Determination of bithionol, bromophen, nitroxynil, oxyclozanide, and tribromsalan in milk with liquid chromatography coupled with tandem mass spectrometry.

LC/MS/MS was developed to determine the residues of bithionol (BTN), bromofen (BMF), nitroxynil (NTX), oxyclozanide (OCZ), and tribromsalan (TBS) in milk. Samples were extracted with ethyl acetate and cleaned up by liquid-liquid separation with acetonitrile and n-hexane. The compounds were determined by RP-LC using a C18 column with 0.1% formic acid-methanol. Mass spectral acquisition was performed in the negative mode by applying selected-reaction monitoring. The method was validated in milk spiked with these compounds at 5-600 microg/kg; average recoveries were in the range 83.8-97.1%, with RSD values of 1.4-8.0%. The interassay RSDs were less than 11%. The LODs of these compounds in milk were 0.1 microg/kg. The method was applied to 24 raw milk samples. The concentration of these compounds in all samples was lower than the Japanese maximum residue limits. The method is rapid, sensitive, and specific for monitoring residues of BTN, BMF, NTX, OCZ, and TBS in milk.

[Determination of hymexazol in agricultural products by GC-NPD].

A method for the determination of hymexazol in agricultural produdcts by gas chromatography with a highly sensitive nitrogen-phosphorus detector (GC-NPD) was investigated. Hymexazol was extracted with acetonitrile, and the acetonitrile layer was separated by salting-out. The water layer was loaded onto a Chem-Elut column. Hymexazol in the water layer was adsorbed on the column, and eluted with ethyl acetate. The acetonitrile layer and the eluate were mixed and evaporated. The residue was dissolved in ethyl acetate, and the sample solution was cleaned up on a C18 column. Hymexazol in the eluate was analyzed by GC-NPD with a high-polarity capillary column (DB-FFAP) and highly deactivated inlet liner. Recoveries of hymexazol spiked in agricultural products (tomato, lemon, soybean and other samples) at the level of 0.1 mug/g ranged from 65.0 to 84.7%. The limit of detection was 0.02microg/g.

[Survey of pesticide residues in imported cereal products (1994.4 approximately 2006.3)].

A survey of pesticide residues in 490 imported cereal products on the Tokyo market from April 1994 to March 2006 was carried out. Eight kinds of organophosphorus pesticides (chlorpyrifos, chlorpyrifos-methyl, DDVP, diazinon, etrimfos, malathion, MEP and pirimiphos-methyl) were detected at levels between Tr (below 0.01 ppm) and 0.82 ppm from 91 samples. In our investigations, chlorpyrifos-methyl and malathion tended to be detected in samples from America, pirimiphos-methyl in those from Europe, and MEP in those from Oceania. Thus, pesticide residues seemed to be different in produce from different areas. Residue levels of these pesticides were calculated as between 0.08 and 13.2% of their ADI values according to the daily intake of cereal products. Therefore, these cereal products should be safe for normal usage.

[Rapid determination of residues of 4 tetracyclines in meat by a microbiological screening, HPLC and LC/MS/MS].

A rapid and precise determination residues of 4 tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DOXY)) in meat was developed by employing three analyses; a microbiological screening, HPLC and LC/MS/MS. TCs were extracted with pH 4.0 McIlvaine buffer containing 0.01 mol/L EDTA from a meat sample, and then purified using a mixed mode, reversed-phase and cation-exchange cartridge. The mean recoveries (n=5) of 0.2 microg/g OTC, TC and CTC, 0.05 microg/g DOXY spiked in meat samples were 76.6-99.0% (C.V. 1.6-5.4%). In 13 meat samples in which the microbiological screening indicated the presence of TCs, CTC (9 samples) and DOXY (4 samples) were identified with HPLC and LC/MS/MS.

Clean-up method of forchlorfenuron in agricultural products for HPLC analysis.

A simplified method for the determination of forchlorfenuron in agricultural products by HPLC with UV detection was investigated. A chopped sample homogenate from agricultural products was extracted with acetone. The extract was filtrated and concentrated. The residues was loaded onto a Chem Elut column and extracted with ethyl acetate. The crude extract was purified on Oasis HLB and Bond Elut PSA mini-columns using a mixture of methanol and ethyl acetate. Forchlorfenuron was analyzed by HPLC with UV detection (263 nm). HPLC separation was performed on an ODS column with methanol-water as the mobile phase. Recoveries of forchlorfenuron from several agricultural products fortified at the level of 0.1 microg/g were in the range of 87.6-99.5%. The limit of detection (S/N=3) was 0.005 microg/g in the sample.

[Survey of pesticide residues in baby foods (1999.4-2005.6)].

A survey of pesticide residues in 316 baby foods on the Tokyo market from April 1999 to June 2005 was carried out. Fifteen kinds of pesticides, including organophosphorus, organochlorine, carbamate, pyrethroid and others, were detected between at levels Tr (below 0.01 ppm) and 0.55 ppm from 28 samples. The baby foods in which residual pesticides were detected were produced from flour, leaf vegetable and fruits. The pesticides were detected in baby foods produced with minimal processing, such as straining and squeezing. For the baby foods containing pesticides, the amounts of intake of the pesticides were calculated from the recommended servings, and compared with the ADI values. Residual pesticide levels in baby food were between 0.06 and 16.6% of the ADI. Therefore, these foods should be safe in normal usage.

[Determination of vitamin K in aojiru (green juice) products by HPLC].

A survey of vitamin K contents was carried out on 41 aojiru products and 10 vegetable juice products that were purchased from local markets. Aojiru is a health food made from green vegetables such as kale, ashitaba, mulberry leaf, barley grass and the like. The products are usually provided in various forms, such as frozen, powder and tablet. Vitamin K in samples was extracted with n-hexane, and separated on a C18 column with methanol-ethanol (95 : 5). After separation, vitamin K was converted to the hydroquinone form on a reduction column and determined with a fluorescence detector at lambdaex 240 nm and lambdaem 430 nm. The contents of vitamin K1 (phylloquinone) in frozen samples (n = 8), powder samples (n = 26) and tablet samples (n=7) were 90-190, 410-3,300, and 640-3,100 microg/100 g, respectively, and that in vegetable juice (n= 10) was 1-12 microg/100 g. Vitamin K2 (menaquinone) was not detected. The daily intake of vitamin K from aojiru products was estimated to be 99-380, 20-250 and 27-210 microg/day for frozen, powder and tablet types, respectively. These results suggest that patients prescribed warfarin should take care about their intake of vitamin K from aojiru products.

[Analysis of imazalil and its major metabolite in citrus fruits by GC-FTD].

A simplified simultaneous analytical method of imazalil (IZ) and its major metabolite, alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (IZM), in citrus fruits was developed, and commodities samples were investigated. A homogenate of citrus fruits was extracted with ethyl acetate under basic conditions. The crude extract was partitioned between 0.025 mol/L of sulfuric acid and ethyl acetate. The analytes were extracted from the aqueous fraction under basic conditions with ethyl acetate. The extract solution was purified with an ENVI-Carb cartridge, and then analyzed by GC-FTD and GC/MS. Recoveries of IZ and IZM added to grapefruit at the level of 0.05 microgram/g were 90.0 and 108.7%, and those in the case of lemon were 100.4 and 93.0%, respectively. The detection limits were 0.01 microgram/g in samples. By this method, IZ and IZM were analyzed in 46 citrus fruits on the market and were detected simultaneously in some samples.

[Simultaneous determination of N-methylcarbamate pesticides and their metabolites in agricultural products].

We investigated the determination of N-methylcarbamate pesticides and their metabolites in agricultural products by HPLC with post-column fluorescence detection after clean-up with an SPE cartridge. The homogenate of agricultural products was extracted with acetone. The crude extract was partitioned between 5% sodium chloride solution and dichloromethane, and the dichloromethane layer was concentrated to dryness. The residue was dissolved with a mixture of acetone and n-hexane, and purified by using Supelclean ENVI-Carb SPE, Bond Elut Extraction Cartridge PSA and SAX in series with a mixture of acetone and n-hexane. N-Methylcarbamate pesticide was analyzed by HPLC with post-column reaction and fluoresce detection. N-Methylcarbamate pesticide in citrus fruits and various kinds of agricultural products could be analyzed accurately by the presented method. Recoveries of N-methylcarbamate pesticides added to several agricultural products at the level of 0.10 ppm were mostly in the range of 60-110%. The limit of detection was 0.005 ppm.

[Simple analysis of maleic hydrazide in agricultural products by HPLC].

A simplified HPLC determination method for maleic hydrazide in agricultural products was developed, and commercial agricultural crops were investigated. The homogenate of agricultural products was extracted with water. The crude extract was purified on an ACCUCAT Bond Elut extraction cartridge using water. Maleic hydrazide was analyzed by HPLC with UV detection (303 nm). The HPLC separation was performed on a ZORBAX SB-Aq column with acetonitrile-water-phosphoric acid(5:95:0.01) as the mobile phase. Recoveries of maleic hydrazide from 15 agricultural products fortified at 1.0 and 10 micrograms/g were in the ranges of 92.6-104.9% and 94.2-101.3%, respectively. The limit of detection was 0.5 microgram/g in samples. The proposed method was applied to the determination of 242 commercial vegetables and fruits. Maleic hydrazide was detected in 2 samples of imported onion at the levels of 4.9 and 7.2 micrograms/g.

[Identification of chemical detected in kiwi fruit during analysis for residual pesticide].

An unknown peak was detected in a GC chromatogram of many kiwi fruit extracts during analysis for pesticide residues. It was identified by GC/MS as diphenyl 2-ethylhexyl phosphate (DPEHP), used as a plasticizer and flame retardant. The concentration of DPEHP was investigated in 15 samples of kiwi fruit, and it was detected at between 0.02 and 0.14 microgram/g in 10 of the samples. It might be due to migration of DPEHP into the fruit from the printed portion of the polyethylene terephthalate (PET) package.

[Performance study of analytical method for ethoxyquin in fruit].

A performance study of an analytical method of ethoxyquin (ET) in apples and pears was conducted. In the proposed method, the sample was homogenized with thiourea and ET was extracted with acetone instead of hexane used in the official analytical method for ET, in order to improve the extraction efficiency. Furthermore, dibutyl hydroxytoluene was added in the test solution to prevent the decomposition of ET. For evaluation of the method. ET spiked into apples and pears at the level of 0.2 microgram/g was determined in replicate in six laboratories. Mean recoveries from apple, pear and Japanese pear were 85.3, 83.0 and 83.9%, respectively. Repeatability relative standard deviation values were 3.8-4.7% and reproducibility relative standard deviation values were 7.8-11.3%. The detection limits were 0.001-0.025 microgram/g in the six laboratories; this value may reflect instrument performance. ET spiked into apples and pears at the level of 0.2 microgram/g was detected by both LC/MS and GC/MS.

[Determination of captafol, cyhexatin, 1-naphthylacetic acid and quintozene in apple, Japanese pear and melon by simultaneous extraction].

A simple and rapid method is described for the determination of the non-registered pesticides, captafol, quintozene (PCNB), cyhexatin and 1-naphthylacetic acid (NAA), in fruits. These pesticides were extracted with acidified acetone, then captafol and PCNB were purified with a Florisil mini column and analyzed by GC-ECD. Cyhexatin was ethylated with ethylmagnesium bromide, and the ethyl derivative was analyzed by GC-FPD (Sn filter). NAA was purified with liquid-liquid extraction and determined by HPLC equipped with a fluorescence detector. These analytes were identified with GC/MS or LC/MS. The minimum identified concentration of the pesticides was below 0.2 ng per injection, which corresponds to a detection limit of below 0.02 microgram/g in the original samples. Recoveries of the pesticides spiked at 0.1 microgram/g into apple, Japanese pear and melon were greater than 61%.

[Analysis of pesticides including chlorine in welsh onions and mushrooms using gas chromatograph with an atomic emission detector (GC-AED)].

An analytical method for the determination of 32 kinds of pesticide residues in onions, Welsh onions and mushrooms using gas chromatograph with an atomic emission detector (GC-AED) was developed. The pesticides were extracted with acetone-n-hexane (2:3) mixture. The crude extract was partitioned between 5% sodium chloride and ethyl acetate-n-hexane (1:4) mixture. The extract was passed through a Florisil mini-column for cleanup with 10 mL of acetone-n-hexane (1:9) mixture. Although the sensitivity of GC-AED was inferior to that of GC-ECD, GC-AED has a superior element-selectivity. Therefore pesticide residues in foods could be analyzed more exactly by using GC-AED. Thirty-two pesticides including chlorine in onion, Welsh onion and shiitake mushroom were detected without interference. Recoveries of these pesticides from samples determined by GC-AED were 64-114%, except for a few pesticides.

[Identification of unknown peaks in foods during residual pesticide analysis].

Four unknown peaks were detected in GC chromatograms during analysis of organophosphorus pesticide residues in foods. They were identified by using GC/MS as triethyl phosphate (TEP), tributyl phosphate (TBP), diphenyl 2-ethylhexyl phosphate (DPEHP) and N-ethyltoluenesulfoneamide (NETSA), which are used as plasticizers or flame retardants in food packaging. These chemical substances were detected in the range of tr. (below 0.01 microg/g) to 11 micro/g from 29 samples, and they were also detected in the packaging. It was supposed that they were transferred to the foods from the packaging. Furthermore, they were detected in some cereals and cereal products which contain fat and had been preserved for a long time.

Inter-laboratory study of an LC-MS/MS method for simultaneous determination of deoxynivalenol and its acetylated derivatives, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in wheat.

To validate an LC-MS/MS method for simultaneous determination of deoxynivalenol (DON) and its acetylated derivatives, 3-acetyl-deoxynivalenol (3ADON) and 15-acetyl-deoxynivalenol (15ADON), in wheat using a multifunctional column, an inter-laboratory study was performed in 9 laboratories using one blank wheat sample, three spiked wheat samples (10, 50, 150 µg/kg) and one naturally contaminated wheat sample. The recoveries ranged from 98.8 to 102.6% for DON, 89.3 to 98.7% for 3ADON, and from 84.9 to 90.0% for 15ADON. The relative standard deviations for repeatability (RSDR) and reproducibility (RSDR) of DON were in the ranges of 7.2-11.3% and 9.5-22.6%, respectively. For 3ADON, the RSDR ranged from 5.3 to 9.5% and the RSDR ranged from 16.1 to 18.0%, while for 15ADON, the RSDR ranged from 6.2 to 11.2% and the RSDR ranged from 17.0 to 27.2%. The HorRat values for the three analytes ranged from 0.4 to 1.2. These results validate this method for the simultaneous determination of DON and its acetylated derivatives, 3ADON and 15ADON.

[Survey of pesticide residues in imported tea (1992.4-2010.3)].

A survey of pesticide residues in 116 imported tea samples on the Tokyo market from April 1992 to March 2010 was carried out. Twenty-two kinds of pesticides, including organophosphorus, organochlorine, pyrethroid and others, were detected at levels between trace (below 0.01 ppm) and 4.0 ppm in 76 samples. The rate of detection was 90% in non-fermented tea, 89% in semi-fermented tea, and 49% in fermented tea. Organophosphorus pesticide was not detected in puer tea, which is fermented with bacteria. The pesticide residues tended to be decreased by a fermentation process. However, organochlorine and pyrethroid pesticides appeared to be chemically stable, so they were still detected in fermented teas. Residue levels of these pesticides were calculated as less than 1% of ADI, except for ethion (45% of ADI) based on the daily intake of tea. Therefore, these teas should be safe when drunk in customary amounts.

Inter-laboratory study of an LC-MS/MS method for simultaneous determination of fumonisin B1, B2 and B3 in corn.

To validate an LC-MS/MS method using a strong anion exchange cartridge for simultaneous determination of fumonisin B1, B2 and B3 in corn, an inter-laboratory study was performed in 9 laboratories using one fumonisin-negative corn sample, three spiked corn samples (FB1: 100-1,000 µg/kg, FB2 and FB3: 10-100 µg/kg) and two naturally contaminated corn samples. The recoveries were in the ranges of 79.7-87.2% for FB1, 78.6-103.2% for FB2 and 80.1-92.8% for FB3. The relative standard deviations for repeatability (RSDr) ranged from 3.7 to 8.0% for FB1, from 2.6 to 15.3% for FB2 and from 4.3 to 9.7% for FB3. The relative standard deviations for reproducibility (RSDR) for FB1, FB2 and FB3 were in the ranges of 6.3-10.1%, 5.9-18.7% and 9.3-16.0%, respectively. The HorRat values for all analytes ranged from 0.2 to 0.9. The difference of the trueness between the two kinds of commercially available anion exchange cartridges used in this study was not significant (p>0.05). Surveillance for fumonisins in corn grits was performed using the validated method. All of the samples were contaminated with fumonisins and the mean concentrations for FB1, FB2 and FB3 were 118.1, 37.3 and 17.9 µg/kg, respectively. These results indicated that the method for simultaneous determination of FB1, FB2 and FB3 in corn was successfully developed and validated.

[Survey of pesticide residues in imported spices and herbs (1997.4-2011.3)].

A survey of pesticide residues in 313 samples of imported spices and herbs on the Tokyo market from April 1997 to March 2011 was carried out. Thirty-seven kinds of pesticides, including organophosphorus, organochlorine, pyrethroid, carbamate and others, were detected between levels of trace (below 0.01 ppm) and 3.3 ppm from 64 samples. The rate of detection was highest in peel (100%) followed by stem (66.7%), fruit (34.5%), bark (33.3%), flower (31.3%) and leaf (14.7%). No residues were detected in root, seed or whole grass. Organochlorine pesticides were detected in all plant parts. The insecticides were detected in products from all production areas, suggesting that their use is common. Residue levels of these pesticides were calculated as less than 1% of their ADI values, based on the daily intake of spices and herbs. Therefore, these spices and herbs should be safe when consumed in customary amounts.

Interlaboratory study of LC-UV and LC-MS methods for the simultaneous determination of deoxynivalenol and nivalenol in wheat.

To evaluate LC methods with UV or MS detection for simultaneous analysis of deoxynivalenol (DON) and nivalenol (NIV) in wheat, an interlaboratory study was conducted in 11 laboratories. DON and NIV were purified using a multifunctional column, and their concentrations were determined using LC-UV or LC-MS(/MS). No internal standards were used. Three fortified wheat samples (0.1, 0.5 and 1 mg/kg), one naturally contaminated wheat sample, and one blank wheat sample were used. The recoveries ranged from 90% to 110% for DON and from 76% to 83% for NIV. For DON, the relative standard deviations for repeatability (RSDr) ranged from 1.1% to 7.6%. The relative standard deviations for reproducibility (RSDr) ranged from 7.2% to 25.2%. For NIV, the RSDr ranged from 2.0% to 10.7%, and the RSDr ranged from 7.0% to 31.4%. Regardless of sample and detector, the HorRat values for DON and NIV ranged from 0.4 to 1.4. Both LC-UV and LC-MS(/MS) methods were considered to be suitable for application as an official method.