Genotyping of Toxic Pufferfish Based on Specific PCR-RFLP Products As Determined by Liquid Chromatography/Quadrupole-Orbitrap Hybrid Mass Spectrometry.

A method based on liquid chromatography-electrospray mass spectrometric analysis of the enzymatically digested amplicons derived from the mitochondrial 16S rRNA gene was established for the discrimination of toxic pufferfish. A MonoBis C18 narrow-bore silica monolith column (Kyoto Monotech) and a Q Exactive mass spectrometer (Thermo Fisher) were employed for separation and detection, respectively. Monoisotopic masses of the oligonucleotides were calculated using Protein Deconvolution 3.0 software (Thermo Fisher). Although a lock mass standard was not used, excellent accuracy (mass error, 0.83 ppm on average) and precision (relative standard deviation, 0.49 ppm on average) were achieved, and a mass accuracy of <2.8 ppm was maintained for at least 180 h without additional calibration. The present method was applied to 29 pufferfish samples, and results were consistent with Sanger sequencing.