Development and Validation of an LC-MS/MS Method for the Ultra-Trace Analysis of Pacific Ciguatoxins in Fish.

BACKGROUND: Ciguatera fish poisoning (CFP) poses a serious threat to both public health and the use of aquatic resources from the various warm-water regions of the world. Hence, a process for the efficient determination of the relevant toxins is required. OBJECTIVE: We sought to develop and validate the first LC-MS/MS method to quantify the major toxins prevalent in fish from the Pacific Ocean. METHOD: Toxins were extracted from fish flesh (2 g) using a methanol-water mixture (9:1, v/v). The extract was heated at 80°C, and low-polarity lipids were eliminated using hexane, initially from the basic solution and later from the acidic solution. The cleanup was performed using solid-phase extraction, Florisil, silica, reversed-phase C18, and primary secondary amine columns. A validation study was conducted by spiking fish flesh with two representative toxins having different skeletal structures and polarities and was calibrated by NMR (qNMR) spectroscopy. RESULTS: The validation parameters for the ciguatera toxins CTX1B and CTX3C at spiked levels of 0.1 µg/kg were as follows: repeatabilities of 2.3-3.5% and 3.2-5.3%; intermediate precisions of 6.3-9.8% and 6.0-7.4%; recoveries of 80-107% and 95-120%, respectively. The lowest detection levels were 0.004 µg/kg for CTX1B, 0.005 µg/kg for 51-hydroxyCTX3C, and 0.009 µg/kg for CTX3C. CONCLUSIONS: The described method practically clears the international action level of 0.01 µg/kg CTX1B equivalents set by the U.S. Food and Drug Administration and the European Food Safety Authority and satisfies the global standards set by Codex and AOAC INTERNATIONAL. HIGHLIGHTS: A validation study for an LC-MS/MS method for ciguatoxin detection was completed for the first time using calibrated toxin standards.

Determination of carbendazim, thiophanate, thiophanate-methyl and benomyl residues in agricultural products by liquid chromatography-tandem mass spectrometry.

A simple and reliable liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for carbendazim (MBC), thiophanate (TE), thiophanate-methyl (TM) and benomyl (BM) in agricultural products. These compounds were extracted from agricultural products with methanol after addition of sodium L-ascorbate. BM was hydrolyzed to MBC during the extraction with methanol. TE and TM were cyclized to ethyl 2-benzimidazole carbamate (EBC) and MBC by refluxing at 120 °C for 30 min with copper acetate in 50% acetic acid. MBC and EBC were cleaned up by an n-hexane wash and extraction with ethyl acetate and determined by LC-MS/MS. The mean recoveries from 10 agricultural products were in the range of 75.8-100.0%, and the relative standard deviations of 5 experiments were in the range of 1.5-9.2% at concentrations equal to the maximum residue limits (MRLs). The calibration curves were made by using commercial MBC and EBC as reference analytical standards without refluxing. The quantification limits were 0.01 mg/kg (as MBC), which is the uniform limit in the positive list system for agricultural chemical residues in foods in Japan.

Development of green onion and cabbage certified reference materials for quantification of organophosphorus and pyrethroid pesticides.

Green onion and cabbage certified reference materials for the analysis of pesticide residues were issued by the National Metrology Institute of Japan, part of the National Institute of Advanced Industrial Science and Technology. Green onion and cabbage samples were grown so as to contain several kinds of organophosphorus and pyrethroid pesticides, and those were collected from a field in the Kochi Prefecture in Japan. The certification was carried out by using multiple analytical methods to ensure the reliability of analytical results; the values of target pesticides (diazinon, fenitrothion, cypermethrin, etofenprox, and permethrin for green onion and chlorpyrifos, fenitrothion, and permethrin for cabbage) were obtained by isotope dilution mass spectrometry. Certified values of target pesticides were 0.96-13.9 and 2.41-6.9 mg/kg for green onion and cabbage, respectively. These are the first green onion and cabbage powder certified reference materials in which organophosphorus and pyrethroid pesticides are determined.

Determination of dithiocarbamates and milneb residues in foods by gas chromatography-mass spectrometry.

A highly sensitive gas chromatographic-mass spectrometric (GC-MS) method was developed for dithiocarbamates (DTCs) and milneb in foods. DTCs and milneb were extracted from foods with cysteine-EDTA solution as sodium salts, and methylated with methyl iodide. Methyl derivatives of DTCs and milneb were cleaned up on a neutral alumina mini column and determined by GC-MS. The mean recoveries of DTCs and milneb were in the range of 72-120%, except for methiram. The quantification limits were 0.01 mg/kg (as CS(2)) in foods except tea (0.1 mg/kg as CS(2)). The developed method was applied to 10 compounds (4 dimethyldithiocarbamates, 3 ethylenebisdithiocarbamates, polycarbamates, propineb and milneb).