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

Saxitoxin (STX) and its analogues produced by toxic dinoflagellates in marine environments are known as specific voltage-gated sodium channel blockers and can be detected by the mouse bioassay (MBA) that has been used as the official testing method for paralytic shellfish toxin monitoring systems worldwide. Considering animal welfare issues and improved performance of analytical instruments, it would be better to replace MBA with non-animal testing methods such as LC-MS/MS which have been recently reported for the detection of STXs in shellfish. However, STX itself is regulated by the Act on the Prohibition of Chemical Weapons, so the domestic use of the reference material is required with permission from the relevant government department. Therein, nontoxic enantiomeric STX (ent-STX) was developed as an alternative reference material for STX. In this study, the LC-MS/MS method using ent-STX-fortified scallops homogenate was validated in accordance with a previous report and CODEX-STAN 292-2008. The spiked concentrations of ent-STX were set to the regulatory limits of CODEX (0.8 mg of STX-2HCl equivalents/kg) and approximately half of the limits. The calibration standard solutions were prepared by dilution with solvent and non-toxic scallop extract, respectively, and were used to quantify the toxins by absolute calibration method. As a result of the validation, the results with ent-STX were found falling within all the guideline criteria (the trueness, repeatability, within laboratory re-producibility, and minimum applicable range). Moreover, ent-STX was used to confirm and quantify STX in mussels, scallops and noble scallops. The ent-STX was confirmed to be utilized as reference material. However, it can be sometimes detected an unidentified STX analogue in Japanese bivalve molluscs with retention time close to STX under the qualitative and quantitative selected reaction monitoring (SRM) conditions of STX which are often used in the previous reports. To avoid the assignment of the unidentified STX analogue to STX, appropriate SRM conditions of m/z 300.1>282.0 need to be selected.