Determination of Tetrodotoxin in Human Urine and Plasma after Immunoaffinity Column Clean-up Using Ultra-Performanc Hydrophilic Interaction Chromatography Coupled with Triple Quadrupole Mass Spectrometry

An ultra-performance hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometric (UPLC-MS/MS) method was developed for the determination of tetrodotoxin (TTX) in human urine and plasma. After the sample was cleaned-up and concentrated by immunoaffinity column, the separation of the TTX was carried out on an Acquity UPLC BEH amide column (100 mm x 2.1 mm, 1.7 mu m) with gradient elution using mobile phases of 0.1% (V/V) formic acid in water and acetonitrile. The analyte was detected by positive electrospray ionization mass spectrometry in the multiple reaction monitoring (MRM) mode, and quantified by external solvent standard calibration. The measuring ranges of TTX in urine and plasma were 0.05 - 400 mu g/L. The average recoveries were 92% - 95% and 91% - 96% for TTX respectively spiked in urine and plasma with relative standard deviations of 3.3% - 7.2% and 3.9% - 7.8% ( n = 5). The limits of detection (LOD, S/N = 3) and limits of quantitation (LOQ, S/N = 10) of TTX were 0.02 mu g/L and 0.05 mu g/L for urine and plasma, respectively. This method is suitable for the detection of TTX in urine and plasma for both forensic and clinical purposes.