[Enhancement of Pesticide Peak Response in GC-MS in the Presence of Multiple Co-Existing Reference Pesticides].

We carried out a collaborative study in six laboratories to confirm the universality of the enhancing effect of co-existing reference pesticides on the GC-MS peak response to a target pesticide (malathion, procymidone, or flucythrinate). First, we confirmed the response enhancement of the target pesticides with increasing numbers of co-existing reference pesticides in solution. Then, using diluted green soybean matrix, we analyzed the target pesticides with two types of matrix-matched calibration, containing the target pesticides or 166 other pesticides. In both cases, the response-enhancing effect of co-existing pesticides was confirmed in all laboratories. The enhancement was reduced by addition of green soybean matrix to the sample and calibration solutions. Our results show that it is necessary to estimate the peak response-enhancing effect of co-existing pesticides in the calibration solution to obtain accurate results with GC-MS determination. The enhancing effect could be reduced by addition of food matrix to the sample and calibration solutions.

Evaluation of the matrix-like effect in multiresidue pesticide analysis by gas chromatography with tandem mass spectrometry.

In multiresidue pesticide analysis using gas chromatography, it has long been recognized that an increase in the number of pesticides present in a standard solution can result in an enhancement of the peak responses of certain pesticides. Despite being widely acknowledged, this phenomenon has been rarely studied and is poorly understood. In this study, the authors have tentatively called this phenomenon the "matrix-like effect" and demonstrated it clearly using gas chromatography with tandem mass spectrometry. Five selected pesticides, namely, omethoate, terbufos, malathion, procymidone, and permethrin, and four internal standard candidates, namely, triphenyl phosphate, naphthalene-d8 , phenanthrene-d10 , and fluoranthene-d10 , were used to evaluate the matrix-like effect following the addition of 58, 108, and 166 other pesticides. With the exception of naphthalene-d8 , the responses of all evaluated pesticides and internal standard candidates were dramatically enhanced by the addition of up to 166 coexisting pesticides. The relative response factors of the five pesticides to each internal standard candidate were not constant under the conditions studied, meaning that these internal standard candidates did not adequately compensate for the matrix-like effect, at least for the five evaluated pesticides. The results revealed that the presence of various mixtures of pesticides in standard solutions might act as an unintentional analyte protectant, that is, some sort of troublesome "quasi-matrix."

Estimation of Irradiation Doses of Raw Beef Liver Samples using 5,6-Dihydrothymidine as an Irradiation Marker.

The quantities of (5S)- and (5R)-5,6-dihydrothymidine (total DHdThd) in frozen beef liver samples were compared between those irradiated by an electron beam (EB) and those subjected to γ-irradiation. DNA extracted from the samples was enzymatically digested to nucleosides and analyzed by liquid chromatography-tandem mass spectrometry for total DHdThd and 2'-deoxythymidine (dThd). Total DHdThd was formed radiospecifically and dose-dependently and the dose-response curves of the ratio of total DHdThd to dThd (total DHdThd/dThd) were similar for both the EB- and γ-irradiated samples. The total DHdThd/dThd was stable after long-term storage (4 months) at -20 °C and the followed heat treatment in a microwave oven. The total DHdThd/dThd could be a robust marker and is equally effective at quantitating both EB- and γ-ray irradiation history. The irradiation doses of raw beef liver samples were estimated using the dose-response curves of the total DHdThd/dThd of other irradiated samples. The ratio of the estimated dose to the actual dose was 0.74-1.30 in the irradiation range of 4.67-7.62 kGy.

Simultaneous analysis of 17 organochlorine pesticides in natural medicines by GC/MS with negative chemical ionization.

Many methods for the determination of pesticide residues in food have been reported. Although natural medicines should be confirmed to be as safe as food, few methods for the determination of pesticide residues in natural medicines have been reported. In this study, 17 organochlorine pesticides were detected in natural medicines using GC/MS with negative chemical ionization (NCI). GC/MS with NCI can detect halogenated pesticides selectively and thus is suitable for the detection of organochlorine pesticides. This study indicates that GC/MS with NCI is useful for analyzing organochlorine pesticides in natural medicines.

Detection of irradiated meat, fish and their products by measuring 2-alkylcyclobutanones levels after frozen storage.

2-Alkylcyclobutanones, such as 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone, were analyzed to assess the irradiation history of irradiated meats or fish, and cooked foods with irradiated ingredients, which had been stored frozen for up to one year. The purpose of the study was to show that irradiated meats could be detected even after having been stored in the distribution system. 2-Alkylcyclobutanones showed a small decrease in irradiated raw meats that had been stored frozen for one year. Cooked foods, such as pancake and fried chicken made with irradiated eggs and chicken, respectively, contained detectable levels of 2-alkylcyclobutanones after storage frozen for one year. The 2-alkylcyclobutanones became undetectable in highly dried samples, such as feed for lab animals, during the same period.

Determination of irradiation histories of raw beef livers using liquid chromatography-tandem mass spectrometry of 5,6-dihydrothymidine.

A method for detecting irradiation histories of raw beef livers was developed by measuring 5,6-dihydrothymidine (DHdThd) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liver DNA was extracted using phenol-chloroform extraction followed by precipitation in 50% ethanol. DNA was then enzymatically digested and nucleosides were purified using an OASIS MCX column. DHdThd and thymidine (dThd) contents of resulting test solutions were analyzed using LC-MS/MS. DHdThd was detected specifically after γ-irradiation. Concentration ratios of DHdThd to dThd in the test solutions increased dose-dependently after irradiation at 1.0-11.3kGy, which included the practical dose for sterilization of 2-7kGy. Dose-response curves from beef livers of individual animals almost overlapped. Thus, this method is a candidate for the detection of irradiation histories of foods from which DNA can be extracted.

Rapid and Reliable Method for Determining Irradiation Histories of Ground Beef and Prawns by Measuring 5,6-Dihydrothymidine.

A rapid and reliable method for determining irradiation histories of ground beef and prawns was developed on the basis of a method for determining the irradiation history of beef liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of 5,6-dihydrothymidine (DHdThd). Improvements in the method included the following: (1) 50% ethanol precipitation in the DNA extraction step was conducted before the RNase step, (2) snake venom phosphodiesterase I was used for DNA digestion to boost liberation of DHdThd, and (3) a matrix-matched calibration curve was used for determining DHdThd by LC-MS/MS analysis. This method successfully determined irradiation histories of ground beef and prawns. Furthermore, a close correlation between the formation of DHdThd and 2-alkylcyclobutanones, which are an established index of irradiation histories, was observed in ground beef. DHdThd in DNA could be a promising candidate for a new index of irradiation histories of various foods.

Analysis of 2-alkylcyclobutanones with accelerated solvent extraction to detect irradiated meat and fish.

A new analytical procedure has been developed to analyze 2-alkylcyclobutanones to detect gamma-ray-irradiated fat-containing foodstuffs. Samples were extracted with an accelerated solvent extraction system via hot and pressurized ethyl acetate in cells. A large amount of fat in the extract was precipitated and removed with filtration by standing at -20 degrees C after the addition of acetonitrile. The extract was further cleaned with a 1 g silica gel mini column, and the radiolytic compounds of 2-docecylcyclobutanone (2-DCB) and 2-tetradecylcyclobutanone (2-TCB) were determined with gas chromatography with mass spectrometry (GC/MS). Sample preparation time before GC/MS was 7-8 h. At first, the procedure was evaluated with a recovery test in eight samples spiked with 2-DCB and 2-TCB at 20 ng/g, resulting in 70-105% recoveries with mostly less than 10% relative standard deviations. The procedure was further evaluated with beef, pork, chicken, and salmon samples irradiated with gamma-rays from 0.7 to 7.0 kGy at -19 degrees C. Both 2-DCB and 2-TCB in most samples were detected with good dose-response relations at all doses, while salmon was detected more than 2 kGy irradiation. The amounts of 2-alkylcyclobutanones produced reflected precursor fatty acids levels in samples, especially for the combination of 2-TCB and stearic acid. The results indicated that the production rate of 2-TCB to stearic acid was more obvious than that of 2-DCB to palmitic acid in frozen samples with gamma-ray irradiation.

[Validation Study on a Multi-Residue Method for Determination of Pesticide Residues in Vegetables and Fruits by using General Matrix Standard Solutions].

Quantitative methods using the matrix-matched standard solutions approach are widely used for multi-residue pesticide determination by GC-MS/MS to deal with the issue of matrix effects. However, preparing matrix-matched standard solutions in analyses of many kinds of samples is very time-consuming. In order to solve this problem, a method that employs general matrix standard solutions has been developed using polyethylene glycol (PEG), extract of vegetables-fruit juice (VFJm) and triphenyl phosphate (named the PEG-VFJm method). Here, a validation study for 168 pesticides was performed on three kinds of samples [potato, spinach and apple] at concentrations of 0.010 and 0.050 µg/g. In these three commodities, 144 to 158 pesticides satisfied the required criteria using the matrix-matched method and 129 to 149 pesticides satisfied the same criteria using the PEG-VFJm method. Our results suggest that application of general matrix standard solutions would enable rapid and effective analyses of pesticides.

Determination of acetamiprid, imidacloprid, and nitenpyram residues in vegetables and fruits by high-performance liquid chromatography with diode-array detection.

Determination of 3 neonicotinoid insecticides, nitenpyram, imidacloprid, and acetamiprid, was studied. Vegetables and fruits were extracted with acetonitrile. The crude extract was passed through a weak anion-exchange cartridge (PSA). The effluent was subjected to silica gel cartridge. Imidacloprid and acetamiprid were eluted with 10 mL of 4:6 (v/v) acetone/hexane, followed by nitenpyram with acetone (20 mL). Pesticides were determined by HPLC with a C-18 column and diode-array detection system. Imidacloprid and acetamiprid were recovered at about 90% at the spike levels with 0.2 and 2 mg/kg in cucumber, potato, tomato, eggplant, Japanese radish, and grape. Nitenpyram was recovered at 64-80%. Relative standard deviations were less than 10% throughout all the recovery tests. In the residue analysis, agriculturally incurred pesticides at 0.08-0.14 mg/kg were designated with UV spectra compared with respective reference standards.

Determination of neonicotinoid pesticide residues in vegetables and fruits with solid phase extraction and liquid chromatography mass spectrometry.

A rapid and simple extraction method for the simultaneous analysis of five neonicotinoid insecticides has been developed. Twelve different fruit and vegetable matrixes were extracted with methanol and cleaned up using a graphitized carbon solid phase extraction cartridge loading with a 20% methanol solution. The concentrated eluate after methanol elution was then analyzed for pesticide residues by liquid chromatography/mass spectrometry in the APCI positive mode. The five pesticides including nitenpyram, thiamethoxam, imidacloprid, acetamiprid, and thiacloprid were recovered at 70-95% at spike levels of 0.1 and 1 mg/kg in bell pepper, cucumber, eggplant, grape, grapefruit, Japanese radish, peach, pear, potato, rice, and tomato. Relative standard deviations were less than 10% for all of the recovery tests. The proposed method is fast, easy to perform, and could be utilized for regular monitoring of pesticide residues.

Determination of seventeen pesticide residues in agricultural products by LC/MS.

Residues of 17 pesticides in agricultural products were determined by LC/MS with an atmospheric pressure chemical ionization (APCI) interface in both positive and negative ion modes. Pesticides were extracted with acetonitrile, and the extracts were cleaned-up with a primary and secondary amine (PSA) mini-column eluted with acetone-hexane (1:1). Rice, orange and potato were spiked with the 17 pesticides at 0.1 microgram/g and analyzed by the proposed method. The average recoveries of these pesticides usually ranged from 70 to 98% and the relative standard deviations were usually around 10%. These results suggested that LC/MS with APCI could be used to determine the residue levels of the 17 pesticides in these crops.

[Trial for estimating the concentration of pesticide residues in the ingredients of processed foods].

If pesticide residues are detected at over the uniform limit (0.01 ppm) in processed foods, the maximum residue limits of pesticides in each ingredient should be considered. For that purpose, analysis of pesticide residues in the ingredients would be required, but in many cases it is difficult to obtain ingredients from the same lot, or the processed foods may have been manufactured overseas. Thus, a means of estimating pesticide concentrations in ingredients before processing would be important. In this study, we estimated the concentration of paclobutrazol, which was detected in LM pickles (LMPs), that would have been present in Takana (LM; mustard leaf) before processing. The LMPs mainly consisted of LM and seasoning liquid (SL), and other ingredients (sesame seeds or red pepper) accounted for less than 0.2% (w/w) of the LMPs. The LM and SL were separated and their paclobutrazol concentrations were determined. The concentration of paclobutrazol in the SL was approximately 1/10 of that detected in LM. Loss of water from LM during processing was taken into account to calculate the content of paclobutrazol in LM before processing, based on the analytical result of processed LM.

[Study of multi-residue method for determining pesticide residues in processed foods manufactured from agricultural products by LC-MS/MS].

A rapid multi-residue method for determination of pesticide residues in processed foods manufactured from agricultural products was examined. Five mL water was added to 5 g sample in a polypropylene tube, and the tube was left to stand at room temperature for 30 min. Then, 20 mL acetonitrile was added to the sample. The mixture was homogenized in a high-speed homogenizer, followed by salting out with 1 g NaCl and 4 g anhydrous MgSO4. After centrifugation, the organic layer was purified on a graphitized carbon/PSA cartridge column. After removal of the solvent, the extract was resolved in methanol-water and analyzed with LC-MS/MS. The recoveries of 93 pesticides fortified into 5 kinds of processed foods [Chinese cabbage kimchi, marmalade, raisin, umeboshi (pickled plum) and worcester sauce] were examined at the concentrations of 0.02 and 0.1 µg/g (n=5). The recoveries of 61 pesticides in all foods were 70-120% with relative standard deviation below 20% at both concentrations. Seventy-four processed foods obtained from markets in Japan were examined with this method. Pesticide residues over the maximum residue limit (0.01 µg/g) were detected in 2 processed foods.

A simple and selective detection method for aristolochic acid in crude drugs using solid-phase extraction.

The official Japanese method for analyzing aristolochic acid I (AA-I) in Asiasarum root using conventional high-performance liquid chromatography (HPLC) is described in the Japanese Pharmacopoeia, Sixteenth Edition. Interfering peaks of AA-I sometimes appear after HPLC analysis of crude drugs. A selective analytical method is needed to determine definitively whether AA-I is present in crude drugs. In this study, we developed a selective method that combined solid-phase extraction and liquid chromatography/mass spectrometry (LC/MS) which may be useful for identifying AA-I in crude drugs and for quality control.

[Validation study on a rapid multi-residue method for determination of pesticide residues in vegetables and fruits by LC-MS/MS].

A validation study was conducted on a rapid multiresidue method for determination of pesticide residues in vegetables and fruits by LC-MS/MS. Pesticide residues in the vegetables or fruits were extracted with acetonitrile in a disposable tube using a homogenizer, followed by salting out with anhydrous magnesium sulfate and sodium chloride in the presence of citrate salts for buffering. The extract was purified with a double-layered cartridge column (graphite carbon black/primary secondary amine silica gel; GCB/PSA). For citrus fruits a purification step with a C18 column was added (this column was connected to the GCB/PSA column). After removal of the solvent, the extract was resolved in methanol/water and analyzed by means of LC-MS/MS. The method was validated according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan; recovery tests were performed on 8 kinds of vegetables and fruits [cabbage, cucumber, Japanese radish, onion, potato, spinach, Amanatsumikan (a citrus fruit) and apple] by fortification of 161 pesticide residues at the concentrations 0.01 and 0.05 µg/g (each concentration of pesticide residue was extracted from 2 samples on 5 separate days). The trueness of the method for 127 pesticides in all 8 commodities was 70-120% with satisfactory repeatability and within-run reproducibility. This method is concluded to be applicable for determination of pesticide residues in vegetables and fruits.

[Application of a rapid and simple multi-residue method for determination of pesticide residues in drinking water and beverages using liquid chromatography-tandem mass spectrometry].

A rapid and simple multi-residue method for determination of pesticides has been applied to drinking water and beverages. To a disposable polypropylene tube containing 10.0 g sample, 20 mL acetonitrile was added and the mixture was shaken vigorously for 1 min to extract pesticides. Then, 1 g sodium chloride and 4 g magnesium sulfate anhydrous were added, followed by vigorous shaking for 1 min and centrifugation to obtain the organic phase. The organic phase was processed with a graphite carbon black/PSA solid phase column. After concentration and reconstitution with 25% methanol containing aqueous solution, the test solution was analyzed with LC-MS/MS. Recovery tests of 91 pesticides fortified (0.02 µg/g) in 35 kinds of drinking water and beverages were conducted. The decline of recoveries in alcoholic beverages is considered to be due to the increase of organic phase volume owing to ethanol included in the alcoholic beverages. A simulation study was carried out with simulated alcoholic beverages, which consisted of 50% grape juice, with various amounts of ethanol and water, to examine pesticides recoveries and volume of the organic phase. The results suggested this method would be applicable both to alcoholic beverages containing less than 10% ethanol and to alcoholic beverages containing over 10% ethanol after dilution with water to below 10% ethanol prior to the addition of acetonitrile. A sample could be processed and analyzed by LC-MS/MS within 2 h. Thus, this method should be useful for monitoring and screening pesticide residues in drinking water and various beverages.

[Determination of histamine in fish and fish products by tandem solid-phase extraction].

A simple and practical method was developed for the determination of histamine in fish and fish products by solid-phase extraction and fluorescence derivatization. Histamine was extracted with trichloroacetic acid. The extract was neutralized and diluted with phosphate buffer (pH 6.8), and cleaned up with a tandem-connected octadecyl silica (ODS) and strong cation exchange silica (SCX) cartridge. After removal of the solvent, histamine was derivatized with fluorescamine and analyzed by ion-paired reversed-phase high-performance liquid chromatography with fluorescence detection. Recovery tests of histamine from six kinds of fish and fish products showed acceptable recovery (83-92%) with low relative standard deviation (less than 5%). This method could be useful for determination of histamine in fish.

[Simultaneous determination of polyether antibiotics and macrolide anthelmintics in livestock products by liquid chromatography/tandem mass spectrometry].

A method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of polyether antibiotics and macrolide anthelmintics in livestock products. The polyether antibiotics and macrolide anthelmintics were extracted from livestock products with acetonitrile and cleaned up with dispersive solid-phase extractions and a silica gel column. The quantification limits of polyether antibiotics and macrolide anthelmintics were 0.00005-0.0005 µg/g. Except for narasin and lasalocid in bovine liver and milk, the recoveries were 70 to 117%. The relative standard deviations met the required guideline. The developed method was applied to six kinds of livestock products.

[Preparation of samples for proficiency testing of pesticide residue analysis in processed foods].

To conduct proficiency testing for the analysis of pesticide residues in processed foods, fortified samples of retort curry and pancake were examined. In the case of retort curry, heating and mixing were necessary at the time of preparation to provide a homogenous analytical sample. A mixture of 4 carbamates and 11 organophosphorus pesticides was spiked and 14 of them showed consistent results in the samples. In the case of pancake, 10 kinds of pesticides were added to the pastry. The prepared pastry was them cooked. The relative concentrations of most of the pesticides in the pancake were not affected and all the pesticides showed consistent results in the samples. These results showed that the two tested samples were suitable for proficiency testing.