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Since amantadine, rimantadine, arbidol, laninamvir, oseltamivir, peramivir, and zanamivir may be used as antiviral agents to treat avian influenza, we herein developed a simultaneous assay using LC-MS/MS. This method was applied to chicken products (including yakitori (grilled chicken), fried chicken, chicken steak, and boiled eggs) as well as chicken tissues (muscle, fat, the liver, gizzards, and heart) and eggs.Samples were extracted with methanol-water (9 : 1), purified by a tandem column with an InertSep ® MAX cartridge (upper part) and InertSep ® MCX cartridge (lower part), and then measured by LC-MS/MS. The sample matrix had no effect on the identification of compounds. Chromatographic separation was performed on a ZIC-HILIC column using a mobile phase of 1% acetic acid solution and 1% acetic acid solution in acetonitrile, resulting in complete separation and other obstructive peaks from the sample matrices. An external solvent calibration curve was used for quantification.The application of the method to 6 samples of chicken tissues and eggs achieved good results of between 77.9 and 97.5% for trueness and between 1.7 and 9.2% for concurrent accuracy. The method was also applied to 9 samples of processed products, including grilled chicken and fried chicken, and achieved good results with true percentages ranging between 72.6 and 99.2% and concurrent accuracies between 3.0 and 11.2%. Therefore, the developed method may also be applied to processed products.The limit of quantification (LOQ) of the developed method was 0.01 mg/kg.The method was then applied to 42 types of commercial processed products, including yakitori, fried chicken, steamed chicken, chicken steak, and boiled eggs, and no antiviral agents were detected.Collectively, the present results confirmed that the method developed herein is applicable to not only chicken tissues, but also their processed products.

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

An LC-MS/MS method for the determination of diniconazole in agricultural products, livestock and marine products was developed. Diniconazole in agricultural products was extracted with acetone. The extract was concentrated and partitioned with n-hexane and 10% sodium chloride solution. Agricultural products such as grains and beans were defatted using n-hexane-acetonitrile. Livestock and marine products were extracted with a mixture of acetone and n-hexane, and the organic layer was evaporated to dryness. The residue was defatted using n-hexane-acetonitrile. Cleanup was carried out using a Florisil cartridge column and a graphitized carbon cartridge column for these samples. The LC separation was carried out on an Inertsil ODS-3 column with a linear gradient of 0.1% formic acid and acetonitrile containing 0.1% formic acid. MS was carried out in the positive ion electrospray ionization mode. The calibration curve was linear between 0.00125 to 0.00750 mg/L. Average recoveries (n=5) of diniconazole from 16 kinds of agricultural products, livestock and marine products fortified at the MRLs (0.01 ppm) were 88.3-108%, and the relative standard deviations were 0.5-5.1%. The limits of quantitation were 0.01 mg/kg.

Azodicarbonamide (ADA) is used in some countries as a flour bleaching agent and a dough conditioner. However, ADA is prohibited for use as a food additive in Japan. Therefore, it is necessary to establish an efficient and sensitive method to determine ADA in wheat flour. A simple and practical procedure to analyze ADA in wheat flour and prepared flour mixes was developed. ADA was extracted from samples by ultrasonication with acetone. ADA in the solution was derivatized with triphenylphosphine (TPP). The ADA-TPP derivative was concentrated and cleaned up using a reversed-phase solid-phase extraction cartridge, and the ADA-TPP derivative was analyzed using HPLC for determination and LC-MS/MS for identification. Good linearity was achieved over the concentration range of 0.25-100 ppm ADA in wheat flour and prepared flour mixes. The mean recoveries from wheat flour and prepared flour mixes fortified at the levels of 1 and 10 ppm ranged from 86.9 to 101.0%, and the coefficients of variation ranged from 1.9 to 3.4%.

The aluminium (Al) content of 105 samples, including bakery products made with baking powder, agricultural products and seafoods treated with alum, was investigated. The amounts of Al detected were as follows (limit of quantification: 0.01 mg/g): 0.01-0.37 mg/g in 26 of 57 bakery products, 0.22-0.57 mg/g in 3 of 6 powder mixes, 0.01-0.05 mg/g in all three agricultural products examined, 0.03-0.90 mg/g in 4 of 6 seafood samples, 0.01-0.03 mg/g in 3 of 11 samples of instant noodles, 0.04-0.14 mg/g in 3 of 4 samples of vermicelli, 0.01 mg/g in 1 of 16 soybean products, but none in soybeans. Amounts equivalent to the PTWI of a 16 kg infant were detected in two samples of bakery products, two samples of powder mixes and one sample of salted jellyfish, if each sample was taken once a week. These results suggest that certain foods, depending on the product and the intake, might exceed the PTWI of children, especially infants.

We examined two unknown red dyes (designated as red dyes "A" and "B") from a dried strawberry package with a label that indicated the presence of food red No. 40 (R40). Red dye "A" was identified as trisodium 3-hydroxy-4-[(2'-methoxy-5'-methyl-4'-sulfonatophenyl)azo]-2,7-naphthalenedisulfonate (CSA-R) by HPLC, UV-VIS spectra and MS spectra. This compound is one of the four reported subsidiary colors of R40. Detailed analyses of red dye "B" by MS and NMR demonstrated that its structure was disodium 3-hydroxy-4-[(2'-methoxy-5'-methyl-4'-sulfonatophenyl)azo]-2-naphthalenesulfonate. Red dye "B" is a structural isomer of R40, that has not been reported previously. Our results suggest that the two minor red dyes were subsidiary colors contained in R40, which had been added to the dried strawberries.

Pollution levels of toxic heavy metals (Pb, Cd, Hg) and arsenic in existing food additives used as food colors (40 samples of 15 kinds) were investigated. Heavy metals were detected in 8 samples; Pb in 1 sample (2.8 microg/g), Hg in 8 samples (0.1-3.4 microg/g) and arsenic in 2 samples (1.7, 2.6 microg/g). The Pb level in 1 sample of lac color (2.8 microg/g) exceeded the limit of 2 microg/g proposed by JECFA and Hg levels in 3 samples of cacao color (1.2-3.4 microg/g) exceeded the limit of 1 microg/g in the EU specification.

Interactions between grapefruit juice and medications have long been recognized. In recent years, several furanocoumarins (FCs) that inhibit P450 activity in intestinal microsomes have been isolated from grapefruit juice. FCs, i.e., bergamottin (BG), bergapten (BP), bergaptol (BT) and 6',7'-dihydroxybergamottin (DHB), in samples was extracted with acetonitrile, and separated on a Phenyl column using 0.1% phosphoric acid-acetonitrile gradient as a mobile phase, with monitoring at 311 nm. The recoveries of BG, BP, BT and DHB from lemon juice spiked at the level of 5.0 microg/g were 103+/-0.7%, 99.5+/-0.2%, 96.5+/-0.2% and 90.1+/-0.2%, respectively. The quantification limits were 1.0 microg/g in samples. The contents of BG, BP and DHB in grapefruit juice (n=13), citrus fruit of 20 species and health food (n=16) were measured. The contents of BG were 0-16 microg/g, 0-16 microg/g and 0-5.6 microg/g, BT were 0-39 microg/g, 0-13 microg/g and 0-28 microg/g, DHB were 0-10 microg/g, 0-35 microg/g and 0-6.2 microg/g, respectively. BP was not detected. These results suggest that patients prescribed calcium antagonists or antiallergic agents should be cautions about their intake of FCs from grapefruit juice, citrus and health foods.

Ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in commercial foods were simultaneously determined and confirmed with high-performance liquid chromatography (HPLC) and liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The samples examined were made up of cereal, fruit, coffee, and cacao products. The limits of quantification (S/N> or =10) of OTA, OTB and CIT were 0.1 microg/kg or less. Aflatoxins (AF), deoxynivalenol (DON) and fumonisins were also surveyed. Of 157 samples examined, 44 were contaminated with OTA at levels of 0.11 to 4.0 microg/kg. At least 2 positive samples were labeled as domestics. In most positive samples, the OTA level was low, less than 1 microg/kg. The highest incidence of OTA was observed in cacao powder (10/12), followed by instant coffee (5/7), cocoa (5/8) and raisin (6/13). OTB was found in fruit and cacao products containing relatively high levels of OTA. Co-occurrence of OTA, CIT and DON was found in cereal products, and co-occurrence of OTA and AF was found in cacao products. Approximately 30% of naturally contaminated OTA in roasted coffee bean moved into the extract solution when brewed with paper filter.

Methods using high-performance liquid chromatography with fluorescence detection (HPLC-FL) and using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for simultaneous determination of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in cereal, fruit, and coffee products. The samples were extracted with ethyl acetate under an acidic condition, and then cleaned up with liquid-liquid separation. The test solutions were analyzed by reverse-phase HPLC-FL and LC/MS/MS. Mass spectral acquisition was performed in positive ion mode by applying multiple reaction monitoring. The performances of both detectors were almost equivalent. The recoveries of OTA and OTB were 87-111%, and that of CIT were 70-88%. The limits of quantification (S/N> or =10) of OTA, OTB and CIT was 0.1 mug/kg or less. These methods were considered to be useful for the determination of the three mycotoxins at low levels (0.1 microg/kg).

An extraction and analytical method was developed for determination of the content and profiles of anthocyanins in commercial dietary supplements containing blueberry extract. Dietary supplements were refluxed with hydrochloric acid-methanol solution, and spectrophotometric assay was performed to evaluate the total anthocyanidin content in extracted solutions as delphinidin, which is one of the anthocyanidins in blueberry extract. Twenty compounds (15 anthocyanins and 5 anthocyanidins) in extracted solutions were separated by HPLC analysis with gradient elution using formic acid and methanol-acetonitrile as the mobile phase, and each peak was confirmed by LC/MS analysis. The proposed method was applied to 25 kinds of commercial dietary supplements, and one supplement whose profile was different from that of the fresh bilberry extract was found.

Organic solvent residue levels in "Existing Food Additives" (n=145), health food materials (n=23), and commercial health food products (n=19) were surveyed. Ethanol was the dominant solvent found in the samples, suggesting its use in the manufacturing process. Methanol, acetone, 2-propanol and ethyl acetate was also found. No residual solvent exceeded the limits set by the Food Sanitation Law.

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.

A method for the determination of 4-hexylresorcinol residues in prawn and crab meat by HPLC was developed. 4-Hexylresorcinol in prawn and crab meats was extracted with methanol using a homogenizer. The extract was diluted 4 times with water, and the diluted solution was passed through a C18 cartridge. The cartridge was washed with water and methanol-water (4 : 6), and then 4-hexylresorcinol was eluted with acetonitrile-0.1% phosphoric acid (55 : 45). The eluate was separated on a Capcell Pak C18 MG column with a mobile phase of acetonitrile-0.1% phosphoric acid (6 : 4) and 4-hexylresorcinol was determined with a UV detector (210 nm). Recoveries of 4-hexylresorcinol from commercial prawn and crab meats spiked at 1.0 and 10 microg/g were 82.4-92.2 and 88.9-91.8%, respectively. The determination limit of 4-hexylresorcinol was 1.0 microg/g in the samples.

A method for the simultaneous determination of stevioside (Stev), rebaudioside A (RebA) and glycyrrhizic acid (GA) in foods was developed. These sweeteners were extracted from foods, except for dried fishes and shellfishes, by dialysis against Tris-HCl buffer (pH 9.0). Dried fishes and shellfishes were extracted with Tris-HCl buffer--methanol (2:8). The extracts were cleaned up with an Oasis MAX cartridge. The cartridge was washed with 0.05 mol/L sodium acetate (pH 4.0)--methanol (19:1), and the three sweeteners were eluted with 0.1 mol/L phosphoric acid--acetonitrile (1:1). Stev, RebA and GA in the eluate were chromatographed on a Develosil RPAQUEOUS-AR-5 (4.6 mm i.d. x 250 mm) column with 0.02 mol/L phosphoric acid-acetonitrile--methanol (90:55:5) as a mobile phase and monitored at 210 nm for Stev and RebA, and at 254 nm for GA. The recoveries of Stev, RebA and GA from 8 kinds of foods spiked at the level of 0.1 g/kg were 81.7-101%, 81.5-100% and 78.6-95.0%, respectively. The determination limits were 0.01 g/kg in samples.

The development of a sensitive pre-column derivatization high-performance liquid chromatography (HPLC) method for determination of sucralose is reported. Sucralose is converted into a strongly ultraviolet (UV)-absorbing derivative, possessing strong absorption at 260 nm, by treatment with p-nitrobenzoyl chloride (PNBCl). Homogenized samples were dialyzed and washed with a Bond Elut ENV cartridge, then the eluate was evaporated to dryness and the residue was derivatized. Subsequently, the sucralose derivative was purified with hexane-ethyl actate (9:1) in a silica cartridge, and then the sucralose derivative was eluted with acetone. HPLC was performed on a phenyl column, using acetonitrile-water (73:27) as a mobile phase with UV detection (260 nm). The calibration curve was linear in the range of 1 microgram/mL to 50 micrograms/mL of sucralose. The recoveries of sucralose from eight kinds of foods spiked at the levels of 0.20 and 0.05 g/kg of sucralose were more than 76.2% with SD values in the range from 0.90% to 4.31%. The quantitative limit of the developed method was 0.005 g/kg for sucralose in samples.

A method for the determination of sucralose in various foods by RI-HPLC and ion chromatography with a pulsed amperometric detector (PAD-IC) was developed. Chopped or homogenized samples were packed into cellulose tubing with 0.01 mol/L hydrochloric acid containing 10% sodium chloride and were dialyzed against 0.01 mol/L hydrochloric acid for 24 hours. The dialyzate was passed through a Bond Elut ENV cartridge, and the cartridge was washed with 0.2 mol/L NaOH and water. Sucralose was eluted from the cartridge with methanol. The extract was taken to dryness in an evaporator and the residue was re-dissolved in water. Sucralose was separated on an Inertsil ODS-3V column with a mobile phase of acetonitrile-water (15:85) and an RI detector. It was also determined on a CarboPak PA1 column with a mobile phase of 100 mmol/L NaOH-75 mmol/L CH3COONa, using a PAD detector. The recoveries of sucralose from various kinds of foods spiked at 50 micrograms/g and 200 micrograms/g ranged from 88-105%. The detection limit in samples was 10 micrograms/g for RI-HPLC and 1 microgram/g for PAD-IC.

An HPLC method was developed for the determination of neohesperidin dihydrochalcone (NHDC) in foods. A solid sample was extracted with methanol. The extract was evaporated and the residue was dissolved in methanol-water (2:8) mixture. In the case of fluid sample, the sample solution was prepared by dilution with methanol-water (2:8) mixture. The sample solution was cleaned up on a C18 cartridge. The cartridge was washed with water and methanol-water (3:7) mixture, and NHDC was eluted with methanol-water (7:3) mixture. NHDC in the eluate was separated on an ODS column and determined with a UV detector (282 nm). The identity of NHDC in foods was confirmed by means of HPLC with a photodiode-array detector. The recoveries of NHDC added to various kinds of foods were 75.2-134%. The determination limit of NHDC was 1 microgram/g.