title: Amnesic shellfish poisoning toxins in shellfish: estimation of uncertainty of measurement for a liquid chromatography/tandem mass spectrometry method.
abstract: A liquid chromatography/mass spectrometry (LC/MS) method for amnesic shellfish poisoning toxins in shellfish was developed and validated. Tissue homogenate (4 g) was extracted with 16 mL methanol-water (1 + 1, v/v). Dilution into acetonitrile-water (1 + 9, v/v) was followed by C18 solid-phase extraction cleanup. Domoic acid (DA) and epi-domoic acid were determined by LC/MS/MS with electrospray ionization and multiple reaction monitoring. External calibration was performed with dilutions of a certified reference standard. Advantages of this method include speed, lower detection limits, and a very high degree of specificity. The LC/MS response was highly linear, and there were no significant interferences to the determination of DA. Formal method validation was performed on 4 shellfish species. Fortification studies gave recoveries (mean +/- SD; n = 24) of 93 +/- 14% at 1 mg/kg, and 93.3 +/- 7.6% at 20 mg/kg over all the species. Analysis of a mussel certified reference material showed the bias as < 5%. The limits of detection and quantitation were 0.15 and 0.5 mg/kg, respectively. Routine application of the method over 4 months gave a recovery for the QC sample (1 mg/kg fortified blank mussel homogenate) run with each batch of 88.9 +/- 5.5% (mean +/- SD; n = 37). The total uncertainty of measurement results were estimated as 0.12 (12%) at 0.25-5 mg/kg and 0.079 (7.9%) at 5-50 mg/kg. The major contribution to the uncertainty was the repeatability of the LC/MS determination, probably arising from subtle matrix effects.
authors: Cawthron Institute, Private Bag 2, 98 Halifax St E., Nelson, New Zealand. patrick.holland@cawthron.org.nz
authors: Patrick T
authors: PT
authors: Holland
authors: Paul
authors: P
authors: McNabb
authors: Andrew I
authors: AI
authors: Selwood
authors: Tracey
authors: T
authors: Neil
mesh_terms: D000647:Amnesia; D000818:Animals; D049872:Bivalvia; D002853:Chromatography, Liquid / Q000379:methods*; D007536:Isomerism; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000032:analysis; D008387:Marine Toxins / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D008433:Mathematics; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D035501:Uncertainty
chemical_list: D008387:Marine Toxins; C012301:domoic acid; D007608:Kainic Acid