Development and Application of High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Analysis of Domoic Acid and Its Isomers in Shellfish

Domoic acid (DA) is the principal neurotoxin responsible for amnesic shellfish poisoning (ASP), and exhibits escalating prevalence in China′s coastal ecosystems with widespread contamination by DA and its structural isomers. To enhance regulatory control of these contaminants, an high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for detection of DA and its isomers was developed and validated through optimization of sample preparation and chromatographic conditions. The samples were extracted with 50.0% (V/V) aqueous methanol, cleaned up using Bond Elut SAX solid-phase extraction cartridges, volume-adjusted, and filtered through 0.22 μm aqueous-phase membranes. Chromatographic separation employed a Luna C18(2)-HST 100A column (100 mm×2 mm, 2.5 μm) with gradient elution (Mobile phase A: 1% formic acid in water; Mobile phase B: 1% formic acid in acetonitrile). Method validation demonstrated that the recoveries were 77.69%–115.84% (Relative standard deviations were 1.65%–8.96%), inter-batch recoveries were 79.39%–110.43% (RSD 1.04%–8.07%, n=6) across four shellfish matrices, with good linearity (R2≥0.999) and high sensitivity (LOD/LOQ: 0.1/0.3 μg/kg). Analysis results of coastal shellfish samples of Shandong Province in 2023 revealed universal DA/isomer detection during July to September, with DA constituting 92.9%, 88.7% and 89.1% of total analogs, respectively. Risk assessment results indicated negligible dietary exposure across age demographics (THQ