Ibotenic acid (IBA) is an amino acid and muscimol (MUS) is the decarboxyl derivative of IBA. They are mushroom neurotoxins with high polarity and low molecular weight. Only one transition (159->113 for IBA and 115->98 for MUS) can be found when directly measured by high performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Therefore, the identification and quantification of trace amount of the toxins in biomaterial are difficult. A highly sensitive and accurate analytical method for IBA and MUS in plasma was developed by LC-MS/MS with the application of bimolecular dansylation and internal standard calibration. Acetonitrile was used for protein precipitation and for toxin extraction from plasma. The toxins and internal standards (L-tyrosine-13C9,15N for IBA and tyramine-d4 for MUS) were derivatized with dansyl chloride (DNSCl). The reaction conditions of the bimolecular dansylation were optimized and the fragmentation pathways of the derivatives in MS/MS were studied. Method validation was carried out according to the Bioanalytical Method Validation Guidance for Industry (FDA, USA, 2018). The limits of detection for IBA and MUS in plasma were 0.3 ng mL-1 and 0.1 ng mL-1, respectively. The linear ranges in plasma were 1-500 ng mL-1 and 1-200 ng mL-1 with the correlation coefficients of 0.998 and 0.999 for IBA and MUS, respectively. The recoveries at three spiked levels were 90.7-111.4% with relative standard deviations (RSDs) of 6.4-10.3% for IBA and the results were 85.1-94.2% with RSDs of 5.0-8.9% for MUS. The toxin levels in patients' plasma samples under different poisoning degree were presented.
Keywords: Bimolecular dansylation; Ibotenic acid; Internal standard calibration; Liquid chromatography-triple quadrupole mass spectrometry; Muscimol; Plasma.
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