Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.

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1 AbstractThe production and certification of a series of azaspiracid (AZA) calibration solution reference materials is described. Azaspiracids were isolated from contaminated mussels, purified by preparative liquid chromatography and dried under vacuum to the anhydrous form. Purity was assessed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Final concentration of each AZA in a CD 3 OH stock solution was determined accurately by quantitative NMR spectroscopy. This solution was then diluted very accurately in degassed, high purity methanol to a concentration of 1.47 ± 0.08 μmol/L for AZA1, 1.52 ± 0.05 μmol/L for AZA2, and 1.37 ± 0.13 μmol/L for AZA3. Aliquots were dispensed into argon-filled glass ampoules, which were immediately flame-sealed. The calibration solutions are suitable for method development, method validation, calibration of liquid chromatography or mass spectrometry instrumentation and quality control of shellfish monitoring programs.2

No abstract

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.

Reversed-phase liquid chromatography (RPLC) was combined with atmospheric pressure chemical ionization mass spectrometry (APCI-MS), via a heated pneumatic nebulizer interface, for the determination of the polycyclic aromatic sulfur heterocycle (PASH) content of samples obtained by the fractionation of an extract of a pond sediment contaminated by coke-oven residues. Some of the samples produced by the fractionation procedure contained large amounts of other polycyclic aromatic compounds (PACs) which co-eluted with the compounds of interest, making it difficult to obtain mass spectra suitable for compound identification and verification. Therefore, the use of tandem mass spectrometry (MS/MS), as a selective method for the identification of target analytes in complex matrices, was investigated. Initially, PASH standards were injected into the mass spectrometric system by flow injection and their collisionally induced dissociation mass spectra recorded. From these results, it was possible to select ions suitable for selected reaction monitoring (SRM) experiments on both the PASH standards (to establish detection limits and also retention times which could be used to identify these compounds) and the fractions (to establish the possible presence of the selected PASHs in the fractions). The RPLCSRM experiments led to a tentative identification of some of the PASH standards in the fractions. However, the use of multiple reaction monitoring experiments allowed the positive identification of dibenzothiophene, phenanthrol4,5-bcdl thiophene, phenanthro [3,4-b] thiophene and benzo [ bl naphthol 2,341 thiophene in the fractions, along with several of their isomers. Quantification of the PASH standards by RPLC-SRM in the extracts found them to be present at high levels.

The implementation of instrumental analytical methods such as LC-MS for routine monitoring of toxins requires the availability of accurate calibration standards. This is a challenge because many toxins are rare, expensive, dangerous to handle, and/or unstable, and simple gravimetric procedures are not reliable for establishing accurate concentrations in solution. NMR has served as one method of qualitative and quantitative characterization of toxin calibration solution Certified Reference Materials (CRMs). LC with chemiluminescence N detection (LC-CLND) was selected as a complementary method for comprehensive characterization of CRMs because it provides a molar response to N. Here we report on our investigation of LC-CLND as a method suitable for quantitative analysis of nitrogenous toxins. It was demonstrated that a wide range of toxins could be analyzed quantitatively by LC-CLND. Furthermore, equimolar responses among diverse structures were established and it was shown that a single high-purity standard such as caffeine could be used for instrument calibration. The limit of detection was approximately 0.6 ng N. Measurement of several of Canada's National Research Council toxin CRMs with caffeine as the calibrant showed precision averaging 2% RSD and accuracy ranging from 97 to 102%. Application of LC-CLND to the production of calibration solution CRMs and the establishment of traceability of measurement results are presented.