Method for determining sn-1-site LPC-DHA in krill oil extract

Abstract

The invention discloses a method for determining sn-1-site LPC-DHA in a krill oil extract, and belongs to the field of food detection. According to the method disclosed by the invention, separation content determination is carried out on sn-1-site LPC-DHA in the krill oil extract by adopting high performance liquid chromatography, and identification is carried out by using a liquid chromatograph. The method for detecting sn-1-site LPC-DHA in the krill oil extract is provided for the first time; the krill extract contains about 15 components, the detection method can separate target substances from other impurities in a complex sample, and the separation effect is good; methanol is adopted for pretreatment, so that the use of toxic substances such as chloroform is avoided; the elution gradient used by the detection method is less in change, and the method is simpler and more convenient; and the detection method has the advantages of good precision, high recovery rate and high accuracy.

Description

Technical Field

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

[0002] Antarctic krill resources are abundant and rich in nutrients such as protein, phospholipids and astaxanthin. Unlike triglycerides in fish oil, most of the fatty acids in Antarctic krill oil exist in the form of phospholipids. Phospholipids are the best transmission form of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Therefore, Antarctic krill oil is more easily absorbed by the human body and has a high development and utilization value. At present, the research on phospholipids is mainly focused on soybeans and egg yolks, and there is less research on phospholipids in aquatic products, and even less research on phospholipids in Antarctic krill oil. Therefore, establishing a method for detecting phospholipids in Antarctic krill oil is conducive to improving the quality evaluation system of Antarctic krill oil and is of great significance to 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 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 prior art 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 sn-1 LPC-DHA in a krill oil extract, comprising the following steps:

[0007] (1) mixing the krill oil extract, performing ultrasonic extraction with an organic solvent A, filtering the extract, and obtaining a sample;

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

[0009] (3) The sample in step (1) is tested by high performance liquid chromatography to separate the sn-1 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: krill oil and water are mixed into a uniform emulsion state, preheated, lipase is added and stirred, and after stirring, the temperature is raised to 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 vacuum concentrating and dehydrating after demulsification to obtain a dehydrated product;

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

[0015] S4: removing free fatty acids: the crude product in step S3 is dissolved in an organic solvent, sodium hydroxide ethanol solution is added dropwise to adjust the pH to 8, and then reacted. After the reaction, sodium hydroxide ethanol solution is added dropwise to adjust the solution pH to 8. After the reaction is completed, 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 of the 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, and more preferably a diameter of 2.7 mm and a column length of 150 mm.

[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 HPLC 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 in the liquid chromatography-mass spectrometer is 50-2000, preferably 100-1000.

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

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

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

[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 further 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 temperature of the high-resolution mass spectrometer in the LC-MS instrument is 350-750°C, preferably 450-650°C, and further 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 further preferably 80V.

[0036] In some embodiments, the collision energy CE of the high-resolution mass spectrometer in the LC-MS instrument 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. Provide a method for detecting sn-1 LPC-DHA in krill oil extract for the first time;

[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 adopts methanol for pretreatment, thus 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 the HPLC chart of krill oil extract sample detection under the conditions of Example 3.

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

[0047] Figure 5 This is the HPLC chart of krill oil extract sample detected 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 It is the high-resolution mass spectrum of sn-1 LPC-DHA standard and sample in positive ion mode.

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

[0051] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes 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 state was obtained. After preheating to 50° C., lipase in an amount of 3% by mass of the krill oil was added and reacted for 60 min. After the reaction was completed, 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 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0056] (3) Add acetone 5 times the mass of krill oil to the product dehydrated 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 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 95% ethanol in an amount equal to 1% by weight, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution 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] Accurately weigh the krill oil extract obtained in Example 1-1 and mix well. Take 0.1 g into a 25 mL volumetric flask, add 20 mL of methanol, sonicate for 20 min, cool to room temperature, adjust the volume to 25 mL, pass through a 0.45 μm organic membrane, and obtain the filtrate for analysis.

[0061] Example 1-3 Liquid chromatograph for elution

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

[0063] (1) Chromatographic column: Ailent InfinityLab Poroshell 120Hilic, 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 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 peak time of sn-1 LPC-DHA is 32.005, which 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] The blank reagent group, the 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 the corresponding solution. The results are shown in Table 2, indicating that the sn-1 LPC-DHA has no other interference 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 is drawn, and the linear regression equation and correlation coefficient of the concentration-peak area of ​​each target compound are calculated, such as 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 being 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 of the signal-to-noise ratio as the detection limit and 10 times of 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 carried out 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, mobile phase A was acetonitrile, mobile phase B was 0.9 mol / L ammonium acetate aqueous solution, and the elution conditions were as shown in Table 5. The spectrum measured 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, mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution, and the elution conditions were as shown in Table 6. The spectrum measured 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 acetonitrile, mobile phase B being 0.1% acetic acid aqueous solution, and isocratic elution; A:B=90:10 (running time 150 min), and the spectrum measured was as follows Figure 5 .

[0102] Example 6 Mass spectrometer identification

[0103] The sample separated by HPLC in Example 1 enters the liquid chromatography-mass spectrometer SCIEX Zeno 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 positive and negative ion modes (ESI, scanning range was 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 graph, which refers to the total ion current graph, which is a graph of the sum of all ion intensities over time or scan times within the selected mass range, and the right side is the corresponding ion graph of the extracted sn-1 LPC-DHA. The red one is the sample graph, and the blue one is the standard graph. 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 ion mode and negative ion mode. 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 Agilent chromatograph was used before, the AB HPLC was used here, so some differences in peak time 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 by means of the preferred embodiments is illustrative rather than restrictive. A person skilled in the art may modify the technical solutions described in the embodiments, or replace some of the technical features by equivalents, based on reading the specification of the present invention; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining sn-1 LPC-DHA in krill oil extract, characterized in that: The following steps are involved: (1) mixing the krill oil extract, performing ultrasonic extraction with an organic solvent A, filtering the extract, and obtaining a sample; (2) preparing a standard stock solution and a 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 substance and measure its content.

2. The method according to claim 1, characterized in that The sn-1 LPC-DHA separated in step (3) was identified using liquid chromatography-mass spectrometry.

3. The method according to claim 1, characterized in that The method for preparing the krill oil extract in step (1) comprises the following steps: S1 Enzymatic hydrolysis: krill oil and water are mixed into a uniform emulsion state, preheated, lipase is added and stirred, and after stirring, the temperature is raised to inactivate the enzyme to obtain a mixed solution; S2 dehydration: adding an organic solvent to the mixed solution in step S1 to demulsify, and vacuum concentrating and dehydrating after demulsification to obtain a dehydrated product; S3 purification: adding acetone to the product after dehydration in step S2, stirring and standing to remove the supernatant; adding acetone again, stirring and standing to remove the supernatant, and vacuum concentrating to obtain a crude product; S4: removing free fatty acids: the crude product in step S3 is dissolved in an organic solvent, sodium hydroxide ethanol solution is added dropwise to adjust the pH to 8, and then reacted. After the reaction, sodium hydroxide ethanol solution is added dropwise to adjust the solution pH to 8. After the reaction is completed, the solution is filtered 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, characterized in that The organic solvent A in step (1) is one or a combination of methanol, ethanol, tetrahydrofuran, chloroform, isopropanol, cyclohexane, and ether.

5. The method according to claim 1, characterized in that The chromatographic column of the high performance liquid chromatography in step (3) is a hydrophilic interaction chromatography column.

6. The method as claimed in claim 5, characterized in that The diameter and column length of the hydrophilic interaction chromatography column are 1.7-3.7 mm and 50-250 mm respectively.

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

8. The method according to claim 1, characterized in that In step (3), the mobile phase A of the high performance liquid chromatography is an organic solvent B, and the mobile phase B is an acid-containing aqueous solution.

9. The method as claimed in claim 8, characterized in that In step (3), the organic solvent B is any one or a combination of acetonitrile, methanol, ethanol and isopropanol; the acid is any one or a combination of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid; preferably, the pH value of the acid-containing aqueous solution is 1-4.

10. The method according to claim 8, characterized in that The elution condition of high performance liquid chromatography is gradient elution, and the elution gradient is:

11. The method according to claim 2, characterized in that The mass spectrometry scanning range in the LC-MS instrument is 50-2000.

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