[Validation Study of LC-MS/MS by Using a Saxitoxin Enantiomer]

Area of Science:

• Marine Toxinology
• Analytical Chemistry
• Food Safety

Background:

• Saxitoxin (STX) and its analogues are potent neurotoxins produced by marine dinoflagellates.
• The mouse bioassay (MBA) is the global standard for STX detection in shellfish but raises animal welfare concerns.
• LC-MS/MS offers a sensitive, non-animal alternative for STX detection.

Purpose of the Study:

• To validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for STX detection using a nontoxic enantiomeric analogue (ent-STX) as a reference material.
• To assess the suitability of ent-STX as a replacement for STX reference material, considering regulatory requirements and analytical performance.
• To investigate potential interferences from unidentified STX analogues in shellfish analysis.

Main Methods:

• Validation of an LC-MS/MS method using ent-STX-fortified scallop homogenate according to CODEX-STAN 292-2008 guidelines.
• Preparation of calibration standards using solvent and non-toxic scallop extract for absolute calibration.
• Application of the validated method to quantify STX in various bivalve molluscs, including mussels and scallops.

Main Results:

• The LC-MS/MS method using ent-STX met all validation criteria for trueness, repeatability, reproducibility, and applicability range.
• Ent-STX was successfully employed to confirm and quantify STX levels in commercially important shellfish species.
• An unidentified STX analogue was detected in some samples, necessitating careful selection of LC-MS/MS conditions to avoid misidentification.

Conclusions:

• Nontoxic enantiomeric STX (ent-STX) is a validated and effective alternative reference material for LC-MS/MS-based detection of paralytic shellfish toxins.
• The developed method offers a viable, animal-free approach for regulatory monitoring of STX in shellfish.
• Further optimization of LC-MS/MS selected reaction monitoring (SRM) conditions is crucial for distinguishing STX from potential interfering analogues.