[Method Improvement for Quantitative Analysis of Chlorophyll Degradation Compounds, Including Pheophorbide, in Chlorella Products].

An official analytical method for chlorophyll degradation compounds, including pheophorbide, in chlorella products, is described in notification Kanshoku No. 99 (May 8, 1981). However, this method has several operational issues, such as the formation of emulsion during liquid-liquid partitioning. Additionally, impurities present in the reagents (sodium sulfate decahydrate or anhydrous sodium sulfate) used to prepare saturated sodium sulfate solution can degrade pheophorbide and other related compounds, resulting in a significant decrease in analytical values. In this study, we thoroughly examined each step of the official method to enhance the operability and develop an alternative method that eliminates the need for saturated sodium sulfate solution. The developed method was evaluated for pheophorbide a and pyropheophorbide a at 100 mg%. Satisfactory analytical performance was achieved with trueness of 100% for pheophorbide a and 90% for pyropheophorbide a, and relative standard deviations of intra- and inter-day precision below 5% for both compounds. The proposed method is considered suitable for regulatory analysis of chlorophyll degradation compounds and would be useful for quality control of chlorella products.

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.

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.

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.

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.

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.

Multiresidue determination of pesticides in tea by liquid chromatography-high-resolution mass spectrometry: Comparison between Orbitrap and time-of-flight mass analyzers.

Liquid chromatography (LC)-Orbitrap mass spectrometry (MS) and LC-time-of-flight (TOF) MS operating in full scan mode at a mass resolution of 140,000 (m/z 200) and 30,000 (m/z 556), respectively, were compared for quantification of pesticide residues in tea. Both methods were validated for 146 pesticides at spike levels of 0.1 and 0.01 mg/kg and compared in terms of recovery, intra- and inter-day precisions, selectivity, linearity, and matrix effect. The results of both analyses were comparable, and recovery and intra- and inter-day precisions were within the acceptable ranges for most pesticides. LC-Orbitrap MS was slightly superior to LC-TOF MS in terms of sensitivity and selectivity due to its higher resolution. However, even using high-resolution LC-Orbitrap MS with a narrow mass window of ±3 ppm, interference by coeluting matrix components was observed, indicating that full scan data are insufficient for unequivocal identification, and additional information such as fragment ions is necessary.

Determination of total avilamycin residues as dichloroisoeverninic acid in porcine muscle, fat, and liver by LC-MS/MS.

A sensitive and reliable method for determining the total avilamycin residues was developed using LC-MS/MS. Avilamycin (consisting of avilamycin A and 15 other minor factors) and its metabolites in porcine muscle, fat, and liver were analysed as the marker residue dichloroisoeverninic acid (DIA), in accordance with the maximum residue limit (MRL) established by international organisations such as Codex Alimentarius Commission and other regulatory bodies. The analytes were extracted from samples with acetone, hydrolysed to DIA, partitioned into ethyl acetate, and cleaned up prior to the LC-MS/MS analysis. The method was validated at Codex MRL and 0.01 mg/kg. The results show excellent recoveries ranging from 100 to 108%, with the relative standard deviations <6%. Matrix effects were negligible for all types of samples, indicating effective sample clean-up. The absence of interfering peaks close to the retention time in blank samples demonstrates high selectivity. Overall, this method is reliable and suitable for regulatory-purpose analysis.

Quantitative analysis of veterinary drugs in bovine muscle and milk by liquid chromatography quadrupole time-of-flight mass spectrometry.

A simple and reliable multiresidue method for quantitative determination of veterinary drugs in bovine muscle and milk using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was developed. Critical MS parameters such as capillary voltage, cone voltage, collision energy, desolvation gas temperature and extraction mass window were carefully optimised to obtain the best possible sensitivity. Analytical samples were prepared using extraction with acetonitrile and hexane in the presence of anhydrous sodium sulphate and acetic acid, followed by ODS cartridge clean-up. The developed method was validated for 82 veterinary drugs in bovine muscle and milk at spike levels of 0.01 and 0.1 mg kg-1. With the exception of cefoperazone and phenoxymethylpenicillin, all these compounds exhibited sufficient signal intensity at 0.01 µg ml-1 (equivalent to 0.01 mg kg-1), indicating the high sensitivity of the developed method. For most targets, the determined accuracies were within 70-120%, with repeatability and reproducibility being below 20% at both levels. Except for sulfathiazole in bovine muscle, no interfering peaks at target compound retention times were detected in the blank extract, indicating that the developed method is highly selective. The absence of sulfathiazole in bovine muscle was confirmed by simultaneous acquisition at low and high collision energies to afford exact masses of molecular adduct and fragment ions. Satisfactory linearity was observed for all compounds, with matrix effects being negligible for most targets in bovine muscle and milk at both spike levels. Overall, the results suggest that the developed LC-QTOF-MS method is suitable for routine regulatory-purpose analysis of veterinary drugs in bovine muscle and milk.

Simultaneous determination of seven anticoagulant rodenticides in agricultural products by gel permeation chromatography and liquid chromatography-tandem mass spectrometry.

A sensitive and reliable method for the simultaneous determination of hydroxycoumarin-type (brodifacoum, bromadiolone, coumatetralyl, and warfarin) and indandione-type (chlorophacinone, diphacinone, and pindone) rodenticides in agricultural products by gel permeation chromatography (GPC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The procedure involved extraction of the rodenticides from samples with acetone, followed by liquid-liquid partitioning with hexane/ethyl acetate (1:1, v/v) and 10% sodium chloride aqueous solution, then cleanup using GPC, and finally, analysis using LC-MS/MS. High recoveries from the GPC column were obtained for all rodenticides tested using a mobile phase of acetone/cyclohexane/triethylamine (400:1600:1, v/v/v). An ODS column, which contains low levels of metal impurities, gave satisfactory peak shapes for both hydroxycoumarin- and indandione-type rodenticides in the LC-MS/MS separation. The average recoveries of rodenticides from eight agricultural foods (apple, eggplant, cabbage, orange, potato, tomato, brown rice, and soybean) fortified at 0.0005-0.001 mg/kg ranged from 76 to 116%, except for bromadiolone in orange (53%) and diphacinone in soybean (54%), and the relative standard deviations ranged from 1 to 16%. The proposed method effectively removed interfering components, such as pigments and lipids, and showed high selectivity. In addition, the matrix effects were negligible for most of the rodenticide/food combinations. The results suggest that the proposed method is reliable and suitable for determining hydroxycoumarin- and indandione-type rodenticides in agricultural products.

Determination of Rodenticide Tetramethylenedisulfotetramine (Tetramine) in Processed Foods by Gas Chromatography-Tandem Mass Spectrometry.

A GC-MS/MS method for determination of the rodenticide tetramethylenedisulfotetramine was developed. Tetramethylenedisulfotetramine was extracted from the sample with ethyl acetate in the presence of anhydrous sodium sulfate. Then, an aliquot of the extract was evaporated under vacuum, followed by acetonitrile/hexane partitioning, and cleanup with a tandem graphitized carbon/primary secondary amine (PSA) column, prior to GC-MS/MS analysis. The recoveries from 10 processed foods, all of which were fortified at 0.1 mg/kg, were in the range of 85-96%, and the relative standard deviations were less than 7%. The proposed method effectively removed co-extracted matrix components, and matrix effects were negligible in the GC-MS/MS analysis. In addition, no interfering peaks were found in the chromatograms of the blank samples at the retention time of tetramethylenedisulfotetramine, indicating that the method is highly selective. Overall results suggest that the proposed method is suitable for determining tetramethylenedisulfotetramine contained in processed foods.

Quantitative analysis of pesticide residues in vegetables and fruits by liquid chromatography quadrupole time-of-flight mass spectrometry.

The applicability of liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) for the quantitative analysis of pesticide residues in vegetables and fruits was demonstrated. The LC-QTOF-MS parameters, such as cone voltage, capillary voltage, collision energy and mass extraction window, were carefully optimised for the analysis of pesticide residues. The LC-QTOF-MS method was validated for 149 pesticides in four vegetables and fruits, i.e. apple, potato, cabbage and spinach, at a spiking level of 0.01 mg kg(-1). The samples were prepared according to the Japanese official multi-residue method with a modification to the column clean-up procedure. Of the 149 pesticides, recoveries in the range of 70-120% were achieved for 147 pesticides in apple, 145 in potato, 141 in cabbage and 131 in spinach, with intra-day precisions (RSDs) of < 25% and inter-day precisions (RSDs) of < 30%, which are within the acceptable range given in the Japanese method validation guideline. Matrix effects were negligible for the majority of the target pesticides. Except for spiroxamine in spinach, no interfering peaks were observed in the blank samples. The target pesticides, except those with low sensitivity, achieved calibration curves with satisfactory linearity, with correlation coefficients (r) greater than 0.995 in the concentration range of 0.002-0.1 µg ml(-1). Furthermore, the majority of the target pesticides provided more than one fragment ion or isotope ion that could be used for confirmation. The overall results suggest that LC-QTOF-MS is a powerful tool for the quantification of pesticide residues in vegetables and fruits at the level of 0.01 mg kg(-1).

Multiresidue determination of pesticides in tea by gas chromatography-tandem mass spectrometry.

An efficient and reliable GC-MS/MS method for the multiresidue determination of pesticides in tea was developed by modifying the Japanese official multiresidue method. Sample preparation was carefully optimized for the efficient removal of coextracted matrix components. The optimal sample preparation procedure involved swelling of the sample in water; extraction with acetonitrile; removal of water by salting-out; and sequential cleanup by ODS, graphitized carbon black/primary secondary amine (GCB/PSA) and silica gel cartridges prior to GC-MS/MS analysis. The recoveries of 162 pesticides from fortified (at 0.01 mg kg(-1)) green tea, oolong tea, black tea and matcha (powdered green tea) were mostly (95-98% of the tested pesticides) within the range of 70-120%, with relative standard deviations of <20%. Poor recovery of triazole pesticides was considered to be due to low recovery from the silica gel cartridges. The test solutions obtained by the modified method contained relatively small amounts of pigments, caffeine and other matrix components and were cleaner than those obtained by the original Japanese official multiresidue method. No interfering peaks were observed in the blank chromatograms, indicating the high selectivity of the modified method. The overall results suggest that the developed method is suitable for the quantitative analysis of GC-amenable pesticide residues in tea.

Multiresidue analysis of pesticides in vegetables and fruits by supercritical fluid extraction and liquid chromatography-tandem mass spectrometry.

A multiresidue method for analyzing pesticides in vegetables and fruits by supercritical fluid extraction (SFE) and LC-MS/MS was developed. The sample preparation and SFE parameters were optimized for extracting LC-amenable polar and medium-polarity pesticides. High recoveries were achieved for most of the tested pesticides by extracting a 1 : 1 : 1 sample-Celite-anhydrous magnesium sulfate mixture with supercritical carbon dioxide at 16.4 MPa at 40℃ for 30 min with methanol added as a modifier. The recoveries of 117 pesticides fortified with 0.01 mg/kg of each pesticide were 70-120%, and the relative standard deviations were less than 25% for 112 pesticides in tomato and 103 pesticides in cucumber. No significant differences were observed in the residue concentrations determined in real samples by the SFE method and the liquid extraction method (the modified Japanese official method). Higher recoveries of polar pesticides, such as acephate and methamidophos, were achieved by the developed SFE method than by the liquid extraction method.