Development of Certified Reference Materials for Diarrhetic Shellfish Poisoning Toxins, Part 1: Calibration Solutions

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.

Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish

“…Since the original descriptions of OA, DTX1, and DTX2, a limited number of additional DST-derivatives have been described, although they have never been found to be present in shellfish in sufficient quantities to cause DSP. 2-Deoxy-OA and 7-deoxy-OA were both isolated as minor constituents from the dinoflagellate P. lima [18,19], 19-epi-OA was isolated as a minor constituent from P. belezianum [20], 19-epi-DTX1 and 19-epi-DTX2 were identified as minor impurities during the production of DSP-certified reference materials [21], while 14,15-dihydro-OA was isolated as a minor constituent from the marine sponge Halichondria okadai during the original isolation of OA [22]. Using a similar bioassay-guided isolation approach as was used to first identify OA from H. okadai, with the goal of identifying novel biologically active compounds from marine organisms, 14,15-dihydrodinophysistoxin-1, along with roughly equivalent amounts of OA and DTX1, was isolated from marine sponges (Phakellia sp.)…”

Semiquantitation of Paralytic Shellfish Toxins by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry Using Relative Molar Response Factors

“…Purified algal toxin material is usually quantitated as a concentrated stock solution by 1 H-NMR spectroscopy for the production of CRMs [ 28 , 29 , 30 ]. With only low μg amounts of material available as mixed solutions in this work, an alternative method of assigning concentrations to the M-toxins was needed.…”

“…As with relative toxicity measurements, the quality of RMR measurements depends on accurate quantitation of the standards used to determine them, which can be confounded by unknown purity or salt form. For toxin certified reference materials (CRMs), the preferred approach is to quantitate limited amounts of scarce toxin as concentrated stock solutions by 1 H-NMR spectroscopy [28][29][30]. However, accurate quantitative NMR still typically requires mg quantities of pure toxin, which can be difficult to obtain for many compounds.…”

Relative molar response of lipophilic marine algal toxins in liquid chromatography/electrospray ionization mass spectrometry

“…Reference materials (RMs) are vital for calibration in LC/MS to ensure accurate analytical results . Considerable efforts have been made to produce certified standards for most of the regulated lipophilic toxins . Focus has primarily been on producing standards for toxins that are specifically regulated or linked to poisoning events, with a reduced availability for related toxins (e.g.…”