[Surveillance of the Naturally Derived Benzoic Acid Levels in Fruits and Fruit Products, and Comparison of Analytical Methods].

Benzoic acid (BA) is typically found in natural food; therefore, naturally occurring BA must be distinguished from added BA preservatives. In this study, we investigated BA levels in 100 samples of fruit products and their fresh fruits as raw materials using dialysis and steam distillation approaches. BA was detected in the range (minimum-maximum) of 2.1-1380 µg/g and 2.2-1950 µg/g in dialysis and steam distillation, respectively. Steam distillation indicated higher BA levels than dialysis.

[Study of Rapid HPLC Method for the Determination of Antioxidants in Foods, and Standard Solution Stability during Storage].

In this study, we simultaneously determined three antioxidants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), using HPLC equipped both with a photodiode array detector and a fluorescence detector in 25 minutes per sample. Due to the combined use of the two detectors, we could achieve improved target selectivity. Further, quantification at the specific wavelengths for each target substance particularly increased BHT detection sensitivity. This approach enabled us to avoid repeated measurements during daily inspections. Furthermore, detections were performed using LC-MS/MS instead of GC-MS to overcome the problem of helium gas shortage.In addition, we investigated antioxidant stability in standard solutions during storage. Although TBHQ was stable in methanol with ascorbic acid at -20℃, ascorbic acid storage has possibility to lead to decrease in BHT and BHA concentrations. We recognized that the mixture of BHT and BHA dissolved in methanol at 4℃ and that of BHT, BHA and TBHQ dissolved in methanol with ascorbic acid at -20℃ were suitable for about one year.

[Attempt to Calculate the Efficiency of Methylation Using 1H-NMR for Analyzing Brominated Vegetable Oil].

We studied the efficiency of methylation for analyzing brominated vegetable oil (BVO). In this report, we investigated whether 1H-NMR is an applicable method for assessing the efficiency of methylation to analyze BVO. 1H-NMR sufficiently calculated the efficiency of methylation using each integral and the numbers of protons derived from the methyl group, which is characteristic in products, and the methine group, which is characteristic in unreacted substances. Additionally, the efficiency of methylation calculated via 1H-NMR was in good agreement with changes in the peak area of BVO fatty acid methyl esters (BVOFAMEs) after various heating times obtained from GC-FID analysis. Therefore, 1H-NMR is applicable for calculating the efficiency of methylation to analyze BVO.

[Determination of Nitrate Ion in Cheese and Sake].

The Japanese official analysis method for determination of nitrate ions in food products used as food additives is associated with various challenges. In some kinds of cheese, the extract becomes suspended. The volume of extracted solution is often not accurate owing to the presence of residues in the solution. Moreover, the determination with liquid chromatography-ultraviolet detection (HPLC-UV) is difficult owing to the influence of impurities. Sake usually does not contain lipids or proteins ; therefore, its analysis can be simplified by omitting the co-precipitation steps to remove them. In the present study, for cheese, the amount of sodium hydroxide solution that causes suspension was reduced, and the influence of residues was removed by adjusting the volume after suction filtration. Whereas, sake was diluted with water and centrifuged. Furthermore, solid-phase extraction (SPE) method using cartridge containing carbon molecular sieve to remove the influence of impurities on the chromatogram was successfully established. The recoveries of the nitrate ions were good outcomes of 91.3-99.6% (CV 0.9-4.5%) (n=5). The analysis range was 0.010-0.20 g/kg for cheese, 0.010-0.20 g/L for milk, and 0.010-0.10 g/kg for sake. The developed analysis methods are considered useful, because various challenges of the official analysis method can be solved and the operation are efficient.

[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.

[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.

[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.

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.

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%.

[Contents of furanocoumarins in grapefruit juice and health foods].

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.

[Simultaneous determination of neotame, alitame and aspartame in foods by HPLC].

Simultaneous determination of three artificial sweeteners, neotame (NE), alitame (AL) and aspartame (APM) in various foods by high-performance liquid chromatography (HPLC) was developed. Chopped or homogenized samples were packed into cellulose tubing with 0.01 mol/L hydrochloric acid containing 10% sodium chloride, and dialyzed against 0.01 mol/L hydrochloric acid for 24-48 hours. The dialyzate was passed through an Oasis MCX cartridge, and the cartridge was washed with water and methanol. Then the three sweeteners were eluted from the cartridge with a mixture of 0.5 mol/L ammonium chloride-acetonitrile (3 : 2). The sweeteners were separated on a Cosmosil 5C18-AR column using a gradient mode with a mobile phase of 0.01 mol/L phosphate buffer (pH 4.0)-acetonitrile and were detected at 210 nm. The recoveries of NE, AL and APM from 8 kinds of foods spiked with 10 and 100 microg/g were 86-100% and 89-104%, respectively. The detection limits of NE, AL and APM were 1 microg/g in samples. Furthermore, the three sweeteners were successfully identified by using liquid chromatography with tandem mass spectrometry.

[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.

[Simultaneous determination of stevioside, rebaudioside A and glycyrrhizic acid in foods by HPLC].

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.