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Residual antibacterial agents in 5909 animal and fishery products in Tokyo, Japan, were investigated over 17 consecutive years (2003-2019). Monitoring of 32 antibacterial agents (lincosamides, macrolides, penicillins, quinorones and tetracyclines) per product was accomplished via two steps: screening (by microbiological methods) and confirmation (by instrumental methods). Microbiological screening methods identified presumptive groups and determined semi-quantitative values. The instrumental methods quantified 81 residues of 11 different antibacterial agents in 72 samples. The screening strategy based on microbiological methods demonstrated the following: (i) the majority of the samples (over 99%) met Japanese regulations, (ii) using multiple methods provided a reliable inspection system with accurate quantitative values and (iii) there was a constant presence of tetracyclines and unexpected residues (lincomycin and norfloxacin) in various products. Thus, this long-term monitoring and screening strategy provided evidence that the frequencies and trends of residual antibacterial agents not only enhance food safety but also help to prevent antimicrobial resistance.

An analytical method has been developed and validated for determining 107 pesticide residues in dried red pepper using LC-MS/MS. LC method, the clean-up and sample dilution processes were examined to determine their impact on reducing the matrix effects. Clean up was performed using an ENVI-CarbIITM/PSA (300/600 mg, 6 mL) SPE cartridge. In the sample dilution process, eight-fold dilution was used. In the validation of the developed method at two concentrations (0.01 and 0.1 μg/g) for 107 pesticides, 96 pesticides showed recovery rates in the range of 70.1 to 112.6%, RSDs of repeatability of ≤11.5 and 3.4%, and RSDs of within-laboratory reproducibility of ≤24.3 and 19.9%. These values fulfill the criteria of the validation guidelines for pesticide residues in Japan. It is concluded that matrix effects and low recovery rates in the process of extraction are the main factors for values that do not conform to the criteria.

We examined the expansion of target food products for the analysis of polychlorinated biphenyls (PCBs). From our studies in fish using an accelerated solvent extractor (ASE) and GC-MS/MS, we found that recovery of low-chlorinated PCBs (some isomer of 3 or 4 chlorinated PCBs) in specific food products was low. Therefore, we attempted to improve the analytical method. In Japanese sea perch (Suzuki) and milk, freeze drying before extracting with ASE did not sufficiently improve the recovery of low-chlorinated PCBs; however, it was significantly improved by changing the column to a silica gel/H2SO4 silica gel multilayer column. To evaluate this method for other food products, PCBs in fish, meat, eggs, milk, and dairy products were analyzed. The samples were extracted using ASE at 125℃ with n-hexane, cleaned up using a silica gel/H2SO4 silica gel multilayer column, and analyzed by GC-MS/MS for 3-7 chlorinated PCBs. The limit of quantification for this method was was 0.03-0.16 μg/kg for the 3-7 chlorinated PCBs. The recovery and the coefficient of variation of 3-7 chlorinated PCBs from each of the food products (n=5) was in the range of 84%-112% and 1%-9%, respectively.

In the field of food analysis and regulation, different instruments are used to determine the accuracy of quantification values. This is essential, as inconsistencies in values are commonly encountered. To visualize the degree of these discrepancies in each food matrix, we compiled a validation study based on a routine method developed in our laboratory, for 121 pesticides in six agricultural products, namely the grapefruit, potato, paprika, cabbage, spinach, and brown rice. These were analyzed by GC-MS/MS and LC-MS/MS, and the results were compared mainly on the basis of trueness. According to the results of the validation study when using GC-MS/MS, of the 121 pesticides tested in each product class, the number of analytes that satisfied the criteria of the Japanese validation guidelines was 97 in grapefruit, 111 in potato, 110 in paprika, 118 in cabbage, 111 in spinach, and 63 in brown rice. In contrast, in the analysis of the same samples by using LC-MS/MS, the number of analytes that satisfied the criteria of the validation guidelines was 50 in grapefruit, 114 in potato, 103 in paprika, 112 in cabbage, 100 in spinach, and 103 in brown rice. Inconsistences in the differences of trueness were mainly attributed to matrix effects of each instrument, as well as to food matrices, of which the most diverged matrix was that of brown rice (over 20%).

The determination of antibacterial agents for animals in swine muscles was improved by microbiological screening and liquid chromatography-mass spectrometry (LC-MS/MS) analyses. In the first instance, the residual drugs were extracted from the samples using the Na2EDTA-McIlvaine buffer (pH 6.0). Subsequently, the agents were purified utilizing a PLS-3 cartridge and extracted with acetonitrile. Considering the microbiological methods, the sensitivities of the investigated drugs were higher and the test plate conditions were improved using a new reference organism Geobacillus stearothermophilus. As a result, a microbiological screening approach able to detect 33 drugs at MRL was developed in Japan. Remarkable, no false positives were detected. Moreover, the same preparation method enabled rapid and reliable microbiological screening, resulting in efficient screening with no undeterminable results.

In this study, the staphylococcal enterotoxin type A (SEA) contaminant was quantified in cow milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the use of a stable isotope-labelled peptide of SEA as an internal standard. SEA was cleaned up in a two-step process that included pH control and trichloroacetic acid (TCA) precipitation. The pH control phase eliminated other proteins. TCA precipitation cleaned up SEA without special equipment. An appropriate enzyme-to-protein ratio maximised tryptic digestion. A desalting process guaranteed the stable retention of SEA-digested peptides. The coverage of amino-acid sequences (>10%) clearly identified the toxin's presence. SEA was accurately quantified using LC-MS/MS based on a multiple-reaction monitoring mode. The developed method was validated based on spiked recovery tests at 50 and 100 µg kg-1 conducted with two samples collected on a daily basis for five days based on Japanese validation guidelines. The new method exhibited good accuracy which ranged from 80% to 82%. The relative standard deviations of repeatability were 13-14% and the relative standard deviations of within-laboratory reproducibility were 13-18%. These standard deviations satisfied the criteria of the Japanese validation guidelines. The quantification limit was estimated to be 10 µg kg-1.

A LC-MS/MS simultaneous analytical method for screening 191 pesticide residues in limes had been developed and validated. Pesticides were extracted with acetonitrile from samples. Then mixed salts, which were anhydrous magnesium sulfate for dehydration, sodium carbonate for adjusting pH, and sodium chloride for salting out, were added to the sample. After centrifugation, supernatant was transferred to a tube. The sample solution was cleaned up using solid phase extraction (SPE) with C18/GC/PSA for the determination by LC-MS/MS. The developed method was improved the recovery rate of thiabendazole, which had a low recovery rate by the conventional method. Validation study, which was following the guidelines of the Ministry of Health, Labor and Welfare, were carried out at 0.01 and 0.1 μg g-1 to evaluate the method. The results of 175 pesticides out of 191 were with satisfactory. A total of 19 imported lime samples sold in Tokyo was analyzed to evaluate the method, then 18 samples contained pesticide residues below MRLs. The developed method is applicable for detection of pesticide residues in lime.

A determination method for tributyltin (TBT) and triphenyltin (TPT) in fish and shellfish using an accelerated solvent extractor (ASE) and LC-MS/MS was developed. The chromatographic separation was conducted on a Poroshell 120 EC-C18 column using an isocratic mobile phase of 0.1% formic acid in 70% methanol. Sample preparation was performed using ASE at 125℃ with n-hexane and a cleanup using a Florisil cartridge. Internal calibration curves using deuterium-labeled TBT and TPT were employed for quantification. For both TBT and TPT, the calibration curves were linear in the range of 0.2-250 ng/mL, and the method quantification limits were 0.8 ng/g for both TBT and TPT. A National Institute for Environmental Studies certified reference material, No. 15 (adductor muscle of scallop), was analyzed to assess the performance of the developed method. The trueness, relative standard deviations of repeatability, and within laboratory reproducibility of this method, evaluated using a recovery test with four spiked fish species and one shellfish, ranged from 89.3 to 105.3%, 1.0 to 4.5%, and 1.3 to 7.6%, respectively.

In this study, we developed a reference labelled protein containing the partial amino acid sequence of botulinum neurotoxin type A (BoNTA). We also applied it as an internal standard to detect specific and non-toxic peptides originated from BoNTA in honey with the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Original proteins in the honey sample were collected through a two-step process that included solubilisation and trichloroacetic acid (TCA) precipitation. Solubilisation by adding water enabled processing of proteins in honey. TCA precipitation collected proteins without specific binding. The combination of protein alkylation and an appropriate enzyme-to-protein ratio ensured feasibility of tryptic digestion. A desalting process eliminated a large amount of salts and other tryptic peptides in the honey sample. The use of the reference labelled protein enabled compensation for tryptic digestion efficiency and electrospray ionisation efficiency based on LC-MS/MS measurement. After the peptide selection and protein BlastP analysis, five unique peptides were chosen. The non-toxic peptides originating from BoNTA were reliably detected using LC-MS/MS based on a multiple-reaction monitoring mode. Detection of several peptides ensured screening of BoNTA in honey samples. Based on the responses, the proteotypic peptide LYGIAINPNR was selected as the quantitative peptide. Due to maintaining the relative ion ratios, the selective transition completely identified the non-toxic peptides. The intensity of the transitions established a detection limit of BoNTA estimated to be 9.4 ng mL

We developed a method for the simultaneous determination of acaricides in comb honey using LC/MS/MS. Because methods for honey analysis had not previously been applied to comb honey, we modified three techniques for sample preparation and LC/MS/MS conditions. First, we used a modified QuEChERS method that changed the extraction solution from ethyl acetate to acetonitrile. Second, we replaced the InertSep® MA-1 (30 mg, 1 ml) clean-up cartridge with an Oasis® HLB (60 mg, 3 ml). Third, we changed the ionisation mode from ESI to atmospheric pressure chemical ionisation (APCI). With these modifications, sample matrices had no effect on the identification and quantification of analytes, using an external solvent calibration curve. We verified this new method with nine acaricides and two metabolites on comb honey and honey samples from three different honey origins. The trueness ranged from 74.0 to 99.4%. The relative standard deviation of repeatability (RSD

We measured the residual amounts of chlorantraniliprole in various vegetables and fruits. Sample solutions were prepared according to our routine procedure based on the QuEChERS method and analyzed by LC-MS/MS. Performance characteristics were evaluated for 8 kinds of food samples by means of recovery tests of 5 replicates at the concentration of 10 ng/g. Recoveries and RSDs (％) ranged from 50.2 to 93.4％ and from 2.1 to 9.7％, respectively. Application of this method to survey 207 vegetables and 163 fruits gave detection rates of 8.2 and 1.2％, respectively. In vegetables, detection rates were high in okra (4 out of 10 samples), paprika (4 out of 23 samples) and tomato (2 out of 6 samples), and leaf vegetables such as lettuce, mizuna, spinach and wrinkled greens also contained high concentrations of chlorantraniliprole. The highest residual concentration was 571 ng/g in mizuna. The samples containing chlorantraniliprole seemed to be mainly from Asian countries, including samples of domestic Japanese origin. However, none of them contained more than the MRL, which suggests that the use of chlorantraniliprole has been properly controlled.

We developed an analytical method for determining 15 antifungal drugs, 2 antiparasitic drugs, and 3 veterinary drugs in fish and livestock products using LC-MS/MS. First, 50% ethanol was added to their products, and the mixture was homogenized to reduce drug degradation. Thereafter, 20 drugs were extracted from the pretreated sample mixture using acetonitrile. Cleanup was performed using an alumina-N SPE cartridge. Finally, chromatographic separation was performed using a fully porous octadecyl silanized silica column. The new method is applicable to fish in which the matrix hampers accurate analysis. It was validated on 8 fish and livestock products. Drug recovery rates ranged from 70.2 to 109.3%, RSDs of repeatability were

In this study, the presence of cereulide in cow's milk was identified and quantified using our validated method with liquid chromatography-tandem mass spectrometry. Cereulide was concentrated using protein acid-precipitation and extracted from the precipitate by using acetonitrile twice. The combination of protein acid-precipitation and extraction sufficiently eliminated the matrix compounds from the milk and a further clean-up step utilising solid-phase extraction could be omitted. For robustly measuring the samples and keeping the MS devices clean, the extraction solution was diluted 10-fold using methanol. Owing to the minimisation of the interferences caused by fragmentation patterns, multiple reaction monitoring information-dependent acquisition-enhanced product ion spectra enabled the characterisation and identification of cereulide. Besides the matrix effect (-4%), an external solvent calibration curve was adapted for accurate quantification. The method was validated using fortified recovery tests, at two concentrations (10 and 50 µg kg

Cyromazine in livestock products was determined using a validated LC-MS/MS method. There are three key points in our methods. First, the extraction was performed with two solutions, methanol and pH 3.0 McIlvaine buffer. The process was optimized for each type of sample. Secondly, cleanup was performed using a reversed-phase and strong cation exchange mixed-mode cartridge. The cartridge was washed with 0.14％ ammonium solution. Thirdly, the chromatographic separation was performed on an anion-cation exchange mode ODS column. There was no matrix effect on the extraction and determination for five livestock products. The quantification was carried out using an external standard calibration curve. This new method satisfies the Japanese guideline criteria. Recovery ranged from 77.2 to 92.1％, the relative standard deviation of repeatability （RSDr） was under 2.2％, and RSDwr was under 6.1％. Residual cyromazine was detected in raw milks and eggs.

An analytical method for PCBs in fishes using an accelerated solvent extractor (ASE) and GC-MS/MS was evaluated. After the extraction of ASE at 125℃ with n-hexane and clean-up with an AgNO3 silica gel/H2SO4 silica gel multilayer column, samples were analyzed by GC-MS/MS. This method was fast, effective and easy to operate. The limit of quantitation of the method was calculated to be 0.78 μg/kg for total PCBs. The recovery and the coefficient of variation of PCBs (n=5) from 6 fishes (Japanese sea perch, chub mackerel, yellowtail, salmon, pacific saury, and sardine) of total PCBs were 91-108% and 1-3%, respectively.

A simple analytical method for the determination of hydrocortisone and progesterone in bovine, swine, and chicken muscle and eggs was developed. Hydrocortisone and progesterone were extracted with acetonitrile and subsequently cleaned-up using an Oasis HLB mini-cartridge. The method was validated in accordance with Japanese guidelines and exhibited trueness from 86.6% to 104.3% and precision (relative standard deviations (RSDs) of repeatability and within reproducibility were under 8.7% and 11.7%, respectively). The method was applied to 103 bovine muscle, 137 swine muscle, 69 chicken muscle and 52 egg samples that were commercially available in Tokyo, Japan. The hydrocortisone concentration was 0.9-41.2 µg kg(-1) in all bovine muscle samples, with an average of 7.7 µg kg(-1) and a median of 6.2 µg kg(-1). The progesterone concentration in 50 samples exceeded the limit of quantification (LOQ) and reached a maximum of 95.4 µg kg(-1). Hydrocortisone was also detected in all swine muscle samples at concentrations of 2.0-56.0 µg kg(-1). Its average and median concentrations amounted to 13.1 and 11.3 µg kg(-1), respectively. Twenty-three samples contained progesterone levels surpassing the LOQ, with a maximum concentration of 107.0 µg kg(-1). No chicken muscle samples contained any of the analytes. The progesterone concentration was 15.5-200.0 µg kg(-1) in all egg samples, with an average of 95.4 µg kg(-1) and a median of 90.5 µg kg(-1).

An accurate and selective analytical method for amantadine, which is used as antiviral drug to treat influenza A virus infection, was developed using LC-MS/MS. Residual amantadine was extracted from 4 kinds of food sample (poultry muscle, liver, gizzard and egg) with acetonitrile-pH 3.0 McIlvaine buffer (7 : 3), then cleaned up with an Oasis® MCX mini-cartridge. An external standard calibration curve was used for quantification, after sample purification by the combination of a reverse-phase strong cation exchange mixed mode cartridge for cleanup and a HILIC column for HPLC. The method was validated by performing recovery tests in accordance with Japanese guidelines for the validation of analytical methods for residual agricultural chemicals in food. Recovery ranged from 79.3% to 91.7%, RSDs of repeatability were under 3.3%, and RSDs of within-laboratory reproducibility were under 8.4%. This new method was applied to samples of poultry and egg purehased in Tokyo, but residual amantadine was not detected at all.

We developed a simultaneous determination method for 37 veterinary drugs in two chicken processed foods (deep-fried chicken and non-fried chicken cutlet) and muscle via liquid chromatography-mass spectrometry. The veterinary drugs belong to 7 different classes, including 4 antifolics, 4 benzimidazoles, 5 macrolides, 7 polyethers, 2 quinolones, 7 sulfonamides, and 8 other classes. The samples were extracted with ethyl acetate followed by acetonitrile with salt and buffers extraction. The two-step extraction enabled analyte extraction from highly lipid samples. The clean-up procedure, a solid-supported liquid extraction clean-up using a diatomaceous earth mini-cartridge, eliminated lipid co-extraction. The prepared sample matrix did not have an effect on the 36 analytes. The method was validated in accordance with the requirements of Japanese validation guidelines. Almost all targeted veterinary drugs successfully satisfied the guideline criteria in the three types of food matrices. The method exhibited recoveries of 70-105%, and the precision of repeatability and within-laboratory reproducibility ranged from 1 to 11% and 1 to 15%, respectively. The limits of quantification were estimated to range from 0.2 to 1.0μg/kg. Applying this method to samples commercially available in Tokyo, residues were detected in 3 out of 26 deep-fried chickens, 5 out of 20 non-fried chicken cutlets, and 17 out of 39 chicken muscles.

A method of rapid analysis of multi-class residual veterinary drugs in milk, fish and shellfish was validated in accordance with Japanese guidelines for the validation of analytical methods for residual agricultural chemicals in food. Using LC-MS/MS, 43 multi-class veterinary drugs, including sulfonamides, quinolones, coccidiostats and antiparasites, could be analyzed in one injection. Analytes were extracted from samples with two kinds of solvent, acetonitrile containing 1 vol% formic acid and anhydrous acetonitrile, and salted out with 4.0 g of magnesium sulfate, 1.5 g of trisodium citrate and 2.0 g of sodium chloride. This method was assessed by performing recovery tests in retail milk and 4 kinds of fresh cultured fish and shellfish (salmon, tiger shrimp, red sea bream and bastard halibut) spiked with the 43 target analytes at the levels of 10 and 100 μg/kg. Using this method, 40 out of 43 drugs satisfied the guideline criteria in milk, 37 drugs in salmon, 42 drugs in tiger shrimp, 41 drugs in red sea bream and 39 drugs in bastard halibut.

A simple analytical method was developed for the determination of total bromine in fruits and grain products by means of wavelength dispersive X-ray fluorescence spectrometry (WDXRF).Five gram samples of fresh fruits, frozen fruits, dried fruits and grain products were extracted with distilled water twice and diluted to 25 mL with distilled water. The sample solution (0.5 mL) was dripped onto the filter paper, which was dried and analyzed by WDXRF. The working curve was linear in the range of 0 to 10 microg/mL. Recoveries at the level of 5 microg/g were 76-104%. The detection limit was 0.5 microg/g and the determination limit was 1.5 microg/g in foods. Compared to the GC-ECD method, this method gave equivalent results for fresh fruits, frozen fruits and grain products. In addition, some dried fruits, in which a slightly high level was detected, gave almost the same results with the GC-ECD method. Therefore, this method is considered to be available as an alternative to the GC-ECD method.

Residues of 37 polar veterinary drugs belonging to six families (quinolones, tetracyclines, macrolides, lincosamides, sulfonamides, and others) in livestock and fishery products were determined using a validated LC-MS/MS method. There were two key points insample preparation. First, extraction was performed with two solutions of different polarity. Highly polar compounds were initiallyextracted with Na2EDTA-McIlvaine's buffer (pH 7.0). Medium polar compounds were then extracted from the same samples with acetonitrile containing 0.1% formic acid. Secondly, cleanup was performed using a single SPE polymercartridge. The first extracted solution was applied to the cartridge. Highly polar compounds were retained on the cartridge. Then, the second extracted solution was applied to the same cartridge. Both highly and medium polar compounds were eluted from the cartridge. This method satisfied the guideline criteria for 37 out of 37 drugs in swine muscle, chicken muscle, bovine muscle, prawn, salmon trout, red sea bream, milk, and honey; 35 out of 37 in egg; and 34 out of 37 in flounder. The LOQ ranged from 0.1 to 5 μg/kg. Residues were detected in 24 out of 110 samples and analyzed using the validated method.

A study of the dietary intake of dioxins, consisting of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (Co-PCBs) through baby foods in the metropolitan Tokyo area was carried out. The samples were homemade baby foods prepared by the total diet-market basket method and baby foods (mainly commercial items) prepared by the duplicate portion method. The daily intake of dioxins per kg of body weight from baby foods in each weaning stage for the case of half intake of homemade baby foods and the other half of mainly commercial items was 0.32 pg TEQ/kg/day in the early stage, 0.45 pg TEQ/kg/day in the intermediate stage, 0.58 pg TEQ/kg/day in the late stage and 1.25 pg TEQ/kg/day in the completed stage. While the daily intakes increase with the weaning progress, they were less than the tolerable daily intake (TDI) of 4 pg TEQ/kg/day for dioxins established in Japan. Dioxins were mainly taken through formula milk in early stage of weaning period, but then were taken through protein-based foods in the subsequent weaning stage.

Natural sex hormones, estradiol-17 beta (EST), progesterone (PRO) and testosterone (TES), in sixty domestic bovine muscles and forty bovine muscles imported from USA and Australia were determined by radioimmunoassay. The EST, PRO and TES levels (mean +/- standard deviation) in domestic samples (n = 60) was 1.15 +/- 1.87 ppt, 3.19 +/- 5.80 ppb and 30.9 +/- 122.1 ppt on a whole basis, respectively. The hormone levels in muscles was presumed to reflect sex, estrous cycle, and so on. The hormone levels in imported bovine muscles (n = 40) were 3.33 +/- 2.83 ppt (EST), 0.52 +/- 0.50 ppb (PRO) and 8.8 +/- 13.0 ppt (TES), respectively.

In the official method for determination of total bromine in fruit and grain foods, bromine is derivatized with 3-pentanone for GC analysis. Co-existing substances sometimes interfere with measurement of the derivative, though the method is highly selective. In this study, the notification method was modified to reduce impurity peaks by applying 3-hexanone. Samples were alkalized and reduced to ash in an electric furnace. After ashing, samples were oxidized with potassium permanganate solution and derivatized with 3-hexanone. The calibration curve was linear from 0.1 microg/mL up to 5.0 microg/mL. The detection limit (S/N = 10) was 0.1 microg/mL, i.e., 5 microg/g for herb, 2.5 microg/g for grains and 1.0 microg/g for fruits. The recoveries of bromine from fruit, grain foods and herbs added at the levels of 5 to 25 microg/g ranged from 84.2 to 96.9%. The values of relative standard deviation (RSD) were from 1.4 to 6.3%. This method should be useful for routine examination of total bromine in foods.

A method for accurate and simple determination of 28 kinds of organonitrogen and 7 kinds of carbamate pesticides in foods was developed. The pesticides in foods were extracted using a method for rapid simultaneous determination of pesticide residues recommended by the Ministry of Health and Welfare. The pesticides co-injected with retention index standards (M-Series) were determined with a dual-column gas chromatograph equipped with ATD. The identification was based on retention indices and the quantitation was done by a multi-internal standard method. This technique provides superior reproducibility of identification and accuracy of quantitation.

An analytical method for determination of sulfonylurea herbicides, chlorimuron ethyl (CE) and tribenuron methyl (TM), in crops by HPLC was developed.The sample was homogenized with acetone followed by extraction with ethyl acetate, and the extract was evaporated. The residue was defatted by acetonitrile-n-hexane partitioning for soybean, peanut and corn grits. The residue was cleaned up with a Florisil column and a Bond Elut® SAX cartrigde column. CE and TM were separated on an HPLC column (Inertsil ODS-2) using acetonitrile-water-phosphoric acid (50:50:0.01) as the mobile phase. These compounds were detected at 240nm.The mean recoveries of these compounds added to crops at the level of 0.1ppm were 59-84% (CE) and 54-79%, except for peanut (TM). The detection limits of CE and TM in crops were 0.01ppm.

A simplified determination of methabenzthiazuron in agricultural products by HPLC with a UV detector was investigated.Each agricultural product sample was homogenized and extracted with acetone. The crude extract was purified on a Bond Elut® Extraction Cartridge PSA using a mixture of acetone and n-hexane. Methabenzthiazuron was analyzed by HPLC with UV detection (275nm). The HPLC separation was performed on an ODS column with acetonitrile-water (1:1) as the mobile phase.Recoveries of methabenzthiazuron from several agricultural products fortified at the level of 0.1ppm were in the range of 93-104%. The limit of detection was 0.005ppm in samples.

A high performance liquid chromatographic procedure with photodiode array detection was developed for determination of 4 chitin biosynthesis inhibitors (diflubenzuron, teflubenzuron, flufenoxuron, chlorfluazuron) and linuron in beef.These agents were extracted with hexane-acetone (2:1). The extract was cleaned up by liquid-liquid partition between MeCN and hexane, followed by column chromatography on Bond Elut® SI. HPLC was carried out on a Wakosil-II 5C18 HG column (4.6mm i. d.×250mm) with MeCN-H2O (5:2) as the mobile phase. These compounds were detected at 250nm and 260nm. The mean recoveries of these compounds added to beef at 0.2μg/g were 82.9-96.7%. The detection limits of these compounds were 0.1μg/g in beef fat.

Although chlordane had been widely used in Japan as a termicide for the protection of wooden structures, its use was prohibited owing to its persistence in 1986. The residues of 19 chlordane compounds (main components in technical chlordane and their metabolites) were measured in shellfish (short-necked clam, Tapes philippinarum) from Tokyo Bay during 1982-1993. Chlordane compounds were extracted with McCN-MeOH (1:1) from shellfish, cleaned-up on a Florisil column and by GPC chromatography, then determined by GC/MS (SIM). The level of total chlordane during 1982-1986, was 11.3-22.7ppb. After the prohibition of chlordane use in 1986, the level in 1991-1993 decreased to ca. 1/5 of that in 1982-1986. Component patterns of chlordane residues in shellfish were different from that in technical chlordane. The ratios of highly chlorinated components (trans-, cis-nonachlor, etc.) were high in comparison with that of technical chlordane, and some metabolites (oxychlordane & heptachlor epoxide) were found in shellfish.

A method for fluorometric determination of salbutamol (SALB) in meat by HPLC with solid-phase extraction was developed.SALB was extracted from meat with 5% metaphosphoric acid solution in the presence of dichloromethane. The extract was adjusted to pH 11-12 and then re-extracted with ethyl acetate-tert-butanol (7:3). The extracted solution was evaporated to dryness. The residue was cleaned up with Bond Elut LRC AccuCAT and Mega Bond Elut C18 cartridges. SALB was separated by HPLC on an ODS column (TSKgel-ODS 80Ts) using 0.05M phosphate buffer (pH 3.0)-MeCN (92:8) as a mobile phase. The fluorescence detector was operated at the excitation wavelength of 225nm and emission wavelength of 310nm.The recoveries from meat (beef, pork and liver) spiked at the level of 100ppb were in the range of 77.6-81.5%, and the detection limit was 5ppb.

A method was developed for assay of progesterone used as an anabolic agent by applying a commercially available enzyme immunoassay (ETA) kit for diagnosis of pregnancy in cattle (by measuring progesterone in whole milk) to the measurement of progesterone in beef. Beef was homogenized with acetonitrile, and progesterone was extracted into dichloromethane. The dichloromethane phase was concentrated and purified by Sephadex LH-20 column chromatography. The concentrated hexane-benzene-methanol (85:10:5) eluate was taken up in buffer solution, and the solution was subjected to enzyme immunoassay using the kit.The mean recoveries of progesterone in beef fortified with 1-10ppb were 116-130%. A good correlation between the values obtained by the ETA method and by HPLC was obtained for progesterone added to beef. The detection limit of progesterone was about 1ppb in the samples. This procedure is useful for initial screening of residual progesterone in beef.