Determination of sulfite in foods and beverages by ion exclusion chromatography with electrochemical detection: collaborative study

A liquid chromatographic (LC) method for determination of total sulfite in foods and beverages by alkali extraction followed by ion exclusion chromatographic separation and electrochemical detection (IEC-EC) was collaboratively studied by 9 laboratories. Blind duplicate samples of starch, diluted lemon juice, wine cooler, dehydrated seafood, and instant mashed potatoes were analyzed without spiking and with added sulfite at 2 levels. The initial sulfite levels varied from 0 to 384 ppm SO2, and the levels added varied from 10 to 400 ppm. The initial sulfite levels determined by the IEC-EC method and the Monier-Williams method were in good agreement. Recovery of added sulfite by the IEC-EC method was generally higher than that by the Monier-Williams method. Within-laboratory repeatability (RSDr) for the IEC-EC method varied from 4.4 to 26.0%, and overall reproducibility (RSDR) varied from 8.5 to 39.3%. The collaborators found the method to be fast, sensitive, and easy to use, which makes it a useful alternative to the Monier-Williams method. The method has been adopted official first action.     

Sulfite analysis of fruits and vegetables by high-performance liquid chromatography (HPLC) with ultraviolet spectrophotometric detection

Free and total sulfite were analyzed in acidified vegetable products, instant mashed potatoes, and dried apples. Sulfite was separated by HPLC and quantified with a UV-vis detector. Resolution from components of food samples was achieved by varying the acid concentration of the eluant solution and by appropriate choice of the analytical wavelength. The minimum detectable levels for sulfite were 0.5 mg/L for a 10-cm analytical column and 1.5 mg/L for a 30-cm column. For analyses done with a 30-cm column, the coefficient of variation was <2% for analysis of free sulfite and total sulfite in acidified vegetables. For dried apples and instant potatoes, it ranged from 1 to 6.5%. The corresponding analytical errors were <4% and 1.2-5.6%, respectively, for the 10-cm column.     

Determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow injection analysis: collaborative study

A method for the determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow injection analysis (FIA) was collaboratively studied by 8 laboratories. In the method, the sample solution is reacted with sodium hydroxide to liberate aldehyde-bound sulfite. The sample stream is acidified to produce SO2 gas, which diffuses across a Teflon membrane in the gas diffusion cell into a flowing stream of malachite green. The degree of discoloration of the malachite green is proportional to the amount of sulfite in the sample solution. Red wine was included in the study but interlaboratory precision for these samples was not satisfactory and correlation with Monier-Williams results was poor. The present method is not recommended for use with these samples. For shrimp, potatoes, dried pineapple, and white wine, average reproducibility (RSDR) of results was 25% for samples at 10 ppm SO2 and 10% for samples at greater than 50 ppm. Overall average reproducibility was 14%. Recoveries of sulfite added to samples averaged 80%. Comparison of FIA with the Monier-Williams method indicated comparable results by the 2 methods. The FIA method has been adopted official first action for determination of greater than or equal to 5 ppm total sulfite in shrimp, potatoes, dried pineapple, and white wine.     

Reevaluation of Monier-Williams method for determining sulfite in food

The Monier-Williams distillation procedure has a long history of successful use for determining sulfite in fruit products and wine; however, a systematic evaluation of its accuracy and precision with other food matrices has not been undertaken. We found that the Monier-Williams distillation yielded greater than 90% recovery of sulfite added to foods such as table grapes, hominy, dried mangoes, and lemon juice. Less than 85% recovery was obtained with broccoli, soda crackers, cheese-peanut butter crackers, mushrooms, and potato chips. These results may, in fact, accurately reflect the residual levels of sulfite if a portion of the sulfite undergoes irreversible reaction with some food components. Analysis of commercial food products gave sulfite levels ranging from 1400 ppm in dried apple slices to 25 ppm in cream sherry.     

Effect of oil marinades with garlic, onion, and lemon juice on the formation of heterocyclic aromatic amines in fried beef patties

Effects of the application of oil marinades with garlic, onion, and lemon juice on the formation of heterocyclic aromatic amines (HAAs) in fried beef patties were investigated. Two different statistical design models were used. In the screening experiment, a significant inhibition of formation of MeIQx was determined only by addition of garlic (p < 0.05). When the amount of garlic was changed from 2 to 20 g/100 g of marinade, the estimated MeIQx content in patties was reduced about 70%. MeIQx (0.38-1.22 ng/g), 4,8-DiMeIQx (n.d.-0.45 ng/g), PhIP (n.d.-0.09 ng/g), norharman (0.76-13.5 ng/g), and harman (2.9-21.5 ng/g) were found in fried patties. The results of two-level and three-level fractional factorial design experiments confirmed the first investigation. They showed a stronger reduction in MeIQx in patties with the addition of increasing amounts of garlic (p < 0.01) and onion (p < 0.05) in marinades. A higher content of lemon juice in marinades led to only a marginal reduction in MeIQx (p > 0.05). The optimum amounts of onion, garlic, and lemon juice that achieved a maximum reduction of HAAs were calculated as 31.2%, 28.6%, and 14.6% in marinade.     

Determination of organic acids by high-performance liquid chromatography with electrochemical detection during wine brewing

Voltammetric determination of acids by means of the electrochemical reduction of quinone was applied to high-performance liquid chromatography (HPLC) with electrochemical detection (ED) for determining organic acids in fruit wines. A two-channel HPLC-ED system was fabricated by use of an ion-exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solution of 0.1 mM HClO(4) and ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and reagent solution, respectively. Determination of acetic, citric, lactic, malic, succinic, and tartaric acids was made by measuring the peak areas of the flow signals due to the reduction current of quinone caused by the eluted acids. The peak area was found to be linearly related to the acid amount ranging from 0.1 to 40 nmol per 20 microL injection. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes. The method was shown as an effective means for following acid contents in fruit juices during fermentation with wine yeast.     

Estimating bergamot juice adulteration of lemon juice by high-performance liquid chromatography (HPLC) analysis of flavanone glycosides

The chemical composition of 30 samples of juices obtained from bergamot (Citrus bergamia Risso and Poit.) fruits is reported and compared to the genuineness parameters adopted by Association of the Industry of Juice and Nectars (AIJN) for lemon juice. It was found that the compositional differences between the two juices are distinguishable, although with difficulty. However, these differences are not strong enough to detect the fraudulent addition of bergamot juice to lemon juice. Instead, we found the high-performance liquid chromatography (HPLC) analysis of the flavanones naringin, neohesperidin, and neoeriocitrin, which are present in bergamot juice and practically absent in the lemon juice, is a convenient way to detect and quantify the fraudulent addition of bergamot juice. The method has been validated by calculating the detection and quantification limits according to Eurachem procedures. Employing neoeriocitrin (detection limit = 0.7 mg/L) and naringin (detection limit = 1 mg/L) as markers, it is possible to detect the addition of bergamot juice to lemon juice at the 1% level. When using neohesperidin as a marker (detection limit = 1 mg/L), the minimal percentage of detectable addition of bergamot juice was about 2%. Finally, it is reported that the pattern of flavonoid content of the bergamot juice is similar to those of chinotto (Citrus myrtifolia Raf) and bitter orange (Citrus aurantium L.) juices and that it is possible to distinguish the three kinds of juices by HPLC analysis.     

Determination of nitrite in cured meats by ion-exclusion chromatography with electrochemical detection

A rapid liquid chromatographic (LC) method was developed for a sensitive determination of nitrite in cured meats, using ion-exclusion chromatographic separation and electrochemical detection (IEC-EC). The current AOAC colorimetric method requires 2 h shaking in a steam bath to eliminate interference from reducing compounds such as ascorbic acid. In the present method, nitrite was analyzed in the presence of ascorbic acid without interference, and the extraction time was reduced to 1 min. The extracted nitrite was determined by ion chromatography using anion-exclusion/HS column and amperometric detector equipped with platinum or glassy carbon electrode operating at +1.0 V vs Ag/AgCl reference electrode. The detection limit was 1 ppb as NO2-. The recoveries of 50 ppm nitrite added to frankfurter and meat stick were 103 and 99.6%, respectively, with relative standard deviations less than 4%. The high speed, sensitivity, and selectivity make the new method a useful alternative to the AOAC colorimetric method.     

Occurrence of fumonisins B(2) and B(4) in retail raisins

Concerns that raisins may be contaminated by fumonisins stem from the persistent occurrence of Aspergillus niger spores on raisins and the recent discovery of fumonisin production by A. niger on grapes, which leads to the widespread occurrence of fumonisin B(2) in wine. This study presents an LC-MS/MS survey of fumonisins in retail raisins. In 10 of 21 brands collected in Denmark, Germany, and The Netherlands, fumonisins B(2) and B(4) were detected at levels up to 13 and 1.3 μg/kg, respectively. Only fumonisin B(2) has been detected in wine, so the presence of fumonisin B(4) in raisins suggests that the fumonisins are produced mainly during the drying process concomitant with the decreasing water activity. Analysis of multiple packages from one manufacturer showed a 3-fold package-to-package variation, suggesting that a few raisins per package are contaminated.     

不同品种红葡萄酒花色苷高效液相色谱指纹图谱识别

利用高效液相色谱法建立了9个品种葡萄酒的花色苷指纹图谱,对不同品种葡萄酒进行识别。HPLC检测：采用反相C18柱,调整流动相pH值1.6,二元梯度洗脱,检测波长518 nm。指纹图谱建立方法：计算HPLC色谱峰的相对保留时间和相对峰面积,按相对保留时间排列相对应的相对峰面积。采用夹角余弦法计算相关系数,并使用SPSS11.0统计分析软件对指纹图谱进行系统聚类分析。结果表明：不同品种葡萄酒花色苷的HPLC指纹图谱存在着差异,山葡萄种及杂种葡萄酒具有区别于欧亚种葡萄酒的特征色谱峰;不同品种葡萄酒花色苷指纹图谱的相似性不同,不同种间品种葡萄酒相似性较差;系统聚类分析初步建立了不同品种葡萄酒花色苷识别模式,对葡萄酒进行了较好的识别。指纹图谱技术结合聚类分析是识别不同酿造品种葡萄酒的有效方法之一。     

Determination of sulfite in foods by headspace liquid chromatography

Sulfite was determined in a variety of foods by liquid chromatography (LC) after the samples were mixed with a solution containing mannitol, FeSO4, and Na2HPO4, adjusted to pH 11, and left to stand for 15 min at room temperature. An aliquot of the mixture was placed in a headspace vial and mixed with 50% H3PO4. After 15 min, a portion of the headspace was removed with a syringe containing LC mobile phase without acetonitrile. The syringe was shaken and an aliquot of the solution was analyzed on an anion exchange column with a mobile phase of 0.03M methane sulfonate (pH 10.8) containing 5% acetonitrile. Sulfite was detected amperometrically (glassy carbon electrode) at +0.7 V. The method was successfully compared to the FDA-modified Monier-Williams procedure for a variety of foods. Minimum detectable levels were about 1 microgram/g, based on a 15 g sample.     

Liquid chromatographic determination of sulfite in grapes and selected grape products

A liquid chromatographic (LC) method is described for the determination of sulfite in grapes and certain grape products. Sulfite is extracted from grapes with aqueous formaldehyde solution buffered at pH 5; free sulfite is converted to hydroxymethylsulfonate (HMS), which is extremely stable at pH 3-7. Subsequent heating to 80 degrees C for 30 min converts reversibly bound forms of sulfite to HMS. The extract is then analyzed by reverse-phase ion-pairing liquid chromatography, using a C18 column and a mobile phase of aqueous 0.005 M tetrabutylammonium ion in 0.05 M acetate, pH 4.7, and a flow rate of 1 mL/min. Aqueous KOH is added to the eluate to convert HMS to free sulfite, which is then treated with 5,5'-dithiobis[2-nitrobenzoic acid]. This reaction produces the 3-carboxy-4-nitrothiophenolate anion, which is determined by measurement of electronic absorption at 450 nm. For grapes spiked with HMS at 5-20 ppm (as SO2), recoveries ranged from 92 to 112%, with a coefficient of variation of 4.6%. The method was also used to determine sulfite in various grape products. Results were comparable to those obtained by the AOAC official Monier-Williams method.     

Liquid chromatographic methodology for the characterization of orange juice

Liquid chromatographic (LC) methodology potentially useful for the characterization of orange juice, with particular regard to detecting adulteration of orange juice by computer pattern recognition analysis, has been developed. After dilution with methanol the juice is extracted with hexane to remove the carotenoids, which are chromatographed on a C18 column with an acetonitrile-methanol-methylene chloride mobile phase and detection at 450 nm. Further extraction of the juice with methylene chloride isolates the methoxylated flavones, which are chromatographed by reverse phase LC with an acetonitrile-methanol-water mobile phase and detection at 280 nm. The flavanone glycosides remaining in solution are chromatographed on a C18 column with an acetonitrile-water mobile phase and detection at 280 nm. The precisions of the heights of the 32 LC peaks selected for pattern recognition analysis were determined from 5 replicate analyses of a single juice. Coefficients of variation of the replicates ranged from 0.3 to 4.5%, with an average of 2.1%. Adulteration of products with sodium benzoate-fortified pulpwash or grapefruit juice can be detected by this method. Pattern recognition analysis of the data obtained for 80 authentic and 19 adulterated orange juices showed that the method is potentially useful for distinguishing between authentic and adulterated products.     

Thermal stability of alpha-amylase from Aspergillus oryzae entrapped in polyacrylamide gel

To determine the suitability as a time-temperature indicator for dielectric pasteurization processes, the thermal stability (50-75 degrees C) of Aspergillus oryzae alpha-amylase immobilized in polyacrylamide gel in phosphate buffer, mashed potatoes, and minced shrimp was examined. Changing the cross-linking agent concentration from 3.3 to 5.3% and adding 2% salt did not markedly affect the thermal stability of the immobilized alpha-amylase. Thermal inactivation was first order, and immobilization generally improved the thermal stability of alpha-amylase. z values of the immobilized system in test food systems were 10.2 degrees C (phosphate buffer), 8.45 degrees C (minced shrimp), and 7.78 degrees C (mashed potatoes).     

Effect of the rootstock and interstock grafted in lemon tree (Citrus limon (L.) Burm.) on the flavonoid content of lemon juice

The grafting of the rootstock with the lemon tree is an agronomical technique used to improve production and/or quality of the fruit. The interstock has been used with different fruit trees to modulate the tree size, fruit production and quality, and the aging of the tree. The lemon trees grafted with interstocks increase their longevity, lemon production and quality; interstocks are also used to decrease the thickness of the trunk at the grafting point. This enlarging of the trunk provokes a decrease of the sap flow. In our study, "Verna" lemon trees were grafted with interstock between the rootstock and the lemon tree to follow the flavonoid content of the lemon juice. The lemon juice was obtained from the lemons collected of the grafted lemon trees. Two types of rootstocks were used: Citrus aurantium L. and Citrus macrophylla L. Seven interstocks from five cultivars of orange tree, one cultivar of lime tree, and one cultivar of tangerine tree were used. "Verna" lemon trees were also grafted directly to the rootstock. The rootstock was more important agronomic factor than the interstock on the total flavonoid content of lemon juice. The interstock grafting had only a small influence on the flavonoid content of the lemon juice, and it modulated the individual flavonoid content. Citrus aurantium L. rootstock and "Berna" and "Washington Navel" interstocks were the most appropriate to graft in the lemon tree. This interstock grafting technique does not increase the flavonoid content of the lemon juice. Regarding the individual flavonoids, the 6,8-di-C-glucosyl diosmetin was the most affected flavonoid by the type of rootstock used. The interstock used is able to alter the individual quantitative flavonoid order of eriocitrin, diosmin, and hesperidin. In addition, the HPLC-ESI/MS(n) analyses provided the identification of two new flavonoids in the lemon juice: Quercetin 3-O-rutinoside-7-O-glucoside and chrysoeriol 6,8-di-C-glucoside (stellarin-2). The occurrence of apigenin 6,8-di-C-glucoside (vicenin-2), eriodictyol 7-O-rutinoside, 6,8-di-C-glucosyl diosmetin, hesperetin 7-O-rutinoside, homoeriodictyol 7-O-rutinoside and diosmetin 7-O-rutinoside was also confirmed in lemon juice by this technique.     

New beverages of lemon juice enriched with the exotic berries maqui, açaı́, and blackthorn: bioactive components and in vitro biological properties

Following previous research on lemon juice enriched with berries, the aim of this work was to design new blends based on lemon juice mixed with different edible berries of exotic and national origin: maqui ( Aristotelia chilensis (Molina) Stuntz), a?a?? ( Euterpe oleracea Mart.), and blackthorn ( Prunus spinosa L.). The phytochemical characterization of controls and blends was performed by HPLC-DAD-ESI/MS(n). Their antioxidant capacity against DPPH, superoxide, and hydroxyl radicals and hypochlorous acid and their potential to inhibit cholinesterases were also assessed. The profiling of the red fruits and lemon revealed a wide range of bioactive phenolics. The novel beverage based on lemon juice and maqui berry (LM) was the most interesting blend in terms of antioxidant capacity. Berry control samples displayed reduced effects on acetylcholinesterase and butyrylcholinesterase, the lemon juice control being always the most active. This activity was also remarkable for lemon-blackthorn (LB) and lemon-a?a?? (LA) blends, the last being the most effective inhibitor of cholinesterases among all samples. The results suggested that lemon juice enriched with berries could be of potential interest in the design of new drinks with a nutritive related function on health for chronic diseases.     

Identification of impact odorants in Bordeaux red grape juice, in the commercial yeast used for its fermentation, and in the produced wine

The aroma extract dilution analysis method was used to detect the impact odorants of Bordeaux Cabernet Sauvignon and Merlot wines extracts, as well as those of the extracts of the corresponding Cabernet Sauvignon juice and dry yeasts used for its fermentation. The wines and the yeasts were extracted using dichloromethane, and the juice was extracted using Amberlite XAD-2. Structural identification of the impact odorants using gas chromatography-mass spectrometry and atomic emission detection (sulfur acquisition) was achieved after enrichment of these extracts by silica gel and Affi-Gel 501 chromatography. The same odorants (with the exception of dimethyl sulfide among 48) were detected in both wine extracts, with about the same flavor dilution (FD) factors. The 18 impact odorants detected in the Cabernet Sauvignon juice and dry yeast extracts were also found in the wine extracts. The odorants with the highest FD factors were 3-(methylsulfanyl)propanal, (E,Z)-nona-2, 6-dienal, and decanal in the juice extract, 2-methyl-3-sulfanylfuran, 3-(methylsulfanyl)propanal, 2-/3-methylbutanoic acids, and phenylethanal in the dry yeast extract, and 2-/3-methylbutanols, 2-phenylethanol, 2-methyl-3-sulfanylfuran, acetic acid, 3-(methylsulfanyl)propanal, 2-/3-methylbutanoic acids, beta-damascenone, 3-sulfanylhexan-1-ol, Furaneol, and homofuraneol in the wine extracts. Determination of the odor thresholds of some of these impact odorants was carried out.     

Analysis of antioxidants from orange juice obtained by countercurrent supercritical fluid extraction,using micellar electrokinetic chromatography and reverse-phase liquid chromatography

Antioxidants from orange juice were determined by the combined use of countercurrent supercritical fluid extraction (CC-SFE) prior to reverse-phase liquic chromatography (RP-LC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published LC procedure, both using diode array detection. The characterization of the different antioxidants was further done by LC-mass spectrometry. Advantages and drawbacks of LC and MEKC for analyzing the antioxidants found in the different orange extracts are discussed. Although LC yields higher peak area and slightly better reproducibility than MEKC, the latter technique provides information about the CC-SFE extracts in analysis times 7 times faster than by LC. This analysis advantage can be used for the quick adjustment of CC-SFE conditions, thus providing a fast way to obtain orange fractions of specific composition.     

Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice

We evaluated 17 plant essential oils and nine oil compounds for antibacterial activity against the foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica in apple juices in a bactericidal assay in terms of % of the sample that resulted in a 50% decrease in the number of bacteria (BA(50)). The 10 compounds most active against E. coli (60 min BA(50) range in clear juice, 0.018-0.093%) were carvacrol, oregano oil, geraniol, eugenol, cinnamon leaf oil, citral, clove bud oil, lemongrass oil, cinnamon bark oil, and lemon oil. The corresponding compounds against S. enterica (BA(50) range, 0.0044-0.011%) were Melissa oil, carvacrol, oregano oil, terpeineol, geraniol, lemon oil, citral, lemongrass oil, cinnamon leaf oil, and linalool. The activity (i) was greater for S. enterica than for E. coli, (ii) increased with incubation temperature and storage time, and (iii) was not affected by the acidity of the juices. The antibacterial agents could be divided into two classes: fast-acting and slow-acting. High-performance liquid chromatography analysis showed that the bactericidal results are related to the composition of the oils. These studies provide information about new ways to protect apple juice and other foods against human pathogens.     

Determination of pyrethroid residues in agricultural products by an enzyme-linked immunosorbent assay

To determine cypermethrin and permethrin in agricultural products, a competitive enzyme-linked immunosorbent assay (ELISA) method was employed. The matrix interferences were minimized by direct dilution of the extracts. No further cleanup was needed. A minimum matrix effect with a 1:10 dilution of white wine for cypermethrin and a 1:200 dilution of red and white wines, fruits, and vegetables for permethrin was found when phosphate-buffered saline containing 40% methanol was employed as the diluent. Good recoveries of spiked levels were observed. The mean percentage recoveries of cypermethrin spiked in white wine and permethrin spiked in red and white wines were 99.7, 74, and 78%, respectively. The mean percentage recoveries of permethrin spiked in apple, banana, cucumber, lettuce, onion, and peach were 99.2, 105, 70.2, 97.5, 94.4, and 89.4%, respectively. Validation of the ELISA method with permethrin-spiked lettuce and peach was carried out using gas chromatography with mass spectrometry, resulting in a good recovery and correlation.