Multiresidue Method for Determination of Algal Toxins in Shellfish: Single-Laboratory Validation and Interlaboratory Study

Abstract

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05–0.20 mg/kg, recoveries were 71–99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10–24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSDr) of 8–12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8–2.0).

Marine biotoxins in shellfish - okadaic acid and analogues - Scientific Opinion of the Panel on Contaminants in the Food chain

“…Liquid chromatography-mass spectrometry (LC-MS) as well as liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), which together in the following text are termed LC-MS/(MS), methods are increasingly being used in monitoring programs. One method (McNabb et al, 2005), with a LOQ of 40 µg OA/kg shellfish tissue, has undergone an intensive single-laboratory validation and a limited inter-laboratory study, although this study did not include real samples. Some EU Member States are currently using LC-MS/(MS) data to supplement information generated by the MBA by parallel testing.…”

“…For both matrices, 11 out of 13 laboratories achieved satisfactory z-scores for the total OA-equivalents. Some of the methods in use have been developed to allow multitoxin group detection (Stobo et al 2005, McNabb et al 2005, Fux et al 2007). The available data from in-house and interlaboratory studies suggested that between-laboratory variability was lower when laboratories used their own in-house validated method, than when they adhered to a strictly standardized protocol.…”

Marine biotoxins in shellfish - Yessotoxin group - Scientific Opinion of the Panel on Contaminants in the Food chain

“…One multi-toxin LC-MS/MS protocol that included some YTXs (YTX, 45-hydroxyYTX, 1a-homoYTX, carboxyYTX) has been subjected to a limited interlaboratory validation study (McNabb et al, 2005). The limit of quantification (LOQ) for this method was 0.017 mg/kg shellfish.…”

Marine biotoxins in shellfish – Pectenotoxin group

“…The mass spectrometric detection of PTX2 in shellfish extracts has been studied in detail and has been shown to be affected by matrix effects, like many other marine toxins. A collaborative study by McNabb et al (2005) focused on the analysis of extracts and found good apparent recovery. Stobo et al (2005), found little matrix effect in the determination of PTX2 by LC-MS, while Fux et al (2008) found between 0 and 92 % signal enhancement, depending on chromatographic conditions.…”