of d(GTIAAC) were required for frit-FAB mass spectrometry and 62 pmol were required for standard probe FAB mass spectrometry. The largest difference between frit-FAB and standard probe FAB was observed for d(pC)₅, for which the limit of detection by frit-FAB was approximately 11-fold lower than by standard FAB mass spectrometry. Adjustment of the mobile phase to pH 7 with trifluoroacetic acid increased the limit of detection (reduced sensitivity) a minimum of sixfold. Equimolar mixtures of two or three oligonucleotides produced deprotonated molecules in identical relative abundances whether analyzed by frit-FAB or standard probe FAB mass spectrometry. Finally, frit-FAB liquid chromatography mass spectrometry was demonstrated by separating mixtures of oligonucleotides on a β -cyclodextrin high-performance liquid chromatography column with a mobile phase containing methanol, water, and triethanolamine.     

Use of fast atom bombardment mass spectrometry for the characterization of nitroaromatic isomers

Positive and negative ion fast atom bombardment (FAB) mass spectra of some monosubstituted nitroaromatic isomers are reported. Generally ions carresponding to [M + H]⁺ and M⁺ are observed in the positive ion FAB spectra; ions such as [M ? H] ^? and M^?˙ are observed in the negative ion FAB spectra. The use of FAB mass spectra to distinguish the isomers is discussed. Comparisons of FAB, chemical ionization and electron impact mass spectra of the same isomers (wherever possible) are reported. The structural information obtained in the negative ion FAB spectra complement those obtained in the positive ion FAB spectra.     

Structural characterization of isoxazolidinyl nucleosides by fast atom bombardment tandem mass spectrometry.

4'-Aza-2',3'-dideoxyerythrofuranosyl derivatives of thymine (AdT, 1) and uracil (AdU, 2) are analogues of 2',3'-dideoxyribofuranosyl thymine (ddT, 3) and uracyl (ddU, 4). Compounds 1 and 2 are representative of a new class of antiviral agents where the sugar moiety is replaced by an isoxazolidine ring. The increasing importance of isoxazolidinyl nucleosides has encouraged the exploitation of simple mass spectrometric rules for unambiguously assigning their structure. The species 1, 2, 5 and 6 were therefore synthesized in order to evaluate the role of the basic centre of the modified sugar moiety in their gas-phase chemistry. The tandem mass spectra of these compounds are similar to those of the wild-type nucleosides and display fragment ions corresponding to [B + 2H](+),[M - BH](+) and [B + 27](+) species, where B is the nucleobase. The last species derives from a retrocycloaddition process which is less evident in 2'-deoxyribosides. This behaviour is consistent with protonation of the analytes at the pyrimidine rings. Model isoxazolidines, in which the nucleobase was replaced by a phenyl or a naphthyl moiety, displayed the expected behaviour of species with a localized charge on the N-O moiety of the isoxazolidine ring.     

High-performance liquid chromatographic separation of enantiomeric amines

Summary Separations of twelve different racemic amines were studied by high-performance liquid chromatography (HPLC) in several column-solvent combinations. Relative retentions, which show some dependence upon the substitution at the asymmetric centers, are reported for the amines which were examined as the (+)-10-camphorsulfonamides.     

High-performance liquid chromatographic resolution of p-nitrobenzyloxyamine derivatives of brain gangliosides

A new quantitative procedure for the high-performance liquid chromatographic (HPLC) resolution of human brain gangliosides employing reversed-phase chromatography is described. To provide a derivative which can be determined by UV absorption techniques, p-nitrobenzyloxyamine was coupled to the gangliosides. Derivatization involves ozonation and cleavage of the ceramide double bond followed by oxime formation to the nascent aldehyde. Individual gangliosides, as they were resolved by HPLC, were collected. These fractions were then identified by thin-layer chromatography (TLC) and by gas chromatography of their monosaccharides. Quantitative results were obtained along with a marked increase in sensitivity over conventional resorcinol-hydrochloric acid quantitation of TLC-resolved gangliosides.     

Automated peak tracking for comprehensive impurity profiling in orthogonal liquid chromatographic separation using mass spectrometric detection

The presence and quantity of impurities in pharmaceutical drugs can have a significant impact on their quality and safety. With the continuous pressure for increased industry productivity, there is urgent need for a systematic and comprehensive drug impurity profiling strategy. We report here our development of the fully automated Comprehensive Orthogonal Method Evaluation Technology (COMET) system. The system includes five columns, seven orthogonal HPLC methods, and hyphenated UV-MS detections, which provides automated generic impurities screening for any drug sample. An automated MS peak tracking approach by program-based mass spectral interpretation is devised to unambiguously track impurities among all orthogonal HPLC methods. The program passes electro-spray ionization mass spectra (ESI-MS) through four sequential decision-making mass ion tests and determines molecular weights for every peak. The system reduces the time required to obtain impurity profile from weeks to days, while the automated MS peak tracking takes only minutes to interpret all MS spectral data of interest. Up-to-date, impurity contents of 56 in-development drug candidate samples have all been successfully illustrated by COMET, which contained more than 500 chemical entities. The program is able to track more than 80% of the compounds automatically with majority of the failure due to insufficient ionization for some impurities by ESI. This system is well suited for efficient drug development and ensuring the quality and safety of drug products.     

On-line solid-phase extraction liquid chromatography-continuous flow frit fast atom bombardment mass spectrometric and tandem mass spectrometric determination of hydrolysis products of nerve agents alkyl methylphosphonic acids by p-bromophenacyl derivatization

For proof of the presence of chemical warfare agents sarin, soman and VX, a rapid, accurate and sensitive method which allows us to determine their hydrolysis products ethyl methylphosphonic acid, isopropyl methylphosphonic acid and pinacolyl methyl phosphonic acid was explored by using continuous flow frit fast atom bombardment (FAB) LC-MS and LC-MS-MS. After derivatization of analytes with p-bromophenacyl bromide, LC-MS-MS analyses for screening were performed by a flow injection method. The three alkyl methylphosphonic acids (AMPAs) were eluted within 5 min, and the detection limits for the three AMPAs ranged from 1 to 5 ng/ml. For confirmation of the screening results, LC-MS-MS analysis with chromatographic separation was conducted by using a narrow bore column. The three AMPAs were all eluted with excellent separation within 25 min, and the detection limits ranged from 1 to 20 ng/ml. Quantitative measurement was performed by LC-MS in selected ion monitoring (SIM) mode with chromatographic separation. Linear calibration curves were obtained for the three AMPAs and the detection limits ranged from 0.5 to 3 ng/ml. The relative standard deviation for peak area ranged from 3.4 to 6.0% at 50 ng/ml for the three AMPAs.     

Negative-ion fast atom bombardment mass spectrometry of N-phosphoamino acids

The fragmentation patterns of N-phosphoamino acids in negative-ion fast atom bombardment mass spectrometry (FABMS) showed different characteristics to those in positive-ion FABMS. Six typical N-diisopropyloxyphorphorylamino acids all had intense [M ? 1]^? peaks, and they underwent similar fragmentation pathways. In general, the elimination of one alkene molecule followed by the loss of one molecule of alcohol occurred. They also favoured an N → O rearrangement reaction, followed by fragmentation to (RO)₂ PO₂^? and (RO) (HO)PO₂^?.