Isotope-labelling derivatisation: a broadly applicable approach to quantitation of algal toxins by isotope dilution LC-MS/MS

Abstract

Two methods were developed for the analysis of algal biotoxins in complex biological and environmental samples to demonstrate the concept of isotope-labelling derivatisation for quantitation. These methods are based on dansyl chloride derivatisation of samples and dansyl-d₆ chloride derivatisation of toxin standards. Derivatised sample and standard are then mixed to achieve isotope dilution calibration in liquid chromatography–tandem mass spectrometry analyses. Quantitation of the marine toxin domoic acid (DA) in mussel tissues and the freshwater toxins anatoxin-a (ATX) and homoanatoxin-a (hATX) in cyanobacteria is demonstrated. For DA, isotope-labelling was incorporated into existing dansylation methodology using inexpensive and commercially available reagents. For ATXs, a novel sample preparation procedure is presented that involves solid phase extraction on a mixed reverse phase/weak anion exchange column that facilitates simultaneous clean-up of the derivatised toxins and removal of excess dansylation reagent through covalent bonding. The challenge of achieving co-elution in LC between deuterated and non-deuterated dansylated toxins was addressed by modifying separation conditions from the usual reverse phase (RP) separation to hydrophilic interaction liquid chromatography in the case of DA and a shortened RP separation with high organic modifier content in the case of the ATXs. The new methods gave limits of detection between 10 and 60 μg kg⁻¹ and allowed for precise, accurate and fast determination of toxins in spiked control samples and matrix reference materials. This work demonstrates that isotope-labelling derivatisation is broadly applicable to the field of algal toxin analysis where derivatisation is well established but isotopically-labelled standards are not available.