A method for analyzing glycerides and isomers thereof

Abstract

The application belongs to the field of light industry grease, and discloses a method for detecting and analyzing the content of positional isomer of same-acid glyceride by high performance liquid chromatography tandem mass spectrometry. Based on the derivatization treatment of same-acid glyceride, the hydroxyl group in the glyceride not combined with the fatty acid is connected with a new group (isocyanate), and the corresponding 1,2-DAG isocyanate derivative or 1,3-DAG isocyanate derivative is generated. The same-acid glyceride derivative is analyzed by using a liquid chromatograph-mass spectrometer, and the content of the positional isomer of same-acid glyceride is quantitatively analyzed by the relative content difference of two daughter ions generated under collision-induced dissociation of the derivative. The method can realize the quantification of the positional isomer of glyceride without separating the positional isomer of glyceride by chromatography, and only the conventional C18 chromatography is used, so that the development of special chiral chromatographic column or specific chromatographic separation technology is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of light industry oil and fat, and relates to an analysis method of glycerides and isomers thereof, in particular to a method for detecting and analyzing the content of positional isomers of same-acid glycerides by high performance liquid chromatography tandem mass spectrometry. BACKGROUND

[0002] Diacylglycerols (DAGs) are lipid substances with one glycerol skeleton and two fatty acid acyl groups, which are generated by hydrolysis of one fatty acid ester group in triacylglycerols (TAGs). DAGs are trace components of natural vegetable oil and endogenous intermediates of fat metabolism in vivo, and have applications in the fields of food industry, chemical industry and pharmaceutical industry. The reported effects include inhibition of triacylglycerol accumulation in vivo, reduction of visceral fat, weight loss, prevention of hypertension, coronary heart disease, diabetes, myocardial infarction and other cardiovascular diseases.

[0003] Glycerides have two isomers, which are divided into 1,3-DAGs and 1,2-DAGs. The two isomers are formed due to the different connection positions of fatty acids on the glycerol backbone. Since pancreatic lipase has 1,3 hydrolysis specificity, 1,3-DAGs can be completely hydrolyzed to free fatty acids in theory during small intestinal digestion and absorption, while 1,2-DAGs produce monoglycerides in addition to hydrolysis. After being absorbed, they can be re-esterified to triacylglycerols and accumulated in vivo. Therefore, the former is also the best configuration of functional oil glycerides.

[0004] In order to further study the functionality of glycerides, it is necessary to accurately identify the composition of glyceride positional isomers. Conventional chromatographic separation techniques are difficult to separate and identify the isomers of DAGs, so it is difficult to quantify them. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide an analysis method of glycerides and isomers thereof, in particular to a method for detecting and analyzing the content of positional isomers of same-acid glycerides by high performance liquid chromatography tandem mass spectrometry. Based on the derivatization of same-acid glycerides, the hydroxyl group in glycerides not combined with fatty acids is connected to a new group (isocyanate) to generate corresponding 1,2-DAG isocyanate derivatives or 1,3-DAG isocyanate derivatives. The same-acid glyceride derivatives are analyzed by liquid chromatography-mass spectrometry, and the content of positional isomers of same-acid glycerides is quantitatively analyzed by the relative content difference of two daughter ions generated under collision-induced dissociation of the derivatives.

[0006] The purpose of the present application is achieved by the following scheme:

[0007] An analytical method for diglycerides and their isomers includes the following steps:

[0008] (1) Mix isocyanate solution and homoglyceride, add stabilizer, and perform derivatization treatment. After the derivatization treatment is completed, remove solvent to obtain a derivative of homoglyceride.

[0009] (2) The obtained homoglyceride derivative was dissolved in an organic solvent and then purified by thin-layer chromatography to obtain a pure homoglyceride derivative.

[0010] (3) The purified isocyanate diglyceride derivative was dissolved in a solvent, and phosphate buffer was added. The solution was then injected into a high-performance liquid chromatography-tandem mass spectrometer for detection and analysis to obtain [AA]. + and [AB] + The percentage content of the two fragment ions (in the ion spectrum, the highest content ion is shown as 100% as a reference, and the other content is shown as a percentage), where A represents fatty acid and B represents isocyanate, then calculate the corresponding [AB]. + Relative ion content, of which [AB] + Relative ion content (%) = [AB] + Ion percentage content / ([AB]) + Ion percentage content + [AA] + (Ion percentage content);

[0011] (4) Replace the isocyanate diglyceride in step (1) with pure 1,2-DAG and pure 1,3-DAG, or with pure 1,2-DAG, pure 1,3-DAG, and a mixture of pure 1,2-DAG and pure 1,3-DAG, respectively. Then repeat steps (1)-(3) to obtain the corresponding [AB]. + The relative content of ions is calculated, along with the corresponding relative content of sn-ABA. The relative content of sn-ABA (%) is calculated as: sn-ABA content / (sn-AAB content + sn-ABA content), where sn-ABA is a 1,3-DAG isocyanate derivative, sn-AAB is a 1,2-DAG isocyanate derivative, A represents fatty acid, and B represents isocyanate; then [AB] is used as the basis for further calculation. + A standard curve was constructed with the relative content of ions as the ordinate and the relative content of sn-ABA as the abscissa. Different standard curves were obtained for different fatty acids.

[0012] (5) Replace the isocyanate diglyceride in step (1) with the diglyceride to be tested, and then repeat steps (1)-(3) to obtain the corresponding [AB]. + The relative content of ions, then [AB] +The relative content of the ions is substituted into the standard curve of the corresponding fatty acid in step (4) to obtain the relative content of sn-ABA, i.e. the relative content of 1,3-DAG in the glycerol diester to be tested.

[0013] The isocyanate in the isocyanate solution in step (1) is preferably 2-naphthyl isocyanate, the solvent of the isocyanate solution is at least one of chloroform, toluene, xylene, preferably chloroform; the mass concentration of the isocyanate in the isocyanate solution is 20%-40%.

[0014] To save costs, the isocyanate solution in step (1) is preferably prepared by the following method: 2-naphthylamine is dissolved and reacted with triphosgene in an organic solvent to generate isocyanate, and then extracted with a solvent to obtain an isocyanate solution. The organic solvent is at least one of chloroform, acetone, acetonitrile, preferably acetonitrile; the extraction solvent is at least one of chloroform, toluene, xylene, preferably chloroform.

[0015] In the preparation of the isocyanate solution in step (1), the concentration of 2-naphthylamine in the mixed solution formed by dissolving 2-naphthylamine and triphosgene in an organic solvent is preferably 0.1-2M, more preferably 0.2M, and the concentration of triphosgene is preferably 0.1-0.5M, more preferably 0.14M; the reaction refers to a reaction under nitrogen protection and in the dark, the reaction temperature is preferably 60-105°C, more preferably 80°C, and the reaction time is preferably 30-120min, more preferably 40min; the solvent used for extraction is one or more of chloroform, toluene, xylene, preferably chloroform.

[0016] The mass ratio of the isocyanate solution in step (1) to the same acid glycerol diester is 20-50:1, preferably 40:1.

[0017] The stabilizer in step (1) is methyl paraben, and the addition amount is 1-10‰wt of the mass of the isocyanate solution, more preferably 4‰wt. In the derivatization process, the addition of the methyl paraben stabilizer is beneficial to the formation of glycerol diester isocyanate derivatives and the stability under weak alkaline conditions, so that in the collision-induced dissociation, more stable two daughter ion fragments are produced, and the difference in the occurrence of steric hindrance effect is more significant.

[0018] The conditions for the derivatization treatment in step (1) are as follows: the reaction temperature is preferably 50-70°C, more preferably 60°C, the reaction time is preferably 15-30min, more preferably 20min, the vacuum condition is that the absolute pressure is preferably 2000-20000pa, more preferably 3000pa, and the entire reaction condition is in a light-proof environment.

[0019] The hydroxyl sites of the derivatized diglycerides are introduced with new groups, and the structure is more stable, which can avoid the inaccurate results caused by acyl migration (1,3-DAG and 1,2-DAG are converted into each other, and finally tend to a specific ratio, and the higher the temperature, the easier it is to occur).

[0020] The method for removing the solvent in step (1) is nitrogen blowing.

[0021] The organic solvent used in step (2) is at least one of n-hexane, chloroform, petroleum ether, and diethyl ether, and more preferably n-hexane.

[0022] In the thin layer chromatography separation condition in step (2), the developing agent is a mixture of n-hexane, diethyl ether, and anhydrous formic acid, and the ratio of n-hexane, diethyl ether, and anhydrous formic acid is preferably 60-80:10-20:5-15 (v / v), and more preferably 73:15:12 (v / v). After scraping the band of the diglyceride isocyanate derivative, the elution is performed with diethyl ether, and then the nitrogen blowing concentration treatment is performed.

[0023] The solvent used in step (3) is n-hexane:methanol (the ratio is preferably 2-6:1, and more preferably 4:1, v / v), and the concentration of the obtained solution after dissolution is 3-10 mg / ml, and preferably 5 mg / ml.

[0024] The phosphate buffer solution used in step (3) is a solution prepared by proportioning Na2HPO4 and NaH2PO4 to 0.1-0.5 M (the concentration of phosphate), and the pH is preferably 6.8-8.4, and more preferably a buffer solution of 0.1 M and pH 7.8.

[0025] The amount of the phosphate buffer solution added in step (3) is 0-20% of the volume of the solution after dissolution, and more preferably 10%.

[0026] The addition of the phosphate buffer solution in the pretreatment process maintains the weak alkalinity of the solution and supplements sodium ions, which helps the diglyceride isocyanate derivative enter the collision-induced dissociation cell and can be ionized into a sodium adduct, which is beneficial to the generation of neutral loss daughter ions and improves the ionization efficiency.

[0027] In the liquid chromatography tandem mass spectrometry analysis condition in step (3), the mobile phase A is methanol, and sodium acetate or ammonium formate is added, and more preferably sodium acetate, and the concentration of the prepared solution is 0.001-0.01 mol / L, and more preferably 0.006 mol / L; and the mobile phase B is acetonitrile or acetonitrile-water, and more preferably acetonitrile.

[0028] In the liquid chromatography tandem mass spectrometry analysis condition in step (3), the liquid chromatography column is a conventional C18 chromatography column.

[0029] The collision-induced dissociation voltage in the mass spectrometry condition of the liquid chromatography tandem mass spectrometry analysis condition described in step (3) is preferably 10-40 eV, more preferably 15 eV, and the relevant parent ions are cracked, and the types and regioisomer contents of the sodiumated adduct parent ion peaks and the corresponding fragment ion peaks of the same-acid diglyceride derivatives are identified and quantified.

[0030] The corona voltage in the mass spectrometry condition of the liquid chromatography tandem mass spectrometry analysis condition described in step (3) is preferably 4.4-5.2 kV, more preferably 4.8 kV, the cone voltage is preferably 40-60 V, more preferably 50 V, the capillary voltage is preferably 3.1-3.8 kV, more preferably 3.4 kV, the source temperature is preferably 80-120 °C, more preferably 100 °C, the probe temperature is preferably 450-550 °C, more preferably 500 °C, the desolvation temperature is preferably 440-530 °C, more preferably 500 °C, the cone gas flow is preferably 40-50 L / h, more preferably 45 L / h, the desolvation gas flow is preferably 380-410 L / h, more preferably 400 L / h, and the gas flow rate is preferably 0.5-0.8 mL / min, more preferably 0.6 mL / min.

[0031] The pure 1,2-DAG and the pure 1,3-DAG in step (4) are both same-acid diglycerides, and the fatty acids in the same-acid diglycerides are preferably one of palmitic acid, stearic acid, and oleic acid;

[0032] In step (4), the same-acid diglycerides in step (1) are replaced by pure 1,2-DAG and pure 1,3-DAG, respectively, and after derivatization treatment, two sn-ABA relative contents, i.e., two x values, can be obtained, and two corresponding [AB] + ion relative contents, i.e., two y, can be obtained by high-performance liquid chromatography tandem mass spectrometry, and then the corresponding linear standard curve is obtained by taking the two sets of data as the starting point and the end point.

[0033] In step (4), the same-acid diglycerides in step (1) are replaced by pure 1,2-DAG, pure 1,3-DAG, and a mixture of pure 1,2-DAG and pure 1,3-DAG, respectively, so that multiple sets of x and y can be obtained, and the standard curve obtained is more accurate.

[0034] According to the types of fatty acids in the same-acid diglycerides, different standard curves of the same-acid diglycerides of different fatty acids can be obtained in step (4).

[0035] When the test is performed on the test DAG in step (5), the liquid chromatography can separate the DAGs with different equivalent carbon numbers (such as dipalmitin and distearin) in the process of injecting into the high performance liquid chromatography tandem mass spectrometer for detection and analysis in step (3), that is, the homologous DAGs of different fatty acids are separated, and then the mass spectrometry is performed on the homologous DAGs of different fatty acids respectively, and the [AB] + ion relative content (%) is substituted into the standard curve of the corresponding fatty acid.

[0036] The mechanism of the present application is as follows:

[0037] The isomers of DAGs include regioisomers and chiral isomers, for example, 1,3-DAG and 1,2-DAG are a pair of regioisomers, and 1,2-DAG further includes a pair of stereoisomers (1,2-DAG, 2,3-DAG), the present application only identifies and quantifies the regioisomers of DAGs, and only identifies the homologous DAGs (the compound formed by derivatization of the homologous DAG can produce two fragment ions under low-energy collision-induced dissociation), that is, the two fatty acids connected to the glycerol skeleton of the DAG are the same, by chemical modification of the DAG, a new group is introduced and combined with the hydroxyl group in the DAG which is not esterified with the fatty acid to form a new compound. Then, separation and identification are performed by high performance liquid chromatography tandem mass spectrometry, wherein the separation purpose of the liquid chromatography is to separate the DAGs with different equivalent carbon numbers (such as dipalmitin and distearin), and the mass spectrometry not only provides the molecular weight information of the DAG for identification, but also quantifies the relative content of the daughter ion fragments, the principle is based on that under the condition of selecting a suitable voltage for collision-induced dissociation (CID), two fragment ions are produced, [AA] + and [AB] + Due to the steric hindrance effect, the group at the sn-2 position is more difficult to lose than the group at the sn-1,3 position, thereby resulting in that the [AB] + fragment ion content is relatively higher, and the [AA] +The content of fragment ions is relatively low. Therefore, a linear calibration curve is constructed according to the content of fragment ions generated by pure 1,2-DAG isocyanate and pure 1,3-DAG isocyanate, with the relative content of sn-ABA (%) (sn-ABA / (sn-AAB+sn-ABA)) as the x-axis (sn-ABA is a 1,3-DAG isocyanate derivative, sn-AAB is a 1,2-DAG isocyanate derivative, A represents a fatty acid, and B represents an isocyanate; the conversion rates of 1,3-DAG and 1,2-DAG after derivatization with isocyanate are both 100%, so the content of sn-AAB and sn-ABA can be calculated according to the content of 1,3-DAG and 1,2-DAG in the raw material), and the relative content of [AB] + ion (%) ([AB] + / ([AB] + +[AA] + ) as the y-axis. The content of [AB] + and [AA] + ions obtained by high-performance liquid chromatography tandem mass spectrometry of the isocyanate derivative generated by derivatization of the homodag in the sample is substituted into the corresponding linear calibration curve, and the percentage content of the homodag region isomer in the sample can be calculated.

[0038] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0039] The present method can realize the quantification of diglyceride region isomers without the need for chromatographic separation of diglyceride region isomers, and only conventional C18 chromatographic analysis is used, thereby reducing the use of special chiral chromatographic columns or the development of specific chromatographic separation techniques. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure 1 is the ion spectrum of the sodium addition compound of the 1,2-DAG isocyanate derivative in Example 1 of the present application, wherein [1] is the [AB] + ion, [2] is the [AA] + ion, and [M+Na] + is the parent ion of the sodium addition compound of the diolein isocyanate derivative.

[0041] Figure 2 Figure 2 is the ion spectrum of the sodium addition compound of the 1,3-DAG isocyanate derivative in Example 1 of the present application, wherein [1] is the [AB] + ion, [2] is the [AA] + ion, and [M+Na] + is the parent ion of the sodium addition compound of the diolein isocyanate derivative.

[0042] Figure 3 Conversion of isocyanate to the corresponding glycerol diester isocyanate derivative in the present invention.

[0043] Figure 4 Conversion of isocyanate to the corresponding glycerol diester isocyanate derivative in the present invention. DETAILED DESCRIPTION

[0044] The present invention is further described in detail by the following examples and drawings, but the embodiments of the present invention are not limited thereto. In the examples, the specific conditions not mentioned are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not mentioned by the manufacturer, and are all conventional products that can be purchased on the market.

[0045] The instrument used in the examples is Agilent 1290 liquid chromatograph-tandem 6490 triple quadrupole mass spectrometer.

[0046] In the examples, the phosphate buffer is prepared by the following method: 1 mol / L Na2HPO4 and 1 mol / L NaH2PO4 are mixed at a volume ratio of 89.6:10.4, and then diluted to 1 L with deionized water to obtain a 0.1 M sodium phosphate solution, pH = 7.8.

[0047] Example 1

[0048] (1) Dissolve 1 mmol of 2-naphthylamine and 0.7 mmol of triphosgene in 5 ml of acetonitrile, protect with nitrogen, and react at 80°C for 40 min under light shielding conditions to generate isocyanate. After removing the solvent by nitrogen blowing, extract the isocyanate with chloroform.

[0049] (2) Take 8 g of the chloroform solution (mass concentration 30%) of the extracted isocyanate and mix with 200 mg of glycerol diester sample, and add 30 mg of methyl p-hydroxybenzoate, and react at 60°C for 20 min. The reaction is carried out under light shielding, water circulating pump vacuum (absolute pressure = 3000 Pa) conditions for derivatization reaction.

[0050] (3) After removing the solvent from the product generated after the above derivatization reaction, dissolve it with 5 mL of n-hexane, separate and purify it by thin layer chromatography, and develop it with n-hexane / ether / anhydrous formic acid (73:15:12, v / v). Scrape off the glycerol diester isocyanate derivative band, elute it with ether, and remove the solvent.

[0051] (4) The separated diglyceride isocyanate derivative 25 mg is dissolved using 5 ml of n-hexane / methanol (4:1, v / v), and 500 ul of phosphate buffer with pH = 7.8 is added. After filtration, it is injected into a high-performance liquid chromatograph-mass spectrometer for analysis. The liquid chromatograph conditions are as follows: a diamonsil plus C18 chromatographic column; specifications: 250 mm x 4.6 mm i.d., particle size 4 μm; source: DIKMA, China; mobile phase: phase A is a methanol solution containing sodium acetate (0.006 mol / L), phase B is acetonitrile, column temperature 25 °C, sample injection amount 1 μL; solvent gradient elution program and flow rate: see Table 1.

[0052]

[0053] Mass spectrometry conditions: positive ion mode (ESI + ), corona voltage = 4.8 kV, capillary voltage = 3.4 kV, cone voltage = 50 V, source temperature = 100 °C, probe temperature = 500 °C, desolvation temperature = 500 °C, cone gas flow = 45 L / h, desolvation gas flow = 400 L / h. Gas flow rate is 0.6 mL / min, CID voltage: 15 eV.

[0054] (5) Pure 1,3-dipalmitin, pure 1,2-dipalmitin are respectively used instead of the diglyceride sample in step (2), and then steps (2)-(4) are repeated to obtain 2 groups of [AB] + ion relative content (%, + / ([AB] + +[AA] + )), and 3 repeated experiments are obtained, i.e. 6 groups of [AB] + ion relative content, and the corresponding sn-ABA relative content (%) (sn-ABA / (sn-AAB+sn-ABA)) is calculated (as shown in Figure 3 , after 20 min of reaction, the conversion rate can basically reach 100%, so the corresponding sn-ABA relative content can be calculated according to the content of 1,3-DAG and 1,2-DAG in the raw material), wherein A is palmitic acid, B is isocyanate, sn-ABA is 1,3-DAG isocyanate derivative, and sn-AAB is 1,2-DAG isocyanate derivative. The sn-ABA relative content is taken as the x-axis, and the [AB] + ion relative content is taken as the y-axis, and a linear standard curve of the same acid diglyceride containing palmitic acid (hereinafter referred to as PP diglyceride) is obtained by 6 groups of data obtained by repeating three experiments.

[0055] The fatty acid palmitic acid in step (5) is replaced by stearic acid and oleic acid respectively to obtain the linear standard curve of the corresponding homoglyceride containing stearic acid (hereinafter referred to as OO glyceride) and the linear standard curve of the homoglyceride containing oleic acid (hereinafter referred to as SS glyceride).

[0056] The PP and SS, OO glyceride linear calibration curves obtained by the method described in Example 1 are y = 0.2396x + 53.3, y = 0.1611x + 64.073, y = 0.1764x + 54.622, R 2 respectively 0.99, 0.99, 0.98.

[0057] The accuracy and precision are verified by 1:1 mass ratio (i.e. molar ratio) of pure 1,3-DAG and pure 1,2-DAG to configure 1,2-DAG and 1,3-DAG mixed samples with known regional isomer content (total concentration of 50 μg / mL-5000 μg / mL), and then the reliability of the detection results and the method under different total concentrations is verified by linear calibration curve after derivatization. The test is 5 times, and the specific data is shown in Table 1:

[0058] Table 1 Stability test

[0059]

[0060] Because the sample cannot be tested immediately after being prepared in the actual process, it will generally be waiting for a period of time due to time problems, so the present application selects 2h at room temperature after derivatization before sampling analysis. In order to avoid the influence of high temperature storage, the present application also sets up 80℃ storage for 2h after derivatization before sampling analysis, and the specific data is shown in Table 1. From Table 1, it can be seen that the performance parameters of the method of the present application are excellent, the linear calibration curve related coefficient for quantifying homoglyceride regional isomer constructed by the method of the present application is high, the reproducibility is good, the accuracy and precision are good under the concentration of 50-5000ug / mL, and the detection results of the sample after derivatization have good stability, so the method has high practicability and reliability. In the subsequent test, if there is no special instruction, the sample is selected for analysis after 2h at room temperature after derivatization.

[0061] Example 2

[0062] The present embodiment is the same as Example 1 except for the following technical features: 8g of the above chloroform solution containing isocyanate (concentration 30%) is mixed with 200mg of glyceride sample, and 20mg of methyl p-hydroxybenzoate is added, and the reaction is carried out at 70℃ for 25min. The reaction is carried out under the conditions of light shielding, water circulating pump vacuum (absolute pressure = 3000Pa) and derivatization reaction.

[0063] The measured PP and SS, OO diglyceride linear calibration curves obtained by the method described in Example 1 are y = 0.2257x + 53.338, y = 0.1673x + 54.956, y = 0.1835x + 62.11, R 2 respectively 0.98, 0.99, 0.96.

[0064] Example 3

[0065] This example is the same as Example 1 except for the following technical features: in step (4), the mobile phase B is an acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 80:20.

[0066] The measured PP and SS, OO diglyceride linear calibration curves obtained by the method described in Example 1 are y = 0.2457x + 54.005, y = 0.1973x + 54.289, y = 0.1935x + 60.443, R 2 respectively 0.95, 0.98, 0.97.

[0067] Example 4

[0068] This example is the same as Example 1 except for the following technical features: in step (4), the sodium acetate in the mobile phase A is replaced by the same molar concentration of ammonium formate.

[0069] The measured PP and SS, OO diglyceride linear calibration curves obtained by the method described in Example 1 are y = 0.1976x + 55.84, y = 0.2099x + 55.022, y = 0.1702x + 62.11, R 2 respectively 0.95, 0.96, 0.97.

[0070] Example 5

[0071] This example is the same as Example 1 except for the following technical features: in step (4), 500ul of phosphate buffer is not added for treatment.

[0072] The measured PP and SS, OO diglyceride linear calibration curves obtained by the method described in Example 1 are y = 0.1909x + 57.507, y = 0.2333x + 53.356, y = 0.1868x + 62.443, R 2 respectively 0.95, 0.97, 0.94.

[0073] Comparative Example 1

[0074] This example is the same as Example 1 except for the following technical features: no derivatization treatment of the diglyceride is performed, the diglyceride sample is directly injected into high performance liquid chromatography tandem mass spectrometry after the pretreatment of step (4).

[0075] The homodiglyceride without derivatization treatment cannot produce two different daughter ion fragments after neutral loss due to having the same fatty acyl group, so it is impossible to realize the quantification of the homodiglyceride regioisomer.

[0076] Comparative Example 2

[0077] This example is the same as Example 1 except for the following technical features: the sodium acetate in the mobile phase A in step (4) is replaced by lithium acetate of the same molar concentration.

[0078] According to the method described in Example 1, the measured PP and SS, OO diglyceride linear calibration curves obtained in this example are y = 0.2242x + 55.84, y = 0.1971x + 60.356, y = 0.1468x + 64.777, R 2 respectively 0.85, 0.81, 0.73.

[0079] Ionization is a lithium adduct parent ion of diglyceride isocyanate, which can also produce two daughter ions after neutral loss. However, it is less effective than the sodium adduct.

[0080] Comparative Example 3

[0081] This example is the same as Example 1 except for the following technical features: no methyl paraben stabilizer is added in step (2).

[0082] According to the method described in Example 1, the measured PP and SS, OO diglyceride linear calibration curves obtained in this example are y = 0.2842x + 49.173, y = 0.1671x + 62.022, y = 0.1302x + 64.777, R 2 respectively 0.85, 0.81, 0.73.

[0083] Comparative Example 4

[0084] This example is the same as Example 1 except for the following technical features: no thin layer chromatography separation and purification treatment of the derivatization product is performed in step (3).

[0085] According to the method described in Example 1, the measured PP and SS, OO diglyceride linear calibration curves obtained in this example are y = 0.2242x + 55.84, y = 0.1971x + 60.356, y = 0.1468x + 64.777, R 20.85, 0.85, 0.79, respectively.

[0086] Comparative Example 5

[0087] The present comparative example is the same as Example 1 except for the following technical features: in the high performance liquid chromatography tandem mass spectrometry analysis condition of step (4), the CID voltage is selected as 50 eV.

[0088] According to the method described in Example 1, the measured PP and SS, OO diglyceride linear calibration curves obtained in the present example are y = 0.2009x + 57.173, y = 0.0999x + 62.689, y = 0.1435x + 63.777, respectively, R 2 0.82, 0.74, 0.82, respectively.

[0089] Due to the excessive collision-induced dissociation voltage, the degree of neutral loss is high, which weakens the difference in the content of fragment ions caused by the loss of 1,3 and 2 positions due to steric hindrance effect, resulting in a lower correlation coefficient of the linear calibration curve and a poorer method accuracy.

[0090] Comparative Example 6

[0091] The present example is the same as Example 1 except for the following technical features: step (1) is not performed, and the isocyanate in step (2) is 2-phenylethyl isocyanate.

[0092] The reaction conversion rate of PP, SS, OO and 2-phenylethyl isocyanate is low (see Table 6), so it is not suitable for synthesizing diglyceride isocyanate derivatives and subsequent isomer content analysis.

[0093] Table 2 is the linear calibration curve data of the method of the examples and comparative examples.

[0094] Table 3 is the precision and accuracy test data of the method of the examples and comparative examples.

[0095] Table 4 is the structure of some related compounds involved in the derivatization process.

[0096] Table 5 is the secondary mass spectrum and sub-ion structure of the diglyceride isocyanate derivative of diolein.

[0097] Table 6 is the conversion rate of 2-phenylethyl isocyanate and diglyceride to corresponding diglyceride isocyanate derivative.

[0098] Table 2

[0099]

[0100]

[0101] Table 3

[0102]

[0103]

[0104] Accuracy, RE: relative error; Precision, RSD: relative standard deviation.

[0105] Table 4

[0106]

[0107] Table 5

[0108]

[0109] Table 6

[0110]

[0111] Conclusion: By derivatization of homoglyceride, the hydroxyl sites not combined with fatty acyl are connected with new groups, then ionized as sodium adducts under the analysis condition of high performance liquid chromatography tandem mass spectrometry, two kinds of daughter ions are produced by neutral loss cleavage under suitable collision induced dissociation voltage, and the content of homoglyceride regioisomer is quantified by the difference of relative content of daughter ions,

[0112] In all examples, the method of Example 1 obtains the best effect, the linear calibration curve for quantifying DAG regioisomer by the method of the application is linear well in the range of 50-5000 μg / mL, has high accuracy, good reproducibility, high practicability and reliability.

[0113] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for analyzing diglycerides and isomers thereof, characterized in that The method comprises the following steps: (1) mixing the isocyanate solution and the homoglyceride, adding a stabilizer, and performing derivatization treatment, removing the solvent after the derivatization treatment, and obtaining a derivative of the homoglyceride; (2) dissolving the obtained derivative of the homoglyceride with an organic solvent, and performing thin layer chromatography separation and purification, and obtaining a pure derivative of the homoglyceride; (3) The purified homodiglyceride derivative is dissolved with a solvent, and phosphate buffer is added, and then injected into a high performance liquid chromatography tandem mass spectrometer for detection and analysis to obtain [AA] + and [AB] + The percentage content of two fragment ions, wherein A represents a fatty acid, and B represents an isocyanate; then the corresponding relative content of [AB] + ion is calculated, wherein the relative content of [AB] + ion (%) = [AB] + ion percentage content / ([AB] + ion percentage content + [AA] + ion percentage content) ; (4) Replacing the same acid glycerol diester in step (1) with pure 1,2-DAG, pure 1,3-DAG, or a mixture of pure 1,2-DAG and pure 1,3-DAG, respectively, and then repeating steps (1)-(3) to obtain the corresponding [AB] + ion relative content, and the corresponding sn-ABA relative content was calculated, and the sn-ABA relative content (%) = sn-ABA content / (sn-AAB content+sn-ABA content), wherein sn-ABA is 1,3-DAG isocyanate derivative, sn-AAB is 1,2-DAG isocyanate derivative, A represents a fatty acid, and B represents isocyanate; and then taking the ion relative content as the vertical coordinate and the sn-ABA relative content as the horizontal coordinate to construct a standard curve, and different fatty acids correspond to different standard curves; and + ion relative content as the vertical coordinate and the sn-ABA relative content as the horizontal coordinate to construct a standard curve, and different fatty acids correspond to different standard curves; and (5) Replacing the same diglyceride in step (1) with the diglyceride to be tested, and then repeating steps (1)-(3) to obtain the corresponding [AB] + ion relative content, then substituting the [AB] + ion relative content into the standard curve of the corresponding fatty acid in step (4) to obtain the sn-ABA relative content, i.e. the relative content of the corresponding 1,3-DAG in the diglyceride to be tested.

2. The method according to claim 1, wherein the isocyanate in the isocyanate solution in step (1) is 2-naphthyl isocyanate.

3. The method according to claim 1, wherein the solvent of the isocyanate solution in step (1) is at least one of chloroform, toluene and xylene.

4. The method according to claim 1, wherein the mass concentration of the isocyanate in the isocyanate solution in step (1) is 20%-40%.

5. The method according to claim 1, wherein the mass ratio of the isocyanate solution to the homoglyceride in step (1) is 20-50:

1.

6. The method according to claim 1, wherein the stabilizer in step (1) is methyl paraben, and the addition amount is 1-10‰wt of the mass of the isocyanate solution.

7. The method according to claim 1, wherein the reaction temperature of the derivatization treatment in step (1) is 50-70℃, the reaction time is 15-30min, the vacuum condition is used, and the whole reaction condition is in a light-proof environment.

8. The method according to claim 1, wherein the organic solvent used for dissolving in step (2) is at least one of n-hexane, chloroform, petroleum ether and diethyl ether.

9. The method according to claim 1, wherein the developing agent in the thin layer chromatography separation condition in step (2) is a mixture of n-hexane, diethyl ether and anhydrous formic acid, the ratio of n-hexane, diethyl ether and anhydrous formic acid is 60-80:10-20:5-15 (v / v), after the glyceride isocyanate derivative band is scraped, diethyl ether is used for elution, and then nitrogen blowing concentration treatment is performed.

10. The method according to claim 1, wherein the solvent in step (3) is n-hexane:methanol, the volume ratio is 2-6:1, the concentration of the obtained solution after dissolving is 3-10mg / ml.

11. The method according to claim 1, wherein the phosphate buffer solution in step (3) is a solution of Na2HPO4 and NaH2PO4 configured in proportion to 0.1-0.5M, and the pH is 6.8-8.

4.

12. The method according to claim 1, wherein the addition amount of the phosphate buffer solution in step (3) is 0-20% of the volume of the solution after dissolving.

13. The method according to claim 1, wherein the mobile phase A in the liquid chromatography tandem mass spectrometry analysis condition in step (3) is methanol, and sodium acetate or ammonium formate is added, the concentration of the sodium acetate or ammonium formate is 0.001-0.01mol / L; and the mobile phase B is acetonitrile or acetonitrile-water.

14. The method according to claim 1, wherein the mass spectrometry analysis condition in step (3) is as follows: the ion source is an electrospray ion source, the ionization mode is positive ion electrospray ionization, the collision gas is argon, the collision energy is 20-50eV, the spray voltage is 3-5kv, the capillary voltage is 30-50V, the capillary temperature is 350-450℃, the sheath gas flow rate is 300-600, the auxiliary gas flow rate is 50-100, the scan range is 100-1000, and the scan time is 0.1-1s. ​ ​ ​ ​ ​ ​ ​ The collision-induced dissociation voltage in the mass spectrometry condition of the liquid chromatography tandem mass spectrometry analysis condition in step (3) is 10-40 eV. The corona voltage in the mass spectrometry condition of the liquid chromatography tandem mass spectrometry analysis condition in step (3) is 4.4-5.2 kV, the cone voltage is 40-60 V, the capillary voltage is 3.1-3.8 kV, the source temperature is 80-120 DEG C, the probe temperature is 450-550 DEG C, the desolvation temperature is 440-530 DEG C, the cone gas flow is 40-50 L / h, the desolvation gas flow is 380-410 L / h, and the gas flow rate is 0.5-0.8 mL / min.

10. The method of claim 1, wherein The method comprises the following steps: The pure 1,2-DAG and the pure 1,3-DAG in step (4) are both homoglycerides, and the fatty acids in the homoglycerides are one of palmitic acid, stearic acid and oleic acid.

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