A method for determining the stable isotope ratio of non-exchangeable hydrogen sites of glycerol in edible oils and fats

Abstract

The present invention proposes a method for determining the stable isotope ratio of non-exchangeable hydrogen sites of glycerol in edible oils and fats, belonging to the field of stable isotope analysis technology. The method comprises the following steps: a) preparing a preliminary glycerol sample; b) determining the stable hydrogen isotope ratio of glycerol in the preliminary glycerol sample using a gas chromatography-pyrolysis-stable isotope ratio mass spectrometer; c) selecting glycerol with a known stable isotope ratio of non-exchangeable hydrogen sites as a working standard substance, processing according to step a), and determining according to step b) to obtain the stable hydrogen isotope ratio of the glycerol working standard substance; and calculating the stable isotope ratio of the non-exchangeable hydrogen sites of glycerol in the edible oil to be tested by comparing the measurement result of the stable hydrogen isotope ratio of the glycerol working standard substance with the known stable isotope ratio of the non-exchangeable hydrogen sites of the glycerol working standard substance. The method is rapid and accurate, providing a new analytical method for adulteration detection and authenticity control of oils and fats.

Description

Technical Field

[0001] The invention belongs to the technical field of stable isotope analysis, and particularly relates to a method for determining the stable isotope ratio of non-exchangeable hydrogen sites of glycerol in edible oils and fats. Background Art

[0002] Oils and fats are an essential part of the human diet and, as daily necessities, are crucial to public health. With the nation's health policies and the nation's growing health needs, the edible oil industry is facing both new opportunities and challenges, placing higher demands on the authenticity and quality of edible oils.

[0003] Domestic and international research indicates that, unlike current characterization analysis focused on molecular content, stable isotope technology studies information at the atomic level of compounds. It has been successfully applied to adulteration detection and authenticity verification of fruit juices, honey, and alcoholic beverages. Several studies have also been reported in the edible oil field. For example, S. Kelly studied the differences between peanut oil, palm oil, and sunflower oil based on the carbon isotope characteristics of fatty acids. Jorge E. Spangenberg studied the stable carbon isotope ratios of olive oil fatty acids. Fronza distinguished C3 and C4 vegetable oils based on the carbon isotope ratios of glycerol in edible oils. Fronza, G., identified the variety and origin of vegetable oils by measuring the stable oxygen isotope ratios of glycerol. In China, Jin Qingzhe et al. used carbon isotope ratios to detect blends of peanut oil and corn oil. Camin et al. studied the origin and variety characteristics of olive oil based on the stable hydrogen isotope ratios of fatty acids.

[0004] It can be seen that most of the research on stable isotopes is mainly focused on the carbon and oxygen isotope analysis and utilization of oils and fats as a whole, glycerol and fatty acids, and there has been no report on the application of glycerol hydrogen isotope ratios in oils and fats. Summary of the Invention

[0005] In order to solve the problem of difficulty in determining the stable isotope ratio of non-exchangeable hydrogen sites of glycerol in edible oils and fats, the present invention establishes a method that can process samples in batches and complete rapid analysis in a short time compared to the nuclear magnetic resonance method.

[0006] The main component of oils and fats is edible oils and fats, which are mainly composed of glycerol as the backbone and various fatty acids. According to the physical and chemical characteristics of oils and fats, not only are the types and contents of fatty acids different in different types of oils and fats, but even for the same type of oils and fats, the stable isotope ratios of different substances in the biological metabolism process are inconsistent. In addition, oils and fats are prone to moisture absorption, and the moisture in the oil matrix easily affects the determination of glycerol hydrogen isotope ratios. Therefore, the hydrogen isotope ratio of the entire oil and fat is difficult to use for accurate analysis of oil adulteration detection. Fatty acids (methyl esters) are a better choice, but the fatty acid content of different samples can vary, resulting in strict injection volume during the determination (commonly used GC-C-IRMS system) to produce a suitable signal intensity. Once the signal intensity does not meet the standard, it needs to be re-measured, affecting the analysis efficiency and accuracy of the results.

[0007] In comparison, isotope analysis of glycerol offers a significant advantage: as it is a backbone component of edible oils, the injection volume can be easily controlled. Therefore, determining the isotope ratio of glycerol's non-exchangeable hydrogen is crucial to determining its application.

[0008] One embodiment of the present invention provides a method for determining the stable isotope ratio of non-exchangeable hydrogen sites of glycerol in edible oils and fats, comprising the following steps:

[0009] a) Preparation of glycerol sample

[0010] a1) adding a reaction aid to the edible oil, performing a hydrolysis reaction, centrifuging, removing the lower layer, and adding hydrochloric acid solution to adjust the pH to 5-6;

[0011] a2) adding an alcohol reagent to the solution obtained in step a1) to dissolve the solution and then drying the solution, repeating the addition of the alcohol reagent to dissolve the solution and then drying the solution at least once to obtain a glycerol solid;

[0012] a3) dissolving the glycerol solids obtained in step a2) with an organic reagent to obtain a preliminary glycerol sample;

[0013] b) determining the stable hydrogen isotope ratio of glycerol in the initial glycerol sample using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry;

[0014] c) selecting glycerol with a known stable hydrogen isotope ratio at non-exchangeable hydrogen sites as a working standard substance, processing according to steps a2) to a3), and measuring according to step b) to obtain the stable hydrogen isotope ratio of the glycerol working standard substance;

[0015] d) By comparing the measured stable hydrogen isotope ratio of the glycerol working standard substance with the known stable isotope ratio of the non-exchangeable hydrogen sites of the glycerol working standard substance, combined with the measured stable hydrogen isotope ratio of the glycerol primary sample, the stable isotope ratio of the non-exchangeable hydrogen sites of glycerol in the edible oil to be tested is calculated accordingly.

[0016] In one embodiment of the present invention, in step a1), the reaction auxiliary agent is an organic solution of an alkaline reagent.

[0017] In one embodiment of the present invention, in step a1), the alkaline organic solution is a methanol solution of potassium hydroxide. Specifically, the concentration of the methanol solution of potassium hydroxide is 1-50 g / 200 ml. Preferably, the concentration of the methanol solution of potassium hydroxide is 23.2 g / 200 ml. The methanol solution of potassium hydroxide is primarily used to hydrolyze the edible oil to be tested to obtain the glycerol to be tested.

[0018] In the embodiment of the present invention, in step a1), the hydrochloric acid solution is added mainly to neutralize the potassium hydroxide in the reaction auxiliary agent.

[0019] In one embodiment of the present invention, in step a1), the reaction aid further comprises isooctane. The isooctane is primarily used to dissolve the fatty acids produced after hydrolysis, separating them from the glycerol and methanol, and facilitating centrifugal separation. Preferably, the ratio of edible oil, methanolic potassium hydroxide solution, and isooctane can be 0.4 g:1 mL:8 mL.

[0020] In one embodiment of the present invention, in step a1), the edible oil comprises at least one of olive oil, soybean oil or peanut oil.

[0021] In one embodiment of the present invention, in step a2), the alcohol reagent comprises at least one of methanol, ethanol, or propanol. The alcohol reagent is added primarily to replace the hydroxyl groups of glycerol with the hydroxyl groups in the alcohol reagent to achieve equilibrium, thereby eliminating the effect of glycerol's exchangeable hydrogens. The ratio of alcohol reagent to edible oil can be 10 ml: 0.1-0.5 g.

[0022] In the embodiment of the present invention, in step a2), the purpose of drying is to remove water from the sample, including water produced during fat hydrolysis and water in the hydrochloric acid solution, to prevent the chromatographic column from having difficulty separating water and glycerol when the sample is fed into the chromatographic column. Preferably, the drying is performed in a constant temperature drying oven.

[0023] In one embodiment of the present invention, in step a3), anhydrous acetone is used for dissolution, thereby eliminating the influence of water and alkaline reagents in the initial glycerol sample.

[0024] It should be noted that in step c) of the embodiment of the present invention, the glycerol working standard substance is pretreated using exactly the same reagents as in steps a2) and a3), and then the stable hydrogen isotope ratio of the entire molecule of the glycerol working standard substance is measured to ensure that the stable hydrogen isotope ratio in the solution system is stable and fixed.

[0025] In one embodiment of the present invention, in step d), specifically, by comparing the deviation between the stable hydrogen isotope ratio of the glycerol working standard substance and the known stable isotope ratio of the non-exchangeable hydrogen sites of the glycerol working standard substance, the same deviation between the stable hydrogen isotope ratio measurement result of the glycerol initial sample and the stable isotope ratio of the non-exchangeable hydrogen sites of glycerol in the edible oil to be tested is removed accordingly, and the stable isotope ratio of the non-exchangeable hydrogen sites of glycerol in the edible oil to be tested is calculated.

[0026] The present invention has the following advantages:

[0027] This invention provides a method for determining the stable isotope ratio of non-exchangeable hydrogen sites of glycerol in edible oils and fats. By hydrolyzing the edible oils and fats to eliminate uncontrolled interference from hydrogen-containing components in the solution system, the stable hydrogen isotope ratios in the sample and a glycerol working standard are measured using a stable isotope ratio mass spectrometer. Finally, the stable isotope ratio of the non-exchangeable hydrogen sites of glycerol in the edible oils and fats is calculated. This method is rapid and accurate, providing a new analytical approach for adulteration detection and authenticity control of oils and fats. DETAILED DESCRIPTION

[0028] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0029] Example 1 :Determination of stable hydrogen isotope ratios of glycerol in edible oils (olive oil, soybean oil, peanut oil)

[0030] a) Prepare KOH-methanol solution: weigh 23.2 g of potassium hydroxide solid and dissolve it in 200 ml of methanol.

[0031] b) Weigh 0.4 g of each edible oil (olive oil, soybean oil, peanut oil) into a 10 mL centrifuge tube. Add 1 mL of KOH-methanol solution to each tube. Shake for 2 minutes, then add 8 mL of isooctane. After standing for 12 hours, centrifuge at 10,000 rpm and remove the lower layer to obtain a methanol solution containing glycerol and KOH.

[0032] c) adjusting the pH of the methanol solution containing glycerol and KOH obtained in step b) to 5.0 with a dilute hydrochloric acid (1 mol / L) solution to obtain an acidic solution containing glycerol.

[0033] d) dissolving the glycerol-containing acidic solution obtained in step c) with 10 mL of methanol, allowing it to stand for 1 hour and then drying it. The solution was then redissolved with 1 mL of anhydrous acetone and dried again. The dissolution and drying process was repeated three times to completely replace the exchangeable hydrogen in the glycerol and remove any residual isooctane, thereby obtaining a glycerol-containing solid.

[0034] e) The solid material containing glycerol was dissolved in 1 mL of anhydrous acetone, and the stable hydrogen isotope ratio of glycerol was determined using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry. The results are shown in Table 1.

[0035] Table 1 Determination of stable hydrogen isotope ratios of glycerol in oil samples δD (‰)

[0036]

[0037] The stable hydrogen isotope ratios of glycerol in olive oil, soybean oil, and peanut oil were determined according to the method described in Example 1. After three treatments, the deviations of the measurement results were all better than 2‰, indicating that the method proposed in the present invention can accurately determine the stable hydrogen isotope ratios of glycerol in edible oils and fats.

[0038] Example 2 :Determination of Stable Hydrogen Isotope Ratios of Glycerol Working Standard

[0039] a) Glycerol working standard material was selected as the research object, and its non-exchangeable hydrogen site stable isotope ratio was -36.81‰;

[0040] b) Three 0.1 g portions of glycerol working standard material were weighed, dissolved in 1 mL of anhydrous acetone, and dried; the mixture was treated according to steps d) and e) of Example 1, dissolved in 10 mL of methanol, allowed to stand for 1 hour, and then dried. The dissolution and drying procedures were repeated three times, and finally each portion was re-dissolved in 1 mL of anhydrous acetone. The stable hydrogen isotope ratio of glycerol was determined by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry. The results are shown in Table 2.

[0041] Table 2 Determination of stable hydrogen isotope ratios after eliminating the influence of exchangeable hydrogen in glycerol δD (‰)

[0042] Simulation sample-1 Simulation sample-2 Simulation sample-3 Glycerol δD measured value -49.59 -47.74 -51.29

[0043] As shown in Table 2, the maximum difference in the measured values ​​of glycerol δD among the three simulated samples is only 2.55‰, which is within the allowable error range of hydrogen isotope determination and can be considered to be the same result.

[0044] It can be seen that no matter what the distribution characteristics of the hydrogen isotope ratio of the glycerol hydroxyl site are, its influence on the determination difference can be eliminated after treatment with methanol.

[0045] To further verify the stability of the stable hydrogen isotope ratio determination of glycerol, the glycerol working standard substance in step a) of Example 2 was taken and treated according to the pretreatment process in Example 1. The stable hydrogen isotope ratio of glycerol was measured using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry. The results of three measurements are shown in Table 3.

[0046] Table 3 Determination of stable hydrogen isotope ratios after eliminating the influence of exchangeable hydrogen in glycerol δD NE (‰)

[0047] Repeat 1 Repeat 2 Repeat 3 Glycerol δD measured value -49.25 -46.71 -50.26

[0048] Comparing Table 3 with Table 2, it can be seen that the average values ​​of the two groups of data (the average value of the three groups of data in Table 2 is -49.54‰ and the average value of the three groups of data in Table 3 is -48.74‰) differ by only 0.80‰, which is within the allowable error range and can be considered to be the same result.

[0049] Example 3 :Analytical method for stable isotope ratio of non-exchangeable hydrogen sites of glycerol in edible oils and fats

[0050] The difference between the true value of the known stable isotope ratio of glycerol non-exchangeable hydrogen sites and the measured average value of the stable hydrogen isotope ratio of glycerol obtained in Table 3 of Example 2 is (-36.81‰)-(-48.74‰)=11.93‰.

[0051] Therefore, the corresponding stable isotope ratios of non-exchangeable hydrogen sites of glycerol in olive oil, soybean oil, and peanut oil can be calculated as -73.59‰ (-85.52‰ + 11.93‰ = -73.59‰), -111.80‰, and

[0052] (-123.73‰+11.93‰=-111.80‰) and -162.82‰(-174.75+11.93‰=-162.82‰).

[0053] Comparative Example 1

[0054] The same as Example 2, the glycerol working standard substance was selected as the research object, except that it was first dissolved in three kinds of water with different stable hydrogen isotope ratios (see Table 4), step d) of Example 1 was omitted, and after drying, step e) was directly performed.

[0055] Table 4 Determination of stable hydrogen isotope ratios of glycerol standard materials after different water treatments δD (‰)

[0056] Simulation sample-1 Simulation sample-2 Simulation sample-3 δD value in water -31.63 -81.35 -142.37 Glycerol δD measured value -48.42 -54.32 -67.43

[0057] As shown in Table 4, the results of the determination of the same glycerol working standard material after dissolving it in water with different stable hydrogen isotope ratios (δD) and then drying it showed significant differences, and were significantly positively correlated with the δD value in water (correlation coefficient R 2 =0.9756), which shows that water affects the determination of glycerol. The reason is that glycerol contains three hydroxyl groups, which are easy to exchange hydrogen isotopes with other hydroxyl donors, thus affecting the hydrogen isotope ratio analysis of the entire glycerol molecule.

[0058] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining the stable isotope ratio of non-exchangeable hydrogen sites of glycerol in edible oils and fats, comprising the following steps: a) Preparation of glycerol sample a1) adding a reaction aid to edible oils and fats, performing hydrolysis reaction, centrifuging, removing the lower layer solution, and adding hydrochloric acid solution to adjust the pH to 5-6; wherein, The reaction aid comprises an organic solution of an alkaline reagent, wherein the organic solution of the alkaline reagent is a methanol solution of potassium hydroxide, and the concentration of the methanol solution of potassium hydroxide is 1-50 g / 200 ml; a2) adding an alcohol reagent to the solution obtained in step a1), dissolving the solution and then drying the solution, repeating the addition of the alcohol reagent, dissolving the solution and then drying the solution at least once to obtain a glycerol solid; the alcohol reagent comprises at least one of methanol, ethanol, or propanol; and the ratio of the alcohol reagent to the edible oil is 10 ml: 0.1-0.5 g; a3) dissolving the glycerol solids obtained in step a2) in anhydrous acetone to obtain a preliminary glycerol sample; b) determining the stable hydrogen isotope ratio of glycerol in the initial glycerol sample using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry; c) selecting glycerol with a known stable hydrogen isotope ratio at non-exchangeable hydrogen sites as a working standard substance, processing according to steps a2) to a3), and measuring according to step b) to obtain the stable hydrogen isotope ratio of the glycerol working standard substance; d) By comparing the measured stable hydrogen isotope ratio of the glycerol working standard substance with the known stable isotope ratio of the non-exchangeable hydrogen sites of the glycerol working standard substance, combined with the measured stable hydrogen isotope ratio of the glycerol primary sample, the stable isotope ratio of the non-exchangeable hydrogen sites of glycerol in the edible oil to be tested is calculated accordingly.

2. The method according to claim 1, characterized in that In step a1), the reaction auxiliary agent further includes isooctane.

3. The method according to claim 1, characterized in that In step a1), the edible oil comprises at least one of olive oil, soybean oil or peanut oil.

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