A method for determining sn-1 LPC-DHA in krill oil extract

Abstract

The present invention discloses a method for determining the sn-1 LPC-DHA in krill oil extract, which belongs to the field of food testing. The present invention uses high performance liquid chromatography to separate and determine the sn-1 LPC-DHA in the krill oil extract, and uses a liquid chromatography-mass spectrometer for identification. For the first time, a method for detecting the sn-1 LPC-DHA in krill oil extract is provided; the krill extract contains about 15 components, and the present detection method can separate the target substance from other impurities in a complex sample, with good separation effect; methanol is used for pretreatment, avoiding the use of toxic substances such as chloroform; the elution gradient used in the detection method changes less, and the method is simpler; and the detection method has good precision, high recovery rate, and high accuracy.

Description

Technical Field

[0001] The invention belongs to the field of food testing, and in particular relates to a method for determining the sn-1 LPC-DHA in a krill oil extract. Background Art

[0002] Antarctic krill is an abundant resource rich in nutrients such as protein, phospholipids, and astaxanthin. Unlike the triglycerides in fish oil, the majority of fatty acids in Antarctic krill oil are present in the form of phospholipids. Phospholipids are the optimal transport form for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), making Antarctic krill oil more readily absorbed by the human body and highly valuable for its development and utilization. Currently, research on phospholipids has primarily focused on soybeans and egg yolks, with limited research on phospholipids in aquatic products, and even less on phospholipids in Antarctic krill oil. Therefore, establishing a method for detecting phospholipids in Antarctic krill oil will contribute to improving the quality evaluation system for Antarctic krill oil and is of great significance for promoting the development of the Antarctic krill industry.

[0003] The best form of DHA for the brain to absorb is lysophosphatidylcholine LPC-DHA. The DHA in most supplements is absorbed in the form of triacylglycerols. These dietary DHA substances are easily enzymatically broken down into free DNA by pancreatic enzymes. However, the sn-1 position LPC-DHA in LPC-DHA can resist enzymatic breakdown by these enzymes and be absorbed by the brain because of the presence of A2 enzyme in pancreatic enzymes.

[0004] Currently, there is no method for detecting sn-1 LPC-DHA. This method develops a liquid phase detection method for sn-1 LPC-DHA in krill oil extract and uses liquid chromatography-mass spectrometry for qualitative confirmation. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the existing technology and provide a liquid phase detection method for sn-1 LPC-DHA in the reaction product of krill oil extract, and to perform qualitative confirmation by liquid chromatography-mass spectrometry.

[0006] In one aspect, the present invention provides a method for determining the sn-1 LPC-DHA in a krill oil extract, comprising the following steps:

[0007] (1) mixing the krill oil extract, ultrasonically extracting it with an organic solvent A, and filtering the extract to obtain a sample;

[0008] (2) Prepare the standard stock solution and standard test solution of sn-1 LPC-DHA;

[0009] (3) The sample in step (1) is detected by high performance liquid chromatography to separate the sn-1 position LPC-DHA substance and measure its content.

[0010] In some embodiments, the sn-1 LPC-DHA obtained in step (3) is identified using liquid chromatography-mass spectrometry.

[0011] In some embodiments, the method for preparing the krill oil extract in step (1) comprises the following steps:

[0012] S1 Enzymatic hydrolysis: Mix krill oil and water until a uniform emulsion is formed. After preheating, add lipase and stir. After stirring, heat and inactivate the enzyme to obtain a mixed solution.

[0013] S2 dehydration: adding an organic solvent to the mixed solution in step S1 to demulsify, and then vacuum concentrating and dehydrating after demulsification to obtain a dehydrated product;

[0014] S3 purification: adding acetone to the product dehydrated in step S2, stirring and standing, and removing the supernatant; adding acetone again, stirring and standing, and removing the supernatant, and vacuum concentrating to obtain a crude product;

[0015] S4: Removal of free fatty acids: The crude product from step S3 is dissolved in an organic solvent, and sodium hydroxide ethanol solution is added dropwise to adjust the pH to 8, followed by reaction. After the reaction, sodium hydroxide ethanol solution is added dropwise to adjust the pH of the solution to 8. After the reaction, the solution is filtered to obtain a filtrate.

[0016] S5 Concentration and Drying: The filtrate is concentrated in vacuo to obtain a brown-red paste product, which is the krill oil extract.

[0017] In some embodiments, the organic solvent A in step (1) includes but is not limited to methanol, ethanol, tetrahydrofuran, chloroform, isopropanol, cyclohexane, and diethyl ether, preferably methanol, ethanol, and chloroform, and more preferably methanol.

[0018] In some embodiments, the chromatographic column for high performance liquid chromatography in step (3) is a hydrophilic interaction chromatography column.

[0019] In some embodiments, the diameter and column length of the hydrophilic interaction chromatography column are 1.7-3.7 mm and 50-250 mm, respectively, preferably 1.9-3.5 mm and 100-200 mm, more preferably 2.7 mm in diameter and 150 mm in length.

[0020] In some embodiments, the flow rate of the HPLC in step (3) is 0.1-1.1 mL / min, and the column temperature is 10-50°C.

[0021] In some embodiments, the flow rate in the high performance liquid chromatography in step (3) is 0.3-0.9 mL / min, and the column temperature is 20-40°C.

[0022] In some embodiments, the flow rate in the high performance liquid chromatography in step (3) is 0.6 mL / min and the column temperature is 30°C.

[0023] In some embodiments, the mobile phase A of the high performance liquid chromatography in step (3) is an organic solvent B, and the mobile phase B is an acid-containing aqueous solution.

[0024] In some embodiments, the organic solvent B in step (3) includes but is not limited to acetonitrile, methanol, ethanol and isopropanol, preferably acetonitrile; the acid includes but is not limited to phosphoric acid, formic acid, acetic acid and trifluoroacetic acid, preferably formic acid; preferably, the pH value of the acid-containing aqueous solution is 1-4, preferably 2.5.

[0025] In some embodiments, the elution condition of the HPLC is gradient elution, and the elution gradient is:

[0026] Running time / min Mobile phase A% Mobile phase B% 0 90 10 5 90 10 30 80 20 45 80 20 46 90 10 50 90 10 .

[0027] In some embodiments, the liquid phase conditions in the LC-MS instrument are the same as the liquid phase conditions in step (3).

[0028] In some embodiments, the scanning range of high-resolution mass spectrometry analysis in liquid chromatography-mass spectrometry is 50-2000, preferably 100-1000.

[0029] In some embodiments, the high-resolution mass spectrometer scanning time in the liquid chromatography-mass spectrometer is 0.1-0.5 s, further 0.1 s, 0.2 s, 0.3 s, 0.4 s, and 0.5 s.

[0030] In some embodiments, the high-resolution mass spectrometry spray gas flow rate in the liquid chromatography-mass spectrometer is 30-80 psi, preferably 40-70 psi, and more preferably 55 psi.

[0031] In some embodiments, the auxiliary heating gas for high-resolution mass spectrometry in the liquid chromatography-mass spectrometer is 40-80 Si, preferably 50-70 Si, and more preferably 60 Si.

[0032] In some embodiments, the high-resolution mass spectrometry curtain gas flow rate in the liquid chromatography-mass spectrometer is 20-60 psi, preferably 30-50 psi, and more preferably 40 psi.

[0033] In some embodiments, the ionization voltage of the high-resolution mass spectrometer in the liquid chromatography-mass spectrometer is 5500 / -4500V.

[0034] In some embodiments, the high-resolution mass spectrometer temperature in the liquid chromatography-mass spectrometer is 350-750°C, preferably 450-650°C, and more preferably 550°C.

[0035] In some embodiments, the de-clustering voltage DP of the high-resolution mass spectrometer in the liquid chromatography-mass spectrometer is 60-100V, preferably 70-90V, and more preferably 80V.

[0036] In some embodiments, the collision energy CE of the high-resolution mass spectrometer in the liquid chromatography-mass spectrometer is 25-65V / (-25)-(-65)V.

[0037] Beneficial effects of the present invention:

[0038] The detection method of the present invention has the following advantages:

[0039] 1. Provides a method for the first time to detect sn-1 LPC-DHA in krill oil extract;

[0040] 2. Good separation effect. Krill extract contains about 15 components. This detection method can separate the target substance from other impurities in complex samples;

[0041] 3. The method used in the present invention uses methanol for pretreatment, avoiding the use of toxic substances such as chloroform;

[0042] 4. The elution gradient used in this detection method changes less, and the method is simpler; and the detection method has good precision, high recovery rate and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the HPLC chart of krill oil extract sample testing.

[0044] Figure 2 This is the standard curve of sn-1 PC-DHA.

[0045] Figure 3 This is an HPLC chart of krill oil extract sample detection under the conditions of Example 3.

[0046] Figure 4 This is an HPLC chart of krill oil extract sample detection under the conditions of Example 4.

[0047] Figure 5 This is an HPLC chart of krill oil extract sample detection under the conditions of Example 5.

[0048] Figure 6 It is the extracted ion chromatogram of the target compound in the sn-1 position LPC-DHA standard and sample.

[0049] Figure 7 This is the high-resolution mass spectrum of sn-1 LPC-DHA standard and sample in positive ion mode.

[0050] Figure 8 This is the high-resolution mass spectra of sn-1 LPC-DHA standards and samples in negative ion mode. DETAILED DESCRIPTION

[0051] In order to facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the embodiments. The contents mentioned in the embodiments are not intended to limit the present invention.

[0052] Example 1 Detection Method of Sn-1 LPC-DHA in Krill Oil Extract

[0053] Example 1-1 Preparation method of krill oil extract

[0054] (1) 100 g of krill oil and 100 g of water were mixed and stirred until a uniform emulsion was formed. The mixture was preheated to 50° C., and lipase (3% by weight of the krill oil) was added and reacted for 60 min. After the reaction, the mixture was heated to 90° C. to inactivate the enzyme and obtain a mixed solution.

[0055] (2) adding 95% ethanol in an amount of 1-2 times the volume of water in step (1) to the mixed solution in step (1) to break the emulsion, and concentrating in vacuo to an anhydrous state after breaking the emulsion to obtain a dehydrated product;

[0056] (3) Add acetone 5 times the mass of the krill oil to the dehydrated product in step (2), stir for 60 minutes, cool to below 0°C, let stand for 8 hours, and remove the supernatant. Then add acetone 3 times the mass of the krill oil, stir for 60 minutes, cool to below 0°C, let stand for 3 hours, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0057] (4) The crude product obtained in step (3) is dissolved in 1 times its mass of 95% ethanol, and a 1% sodium hydroxide ethanol solution is added dropwise until the solution pH reaches 6, and then reacted for 60 minutes. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise until the solution pH reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

[0058] (5) The filtered solution in step (4) is vacuum concentrated to remove ethanol, and the moisture content of the material is controlled to be below 2%, and the residual acetone and ethanol content is below 5000 ppm. Finally, a brown-red paste product is obtained, which is the krill oil extract.

[0059] Example 1-2 Pretreatment of krill oil extract

[0060] The krill oil extract obtained in Example 1-1 was accurately weighed and mixed. 0.1 g was placed in a 25 mL volumetric flask, 20 mL of methanol was added, and the mixture was sonicated for 20 min. The mixture was cooled to room temperature, and the volume was adjusted to 25 mL. The mixture was passed through a 0.45 μm organic membrane to obtain a filtrate for analysis.

[0061] Example 1-3 Liquid chromatograph for elution

[0062] The pre-treated samples were injected into the HPLC instrument. The parameters were as follows:

[0063] (1) Chromatographic column: Ailent InfinityLab Poroshell 120 Hilic, 4.6 × 150 mm, 2.7 μm;

[0064] (2) Flow rate: 0.6 mL / min;

[0065] (3) Column temperature: 30°C;

[0066] (4) Injection volume: 10 μL;

[0067] (5) Detection wavelength: 205 nm;

[0068] (6) Mobile phase A: acetonitrile;

[0069] (7) Mobile phase B: pH 2.5 formic acid aqueous solution;

[0070] (8) Gradient elution:

[0071] Table 1

[0072]

[0073]

[0074] The measured spectrum is as follows Figure 1 As shown, the components in krill oil extract are concentrated in 23-26min and 30-34min, and there are about fifteen components. The sn-1 position LPC-DHA peaks at 32.005 and is well separated from other components.

[0075] Example 2 Liquid Phase Methodology Verification

[0076] According to the conditions and operating steps in Example 1, the analytical method for the target compound was confirmed.

[0077] 1. Specificity experiment

[0078] A blank reagent group, a standard of sn-1 LPC-DHA, and the krill oil extract sample prepared in Example 1-1 were prepared according to the method of Example 1-3 to obtain corresponding solutions. The results are shown in Table 2, indicating that there is no other interference with the sn-1 LPC-DHA at the corresponding position.

[0079] Table 2

[0080] Group sn-1 LPC-DHA Blank reagent group No peak sn-1 LPC-DHA standard 32.034 Sample group in Example 1 32.034

[0081] 2. Linear range

[0082] With the reference substance concentration c (μg / mL) as the horizontal axis and the peak area Area of ​​each reference substance as the vertical axis, the standard curve of each reference substance was drawn, and the linear regression equation and correlation coefficient of the concentration-peak area of ​​each target compound were calculated, as shown in the figure: Figure 2 The linear regression equation is: y = 34.58x-41.45. The linear range of the target compound is: 16.2791 ~ 174.0018 μg / mL, and the linear correlation coefficients are 0.99925, which meets the requirement of greater than 0.99, indicating that the linear relationship is good within this linear range.

[0083] 3. Limit of detection and limit of quantification

[0084] Taking 3 times the signal-to-noise ratio as the detection limit and 10 times the signal-to-noise ratio as the quantification limit, the detection limit was 0.084 μg / mL and the quantification limit was 0.279 μg / mL.

[0085] 4. Precision and accuracy

[0086] The precision and accuracy tests were performed according to the method of Example 1. The test results are shown in Tables 3 and 4, indicating that the precision and accuracy of this method are good and meet the requirements of GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for National Food Safety Standards".

[0087] Table 3 Precision

[0088]

[0089]

[0090] Table 4 Accuracy

[0091]

[0092] Example 3

[0093] The other conditions were the same as those in Example 1. The mobile phase A was acetonitrile, the mobile phase B was 0.9 mol / L ammonium acetate aqueous solution, and the elution conditions were as shown in Table 5. The spectrum was as follows: Figure 3 .

[0094] Table 5

[0095] Running time / min Mobile phase A% Mobile phase B% 0 90 10 5 90 10 15 80 20 20 80 20 22 90 10 30 90 10

[0096] Example 4

[0097] The other conditions were the same as those in Example 1. The mobile phase A was acetonitrile, the mobile phase B was 0.1% phosphoric acid aqueous solution, and the elution conditions were as shown in Table 6. The spectrum was as follows: Figure 4 .

[0098] Table 6

[0099] Running time / min Mobile phase A% Mobile phase B% 0 90 10 5 90 10 15 80 20 20 80 20 22 90 10 35 90 10

[0100] Example 5

[0101] The other conditions were the same as those in Example 1, with mobile phase A being 0.1% acetic acid in acetonitrile and mobile phase B being 0.1% aqueous acetic acid, and isocratic elution; A:B=90:10 (running time 150 min). The spectrum obtained was as follows: Figure 5 .

[0102] Example 6 Mass spectrometer identification

[0103] The sample separated by HPLC in Example 1 was fed into the SCIEX Zeno LC-MS instrument. 7600 for identification and quantitative analysis. The liquid phase conditions were the same as those mentioned above. The experimental conditions of mass spectrometry were as follows: electrospray ionization positive and negative ionization modes (ESI, scanning range: m / z 100-1000, scanning time: 0.2 s, spray gas: 55 psi, auxiliary heating gas: 60 si, curtain gas: 40 psi, ionization voltage: 5500 / -4500 V, temperature: 550°C, DP (declustering voltage): 80 V, CE (collision energy): 45±20 V / -45±20 V).

[0104] Example 7 Liquid Quality Qualitative Confirmation Method

[0105] from Figure 6 As can be seen, the left side is the TIL diagram, which refers to the total ion current diagram. It is a diagram showing the sum of all ion intensities over time or scan times within the selected mass range. The right side is the diagram of the corresponding ions of the extracted sn-1 LPC-DHA. The red one is the sample diagram, and the blue one is the standard diagram. The peak time of the sn-1 LPC-DHA standard and the target compound in the sample is 30.417min, which is consistent with the peak time. The separated substances were tested in positive and negative ion modes. Figure 7 and Figure 8 It can be seen that the primary and secondary mass-to-charge ratios of the sample and the target product match well. (Because the previous chromatograph was an Agilent chromatograph, the LC-MS here uses an AB, so some differences in peak times are normal.)

[0106] By matching the retention time and the primary and secondary mass spectra, it can be confirmed that the target compound in the sample is consistent with the provided standard sn-1 position LPC-DHA.

[0107] It should be understood that the detailed description of the technical solutions of the present invention using the preferred embodiments above is illustrative and not restrictive. A person skilled in the art, after reading the present specification, may modify the technical solutions described in the embodiments or replace some of the technical features therein with equivalents; such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining the sn-1 position LPC-DHA in krill oil extract, characterized in that: The following steps are involved: (1) The krill oil extract is mixed and ultrasonically extracted with an organic solvent A, and the extract is filtered to obtain a sample; wherein the organic solvent A is methanol and / or ethanol; (2) Prepare the standard stock solution and standard test solution of sn-1 LPC-DHA; (3) The sample in step (1) is tested by high performance liquid chromatography to separate the sn-1 LPC-DHA and measure its content; The chromatographic conditions used for high performance liquid chromatography detection were: The chromatographic columns used in the high performance liquid chromatography were hydrophilic interaction chromatography columns: Ailent InfinityLab Poroshell 120Hilic, 4.6 × 150 mm, 2.7 μm; The detection wavelength was 205 nm; The mobile phase A of the HPLC was acetonitrile, and the mobile phase B was a pH 2.5 formic acid aqueous solution; The elution condition of high performance liquid chromatography is gradient elution, and the elution gradient is: 。 2. The method according to claim 1, wherein The sn-1 LPC-DHA separated in step (3) was identified using liquid chromatography-mass spectrometry.

3. The method according to claim 1, wherein The preparation method of the krill oil extract in step (1) comprises the following steps: S1 Enzymatic hydrolysis: Mix krill oil and water until a uniform emulsion is formed. After preheating, add lipase and stir. After stirring, heat and inactivate the enzyme to obtain a mixed solution. S2 dehydration: adding an organic solvent to the mixed solution in step S1 to demulsify, and then vacuum concentrating and dehydrating after demulsification to obtain a dehydrated product; S3 purification: adding acetone to the product dehydrated in step S2, stirring and standing, and removing the supernatant; adding acetone again, stirring and standing, removing the supernatant, and vacuum concentrating to obtain a crude product; S4: removing free fatty acids: dissolving the crude product in step S3 in an organic solvent, adding sodium hydroxide ethanol solution dropwise to adjust the pH to 8, and reacting. After the reaction, further adding sodium hydroxide ethanol solution dropwise to adjust the solution pH to 8; after the reaction, filtering to obtain a filtrate; S5 Concentration and Drying: The filtrate is concentrated in vacuo to obtain a brown-red paste product, which is the krill oil extract.

4. The method according to claim 1, wherein In step (3), the flow rate of the HPLC is 0.1-1.1 mL / min, and the column temperature is 10-50°C.

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