Spectroscopy Solutions

Absolute Quantification of Lipophilic Shellfish Toxin Standards by Quantitative 1H NMR

Learn about accurate quantification of lipophilic toxins in shellfish and why it is important in food safety and often hampered due to lack or limited availability of authentic standards. To solve the problem, we investigated calibration of representative lipophilic shellfish toxin standards, DTX1, OA and YTX, by quantitative NMR (qNMR) using Certified Reference Material (CRM) as internal standard (IS). 


This method is nondestructive and produces results traceable to the International System of Units. To avoid contamination due to an IS, we employed the indirect methods that use the residual proton signals in deuterated solvent as removable IS. Therefore, ISs are quantified by qNMR using CRM in advance. Dried toxin standards were dissolved in CD3OD or pyridine-d5.

The concentration of a toxin standard in solution was determined by 1H NMR, using the following conditions: data acquisition period, 4.0 sec; relaxation delay, 60 sec; spectral width, 40 ppm region; scans, 8 times; pulse angle, 90º; temperature, 5 or 7ºC; S/N >100.

In the spectra thus obtained, the separation of signals for oxymethine, oxymetylene, and olefinic protons was clearer than that for protons in other regions and judged suitable for use in quantification.

In addition, the signals with better reproducibility throughout the data processing such as manually operated phase correction were chosen.

In this study, the uncertainty of quantitative value of respective toxin standards was about 1%. The optimized qNMR conditions were applicable to the accurate quantification of lipophilic shellfish toxins. The method has the merit of simple manipulation and low risks of contamination.


Tsuyoshi KatoTsuyoshi Kato is the study director of quantitative and qualitative analysis using NMR at Japan Food Research Laboratories. He studied at Mie University and obtained his MSc in the Graduate School of Bioresources. He has presented on the absolute quantification of lipophilic shellfish toxin standards by quantitative 1H NMR at the 127th International Annual Meeting and Exposition in Chicago, USA and also on the accurate valuation of DTX1 standard by quantitative nuclear magnetic resonance at the 15th International Conference on Harmful Algae in Changwon, Korea. 

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