Simple and fast determination of tetrodotoxin in human plasma based on hydrophilic-interaction/ion-exchange mixed-mode solid phase extraction combined with liquid chromatography-tandem mass spectroscopy

In this study, we developed and validated a simple, fast and sensitive LC-MS/MS method for the mea-surement of tetrodotoxin (TTX) in human plasma. Three HILIC-type solid phase extraction (SPE) carriers (PSA, silica, Siphila i HILIX) with different stationary phase functional groups were compared. The Siphila i HILIX SPE plate containing multi-carboxyl groups was finally selected due to obviously better extraction recovery of TTX (about 80% of recovery from plasma samples) than the other two and no significant ma-trix effects were observed, which was speculated to have mixed-mode synergistic effects of hydrophilic interaction and ion exchange. 100 mu L plasma sample was precipitated rapidly with acetonitrile containing 1% trichloroacetic acid, and filtrates were loaded onto Siphila i HILIX 96 well SPE plate. After washed with 95% acetonitrile, TTX was eluted with 200 mu L of 50% acetonitrile containing 1% trichloroacetic acid. 2 mu L of elution solution was directly injected into LC-MS/MS and the total run time on a BEH amide col-umn was 4.5 min. The method avoids the evaporation and ultrafiltration processes which is simple and timesaving ( < 30 min). TTX and internal standard (arginine-15N4) were monitored in positive mode using m/z 320.3 & RARR; 162.2 (quantification transition for TTX), 320.3 & RARR; 284.1 (confirmation transition for TTX) and 179.2 & RARR; 63.0 (transition for IS), respectively. The method was linear in the range of 0.1-20 ng/mL for TTX with the low limit of quantification (S/N > 10) of 0.1 ng/mL; the intra-and inter-assay accuracies were in the range of 98.5%-99.8% (relative standard deviations, RSDs < 5.92%) and 98.8-99.5% (RSDs < 6.23%), re-spectively. Biases of spiking analysis were ranged from-7.00% to 7.43% for healthy human plasma samples (RSDs < 8.83%) and from-5.00% to 3.93% for hemolytic, high triglyceride, high cholesterol and high biliru-bin plasma samples (RSDs < 6.40%), which proved the good anti-interference property of the method. The results showed that the method is sensitive, accurate, specific, reliable, and can be used to monitor the concentration of TTX in plasma to meet the needs of clinical research and poisoning screening.(c) 2022 Published by Elsevier B.V.

Automated summary

In this study, a simple, fast, and sensitive LC-MS/MS method was developed and validated for measuring tetrodotoxin (TTX) in human plasma. Different solid phase extraction (SPE) carriers with different functional groups were compared, and the Siphila i HILIX SPE plate with multi-carboxyl groups was selected for better extraction recovery of TTX. The method avoids the evaporation and ultrafiltration processes, making it simple, time-saving, and effective for analyzing TTX in plasma.