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Rapid quantitative method for simultaneous determination of benzoic acid, sorbic acid, and four parabens in meat and nonmeat products by liquid chromatography.

A liquid chromatographic (LC) method for the simultaneous determinations of benzoic acid, sorbic acid, and methyl, ethyl, propyl, and butyl parabens (methyl, ethyl, propyl, and butyl-p-hydroxybenzoates) in meat and nonmeat products was developed. Benzoic acid, sorbic acid, and parabens were extracted from meat and nonmeat products with 70% ethanol. After filtration, extracts were analyzed by reverse phase liquid chromatography. Homogeneously ground samples of fresh sausage and hamburger were fortified with benzoic acid, sorbic acid, and each paraben at 5 different concentrations. Average recovery (after discarding outliers) for each preservative at all 5 levels was greater than 95% with a coefficient of variation less than 5%.

Animals↗

Sorption of benzoic acid, sorbic acid, benzyl alcohol, and benzalkonium chloride by flexible tubing.

Earlier studies using methylparaben, propylparaben, and several types of flexible tubing showed that the parabens are sorbed by a number of commonly used tubing. The sorption increased with increase in time of contact and the tubing surface area. The temperature and pH ranges tested did not affect sorption. Current studies evaluated other commonly used antimicrobial preservatives used in oral, parenteral, opthalmic, nasal, and other pharmaceutical products. These preservatives include benzoic acid, sorbic acid, benzyl alcohol, and benzalkonium chloride. Results show that all preservatives, except benzalkonium chloride, are sorbed by several types of flexible tubing. After 24 hr, some types of tubing sorbed over 40% of benzoic acid, benzyl alcohol, and about 30% of sorbic acid. Significant losses were observed within a few hours. No sorption of benzalkonium chloride occurred in the tubing tested. Three different types of fluoropolymer resin tubing, Teflon (flourinated ethylene propylene (FEP), Teflon perfluoroalkoxy) (PFA) and Teflon polytetrafluoroethylene (PTFE, NXT), fluoropolymer FEP laminated Tygon tubing, and Zelite do not sorb any of the preservatives. Silicone tubing shows the highest sorption of preservatives. After 120 hr, as much as 64% of the preservative is sorbed by some tubing. Detailed studies using silicone tubing show that the sorption increases with increase in surface area at 25 degrees C, and increasing the temperature to 40 degrees C shows little change in sorption. At the concentration range generally used in formulations, the concentration of the preservative has no significant effect on sorption. Refilling the tubing with fresh preservative solution after contact with preservative solution for 168 hr still causes extensive sorption of the preservative. Desorption studies indicate that the sorbed preservatives are desorbed in water to a limited extent over 120 hr. Results are important in situations where short or extended interruptions occur in the processing and filling of products. This can lead to decreased and variable results due to residence of the solution in the tubing.

Adsorption↗

Extraction of organic acids by ion-pair formation with tri-n-octylamine. Part V. Simultaneous determination of synthetic dyes, benzoic acid, sorbic acid, and saccharin in soft drinks and lemonade syrups.

Synthetic dyes, benzoic acid, sorbic acid, and saccharin are extracted simultaneously from soft drinks with 0.01M tri-n-octylamine at pH = 5.5 and are back-extracted to an aqueous phase with 0.1M sodium perchlorate. The perchlorate solution is injected directly into the reverse phase liquid chromatographic system which permits the separation of all the substances investigated. Forty-six commercial samples of soft drinks and 11 lemonade syrups were analyzed. All samples conformed to the legal prescriptions.

Acids↗

Ferrous Iron Oxidation by Thiobacillus ferrooxidans: Inhibition with Benzoic Acid, Sorbic Acid, and Sodium Lauryl Sulfate.

Benzoic acid, sorbic acid, and sodium lauryl sulfate at low concentrations (5 to 10 mg/liter) each effectively inhibited bacterial oxidation of ferrous iron in batch cultures of Thiobacillus ferrooxidans. The rate of chemical oxidation of ferrous iron in low-pH, sterile batch reactors was not substantially affected at the tested concentrations (5 to 50 mg/liter) of any of the compounds.

Journal Article↗

Simultaneous analysis of dehydroacetic acid, benzoic acid, sorbic acid and salicylic acid in cosmetic products by solid-phase extraction and high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of dehydroacetic acid (DHA), benzoic acid (BA), sorbic acid (SOA) and salicylic acid (SA) was developed for application to cosmetic products. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-n-butylammonium (TBA) hydroxide as an ion-pair reagent. Cosmetic samples were purified by solid-phase extraction using Bond-Elut SI cartridges. Four acidic preservatives were eluted with methanol from cartridges. The HPLC assay was carried out using TSK gel ODS-80TM column (5 microm, 150 x 4.6 mm I.D.). The mobile phase consisted of a mixture of water and methanol (65:35, v/v) containing 2.5 mM TBA hydroxide adjusted with phosphoric acid to pH 7.0. The calibration curves of these preservatives showed good linearity with UV detection (235 nm). The correlation coefficients were better than 0.999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2.5 ng for DHA, 4.0 ng for BA, 2.0 ng for SOA and 5.5 ng for SA. The procedure described here is simple, selective and is suitable for quality control of finished cosmetic products.

Benzoic Acid↗

Simultaneous determination of preservatives (benzoic acid, sorbic acid, methylparaben and propylparaben) in foodstuffs using high-performance liquid chromatography.

A reversed-phased HPLC method that allows the separation and simultaneous determination of the preservatives benzoic (BA) and sorbic acids (SA), methyl- (MP) and propylparabens (PP) is described. The separations were effected by using an initial mobile phase of methanol-acetate buffer (pH 4.4) (35:65) to elute BA, SA and MP and changing the mobile phase composition to methanol-acetate buffer (pH 4.4) (50:50) thereafter. The detector wavelength was set at 254 nm. Under these conditions, separation of the four components was achieved in less than 23 min. Analytical characteristics of the separation such as limit of detection, limit of quantification, linear range and reproducibility were evaluated. The developed method was applied to the determination of 67 foodstuffs (mainly imported), comprising soft drinks, jams, sauces, canned fruits/vegetables, dried vegetables/fruits and others. The range of preservatives found were from not detected (nd)--1260, nd--1390, nd--44.8 and nd--221 mg kg(-1) for BA, SA, MP and PP, respectively.

Benzoic Acid↗

Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid.

The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.

Adaptation, Physiological↗

Gas-liquid chromatographic determination of benzoic acid and sorbic acid in foods: NMKL collaborative study.

A gas-liquid chromatographic method for the simultaneous determination of benzoic acid and sorbic acid in foods was collaboratively studied by 8 laboratories. Benzoic and sorbic acids are isolated from food by successive extractions with ether, sodium hydroxide, and methylene chloride, converted to trimethylsilyl (TMS) esters, and determined by gas-liquid chromatography. Phenylacetic acid and caproic acid are used as internal standards for benzoic acid and sorbic acid, respectively. Seven samples were collaboratively studied: almond paste, fish homogenate, and apple juice with benzoic and sorbic acid levels from 0.04 to 2 g/kg. Average recoveries (%) for benzoic and sorbic acids were as follows: almond paste, 99.6 and 101.2; fish homogenate, 99.2 and 97.4; and apple juice 98.2 and 106.6. The reproducibility coefficients of variation (%) for benzoic and sorbic acids at 0.5-2 g/kg levels were 3.5-6.1 and 5.2-9.0; and at the 0.04 g/kg level, 14.7 and 23.3, respectively. The method has been adopted official first action at 0.5-2 g/kg levels.

Benzoates↗

The weak acid preservative sorbic acid inhibits conidial germination and mycelial growth of Aspergillus niger through intracellular acidification.

The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 10(5)/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using (31)P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pH(cyt)) by more than 1 pH unit and a depression of vacuolar pH (pH(vac)) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pH(cyt). NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.

Aspergillus niger↗

Genotoxicity study of reaction products of sorbic acid.

Sorbic acid (E200) and its salts (potassium and calcium sorbate: E202 and E203) are allowed for use as preservatives in numerous processed foods. Sorbic acid has a conjugated system of double bonds which makes it susceptible to nucleophilic attack, sometimes giving mutagenic products. Under conditions typical of food processing (50-80 degrees C), we analyzed the cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines. Mutagenesis studies, involving the Ames test and genotoxicity studies with HeLa cells and plasmid DNA, showed that none of the products studied presented either mutagenic or genotoxic activities.

Mutagenicity Tests↗

Effects of sorbic acid and sorbic acid-nitrite in vivo on bone marrow chromosomes of mice.

The effect of sorbic acid alone and in combination with sodium nitrite has been studied on bone marrow chromosomes of mice following 30 days oral treatment. Bone marrow of mice exposed to sorbic acid (15 mg/kg) and sorbic acid nitrite (7.5-1 mg/kg) showed an increase in mitotic index indicating that the drugs had an effect on spindle apparatus. Sorbic acid effected spindle activity but did not damage chromosomes, whereas nitrite itself was clastogenic. However, a combination of half the concentration each of sorbic acid (15 mg/kg) and sodium nitrite (2 mg/kg) together gave synergistic effects, which may be ascribed to the formation of some genotoxic compound in vivo.

Administration, Oral↗

Sister chromatid exchanges and micronuclei formations induced by sorbic acid and sorbic acid-nitrite in vivo in mice.

The in vivo induction of sister chromatid exchanges and micronuclei formations by acute treatment with different concentrations of sorbic acid and by nitrite, individually and in combination, was studied in bone marrow cells of mice. A significant increase in the frequency of sister chromatid exchanges was only observed with the three higher concentrations of sorbic acid when compared to a distilled water control. Sodium nitrite produced a significant increase at all doses tested. A combination of half the concentration of sorbic acid and of sodium nitrite gave an additive effect over that of sorbic acid or sodium nitrite alone. In the micronucleus assay, the highest dose of sorbic acid (150 mg/kg body weight) produced a significant increase in micronuclei formations compared to the distilled water control. Sodium nitrite alone induced significant numbers of micronuclei at all concentrations tested when compared to the negative control. However, a combination of half the concentration of sorbic acid and of sodium nitrite gave synergistic effects which could possibly be ascribed to the formation of certain genotoxic compounds in vivo.

Animals↗

Effect of surfactants and dispersed components on the activity and reactivity of sorbic acid.

In common with many other carboxylic acids, sorbic acid shows significant solubility in aqueous and non-aqueous solvents. The presence of a non-aqueous phase (e.g. fat) can markedly affect the concentration of the preservative in the aqueous phase. Solute distribution between the two phases is pH- and concentration-dependent. The presence of dissolved surfactants in the aqueous phase will also affect the activity of sorbic acid. This effect is due to the partitioning of the solute into surfactant micelles. The presence of dispersed components and surfactant micelles also has a marked effect on the reactivity of sorbic acid. Whereas thiols react slowly with sorbic acid, the rate of reaction is increased many-fold by the addition of low molecular weight surfactants. The mechanism of this catalysis will be explained. It has been suggested that sorbic acid inhibits enzymes by reacting with sulphydryl groups of the proteins. Kinetic data from model system studies suggest that the sorbic acid-thiol reaction may be too slow for it to be an obvious means of enzyme inhibition. However, this does not take account of possible catalysis of the reaction in the microenvironment of the protein, perhaps in a manner similar to that identified with low molecular weight surfactants.

Food Preservation↗

High performance liquid chromatographic determination of sorbic acid in wine.

A method is described for the qualitative and quantitative determination of 2,4-hexadienoic acid (sorbic acid) by high performance liquid chromatography (HPLC). The results show excellent reproducibility (+/- 2.55%) and agree well with values obtained using the official AOAC ultraviolet method for both fortified wine and commercial wines. Sorbic acid is separated by HPLC, using a strong anion exchange resin, Zipax SAX, eluted with 0.01M Na2B4O7, and detected using UV (254 nm) detector. Sodium benzoate is used as an internal standard.

Chromatography, High Pressure Liquid↗

Urinary excretion of mutagens and the effects of sorbic acid on the lipid peroxide level in the mice fed on a 15% sorbic acid diet.

The urine of the mice fed on a 15% sorbic acid diet was treated with or without beta-glucuronidase and was fractionated by XAD-2 column chromatography. The non-polar urine fraction was slightly mutagenic towards TA 98 when metabolically activated, but not towards TA 100. From the comparison of thin-layer chromatograms between the intestinal and urinary samples, it was suggested that a part of the mutagens produced in the intestine was excreted in the urine. As for the lipid peroxidation, the levels of lipid peroxide in the liver of the mice fed on a 15% sorbic acid diet were lower than those in the control over the feeding period of 15 months. Moreover, there was a correlation between the concentration of sorbic acid (X) in the diet and the lipid peroxide level (Y) in mice fed on potassium sorbate diets, obeying the linear equation, Y=-8.39X+ 341 (p less than 0.01). However, the lipid peroxide levels of 15% sorbic acid group did not fit with the above equation, and was higher than those of 20.1% potassium sorbate group, which was equivalent to 15% sorbic acid group in respect of sorbic acid concentration. Accordingly, the difference of lipid peroxide levels between the two groups (15% sorbic acid and 20.1% potassium sorbate group) might reflect productive difference of the mutagens.

Administration, Oral↗