CN108663462A - Vitamin A in a kind of measurement milk powder, the method for D, E - Google Patents
Vitamin A in a kind of measurement milk powder, the method for D, E Download PDFInfo
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- CN108663462A CN108663462A CN201810882226.7A CN201810882226A CN108663462A CN 108663462 A CN108663462 A CN 108663462A CN 201810882226 A CN201810882226 A CN 201810882226A CN 108663462 A CN108663462 A CN 108663462A
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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
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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
- G01N30/04—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
- G01N30/60—Construction of the column
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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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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The present invention relates to vitamin A in a kind of measurement milk powder, the method for D, E include the following steps：By powdered milk sample mixing, appropriate, addition water, absolute ethyl alcohol, ascorbic acid, BHT and KOH aqueous solutions are weighed, saponification is heated, postcooling constant volume is completed in saponification；Vitamin A in solution, the content of D, E are measured using the high performance liquid chromatograph equipped with bivalve three pump system；By comparing the retention time of sample and standard substance in one-dimensional liquid chromatogram, whether contain vitamin A, E in confirmatory sample；By comparing the retention time of sample in two-dimensional liquid chromatography and standard substance, whether contain vitamin D in confirmatory sample；Finally according to the vitamin A of gained, the amount of D, E calculate vitamin A in sample, the content of D, E after conversion；This invention simplifies operating procedures, greatly shorten the pre-treatment time, save manpower and reagent cost, realize automatic on-line purification detection, improve sensitivity, ensure that the accuracy and reproducibility of test result.
Description
[technical field]
The invention belongs to technical field of analytical chemistry, vitamin A in specifically a kind of measurement milk powder, the method for D, E.
[background technology]
Vitamin is widely present in various organisms, have now been found that there are about tens of remaining classes, chemical constitution is different,
There are aliphatic, aromatic series, alicyclic, heterocycle and steroid.In physiological function, they are both nor constitute various tissues
Primary raw material, nor the source of energy i (in vivo), but they are to participate in reconciling the essential small molecule of substance metabolism process
Organic compound.Vitamin A, D, E are the isoprene derivatives of nonpolar hydrophobic, dissolve in lipid and fat solvent, and
It is not soluble in water, belong to liposoluble vitamin.Intake is excessive, intake is unbalanced or shortage can all cause corresponding symptom, influences body
The growth and development of body and our life.
In order to adapt to the nutritional need of different people, prevents diseases, the condensed foods such as malnutrition and progress into ours
Life.Vitamin enrichment agent is also to be added into numerous food using earliest and most widely used nutrition fortifier, is people
Body replenishing vitamins." food enrichment uses sanitary standard (tentative) " and food nutrient fortifying were issued in 1986 in China
Agent administration of health method.GB14880 is issued within 1994《Food enrichment uses standard》, it is updated to 2012 editions at present,
To the food nutrient fortifying in specification China, enterprise is instructed to produce, ensures that consumer health plays safely positive effect.It is wherein right
The usage amount of vitamin A is 600 μ g/kg-17000 μ g/kg according to food class requirement, and vitamin D requires to be 2 μ g/kg-112 μ
G/kg, vitamin E require to be 5 μ g/kg-1450 μ g/kg.
Vitamin A, D, E are unstable in external environment, it is easy to be illuminated by the light, air etc. influence and by Oxidative demage, together
When food substrate complicated component, content is low in the product for vitamin D, and especially content is extremely low in breast milk and milk, thus to inspection
The sensitivity requirement of survey method is high.Based on above-mentioned difficult point, the pre-treating method of general sample includes saponification broken wall, organic solvent extraction
Take phase inversion, concentration and purification.Pretreatment process very complicated takes consumption manpower, largely effects on the analysis efficiency of sample.
Currently, national food safety standard method is only limitted to vitamin A, E is carried out while being measured.Vitamin A, D, E while the side of test
The research of method also has been reported that, predominantly liquid chromatography and a small number of liquid chromatography tandem mass spectrometries.
2013 Wang An groups be equal to《Environment monitoring management and technology》Periodical reports Feeds by HPLC
Middle vitamin A, the method for D3, E, after method is impregnated using 90% ethyl alcohol, then with the extraction of 95% EtOH Sonicate, but due to feed base
Matter is complicated, is damaged to chromatographic column serious, while VD contents are low is easy to can't check by matrix interference, it is difficult to promote.Then, Sai Mo
Fly to have issued online two-dimensional columns switching method while measuring vitamin A in feed, the solution of D, E, and in 2018 by method
The scope of application is expanded in milk powder matrix.But its pre-treatment evitable must be needed by saponification extraction, and organic solvent turns
Phase concentrates these cumbersome processes.Switch method using two-dimensional columns, although solving the problems, such as matrix interference, due to object
Diffusion phenomena in chromatography assigning process are susceptible to chromatography peak stretching and examine the case where not measuring, in Two way chromatograms column
This influence becomes apparent from, therefore the detection limit of VD is difficult to reduce, and cannot be satisfied the testing requirement of low content sample.Yangzhou in 2015
Vitamin A in the reported high performance liquid chromatography tandem mass spectrometry test milk powder of quality inspection, the method for D, E, although improving method
Sensitivity, but pretreatment process can not still avoid saponification and organic solvent phase inversion from concentrating.
[invention content]
Vitamin A in a kind of measurement milk powder, the side of D, E are provided present invention aim to solve above-mentioned deficiency
Method simplifies operating procedure, substantially reduces the time of pre-treatment, saves manpower and reagent cost, realizes automatic on-line
Purification detection, improves sensitivity, ensure that the accuracy and reproducibility of test result.
Vitamin A in a kind of measurement milk powder is designed to achieve the above object, and the method for D, E include the following steps：
1) sample treatment：By powdered milk sample mixing, appropriate, addition water, absolute ethyl alcohol, ascorbic acid, BHT and KOH are weighed
Aqueous solution, heats saponification, and postcooling constant volume is completed in saponification；
2) using equipped with bivalve three pump system high performance liquid chromatograph determination step 1) obtained by solution in whether contain
Vitamin A, D, E；If one-dimensional liquid chromatography results show that sample is doubtful containing vitamin A, when E, then comparative sample and vitamin
The retention time of A, E standard substance, whether to contain vitamin A, E in confirmatory sample；If two-dimensional liquid chromatography result shows sample
Product it is doubtful containing vitamin D when, then the retention time of comparative sample and vitamin D standard substance, whether to contain in confirmatory sample
There is vitamin D；
If 3) contain vitamin A in confirmatory sample, D, E then calculate vitamin A in sample, the content of D, E by formula.
Further, in step 1), it is 2g to weigh sample size, and 20mL warm water, 30mL absolute ethyl alcohols are added in sample, and 1g resists
Bad hematic acid, 1gBHT and 20mL50%KOH aqueous solutions, 80 DEG C of heating water bath 30min are cooled to room temperature after the completion, with 50% ethyl alcohol
Aqueous solution is settled to 100mL, and 2mL is taken to cross 0.45 μm of filter membrane, waits for that machine is analyzed.
Further, it in step 1), needs that appropriate amount of starch enzyme is added if containing starch in sample, before saponification.
Further, in step 2), in the high performance liquid chromatograph determination condition equipped with bivalve three pump system, online solid phase
Extraction part service condition be：(1) vitamin A, it is 4.6 × 12.5mm that D, E, which are enriched with decontaminating column and use PLRP-S columns, specification,
Aperture 15-20um；(2) mobile phase is 40% ethanol water, run time 22min；Flow rate of mobile phase：0-4min is 1mL/
min；4-15min is 0.2mL/min；15-22min is 1mL/min, and sampling volume is 200 μ L；(3) flow path of external six-way valve 3
It is set as：0-4min is 4 → 5 → 7 → 6；8→9；4-18min is 8 → 7 → 5 → 9；4→6；18-22min is 4 → 5 → 7 → 6；
8→9。
Further, in step 2), in the high performance liquid chromatograph determination condition equipped with bivalve three pump system, one-dimensional liquid phase
The service condition of chromatography is：(1) it is 4.6 × 100mm, grain size 4 that liquid-phase chromatographic column, which uses Poroshell 120EC-C8 columns, specification,
μm；(2) mobile phase is water and acetonitrile, using gradient elution, run time 22min, flow rate of mobile phase 1.5mL/min；(3)
Using UV detector, 0-11min wavelength is set as 325nm, and 11-22min wavelength is set as 294nm；(4) six-way valve 25 in case
Flow path be set as：0-12.4min is 17 → 16 → 19 → 18；14→15；12.4-13.1min is 14 → 19 → 16 → 15；17
→18；13.1-22min is 17 → 16 → 19 → 18；14→15.
Further, in step 2), in the high performance liquid chromatograph determination condition equipped with bivalve three pump system, two-dimentional liquid phase
The service condition of chromatography is：(1) vitamin D trapping column uses Poroshell 120EC-C18 columns, specification 4.6*5mm, grain size
4μm；(2) liquid-phase chromatographic column uses Eclipse PAH columns, specification 2.1*100mm, 3.5 μm of grain size；(3) mobile phase is methanol
And acetonitrile, using gradient elution, run time 22min, flow velocity 0.4mL/min；(4) UV detector, wavelength is used to set
It is set to 264nm.
Further, it in step 2), compares with the retention time at standard items peak through sample peak, when confirming that chromatographic peak retains
Between it is whether consistent, so that it is determined that whether detect determinand vitamin A in sample, D, E, if it does, then using standard curve external standard
Method is quantified.
Further, in step 2), by comparing the retention time of sample and standard substance in one-dimensional liquid chromatogram, confirm
Whether vitamin A, E are contained in sample；By comparing the retention time of sample in two-dimensional liquid chromatography and standard substance, sample is confirmed
Whether contain vitamin D in product.
Further, in step 3), vitamin A in the sample extracting solution obtained by step 2), the content of D, E, conversion
Vitamin A in sample, the content of D, E are calculated afterwards.
The present invention compared with the existing technology, has the following advantages that：
(1) present invention establishes a kind of saponification of the multi-dimensional chromatograph pattern to milk powder of a variety of separating mechanisms of use combination
Liquid directly carries out in-line purification, and test wherein vitamin A, the high performance liquid chromatography of D, E simplifies operating procedure, contract significantly
The short time of pre-treatment, manpower and reagent cost are saved, realizes automatic on-line purification detection, improve sensitivity, protect
The accuracy and reproducibility for having demonstrate,proved test result prevent vitamin enrichment agent to instruct food enterprise production to adjust from source
The adverse effect brought to consumer health is not allowed in amount.
(2) in the pre-treatment of existing method, sample saponification liquor is needed by the repeated multiple times extraction phase inversion of organic solvent, extraction
After being repeatedly washed to solution and being in neutrality, rotary evaporation is concentrated into close dry organic solvent afterwards.And before this process is exactly entire
In processing the step of most time-consuming and consumption manpower, the efficiency and production capacity of entire method are limited.And the present invention is direct using saponification liquor
The step of upper machine analysis, cleverly avoids organic solvent phase inversion, concentrate after constant volume, redissolution, greatly simplifies sample pre-treatments
Operating procedure significantly improves the analysis efficiency of sample.Simultaneously as pretreatment process simplifies, vitamin A is reduced, D, E are dividing
The risk of oxidative degradation during analysis, it is ensured that the accuracy and reproducibility of test result.Its comparison diagram such as 8 institute of Figure of description
Show.
(3) present invention is using upper machine analysis after the direct constant volume of saponification liquor, i.e., saponification liquor need not adjust pH, avoid due to
Sample solution object caused by having solid precipitation phenomenon during adjusting pH loses, and expands the scope of application of method.
(4) present invention using the high performance liquid chromatography equipped with bivalve three pump system while detecting the vitamin A in sample, D,
E, Solid Phase Extraction and highly effective liquid phase chromatographic system on-line joining process, realize the purification of sample solution and Two way chromatograms analyze it is automatic
Connection.One-dimensional chromatography vitamin A, E, Two way chromatograms analyze vitamin D, while having used vitamin in Two way chromatograms for the first time
D trapping columns efficiently solve object wide spirit of chromatographic peak caused by diffusion phenomena during chromatography distribution behavior
The problem of sensitivity reduces.
(5) present invention realizes strong basicity sample solution using the decontaminating column and chromatographic column of special filler and specification
Efficient liquid phase chromatographic analysis.
(6) it after object is enriched in decontaminating column and trapping column in the present invention, is all made of and purification process flow path opposite direction
The mode of elution, object is transferred in analytical column, and the chromatographic peak of acquisition, peak width is moderate, and symmetry is good.
(7) vitamin A in the present invention, the quantitative limit of α vitamin Es, δ vitamin Es, γ vitamin Es is 80 μ g/100g,
Quantifying for calciferol is limited to 2 μ g/100g, and quantifying for vitamine D3 is limited to, 2 μ g/100g.
[description of the drawings]
Fig. 1 is vitamin A, the chromatogram of E in one-dimensional chromatography in the embodiment of the present invention 1；
Fig. 2 is the chromatogram of vitamin D in Two way chromatograms in the embodiment of the present invention 1；
Fig. 3 is analytical solution flow path schematic diagram when being furnished with the SPE loadings of bivalve three pump system in the embodiment of the present invention 1；
When Fig. 4 is the SPE elutions equipped with bivalve three pump system in the embodiment of the present invention 1, VA/VD/VE is transferred to one-dimensional color
Analytical solution flow path schematic diagram when composing column；
When Fig. 5 is the SPE cleanings equipped with bivalve three pump system in the embodiment of the present invention 1, VA/VD/VE is from one-dimensional chromatographic column
Analytical solution flow path schematic diagram when elution；
When Fig. 6 is the SPE cleanings equipped with bivalve three pump system in the embodiment of the present invention 1, VD is transferred to from one-dimensional chromatographic column
Analytical solution flow path schematic diagram when trapping column；
When Fig. 7 is the SPE balances equipped with bivalve three pump system in the embodiment of the present invention 1, it is two dimension that VD is shifted from trapping column
Analytical solution flow path schematic diagram when analytical column；
Fig. 8 is the comparison diagram of existing method flow and saponification liquor direct analyzing method flow of the present invention；
In Fig. 3 to Fig. 7：1, Solid Phase Extraction sampling pump 2, autosampler 3, external six-way valve 4, external first valve port
5, external second valve port 6, external third valve port 7, external 4th valve port 8, external 5th valve port 9, external 6th valve port
10, waste collecting device 11, solid-phase extraction column 12, One Dimension Analysis pump 13, One Dimension Analysis column 14, the first valve port 15 in case,
Second valve port 16 in case, third valve port 17 in case, the 4th valve port 18 in case, the 5th valve port 19 in case, the 6th valve port in case
20, variable-wavelenght detector 21, two-dimension analysis pump 22, two-dimentional trapping column 23, two-dimension analysis column 24, Diode Array Detector
Six-way valve in device 25, case.
[specific implementation mode]
The invention belongs to analytical chemistry fields to vitamin A in milk powder, the measurement of D, E, and principle is：By the dimension in sample
Raw element A, D, E water, absolute ethyl alcohol, ascorbic acid, BHT and KOH aqueous solutions heat saponification, and postcooling constant volume, warp are completed in saponification
High performance liquid chromatograph equipped with bivalve three pump system measures content, by comparing sample and standard substance chromatographic peak retention time
Testing result is confirmed.
Vitamin A in the measurement milk powder, the method for D, E include the following steps：1) sample treatment：By powdered milk sample mixing,
Appropriate, addition water, absolute ethyl alcohol, ascorbic acid, BHT and KOH aqueous solutions are weighed, saponification is heated, postcooling constant volume is completed in saponification；
2) using equipped with bivalve three pump system high performance liquid chromatograph determination step 1) obtained by solution in whether contain vitamin A,
D,E；If one-dimensional liquid chromatography results show that sample is doubtful containing vitamin A, when E, then comparative sample and vitamin A, E reference substances
The retention time of matter, whether to contain vitamin A, E in confirmatory sample；If two-dimensional liquid chromatography result show sample it is doubtful containing
When vitamin D, then the retention time of comparative sample and vitamin D standard substance, whether to contain vitamin D in confirmatory sample；
If 3) contain vitamin A in confirmatory sample, D, E then calculate vitamin A in sample, the content of D, E by formula.
Wherein, it in step 1), needs that appropriate amount of starch enzyme is added if containing starch in sample, before saponification.In step 2), through sample
Product peak compares with the retention time at standard items peak, confirm chromatographic peak retention time it is whether consistent, so that it is determined that in sample whether
Detect determinand vitamin A, D, E, if it does, then being quantified using standard curve external standard method.It specifically can be by comparing one-dimensional
Whether the retention time of sample and standard substance in liquid chromatogram contains vitamin A, E in confirmatory sample；By comparing Two-dimensional Liquid
Whether the retention time of sample and standard substance in phase chromatography contains vitamin D in confirmatory sample.In step 3), according to step
2) vitamin A in the sample extracting solution obtained by, the content of D, E calculate vitamin A in sample, the content of D, E after conversion.
High performance liquid chromatograph of the present invention equipped with bivalve three pump system, that is, bivalve three pumps high performance liquid chromatography
System, including Solid Phase Extraction sampling pump 1, autosampler 2, external six-way valve 3, waste collecting device 10, solid-phase extraction column 11,
One Dimension Analysis pump 12, One Dimension Analysis column 13, variable-wavelenght detector 20, two-dimension analysis pump 21, two-dimentional trapping column 22, two-dimension analysis
Column 23, diode array detector 24, six-way valve 25 and control system in case, external six-way valve 3 are equipped with external first valve port
4, external second valve port 5, external third valve port 6, external 4th valve port 7, external 5th valve port 8, external 6th valve port 9, in case
Six-way valve 25 is equipped with the first valve port 14 in case, the second valve port 15 in case, third valve port 16 in case, the 4th valve port 17, case in case
6th valve port 19 in interior 5th valve port 18, case；Solid Phase Extraction sampling pump 1 is connect with autosampler 2, for realizing sample analysis
The absorption of solution and sample introduction；2 other end of autosampler is connect with external first valve port 4；11 both ends of solid-phase extraction column respectively with
External second valve port 5 and the connection of external 4th valve port 7, positive flow path for realizing sample analysis solution primary purification, instead
To flow path for realizing the elution sample introduction of enrichment of analyte；Waste collecting device 10 is connect with external third valve port 6, for collecting
Eluent；One Dimension Analysis pump 12 is connect with external 5th valve port 8 of external six-way valve 3, is balanced each other one-dimensional point for transporting flowing
Analysis column 13 and reversed elution are enriched in the object of solid-phase extraction column 11；13 one end of One Dimension Analysis column connects with external 6th valve port 9
It connects, 13 other end of One Dimension Analysis column is connect with the first valve port 14 in case, is used for analysis part object；Variable-wavelenght detector 20
It is connect with the second valve port 15 in case, for detecting object；22 both ends of two-dimentional trapping column respectively with third valve port 16 and case in case
Interior 6th valve port 19 connection, positive flow path is for realizing the elution sample introduction of enrichment of analyte, and reversed flow path is for realizing sample
The double purification of analytical solution；Two-dimension analysis pump 21 is connect with the 4th valve port 17 in case, is balanced each other two-dimentional point for transporting flowing
Analysis column 23 and positive elution are enriched in the object of two-dimentional trapping column 22；23 one end of two-dimension analysis column connects with the 5th valve port 18 in case
It connects, 23 other end of two-dimension analysis column is connect with diode array detector 24, for detecting object.
The present invention uses particular fillers, the decontaminating column of specification and chromatographic column, realizes the direct constant volume of strong basicity saponification liquor for the first time
Upper machine analysis afterwards, i.e., saponification liquor need not adjust pH, and avoid has solid to be precipitated now due to sample solution during adjusting pH
Object loses as caused by, expands the scope of application of method, cleverly avoids organic solvent phase inversion, concentration, redissolution
The step of, greatly simplify sample pre-treatments operating procedure, significantly improves the analysis efficiency of sample.Simultaneously as pre-treatment
Journey simplifies, and reduces vitamin A, the risk of D, E oxidative degradation in the analysis process, it is ensured that the accuracy of test result and reproduction
Property.Vitamin D trapping column has been used in Two way chromatograms column, has been efficiently solved since object is during chromatography distribution behavior
Caused by diffusion phenomena the problem of chromatographic peak wide sensitivity decrease.
The present invention is made with reference to specific embodiment further explained below：
Embodiment 1：Vitamin A in milk powder, the measurement of D, E
1. the preparation of sample：
The sample 2g after mixing is accurately weighed in 150mL boiling flasks, addition 20mL distilled water, 30mL absolute ethyl alcohols,
1g ascorbic acid, 1g BHT, 20mL50%KOH aqueous solutions, 80 DEG C of heating water bath 30min are cooled to room temperature, after the completion with 50%
Ethanol water is settled to 100mL, and 2mL is taken to cross 0.45 μm of filter membrane, upper machine analysis.
2. setting instrument parameter：
The service condition of on-line solid phase extraction part is：
(1) vitamin A, it is 4.6 × 12.5mm, aperture 15-20um that D, E, which are enriched with decontaminating column and use PLRP-S columns, specification,；
(2) mobile phase is 40% ethanol water, run time 22min；Flow rate of mobile phase：0-4min is 1mL/min；
4-15min is 0.2mL/min；15-22min is 1mL/min, and sampling volume is 200 μ L；
(3) flow path of external six-way valve 3 is set as：0-4min is 4 → 5 → 7 → 6；8→9；4-18min be 8 → 7 → 5 →
9；4→6；18-22min is 4 → 5 → 7 → 6；8→9.
The service condition of one-dimensional liquid chromatogram is：
(1) it is 4.6 × 100mm, 4 μm of grain size that liquid-phase chromatographic column, which uses Poroshell 120EC-C8 columns, specification,；
(2) mobile phase is water and acetonitrile, using gradient elution, run time 22min；Flow rate of mobile phase is 1.5mL/
min；
(3) UV detector, 0-11min wavelength is used to be set as 325nm, 11-22min wavelength is set as 294nm；
(4) flow path of six-way valve 25 is set as in case：0-12.4min is 17 → 16 → 19 → 18；14→15；12.4-
13.1min is 14 → 19 → 16 → 15；17→18；13.1-22min is 17 → 16 → 19 → 18；14→15.
The service condition of two-dimensional liquid chromatography is：
(1) vitamin D trapping column uses Poroshell 120EC-C18 columns, specification 4.6*5mm, 4 μm of grain size；
(2) liquid-phase chromatographic column uses Eclipse PAH columns, specification 2.1*100mm, 3.5 μm of grain size；
(3) mobile phase is methanol and acetonitrile, using gradient elution, run time 22min；Flow rate of mobile phase is 0.4mL/
min；
(4) UV detector, wavelength is used to be set as 264nm.
3. qualitative：
It compares with the retention time at standard items peak through sample peak, confirms whether chromatographic peak retention time is consistent, to really
Whether determinand is detected in random sample product.
4. quantitative：
Using standard curve quantified by external standard method.
5. calculating：
According to vitamin A in sample extracting solution, the content of D, E carry out vitamin A in sample, D, the calculating of E contents.
The present invention is simultaneously not limited to the embodiments described above, other any Spirit Essences and principle without departing from the present invention
Changes, modifications, substitutions, combinations, simplifications made by lower, should be equivalent substitute mode, be included in the protection model of the present invention
Within enclosing.
Claims (9)
1. vitamin A in a kind of measurement milk powder, the method for D, E, which is characterized in that include the following steps：
1) sample treatment：By powdered milk sample mixing, weighs in right amount, it is water-soluble that water, absolute ethyl alcohol, ascorbic acid, BHT and KOH is added
Liquid, heats saponification, and postcooling constant volume is completed in saponification；
2) using equipped with bivalve three pump system high performance liquid chromatograph determination step 1) obtained by solution in whether containing dimension life
Plain A, D, E；If one-dimensional liquid chromatography results show that sample is doubtful containing vitamin A, when E, then comparative sample and vitamin A, E marks
The retention time of quasi- substance, whether to contain vitamin A, E in confirmatory sample；If two-dimensional liquid chromatography result shows that sample is doubtful
When containing vitamin D, then the retention time of comparative sample and vitamin D standard substance, is given birth to whether containing dimension in confirmatory sample
Plain D；
If 3) contain vitamin A in confirmatory sample, D, E then calculate vitamin A in sample, the content of D, E by formula.
2. the method as described in claim 1, it is characterised in that：In step 1), it is 2g to weigh sample size, and 20mL is added in sample
Warm water, 30mL absolute ethyl alcohols, 1g ascorbic acid, 1gBHT and 20mL50%KOH aqueous solutions, 80 DEG C of heating water bath 30min are completed
Postcooling is settled to 100mL to room temperature, with 50% ethanol water, and 2mL is taken to cross 0.45 μm of filter membrane, waits for that machine is analyzed.
3. the method as described in claim 1, it is characterised in that：In step 1), need to add if containing starch in sample, before saponification
Enter appropriate amount of starch enzyme.
4. the method as described in claim 1, which is characterized in that in step 2), be furnished with the high-efficient liquid phase color of bivalve three pump system
In spectrometer determination condition, the service condition of on-line solid phase extraction part is：
(1) vitamin A, it is 4.6 × 12.5mm, aperture 15-20um that D, E, which are enriched with decontaminating column and use PLRP-S columns, specification,；
(2) mobile phase is 40% ethanol water, run time 22min；Flow rate of mobile phase：0-4min is 1mL/min；4-
15min is 0.2mL/min；15-22min is 1mL/min, and sampling volume is 200 μ L；
(3) flow path of external six-way valve 3 is set as：0-4min is 4 → 5 → 7 → 6；8→9；4-18min is 8 → 7 → 5 → 9；4
→6；18-22min is 4 → 5 → 7 → 6；8→9.
5. the method as described in claim 1, which is characterized in that in step 2), be furnished with the high-efficient liquid phase color of bivalve three pump system
In spectrometer determination condition, the service condition of one-dimensional liquid chromatogram is：
(1) it is 4.6 × 100mm, 4 μm of grain size that liquid-phase chromatographic column, which uses Poroshell 120EC-C8 columns, specification,；
(2) mobile phase is water and acetonitrile, using gradient elution, run time 22min, flow rate of mobile phase 1.5mL/min；
(3) UV detector, 0-11min wavelength is used to be set as 325nm, 11-22min wavelength is set as 294nm；
(4) flow path of six-way valve 25 is set as in case：0-12.4min is 17 → 16 → 19 → 18；14→15；12.4-13.1min
It is 14 → 19 → 16 → 15；17→18；13.1-22min is 17 → 16 → 19 → 18；14→15.
6. the method as described in claim 1, which is characterized in that in step 2), be furnished with the high-efficient liquid phase color of bivalve three pump system
In spectrometer determination condition, the service condition of two-dimensional liquid chromatography is：
(1) vitamin D trapping column uses Poroshell 120EC-C18 columns, specification 4.6*5mm, 4 μm of grain size；
(2) liquid-phase chromatographic column uses Eclipse PAH columns, specification 2.1*100mm, 3.5 μm of grain size；
(3) mobile phase is methanol and acetonitrile, using gradient elution, run time 22min, flow velocity 0.4mL/min；
(4) UV detector, wavelength is used to be set as 264nm.
7. the method as described in claim 1, it is characterised in that：In step 2), the retention time through sample peak and standard items peak
It compares, confirms whether chromatographic peak retention time is consistent, so that it is determined that determinand vitamin A whether is detected in sample, D, E, if
Contain, is then quantified using standard curve external standard method.
8. the method as described in claim 1, it is characterised in that：In step 2), by comparing sample in one-dimensional liquid chromatogram and
Whether the retention time of standard substance contains vitamin A, E in confirmatory sample；By comparing sample in two-dimensional liquid chromatography and mark
Whether the retention time of quasi- substance contains vitamin D in confirmatory sample.
9. the method as described in claim 1, it is characterised in that：In step 3), in the sample extracting solution obtained by step 2)
Vitamin A, the content of D, E calculate vitamin A in sample, the content of D, E after conversion.
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| CN110031573A (en) * | 2019-05-31 | 2019-07-19 | 福建省食品药品质量检验研究院 | The method of two-dimensional columns switching high effective liquid chromatography for measuring vitamin D content |
| CN110031573B (en) * | 2019-05-31 | 2021-08-24 | 福建省食品药品质量检验研究院 | Method for measuring vitamin D content by two-dimensional column switching high performance liquid chromatography |
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| CN112229933A (en) * | 2020-10-23 | 2021-01-15 | 南京玉鹤鸣医学营养科技股份有限公司 | Method for determining vitamin types by using two-dimensional liquid chromatography |
| CN115219603A (en) * | 2021-04-15 | 2022-10-21 | 仙乐健康科技股份有限公司 | Method for simultaneously determining vitamins A, D and E in sample |
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