LC-UV and LC-MS methods for the determination of domoic acid

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Abstract

Under European legislation, domoic acid (DA), the main constituent of amnesic shellfish poisoning, is monitored to protect the shellfish consumer. To ensure comparability amongst analytical data, it was deemed necessary to undertake performance assessments of the methods conducted by monitoring laboratories of the United Kingdom and Ireland.

In phase I of a two-phase inter-comparison, three laboratories used high-performance liquid chromatography and ultraviolet detection (HPLC-UV). Concentration data for a DA standard solution, a crude extract of whole scallops and a scallop-homogenate fell within internationally accepted limits, demonstrating good agreement for these matrices. Between-laboratory analyses of a scallop gonad showed a higher variation (>16%).

In phase II, a second gonad homogenate containing DA one order of magnitude higher in concentration gave results acceptable to internationally set criteria.

The efficiency of the strong anion-exchange cartridges used in sample-extract clean-up should be monitored as part of a laboratory quality control system.

From a recovery study, it is suggested that recovery correction should also be applied.

There was no difference in the quantitation of DA in standard solutions or shellfish using either LC-UV or LC with mass spectrometric (MS) detection, and between-laboratory MS data for a gonad homogenate were also equivalent.

Variations of the published method practised by the monitoring laboratories were found not to compromise results, thus demonstrating an acceptable degree of ruggedness, as well as comparability between the participants.

Introduction

Amnesic shellfish poisoning (ASP) toxins are produced by microalgae (e.g., Pseudo-nitzschia spp.) and can accumulate in shellfish. Consumption of toxic shellfish may cause a number of effects in humans, including vomiting, diarrhea and permanent short-term memory loss [1].

European Union (EU) legislation [2] was amended in 1997 [3], and adopted in the United Kingdom (UK) in 1998, to include domoic acid (DA), the main constituent of ASP toxins, in the suite of biotoxins to be determined in the regulatory monitoring of shellfish. The amendment requires shellfish to contain less than 20 mg/kg of DA as analyzed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection; however, no specific procedure has been officially validated or recommended by the EU.

The International Scientific Community has proposed two methods [4], [5]. The suitability of these methods is being investigated by the European Committee on Normalization (CEN) and by a EU working group. Irish and UK laboratories involved in the regulatory monitoring of shellfish poisons have since adopted the procedure of Quilliam et al. [4]. A performance assessment was deemed necessary to ensure comparability amongst the monitoring laboratories involved in the routine analyses of ASP, so inter-laboratory comparisons were undertaken involving four laboratories whereby concentration data was reviewed on the basis of applied methodologies.

For the purposes of this inter-comparison study reported here, three biotoxin-monitoring laboratories (LAB-1, LAB-2 and LAB-3) were included along with LAB-4, which participated to develop LC with mass spectrometric (LC-MS) method of analysis. The study consisted of two phases. Thus, this article describes results of an inter-laboratory comparison study, as performed by the four laboratories, of the HPLC-UV and LC-MS methods used for the detection and quantification of DA in standard solutions and in real shellfish samples naturally incurred with DA.

Section snippets

Inter-laboratory performance studies

External assessments of the quality of the results generated by individual laboratories and in the form of inter-laboratory comparisons satisfy the requirement to demonstrate comparability of analytical data. By centrally distributing samples, assessments of performance in the inter-laboratory studies were made possible, and the participation in such comparisons proved necessary in method development and refinement, as well as validation.

Preparation of standard solutions, crude scallop extracts and homogenates

Standard solutions containing DA were prepared by weight and by dilution of the certified reference standard (DACS-1C; NRC, Canada) in 10% aqueous acetonitrile. Aliquots (0.5 mL) were then transferred to 2 mL amber ampoules and sealed under nitrogen. The nominal DA concentration was 2 μg/mL, which the participants did not know. Ampoules were stored at 2–8°C. Crude extracts were prepared from homogenized scallops obtained from the Scottish West Coast in 1999. These methanol/water extracts were

Phase II

All four participants were involved in phase II, and both LAB-1 and LAB-4 deployed LC-MS techniques. Phase II involved the analysis of three DA standards (5, 2 and 1 μg/mL), a scallop-gonad homogenate, and a CRM (MUS1-B). The extraction methodology and HPLC conditions are summarized in Table 2, Table 3.

LAB-4 applied a different sample-extraction procedure, which involved vortex mixing (see Table 2) for 2 min, followed by centrifugation. The volume of the extracting solvents also differed from

Results of the inter-comparison exercises

Concentration data and percentage c.v.s are detailed in Table 4, Table 5. Mean DA data are presented as scatter plots, and the error bars represent one standard deviation of the mean (Figure 1, Figure 2, Figure 3, Figure 4).

Summary

It was apparent that there was a need for laboratories involved with the regulatory monitoring of DA in shellfish to demonstrate comparability of concentration data, since each laboratory had adopted a single published method although there were variations in its deployment. A critical review of the evidence emerging from the inter-comparisons enabled suggestions to be made to improve the confidence of the data. This study was limited to four laboratories, although there was an obvious

Acknowledgements

This project was funded by the UK Food Standards Agency, and the Scottish Environment Executive Rural Affairs Department. The authors thank Drs. Ian Davies, Colin Moffat and David Wells of FRS for their comments.

References (10)

  • E.C.D. Todd

    J. Food Protect.

    (1993)
  • W.P. Cofino et al.

    Mar. Pollut. Bull.

    (1994)
  • D.E. Wells et al.

    Mar. Pollut. Bull.

    (1997)
  • European Commission, Council Directive 91/492/EEC of 15 July, Off. J. L 268 (1991)...
  • European Commission, Council Directive 97/61/EC of 20 October, Off. J. L 295 (1997)...
There are more references available in the full text version of this article.

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