[Reduction of Orange Allergen Cit s 2 Levels in Fresh Orange Juice with Pineapple Bromelain Enzymatic Treatment].

Oranges are consumed worldwide; however, they contain Cit s 2, a major profilin allergen. We aimed to reduce Cit s 2 levels by preparing mixed orange fresh juice with pineapple, as a convenient method for any kitchen. Cit s 2 levels in orange extracts digested with pineapple extract and its protease bromelain were evaluated with quantitative enzyme-linked immunosorbent assay. Cit s 2 levels decreased according to reaction temperature and time, which was inhibited by iodoacetic acid. Treatment with pineapple extract diluted 40-fold and 0.1 mg/mL of bromelain at 37℃ for 30 min contributed to reducing residual Cit s 2 levels below the cut-off of 15%, respectively. Since this condition can increase the proportion of orange juice and reduce the risk of ingesting the pineapple allergen bromelain, it is considered to be more practical. Broad utilization of proteases in hypoallergenic food products is expected following clinical studies for verification.

Problems and solutions of polyethylene glycol co-injection method in multiresidue pesticide analysis by gas chromatography-mass spectrometry: evaluation of instability phenomenon in type II pyrethroids and its suppression by novel analyte protectants.

Polyethylene glycol 300 is commonly used as a base material for "analyte protection" in multiresidue pesticide analysis via gas chromatography-mass spectrometry. However, the disadvantage of the co-injection method using polyethylene glycol 300 is that it causes peak instability in α-cyano pyrethroids (type II pyrethroids) such as fluvalinate. In this study, we confirmed the instability phenomenon in type II pyrethroids and developed novel analyte protectants for acetone/n-hexane mixture solution to suppress the phenomenon. Our findings revealed that among the examined additive compounds, three lipophilic ascorbic acid derivatives, 3-O-ethyl-L-ascorbic acid, 6-O-palmitoyl-L-ascorbic acid, and 6-O-stearoyl-L-ascorbic acid, could effectively stabilize the type II pyrethroids in the presence of polyethylene glycol 300. A mixture of the three ascorbic acid derivatives and polyethylene glycol 300 proved to be an effective analyte protectant for multiresidue pesticide analysis. Further, we designed and evaluated a new combination of analyte protectant compounds without using polyethylene glycol or the troublesome hydrophilic compounds. Consequently, we obtained a set of 10 medium- and long-chain saturated fatty acids as an effective analyte protectant suitable for acetone/n-hexane solution that did not cause peak instability in type II pyrethroids. These analyte protectants will be useful in multiresidue pesticide analysis by gas chromatography-mass spectrometry in terms of ruggedness and reliable quantitativeness. Graphical abstract Comparison of effectiveness of the addition of lipophilic derivatives of ascorbic acid in controlling the instability phenomenon of fluvalinate with polyethylene glycol 300.

Persistent organic pollutants in red-crowned cranes (Grus japonensis) from Hokkaido, Japan.

The red-crowned crane (Grus japonensis) from eastern Hokkaido is classified as a Special Natural Monument in Japan. In this study, we determined the concentrations of persistent organic pollutants (POPs) in red-crowned crane muscle tissues (n = 47). Polychlorinated biphenyls (PCBs) had the highest median concentration (240ng/g lipid weight), followed by dichlorodiphenyltrichloroethane and its metabolites (DDTs) (150ng/g lipid weight), chlordane-related compounds (CHLs) (36ng/g lipid weight), hexachlorobenzene (HCB) (16ng/g lipid weight), hexachlorocyclohexanes (HCHs) (4.4ng/g lipid weight), polybrominated diphenyl ethers (PBDEs) (1.8ng/g lipid weight), and finally, Mirex (1.5ng/g lipid weight). Additionally, a positive correlation was found among POP concentrations. No sex differences beyond body parameters were observed. Additionally, red-crowned cranes exhibited a high enantiomeric excess of (+)-alpha-HCH, with enantiomer fractions varying from 0.51 to 0.87 (average: 0.69).

[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."

Size Distribution of Chlorinated Polycyclic Aromatic Hydrocarbons in Atmospheric Particles.

The particle size distribution of chlorinated polycyclic aromatic hydrocarbons (ClPAHs) in particulate matter (PM) in Japan is examined for the first time. PM was collected using a PM0.1 air sampler with a six-stage filter. PM was collected in October 2014 and January 2015 to observe potential seasonal variation in the atmospheric behavior and size of PM, including polycyclic aromatic hydrocarbons (PAHs) and ClPAHs. We found that the concentration of PAHs and ClPAHs between 0.5-1.0 µm and 1.0-2.5 µm markedly increase in January (i.e., the winter season). Among the ClPAHs, 1-ClPyrene and 6-ClBenzo[a]Pyrene were the most commonly occurring compounds; further, approximately 15% of ClPAHs were in the nanoparticle phase (<0.1 µm). The relatively high presence of nanoparticles is a potential human health concern because these particles can easily be deposited in the lung periphery. Lastly, we evaluated the aryl hydrocarbon receptor (AhR) ligand activity of PM extracts in each size fraction. The result indicates that PM < 2.5 µm has the strong AhR ligand activity.

Detection of antibiotics in chicken eggs obtained from supermarkets in Ho Chi Minh City, Vietnam.

The residual levels of antibiotics in Vietnamese eggs were monitored from 2014 to 2015. A total of 111 egg packages, distributed by 11 different companies, were collected from supermarkets in Ho Chi Minh City and the levels of 28 antibiotics were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method. Sixteen samples tested positive for antibiotics; a total of eight compounds (enrofloxacin, ciprofloxacin, norfloxacin, sulfadimethoxine, sulfamethazine, sulfamonomethoxine, tilmicosin and trimethoprim) were detected. Enrofloxacin was detected in eight samples, with two samples exhibiting concentrations exceeding 1,000 µg kg-1. Tilmicosin was detected in three samples at a range of 49-568 µg kg-1. We observed that two of the 11 companies frequently sold antibiotic-contaminated eggs (detection rates of 56 and 60%), suggesting that a number of companies do not regulate the use of antibiotics in egg-laying hens. Our findings indicate that livestock farmers require instruction regarding antibiotic use and that continual antibiotic monitoring is essential in Vietnam.

Identification and characterization of oxidative metabolites of 1-chloropyrene.

Polycyclic aromatic hydrocarbons (PAHs) and chlorinated PAHs (ClPAHs) are ubiquitous contaminants that bind to the aryl hydrocarbon receptor (AhR) and exhibit mutagenic potential. It is difficult to monitor human exposure levels to ClPAHs because the exposure routes are complicated, and environmental concentrations are not always correlated with the levels of PAHs. Urinary PAH metabolites are useful biomarkers for evaluating PAH exposure, and ClPAH metabolites may therefore contribute to the estimation of ClPAH exposure. One of the most abundant ClPAHs present in the environment is 1-chloropyrene (ClPyr), and urinary ClPyr metabolites have the potential to be good biomarkers to evaluate the level of exposure to ClPAHs. Since the metabolic pathways involving ClPAHs are still undetermined, we investigated the effect of human cytochrome P450 enzymes on ClPyr and identified three oxidative metabolites by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance. We found that ClPyr was metabolized most efficiently by the P450 1A1 enzyme, followed by the 1B1 and 1A2 enzymes. Similar to ClPyr, these metabolites were shown to have agonist activity for the human AhR. We detected these metabolites when ClPyr reacted with a pooled human liver S9 fraction as well as in human urine samples. These results suggest that the metabolites may be used as biomarkers to evaluate the extent of exposure to ClPAHs.

Benzotriazole ultraviolet stabilizers show potent activities as human aryl hydrocarbon receptor ligands.

Benzotriazole ultraviolet stabilizers (BUVSs) used in consumer products are raising concerns as new pollutants in the aquatic environment. We determined the agonistic activities of eight BUVSs and a chemically distinct UV absorber (4-methylbenzylidinecamphor) toward the human aryl hydrocarbon receptor (AhR) and thyroid hormone receptors alpha and beta. Although none of the BUVSs showed ligand activity against the thyroid hormone receptors, four of them (UV-P, UV-9, UV-326, and UV-090) showed significant AhR ligand activity. Their half-maximal effective concentrations (EC50) were 130 nM for UV-P, 460 nM for UV-9, and 5.1 µM for UV-090 (a value for UV-326 could not be determined). Of the numerous AhR ligands, it is well-known that those considered nontoxic are quickly metabolized by enzymes such as CYP1A1, which destroys their ability to function as ligands. Accordingly, we established a new yeast assay for simultaneous monitoring of both the strength of AhR ligand activity and ligand degradation by CYP1A1. We found the AhR ligand activities of the above four BUVSs to be stable in the presence of CYP1A1; therefore, they have the potential to accumulate and exert potent physiological effects in humans, analogous to polycyclic aromatic hydrocarbons and dioxins, which are known stable and toxic ligands.

Inhalation and dietary exposure to Dechlorane Plus and polybrominated diphenyl ethers in Osaka, Japan.

This study estimated daily exposure to Dechlorane Plus (DP) and polybrominated diphenyl ethers (PBDE) via inhalation and diet. Samples of atmospheric particles and food (obtained by market basket method) from Osaka, Japan were analyzed for DP (syn-, anti-) and PBDE using gas chromatography-mass spectrometry. DP was detected in both atmospheric particles and food samples. Among the atmospheric particles, DP was detected in all samples. ΣDP concentration was 7.1-15.4 pg m(-3) and anti-DP was the dominant residue among DP isomers. PBDE was also detected in all the atmospheric particles. ΣPBDE concentration was 9.9-23.3 pg m(-3). In the market basket study, DP was detected in Groups Ш (sugar and confectionary), V (legumes and their products), X (fish, shellfish, and their products), and XI (meat and eggs) at concentrations of 3.3, 2.8, 1.9, and 1.5 pg g(-1) wet wt, respectively. PBDE was detected in Groups Ш, IV (oils and fats), V, X, XI, and XШ (seasonings and other processed foods) at concentrations of 153, 79.1, 74.6, 308, 94.8, and 186 pg g(-1) wet wt, respectively. The daily intake of ΣDP (750 pg day(-1)) via inhalation and diet was approximately one percent of that for ΣPBDE (62 ng day(-1)).

[Allergy due to probable contamination of bread used for school meals with milk].

BACKGROUND: In May 2012, two schoolchildren developed allergic symptoms after eating a school meal of fried bread in Osaka, Japan. One specific raw material, milk, should not be present in fried bread. However, we suspected the unintentional mixing of milk in the bread manufacturing process. Our aim was to verify the reason if this was so. METHODS: We first manufactured bread that contained milk as one of its components. We then thoroughly cleaned the manufacturing line except for the dough divider and, as per the revised instruction manual, continuously manufactured bread that did not contain milk. In this manner, we tried to simulate the conditions at the time of the incident. Casein is a major milk protein. We, therefore, determined casein concentration in the milk-free bread, using casein enzyme-linked immunosorbent assay. RESULTS: We observed casein levels higher than 1000 ppm in the initial lots of breads, but lower levels in the later lots. Casein levels also decreased upon frying of bread. CONCLUSION: High casein levels, which were observed in the earlier lots of breads, were lower in the later lots. In the manufacturing line, the remnants of the bread dough that contained milk presumably got mixed with the dough used in the subsequent manufacture of milk-free bread.

Characterization of the decomposition of compounds derived from imidazolidinyl urea in cosmetics and patch test materials.

BACKGROUND: Imidazolidinyl urea releases formaldehyde through decomposition. However, there have been few reports on the chemistry of imidazolidinyl urea in cosmetics. OBJECTIVES: The aim of this study was to characterize imidazolidinyl urea-derived compounds in cosmetics and to determine which compounds are responsible for the cross-reactivity with diazolidinyl urea. METHODS: We analysed imidazolidinyl urea dissolved in aqueous solutions, imidazolidinyl urea patch test materials and imidazolidinyl urea-preserved cosmetics by high-performance liquid chromatography/photodiode array detection and liquid chromatography/mass spectrometry. The results were compared with those obtained with a diazolidinyl urea aqueous solution. RESULTS: In the analysed cosmetic samples and patch test materials, imidazolidinyl urea was primarily composed of allantoin, (4-hydroxymethyl-2,5-dioxo-imidazolidine-4-yl)-urea (HU), (3,4-bis-hydroxymethyl-2,5-dioxo-imidazolidine-4-yl)-urea (3,4-BHU), and (3-hydroxymethyl-2,5-dioxo-imidazolidine-4-yl)-urea. CONCLUSIONS: Two of the imidazolidinyl urea-derived major decomposition compounds - HU and 3,4-BHU - are common in the diazolidinyl urea-decomposed compound present in cosmetics. These compounds are possible causative agents of the cross-reactivity between diazolidinyl urea and imidazolidinyl urea.

The different decomposition properties of diazolidinyl urea in cosmetics and patch test materials.

BACKGROUND: Diazolidinyl urea is a formaldehyde-releasing compound that releases formaldehyde through its decomposition. However, there have been few reports about the decomposition properties of diazolidinyl urea in cosmetics and patch test materials. OBJECTIVES: The aim of this study was to show how diazolidinyl urea decomposes in cosmetics and patch test vehicles, and to determine which cosmetic compounds should be evaluated in patch test studies of diazolidinyl urea. METHOD: We fractionated diazolidinyl urea-dissolving buffers or diazolidinyl urea-containing cosmetics with high-performance liquid chromatography-photodiode array detector (HPLC-PDA), and characterized them in liquid chromatography-mass spectrometry (LC-MS) and (1) H-nuclear magnetic resonance studies. Diazolidinyl urea-containing cosmetics and diazolidinyl urea patch test materials were also analysed with HPLC-PDA and LC-MS. RESULTS: Diazolidinyl urea was decomposed to (4-hydroxymethyl-2,5-dioxo-imidazolidine-4-yl)-urea (HU) and (3,4-bis-hydroxymethyl-2,5-dioxo-imidazolidine-4-yl)-urea (3,4-BHU) in most of the cosmetic samples tested. The peak patterns of the patch test materials analysed with the HPLC-PDA were different from those of the cosmetic samples. CONCLUSIONS. The diazolidinyl urea-derived decomposition products differed between the cosmetics and patch test preparations. To test the contact sensitivity of the diazolidinyl urea present in cosmetics, patch tests with HU and 3,4-BHU in petrolatum should be performed.

Development of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of hen's egg white allergens Gal d 1-4 in fresh and processed eggs.

Hen's egg white allergens, namely Gal d 1-4, cause food allergies worldwide and their intake must be strictly controlled by allergic individuals. However, an efficient method for quantifying these allergens is currently unavailable. We aimed to develop an LC-MS/MS method for simultaneous Gal d 1-4 quantification. Purified Gal d 1-4 proteins were trypsin-digested and the resulting peptides used in LC-MS/MS analysis. The limits of quantification were 9.77-39.1 ng/mL. The Gal d 1-4 recovery in fresh and processed eggs was 68.3-121.3%, and intra- and interassay coefficients of variation were 1.5-15.7% and 2.4-38.1%, respectively, indicating high sensitivity, accuracy, and reproducibility. In addition, the high specificity of this method was confirmed by testing 27 other foods. This newly developed method could provide reliable information to the industrial food and clinical fields, facilitating improved quality of life for individuals with egg allergies.

Determination of pyrethroid pesticides in cinnamomi cortex.

In Japan, maximum residue levels (MRL) have been set for eight pesticides (alpha-BHC, beta-BHC, gamma-BHC, delta-BHC (BHCs), p,p'-DDE, o,p'-DDT, p,p'-DDD, p,p'-DDT (DDTs)) in 14 crude drugs (below 0.2 ppm as total of BHCs, below 0.2 ppm as total of DDTs). There are fears that pesticides present in crude drugs for which MRL are set will be changed from BHCs and DDTs to other pesticides with MRL setting as the turning point. There are few surveys of pyrethroid pesticide in crude drugs distributed in Japan. The actual situation of pyrethroid pesticides in crude drugs distributed in Japan after setting MRL is not unclear and should be clarified. Although a method to analyze permethrin, cypermethrin and fenvalerate in 11 crude drugs was reported, it is not adequate because the recovery rates of permethrin, cypermethrin and fenvalerate from Cinnamomi cortex were very low and the method, including liquid-liquid partition is difficult. In this study, we developed a method using solid-phase extraction to analyze permethrin, cypermethrin and fenvalerate in Cinnamomi cortex with acceptable recovery rates. The sample solution was determined by gas chromatography/mass spectrometry with negative chemical ionization. The recovery rates of permethrin, cypermethrin and fenvalerate from Cinnamomi cortex were between 87.9 and 90.7%. Five samples of Cinnamomi cortex were analyzed according to the proposed method. No samples contained permethrin, cypermethrin and fenvalerate over detection limits. The proposed method could analyze permethrin, cypermethrin and fenvalerate in all crude drugs for which MRL are set.

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.

Concentrations of total mercury and methylmercury in red snow crabs (Chionoecetes japonicus) caught off the coast of Japan.

The total mercury (T-Hg) and methylmercury (MeHg) concentrations in red snow crabs (Chionoecetes japonicus) caught off the coast of Japan were analyzed. The T-Hg concentration ranged from 0.03 to 0.56 mg/kg (mean: 0.21 mg/kg) in the raw muscle, and 0.02 to 0.74 mg/kg (mean: 0.27 mg/kg) in the boiled muscle. The MeHg concentration ranged from 0.04 to 0.54 mg/kg (mean: 0.20 mg/kg) in the raw muscle. The mean ratio of MeHg to T-Hg was 0.88. The crab body weight was found to significantly correlate with the concentrations of T-Hg (r = 0.488) and MeHg (r = 0.490) (p ≤ 0.01). For the general population in Japan, the intake of MeHg from eating red snow crab was estimated to be lower than 0.013 mg/week, which was less than one-sixth of the tolerable MeHg intake (0.08 mg/week).

Determination of the human cytochrome P450 monooxygenase catalyzing the enantioselective oxidation of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183).

2,2',3,5',6-Pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183) possess axial chirality and form the aS and aR enantiomers. The enantiomers of these congeners have been reported to accumulate in the human body enantioselectively via unknown mechanisms. In this study, we determined the cytochrome P450 (CYP) monooxygenase responsible for the enantioselective oxidization of PCB 95 and PCB 183, using a recombinant human CYP monooxygenase. We evaluated 13 CYP monooxygenases, namely CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4F2, and aromatase (CYP19), and revealed that CYP2A6 preferably oxidizes aS-PCB 95 enantioselectively; however, it did not oxidize PCB 183. The enantiomer composition was elevated from 0.5 (racemate) to 0.54. In addition, following incubation with CYP2A6, the enantiomer fraction (EF) of PCB 95 demonstrated a time-dependent increase.

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.

PCR-Based Method for the Detection of Toxic Mushrooms Causing Food-Poisoning Incidents.

In this study, species-specific identification of five toxic mushrooms, Chlorophyllum molybdites, Gymnopilus junonius, Hypholoma fasciculare, Pleurocybella porrigens, and Tricholoma ustale, which have been involved in food-poisoning incidents in Japan, was investigated. Specific primer pairs targeting internal transcribed spacer (ITS) regions were designed for PCR detection. The specific amplicons were obtained from fresh, cooked, and simulated gastric fluid (SGF)-treated samples. No amplicons were detected from other mushrooms with similar morphology. Our method using one-step extraction of mushrooms allows rapid detection within 2.5 hr. It could be utilized for rapid identification or screening of toxic mushrooms.