Micro qNMR in conjunction with IDMS was used to set up new certified reference material solutions for algae toxins such as paralytic shellfish toxins.

The presence of marine toxins in shell fish and sea food has always been a concern, but has spread in recent years. Increasing global temperatures are suspected to be the reason for an observed expansion of algae species that are commonly the origin of these toxic compounds into areas where they have not been found before. Examples are the emerging of ciguatoxin contaminated fish in the canaries or the increasing occurrence of paralytic shellfish toxins in Northern American lakes.

It is therefore very important, that fish and seafood are monitored for contamination by algae toxins. Historically, this has been done by bio-assays such as ELISA or mouse bioassays. However with LC-MS instruments being more sensitive and easier to use, various new methods using LC-MS have been established in recent years. Also, LC-MS is recommended or required as a method by newer regulations such as EC regulation No 15/2011.

Therefore, the access to well characterized reference materials for quantitation of these different toxins has become an increased need in the market. These reference materials should be characterized and prepared according to ISO/IEC 17025 and ISO 17034 to ensure highest precision and accuracy. Unfortunately, isolation, synthesis and purification of those compounds or their stable isotope labeled analogs is very challenging and batch sizes are typically in the range of a few mg. In order to achieve certification of such small batches according to the above mentioned double accreditation a combined setup of quantitative NMR (qNMR) and Isotope Dilution MS (IDMS) was successfully established.

In a first step, the accurate concentration of a dissolved toxin is determined by 1H-High Performance®-qNMR measurements. Gravimetric dilution and ampule filling delivers the final product with a certified concentration and an associated expanded uncertainty, which can be subsequently applied in an HPLC-IDMS experiment that results in a concentration for the stable isotope labeled analog. Gravimetric IDMS experiments are also carried out to determine the homogeneity and stability contribution to the overall uncertainty.

These concepts were successfully adopted for the certification of multiple toxins despite that they tend to undergo rearrangement or deuterium exchange reactions and often show a low stability at ambient temperature.

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