[Simultaneous determination of ochratoxin A, B and citrinin in foods by HPLC-FL and LC/MS/MS].

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).

[Investigation of ochratoxin a, B and citrinin contamination in various commercial 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.

[Survey of glycoalkaloids content in the various potatoes].

The content of potato glycoalkaloid (PGA) was investigated in 27 cultivars of raw potatoes and 31 potatoes in commercial foods with peel. The investigation of the 27 cultivars of potatoes showed different contents of glycoalkaloids. "May queen" and "Sherry" showed high contents of PGA (180 mg/kg and 320 mg/kg, respectively) among the raw potatoes of middle size (ca. 100 g). On the other hand, "Inca red" showed the lowest content of 21 mg/kg. Higher contents of PGA were found in smaller potatoes in this study. The content of PGA varied in the range of 48-350 mg/kg in the potatoes in commercial foods with peel.

[Concentration in plasma and excretion in milk of lactating cows after oral administration of tribromsalan, oxyclozanide and bromofenofos].

The fasciolicides tribromsalan (TBS), oxyclozanide (OCZ) and bromofenofos (BFF) were orally administered to three lactating cows. The concentrations of TBS, OCZ and the BFF metabolite dephosphate bromofenofos (DBFF) in plasma, and the excretion of these compounds in milk were determined by high-performance liquid chromatography. In plasma, the concentrations of TBS, OCZ and DBFF reached maximum at about 1.0 day and the compounds remained detectable until 5.7, 7.4 and 15.1 days after administration, respectively. The detection limits of these compounds in plasma were 10, 2 and 2 ppb, respectively. In milk, the concentrations of TBS, OCZ and DBFF reached maximum at about 24 hours and the compounds remained detectable until 30-47, 30-47 and 78-119 hours after administration, respectively. The detection limits of these compounds in milk were 5.1 and 1 ppb, respectively. The residence times of TBS and BFF were very close to the withdrawal times of the fasciolicides.

[Example of examination of allergic substances (egg, milk) in commercial foods].

Allergic substances (eggs, milk) in commercial foods were measured by means of the notified ELISA methods using FASTKIT ELISA kit (N kit) and "Tokuteigenzairyo sokutei kit" (M kit). Some samples were also analyzed with the notified western blot method using "Tokuteigenzairyo western blot kit". In the methods for detection of eggs, proteins of fish egg and chicken muscle were examined using the ELISA and Western blotting methods to check for cross-reaction. Sujiko was false positive with the M kit. Difficulties were also encountered in the notified ELISA and Western blotting methods with raw chicken. In foods labeled as containing eggs, it was difficult to detected in the heart-processed food using both notified methods. In the method for detection of milk, foods containing thickening polysaccharides, showed cross-reaction with the N kit. However, this cross-reaction was eliminated when an improved N kit was used. Foods labeled as containing milk did not present any difficulty.

Investigation of false-positive reactions for CBH351 maize in screening PCR analysis.

Examination for CBH351 maize was conducted by the qualitative polymerase chain reaction (PCR) method in maize grain and maize processed foods obtained in the Tokyo area. The numbers of samples possibly positive in the screening test were 7 of 22 (31.8%) for maize grain samples, 4 of 14 (28.6%) for semi-processed foods, 11 of 30 (36.7%) for canned products, 3 of 30 (10.0%) for maize snacks, 3 of 4 (75%) for tacos and 1 of 3 (33.3%) for tortillas. However, CBH351 maize was not detected in the confirmation test. Therefore, the results of the screening test were false-positive. Since the reaction might have been caused by the base sequences of the 3'-end of primers CaM03-5' and CBH02-3' used in the screening test, a new primer pair was designed. The PCR products obtained with the new primer pair TMC2-5'--TMS2-3' were specific for CBH351 and were not obtained with barley, wheat, rice, RRS, Bt11, or Event176. Thus, the new primer pair shows high specificity. CBH351 maize was detected from samples containing at least 0.05% CBH 351 maize DNA by using this primer pair.

Modified determination method of total bromine in agricultural products by gas chromatography.

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.

Detection of genetically modified organisms in foreign-made processed foods containing corn and potato.

Investigations of the validity of labeling regarding genetically modified (GM) products were conducted using polymerase chain reaction (PCR) methods for foreign-made processed foods made from corn and potato purchased in the Tokyo area and in the USA. Several kinds of GM crops were detected in 12 of 32 samples of processed corn samples. More than two GM events for which safety reviews have been completed in Japan were simultaneously detected in 10 samples. GM events MON810 and Bt11 were most frequently detected in the samples by qualitative PCR methods. MON810 was detected in 11 of the 12 samples, and Bt11 was detected in 6 of the 12 samples. In addition, Roundup Ready soy was detected in one of the 12 samples. On the other hand, CBH351, for which the safety assessment was withdrawn in Japan, was not detected in any of the 12 samples. A trial quantitative analysis was performed on six of the GM maize qualitatively positive samples. The estimated amounts of GM maize in these samples ranged from 0.2 to 2.8%, except for one sample, which contained 24.1%. For this sample, the total amount found by event-specific quantitative analysis was 23.8%. Additionally, Roundup Ready soy was detected in one sample of 21 potato-processed foods, although GM potatoes were not detected in any sample.

Determination of thyroid hormones in pharmaceutical preparations, after derivatization with 9-anthroylnitrile, by high-performance liquid chromatography with fluorescence detection.

HPLC with fluorescence detection was used for the determination of low levels of liothyronine sodium and levothyroxine sodium in pharmaceutical preparations after fluorogenic derivatization. 9-Anthroylnitrile in dimethyl sulfoxide was used as a precolumn fluorogenic reagent. The 9-anthroylnitrile derivatives of liothyronine sodium and levothyroxine sodium were separated on a reversed-phase column with acetonitrile-0.02 M sodium dodecylsulfate (pH 3.5 with phosphoric acid) as the eluent. The calibration graphs were linear over a sample concentration range of 0.25-2.5 microg/ml. The detection limits for liothyronine sodium and levothyroxine sodium were 0.2 ng per injection. The proposed method was applied to the determination of thyroid hormones in pharmaceutical preparations.

[Microbiological method for the detection of antibiotic residues in meat using mixed-mode, reverse-phase and cation-exchange cartridge].

A microbiological method for screening of residual benzylpenicillin (PCG), oxytetracycline (OTC) and spiramycin (SPM) in meat using a single mixed mode, reversed-phase and cation-exchange cartridge was developed. A meat sample was extracted with 0.1 mol/mL pH 4.5 phosphate buffer and the extract was applied to a MCX cartridge. The cartridge was washed, and adsorbed antibiotic residues were eluted with acetonitrile for acidic fractions and acetonitrile containing 5% ammonia solution-0.1 mol/mL pH 4.5 phosphate buffer (9:1, v/v) for basic fractions. Each eluate was evaporated to dryness and the residue was dissolved in phosphate buffer to prepare test solutions for microbiological assay. When the diameter of the inhibition zone was more than 12 mm, the result was considered positive. In this method, the average recoveries of PCG at 0.05 microg/g, OTC at 0.1 microg/g and SPM at 0.2 microg/g were 70%, 92% and 84%, respectively. Tolerances of the three antibiotics were detected. All the results demonstrate that this method is simple, rapid and useful for screening of these three antibiotic residues in meat.

[Effect of coexisting metals on determination of lead and cadmium in the plastic materials test of the Japanese Food Sanitation Law].

The effect of coexisting metals in a sample on the determination of lead and cadmium in plastics used for food contact materials was investigated. In the official method specified in the Japanese Food Sanitation Law, contents of lead and cadmium are determined by a dry incineration method using sulfuric acid. It was assumed that sometimes, coexisting metals in a sample may form insoluble sulfate and that lead sulfate might be adsorbed into the insoluble sulfate. Therefore, hydrochloric acid was added to the ash, to turn formed insoluble sulfate into soluble compounds (HCl addition method). We found that recoveries of cadmium were not affected in the presence of other metals except when calcium exceeded 20 mg/g in both methods. Recoveries of lead decreased in the presence of barium exceeding 0.1 mg/g or calcium exceeding 10 mg/g in the official method. However, improvement of recoveries was achieved with the HCl addition method and by reducing the sample amount to one-tenth (0.1 g) of that specified in the official method.

Investigation of spectrophotometrically determined substances in Yucca extract by GC/MS, TLC and on-column injection GC.

Spectrophotometrically determined substances in Yucca extract, listed as "Yucca foam extract" in the "List of Existing Food Additives in Japan", were investigated by GC/MS, TLC and GC. A TLC method using an anisaldehyde color developing reagent similar to that employed in spectrophotometry was established for selective detection of sapogenins in Yucca extract. Several steroidal sapogenins were found by GC/MS in the fractions corresponding to spots on the TLC plate, and these were assumed to have contributed to the color development in spectrophotometry. Sarsasapogenin and smilagenin were the dominant sapogenins. An on-column injection GC method to determine these sapogenins in Yucca extract was also developed. The sum of these two sapogenins in Yucca extract was 0.9%. The total amount of sapogenin estimated by GC was approximately 2%, which was similar to that measured by spectrophotometry.

[Analysis of lac color in diets and feces of rats for toxicity studies].

An analytical method was developed for lac color in diets fed to rats and in the feces, and the contents of lac color were determined. After lac color was extracted with 0.05% sodium carbonate and 50% ethanol containing 0.02% sodium lauryl sulfate from the diets and feces, the extracted color solutions were analyzed by HPLC. The recoveries of lac color from diets spiked at 1.25, 5.00% and that from feces spiked at 5.00% were 85.6, 93.4% and 69.5%, respectively. Contents of lac color in diets prepared to contain 1.25 and 5.00% were 1.1 and 5.2%, and dose levels were confirmed by these results. Contents of lac color in feces of male and female rats given lac color were 127.8 mg/g and 138.6 mg/g, respectively. By comparing the HPLC chromatograms of laccaic acids in the diet with those in feces of rats, laccaic acid A, B, C and E were detected in both, and their content ratios were approximately determined.

Determination of low levels of methanol and ethanol in licorice extract by large volume injection head-space GC.

A large volume injection head-space GC method was established for measuring low levels of residual methanol and ethanol in licorice extract used a food additive. A vial was kept at 50 degrees C in the oven of the head-space sampler. Injection of the head-space gas for 0.75 min into a Poraplot Q GC column with a initial oven temperature of 35 degrees C, enabled the determination of low levels (5 micrograms/g) of methanol and ethanol. The standard deviations for five rounds of analysis of methanol and ethanol in licorice extracts were between 0.82 and 2.97. Methanol was found in 6 samples out of 9 collected in 1999, at concentrations exceeding 50 micrograms/g, the limit set by the Japanese Government, established in 1999 and coming into force on April 1, 2000. The highest concentration reached 10,000 micrograms/g. Methanol at a concentration exceeding 50 micrograms/g was found in 2 out of 9 samples collected in 2000. The highest concentration was 270 micrograms/g.

[Simulation of migration from internal can coatings].

Migration from can-coatings into retorted canned food simulants (canned oil and water, 121 degrees C, 30 min) was investigated through HPLC with a fluorescence detector and evaporative light scattering detector, and by measurements of residue on evaporation and consumption of potassium permanganate. HPLC analysis revealed that migration into the canned oil was hundreds of times more than that into n-heptane (25 degrees C, 60 min, the official test conditions according to the Japanese Food Sanitation Law), whereas it was similar to the migration into isooctane-butyl acetate mixtures (60 degrees C, 60 min), and that migration into the canned water was several times more than that into water (95 degrees C, 30 min, the official test conditions). Residue on evaporation for the n-heptane extract was several-fold lower than 30 ppm (the official limit), whereas that for the isooctane-butyl acetate mixtures exceeded 30 ppm. Consumption of potassium permanganate for the canned water was 30 times higher than that for the water extract (95 degrees C, 30 min). The official test conditions for can-coatings, in particular the use of n-heptane as an oil simulant, were suggested to lead to substantial underestimation of migration into canned food.

[Determination of isothiocyanates and related compounds in mustard extract and horseradish extract used as natural food additives].

Amounts of isothiocyanates and related compounds in a mustard extract and a horseradish extract for food additive use were determined by GC, after confirmation of the identity of GC peaks by GC/MS. Amounts of allyl isothiocyanate, which included that of allyl thiocyanate, because most of the allyl thiocyanate detected in the sample was assumed to have been formed from allyl isothiocyanate during GC analysis, were 97.6% and 85.4%, in the mustard extract and the horseradish extract, respectively. Total amounts of the identified isothiocyanates in the mustard extract and the horseradish extract were 98.5% and 95.4%, respectively. Allyl cyanide, a degradation product of allyl isothiocyanate, was found in the mustard extract and the horseradish extract at the levels of 0.57% and 1.73%, respectively. beta-Phenylethyl cyanide, a possible degradation product of beta-phenylethyl isothiocyanate, and allyl sulfides were found in the horseradish extract, at the levels of 0.13% and 0.46%, respectively. Allylamine, which is another degradation product of allyl isothiocyanate, was determined after acetylation, and was found in the mustard extract and the horseradish extract at the levels of 8 micrograms/g and 67 micrograms/g, respectively.

[Determination of dimethylformamide in food additive sucrose esters of fatty acids using solid-phase extraction].

A simple method using Florisil cartridges was developed for the determination of dimethylformamide (DMF) in sucrose esters of fatty acids present in sugar esters (SuE) and sucrose acetate isobutyrate (SAIB) used as food additives. SuE was dissolved in acetone and loaded on a Florisil cartridge. SAIB was dissolved in hexane, loaded on a Florisil cartridge and washed with 10% acetone in hexane. The columns were eluted with acetone and DMF in the eluates was determined by GC with an FID detector. Recoveries of DMF at the level of 0.5-100 micrograms/g were 93.3-102.6%. The determination limit was 0.5 microgram/g.

[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.

C-terminal modification of monoclonal antibody drugs: amidated species as a general product-related substance.

Twelve therapeutic mAbs, comprising 10 IgG1s and 2 IgG4s, were analyzed by a peptide mapping technique to detect and quantify C-terminal modifications. C-terminal amidated structures were found in 8 out of the 12 mAbs. An in vitro study using a commercially available peptidylglycine alpha-amidating monooxygenase (PAM) revealed that both IgG1 and IgG4 can be substrates for PAM. This study showed that C-terminal amidation is a general C-terminal modification on the heavy chains of therapeutic mAbs and that C-terminal amidation of mAbs can be catalyzed by a certain PAM(s) in the Chinese hamster ovary (CHO) cells that are widely used for manufacturing therapeutic mAbs.