[Analytical Method for Melengestrol Acetate in Livestock Products Using LC-MS/MS].

The present study verified that it is possible to analyze melengesterol acetate using the existing multi-residue method. Melengestrol acetate was extracted from livestock products using acidic acetonitrile acidified with acetic acid in the presence of n-hexane and anhydrous sodium sulfate. The crude extracts were cleaned up using an octadecylsilanized silica gel cartridge column. Separation by HPLC was performed using an octadecylsilanized silica gel column with linear gradient elution of 0.1 vol% formic acid and acetonitrile containing 0.1 vol% formic acid. For the determination of the analyte, tandem mass spectrometry with positive ion electrospray ionization was used. In recovery tests using four livestock products fortified with maximum residue limits levels of melengestrol acetate (0.001-0.02 mg/kg), the truenesses ranged from 82% to 100%, and the repeatabilities for the entire procedure ranged from 0.5 RSD% to 5.6 RSD%. In recovery tests using 11 livestock products fortified with 0.0005 mg/kg of melengestrol acetate, the truenesses ranged from 88% to 99%, and the repeatabilities ranged from 1.3 RSD% to 5.4 RSD%. The limit of quantification for melengestrol acetate in livestock products was 0.0005 mg/kg.

[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%.

[Development of Analytical Method and Surveillance ofGibberellic Acid in Banana, Cherry, and Kiwi Fruit].

Gibberellic acid (GA3) is commonly used as a plant growth regulator in many food crops owing to its essential signaling functions during plant growth and development. In Japan, a threshold for administrative action for GA3 content of 0.3 mg/kg applies in produce in which maximum residue limits have not been established. Although the threshold is based on previous studies, the GA3 concentrations in individual foods are still unknown. Thus, we surveyed the concentrations of GA3 in banana, cherry, and kiwi fruit on the Japanese market. We developed and validated a method for the analysis of GA3 using solid-phase extraction and LC-MS/MS in accordance with accepted criteria of trueness, repeatability, and selectivity. The limits of detection and of quantification were determined as 0.005 and 0.05 mg/kg, respectively, in all fruits. Concentrations of GA3 did not exceed 0.3 mg/kg regardless of ripeness, suggesting the reasonability of the current regulation of GA3 in banana, cherry, and kiwi fruit. These findings can support prompt administrative action on these fruits, contributing to the regulation of GA3 in Japan.

[An LC-MS/MS Analytical Method for Moenomycin A in Livestock Products].

A simple and sensitive method for the determination of moenomycin A residues in livestock products using LC-MS/MS was developed. Moenomycin A, a residual definition of flavophospholipol, was extracted from samples with a mixture of ammonium hydroxide and methanol (1 : 9, v/v) preheated at 50℃. The crude extracted solutions were evaporated and purified by liquid-liquid partitioning between a mixture of ammonium hydroxide, methanol and water (1 : 60 : 40, v/v/v) and ethyl acetate. The alkaline layer was taken, and cleaned up using a strong anion exchange (InertSep SAX) solid phase extraction cartridge. The LC separation was performed on an Inertsil C8 column with liner gradient elution using 0.3 vol% formic acid and acetonitrile containing 0.3 vol% formic acid. Moenomycin A was detected using tandem mass spectrometry with negative ion electrospray ionization. Recovery tests were conducted using three porcine samples (muscle, fat and liver) and chicken eggs. Samples were spiked with moenomycin A at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The trueness ranged from 79 to 93% and precision ranged from 0.5 to 2.8%. The limit of quantification (S/N≥10) of the developed method is 0.01 mg/kg. The developed method would thus be very useful for regulatory monitoring of flavophospholipol in livestock products.

[Determination of Chlorothalonil Metabolite I in Livestock Products by LC-MS/MS].

An analytical method based on LC-MS/MS was developed for the determination of chlorothalonil metabolite I in livestock products. Chlorothalonil metabolite I in livestock products was extracted with acetone. The crude extracts were defatted by acetonitrile and n-hexane partitioning. Cleanup was carried out using a combination of ethylene diamine-N-propyl silylation silica gel (PSA) and silica gel (SI) mini columns with acidic condition. The sample solution was subjected to LC-MS/MS using an external solvent calibration curve. The average recovery (n=5) of chlorothalonil metabolite I from five types of livestock products (cattle muscle, cattle fat, cattle liver, milk and egg) spike at the maximum residue limits (MRLs) or at a uniform limit of 0.01 mg/kg was 97.1-102.9%, with a relative standard deviation of 1.4-6.8%. The limit of quantitation of the developed method was calculated to be 0.01 mg/kg.

[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 Asulam in Livestock Products by LC-MS/MS].

An analytical method based on LC-MS/MS was developed for the determination of asulam in livestock products. Asulam in livestock products was extracted with acetone. The crude extracts were defatted by acetonitrile and n-hexane partitioning. Cleanup was carried out using a combination of ethylene diamine-N-propyl silylation silica gel (PSA) and octadecyl silylated silica gel (C18) mini columns with acidic condition. The sample solution was subjected to LC-MS/MS using an external solvent calibration curve. The average recovery (n=5) of Asulam from four types of livestock products (bovine muscle, bovine fat, bovine liver and milk) spike at the maximum residue limits (MRLs) or at a uniform limit of 0.01 mg/kg was 92.7-98.7%, with a relative standard deviation of 3.1-11.6%. The limit of quantitation of the developed method was calculated to be 0.01 mg/kg.

Multiresidue method for determining multiclass acidic pesticides in agricultural foods by liquid chromatography-tandem mass spectrometry.

A reliable multiresidue method was developed for determining multiclass acidic pesticides in cereal grains, legumes, vegetables, and fruits. The target pesticides comprise 75 compounds, including phenoxy acid, sulfonylurea, imidazoline, and triazolopyrimidine herbicides, with acidic dissociation constant (pKa) values of 1.9-5.9. The method includes extraction with acidified acetonitrile, salting out, cleanup with octadecyl silica and primary secondary amine cartridges, and subsequent liquid chromatography-tandem mass spectrometry. The analytical performance of the developed method was validated for nine foods (i.e., brown rice, soybeans, peanuts, spinach, cabbage, eggplant, potatoes, apples, and oranges) at a concentration of 0.01 mg kg-1. Because matrix effects were negligible for most pesticide and food combinations, solvent-based calibration curves were used for quantification purposes. Most of the target compounds exhibited satisfactory analytical performance with trueness values of 70-100% and relative standard deviations below 14%. The high selectivity of the developed method was evidenced by the absence of interfering peaks near those of the target analytes. With the exception of 1-naphthaleneacetic acid, for which linearity was observed at 2.5-100 ng mL-1, linear calibration curves were constructed for the target compounds in the 1-100 ng mL-1 range, with coefficients of determination exceeding 0.995. The limits of detection were 3 µg kg-1 or below in the examined matrices. The results demonstrate that the developed method is suitable for monitoring acidic pesticides in a variety of foods.

Quantitative and Confirmatory Analysis of Pesticide Residues in Cereal Grains and Legumes by Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

For controlling pesticide residues in food and ensuring food safety, multiresidue methods that can monitor a wide range of pesticides in various types of foods are required for regulatory monitoring. In this study, to demonstrate the applicability of liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) for quantitative and confirmatory analysis of pesticide residues in cereal grains and legumes, the LC-QTOF-MS method using full-scan acquisition was validated for 151 pesticides in brown rice, soybeans, and peanuts at a spiked level of 0.01 mg/kg. With the exception of 5 out of 151 target pesticides, sufficiently high signal intensities were obtained at 0.005 µg/mL (corresponding to 0.01 mg/kg). Trueness was in the range 70-95%, with intra- and inter-day precisions below 16% and 24%, respectively, with the exception of 7 pesticides in brown rice, 10 pesticides in soybeans, and 9 pesticides in peanuts. No interfering peaks were observed near the retention times of the target pesticides. Furthermore, information on accurate fragment-ion masses obtained by a data-independent acquisition enabled unambiguous confirmation. The results suggest that the LC-QTOF-MS method is suitable for pesticide residues' analysis of cereal grains and legumes, and can be utilized for regulatory routine analysis.

Multi-residue determination of pesticides in green tea by gas chromatography-tandem mass spectrometry with atmospheric pressure chemical ionisation using nitrogen as the carrier gas.

Helium is commonly used as a carrier gas in gas chromatography-tandem mass spectrometry (GC-MS/MS); however, there are growing concerns regarding its global shortage and the resulting limited supply and high cost. Using nitrogen as an alternative carrier gas in GC-MS/MS with the widely used electron ionisation (EI) technique leads to a significantly lower sensitivity; thus, in this study, we explored the use of atmospheric-pressure chemical ionisation (APCI) as the ionisation method and examined the applicability of GC-(APCI)MS/MS with nitrogen gas for the determination of pesticide residues. GC-(APCI)MS/MS using nitrogen provided slightly wider peaks, and poorer isomeric separation compared to those using helium under identical conditions; however, the peak intensities were comparable. GC-(APCI)MS/MS using nitrogen was validated for 166 pesticides in green tea at a spiking level of 0.01 mg/kg and was compared with the conventional GC-(EI)MS/MS using helium gas. Except dimethomorph and resmethrin, GC-(APCI)MS/MS showed satisfactory results that were comparable to those of GC-(EI)MS/MS for most compounds, with trueness in the range of 73%-95% and relative standard deviations of <11%. The sensitivity and selectivity of GC-(APCI)MS/MS with nitrogen were superior to those of GC-(EI)MS/MS with helium. Therefore, GC-(APCI)MS/MS using nitrogen as the carrier gas, which has minimal concerns related to availability, could be a promising alternative to the conventional GC-(EI)MS/MS technique that employs helium.

[Analytical Method for Fipronil and Fipronil Sulfone in Livestock Products Using LC-MS/MS].

A rapid and sensitive method for the simultaneous determination of fipronil and fipronil sulfone (metabolite B) in livestock products was developed. The analytes were extracted from samples with acidic acetonitrile. The crude extracts were subjected to clean-up step using neutral alumina cartridge column. The HPLC separation was performed on a C18 column with isocratic elution of acetonitrile and ammonium formate solution. For the determination of the analytes, a tandem mass spectrometry with negative ion electrospray ionization was used. In the recovery tests using 6 livestock products fortified with MRLs levels of analytes, the truenesses for fipronil and fipronil sulfone were 95 to 115 and 94 to 101% with the repeatabilities of 0.8 to 4.1 and 0.9 to 5.1 RSD%, respectively. The limits of quantification for both analytes were estimated to be 0.001 mg/kg. The developed method is considered suitable for regulatory analysis of fipronil and fipronil sulfone.

Quantitative analysis of pesticide residues in tea by gas chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization.

In this study, gas chromatography-tandem mass spectrometry (GC-MS/MS) using an atmospheric pressure chemical ionization (APCI) source was applied for the quantitative analysis of pesticide residues in tea. To determine the optimum ionization conditions for multiresidue analysis, the full-scan mass spectra and peak intensities of pesticides were compared in the presence and absence of water as a modifier. When water was added as a modifier in the ion source, most of the target compounds formed [M+H]+ ions and exhibited enhanced intensities. However, compounds consisting of only carbon, hydrogen, and chlorine, such as aldrin, γ-hexachlorocyclohexane, and p,p'-dichlorodiphenyldichloroethane, typically formed M+· or fragment ions, whose intensities were significantly decreased by the addition of water. GC-MS/MS methods using APCI (without modifier addition) and electron ionization (EI) were validated for 16 pesticides in tea at spiking levels of 0.01 and 0.1 mg/kg. Unlike EI, signal suppression was observed for most compounds at a spiking level of 0.01 mg/kg using APCI; however, dilution of the samples minimized this effect. Using APCI, the trueness of the target compounds ranged from 77% to 121% at both spiking levels, except for pyrethrins owing to matrix effects, with relative standard deviations of less than 14%. For most compounds, these results were comparable with those obtained using EI. However, because the use of APCI limited fragmentation, this ionization technique offered significantly higher sensitivity and specificity than EI. Using APCI, linear calibration curves with coefficients of determination greater than 0.998 were obtained in the range of 0.0005-0.5 µg/mL for all compounds. These findings indicated that GC-MS/MS with APCI is applicable for the routine monitoring of pesticide residues, even in complex samples such as tea.

Development of an analytical method for determination of total ethofumesate residues in foods by gas chromatography-tandem mass spectrometry.

Analytical method was developed for determining the total residue of ethofumesate (ET) herbicide using GC-MS/MS. The ET residues were analyzed as a sum of ET, 2-keto-ethofumesate (KET), and open-ring-2-keto-ethofumesate (OKET) and its conjugate. The extracted samples were partitioned with hexane and NaOH solution. For ET analysis, the hexane layer was cleaned up by a silica gel cartridge prior to GC-MS/MS analysis. For the analyses of the metabolites, the aqueous layer was heated with HCl to hydrolyze the conjugates, thereafter, heated in acetic anhydride to convert OKET to KET, and cleaned up by a silica gel cartridge prior to GC-MS/MS analysis. The method was validated for ET, KET, and OKET in garlic, onion, and sugar beet at 0.3 and 0.01 mg/kg. The recoveries were 94-113%, with relative standard deviations of <6%. The limits of detection were 0.0005 mg/kg for all analytes. The proposed method is suitable for regulatory analysis.

Total determination of triclabendazole and its metabolites in bovine tissues using liquid chromatography-tandem mass spectrometry.

A reliable LC-MS/MS analytical method for the determination of residual triclabendazole and its principal metabolites (triclabendazole sulfoxide, triclabendazole sulfone and keto-triclabendazole) in bovine tissues was developed, in which triclabendazole and its metabolites are oxidized to keto-triclabendazole as a marker residue. The method involves sample digestion with hot sodium hydroxide, thus releasing the bound residues of various triclabendazole metabolites in bovine tissues. The target compounds are extracted from the digest mixture with ethyl acetate, defatted by liquid-liquid partitioning using n-hexane and acetonitrile, then oxidized with hydrogen peroxide in a mixture of ethanol and acetic acid. The reaction mixture is cleaned up using a strong cation exchange cartridge (Oasis MCX) and the analytes are quantified using LC-MS/MS. The optimal conditions for the complete oxidation of triclabendazole and its metabolites to keto-triclabendazole are an incubation time of 16 h and a temperature of 90 °C. The developed method was evaluated using three bovine samples: muscle, fat, and liver. Samples were spiked with triclabendazole and its principal metabolites at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The validation results show excellent recoveries (81-102%) and precision (<10%) for all target compounds. The limit of quantification (S/N ≥ 10) of the developed method is 0.01 mg/kg. These results suggest the developed method is applicable to quantifying residual triclabendazole in bovine tissues in compliance with the MRLs established by the Codex Alimentarius and EU and Japanese regulations, and thus the proposed method will be a useful tool for the regulatory monitoring of residual triclabendazole and its metabolites.

Determination of total florfenicol residues as florfenicol amine in bovine tissues and eel by liquid chromatography-tandem mass spectrometry using external calibration.

A reliable liquid chromatography-tandem mass spectrometry method was developed to determine total florfenicol residues in bovine tissues and eel. Florfenicol and its metabolites (florfenicol amine, monochloroflorfenicol, florfenicol oxamic acid, and florfenicol alcohol) were analyzed as the marker residue, florfenicol amine, as defined by several regulatory agencies. After hydrolysis with hydrochloric acid, samples were defatted and subjected to solid-supported liquid extraction and Oasis MCX-cartridge cleanup before analysis. The method was validated for florfenicol and its metabolites at two levels in eel and bovine muscle, fat, and liver. Excellent recoveries were obtained (93-104%), with relative standard deviations of <6% for all compounds. Negligible matrix effects and minimal analyte loss during sample preparation enabled accurate quantification by external calibration using solvent standards. No interfering peaks were observed around the retention time of florfenicol amine, indicating the high selectivity of the method. Retention times in the spiked samples corresponding to that of the calibration standard in solvent did not exceed ±0.1 min. Ion ratios from the spiked sample were within ±10% (relative) of the calibration standards. Calibration curves were linear in the range of 0.5 to 100 ng/mL, with coefficients of determination higher than 0.998. The limits of quantification and limits of detection of the proposed method were estimated to be 0.01 mg/kg and 0.0005 mg/kg, respectively, in all food samples. Thus, the developed method is considered reliable and suitable for regulatory use.

Determination of the total tulathromycin residues in bovine muscle, fat, and liver by liquid chromatography-tandem mass spectrometry.

A reliable and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed to quantify total tulathromycin residues in bovine tissues. Specifically, the above method relied on the quantification of CP-60,300, a marker produced by tulathromycin hydrolysis, for which maximum residue limits (MRLs) were established by the European Union and several other countries. Sample preparation and LC-MS/MS conditions were thoroughly optimized to allow for accurate quantification. The optimized procedure involved sample homogenization with 2 mol/L hydrochloric acid and ethyl acetate, heating of the resulting aqueous layer to convert tulathromycin and its metabolites into the marker residue, cleanup by a polymer-based cation-exchange cartridge, and subsequent analysis by LC-MS/MS. The developed method was validated for tulathromycin A and the marker residue in bovine muscle, fat, and liver at two levels, namely at the MRL set in Japan and at 0.01 mg/kg. Excellent analytical performance was observed, with the average recoveries of tulathromycin A and the marker residue ranging from 98 to 107%, and relative standard deviations ranging from 1 to 3%. Matrix effects were negligible, and analyte loss during sample preparation was minimal for all matrices tested, which allowed for accurate determination by external standard calibration using a solvent standard. No interfering peaks were observed close to the retention time of the marker residue for all matrices, which was indicative of high specificity. Overall, the developed method was proven suitable for regulatory purpose analysis of total tulathromycin residues.

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

An analytical method for the determination of zilpaterol in livestock products was developed. The sample was stirred with n-hexane and n-hexane saturated acetonitrile, and zilpaterol in the sample was extracted with acetonitrile. The extract was cleaned up on a ODS cartridge column (1 g) and SCX cartridge column (500 mg). The LC separation was carried out using an Inertsil ODS-4 column and linear gradient elution with 0.1%formic acid and acetonitrile containing 0.1% formic acid as mobile phase. Detection of MS was carried out positive ion electrospray ionization mode. Average recoveries (n=5) of zilpaterol from 6 kinds of livestock products fortified at the MRLs (0.01 mg/kg) were 87.0-99.4%, and the relative standard deviations were 2.4-6.3%. The limits of quantitation were 0.01 mg/kg.

Total determination of residual flutolanil and its metabolites in livestock products and seafood using liquid chromatography-tandem mass spectrometry.

We have developed a simple and sensitive LC-MS/MS analytical method for the determination of residual flutolanil and its principal metabolites, including α,α,α-trifluoro-3'-hydroxy-o-toluanilide (M-4) and its conjugates, in livestock and seafood products. Both flutolanil and its metabolites contain the 2-(trifluoromethyl)benzoic acid (2-TFMBA) moiety. In this method, flutolanil and its metabolites are converted to 2-TFMBA by hydrolysis. The method involves direct hydrolysis with sodium hydroxide at 200°C, acidification, partitioning into a mixture of ethyl acetate-n-hexane (1:9, v/v), clean-up using a strong anion exchange cartridge (InertSep SAX), and then quantification using LC-MS/MS. The optimal conditions for the complete hydrolysis of flutolanil to 2-TFMBA are an incubation time of 6 h and a temperature of 200°C. The developed method was evaluated using seven types of food: bovine samples of muscle, fat, liver and milk, as well as egg, eel, and freshwater clam. Samples were spiked both at 0.01 mg/kg and at the Japanese maximum residue limit (MRL) established for each food type. The validation results show excellent recoveries (88-107%) and precision (< 10%) for flutolanil and M-4. The limit of quantification (S/N ≥ 10) of the developed method is 0.01 mg/kg. The developed method is applicable to the definition of residual flutolanil for animal-based food commodities and MRLs established by the Codex Alimentarius, and will be useful for the regulatory monitoring of residual flutolanil and its metabolites in food products.

[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%.