CN114088826A - Extraction method of vitamin D3 and method for detecting content of vitamin D3 - Google Patents
Extraction method of vitamin D3 and method for detecting content of vitamin D3 Download PDFInfo
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- CN114088826A CN114088826A CN202111254035.4A CN202111254035A CN114088826A CN 114088826 A CN114088826 A CN 114088826A CN 202111254035 A CN202111254035 A CN 202111254035A CN 114088826 A CN114088826 A CN 114088826A
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- 239000011647 vitamin D3 Substances 0.000 title claims abstract description 88
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 title claims abstract description 72
- 238000000605 extraction Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 23
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 title description 7
- 235000005282 vitamin D3 Nutrition 0.000 title description 6
- 229940021056 vitamin d3 Drugs 0.000 title description 6
- 239000000243 solution Substances 0.000 claims abstract description 131
- 239000011710 vitamin D Substances 0.000 claims abstract description 77
- 229930003316 Vitamin D Natural products 0.000 claims abstract description 66
- 235000019166 vitamin D Nutrition 0.000 claims abstract description 66
- 150000003710 vitamin D derivatives Chemical class 0.000 claims abstract description 66
- 229940046008 vitamin d Drugs 0.000 claims abstract description 66
- 238000001514 detection method Methods 0.000 claims abstract description 65
- 239000007788 liquid Substances 0.000 claims abstract description 47
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000002270 dispersing agent Substances 0.000 claims abstract description 42
- 239000000126 substance Substances 0.000 claims abstract description 39
- 238000002156 mixing Methods 0.000 claims abstract description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical group CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 21
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 21
- 238000012360 testing method Methods 0.000 claims abstract description 21
- 235000010354 butylated hydroxytoluene Nutrition 0.000 claims abstract description 20
- 239000011259 mixed solution Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 26
- 238000004811 liquid chromatography Methods 0.000 claims description 18
- 238000009210 therapy by ultrasound Methods 0.000 claims description 12
- 239000012088 reference solution Substances 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 8
- 239000012895 dilution Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000012491 analyte Substances 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000005070 sampling Methods 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000006185 dispersion Substances 0.000 abstract description 3
- 239000000843 powder Substances 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 12
- 239000012085 test solution Substances 0.000 description 11
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 8
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 7
- 239000011575 calcium Substances 0.000 description 7
- 229910052791 calcium Inorganic materials 0.000 description 7
- 229910000019 calcium carbonate Inorganic materials 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- -1 2, 6-di-tert-butyl-p-cresol n-hexane Chemical compound 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 239000013558 reference substance Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000007865 diluting Methods 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 2
- 239000007910 chewable tablet Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 101100001794 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) aps-2 gene Proteins 0.000 description 1
- 230000009102 absorption Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- PGXWDLGWMQIXDT-UHFFFAOYSA-N methylsulfinylmethane;hydrate Chemical compound O.CS(C)=O PGXWDLGWMQIXDT-UHFFFAOYSA-N 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- YUGCAAVRZWBXEQ-WHTXLNIXSA-N previtamin D3 Chemical compound C=1([C@@H]2CC[C@@H]([C@]2(CCC=1)C)[C@H](C)CCCC(C)C)\C=C/C1=C(C)CC[C@H](O)C1 YUGCAAVRZWBXEQ-WHTXLNIXSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007127 saponification reaction Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- 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
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- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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Abstract
The invention belongs to the technical field of chemical detection, and particularly relates to vitamin D3The extraction method of (1) and the detection of vitamin D3The method of content. The vitamin D provided by the invention3The extraction method is carried out under the condition of keeping out of the light, and comprises the following steps: mixing the substance to be tested with dispersant, antioxidant and extractantObtaining solution of the substance to be detected; the dispersing agent is a mixed solution of dimethyl sulfoxide and water, the antioxidant is 2, 6-di-tert-butyl-p-cresol, and the extracting agent is n-hexane; purifying the solution of the substance to be detected to obtain vitamin D3Extracting the liquid. The invention simultaneously carries out the steps of dispersion and extraction so as to ensure the vitamin D in the object to be detected3The vitamin D is avoided by extracting with an extractant immediately after dispersion in the solution3Loss of vitamin D, significantly improved vitamin D3Thereby increasing the vitamin D content in the test substance3The accuracy of the content.
Description
Technical Field
The invention belongs to the technical field of chemical detection, and particularly relates to vitamin D3The extraction method of (1) and the detection of vitamin D3The method of content.
Background
Vitamin D3Is a fat-soluble vitamin, is regarded as a hormone precursor acting on calcium and phosphorus metabolism, helps absorption of calcium and phosphorus in intestinal tracts, promotes bone calcium deposition, increases reabsorption of calcium and phosphorus by kidneys, improves the concentration of calcium and phosphorus in blood, and is beneficial to mineralization of bones. Most of the existing compound preparations for supplementing calcium and vitamins contain vitamin D3Due to vitamin D3Is unstable to light and heat, and is easy to be oxidized and photolyzed into previtamin D in air3Trans-vitamin D3Various products such as photosterols and tachysterols. In order to increase vitamin D in the compound preparation3Will mostly contain vitamin D3The clathrate of the powder is used as a raw material for preparing a compound preparation. However, when the content of vitamin D3 in the compound preparation is detected, the form of the inclusion compound causes vitamin D3The difficulty of extraction increases.
Existing extraction of vitamin D3The method mostly adopts extraction and measurement after saponification of the compound preparation, but loss is generated in extraction, and the extraction efficiency is reduced, so that the accuracy of a detection result is influenced.
Disclosure of Invention
In view of the above, the present invention provides a vitamin D3The extraction method of (1) and the detection of vitamin D3The extraction method provided by the invention can efficiently extract vitamin D in the object to be detected3Ensure vitamin D in the test object3The extraction is more complete, so that the accuracy of the detection result is ensured.
In order to solve the technical problems, the invention provides vitamin D3The extraction method is carried out under the condition of keeping out of the light, and comprises the following steps:
mixing a substance to be detected with a dispersant, an antioxidant and an extractant to obtain a solution of the substance to be detected; the dispersing agent is a mixed solution of dimethyl sulfoxide and water, the antioxidant is 2, 6-di-tert-butyl-p-cresol, and the extracting agent is n-hexane;
purifying the solution of the substance to be detected to obtain vitamin D3Extracting the liquid.
Preferably, the volume ratio of dimethyl sulfoxide to water in the dispersing agent is 9.8-10.2: 2.
preferably, the mixing comprises the steps of:
dissolving part of antioxidant in an extractant to obtain antioxidant-extractant solution;
dissolving the rest antioxidant in the dispersant to obtain antioxidant-dispersant solution;
and mixing the object to be detected, the antioxidant-extractant solution and the antioxidant-dispersant solution to obtain an object solution to be detected.
Preferably, the mass concentration of the 2, 6-di-tert-butyl-p-cresol in the antioxidant-extractant solution is 0.018 to 0.022 percent;
the mass concentration of the 2, 6-di-tert-butyl-p-cresol in the antioxidant-dispersant solution is 0.18-0.22 mg/mL;
vitamin D in the test substance3Quality of (1)The amount and volume ratio of antioxidant-extractant solution was 25 μ g: 23-27 mL;
the volume ratio of the antioxidant-extractant solution to the antioxidant-dispersant solution is 1: 1.8 to 2.2.
Preferably, the mixing of the analyte, the antioxidant-extractant solution and the antioxidant-dispersant solution comprises the following steps:
carrying out vortex or shaking on the obtained mixed system after ultrasonic treatment;
the ultrasonic treatment power is 200-400W, the time is 13-17 min, and the temperature is below 30 ℃;
the vortex or shaking time is independently 13-17 min.
Preferably, the purification process comprises the steps of:
carrying out first centrifugal treatment on the solution of the substance to be detected to obtain supernatant liquid, so as to obtain primary purified solution;
subjecting the primary purified solution to a second centrifugation to obtain a supernatant to obtain vitamin D3Extracting the liquid.
Preferably, the rotation speed of the first centrifugation and the rotation speed of the second centrifugation are respectively more than 4000r/min, and the time is respectively 4-6 min.
The invention also provides a method for detecting vitamin D3The method comprises the following steps:
with vitamin D3The extract is to-be-detected liquid, and vitamin D3The extracting solution is extracted by the extracting method of any one of claims 1 to 7;
injecting the liquid to be detected into a liquid chromatograph to obtain a liquid chromatogram of the liquid to be detected;
mixing vitamin D3Injecting the reference solution into a liquid chromatograph to obtain a reference liquid chromatogram;
calculating vitamin D in the test substance according to the formula 13The content is as follows:
wherein A isTo be treatedIs vitamin D in the solution to be tested3Peak area of (a);
Mto pairIs vitamin D3Sample size of control: mg;
Pto pairIs vitamin D3Vitamin D in control3The content of (A): 99.8 percent
DTo be treatedThe dilution times of the solution to be tested are as follows: 25;
Ato pairAs vitamin D in control solution3Peak area of (a);
Mto be treatedSampling volume for the test substance: g;
Dto pairIs vitamin D3Dilution factor of control solution: 10000.
preferably, the liquid chromatography detection conditions include: the chromatographic column filler is amino bonded silica gel, and the column temperature is 25-35 ℃; taking a mixed solution of n-hexane and isopropanol as a mobile phase; the flow rate is 1.3-1.7 mL/min; the detection wavelength was 265 nm.
Preferably, the volume ratio of n-hexane to isopropanol in the mixed solution of n-hexane and isopropanol is 98.9-99.1: 0.9 to 1.1.
The invention provides vitamin D3The extraction method is carried out under the condition of keeping out of the light, and comprises the following steps: mixing a substance to be detected with a dispersant, an antioxidant and an extractant to obtain a solution of the substance to be detected; the dispersing agent is a mixed solution of dimethyl sulfoxide and water, the antioxidant is 2, 6-di-tert-butyl-p-cresol, and the extracting agent is n-hexane; purifying the solution of the substance to be detected to obtain vitamin D3Extracting the liquid. The vitamin D in the substance to be detected is extracted by mixing the substance to be detected, the antioxidant, the dispersing agent and the extracting agent3The invention simultaneously carries out the steps of dispersion and extraction so as to ensure the vitamin D in the object to be detected3The extract is extracted immediately after dispersing in the solution to avoid vitamin D3Loss of vitamin D, significantly improved vitamin D3Thereby increasing the vitamin D content in the test substance3And (3) the detection accuracy of the content.
Drawings
FIG. 1 is a liquid chromatography spectrum of example 1;
fig. 2 is a linear curve point diagram.
Detailed Description
The invention provides vitamin D3The extraction method is carried out under the condition of keeping out of the light, and comprises the following steps:
mixing a substance to be detected with a dispersant, an antioxidant and an extractant to obtain a solution of the substance to be detected; the dispersing agent is a mixed solution of dimethyl sulfoxide and water, the antioxidant is 2, 6-di-tert-butyl-p-cresol, and the extracting agent is n-hexane;
purifying the solution of the substance to be detected to obtain vitamin D3Extracting the liquid.
Mixing a substance to be detected with a dispersing agent, an antioxidant and an extracting agent to obtain a solution of the substance to be detected; the dispersing agent is a mixed solution of dimethyl sulfoxide and water, the antioxidant is 2, 6-di-tert-butyl-p-cresol, and the extracting agent is n-hexane. The light-shielding condition is not particularly limited, and the light-shielding can be realized. The embodiment of the invention provides a light-proof condition by using a dark room. The invention can avoid vitamin D by preparing the solution of the substance to be detected under the condition of keeping out of the sun3Is oxidized.
In the present invention, the analyte preferably includes vitamin D3Powder and/or containing vitamin D3More preferably vitamin D3The compound preparation of (1). In the present invention, the vitamin D-containing composition3The compound preparation preferably comprises calcium carbonate D3Granules, calcium carbonate D3Chewable tablets or vitamin D calcium chewable tablets.
In the invention, the volume ratio of dimethyl sulfoxide to water in the dispersing agent is preferably 9.8-10.2: 2, more preferably 10: 2. in the invention, the mass of the object to be detected, the volume ratio of the dispersing agent to the extracting agent is preferably 20-30 mug: 40-60 mL: 10-30 mL, more preferably 25 μ g: 50mL of: 25 mL.
In the present invention, the mixing preferably comprises the steps of:
dissolving part of antioxidant in an extractant to obtain antioxidant-extractant solution;
dissolving the rest antioxidant in the dispersant to obtain antioxidant-dispersant solution;
and mixing the object to be detected, the antioxidant-extractant solution and the antioxidant-dispersant solution to obtain an object solution to be detected.
In the invention, part of the antioxidant is dissolved in the extractant to obtain antioxidant-extractant solution. In the invention, the mass concentration of the 2, 6-di-tert-butyl-p-cresol in the antioxidant-extractant solution is preferably 0.018-0.022%, and more preferably 0.02%. The present invention is not particularly limited as long as the dissolution can be completed.
The invention dissolves the rest antioxidant in the dispersant to obtain antioxidant-dispersant solution. In the invention, the mass concentration of the 2, 6-di-tert-butyl-p-cresol in the antioxidant-dispersant solution is preferably 0.18-0.22 mg/mL, and more preferably 0.2 mg/mL. The present invention is not particularly limited as long as the dissolution can be completed.
After the antioxidant-extractant solution and the antioxidant-dispersant solution are obtained, the object to be detected, the antioxidant-extractant solution and the antioxidant-dispersant solution are mixed to obtain the object to be detected solution. In the present invention, vitamin D is contained in the test substance3The ratio of the mass of (a) to the volume of the extractant mixed solution is preferably 25 μ g: 23-27 mL, more preferably 25 μ g: 25 mL. In the present invention, the volume ratio of the antioxidant-extractant solution to the antioxidant-dispersant solution is preferably 1: 1.8-2.2, more preferably 1: 2.
in the present invention, the mixing of the analyte, the antioxidant-extractant solution, and the antioxidant-dispersant solution preferably includes the following steps: and carrying out vortex or shaking on the obtained mixed system after ultrasonic treatment. In the invention, the power of ultrasonic treatment is preferably 200-400W, more preferably 250-300W; the time of ultrasonic treatment is preferably 13-17 min, and more preferably 15 min; the temperature of the ultrasonic treatment is preferably below 30 ℃, and more preferably 25-30 ℃. In the present invention, the ultrasonic treatment is preferably accompanied by shaking.
In the invention, the vortex or shaking time is preferably independent for 13-17 min, and more preferably independent for 15 min; the rotation speed of the shaking is preferably 250r/min or more.
The device for swirling and shaking is not particularly limited, and a device which is conventional in the field can be adopted. In an embodiment of the invention, the vortexing is performed in a micro vortex mixer; the shaking was carried out in a shaker.
After the solution of the substance to be detected is obtained, the solution of the substance to be detected is purified to obtain vitamin D3Extracting the liquid. In the present invention, the purification process preferably comprises the steps of:
carrying out first centrifugal treatment on the solution of the substance to be detected to obtain supernatant liquid, so as to obtain primary purified solution;
subjecting the primary purified solution to a second centrifugation to obtain a supernatant to obtain vitamin D3Extracting the liquid.
According to the invention, the solution of the substance to be detected is preferably subjected to a first centrifugation treatment to obtain a supernatant, so as to obtain a primary purified solution. In the invention, the rotation speed of the first centrifugal treatment is preferably more than 4000r/min, and more preferably 5000-6000 r/min; the time of the first centrifugal treatment is preferably 4-6 min, and more preferably 5 min.
After the primary purified solution is obtained, the invention preferably performs second centrifugation treatment on the primary purified solution to obtain supernatant liquid to obtain vitamin D3Extracting the liquid. In the invention, the rotation speed of the second centrifugal treatment is preferably more than 4000r/min, and more preferably 5000-6000 r/min; the time of the second centrifugal treatment is preferably 4-6 min, and more preferably 5 min.
The invention also provides a method for detecting vitamin D3The method comprises the following steps:
with vitamin D3The extract is to-be-detected liquid, and vitamin D3The extracting solution is extracted by the extracting method in the technical scheme;
injecting the liquid to be detected into a liquid chromatograph to obtain a liquid chromatogram of the liquid to be detected;
mixing vitamin D3Injecting the reference solution into a liquid chromatograph to obtain a reference liquid chromatogram;
calculating vitamin D in the test substance according to the formula 13The content is as follows:
wherein A isTo be treatedIs vitamin D in the solution to be tested3Peak area of (a);
Mto pairIs vitamin D3Sample size of control: mg;
Pto pairIs vitamin D3Vitamin D in control3The content of (A): 99.8 percent
DTo be treatedThe dilution times of the solution to be tested are as follows: 25;
Ato pairAs vitamin D in control solution3Peak area of (a);
Mto be treatedSampling volume for the test substance: g;
Dto pairIs vitamin D3Dilution factor of control solution: 10000.
in the present invention, the vitamin D3The preparation method of the reference substance solution comprises the following steps: mixing vitamin D3Dissolving the reference substance in 0.02 wt% 2, 6-di-tert-butyl-p-cresol n-hexane solution, and diluting to obtain vitamin D3Vitamin D with mass concentration of 1-2 mug/mL3And (4) a control solution.
In the present invention, the liquid chromatography detection conditions preferably include: the chromatographic column filler is amino bonded silica gel, the column temperature is preferably 25-35 ℃, and more preferably 28-30 ℃; taking a mixed solution of n-hexane and isopropanol as a mobile phase; the flow rate is 1.3-1.7 mL/min, and more preferably 1.4-1.5 mL/min; the detection wavelength is 265 nm; in the invention, the volume ratio of n-hexane to isopropanol in the mixed solution of n-hexane and isopropanol is preferably 98.9-99.1: 0.9-1.1, and can be specifically 98.9: 1.1, 99: 1 or 99.1:0.9. in the invention, the liquid inlet amount of the liquid chromatogram is preferably 98-102 mu L, and more preferably 100 mu L. In the present invention, the chromatographic column preferably comprises an APS-2 amino column, XB-NH2Column or ZORBAX NH2Column, more preferably XB-NH2And (3) a column.
In the present invention, the detection limit of the liquid chromatography is preferably 0.315 ng; the limit of quantitation for the liquid chromatography is preferably 0.525 ng.
In the present invention, the vitamin D3Mass concentration of reference substance solution and vitamin D in solution to be detected3Have similar mass concentration and vitamin D3The reference substance solution preferably contains vitamin D in the solution to be detected3The mass concentration of (A) is 80-120%.
In the present invention, the liquid chromatography detection conditions for detecting the control solution are preferably the same as the liquid chromatography detection conditions for detecting the solution to be detected, and the detailed description thereof is omitted here.
In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Respectively adding 13.59mg and 11.57mg of vitamin D in a dark room3Dissolving a reference sample in a 0.02 mass percent n-hexane solution of 2, 6-di-tert-butyl-p-cresol, diluting to 100mL, and diluting 1mL by 100 times to obtain vitamin D with mass concentrations of 1.359 mu g/mL and 1.157 mu g/mL3A control solution;
respectively adding vitamin D3Performing liquid chromatography detection on the reference solution to obtain a reference liquid chromatogram; the detection conditions of the liquid chromatogram are as follows: with XB-NH2The column is a chromatographic column, and the column temperature is 30 ℃; in volume ratio 99: 1, taking a mixed solution of normal hexane and isopropanol as a mobile phase; the flow rate is 1.5 mL/min; the detection wavelength is 265 nm;
dissolving 2, 6-di-tert-butyl-p-cresol in n-hexane to obtain a 0.02 mass percent n-hexane solution of 2, 6-di-tert-butyl-p-cresol;
mixing a mixed solution of dimethyl sulfoxide and water with the volume ratio of the dimethyl sulfoxide to the water being 10:2 with 2, 6-di-tert-butyl-p-cresol to obtain an antioxidant-dispersant solution with the concentration of the 2, 6-di-tert-butyl-p-cresol being 0.2 mg/mL;
mixing 10mg of vitamin D3Powder (available from Dismann (Shanghai) Co., Ltd., lot number UT18020008, vitamin D)30.25 percent of the theoretical content), 25mL of 2, 6-di-tert-butyl p-cresol n-hexane solution and 50mL of antioxidant-dispersant solution are subjected to ultrasonic treatment (30 ℃ and shaking) for 15min under the condition that the power is 200W, and then are mixed for 15min in a micro vortex mixer to obtain the vitamin D3A powder solution;
mixing said vitamin D3Centrifuging the powder solution at 4000r/min for 5min, and centrifuging the upper layer solution at 4000r/min for 5min to obtain vitamin D3Extracting solution;
injecting 100 mu L of vitamin D3 extract into a liquid chromatograph for liquid chromatography detection to obtain a liquid chromatogram of the liquid to be detected; the detection conditions of the liquid chromatogram are as follows: with XB-NH2The column is a chromatographic column, and the column temperature is 30 ℃; in volume ratio 99: 1, taking a mixed solution of normal hexane and isopropanol as a mobile phase; the flow rate is 1.5 mL/min; the detection wavelength is 265 nm;
calculation of vitamin D according to equation 13Vitamin D in powder3The content is as follows:
wherein A isTo be treatedIs vitamin D in the solution to be tested3Peak area of (a);
Mto pairIs vitamin D3Sample size of control: mg;
Pto pairIs vitamin D3Vitamin D in control3The content of (A): 99.8 percent
DTo be treatedThe dilution times of the solution to be tested are as follows: 25;
Ato pairAs vitamin D in control solution3Peak area of (a);
Mto be treatedIs an object to be measuredThe sampling amount of (c): g;
Dto pairIs vitamin D3Dilution factor of control solution: 10000.
example 2
The detection was carried out by the method of example 1 except that the analyte was calcium carbonate D3Granules (each bag containing 0.625g of calcium carbonate, vitamin D)3100IU corresponding to 2.5. mu.g), 10 bags of calcium carbonate D3Granules (equivalent to 25. mu.g vitamin D)3) As the analyte.
Comparative example 1
And taking a 2, 6-di-tert-butyl-p-cresol n-hexane solution with the mass concentration of 0.02% as a blank solvent control.
Comparative example 2
Detection was carried out according to the method of example 1, except that 10mg of vitamin D was added3Powder (available from Dismann (Shanghai) Co., Ltd., lot number UT18020008, vitamin D)30.25 percent of vitamin D) and 50mL of dispersant mixed solution are subjected to ultrasonic treatment (30 ℃ and shaking) for 15min under the condition that the power is 200W, and then are mixed with 25mL of 2, 6-di-tert-butyl-p-cresol n-hexane solution for 15min in a micro vortex mixer to obtain the vitamin D3Powder solution.
Comparative example 3
According to vitamin D3Vitamin D in powder import drug registration standard JX200800963Content detection method for detecting vitamin D in example 13Vitamin D in powder3The content of (a).
The liquid chromatography spectrum in example 1 is shown in fig. 1.
The results of the tests of examples 1 and 2, comparative examples 1 to 3 are shown in Table 1.
TABLE 1 test results of examples 1 and 2, comparative examples 1 to 3
As can be seen from the results of example 1 and comparative examples 2 and 3, the detection method provided by the invention has higher detection accuracy.
From the results of comparative example 1, it is clear that the blank solvent has no influence on the detection results of the present invention.
Detecting the specificity of the detection method:
vitamin D from the control solution of example 13Retention time and vitamin D in test solutions of examples 1 and 23The retention times of (1) and comparative example 1 are shown in Table 2.
TABLE 2 Retention time of control solution, examples 1, 2 and comparative example 1
As can be seen from the results in Table 2, the blank solvent contained no vitamin D3Peak, vitamin D in examples 1, 23The main peak retention time of (A) and vitamin D in the control solution3The main peak retention time is consistent, the blank solvent does not interfere the content detection of the product, and the detection method provided by the invention has good specificity.
Vitamin D3Content system applicability detection:
a control solution was prepared as in example 1, except that the control solution was destroyed at 60 ℃ for 1h to yield previtamin D3And obtaining the liquid to be detected. The resulting test solutions were tested in 6 consecutive runs according to the method of example 1, and the results are shown in Table 3.
TABLE 3 results of 6 consecutive tests
As is clear from the results in Table 3, previtamin D3Peak and vitamin D3The average value of the separation degree of the peak is 15.6 and is more than 5, and the number of theoretical plates is calculated according to vitamin D3Peak counts were 16778, greater than 5000, and vitamin D3Relative Standard Deviation (RSD) (n 6) of peak retention time is 0.16% and peak area RSD (n 6) is 0.39%, both less than 2.0%, and vitamin D is obtained3Content measurementThe system has good applicability.
The detection method comprises the following steps of detection of precision:
the test solutions were prepared according to the method of example 1, and two experimenters were allowed to perform liquid chromatography tests according to the method of example 1 by using two liquid chromatographs of different types, respectively, and the test results are shown in table 4.
TABLE 4 vitamin D in the test solution3Results of content measurement
As can be seen from Table 4, vitamin D was detected by the A laboratory3Vitamin D in powder3Content repeatability results were 0.255%, RSD (n ═ 6) was 0.90%, vitamin D detected by b experimenter3Vitamin D in powder3The content repeatability result is 0.253%, the RSD (n-6) is 1.16%, the content average value of 12 data is 0.254%, and the RSD (n-12) is 1.04%, which proves that the vitamin D provided by the invention is3The content detection method has good precision.
Determining a detection limit:
the detection limit was determined according to a conventional method, and the results thereof are shown in Table 5.
TABLE 5 detection limits of the detection methods provided by the present invention
| Main peak | Signal-to-noise ratio (S/N) | Detection limit | Limit of | About a ratio corresponding to the limit |
| Vitamin D3 | 3.09 | 0.315ng | 100ng | 0.315％ |
As is clear from the results in Table 5, the detection method provided by the present invention is excellent in sensitivity.
And (4) quantitative limit:
vitamin D preparation according to example 13Control solution, except vitamin D3The concentration of the reference solution is 0.525 ng/mL; vitamin D in the control solution was measured 6 consecutive times according to the method of example 13The results are shown in Table 6.
TABLE 6 detection results of quantitative limit of the detection method provided by the present invention
As can be seen from the results in Table 6, the quantitative limit of the detection method provided by the present invention is far less than the limit, and the detection method provided by the present invention can accurately and quantitatively detect vitamin D3And (4) content.
The linear relationship is:
vitamin D preparation according to example 13The difference of the reference solution is that the concentration of the reference solution is respectively 200 percent, 150 percent and 120 percent of the limit,100%, 50% and 20%; detection of vitamin D in control solutions according to the method of example 13The results are shown in Table 7.
TABLE 7 vitamin D3Content detection linearity research result
Linear regression was performed according to the data in table 7 with the amount of sample (ng) as the abscissa and the peak area as the ordinate by the least square method to obtain a linear curve, and the data in table 7 was plotted as a point line graph, as shown in fig. 2.
As can be seen from table 7 and fig. 2, the linear correlation coefficient r of the obtained phenomenon curve was 1.0000, and the peak area and the amount of sample had a good linear relationship.
Stability of the solution to be tested:
the test solutions were prepared according to the method of example 1, and the content of vitamins in the test solutions was measured according to the measurement method of example 1 at 0h, 2h, 4h, and 8h after the test solutions were prepared, respectively, and the results are shown in table 8.
TABLE 8 vitamin D in the test solutions at different test times3Content (wt.)
As can be seen from the results in Table 8, vitamin D was found in the test solutions3The content is degraded within 8 hours, and the content does not change obviously within 2 hours, so that the detection of the liquid to be detected is ensured to be finished within 2 hours as much as possible.
Accuracy:
test sample solutions of 80%, 100%, and 120% of the content detection limit concentration were prepared, respectively, to verify the accuracy.
Preparation of a reference solution: collecting vitamin D3Content reference (content 99.9%) 1 #: 13.26mg, 2 #: 11.04mg, respectively placing in a 100mL brown volumetric flask, adding a proper amount of 2, 6-di-tert-butyl-p-cresol solution with the mass concentration of 0.02% in n-hexane, and ultrasonically dissolvingAnd (4) cooling, keeping the volume constant to 100mL, transferring the solution with the volume constant of 1mL into a 100mL volumetric flask, keeping the volume constant, and shaking up to obtain a reference solution.
Preparation of system suitability solution: taking appropriate amount of control solution, and destroying in water bath at 60 deg.C for 1 hr to obtain previtamin D3Taking out and cooling as a system applicability solution to verify previtamin D3And vitamin D3Do not overlap.
Preparing a test solution: separately weighing vitamin D38.04mg, 8.03mg, 8.01mg, 10.45mg, 10.02mg, 10.30mg, 12.90mg, 12.57mg and 12.95mg of the powder (content: 0.254%) were put in a 100mL brown flask, and a solution (1 mL of a solution containing 0.2mg of 2, 6-di-t-butyl-p-cresol (BHT) was prepared from a dimethyl sulfoxide-water (10:2) mixed solution]Adding 25mL of 2, 6-di-tert-butyl-p-cresol normal hexane solution with the mass concentration of 0.02% into 50mL of the sample solution, performing ultrasonic treatment for 15min, shaking constantly, keeping the temperature of an ultrasonic water bath not to exceed 30 ℃, mixing the mixture by using a micro vortex mixer or shaking the mixture by using a shaking table for 15min, centrifuging the mixture for 5min (at least 4000r/min), separating the upper layer solution, centrifuging the mixture for 5min (at least 4000r/min), and taking the upper layer clear solution as a sample solution; the concentration of the sample solution is about 80%, 100% or 120% of the limit, and 3 parts of each sample solution are prepared by the same method.
Respectively injecting the test solution and the reference solution into a liquid chromatograph with a volume of 100 μ L, recording chromatogram, and verifying vitamin D by calculating recovery rate according to peak area by external standard method3Accuracy of assay.
TABLE 9 vitamin D3Content determination accuracy verification result
According to the data in Table 8, vitamin D3Vitamin D in powder3The content measurement accuracy confirmed that the average recovery rate was 100.6%, the RSD (n ═ 9) was 1.66%, and the recovery rates of 3 samples having similar concentrations were all equal to each otherThe detection method provided by the invention has good accuracy within 92-105%.
Example 3
Vitamin D detection according to example 13Except that the flow rate was 1.3mL as measured by liquid chromatography.
Example 4
Vitamin D detection according to example 13Except that the flow rate was 1.7mL as measured by liquid chromatography.
Example 5
Vitamin D detection according to example 13Except that the column temperature was 25 ℃ as measured by liquid chromatography.
Example 6
Vitamin D detection according to example 13Except that the column temperature was 35 ℃ as measured by liquid chromatography.
Example 7
Vitamin D detection according to example 13The content of (b) is different in that the volume ratio of n-hexane to isopropanol in the mobile phase is 99.1: 0.9.
example 8
Vitamin D detection according to example 13The content of (b) is different in that the volume ratio of n-hexane to isopropanol in the mobile phase is 98.9: 1.1.
example 9
Vitamin D detection according to example 13The content of (A) is different in that the chromatographic column is XB-NH during liquid chromatography detection2And (3) a column.
Example 10
Vitamin D detection according to example 13The content of (A) is different from (B) in that a chromatographic column is ZORBAXNH in liquid chromatography detection2And (3) a column.
The results of two tests conducted according to the test methods of example 1 and examples 3 to 10 are shown in Table 9.
TABLE 9 test results of example 1 and examples 3 to 10
As is clear from the results in Table 9, the detection method provided by the present invention has good durability.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. Vitamin D3The extraction method is carried out under the condition of keeping out of the light, and comprises the following steps:
mixing a substance to be detected with a dispersant, an antioxidant and an extractant to obtain a solution of the substance to be detected; the dispersing agent is a mixed solution of dimethyl sulfoxide and water, the antioxidant is 2, 6-di-tert-butyl-p-cresol, and the extracting agent is n-hexane;
purifying the solution of the substance to be detected to obtain vitamin D3Extracting the liquid.
2. The extraction method according to claim 1, wherein the volume ratio of dimethyl sulfoxide to water in the dispersing agent is 9.8-10.2: 2.
3. the extraction method according to claim 1 or 2, characterized in that said mixing comprises the steps of:
dissolving part of antioxidant in an extractant to obtain antioxidant-extractant solution;
dissolving the rest antioxidant in the dispersant to obtain antioxidant-dispersant solution;
and mixing the object to be detected, the antioxidant-extractant solution and the antioxidant-dispersant solution to obtain an object solution to be detected.
4. The extraction method according to claim 3, wherein the mass concentration of 2, 6-di-tert-butyl-p-cresol in the antioxidant-extractant solution is 0.018-0.022%;
the mass concentration of the 2, 6-di-tert-butyl-p-cresol in the antioxidant-dispersant solution is 0.18-0.22 mg/mL;
vitamin D in the test substance3The mass and volume ratio of the antioxidant-extractant solution is 25 mug: 23-27 mL;
the volume ratio of the antioxidant-extractant solution to the antioxidant-dispersant solution is 1: 1.8 to 2.2.
5. The extraction method according to claim 4, wherein the mixing of the analyte, the antioxidant-extractant solution, and the antioxidant-dispersant solution comprises the steps of:
carrying out vortex or shaking on the obtained mixed system after ultrasonic treatment;
the ultrasonic treatment power is 200-400W, the time is 13-17 min, and the temperature is below 30 ℃;
the vortex or shaking time is independently 13-17 min.
6. The extraction method according to claim 1, characterized in that said purification process comprises the following steps:
carrying out first centrifugal treatment on the solution of the substance to be detected to obtain supernatant liquid, so as to obtain primary purified solution;
subjecting the primary purified solution to a second centrifugation to obtain a supernatant to obtain vitamin D3Extracting the liquid.
7. The extraction method according to claim 6, wherein the rotation speed of the first centrifugation and the rotation speed of the second centrifugation are respectively more than 4000r/min and the time is respectively 4-6 min.
8. Vitamin D detection method3The method comprises the following steps:
with vitamin D3The extract is to-be-detected liquid, and vitamin D3The extracting solution is extracted by the extracting method of any one of claims 1 to 7;
injecting the liquid to be detected into a liquid chromatograph to obtain a liquid chromatogram of the liquid to be detected;
mixing vitamin D3Injecting the reference solution into a liquid chromatograph to obtain a reference liquid chromatogram;
calculating vitamin D in the test substance according to the formula 13The content is as follows:
wherein A isTo be treatedIs vitamin D in the solution to be tested3Peak area of (a);
Mto pairIs vitamin D3Sample size of control: mg;
Pto pairIs vitamin D3Vitamin D in control3The content of (A): 99.8 percent
DTo be treatedThe dilution times of the solution to be tested are as follows: 25;
Ato pairAs vitamin D in control solution3Peak area of (a);
Mto be treatedSampling volume for the test substance: g;
Dto pairIs vitamin D3Dilution factor of control solution: 10000.
9. the detection method according to claim 8, wherein the liquid chromatography detection conditions include: the chromatographic column filler is amino bonded silica gel, and the column temperature is 25-35 ℃; taking a mixed solution of n-hexane and isopropanol as a mobile phase; the flow rate is 1.3-1.7 mL/min; the detection wavelength was 265 nm.
10. The detection method according to claim 9, wherein the volume ratio of n-hexane to isopropanol in the mixed solution of n-hexane and isopropanol is 98.9-99.1: 0.9 to 1.1.
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