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    • 专利摘要:
    Method of determination of glyoxal. The present invention relates to a method for determining glyoxal; the determination is made by derivatization and simultaneous extraction of the glyoxal in an aqueous sample; preferably said determination is made in alcoholic beverages. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2611954A1
    申请号:ES201730211
    申请日:2017-02-20
    公开日:2017-05-11
    发明作者:Nielene María MORA DIEZ；María Isabel ACEDO VALENZUELA；María Isabel RODRÍGUEZ CÁCERES；Mónica PALOMINO VASCO
    申请人:Universidad de Extremadura；
    IPC主号:
    专利说明:

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    GLIOXAL DETERMINATION METHOD
    The present invention relates to a glyoxal determination method. The determination is made by derivatization and simultaneous extraction of glyoxal from an aqueous sample. Preferably said determination is made in alcoholic beverages.
    Background of the invention
    Glyoxal belongs to the family of low molecular weight dicarbonyl compounds. These compounds are important because they can produce toxicological effects in the human body, because they react quickly to form reactive carbonic and oxygen species, which are related to diabetes, cardiovascular problems and age-related diseases.
    Glioxal appears in fermented foods such as wine and beer due to the microbial activity of Saccharomyces cerevisae and Leuconostoc oenos. Dicarbonyl compounds are important due to their sensory impact, their reactivity and their potential microbiological effects. The presence of glyoxal can lead to detriments in the taste of wine and beer, by reacting with amino acids that have sulfur in their structure, since substances with unpleasant odors are generated. For all these reasons, it is important to know and control the amount of glyoxal present in food.
    For example, patent method EP3030904 describes a method of determining alpha dicarbonyl compounds, specifically glyoxal, by derivatizing said compound and detecting the derivatized product by fluorescence. Said method is selective for glyoxal.
    Therefore, it is of great interest to be able to develop new glyoxal determination methods that are precise and selective for this compound.
    Description of the invention
    Due to its structure, glyoxal has neither absorbance nor fluorescence, so it is difficult to determine and its derivatization is necessary.
    As for the state of the art, in the methods described for the glyoxal derivatization the reactions require long times, from two to three hours and high temperatures of 60 ° C to 90 ° C. In the method of the invention the reaction time is reduced because the derivatization of glyoxal and its extraction takes place simultaneously. On the other hand, it takes place at room temperature. The method of the present invention reduces the time and temperature with respect to the methods already known.
    In the method of the invention very small solvent volumes are used which is always favorable from the economic and environmental point of view.
    The method of the invention is selective for glyoxal since the rest of the dicarbonyl compounds do not react under the conditions of the method of the invention.
    The method has good precision.
    The present method derivatizes and extracts glyoxal by means of a dispersive liquid micro-extraction.
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    Therefore, one aspect of the invention relates to a glyoxal determination method characterized in that it comprises the following steps:
    a) mixing an extracting solvent and a dispersing solvent;
    b) adding 3,4 diaminopyridine to an aqueous sample containing glyoxal;
    c) set the pH in a range between 10.5 and 12.5;
    d) introducing the mixture of step a) in the aqueous sample presenting glyoxal and 3,4 diaminopyridine;
    e) homogenize the mixture of step d);
    f) centrifuge and
    g) collect the phase where the glyoxal is and elute it in a chromatographic system, where stages a) and b) can be performed in any order.
    Description of a preferred realization
    As referenced above, the present invention relates to a glyoxal determination method.
    The extracting solvent must be insoluble in water, have a high extraction capacity for the target analyte, have a good performance in chromatography and easily disperse in the aqueous phase.
    Therefore, the extractant solvent is selected from aliphatic alkyl halides of 1 to 6 carbon atoms; preferably, it is selected from: dichloromethane, chloroform, carbon tetrachloride or mixtures thereof. In particular, the extracting solvent is dichloromethane.
    The dispersing solvent must have a good solubility both in the aqueous phase and in the organic phase. Therefore, the dispersing solvent is preferably selected from: methanol, acetone, dimethylformamide, acetonitrile, and aliphatic alcohols of 1 to 6 carbon atoms or mixtures thereof. In particular, the dispersing solvent is methanol, 1-propanol, butan-1-ol and pentan-2-ol or mixtures thereof.
    In particular, the combination of solvents is dichloromethane / butan-1-ol. In particular, the volume used is 0.75 mL of dispersant solvent and 150 pL of extractant solvent.
    Preferably the pH in step c) is adjusted between 10.5 and 12.5. In particular, the pH is 11.6. Particularly in step c) the ratio is 1:12 (in ppm) between glyoxal: 3, 4 diaminopyridine.
    Preferably, stage e) of homogenization is carried out for a time between 0.5 min and 5 min. In particular it is 1 min. In particular, homogenization is performed by agitation. Particularly, step f) of centrifugation is performed at 3000 rpm and for 5 min.
    Preferably step g) is fluorometrically monitored. In particular, the determination is made in an HPLC with a fluorescence detector.
    Preferably in step g) after collecting the phase where glyoxal is found, methanol is added until a homogeneous solution is obtained that prevents evaporation of solvent, the pH is adjusted to 2 in order to maximize the fluorescent signal.
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    Example 1
    On an aqueous solution of glyoxal (wine or beer): 3.4 diaminopyridine in a 1:12 ratio with a final volume of 1.5 mL and a pH of 11.5 to 12, a mixture formed by 750pL was added quickly of 1-butanol and 150pL of CH2Cl2, the whole was homogenized in vortex for 1 minute, centrifuged at 3000 rpm for 5 minutes, expected 10 minutes, 0.5 mL of chloroacetic acid / sodium chloroacetate buffer (ClCH2COOH / ClCH2COONa) (0.3M, pH 2.0) and 1.5 mL of methanol and flush with H2O.
    The sample was introduced into the HPLC equipment and glyoxal was determined.
    Example 2
    The method of the invention was applied to several monovarietal wines belonging to the D.O. "Ribera del Guadiana” and several brands of Spanish beers. The results are shown in Table 1.
    Table 1. Glioxal concentrations (expressed in mg / L) found in the different samples analyzed together with their standard deviation __________________________
     Drink  Type Concentration (mg / L)
     Came  Red wine 1 7.8 ± 0.4
     Red wine 2  7.4 ± 0.3
     White wine  9.5 ± 0.5
     Pink wine  5.8 ± 0.9
     Beer  Beer 1 6.5 ± 0.8
     Beer 2  2.8 ± 0.5
     Beer 3  3.4 ± 0.6
     Black beer  5.57 ± 0.08
     Alcohol-free beer  6.5 ± 0.9
    Glioxal levels range between 5.8 and 9.5 mg / L in wines and between 2.8 and 6.5 mg / L in beers. These results are consistent with the bibliography, which describes higher concentrations of glyoxal in wine than in beer. It is also important to note that these concentrations are low and that they do not pose any risk to health.
    In order to evaluate the accuracy, a sample of red wine was analyzed for five consecutive days. The relative standard deviation calculated was 4.1% for 7.4 mg / L glyoxal.
    权利要求:
    Claims (7)
    [1]
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    1. Method for determining glyoxal characterized by comprising the following stages:
    a) mixing an extracting solvent and a dispersing solvent;
    b) adding 3,4 diaminopyridine to an aqueous sample containing glyoxal;
    c) set the pH in a range between 10.5 and 12.5;
    d) introducing the mixture of step a) in the aqueous sample presenting glyoxal and 3,4 diaminopyridine;
    e) homogenize the mixture of step d);
    f) centrifuge and
    g) collect the phase where the glyoxal is and elute it in a chromatographic system, where stages a) and b) can be performed in any order.
    [2]
    2. Method for determining glyoxal according to revindication 1 characterized in that the extractant solvent is selected from aliphatic alkyl halides of 1 to 6 carbon atoms.
    [3]
    3. Glioxal determination method according to revindication 2 characterized in that the extractant solvent selects between: dichloromethane, chloroform, carbon tetrachloride or mixtures thereof.
    [4]
    4. Method of determination according to any of claims 1-3 characterized in that the dispersing solvent is selected from: aliphatic alcohols of 1 to 6 carbon atoms, methanol, acetone, dimethylformamide, acetonitrile, or mixtures thereof.
    [5]
    5. Method of determination according to revindication 4 characterized in that the dispersing solvent is selected from: methanol, 1-propanol, butan-1-ol and pentan-2-ol or mixtures thereof.
    [6]
    6. Method for determining glyoxal according to any of claims 1-5, characterized in that the pH in step b) is between 10.5 and 12.5.
    [7]
    7. Method for determining glyoxal according to any one of claims 1-6 wherein the eluate from step g) is monitored fluorimetrically.
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