Determination of tetrodotoxin in urine by liquid chromatography-tandem mass spectrometry with internal standard calibration

Abstract

An analytical method was developed for tetrodotoxin(TTX) in urine by liquid chromatography-tandem mass spectrometry(LC-MS/MS) with internal standard calibration. TTX in the sample was extracted with the mixture of acetic acid/methanol/acetonitrile(0.005 mL/0.8 mL/1.8 mL), cleaned by solid phase extraction(SPE) with cation exchange cartridge, eluted with 50% acetonitrile/water containing 0.3% hydrochloric acid, and neutralized with ammonia. The extract was separated by a Waters XBridge~(TM) BEH Amide column(150 mm×3.0mm, 1.7 μm) and measured by MS/MS. By optimizing sample extraction and SPE cleanup conditions, the problems of low recovery and strong suppression effects of MS signal for TTX in urine were resolved when cleaned with cation exchange cartridge. Quantitatively calibrated by the internal standard of Kasugamycin, good linear relationship was found for TTX in urine at the range of 0.2-200 μg/L with the correlation coefficient(r~2) of 0.997. The limits of detection and quantitation for TTX in sample matrix were 0.1 and 0.2μg/L, respectively. The average recoveries at three spiking levels(0.2, 10.0 and 200 μg/L) were 89.3%-95.3% with relative standard deviation(n=6) less than 5.1%. The concentrations of TTX in urine from 11 poisoning patients were 0.4-138 μg/L. The detection rate was 100% in urine collected within 3 days after poisoning. The established method was simple, accurate and sensitive. It can provide reliable technical support for the rapid treatment of TTX poisoning events and the study of toxin metabolism in vivo.