The quantitative analysis of thiamin and riboflavin and their respective vitamers in fermented alcoholic beverages

This research aimed to develop a simple and effective method for analyzing thiamin (B(1)), riboflavin (B(2)) and their respective vitamers by high performance liquid chromatography (HPLC) in fermented alcoholic beverages. The method developed here employs a phosphate buffer/methanol gradient elution on a single reverse phase column, coupled with independent fluorescent detection regimes. It also employs a precolumn derivatization to convert thiamin to thiochrome via an alkaline potassium ferricyanide solution. The method described here allowed a spike recovery of better than 97%, with a typical linear detection range (R(2) ≥ 0.9997) between ≤ 5 and ≥ 500 μg/L for all vitamers studied. Lager style beers were found to contain significantly (p < 0.001) less thiamin than other tested styles of beers (lager, 35.7 μg/L; ale, 88.3 μg/L; stout/porters, 104.4 μg/L; wheat beers, 130.7 μg/L), which may be due to the raw material and extensive processing that occurs for this style. There was no statistical difference (p = 0.608) between the riboflavin content of each beer style. Furthermore, wines and ciders contain less thiamin and riboflavin than beer, which is also likely to be due to the base materials used and the differences in processing steps to produce these beverages.     

Factors influencing benzene formation from the decarboxylation of benzoate in liquid model systems

Benzene may occur in foods due to the oxidative decarboxylation of benzoate in the presence of hydroxyl radicals. This study investigated factors influencing benzene formation in liquid model systems. The type of buffer, other sources of hydroxyl radical formation in food (photo oxidation of riboflavin and lipid oxidation), transition metal ion concentrations, and the inhibitory effect of antioxidants were tested in benzoate containing model systems. Regarding the hydroxyl radical sources tested, the highest benzene formation was observed in light exposed model systems containing ascorbic acid, Cu(2+), and riboflavin in Na-citrate buffer (1250 ± 131 μg kg(-1)). In practice, it seems that the combination ascorbic acid/transition metal ion remains the biggest contributor to benzene formation in food. However, the concentration of Cu(2+) influences significantly benzene formation in such a system with highest benzene yields observed for Cu(2+) 50 μM (1400 μg kg(-1)). The presence of antioxidants with metal chelation or reduction properties could prevent completely benzene formation.     

HPLC Determination of Stability and Distribution of Added Folic Acid and Some Endogenous Folates During Breadmaking

Bread flour was spiked with folic acid (1.40 mg/lb or 3.08 μg/g of flour) and processed into bread by the sponge and dough method. Changes that occurred to added folic acid and endogenous folate contents through different processing stages, including sponge formation, proofing, and baking, were assessed by reversed‐phase ion‐pair HPLC combined with UV and fluorometric detection. Sample extraction required α‐amylase and rat plasma deconjugase digestion, and sample preparation required purification by solid‐phase extraction. Added folic acid was measured by monitoring UV absorption at 280 nm. Four selected forms of endogenous folates including tetrahydrofolate (THF), 5‐formyl‐THF, 10‐formylfolate, and 5‐methyl‐THF were identified and quantified throughout the bread processing using a fluorescence excitation wavelength of 290 nm and emission wavelength of 350 or 450 nm. Data indicate a relatively good stability of added folic acid and native folates to the baking process, and increased endogenous folate contents in dough and bread as compared with the flour from which they were made.     

Dummy molecularly imprinted solid-phase extraction for selective determination of five phthalate esters in plastic bottled functional beverages

In this paper, a highly selective sample cleanup procedure combing dummy molecular imprinting and solid-phase extraction (DMI-SPE) was developed for the simultaneous isolation and determination of five phthalate esters in plastic bottled beverages. The new imprinted microspheres were synthesized via precipitation polymerization using diisononyl phthalate as a dummy template that showed high selectivity and affinity to the five kinds of phthalate esters and were successfully applied as selective sorbents of DMI-SPE for the simultaneous determination of the phthalate esters from plastic bottled beverages. Good linearity was obtained in a range of 5.0-750.0 μg/L, and the average recoveries of the five phthalate esters at three spiked levels ranged from 84.3 to 96.2% with the relative standard deviations less than 5.49%. The developed extraction protocol eliminated the effect of template leakage on quantitative analysis and could be applied for the determination of phthalate esters in complicated functional beverages products.     

Folic Acid,Iron, and Zinc Contents in Chosen Food Products Prepared with Fortified Flours

In 2002, Brazil issued a regulation requiring that corn and wheat flours be fortified by the addition of folic acid and iron. The aim of the present work was to evaluate the levels of folic acid, iron, and zinc in macaroni, pizza, and bread prepared with fortified flour. The methodologies used for the mineral (FAAS) and vitamin (HPLC) determinations were validated and considered adequate. For macaroni, the folic acid content range was 50.0–182.4 μg/100 g. The iron and zinc levels were 1.6–5.4 mg/100 g and 0.7–1.0 mg/100 g, respectively. The pizza and bread samples contained 14.8–271.3 μg/100 g and 47.4–481.4 μg/100 g of folic acid, respectively. The iron and zinc levels were 2.6–7.3 mg/100 g and 0.4–0.9 mg/100 g, respectively, for pizza, and 3.0–12.4 mg/100 g and 0.4–0.8 mg/100 g for bread. The results indicated that the distribution of folic acid and iron in the majority of the samples tested was heterogeneous. This study could be of use to governmental authorities in their food fortification evaluation programs. Moreover, the average iron contents observed in the products could result in problems with zinc absorption, and it is important to consider that iron can induce oxidative stress in cells.     

Rapid and sensitive determination of sulfonamide residues in milk and chicken muscle by microfluidic chip electrophoresis

A new, rapid, and sensitive method was proposed for the determination of sulfonamide residues in milk and chicken muscle samples by microchip electrophoresis with laser-induced fluorescence detection. Separation of fluorescamine-labeled sulfonamides was accomplished by using a buffer containing 5 mmol/L boric acid and 1% (w/v) polyvinyl alcohol (PVA). The pH, amount of PVA, and concentration of boric acid in the running buffer were found to have great influence on the separation. By optimizing these conditions, the separation of four sulfonamides, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, and sulfanilamide, was achieved within 1 min with limits of detection (S/N = 3) of 0.2-2.3 μg/L, which are well below the maximum residue limit. The proposed method also exhibited very good repeatability; the relative standard deviations for both within-day and between-day measurements were ≤3.0%. With a simplified sample pretreatment protocol, fast determination of sulfonamides in real samples was successfully performed with standard addition recoveries of 93.3-100.8 and 82.9-92.3%, respectively.     

Simultaneous liquid chromatographic determination of water-soluble vitamins, caffeine, and preservative in oral liquid tonics

A rapid, simple, and reliable liquid chromatographic method has been developed for the simultaneous determination of nicotinamide (niacinamide), thiamine, riboflavin, riboflavin sodium phosphate, pyridoxine, caffeine, and sodium benzoate in commercial oral liquid tonics. The 7 components are separated on a reverse phase C18 column using a mobile phase of acetonitrile-0.01M potassium dihydrogen phosphate-triethylamine (8 + 91.5 + 0.5 v/v/v) containing 5mM sodium octanesulfonate and adjusted to pH 2.8 with phosphoric acid. Components are detected at 254 nm with attenuation 0.02 AUFS. Acetanilide is used as an internal standard. In addition to the 7 components mentioned, nicotinic acid (niacin), cyanocobalamin, and folic acid are also separated under the same conditions. Sample preparation involves only addition to internal standard solution and dilution with mobile phase and then filtration. Recoveries of the 7 components and cyanocobalamin from spiked preparations ranged from 97 to 104% with coefficients of variation of 0.9-4.2%.     

A quantitative stable-isotope LC-MS method for the determination of folic acid in fortified foods

A stable-isotope liquid chromatography-mass spectrometry (LC-MS) assay was developed for the quantitative determination of folic acid in fortified foods. Folic acid was extracted from food samples into a phosphate buffer, purified on a C-18 Sep-Pak cartridge, and analyzed by LC-MS in the negative ion mode using electrospray ionization. The analyte was quantified using (13)C(5)-folic acid as an internal standard. The coefficient of variation for the precision of the method was 5.6% based on the analysis of four sample replicates. The accuracy of the method was assessed using a standard method of addition of folic acid to a shredded whole-wheat cereal. The quantitative determination of folic acid in this matrix was linear over 1 order of magnitude having a concentration range of 2.4 to 24 microg/g of food (or 0.05 to 0.5 microg of analyte injected into the LC-MS). The overall quantitative efficiency of the method was evaluated using a standard reference material (infant formula SRM 1846). The method was applied to the determination of folic acid in several test samples (fortified breakfast cereals), and the values were in accord with the manufacturer's claim. This method advances a LC-MS technique for the determination of folic acid in fortified foods based on stable-isotope dilution methodology. The specificity of the technique and quantitative accuracy of the method in various food substrates suggests that the method may be adapted for routine analysis in other fortified foods.     

Development of a simplified method for the determination of folates in baker's yeast by HPLC with ultraviolet and fluorescence detection

A simplified HPLC method for rapid determination of folates in yeast with ultraviolet and fluorescence detection without sample purification has been developed. By use of the column Aquasil C(18), specially designed for polar analytes, and gradient elution, it was possible to separate and determine five folate derivatives: tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate with fluorescence detection, and 10-formylfolic acid and folic acid with ultraviolet detection. The sample preparation required only a small amount of dry yeast (25-50 mg) and included an extraction of folates by heat treatment and deconjugation of folate polyglutamates to monoglutamates with the use of rat serum conjugase. Validation involved investigation of matrix effects, determination of recovery by standard addition method, repeatability, and stability tests. The dominating folate forms in commercial dry baker's yeast were found to be tetrahydrafolate and 5-methyltetrahydrofolate with a total folate content of 2890 microg/100 g (63.4 nmol/g). The simplicity of the method makes it suitable for folate screening studies of different yeast strains.     

Assay of riboflavin in sample wines by capillary zone electrophoresis and laser-induced fluorescence detection

To routinely assay the concentration of riboflavin (RF) in wines, a rapid and sensitive method was developed and evaluated. The method is based on a simple sample preparation, capillary zone electrophoretic separation and laser-induced fluorescence detection (CZE-LIF). Sample preparation required only dilution and filtration. Under optimized conditions, the limit of detection of riboflavin was 0.5 micro g/L, using a hydrodynamic sample introduction of 10 s at 54 mbar. The method was fully validated: the recovery of RF in wines was >95%. The concentrations of RF within the three sample types of Italian wines investigated here ranged from 69 to 151 micro g/L with a mean value (+/-SD) of 112 +/- 25 micro g/L, from 74 to 193 micro g/L with a mean value of 115 +/- 45 micro g/L, and from 156 to 292 micro g/L with a mean value of 226 +/- 40 micro g/L, for white, rosé, and red wines, respectively. Such an accurate and highly sensitive CZE-LIF method represents a powerful improvement over previous methods in terms of sensitivity, simplicity, and efficiency. It is well suited to satisfy the demands for accurate and sensitive detection with minimal sample preparation and cleanup.     

Simultaneous determination of eight water-soluble vitamins in supplemented foods by liquid chromatography

A fast, simple, and reliable method for the isolation and determination of the vitamins thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, folic acid, cyanocobalamin, and ascorbic acid in food samples is proposed. The most relevant advantages of the proposed method are the simultaneous determination of the eight more common vitamins in enriched food products and a reduction of the time required for quantitative extraction, because the method consists merely of the addition of a precipitation solution and centrifugation of the sample. Furthermore, this method saves a substantial amount of reagents as compared with official methods, and minimal sample manipulation is achieved due to the few steps required. The chromatographic separation is carried out on a reverse phase C18 column, and the vitamins are detected at different wavelengths by either fluorescence or UV-visible detection. The proposed method was applied to the determination of water-soluble vitamins in supplemented milk, infant nutrition products, and milk powder certified reference material (CRM 421, BCR) with recoveries ranging from 90 to 100%.     

Determination of total riboflavin in cooked sausages.

A simple and rapid method for determining riboflavin content in cooked sausages by ion-pair reversed-phase liquid chromatography has been set up. Samples were subjected to acid and enzymatic hydrolysis. Sample extracts were directly chromatographed, avoiding purification and concentration treatment. Final determination was performed by high-performance liquid chromatography with fluorescence detector (excitation, 227 nm; emission, 520 nm), on a 25 cm x 4 mm i.d. Spherisorb ODS-2 cartridge using a mixture of 5 mM heptanesulfonic acid adjusted to pH 2.7 with phosphoric acid and acetonitrile (75:25, v/v) as mobile phase. Precision of the method was 1.3% (within a day) and 2.6% (between days). The detection limit was 0.015 mg/100 g. The recovery was >95%.     

氢化物原子荧光光谱法测定水溶肥料中的硒

文章介绍了原子荧光光谱法测定水溶肥料中硒含量的方法。该方法具有操作简单、快速、基体干扰少、灵敏度高等优点。该方法检出限为0.24 μg/L,线性范围0 μg/L～300 μg/L,加标回收率96.5%～103.0%。     

Synthesis of a novel composite imprinted material based on multiwalled carbon nanotubes as a selective melamine absorbent

A novel composite imprinted material, on the basis of a multiwalled carbon nanotube (CNT)-incorporated layer using melamine as a template, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linker, was synthesized by a surface imprinting technique. The imprinted/CNT sorbent was characterized by a scanning electron microscope (SEM). Adsorption dynamics and a Scatchard adsorption model were employed to evaluate the adsorption process. The results showed that the imprinted/CNT sorbent displayed a rapid dynamic adsorption and a high adsorption capacity of 79.9 μmol g(-1) toward melamine. Applied as a sorbent, the imprinted/CNT sorbent was used for the determination of melamine in a real sample by online solid-phase extraction-high-performance liquid chromatography (SPE-HPLC). An enrichment ratio of 563-fold, detection limit (S/N = 3) of 0.3 μg L(-1), and quantification limit of 4.5 μg L(-1) were achieved.     

Folic Acid Content of Ready‐to‐Eat Cereals Determined by Liquid Chromatography‐Mass Spectrometry: Comparison to Product Label and to Values Determined by Microbiological Assay

Twelve popular ready‐to‐eat breakfast cereals fortified with folic acid were sampled in the United States in 2006, and the data have been incorporated into the USDA National Nutrient Database for Standard Reference. Cereals were collected from three statistically selected retail outlets in each of four primary census regions, and four composites of each product were prepared using random groupings of three locations each. Folic acid was determined using a validated LC‐MS method, with ¹³C‐folic acid as an internal standard, after trienzyme treatment and solid phase extraction. A cereal reference material (AACC VMA399) was analyzed as a control. Selected samples were also assayed using the standard microbiological method, with and without trienzyme extraction, to generate an estimate of endogenous folate. On average, as shown on the label, folate content was underestimated. In seven cereals, folate was within 5% of the declared value; in four cereals, it was 5–20% higher; and in two cereals, it was >20% greater, representing –75 to +69 μg/serving (mean 17) of the label value, equivalent to –19% to +17% of the 400 μg/daily value. The microbiologically determined folic acid was higher than LC‐MS by 10–67% (mean 40%). Therefore, use of label values might underestimate folate intake from some breakfast cereals.     

Voltammetric determination of the antioxidant capacity in wine samples using a carbon nanotube modified electrode

A direct determination of gallic acid was achieved at a carbon paste electrode modified with carbon nanotubes under differential pulse voltammetry conditions. The values obtained for gallic acid were used to estimate the antioxidant properties of the wine sample based on gallic acid oxidation. The proposed method is based on the gallic acid oxidation process at a modified carbon paste electrode (MCPE) containing 30% (m/m) of carbon nanotubes monitored at 0.35 V versus Ag/AgCl (KCl 3 mol L(-1)). Using the optimized experimental conditions, the calibration curve for gallic acid was linear in the concentration range from 5.0 × 10(-7) to 1.5 × 10(-5) mol L(-1) with a detection limit of 3.0 × 10(-7) mol L(-1). The MCPE was successfully applied for the determination of the antioxidant capacity for red and white wine samples without interference of glucose and ascorbic acid, and the obtained results were compared with the standard spectrophotometric method.     

Antioxidative activity of urate in bovine milk

The antioxidative effects of urate on peroxidase-induced protein oxidation and light-induced riboflavin degradation and lipid oxidation in whole milk were studied. In addition, experiments using ascorbate were conducted to directly compare the antioxidative activity of urate and ascorbate. The presence of urate and/or ascorbate (10-30 mg/L) lowered peroxidase-induced formation of dityrosine by 44-96% in unpasteurized whole milk. No synergistic effect of urate and ascorbate on peroxidase-induced dityrosine formation was registered, but merely an additive effect. Light exposure of pasteurized whole milk showed that ascorbate was oxidized at the expense of urate, which indicated ascorbate-mediated recycling of the urate radical. Moreover, both urate and ascorbate (30 mg/L) retarded light-induced lipid oxidation in pasteurized whole milk as measured by formation of lipid hydroperoxides with urate being the most effective (28% reduction in lipid hydroperoxides) compared with ascorbate (14%). Finally, addition of urate or ascorbate (300 mg/L) to pasteurized whole milk showed a slight protective effect against light-induced degradation of riboflavin with urate being the most effective.     

Optimization and validation of the reversed-phase high-performance liquid chromatography with fluorescence detection method for the separation of tocopherol and tocotrienol isomers in cereals, employing a novel sorbent material

The separation and determination of tocopherols (Ts) and tocotrienols (T3s) by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed and validated after optimization of various chromatographic conditions and other experimental parameters. Analytes were separated on a PerfectSil Target ODS-3 (250 × 4.6 mm, 3 μm) column filled with a novel sorbent material of ultrapure silica gel. The separation of Ts and T3s was optimized in terms of mobile-phase composition and column temperature on the basis of the best compromise among efficiency, resolution, and analysis time. Using a gradient elution of mobile phase composed of isopropanol/water and 7 °C column temperature, a satisfactory resolution was achieved within 62 min. For the quantitative determination, α-T acetate (50 μg/mL) was used as the internal standard. Detection limits ranged from 0.27 μg/mL (γ-T) to 0.76 μg/mL (γ-T3). The validation of the method was examined performing intraday (n = 5) and interday (n = 3) assays and was found to be satisfactory, with high accuracy and precision results. Solid-phase extraction provided high relative extraction recoveries from cereal samples: 87.0% for γ-T3 and 115.5% for δ-T. The method was successfully applied to cereals, such as durum wheat, bread wheat, rice, barley, oat, rye, and corn.     

Role of riboflavin in beer flavor instability: determination of levels of riboflavin and its origin in beer by fluorometric apoprotein titration

A method for the quantitative determination of riboflavin levels in beer was developed. The method is based on the quenching of riboflavin fluorescence, which occurs when riboflavin binds to the aporiboflavin-binding protein from egg white. The method does not require any pretreatment of the beer before analysis, other than dilution, and proved to be simple, reliable, and sensitive. The lowest concentration that could be detected was approximately 10 nM riboflavin. The possible interference of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) with the determination of the riboflavin content of beer was excluded, because beer contains only a very small amount of FAD (0.03 microM) and no FMN. The riboflavin levels of the types and brands of beer investigated were in the range of 0.5-1.0 microM. The origin of the riboflavin in beer proved to be the malt. Hop and yeast hardly contributed to the riboflavin content of beer. Besides its use in the determination of riboflavin levels, the aporiboflavin-binding protein also provides a way to remove riboflavin from beer, which reduces the light sensitivity and the related lightstruck off-flavor formation in beer.     

Microwave-assisted rapid determination of vitamins a and e in beverages

A new rapid procedure for the determination of vitamins A and E in beverages has been developed and validated. Key steps include a microwave-assisted saponification of the sample and a single-step extraction of the vitamins prior to HPLC analysis. All sample preparation steps are carried out consecutively in the same vial. The vitamins are determined using normal-phase (Si-60) HPLC with fluorescence detection. The method is applicable to beverages with a content of all-trans-retinol >0.14 mg/L and/or a content of alpha-tocopherol >1 mg/L. Recoveries determined by spiking experiments ranged from 91.3 to 106.3%. The precision of the method is characterized by relative standard deviations of <2% for alpha-tocopherol and <5% for all-trans-retinol.