Quantification of Saxitoxin and Neosaxitoxin in Human Urine Utilizing Isotope Dilution Tandem Mass Spectrometry

Abstract

Saxitoxin and neosaxitoxin are potent neurotoxins that can cause paralytic shellfish poisoning when consumed. A new assay is presented here to quantify saxitoxin (STX) and neosaxitoxin (NEO) in human urine samples. Sample preparation of 500-microL samples included the use of weak-cation-exchange solid-phase extraction in a multiplexed 96-well format. Extracts were preconcentrated and analyzed via 10-min hydrophilic interaction liquid chromatography followed by electrospray ionization. Protonated molecular ions were quantified via multiple reaction monitoring mode in a Qtrap mass spectrometer. The method uses novel 15N7-isotopically enriched STX and NEO internal standards. Method validation included the characterization of two enriched urine pools. The lowest reportable limits for STX and NEO were 4.80 and 10.1 ng/mL, respectively, using both quantification and confirmation ions. These two toxins were not detected in a reference range of humans who consumed seafood in the preceding 72 h, suggesting that few false positives would occur when trying to identify people exposed to STX or NEO.

Verification and quantification of saxitoxin from algal samples using fast and validated hydrophilic interaction liquid chromatography–tandem mass spectrometry method

“…HILIC-MS method has been used before for PSP toxins [15,16] but the lack of isotopically labelled internal standard for algal toxins has slowed down the development of validated quantification analysis methods. In the study of Johnson et al [17] a novel 15 N 7 -isotopically enriched STX was used as an internal standard when quantifying STX. However, any isotopically labelled standard is still not commercially available.…”

Saxitoxin Analysis

“…(30) As stated above, lack of isotopically labeled internal standards for PSP toxins has slowed the development of validated quantitative analysis methods. In the study of Johnson et al (33) , a novel 15 N 7 -isotopically enriched STX was used as an internal standard when quantifying STX. However, isotopically labeled standard is still not commercially available.…”

Indirect Quantitation of Saxitoxin by HPLC with Post-column Oxidation and Fluorometric Detection

“…Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is one of the most potentially useful methods for the identification and quantification of STX and PSTs. 2,6,[8][9][10][11] In terms of the analysis of STX, it is noteworthy that some of the LC/MS/MS methods have quantified STX in shellfish extract and human urine samples by using gonyautoxin 1 10 and 15 N7-isotopically labeled STX, 11 respectively, as an internal standard. Another instrumental method, high-performance liquid chromatography coupled with fluorescent detection (HPLC-FD), 12,13 has been used in the research field, and the applicability of this method as the official testing method for STX and other STX analogues in shellfish has been demonstrated.…”