Quantitative detection method for SN-2 DHA in algal oil

Abstract

The invention belongs to the technical field of detection, and particularly provides a quantitative detection method for SN-2DHA in algal oil, which comprises the following steps: carrying out specific enzymolysis on fatty glyceride of the algal oil by using immobilized lipase in an absolute ethyl alcohol environment to obtain SN-2DHA in the algal oil; a high performance liquid chromatography diode array detector (DAD) external standard method is used for directly quantifying SN-2DHA after algae oil enzymolysis, and the method specifically comprises the following steps: (1) carrying out enzymolysis on an algae oil sample to be detected; (2) purifying the enzymatic hydrolysate of the algae oil sample; (3) detecting a test sample; and (4) quantifying by an external standard method. Compared with the prior art, the detection method disclosed by the invention is simple and convenient to operate and accurate in quantification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a quantitative detection method of SN-2 DHA in algal oil. BACKGROUND

[0002] Docosahexaenoic acid, namely DHA, is an essential polyunsaturated fatty acid for human body. DHA is an important component of brain cell membrane, participates in the formation and development of brain cells, plays an important role in the extension of nerve cell axon and the formation of new sprouts, can maintain the normal physiological activity of nerve cells, and participates in the process of brain thinking and memory formation. Human body can synthesize a small amount of DHA, but the efficiency is extremely low, which cannot meet the body's needs at all. Therefore, DHA is considered as a "conditionally essential fatty acid", which mainly relies on exogenous intake to ensure sufficient level.

[0003] Microalgae is the original producer of DHA, which is considered as an ideal source of high-quality DHA. In algal oil, DHA usually exists in the form of glycerol fatty acid ester, and three hydroxyl positions in the glycerol molecular structure are combined with one fatty acid respectively to obtain triglyceride, in which one or more fatty acid molecules are DHA. The esterification position corresponding to DHA glyceride has SN-1, SN-2 and SN-3, in which the fatty acids at SN-1 and SN-3 positions are easily hydrolyzed by lipase in the digestive process to become free fatty acids, while the fatty acid at SN-2 position is firmly fixed and not easy to be cut off. The absorption efficiency of free fatty acids is relatively low, and free fatty acids are easy to combine with minerals in the intestinal tract to form insoluble substances. SN-2 DHA refers to the structure form of DHA at the SN-2 position of triglyceride, which makes SN-2 DHA in the middle position of triglyceride, which is not easy to be hydrolyzed by lipase in digestion, and is more easily absorbed by intestinal cells. For infants and young children, after SN-2 DHA is absorbed by the brain, it can promote rapid division and proliferation of brain cells, increase brain capacity, improve intelligence and learning ability, and also has beneficial effects such as regulating central nervous system function.

[0004] Therefore, the SN-2 DHA content is an important index for measuring the quality of algal oil. At present, there is no special standard quantitative detection method for SN-2 DHA. The detection methods related to the degree include GB / T 24894-2010 Animal and Vegetable Fats and Oils - Molecular Species 2-Position Fatty Acid Composition and the patent method CN116990404A, a method for detecting the content of Sn-2 DHA in DHA deployment oil applied by Ji Biyou Biotechnology (Wuhan) Co., Ltd. The detection principles and steps of the two methods are roughly the same: first, the sample oil is purified by an aluminum oxide chromatographic column; second, the purified liquid is added with a buffer solution, pancrelipase, a sodium cholate solution and a calcium chloride solution, and enzymolysis is carried out under a certain temperature and time; third, hydrochloric acid and diethyl ether are added after the enzymolysis is completed, and the diethyl ether extract is separated by thin plate chromatography; finally, the separated fatty acid monoglyceride is scraped from the chromatographic plate, fatty acid methyl ester is prepared according to GB / T17376, and the percentage of fatty acid 2-position monoglyceride is calculated according to GB / T17377 gas chromatography method.

[0005] The experimental steps of the above method are very complicated, and the patience and operation skills of the experimenters are required to be high. A large number of reagents are used in the experimental process, especially the step of separating fatty acid ester by thin plate chromatography, and the organic reagents used are volatile and toxic, which can easily affect the health of experimenters. Moreover, the detection of GB / T 24894-2010 is only qualitative analysis, not quantitative detection method. The patent method of Ji Biyou is to further use percentage calculation on the basis of GB / T 24894-2010. In summary, the steps of the above detection methods related to the degree are very complicated, and it is difficult to carry out quantitative detection and actual detection of a large number of samples with simple steps in practical application. SUMMARY

[0006] Based on the above status, the purpose of the present application is to provide a quantitative detection method for analyzing SN-2 DHA, which is simple in operation, stable and easy to analyze. The technical scheme is as follows:

[0007] A quantitative detection method for SN-2 DHA in algal oil, comprising the following steps:

[0008] (1) Enzymolysis of the algal oil sample to be tested: the algal oil sample to be tested, lipase and anhydrous ethanol are placed in a round-bottom flask, stirred uniformly, and then enzymolysis is carried out to obtain an algal oil sample enzymolysis solution;

[0009] (2) Purification of the algal oil sample enzymolysis solution: the algal oil sample enzymolysis solution of step (1) is taken and added into a volumetric flask, anhydrous ethanol is added for constant volume, and then an organic filter membrane is used for filtration to obtain a test sample solution;

[0010] (3) Test sample detection: the test sample solution of step (2) is taken and detected by using a high performance liquid chromatograph.

[0011] The chromatographic conditions are as follows: using PFP chromatographic column, using 0.1% formic acid aqueous solution as mobile phase A, methanol as mobile phase B, and acetonitrile as mobile phase C for gradient elution, and the gradient elution program is as follows:

[0012]

[0013] The detector is a diode array detector, and the detection wavelength is 210 nm.

[0014] (4) External standard method quantification: a certain amount of DHA monoglyceride is detected by using a high performance liquid chromatograph to determine the content of SN-2 DHA, and the chromatographic conditions are the same as step (3); a gradient amount of DHA monoglyceride is detected by using a high performance liquid chromatograph to establish a standard curve equation of SN-2 DHA, and the content of SN-2 DHA in the to-be-detected algal oil sample is determined by the standard curve.

[0015] The present application adopts lipase to specifically enzymatically hydrolyze fatty acid glyceride in anhydrous ethanol environment to obtain SN-2 fatty acid glyceride, and uses a high performance liquid chromatograph diode array detector to detect the actual enzymolysis concentration of SN-2 DHA in algal oil, so as to calculate the detection method of the content of SN-2 DHA.

[0016] In some specific embodiments, in step (1), the mass / volume ratio of the to-be-detected algal oil sample, the fatty acid enzyme, and the anhydrous ethanol is 1-5 g: 1-5 g: 100-500 ml.

[0017] In some specific embodiments, in step (1), the mass / volume ratio of the to-be-detected algal oil sample, the fatty acid enzyme, and the anhydrous ethanol is 1 g: 1 g: 100-500 ml.

[0018] In some specific embodiments, in step (1), the mass / volume ratio of the to-be-detected algal oil sample, the fatty acid enzyme, and the anhydrous ethanol is 1 g: 1 g: 100 ml.

[0019] In some specific embodiments, in step (1), the stirring is magnetic stirring.

[0020] In some specific embodiments, in step (1), the lipase is an immobilized lipase.

[0021] In some specific embodiments, in step (1), the enzymolysis time is 4-24 h.

[0022] In some specific embodiments, in step (1), the enzymolysis time is 12 h.

[0023] In certain embodiments, in step (2), the organic filter membrane has a pore size of 0.1-0.5 μm.

[0024] In certain embodiments, in step (2), the organic filter membrane has a pore size of 0.25 μm.

[0025] In certain embodiments, in step (3), the PFP chromatographic column has a column length of greater than 100 mm.

[0026] In certain embodiments, in step (3), the PFP chromatographic column has a column length of greater than 200 mm.

[0027] In certain embodiments, in step (3), the PFP chromatographic column has a column length of greater than 250 mm.

[0028] The present application establishes a method for quantitatively detecting SN-2 DHA in algal oil. Compared with the prior art, the detection method of the present application has at least the following beneficial effects:

[0029] (1) The detection method of the present application is simple to operate. First, the pretreatment of the present application is simple, and the algal oil sample to be measured does not need to be purified by an aluminum peroxide chromatographic column; second, in the enzyme hydrolysis process, no buffer solution, sodium cholate solution, calcium chloride solution needs to be added; third, the enzyme hydrolysis solution does not need to be extracted by reagents such as hydrochloric acid and diethyl ether, and the fatty acid glycerol ester is separated by thin layer chromatography; finally, the sample solution does not need to be subjected to methyl esterification treatment, and can be directly subjected to high performance liquid chromatography diode array detector quantitative analysis of the target SN-2 DHA after enzyme hydrolysis.

[0030] (2) The method of the present application uses a high performance liquid chromatography diode array detector, and has a clear absorption peak for detecting SN-2 DHA at a specific wavelength of 210 nm. There is no related literature or data using a high performance liquid chromatography diode array detector to directly detect SN-2 DHA.

[0031] (3) Since there is no SN-2 DHA glycerol monosteride as a control sample, the present application takes DHA glycerol monosteride as a control sample, which includes isomers SN-1 DHA and SN-2 DHA, further uses a PFP chromatographic column specially separating isomers and a specific mobile phase gradient elution condition, effectively determines the content of SN-2 DHA glycerol monosteride in the control sample, and then establishes a SN-2 DHA standard curve equation, so as to accurately quantify SN-2 DHA in the sample. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A blank chromatogram is shown.

[0033] Figure 2Chromatogram of DHA monoglyceride (including SN-1 DHA, SN-2 DHA) is shown.

[0034] Figure 3 Standard working curve of SN-2 DHA is shown.

[0035] Figure 4 Separation of SN-1 DHA and SN-2 DHA under different chromatographic column conditions is shown.

[0036] Figure 5 Separation of SN-1 DHA and SN-2 DHA under different elution conditions is shown. DETAILED DESCRIPTION

[0037] The present application will now be described with reference to the following examples which are intended to illustrate the application (but not to limit the application).

[0038] Unless specifically indicated otherwise, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references.

[0039] In addition, unless otherwise specified in the examples, conventional conditions or manufacturer's recommended conditions were used. Where the manufacturer of reagents or instruments is not indicated, it is intended that the conventional products available from commercial vendors were used. Those skilled in the art know that the examples describe the application by way of illustration and are not intended to limit the scope of the application claimed. All publications and other references mentioned herein are incorporated by reference in their entirety. Example 1

[0040] 1. Instruments

[0041] High performance liquid chromatograph (with DAD detector), magnetic stirring constant temperature water bath, electronic balance.

[0042] 2. Reagents

[0043] Methanol (chromatographic pure), fat immobilized enzyme (Novozyme 435), primary water, acetonitrile (chromatographic pure), anhydrous ethanol, DHA monoglyceride. Unless otherwise specified, the reagents used were of analytical purity.

[0044] 3. Analysis method

[0045] 3.1 Instrument conditions and parameters

[0046] 3.1.1 Chromatographic conditions

[0047] Chromatographic column: FPF chromatographic column (4.0 x 250 mm, 5.0 μm); column temperature: 40 °C; flow rate: 0.5 mL / min; mobile phase and elution gradient are shown in Table 1 below:

[0048] Table 1 Mobile phase and elution gradient

[0049] Time (min) A 0.1% formic acid solution (%) B methanol (%) C acetonitrile (%) 0 10 30 60 6 10 30 60 12 5 35 60 23 5 35 60 23.01 10 30 60 25 10 30 60

[0050] The detector is a diode array detector, and the detection wavelength is 210 nm.

[0051] 3.2 Preparation of solutions

[0052] 3.2.1 Preparation of the control test solution

[0053] About 10.0 mg of DHA monoglyceride reference substance was precisely weighed into a 50 mL volumetric flask, ultrasonically dissolved in anhydrous ethanol, and made up to volume to obtain the control test solution.

[0054] 3.2.2 Preparation of the sample test solution

[0055] About 0.1 g of the algal oil sample to be tested was precisely weighed into a 50 mL round-bottom flask, 100 mg of fat immobilized enzyme was added, 10 ml of anhydrous ethanol was added, the rotor was added, the bottle cap was closed, and stirring was performed in a 50°C constant temperature water bath for 4 hours. The enzyme solution was made up to 100 ml with anhydrous ethanol, filtered through a 0.2 μm organic filter membrane to obtain the sample test solution.

[0056] 3.3 Detection

[0057] 1 μL, 2 μL, 4 μL, 8 μL, and 10 μL of the DHA monoglyceride control test solution were precisely pipetted as the working curve, 2 μL of the sample test solution was added, and injected into the high performance liquid analyzer for analysis and determination to establish the SN-2 DHA standard curve equation for quantitative calculation of the content of SN-2 DHA in the sample.

[0058] 3.4 Calculation results

[0059] 3.4.1 Calibration of the control test solution

[0060] Since the DHA monoglyceride contains SN-1 DHA and SN-2 DHA, the content of SN-2 DHA therein needs to be determined before the standard curve is established. The specific calculation formula is as follows:

[0061]

[0062] In the formula: P—percentage of SN-2 DHA in the control solution;

[0063] A1—peak area of SN-2 DHA in the control solution;

[0064] A total—addition of the peak areas of SN-1 DHA and SN-2 DHA in the control solution.

[0065] 3.4.2 Drawing of the standard curve

[0066] According to the mass concentration of SN-2 DHA in the control test solution and its peak area, a standard curve of the relationship between the mass concentration and the peak area was established.

[0067] 3.4.3 Calculation of SN-2 DHA content in the sample

[0068]

[0069] In the formula: X - SN-2 DHA content in the test sample, g / 100g;

[0070] C - SN-2 DHA concentration in the sample solution, mg / mL;

[0071] V - sample dilution volume, mL;

[0072] K - unit conversion coefficient, K = 0.1;

[0073] M - weight of the sample, g.

[0074] 4. Methodology verification

[0075] 4.1 Specificity test (blank)

[0076] 4.1.1 Test method

[0077] The blank solution was treated according to the sample preparation method in 3.2.2, and the peak time was compared with the DHA monoglyceride (including SN-1 DHA and SN-2 DHA) control solution according to the instrument conditions in 3.1.

[0078] 4.1.2 Test results

[0079] As shown in Figure 1-2 , the blank solution had no chromatographic peak at the peak time of SN-2 DHA, indicating that the blank had little effect on the test results.

[0080] 4.2 Confirmation of linear range

[0081] 4.2.1 Test data

[0082]

[0083] 4.2.2 Standard working curve

[0084] The standard working curve was drawn with concentration as the abscissa and peak area as the ordinate, as shown in Figure 3 .

[0085] 4.2.3 Conclusion of linear test

[0086] Linear evaluation: SN-2 DHA correlation coefficient R is 0.9999, so the method for determining SN-2 DHA presents good linearity between the concentration of 0.0237 mg / mL to 0.38032 mg / mL, which meets the requirements of GB / T 27404-2008 "laboratory quality control specification"

GB / T 27404-2008 requires correlation coefficient R≥0.99

[0087] 4.3 Detection limit and quantification limit

[0088] The detection limit DL and quantification limit QL of the analysis method are calculated by the signal-to-noise ratio (S / N). DL is defined as the concentration to be analyzed corresponding to S / N=3, and QL is defined as the concentration to be analyzed corresponding to S / N=10.

[0089] 4.3.1 Detection limit

[0090] When the signal-to-noise ratio (S / N) is 3, the detection concentration of SN-2 DHA is 0.003 mg / mL, the sample sampling amount is 0.1 g, and the dilution volume is 100 mL, the detection limit of SN-2 DHA of the method is 3 mg / g.

[0091] 4.3.2 Quantification limit

[0092] When the signal-to-noise ratio (S / N) is 10, the detection concentration of SN-2 DHA is 0.01 mg / mL; the sample sampling amount is 0.1 g, and the constant volume is 100 mL, the quantification limit of SN-2 DHA of the method is 10 mg / g.

[0093] 4.4 Precision test

[0094] 4.4.1 Test method

[0095] Weigh 6 samples, process the samples according to the sample preparation method of 3.2.2, detect the content of SN-2 DHA in the sample, and calculate the RSD (%).

[0096] 4.4.2 Test data (see table below)

[0097]

[0098] 4.4.3 Test conclusion

[0099] The RSD of the content of SN-2 DHA of 6 samples is 0.9%, which indicates that the method has good precision and meets the requirements of GB / T 27404-2008 "laboratory quality control specification"

GB / T 27404-2008 requires RSD (%)≤1.3%

[0100] 4.5 Accuracy test (recovery rate)

[0101] 4.5.1 Test method

[0102] Spiked: 0.1 g of sample was precisely weighed 9 times (the SN-2 DHA content in the known sample was 25.3 g / 100 g), divided into 3 groups, 3 in each group, and each group was prepared according to 3.2.2 sample test solution, and about 13 mg, 17 mg, and 21 mg of SN-2 DHA reference substance was precisely added respectively, and then measured under the conditions of 3.1.

[0103] 4.5.2 Test data as follows

Test sample: algal oil soft capsules

[0104] SN-2 DHA recovery test results table

[0105]

[0106] 4.5.3 Test conclusion

[0107] The measured amount of addition = the measured amount of spiked sample - the measured amount of sample; recovery rate (%) = measured amount of addition / theoretical amount of addition x 100%.

[0108] The average recovery rate of SN-2 DHA was 100.6%, which met the requirements of GB / T 27404-2008 "Laboratory Quality Control Specification"

GB / T 27404-2008 requires the recovery rate range to be 95-105%

[0109] In summary, through the linearity, precision, specificity (blank), detection limit, limit of quantification, accuracy (recovery rate) test of the SN-2 DHA content determination method, all meet the requirements of GB / T 27404-2008 "Laboratory Quality Control Specification", proving that the content determination method is scientific and effective, and can achieve the purpose of quality control of SN-2 DHA content in algal oil.

[0110] 5. Methodology optimization

[0111] 5.1 Column optimization

[0112] In this experiment, different column lengths of chromatographic columns were used as chromatographic column condition exploration and optimization tests, in which the separation effects of 250 mm and 100 mm column length chromatographic columns were compared. Figure 5 The separation effect of the 100 mm column length chromatographic column can be seen that the chromatographic peaks of SN-1 DHA and SN-2 DHA are stuck together, and the baseline separation effect is not achieved. Figure 2 The separation effect of the 250 mm column length chromatographic column can be seen that the chromatographic peaks of SN-1 DHA and SN-2 DHA basically achieve the baseline separation effect. It can be known that the column length of the chromatographic column is a key important parameter for separating SN-1 DHA and SN-2 DHA.

[0113] 5.2 Optimization of elution conditions

[0114] To achieve effective separation of isomers SN-1 DHA, SN-2 DHA, the selection of mobile phase and elution program is also one of the key technologies. With volume concentration of 0.1% formic acid aqueous solution as mobile phase A, methanol as mobile phase B, acetonitrile as mobile phase C, gradient and isocratic elution were carried out respectively. The experimental results are shown in Figures Figure 2 and Figure 4 respectively, and isocratic elution cannot effectively separate isomers SN-1 DHA, SN-2 DHA.

Claims

1. A method for quantitatively detecting SN-2 DHA in algal oil, characterized by, It comprises the following steps: (1) Enzymolysis of the sample of the algal oil to be tested: the sample of the algal oil to be tested, lipase and anhydrous ethanol are placed in a round-bottom flask, stirred uniformly, and then enzymolysis is performed to obtain an enzymolysis solution of the sample of the algal oil; (2) Purification of the enzymolysis solution of the sample of the algal oil: the enzymolysis solution of the sample of the algal oil obtained in step (1) is taken into a volumetric flask, anhydrous ethanol is added to make up the volume, and then an organic filter membrane is used to filter to obtain a test sample solution; (3) Detection of the test sample: the test sample solution obtained in step (2) is taken, and a high-performance liquid chromatograph is used to perform detection; The chromatographic conditions are as follows: a PFP chromatographic column is used, a 0.1% formic acid aqueous solution is used as mobile phase A, methanol is used as mobile phase B, and acetonitrile is used as mobile phase C for gradient elution, and the gradient elution program is as follows: A diode array detector is used, and the detection wavelength is 210 nm. (4) Quantitative determination by an external standard method: a certain amount of DHA monoglyceride is taken, a high-performance liquid chromatograph is used to perform detection, the content of SN-2 DHA is determined, and the chromatographic conditions are the same as in step (3); a gradient amount of DHA monoglyceride is taken, a high-performance liquid chromatograph is used to perform detection, a standard curve equation of SN-2 DHA is established, and the content of SN-2 DHA in the sample of the algal oil to be tested is determined through the standard curve.

2. The method of claim 1, wherein, In step (1), the mass / volume ratio of the sample of the algal oil to be tested, the lipase and the anhydrous ethanol is 1-5 g: 1-5 g: 100-500 ml.

3. The method of claim 1, wherein, In step (1), the mass / volume ratio of the sample of the algal oil to be tested, the lipase and the anhydrous ethanol is 1 g: 1 g: 100-500 ml.

4. The method of claim 1, wherein, In step (1), the stirring is magnetic stirring.

5. The method of claim 1, wherein, In step (1), the lipase is immobilized lipase.

6. The method of claim 1, wherein, In step (1), the enzymolysis time is 4-24 h.

7. The method of claim 1, wherein, In step (1), the enzymolysis time is 12 h.

8. The method of claim 1, wherein, In step (2), the pore size of the organic filter membrane is 0.1-0.5 μm.

9. The method of claim 1, wherein, In step (2), the pore size of the organic filter membrane is 0.25 μm.

10. The method of claim 1, wherein, In step (3), the column length of the PFP chromatographic column is greater than 200 mm.

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