CN111721848A - Method for measuring content of phosphatidylserine - Google Patents
Method for measuring content of phosphatidylserine Download PDFInfo
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- CN111721848A CN111721848A CN201910219995.3A CN201910219995A CN111721848A CN 111721848 A CN111721848 A CN 111721848A CN 201910219995 A CN201910219995 A CN 201910219995A CN 111721848 A CN111721848 A CN 111721848A
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- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 title claims abstract description 103
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 title claims abstract description 103
- 238000000034 method Methods 0.000 title claims abstract description 45
- 239000011159 matrix material Substances 0.000 claims abstract description 28
- 239000000203 mixture Substances 0.000 claims abstract description 23
- 239000002904 solvent Substances 0.000 claims abstract description 23
- 239000012736 aqueous medium Substances 0.000 claims abstract description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 204
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 121
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 62
- 235000009508 confectionery Nutrition 0.000 claims description 56
- 239000000499 gel Substances 0.000 claims description 55
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 50
- 108010010803 Gelatin Proteins 0.000 claims description 41
- 102000004142 Trypsin Human genes 0.000 claims description 41
- 108090000631 Trypsin Proteins 0.000 claims description 41
- 239000008273 gelatin Substances 0.000 claims description 41
- 229920000159 gelatin Polymers 0.000 claims description 41
- 235000019322 gelatine Nutrition 0.000 claims description 41
- 235000011852 gelatine desserts Nutrition 0.000 claims description 41
- 239000012588 trypsin Substances 0.000 claims description 38
- 238000000105 evaporative light scattering detection Methods 0.000 claims description 18
- 238000000605 extraction Methods 0.000 claims description 12
- 239000000084 colloidal system Substances 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 229940088598 enzyme Drugs 0.000 claims description 8
- 239000012046 mixed solvent Substances 0.000 claims description 6
- 102000057297 Pepsin A Human genes 0.000 claims description 5
- 108090000284 Pepsin A Proteins 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 229940111202 pepsin Drugs 0.000 claims description 5
- 235000010418 carrageenan Nutrition 0.000 claims description 4
- 229920001525 carrageenan Polymers 0.000 claims description 4
- 150000004676 glycans Chemical class 0.000 claims description 4
- 235000010987 pectin Nutrition 0.000 claims description 4
- 229920001277 pectin Polymers 0.000 claims description 4
- 239000001814 pectin Substances 0.000 claims description 4
- 229920001282 polysaccharide Polymers 0.000 claims description 4
- 239000005017 polysaccharide Substances 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 claims description 2
- 235000010643 Leucaena leucocephala Nutrition 0.000 claims description 2
- 229920000881 Modified starch Polymers 0.000 claims description 2
- 239000004368 Modified starch Substances 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 239000000679 carrageenan Substances 0.000 claims description 2
- 229940113118 carrageenan Drugs 0.000 claims description 2
- 235000019426 modified starch Nutrition 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 2
- 241000220479 Acacia Species 0.000 claims 1
- 229960001701 chloroform Drugs 0.000 description 68
- 239000000243 solution Substances 0.000 description 62
- 238000004587 chromatography analysis Methods 0.000 description 46
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 45
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 45
- 239000000523 sample Substances 0.000 description 40
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 30
- 238000002360 preparation method Methods 0.000 description 30
- 239000012086 standard solution Substances 0.000 description 27
- 239000000706 filtrate Substances 0.000 description 17
- 239000012528 membrane Substances 0.000 description 17
- 239000012085 test solution Substances 0.000 description 16
- 235000010469 Glycine max Nutrition 0.000 description 15
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 15
- 238000003260 vortexing Methods 0.000 description 14
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 13
- 108010059820 Polygalacturonase Proteins 0.000 description 13
- 108010093305 exopolygalacturonase Proteins 0.000 description 13
- 102000004882 Lipase Human genes 0.000 description 10
- 108090001060 Lipase Proteins 0.000 description 10
- 239000004367 Lipase Substances 0.000 description 9
- 235000019421 lipase Nutrition 0.000 description 9
- 239000006185 dispersion Substances 0.000 description 8
- 238000004090 dissolution Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 230000007071 enzymatic hydrolysis Effects 0.000 description 5
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- 238000001914 filtration Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 102000035195 Peptidases Human genes 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- RSBRGUIIKKPRFQ-UHFFFAOYSA-N acetic acid;n,n-diethylethanamine;propan-2-ol;hydrate Chemical compound O.CC(C)O.CC(O)=O.CCN(CC)CC RSBRGUIIKKPRFQ-UHFFFAOYSA-N 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- -1 n-hexane-isopropanol-acetic acid-triethylamine Chemical compound 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 229960001153 serine Drugs 0.000 description 1
- 229940083466 soybean lecithin Drugs 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for determining the content of phosphatidylserine, in particular to a method for determining the content of phosphatidylserine in a composition containing a colloidal matrix and phosphatidylserine, wherein the method comprises dissolving or dispersing the composition in an aqueous medium; extracting phosphatidylserine with a solvent; and determining the content of phosphatidylserine.
Description
Technical Field
The present disclosure relates to a method for measuring the content of phosphatidylserine, and more particularly, to a method for measuring the content of phosphatidylserine in a composition comprising a colloidal matrix and phosphatidylserine.
Background
Phosphatidylserine (PS) is a new food raw material, and can be prepared by converting soybean lecithin and L-serine with phospholipase, purifying, and drying. It has the functions of improving brain function, helping to concentrate attention and improving memory, and is widely applied to various foods and health care products. However, at present, no unified national, local or industrial standard exists for detecting the content of phosphatidylserine in food or health food.
In the prior art, the content of phosphatidylserine in foods such as bread, milk powder, yoghourt, tabletted candies and the like is detected, but the method is lack of a water bath dissolving or dispersing step and is not suitable for a composition containing a colloid matrix and phosphatidylserine, particularly for the content detection of the phosphatidylserine in gel candy. The prior art has the problems of incomplete extraction of phosphatidylserine, low detection rate, poor repeatability of detection results and the like because gel candies are not completely dissolved or dispersed.
A rapid and accurate detection method with strong specificity and high recovery rate is needed to be used for measuring the content of phosphatidylserine in a composition containing a colloid matrix and phosphatidylserine, in particular to gel candy.
Disclosure of Invention
The present disclosure relates to a method for measuring the content of phosphatidylserine, in particular, a method for measuring the content of phosphatidylserine in a composition comprising a colloidal matrix and phosphatidylserine, the method comprising dissolving or dispersing the composition in an aqueous medium; extracting phosphatidylserine with a solvent; and determining the content of phosphatidylserine.
After dissolving or dispersing the composition, an enzyme may be added to enzymatically digest at least a portion of the colloidal matrix to facilitate extraction.
The solvent for extracting phosphatidylserine may be a mixed solvent of chloroform and methanol, preferably wherein the volume fraction of chloroform in the solvent is 50% to 70%.
Detailed Description
The present disclosure relates to a method for measuring the content of phosphatidylserine, in particular, a method for measuring the content of phosphatidylserine in a composition comprising a colloidal matrix and phosphatidylserine, the method comprising dissolving or dispersing the composition in an aqueous medium; extracting phosphatidylserine with a solvent; and determining the content of phosphatidylserine.
In one embodiment, the colloidal matrix and phosphatidylserine in the composition are mixed, preferably homogeneously mixed. The composition may be a gel confection.
In one embodiment, the aqueous medium may be water.
In one embodiment, the dissolving or dispersing step may be facilitated by heating, such as water bath heating. The bath temperature is preferably from 30 ℃ to 100 ℃, more preferably from 40 ℃ to 80 ℃, especially preferably from 45 ℃ to 65 ℃, most preferably 60 ℃.
In one embodiment, the phosphatidylserine content can be determined by high performance liquid chromatography, preferably using an evaporative light scattering detector or an ultraviolet detector, preferably using external standard-log method for quantification.
In one embodiment, after dissolving or dispersing the composition, an enzyme may be added to enzymatically hydrolyze at least a portion of the colloidal matrix to facilitate extraction.
Without being bound by theory, the enzymatic hydrolysis step facilitates the liberation of phosphatidylserine from the colloidal matrix, which is uniformly dissolved or dispersed in the aqueous medium, facilitating the increase in recovery of the assay.
The enzyme is preferably used in an amount effective to enzymatically degrade the colloidal matrix, for example, from about 0.3 to 1.0g, most preferably 0.5g, per 1.5g of the composition (e.g., gel candy).
In one embodiment, the composition is dissolved or dispersed in an aqueous medium by heating, such as in a water bath, and then allowed to stand at room temperature before the enzyme is added.
The enzymatic hydrolysis temperature may be any temperature that allows for effective enzymatic hydrolysis of the colloidal matrix, such as 36.0 ℃ to 38.0 ℃, preferably 36.3 ℃ to 37.7 ℃, more preferably 37 ℃.
The enzymatic hydrolysis time may be any time that allows for effective enzymatic hydrolysis of the colloidal matrix, such as 10 to 120 minutes, preferably 30 to 90 minutes, more preferably 60 minutes.
In one embodiment, the colloidal matrix may comprise a protein colloid or a plant polysaccharide colloid; and the enzyme may be a protease.
In one embodiment, the protein colloid may be gelatin, the polysaccharide colloid may be selected from pectin, carrageenan, starch, modified starch or acacia, or the protease may be trypsin or pepsin.
In one embodiment, the enzymatically digested composition can be allowed to stand at room temperature and the phosphatidylserine can be extracted with a solvent.
In one embodiment, the solvent for extracting phosphatidylserine may be a mixed solvent of chloroform: methanol.
In one embodiment, the hydrophilicity of the mixed solvent may be increased, for example, the ratio of methanol in the mixed solvent may be increased, for example, the volume fraction of chloroform in the solvent may be 40% to 80%, preferably 50% to 70%, and more preferably 68%, which is advantageous for increasing the recovery rate of the measurement method.
When the solvent for extracting phosphatidylserine is located in the lower layer, the lower layer solution may be sucked into the volumetric flask.
The step of extracting phosphatidylserine may be repeated a plurality of times, for example 2, 3, 4, 5 or 6 times, in order to sufficiently extract phosphatidylserine. The extract can be placed in a volumetric flask and the volume can be fixed to the scale with the extraction solvent.
The method comprises the steps of precisely weighing a proper amount of phosphatidylserine standard substance, dissolving or dispersing the phosphatidylserine standard substance by using an extraction solvent such as a chloroform-methanol mixed solvent, fixing the volume to prepare a series of standard solutions of 80 mu g/mL to 400 mu g/mL, drawing a log-log standard curve by taking the peak area of the standard solution as a logarithm and then as a ordinate and taking the concentration of the standard solution as a logarithm and then as an abscissa, and calculating the concentration of the sample solution from the log-log standard curve according to the peak area.
For example, in one embodiment, the gel candy containing phosphatidylserine is dissolved or dispersed in a water bath at 60 ℃, then allowed to stand to room temperature, added with 0.5g protease, and enzymolyzed at 37 ℃ for 1 hour; placing the gel candy after enzymolysis to room temperature, adding a proper amount of chloroform and methanol solvent for extraction, sucking the lower layer solution into a volumetric flask, repeating the operation for 3 times, and then carrying out constant volume to scale by using the chloroform and methanol solvent; filtering with 0.45 μm filter membrane, collecting the filtrate, and loading on machine; precisely weighing a proper amount of phosphatidylserine standard substance, dissolving or dispersing the phosphatidylserine standard substance by using a chloroform-methanol solvent, and fixing the volume to prepare a series of standard solutions of 80 mu g/mL to 400 mu g/mL; and (3) detecting by combining high performance liquid chromatography with an evaporative light diffuser, taking the peak area of the standard solution as a logarithm and then as a vertical coordinate, taking the concentration of the standard solution as a logarithm and then as an abscissa, drawing a double-logarithm standard curve, and calculating the concentration of the sample solution from the double-logarithm standard curve according to the peak area.
Examples
The test sample in the examples was a gel candy in which a colloidal matrix and phosphatidylserine were uniformly mixed.
Example 1
1. Referring to the 'method for detecting the content of phosphatidylserine' disclosed in the specification of the Chinese invention patent application with the publication number of CN107688073A, the extraction solvent is chloroform/methanol solution (90: 10), the phosphatidylserine in the gelatin gel candy is measured, the detection is carried out by combining high performance liquid chromatography with an evaporative light scattering detector, and the quantification is carried out by an external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 phosphatidylserine (soy-derived) controls
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
accurately weighing a proper amount of sample (about 25mg of phosphatidylserine) in a 100mL volumetric flask, adding 10mL of chloroform/methanol solution (90: 10) mixed solution, carrying out ultrasonic treatment for 5min, standing to room temperature, fixing the volume of the chloroform/methanol solution (90: 10) mixed solution to a scale, filtering with a 0.45 mu m filter membrane, and taking a subsequent filtrate and measuring on a machine.
5. Preparation of standard solution:
precisely weighing about 20mg of phosphatidylserine reference substance into a 50mL brown volumetric flask, adding a chloroform/methanol solution (90: 10) mixed solution, performing ultrasonic treatment until the mixed solution is dissolved or dispersed, and fixing the volume to the scale. Precisely transferring standard intermediate working solution into brown volumetric flasks of 2.0mL, 4.0mL, 6.0mL, 8.0mL and 10.0mL to 10mL respectively, diluting the extraction solvent to a constant volume, filtering with a 0.45 μm filter membrane, and measuring the subsequent filtrate on a machine.
6. The chromatographic conditions were as follows:
evaporative light scattering detector parameter settings:
temperature: 70 deg.C
Gas flow rate: 1.5L/min
A chromatographic column: merck LiChrospher100Diol 5 μm 4X 125mm
Flow rate: 1.0mL/min
Column temperature: 35 deg.C
Sample introduction amount: 10 μ L
Mobile phase: mobile phase A: n-hexane-isopropanol-acetic acid-triethylamine (820: 170: 10: 0.8)
Mobile phase B: isopropanol-water-acetic acid-triethylamine (850: 140: 10: 0.8)
And (3) an elution mode: gradient elution
| Step (ii) of | Run time/min | Mobile phase A/%) | Mobile phase B/%) |
| 1 | 0 | 100 | 0 |
| 2 | 20 | 50 | 50 |
| 3 | 25 | 0 | 100 |
| 4 | 29 | 0 | 100 |
| 5 | 30 | 100 | 0 |
| 6 | 35 | 100 | 0 |
7. Sample assay
And respectively taking the standard solution and the sample solution into a chromatographic column, recording a chromatogram, taking the logarithm of the peak area of the standard solution as a vertical coordinate, taking the logarithm of the concentration of the standard solution as a horizontal coordinate, drawing a log-log standard curve, and searching the concentration of the sample solution from the double-logarithm standard curve according to the peak area.
In the formula:
x-content of Phospholserine in the sample (mg/mg)
ρ -Phospholserine concentration in milligrams per milliliter (mg/mL) in the test solution obtained from the standard curve
V-volume of sample solution constant volume, unit is milliliter (mL)
m one-sample mass in milligrams (mg)
8. The experimental results are as follows:
example 2:
1. dissolving or dispersing gelatin gel candy in water bath at 60 deg.C, extracting with chloroform/methanol solution (90: 10), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography combined with evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 phosphatidylserine (soy-derived) controls
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until dissolution or dispersion, shaking is carried out occasionally, and the centrifuge tube is placed to room temperature. Adding chloroform/methanol solution (90: 10) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (90: 10) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
example 3:
1. dissolving or dispersing gelatin gel candy in water bath at 60 deg.C, extracting with chloroform/methanol solution (65: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography combined with evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 phosphatidylserine (soy-derived) controls
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until dissolution or dispersion, shaking is carried out occasionally, and the centrifuge tube is placed to room temperature. Adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
example 4:
1. dissolving or dispersing gelatin gel candy in water bath at 60 deg.C, adding pepsin 0.5g to enzymolyze matrix in gelatin gel candy, extracting solvent is chloroform/methanol solution (65: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography combined with evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Pepsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until dissolution or dispersion, shaking is carried out occasionally, and the centrifuge tube is placed to room temperature. Adding 0.5g of pepsin for enzymolysis for 1 hour at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
example 5:
1. dissolving or dispersing gelatin gel candy in water bath at 60 deg.C, adding 0.5g trypsin for enzymolysis of matrix in gelatin gel candy, extracting with chloroform/methanol solution (65: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography and evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until dissolution or dispersion, shaking is carried out occasionally, and the centrifuge tube is placed to room temperature. Adding 0.5g of trypsin for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
Example 6:
1. dissolving or dispersing gelatin gel candy in water bath at 45 deg.C, adding 0.5g trypsin for enzymolysis of matrix in gelatin gel candy, extracting with chloroform/methanol solution (65: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography and evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) was precisely weighed into a centrifuge tube, 10mL of water was added, and the mixture was dissolved or dispersed in a 45 ℃ water bath, shaken up from time to time, and allowed to stand at room temperature. Adding 0.5g of trypsin for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
Example 7:
1. dissolving or dispersing gelatin gel candy in water bath at 50 deg.C, adding 0.5g trypsin for enzymolysis of matrix in gelatin gel candy, extracting with chloroform/methanol solution (65: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography and evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) was precisely weighed into a centrifuge tube, 10mL of water was added, and the mixture was dissolved or dispersed in a 50 ℃ water bath, shaken up from time to time, and allowed to stand at room temperature. Adding 0.5g of trypsin for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
Example 8:
1. dissolving or dispersing gelatin gel candy in 65 deg.C water bath, adding 0.5g trypsin for enzymolysis of matrix in gelatin gel candy, extracting with chloroform/methanol solution (65: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography and evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) was precisely weighed into a centrifuge tube, 10mL of water was added, and the mixture was dissolved or dispersed in a water bath at 65 ℃ with shaking, and left to stand at room temperature. Adding 0.5g of trypsin for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
example 9:
1. dissolving or dispersing gelatin gel candy in water bath at 60 deg.C, adding 0.5g trypsin for enzymolysis of matrix in gelatin gel candy, extracting with chloroform/methanol solution (55: 40), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography and evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until dissolution or dispersion, shaking is carried out occasionally, and the centrifuge tube is placed to room temperature. Adding 0.5g of trypsin for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (55: 40) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (55: 40) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
example 10:
1. dissolving or dispersing gelatin gel candy in water bath at 60 deg.C, adding 0.5g trypsin for enzymolysis of matrix in gelatin gel candy, extracting with chloroform/methanol solution (68: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography and evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until dissolution or dispersion, shaking is carried out occasionally, and the centrifuge tube is placed to room temperature. Adding 0.5g of trypsin for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (68: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (68: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
example 11:
1. dissolving or dispersing gelatin gel candy in water bath at 60 deg.C, adding 0.4g trypsin for enzymolysis of matrix in gelatin gel candy, extracting with chloroform/methanol solution (65: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography and evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until dissolution or dispersion, shaking is carried out occasionally, and the centrifuge tube is placed to room temperature. Adding about 0.4g of trypsin for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
example 12:
1. dissolving or dispersing gelatin gel candy in water bath at 60 deg.C, adding 1.0g trypsin for enzymolysis of matrix in gelatin gel candy, extracting with chloroform/methanol solution (65: 30), measuring phosphatidylserine in gelatin gel candy, detecting with high performance liquid chromatography and evaporative light scattering detector, and quantifying by external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Phosphatidylserine (soy-derived) control
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until dissolution or dispersion, shaking is carried out occasionally, and the centrifuge tube is placed to room temperature. Adding 1.0g of trypsin for enzymolysis at 37 ℃ for 1 h; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
example 13:
1. dissolving gelatin gel candies in water bath at 60 ℃, respectively adding 0.5g of trypsin, lipase, pectinase and amyloglucosidase to carry out enzymolysis on matrixes in the gelatin gel candies, wherein an extraction solvent is a trichloromethane/methanol solution (65: 30), measuring phosphatidylserine in the gelatin gel candies, detecting by combining high performance liquid chromatography and an evaporative light scattering detector, and quantifying by using an external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Lipase
2.9 pectinases
2.10 Amyloglucosidase
2.11 phosphatidylserine (soy-derived) controls
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until the sample is dissolved, shaking is carried out from time to time, and the centrifuge tube is placed to room temperature. Adding 0.5g of trypsin, lipase, pectinase and amyloglucosidase respectively for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
| trypsin | Lipase enzyme | Pectinase | Amyloglucosidase |
| 51.25mg/2.5g | 40.51mg/2.5g | 27.30mg/2.5g | 40.68mg/2.5g |
Example 14:
1. dissolving pectin gel candies in water bath at 60 ℃, adding 0.5g of trypsin, lipase, pectinase and amyloglucosidase to respectively perform enzymolysis on matrixes in the gel candies, wherein an extraction solvent is a trichloromethane/methanol solution (65: 30), measuring phosphatidylserine in the pectin gel candies, detecting by high performance liquid chromatography and an evaporative light scattering detector, and quantifying by an external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Lipase
2.9 pectinases
2.10 Amyloglucosidase
2.11 phosphatidylserine (soy-derived) controls
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until the sample is dissolved, shaking is carried out from time to time, and the centrifuge tube is placed to room temperature. Adding 0.5g of trypsin, lipase, pectinase and amyloglucosidase respectively for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, 0.45mn filter membrane is used for filtration, and the subsequent filtrate is measured on a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
| trypsin | Lipase enzyme | Pectinase | Amyloglucosidase |
| 50.79mg/2.5g | 40.11mg/2.5g | 16.29mg/2.5g | 43.44mg/2.5g |
Example 15:
1. dissolving carrageenin gel candy in water bath at 60 ℃, adding 0.5g of trypsin, lipase, pectinase and amyloglucosidase to hydrolyze the matrix in the gel candy, wherein the extraction solvent is a trichloromethane/methanol solution (65: 30), measuring phosphatidylserine in the carrageenin gel candy, detecting by high performance liquid chromatography and an evaporative light scattering detector, and quantifying by an external standard-double logarithm method.
2. The reagents used were as follows:
2.1 Hexane (pure chromatography)
2.2 Isopropanol (pure chromatography)
2.3 Triethylamine (pure chromatography)
2.4 acetic acid (analytically pure)
2.5 trichloromethane (analytical purity)
2.6 methanol (analytically pure)
2.7 Trypsin
2.8 Lipase
2.9 pectinases
2.10 Amyloglucosidase
2.11 phosphatidylserine (soy-derived) controls
3. The instrument used was as follows:
high performance liquid chromatography (Agilent model 1260 high performance liquid chromatography, manufacturer: Agilent corporation, USA);
4. preparation of a test solution:
an appropriate amount of sample (about 25mg of phosphatidylserine) is precisely weighed into a centrifuge tube, 10mL of water is added, water bath is carried out at 60 ℃ until the sample is dissolved, shaking is carried out from time to time, and the centrifuge tube is placed to room temperature. Adding 0.5g of trypsin, lipase, pectinase and amyloglucosidase respectively for enzymolysis for 1h at 37 ℃; adding chloroform/methanol solution (65: 30) 10mL, extracting, vortexing for 5min, and centrifuging at 6000r/min for 5 min; sucking the lower layer solution into a 100mL volumetric flask, and repeating the operation for 2 times; chloroform/methanol solution (65: 30) is fixed to the constant volume to the scale, and is filtered by a 0.45 mu m filter membrane, and the subsequent filtrate is measured by a machine.
5. The standard solution preparation, HPLC analysis conditions and sample determination were the same as in example 1.
6. The experimental results are as follows:
| trypsin | Lipase enzyme | Pectinase | Amyloglucosidase |
| 49.53mg/2.5g | 38.81mg/2.5g | 28.64mg/2.5g | 50.35mg/2.5g |
Example 16:
the phosphatidylserine standard was prepared as a series of standard solutions of 80. mu.g/mL, 160. mu.g/mL, 240. mu.g/mL, 320. mu.g/mL, and 400. mu.g/mL, and the detection was performed under the conditions of the chromatography in example 1.
Taking the logarithm of the peak area of the standard solution as an ordinate, taking the logarithm of the concentration of the standard solution as an abscissa, drawing a log-log standard curve, and obtaining a regression equation y of 1.325x +3.931, wherein the R value is 1.000 and the range is 86-431 mu g/mL.
Example 17:
taking 1.5g of gelatin gel confectionary sample without adding phosphatidylserine into a centrifuge tube, adding 45mg of phosphatidylserine raw material into the centrifuge tube to prepare six parallels, detecting according to example 5, and calculating the recovery rate and the average recovery rate, wherein the results are shown in the following table
Results of the Table accuracy test
Example 18:
a phosphatidylserine gelatin gel candy sample is taken and tested according to the example 5, and the precision is measured after 6 times of parallel operation. Another gelatin gel candy sample was taken by different experimenters at different times and tested according to example 5, and the repeated RSD and 12 sets of data RSD of two repeated experiments were calculated by performing 6 parallel operations. The results are shown in the following table.
Results of the Table precision test
Through optimization of an experimental process, the detection rate of phosphatidylserine in the gelatin gel candy is improved, and the table is as follows:
TABLE EXAMPLES 1 TO 5 comparison of phosphatidylserine assay results
The samples to be tested were dissolved or dispersed by different water bath temperatures and tested as in examples 5 to 8, respectively, with the results as follows:
samples to be tested are extracted by mixed solutions of trichloromethane and methanol with different proportions, and are respectively tested according to the examples 5, 9 and 10, and the results are as follows:
| examples | 5 | 9 | 10 |
| Chloroform methanol | 65∶30 | 55∶40 | 68∶30 |
| Results | 50.29mg/3g | 49.90mg/3g | 50.11mg/3g |
The samples to be tested are subjected to enzymolysis by adding trypsin with different dosages, and the determination is respectively carried out according to the embodiment 5, the embodiment 11 and the embodiment 12, and the results are as follows:
| examples | 5 | 11 | 12 |
| Amount of trypsin | 0.5g | 0.4g | 1.0g |
| Results | 50.29mg/3g | 51.89mg/3g | 48.63mg/3g |
Gel candy samples of different matrices were enzymatically hydrolyzed by adding different enzymes, as determined in example 13, example 14, and example 15, respectively, and the results are shown in the following table:
| examples | 13 | 14 | 15 |
| Trypsin | 51.25mg/2.5g | 50.79mg/2.5g | 49.53mg/2.5g |
| Lipase enzyme | 40.51mg/2.5g | 40.11mg/2.5g | 38.81mg/2.5g |
| Pectinase | 27.30mg/2.5g | 16.29mg/2.5g | 28.64mg/2.5g |
| Amyloglucosidase | 40.68mg/2.5g | 43.44mg/2.5g | 50.35mg/2.5g |
。
Claims (10)
1. A method for determining the content of phosphatidylserine in a composition comprising a colloidal matrix and phosphatidylserine, the method comprising dissolving or dispersing the composition in an aqueous medium; extracting phosphatidylserine with a solvent; and determining the content of phosphatidylserine.
2. The method of claim 1, wherein the composition is a gel candy, such as a soft candy.
3. The method of claim 1, wherein the aqueous medium is water.
4. The method of claim 1, wherein the dissolving or dispersing step is facilitated by heating, such as water bath heating.
5. The method according to claim 1, wherein the phosphatidylserine content is determined by high performance liquid chromatography, preferably using an evaporative light scattering detector or an ultraviolet detector, preferably using external standard-log method.
6. The method of claim 1, wherein after dissolving or dispersing the composition, an enzyme is added to enzymatically hydrolyze at least a portion of the colloidal matrix to facilitate extraction.
7. The method of claim 6, wherein the colloidal matrix comprises a protein colloid or a plant polysaccharide colloid; and the enzyme is a protease.
8. The method of claim 7, wherein the protein colloid is gelatin; or the polysaccharide colloid is selected from pectin, carrageenan, starch, modified starch or acacia; or the protease is trypsin or pepsin.
9. The method according to any one of claims 1 to 8, wherein the solvent used for extracting phosphatidylserine is chloroform: and (3) a methanol mixed solvent.
10. The method of claim 9, wherein the volume fraction of chloroform in the solvent is 50% -70%.
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| CN108896680A (en) * | 2018-07-20 | 2018-11-27 | 汤臣倍健股份有限公司 | A method of utilizing the phosphatide of LC-MS technology detection albumen powder |
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