Abstract
Clinical diagnosis of tetrodotoxin (TTX) poisoning can be difficult because of the lack of characteristic morphological findings and a screening test, such as an immunoassay. Here, we present a fully validated method for the analysis of TTX in serum and urine. In this method, serum and urine samples were extracted using MonoSpin CBA or amide columns, followed by LC–MS/MS analysis. The TTX was eluted from the column by 0.1 mL of 10 % acetic acid solution, and was directly injected into LC–MS/MS. An Agilent 1200 HPLC system equipped with a HILIC separation column (Zorbax HILIC Plus 2.1 × 100 mm, 3.5 μm) was used for isocratic elution, with a mobile phase of 10 mM ammonium formate with formic acid (95:5, v/v), along with 5 mM trifluoroacetic acid and 2 % acetonitrile. TTX was detected with an Agilent 6410 mass spectrometer utilizing positive electrospray ionization and multiple reaction monitoring. Limits of quantification for serum and urine were established to be 1 and 0.5 ng mL−1, respectively. Limits of detection for serum and urine were 0.5 and 0.25 ng mL−1, respectively. Intra-day and inter-day precision varied from 1.5 to 8.5 %. The recovery was >86.5 % for both matrices. In this method, the sample preparation process prior to injection into the LC–MS/MS takes approximately 10–15 min, which reduces the extraction time to one-tenth of that of previous methods. The application of this method was further verified by analysis of biological materials from a patient suffering from puffer fish poisoning.
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This work was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 25460878) from the Japan Society for the Promotion of Science.
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Saito, T., Miura, N., Namera, A. et al. A Rapid Sample Preparation Procedure Using MonoSpin CBA and Amide Columns for Tetrodotoxin Detection in Serum and Urine Using LC–MS/MS Analysis. Chromatographia 77, 687–693 (2014). https://doi.org/10.1007/s10337-014-2663-3
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DOI: https://doi.org/10.1007/s10337-014-2663-3