Determination of nine preservatives in processed foods using a modified QuEChERS extraction and quantified by HPLC-PDA.

In this study, a new method was developed for simultaneously determining nine preservatives, that is, benzoic acid (BA), sorbic acid (SOA), dehydroacetic acid (DHA) and PHBAs (methyl p-hydroxybenzoate [PHBA-me], ethyl p-hydroxybenzoate [PHBA-et], isopropyl p-hydroxybenzoate [PHBA-ipro], propyl p-hydroxybenzoate [PHBA-npro]), isobutyl p-hydroxybenzoate [PHBA-ibut] and butyl p-hydroxybenzoate [PHBA-nbut]), in processed foods, employing liquid chromatography (LC). This procedure accelerated sample preparation and improved efficiency by employing modified quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction without clean-up. Samples were prepared with 20 mL of acetonitrile/water (1:1) with the assistance of a ceramic stone. The extract solutions were diluted 10 times or according to the detection amount and then injected into an LC-PDA. This method showed good linearity, and the LOQs were 10 mg/kg for BA, SOA and DHA and 5 mg/kg for the PHBAs. When validating this method, the recoveries of the nine preservatives were in the range 77.0-99.6%, RSDr values were in the range 0.7-5.3% and those of RSDwr were in the range 2.3-8.4%. These results suggest that this new method is highly reproducible.

Development of a highly sensitive liquid chromatography with tandem mass spectrometry method for the qualitative and quantitative analysis of high-intensity sweeteners in processed foods.

A new method for the simultaneous determination of two sweeteners (Advantame and Neotame) in processed foods using liquid chromatography (LC) with tandem mass spectrometry(MS/MS) was developed herein. Chromatographic separations were performed using an ACQUITY UPLC CSH C18 column at 40 °C via a mobile phase comprising 10-mmol/L ammonium formate and methanol. Samples were prepared via rapid dialysis using 30% methanol solution in a thermostatic shaker set at 160 rpm and 50 °C for 1 h. The matrix in the test solution had no effect on the identification and quantification of the compound without a clean-up step using solid-phase extraction (SPE). This method satisfied all validation criteria with a limit of quantification (LOQ) of 0.01 µg/g for all samples. Using this method, the amounts of Advantame and Neotame in 24 processed foods were subsequently investigated, with the results indicating their detection beyond the lower LOQ. Moreover, a multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) method was developed and described to further enhance product-identification ability.

[Determination of Aspartame, Acesulfame Potassium, and Sucralose in Chewing Gum by Dialysis Extraction].

The three sweeteners, aspartame, acesulfame potassium, and sucralose, in chewing gum were determined by using dialysis and direct extraction methods. The results revealed that the previously reported dialysis method tended to show poor extraction of aspartame in comparison with the direct-extraction method. The direct extraction also caused operational problems, such as the gum base adhering to the instruments. Therefore, we attempted to improve the dialysis method by changing the dialysate, to which the three sweeteners were extracted while the sample stayed inside the dialysis tube. By changing the dialysate to 60% methanol and dialyzing for 24 hr at room temperature or 2 hr with shaking while heating at 50℃, all three sweeteners were extracted as good as those with the direct-extraction method.

[A Screening Analysis Method for Stevia Sweetener in Processed Foods by LC-MS/MS].

A LC-MS/MS-based screening method was developed for stevia sweetener in processed foods. After extraction of stevia sweetener from processed foods by dialysis, the dialysate was diluted with water, and stevia sweetener was measured by LC-MS/MS. Recovery from 5 kinds of processed foods spiked with 10 mg/kg of stevioside （SS）, 10 mg/kg of rebaudioside A （RS）, or 100 mg/kg of α-glu-cosyltransferase-treated stevia （Gts） product was excellent, and no interfering peak was observed. Thirty-six commercial processed foods indicated as containing "stevia" were analyzed using this established method. Among them, 33 contained SS, 33 contained RS, and 11 contained Gts. Five products contained both stevia extract and Gts.

[A Rapid and Simple Method for Detection of Colchicum autumnale Using PCR].

Colchicum autumnale is a perennial, toxic plant that originated in Europe and North Africa. Although inedible, it is occasionally consumed accidentally because it resembles the edible Allium victorialis and other related species. This misidentification has led to episodes of food poisoning in Japan. However, determining the causative agent of a food poisoning outbreak by observing the sample visually or analyzing the chemical composition is challenging when dealing with small samples. Therefore, we developed a novel set of PCR primers that anneal to the internal transcribed spacer (ITS) region of C. autumnale ribosomal DNA, designed to detect the presence of C. autumnale in small samples. These primers successfully detected C. autumnale in all samples in which it was present, and did not give a positive PCR band in the 48 other distinct crop species tested, in which it was not present. Further, our method could amplify DNA from samples of C. autumnale that had been heat-treated and digested using artificial gastric fluids. Thus, this PCR strategy is highly specific and can be used to distinguish C. autumnale simply and rapidly from various other crops.

[Method of Quantitative Analysis by HPLC and Confirmation by LC-MS/MS of Erythritol, Maltitol, Lactitol and Trehalose in Foods].

A quantitative determination method of erythritol, maltitol, lactitol and trehalose in foods by HPLC, and confirmation method by LC-MS/MS were developed. HPLC analysis was performed on a separation column packed with amino group-binding polymer with acetonitrile-water (80 : 20) as the mobile phase. The column was operated at room temperature, and the three sugar alcohols and trehalose were quantified. LC-MS/MS confirmation was performed on an amino group-bound column with acetonitrile-ammonium acetate solution as the mobile phase, with detection in the SRM mode. At low sample dilution ratios, the analysis may be affected by matrix derived from the sample, but this can be suppressed by 1,000-fold or greater dilution. Recoveries of the three sugar alcohols and trehalose spiked into food samples, such as tea, jelly, tablets (ramune candy), and chocolate, exceeded 90% (CV≦6.1%) in HPLC and 94% (CV≦4.8%) in LC-MS/MS.

[Quantitative Analysis of L-Ascorbic Acid and Erythorbic Acid in Foods by HPLC, and Confirmation Method by LC-MS/MS].

Analysis of L-ascorbic acid (AsA) and erythorbic acid (ErA) in foods is generally performed by HPLC measurement after extraction with metaphosphoric acid solution. But this method can not always measure the concentrations of AsA and ErA precisely due to the presence of interfering compounds, and the reproducibility of retention time is poor. We considered that quantitative analysis by HPLC and confirmation by LC-MS/MS using an identical extraction solvent might be an effective approach for AsA and ErA analysis. Chelate fiber was added to the sample, followed by extraction with acetic acid solution containing ethylenediaminetetraacetic acid disodium salt, purification with Oasis MCX, and 2-fold dilution with methanol. The resulting solution was used for quantification by HPLC using a ZIC-HILIC column and identification by LC-MS/MS. In recovery tests with 8 kinds of foods, the recovery of AsA was over 91%, and that of ErA was over 88%. The RSD was 5.1% or less for both analytes. Analysis of 8 kinds of foods by both methods showed that this method gave better RSD values than the conventional method. AsA and ErA in all samples were confirmed by product ion scanning and selected reaction monitoring of LC-MS/MS.

[Determination of Nitrite in Meat and Fish Products using Dialysis Extraction].

A simple and useful method for the determination of nitrite in meat and fish products was developed. The sample (2.5 g) was extracted and cleaned up by dialysis in tris hydroxymethyl aminomethane solution with shaking at 80℃ for 2.5 hr. Nitrite in the dialysate was quantified by colorimetric analysis. Furthermore, the dialysate was cleaned up with SPE under cooling, and nitrite in the resulting solution was determined using LC-UV with an anion exchange column for confirmation. The recoveries of nitrite from frankfurter and fish sausage, fortified at the levels of 0.002 g/kg and the maximum usage dose (0.070 g/kg for meat products, 0.050 g/kg for fish sausage) ranged from 82.6 to 104.8% in colorimetric analysis and from 88.3 to 97.6% in LC-UV confirmation analysis. The values determined in various meat and fish products by the developed method and by the Japanese official method were approximately equivalent.

[Method of Quantitative Analysis Using Enzymatic Hydrolysis of α-Glucosyltransferase-Treated Stevia in Foods].

A quantitative analysis by HPLC of α-glucosyltransferase-treated stevia in foods was considered. This analysis is the way which hydrolyzed α-glucosyltransferase-treated stevia in the stevioside (SS) and the rebaudioside A (RS) using a glucoamylase. Recovery (%) of α-glucosyltransferase-treated stevia, spiked at 200 mg/kg in various foods, were more than 80% and the relative standard deviations were less than 5.0% as SS and RS for the rate of collection. A qualitative analysis by LC-MS/MS was performed 36 products of commercial foods containing stevia. We quantified of 11 products in which α-glucosyltransferase-treated stevia was detected. Quantitative value was at most 180 mg/kg as SS, at most 70 mg/kg as RS.

[Analysis of Acidic Tar Dyes in High-Protein Foods: Achievement of Improved Recoveries and Shortened Operation Time by Loading Sample Extract onto Polyamide Columns at pH 8.5].

The recoveries of xanthene dyes in the analysis of acidic tar-dyes in high-protein foods were improved by loading them onto polyamide columns at pH 8.5, instead of using the conventional pH 3-4 solution. The experimental scale was reduced to approximately half that of the conventional method. Furthermore, instead of eliminating the organic solvent in the extract by evaporation, the extract was diluted with water prior to PA column cleanup in order to reduce the ratio of organic solvent so that acidic tar-dyes would be better retained on the column. The above two procedures shortened the operation time and allowed for a simpler protocol. With this method, the recoveries of erythrosine, phloxine, and rose bengal from salted cod roe were 82, 88, and 74%, respectively. The recovery percentages were greatly improved compared to those achieved by conventional column loading at pH 3.5 (26, 44, and 18%, respectively). The recoveries of azo-dyes (Amaranth, New Coccine, Allura Red AC, Tartrazine, Sunset Yellow FCF) were also improved from 41-66 to 79-99%.

[Survey of Aluminium Content of Processed Food Using Baking Powder (2015-2016)].

The aluminium (Al) content of Japanese confectionery and foods containing flour was investigated. Some of these items were investigated in previous studies, which examined foods that made use of baking powder containing aluminium potassium sulfate (Alum). Al was detected in 41 of the 123 samples at levels ranging from 0.01 (limit of quantitation) to 0.40 mg/g. The detection rate of Al in almost all confectionery (except Japanese confectionery) was decreased as compared with previous studies. However, the detection rate of Al in Japanese confectionery and foods containing flour was high. For 4 of the 41 samples tested, consuming one serving once a week would result in an Al intake exceeding the PTWI for young children (body weight=16 kg).

A Rapid Dialysis Method for Analysis of Artificial Sweeteners in Foods (2nd Report).

Following the previous report, a rapid dialysis method was developed for the extraction and purification of four artificial sweeteners, namely, sodium saccharide (Sa), acesulfame potassium (AK), aspartame (APM), and dulcin (Du), which are present in various foods. The method was evaluated by the addition of 0.02 g/kg of these sweeteners to a cookie sample, in the same manner as in the previous report. Revisions from the previous method were: reduction of the total dialysis volume from 200 to 100 mL, change of tube length from 55 to 50 cm, change of dialysate from 0.01 mol/L hydrochloric aqueous solution containing 10% sodium chloride to 30% methanol solution, and change of dialysis conditions from ambient temperature with occasional shaking to 50℃ with shaking at 160 rpm. As a result of these revisions, the recovery reached 99.3-103.8% with one hour dialysis. The obtained recovery yields were comparable to the recovery yields in the previous method with four hour dialysis.

Analyses of Acidic Tar Dyes in High-Protein Foods and Examination of Extraction and Clean-Up Methods for Various Foods.

Extraction and clean-up methods were examined for the analysis of acidic tar dyes in various high-protein foods. 1% Aqueous ammonia followed by ethanol, 1% aqueous ammonia-ethanol (1 : 1) mixture, and 1% aqueous ammonia-tetrahydrofuran (1 : 1) mixture were used in sequence for boiled fish paste (kamaboko), pounded fish cake (hanpen), and sausage. The sausage extract was centrifuged at low temperature to solidify and remove the contained fat. Salted cod roe with red pepper was extracted twice with 1% aqueous ammonia-ethanol (1 : 1) mixture, followed by extraction with 1% aqueous ammonia-tetrahydrofuran (1 : 1) mixture. A divinylbenzene-N-vinylpyrrolidone copolymer column was used for the clean-up of xanthen dyes. In the case of clogging-prone samples, the same type of large-particle-size column was used. A polyamide column was used for clean-up of the other dyes. When each dye was added at 5 µg/g in the foods, recoveries from kamaboko, hanpen, and sausage ranged from 76 to 102%, and the average recovery from the two types of salted cold roe with red pepper ranged from 45 to 98%.

Survey on Metals Contained in Stainless Steel Kitchenware and Tableware.

Stainless steel kitchenware and tableware on sale in Japan were investigated. Surface elemental composition ratios of 172 samples were analyzed by the fluorescence X-ray method. High levels of manganese (9.59-20.03%)were detected in 17 samples. This finding was confirmed by ICP analysis. Next, we conducted migration tests. Samples conformed to the Italian Specific Migration Limits. Moreover, lead and antimony were not detected in these samples, in accordance with the Japanese Food Sanitation Law.

Structures of Two Yellow Dyes Found in Cucumbers Pickled in Soy Sauce Colored with Tartrazine (Y4).

Two yellow dyes, together with tartrazine (Y4), were found in cucumbers pickled in soy sauce, for which the use of tartrazine is permitted, by TLC, LC-DAD, and LC-MS. The retention times on LC chromatograms and the maximum absorbance wavelengths measured by LC-DAD of the two dyes were different from those of tartrazine. Mass spectra of the dyes indicated that these dyes lacked one sulfonyl group of tartrazine. The presence of two less sulfonated dyes in tartrazine has been reported. Hence, the two less sulfonated dyes were synthesized. The two dyes found in cucumbers were compared with the synthesized dyes by LC-DAD and LC-MS. Since the retention times of the dyes in cucumbers on the LC chromatograms, as well as their LC-DAD spectra and mass spectra, were found to be identical with those of the synthesized dyes, we concluded these dyes are the less sulfonated subsidiary dyes of tartrazine.

Simultaneous Analysis of Oil-Soluble, Basic, and Acidic Illegal Dyes in Foods Using Liquid Chromatography-Diode-Array Detection.

A method for simutaneously detecting 8 oil-soluble and 10 water-soluble (3 basic and 7 acidic) illegal dyes in foods was developed. The sample was mixed with water, followed by methanol and tetrahydrofuran. Transesterification with sodium methoxide was applied to the mixture, which allowed the triglycerides in the sample to be converted to fatty acid methyl esters. This treatment resulted in a biphasic mixture. Oil-soluble dyes and fatty acid methyl esters were deposited in the upper organic phase, which was cleaned using a silica-gel solid-phase extraction (SPE) column to remove the fatty acid methyl esters from the solution. The water-soluble dyes were deposited in the aqueous phase, and an Oasis hydrophilic-lipophilic-balanced SPE column was used to remove polar matrix components from the solution. The resulting dyes were subsequently analyzed via LC-diode-array detection using a single method. The practical LODs of the samples were defined as the lowest spiked dye concentrations at which the similarity coefficient for the spectra of the LC test solution and the corresponding reference standard solution were greater than 0.99, thus affording LODs of 0.5-1.0 µg/g. Recoveries of the dyes at a spiking level of 5.0 µg/g from soft drink, chili sauce, and mustard were generally greater than 70%. Recoveries from paprika powder were between 33 and 103%.

Analysis of Acidic Tar Dyes in High Protein Food: Effect of pH of Column Loading Solution for Clean-Up.

The effect of pH of the clean-up process in the analysis of 11 permitted tar dyes in high protein food was investigated by using a handmade polyamide column (PA column) and Oasis HLB. Boiled fish paste spiked with the 11 dyes was extracted with appropriate solvents and the pH of the extract was adjusted to 3.0-7.0 in increments of 0.5, followed by clean-up with the PA column. At pH 3.0-5.5, precipitate formed in the extract clogged the column, and the recoveries of R3, R104 and R105 were 26-68%. At pH 6.0-7.0, clogging was not observed and the recoveries of the 3 dyes were somewhat higher, at 38-79%. The recoveries of other dyes were more than 80% at pH 3.0-7.0. With Oasis HLB, column loading was conducted at pH 11.0, and the recoveries of the 3 dyes improved to 70-83%. In conclusion, all 11 dyes could be cleaned-up with the PA column and Oasis HLB and the recoveries exceeded 70%.

A Rapid Dialysis Method for Analysis of Stevioside and Rebaudioside A in Foods.

A rapid dialysis method for the analysis of stevioside (SS) and rebaudioside A (RS) in foods was developed. Minced samples (10 g) were packed into 30 cm net length dialysis tubing with 30% methanol to increase the dialysis efficiency. The dialysis tubing was put in a 100 mL centrifuge tube, and the total fluid volume was made up to 100 mL with 30% methanol. Dialysis was done with shaking while heating at 50℃. The dialysis times were reduced from 48-72 hr in the conventional method to 2 hr under these conditions. The dialysate was loaded on a C18 solid- phase extraction cartridge, and the cartridge was washed with 40% methanol. SS and RS were eluted from the cartridge with 80% methanol, and separated by reversed-phase HPLC. Recovery yields (%)of SS and RS, spiked at 0.02 g/kg in various foods, were 83.0-105.1% and the relative standard deviations were mostly less than 5%.

Use of Dialysis and Liquid-Liquid Extraction for the Determination of Propionic Acid by Gas Chromatography.

A simple and efficient method for the determination of propionic acid (PA) in foods was developed. The sample was cleaned up by dialysis, and PA in the resulting solution was extracted into ethyl acetate for GC analysis. Sodium sulfate was used as a salting-out agent in the extraction process, and GC-FID and GC-MS were successfully applied to the determination and confirmation of PA, respectively. The recoveries were in the range of 98.9-104.4% at the addition level of 0.2 g/kg from 6 foods, bread, cake, cheese, worcester sauce, vinegar-pickles and yogurt. To evaluate the performance of the developed method, recoveries from bread, cake and cheese were compared with those of the notified method at the maximal allowable addition level of PA as a preservative for each food. Recoveries of 98.2-99.5% for the developed method and 91.2-92.0% for the notified method were obtained. The analytical limit was 0.1 g/kg in samples for both determination and confirmation.

Aluminium content of foods originating from aluminium-containing food additives.

Aluminium (Al) levels of 90 food samples were investigated. Nineteen samples contained Al levels exceeding the tolerable weekly intake (TWI) for young children [body weight (bw): 16 kg] when consuming two servings/week. These samples were purchased multiple times at specific intervals and were evaluated for Al levels. Al was detected in 27 of the 90 samples at levels ranging from 0.01 (limit of quantitation) to 1.06 mg/g. Of these, the Al intake levels in two samples (cookie and scone mix, 1.3 and 2 mg/kg bw/week, respectively) exceeded the TWI as established by European Food Safety Authority, although the level in the scone mix was equivalent to the provisional TWI (PTWI) as established by Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives. The Al levels markedly decreased in 14 of the 19 samples with initially high Al levels. These results indicated reductions in the Al levels to below the PTWI limits in all but two previously identified food samples.