Stephen Pleasance

Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry☆☆☆

The application of capillary electrophoresis-mass spectrometry (CE-MS) to the analysis of compounds of concern to the aquaculture industry is reported. Two different approaches to coupling the CE column to an IonSpray atmospheric pressure ionization (API) interface, viz., a liquid-junction and a coaxial arrangement, are describe and compared with regard to ruggedness, ease of use, sensitivity and electrophoretic performance. The different injection modes used in three commercial capillary electrophoresis systems were also evaluated for their applicability to CE-MS. The use of CE-MS for the analysis of a variety of classes of antibiotics used in the fish aquaculture industry, such as the sulfonamides and their potentiators (e.g., trimethoprim), is demonstrated and was used to confirm the presence of these components in shellfish extracts at the low ppm level. CE-MS was also applied to the analysis of marine toxins such as saxitoxin and its analogues which are associated with paralytic shellfish poisoning, and also the toxins responsible for amnesic and diarrheic shellfish poisoning. Tandem mass spectrometry (MS-MS) was used to provide structural information on these analytes, and the ability to distinguish isomeric compounds based on their different migration and fragmentation characteristics using CE-MS-MS is demonstrated.

Analysis of paralytic shellfish poisons by capillary electrophoresis

A capillary electrophoresis (CE) method with UV detection is described for the separation and determination of underivatized toxins associated with paralytic shellfish poisoning (PSP). Confirmation of the electrophoretic peaks was facilitated by mass spectrometric (MS) detection using an ionspray CE-MS interface and by high-performance liquid chromatography with fluorescence detection. The determination of PSP toxins, such as saxitoxin and neosaxitoxin, in toxic dinoflagellates and scallops is demonstrated and comparisons are made with existing techniques.

An evaluation of atmospheric pressure ionization techniques for the analysis of N-methyl carbamate pesticides by liquid chromatography mass spectrometry

Atmospheric pressure ionization (API) techniques are evaluated for the mass spectral analysis of N-methyl carbamate pesticides. Atmospheric pressure chemical ionization (APCI) using a heated nebulizer interface provided both protonated molecules and abundant, characteristic fragment ions. With ion spray (ISP; pneumatically assisted electrospray ionization), which utilizes a milder “ion evaporation” process, primarily protonated molecules were obtained, although fragment ions similar to those observed in APCI could be induced by variation of the API orifice voltage. Product ion spectra of ISP-derived protonated molecules, generated by tandem mass spectrometry using collision-induced dissociation, are also presented. The APCI and ISP spectra of the carbamates are compared to those obtained with a thermospray interface and also to their electron ionization and methane CI spectra obtained with a particle beam interface. For all four interfaces, combined liquid chromatography mass spectrometry methods using conventional (4.6 mm i.d.) columns are described for the separation and detection of pesticide mixtures. These methods are applied to the confirmatory analysis of three representative carbamate pesticides, spiked at the 0.1-ppm level in green peppers. For those carbamates amenable to gas chromatography mass spectrometry, comparative results are presented.

Determination of sulfonamides by liquid chromatography, ultraviolet diode array detection and ion-spray tandem mass spectrometry with application to cultured salmon flesh☆

Ion-spray mass spectrometry was investigated for the analysis of 21 antibacterial sulfonamide drugs. All of the sulfonamides analyzed gave positive ion mass spectra with abundant protonated molecules and no fragmentation. Tandem mass spectrometry (MS-MS) using collision-induced dissociation provided structural information, allowing the identification of common fragmentation pathways and the differentiation of isomeric and isobaric sulfonamides. A reversed-phase high-performance liquid chromatographic method was developed, using gradient elution and ultraviolet diode-array detection (DAD), enabling the separation of 16 of the sulfonamides. Combined liquid chromatography (LC)-MS was accomplished using the ion-spray interface. Analyses of a mixture of sulfonamide standards were performed with gradient elution and the mass spectrometer configured for full-scan acquisition, selected-ion monitoring, or selected-reaction monitoring. Procedures for the analysis of sulfadimethoxine (SDM), a representative sulfonamide used in the aquaculture industry, are described. The presence of SDM in cultured salmon flesh was confirmed at levels as low as 25 ng/g by a combination of LC-DAD and LC-MS-MS.

Isolation of a new diarrhetic shellfish poison from Irish mussels

A new marine toxin dinophysistoxin-2 (DTX-2)4, isolated from toxic Irish mussels and biogenetically related to the toxins okadaic acid 1 and dinophysistoxin-1 (DTX-1)2, the principal agents responsible for diarrhetic shellfish poisoning (DSP), is reported.

Continuous Flow Fast Atom Bombardment with Packed Microcolumns: A Comparison of Precolumn Versus Coaxial Matrix Delivery

The effect of adding glycerol to the mobile phase on the chromatographic separation of peptides has been investigated using a continuous flow fast atom bombardment (CFFAB) interface coupled with commercial packed microcolumns (25 cm × 320 μm.i.d.). In a comparative study using a UV detector, it was found that chromatographic peak broadening progressively increased with increasing percentage of glycerol in the mobile phase. In the liquid chromatographic FAB mass spectrometric analysis, this effect is compounded by the dynamic mixing of the column effluent on the probe. Improvements of 25–155% in the overall separation efficiencies were obtained by introducing the matrix independently to the probe tip via a coaxial arrangement. Application of this coaxial CFFAB is demonstrated by the analysis of peptide mixtures and tryptic digests.

Characterization of a mixture of lobster digestive cysteine proteinases by ionspray mass spectrometry and tryptic mapping with LC—MS and LC—MS—MS☆

Abstract An inseparable mixture of two cysteine proteinases, isolated from the digestive tract of the American lobster, was investigated by ionspray mass spectrometry (ISP-MS), using a combination of infusion of intact proteins with on-line liquid chromatography—mass spectrometry (LC—MS) and LC—MS—MS analyses of tryptic digests. These data were interpreted by comparisons with predictions from results of molecular cloning of cysteine-proteinase-encoding messenger RNA sequences previously isolated from the lobster hepatopancreas. Investigations of the numbers of free thiol groups and of disulfide bonds were made by measuring the molecular weights of the alkylated proteins with and without prior reduction of disulfide bonds, and comparison with the corresponding data for the native proteins. Identification of tyrptic fragment peptides containing cysteine residues was facilitated by comparing LC—MS analyses of tryptic digests of denatured and of denatured and alkylated proteins, since such tryptic peptides are subject to shifts in both mass and retention time upon reduction and alkylation. Confirmation of amino acid sequences was obtained from fragment ion spectra of each tryptic peptide (alkylated or not) as it eluted from the column. Acquisition of such on-line LC—MS data was possible through use of the entire effluent from a standard 1 mm high performance liquid chromatography (HPLC) column by an IonsSpray ® LC—MS interface (pneumatically assisted electrospray).