[Determination of Flubendazole and Metabolite in Livestock Products Using LC-MS/MS].

This study proposes a method to determine flubendazole and metabolite R35475 in livestock products using tandem mass spectrometry coupled with positive ion electrospray ionization. Acetone is used to extract flubendazole and metabolite R35475 from the livestock samples. These extracts were purified using an SCX cartridge column (500 mg). Furthermore, high-performance liquid chromatography was performed on an Inertsil ODS-4 column with a gradient formed using methanol and water, both of which contain 5 mmol/L of ammonium acetate. The recovery tests using bovine muscle, fat, liver, milk, and egg fortified at the maximum residue limits of analytes or 0.005 mg/kg revealed that the trueness (n=5) of flubendazole and metabolite R35475 ranged from 89.4 to 106.4% with a repeatability rate of 1.7-7.8%.

[Determination of Quinclorac in Livestock Products by LC-MS/MS].

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed for determining quinclorac in livestock products. Quinclorac was extracted from the samples using a solution of acetone and hydrochloric acid mixed in a 99 : 1 ratio. The crude extract was purified with ethyl acetate under basic conditions, followed by quinclorac extraction with ethyl acetate under acidic conditions and analysis using LC-MS/MS. The average recoveries of quinclorac from five livestock products (n=5) fortified at the maximum residue limits or 0.01 mg/kg ranged from 85.6 to 93.5%, with the precision of repeatability ranging from 1.7 to 6.8%. The quantification limit in this analytical method was 0.01 mg/kg. These results suggest that the developed method is useful for analyzing quinclorac in livestock products.

[Determination of Albendazole Metabolite in Livestock Products Using LC-MS/MS].

A method for determining albendazole metabolite (metabolite I) in livestock products using LC-MS/MS was proposed. Livestock samples were hydrolyzed with 6 mol/L HCl at 110℃ for an hour and defatted with ethyl acetate and n-hexane (1 : 1, v/v) mixture. Metabolite I was extracted with acetonitrile from the sample, and the extracts were salted out under basic conditions, allowing the acetonitrile layer to separate. The acetonitrile solution was cleaned up using a cartridge column packed with divinylbenzene-N-vinylpyrolidone copolymer bearing sulfo groups. The HPLC separation was conducted on an Inertsil ODS-4 column with a gradient formed from water containing 0.05% (v/v) formic acid and acetonitrile containing 0.05% (v/v) formic acid. To detect metabolite I, tandem mass spectrometry with positive ion electrospray ionization was used. Truenesses (n=5) of metabolite I from cattle meat, fat, liver, and milk spiked at the maximum residue limits or the 0.01 mg/kg were in the range from 83.6 to 97.9%, and the relative standard deviations were from 1.6 to 6.1%.

[Determination of Hexazinone and Its Major Metabolites in Livestock Products by LC-MS/MS].

A method for the determination of hexazinone and three metabolites (hexazinone metabolite B, hexazinone metabolite C, hexazinone metabolite F) in livestock products by LC-MS/MS was developed. Hexazinone and the three metabolites were extracted from a sample with acetonitrile in the presence of n-hexane, and lipid was removed by acetonitrile/n-hexane partition. The acetonitrile extract was cleaned up using a SAX/PSA cartridge column. Average recoveries (n=5) of hexazinone and the three metabolites from cattle meat, fat, liver and milk spiked at the maximum residue limits (MRLs) or at 0.0025 mg/kg ranged from 85.6 to 96.0%, and the relative standard deviations ranged from 0.8 to 4.9%.

Determination of Diphenylamine in Agricultural Products by HPLC-FL.

A method for the determination of diphenylamine in agricultural products was developed. Diphenylamine was extracted with acetonitrile from a sample under an acidic condition, passed through a C18 cartridge column, re-extracted with n-hexane, cleaned up on a PSA cartridge column, determined by HPLC with fluorescence detector and confirmed by liquid chromatography with tandem mass spectrometry. Average recoveries (n=5) from brown rice, corn, soybeans, potato, cabbage, eggplant, spinach, orange, apple and green tea were in the range from 76.7 to 94.9%, and the relative standard deviations were from 0.6 to 5.8% at concentrations equal to the maximum residue limits (MRLs). The quantification limits were 0.01 mg/kg, which is the uniform limit in the positive list system for agricultural chemical residues in food in Japan.

[Analytical method of clofencet in animal fishery products by LC/MS].

A method for the determination of clofencet in animal and fishery products was developed, using liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The sample was homogenized with water and hexane, and the homogenate was extracted with acetonitrile, then acetonitrile-water (4 : 1). An aliquot of the crude extract was passed through a C18 cartridge column (1,000 mg), and the eluate was concentrated. A solution of 10% sodium chloride and 1% sodium hydrogen carbonate, and ethyl acetate were added to the residue, and the mixture was shaken. After shaking, the aqueous phase was recovered and acidified with hydrochloric acid, and then clofencet was extracted with ethyl acetate. The extract was evaporated to dryness and the residue was dissolved in methanol-water (3 : 7). Clofencet was analyzed by LC/MS. The recoveries of clofencet from ten kinds of animal and fishery products were 77.8-97.8%, and the relative standard deviations were 0.6-5.8% (n=5). S/N of the peak of clofencet was >10 and no interfering peak was found in animal and fishery products fortified at 0.01 mg/kg.

[Analytical method of dodine in agricultural products by LC/MS].

A method for the determination of dodine in agricultural products was developed by using liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS). Dodine was extracted with acetonitrile and then acetonitrile-water (7 : 3) from a sample, and re-extracted with ethyl acetate. The extract was cleaned up on a PSA cartridge column (500 mg), and dodine was analyzed by LC/MS. In the case of oil seeds and nuts, hexane/acetonitrile-hydrochloric acid partition was performed to remove lipids before re-extraction with ethyl acetate. In the case of samples that contained a lot of chlorophyll, the eluate of the PSA cartridge column was further cleaned up on a graphitized carbon cartridge column (500 mg). The calibration curve was linear from 0.0001-0.02 microg/mL of dodine. The recoveries of dodine from sixteen kinds of agricultural products fortified at 0.1 mg/kg were 80.3-100.0%, and their relative standard deviations were 0.3-6.4%. The limits of detection (S/N=3) were 0.0006 mg/kg.

Validation of an HPLC analytical method coupled to a multifunctional clean-up column for the determination of deoxynivalenol.

To evaluate a method using a multifunctional clean-up column coupled with high performance liquid chromatography as an official analytical method for the determination of deoxynivalenol in wheat used as food or feed, an inter-laboratory study was performed in 12 laboratories using four naturally contaminated wheat samples and one spiked sample. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) of naturally contaminated wheat were in the range 5.8-11.3% and 12.0-20.7%, respectively. The HORRAT was less than 1.0 in each sample. From the spiking test, the recovery rate, RSDr, RSDR and HORRAT value were 100.0%, 11.2%, 10.3% and 0.5, respectively. The limit of quantification is 0.10 mg/kg from the range obtained in a linear calibration. Thus, it should be useful as a sensitive and validated analytical method for the determination of deoxynivalenol in wheat intended for use in food and feed.