Development and application of immunoaffinity column purification and ultrahigh performance liquid chromatography-tandem mass spectrometry for determination of domoic acid in shellfish

Abstract

Domoic acid (DA) is a neurotoxin associated with amnesic shellfish poisoning (ASP). Though LC coupled to tandem mass spectrometry (LC-MS/MS) has become the preferred method for DA determination, traditional sample pretreatment is still labor-intensive. In this study, a simple, efficient and selective method for LC-MS/MS analysis of DA in shellfish was established by optimizing clean-up procedures on a self-assembly immunoaffinity column (IAC). Shellfish was extracted with 75% methanol twice and diluted with phosphate buffered saline (PBS, 1:2). The mixture was purified on IAC as follows: preconditioned with PBS, loaded with sample, washed by 50% MeOH, and eluted with MeOH containing 2% ammonium hydroxide. Concentrated analyte was monitored by multiple reaction monitoring (MRM) using electrospray (ESI) positive ion mode throughout the LC gradient elution. Based on the post-extraction addition method, matrix effects for various shellfish matrices were found to be less than 8%. The developed method was fully validated by choosing mussel as the representative matrix. The method had a limit of detection (LOD) of 0.02 µg·g-1, showed excellent linear correlation in the range of 0.05-40 µg·g-1, and obtained ideal recoveries (91-94%), intra-day RSDs (6-8%) and inter-day RSDs (3-6%). The method was successfully applied to DA determination in 59 shellfish samples, with a detection rate of 10% and contaminated content of 0.1-14.9 µg·g-1.