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.

Keywords: domoic acid; immunoaffinity column; purification; shellfish; ultrahigh high performance liquid chromatography tandem mass spectrometry.

Figure 1

Chemical structure of domoic acid.

Figure 2

(A) The influence of collision energy on the product ion intensity and (B) Product ion MS2 spectra of [M + H]⁺ ion (m/z 312.2) at a collision energy of 16 eV.

Figure 3

Coupling efficiency of CNBr- and NHS-activated Sepharose 4B (1 mL) conjugated with different amount of mAb (n = 3).

Figure 4

MRM analyses for DA and its isomers in 1-mL portions of CRM-ASP-Mus-d extract (A) before and (B) after IAC treatment (injection volume, 2 μL).

Figure 5

Evaluation on the capacity and recovery of the IAC column (n = 3).

Figure 6

Representative MRM chromatogram of DA obtained from (A) 25.0 ng·mL⁻¹ of standard solution; (B) blank mussel sample; (C) mussel sample spiked at a concentration of 0.25 µg·g⁻¹; (D) naturally DA-contaminated shellfish sample.