CN109613277A - Method that is a kind of while detecting vitamin D and vitamin K - Google Patents
Method that is a kind of while detecting vitamin D and vitamin K Download PDFInfo
- Publication number
- CN109613277A CN109613277A CN201811535984.8A CN201811535984A CN109613277A CN 109613277 A CN109613277 A CN 109613277A CN 201811535984 A CN201811535984 A CN 201811535984A CN 109613277 A CN109613277 A CN 109613277A
- Authority
- CN
- China
- Prior art keywords
- vitamine
- farnoquinone
- mobile phase
- determinand
- vitamin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Abstract
The invention belongs to detection technique fields, more particularly to a kind of method for detecting vitamin D and vitamin K simultaneously.The present invention provides a kind of methods for detecting vitamin D and vitamin K simultaneously, the following steps are included: the extraction of vitamine D3 and farnoquinone a) is carried out to determinand using the mixed solution of n-hexane and dehydrated alcohol, b) detection of the determinand vitamine D3 and farnoquinone is carried out using liquid chromatography, obtain Liquid Chromatography data, according to the liquid chromatogram standard working curve of vitamine D3 and farnoquinone, the content of the determinand vitamine D3 and farnoquinone is obtained.The experimental results showed that the method for the present invention can detect vitamine D3 and farnoquinone simultaneously, the accuracy rate of detection is high, and the detection of vitamine D3 and farnoquinone is carried out using the method for the present invention, and every batch of sample only needs 8.5h, substantially increases detection efficiency.
Description
Technical field
The invention belongs to detection technique fields, more particularly to a kind of method for detecting vitamin D and vitamin K simultaneously.
Background technique
Currently, vitamin D and vitamin K are separately detected, the detection for vitamine D3, sample treatment needs
4h, and complicated for operation cumbersome, including sample saponification, extract, concentration, washing, constant volume and filtering, the machine time is 6h, inspection on sample
The vitamine D3 for surveying a collection of sample needs 10h altogether；Detection for farnoquinone, sample treatment need 1.5h, the machine time on sample
For 7h, the farnoquinone for detecting a collection of sample needs 8.5h altogether；Vitamine D3 and the farnoquinone detection for completing a collection of sample, need altogether
18.5h is wanted, efficient, the quick detection of product is unfavorable for.Also, in test sample vitamine D3 and farnoquinone, process
Journey needs to be unfavorable for testing staff's to the biggish reagent of human injury using a large amount of potassium hydroxide, petroleum ether, ethyl acetate etc.
Health.
Summary of the invention
In view of this, the present invention provides a kind of method for detecting vitamin D and vitamin K simultaneously, it is existing for solving
Technology detection vitamine D3 and farnoquinone operating process is cumbersome, the time is long, is unfavorable for the problem of testing staff's health.
The specific technical solution of the present invention is as follows:
Method that is a kind of while detecting vitamin D and vitamin K, comprising the following steps:
A) extraction of vitamine D3 and farnoquinone is carried out to determinand using the mixed solution of n-hexane and dehydrated alcohol,
B) detection that the determinand vitamine D3 and farnoquinone are carried out using liquid chromatography, obtains liquid chromatogram number
According to obtaining the determinand vitamine D3 and Wei Sheng according to the liquid chromatogram standard working curve of vitamine D3 and farnoquinone
The content of plain K2.
In the present invention, vitamine D3 and farnoquinone are carried out to determinand using the mixed solution of n-hexane and dehydrated alcohol
Extraction to carry out sample treatment, avoid being conducive to the health of testing staff using the high solvent of toxicity, the experimental results showed that,
The method of the present invention can detect vitamine D3 and farnoquinone simultaneously, and the accuracy rate of detection is high, be tieed up using the method for the present invention
The detection of raw element D3 and farnoquinone, every batch of sample only need 8.5h, substantially increase detection efficiency.
Preferably, the determinand is calcium Vitamin D Vitamin K soft capsule.
Preferably, the volume ratio of n-hexyl alcohol and dehydrated alcohol is (10 in the mixed solution of the n-hexane and dehydrated alcohol
~20): (80~90).
It is furthermore preferred that the volume ratio of n-hexyl alcohol and dehydrated alcohol is 20:80 in the mixed solution of n-hexane and dehydrated alcohol.
Preferably, the concentration of vitamine D3 is 0.128 μ of μ g/ml~1.28 g/ in the liquid chromatogram standard working curve
ml；
The concentration of farnoquinone is 0.513 μ of μ g/ml~5.13 g/ml in the liquid chromatogram standard working curve.
Preferably, the condition of the liquid chromatography are as follows:
Chromatographic column is C18 chromatographic column；
Chromatographic column column temperature is 30 DEG C~50 DEG C；
Detection wavelength is 264nm；
Mobile phase includes mobile phase A and Mobile phase B, and the mobile phase A is 95% methanol-water solution, and the Mobile phase B is
Isopropanol；
Flow rate of mobile phase is 0.5mL/min~1.5mL/min.
It is furthermore preferred that chromatographic column column temperature is 40 DEG C；
Flow rate of mobile phase is 1mL/min.
Preferably, the length of the C18 chromatographic column is 250nm, and the internal diameter of the C18 chromatographic column is 4.6mm, the C18
The packing material size of chromatographic column is 5 μm.
In the present invention, C18 chromatographic column be selected from 250 × 4.6mm of Agilent C18,5 μm；(Town in Shanghai composes limited public affairs to CNW
Department) C18 250 × 4.6mm, 5 μm；Enlightening horse (Diamonsil) Plus C18 250 × 4.6mm, 5 μm or Shimadzu WondaSil
C18 250 × 4.6mm, 5 μm.C18 chromatographic column is preferably 250 × 4.6mm of Agilent C18, and 5 μm.
Preferably, the liquid chromatography uses gradient elution.
Preferably, in the gradient elution, 0.01~20.00min, the volume ratio of the mobile phase A and the Mobile phase B
For 98:2；The volume ratio of 20.01~38.00min, the mobile phase A and the Mobile phase B is 50:50；38.01~
The volume ratio of 42.00min, the mobile phase A and the Mobile phase B is 0:100；42.01~45.00min, the mobile phase A
Volume ratio with the Mobile phase B is 50:50；45.01~53.00min, the volume ratio of the mobile phase A and the Mobile phase B
For 98:2.
Preferably, described to be extracted as ultrasonic extraction.
Preferably, the frequency of the ultrasonic extraction is 20KHz~60KHz；
The temperature of the ultrasonic extraction is 55 DEG C~65 DEG C；
The time of the ultrasonic extraction is 25min~35min.
It is furthermore preferred that the frequency of ultrasonic extraction is 40KHz；
The temperature of the ultrasonic extraction is 60 DEG C；
The time of the ultrasonic extraction is 30min.
In the present invention, vitamine D3 and farnoquinone are carried out to determinand using the mixed solution of n-hexane and dehydrated alcohol
Extraction, carry out constant volume after extraction, vitamine D3 and farnoquinone separated using RP chromatography, examined with UV detector
It surveys, and with quantified by external standard method, obtains the content of vitamine D3 and farnoquinone in determinand.
In conclusion the present invention provides a kind of methods for detecting vitamin D and vitamin K simultaneously, comprising the following steps:
A) extraction of vitamine D3 and farnoquinone, b are carried out to determinand using the mixed solution of n-hexane and dehydrated alcohol) use liquid
Phase chromatography carries out the detection of the determinand vitamine D3 and farnoquinone, Liquid Chromatography data is obtained, according to vitamine D3
With the liquid chromatogram standard working curve of farnoquinone, the content of the determinand vitamine D3 and farnoquinone is obtained.Experiment
The result shows that the method for the present invention can detect vitamine D3 and farnoquinone simultaneously, the accuracy rate of detection is high, using present invention side
Method carries out the detection of vitamine D3 and farnoquinone, and every batch of sample only needs 8.5h, substantially increases detection efficiency, also, this hair
It is bright that the mixed solution of n-hexane and dehydrated alcohol is used to carry out the extraction of vitamine D3 and farnoquinone to determinand to carry out sample
Product processing, avoids the solvent high using toxicity, is conducive to the health of testing staff.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the liquid chromatogram of blank solution in the embodiment of the present invention；
Fig. 2 is the liquid chromatogram of working solution in the embodiment of the present invention；
Fig. 3 is the vitamine D3 standard working curve of liquid chromatogram standard working curve in the embodiment of the present invention；
Fig. 4 is the farnoquinone standard working curve of liquid chromatogram standard working curve in the embodiment of the present invention.
Specific embodiment
The present invention provides a kind of methods for detecting vitamin D and vitamin K simultaneously, for solving prior art detection dimension
Raw element D3 and farnoquinone operating process are cumbersome, the time is long, is unfavorable for the problem of testing staff's health.
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
For a further understanding of the present invention, the present invention will be described in detail combined with specific embodiments below.
Instrument used in the embodiment of the present invention includes high performance liquid chromatograph (containing UV detector), Ultrasound Instrument and electronics day
Flat, the reagent of use includes n-hexane (AR), methanol (chromatographically pure), isopropanol (chromatographically pure), dehydrated alcohol (AR), ethyl acetate
(AR), vitamine D3 reference substance (source: DR, lot number: 100656, purity: 99.9%), farnoquinone reference substance (source: USP,
Lot number: R059X0, purity: 99.8%), determinand is that soup minister is good for calcium Vitamin D Vitamin K soft capsule again.
Embodiment 1
The preparation of the present embodiment progress vitamine D3 stock solution and farnoquinone stock solution.
The preparation of vitamine D3 stock solution: precision weighs vitamine D3 standard items 3.06mg and is placed in 500ml brown volumetric flask
In, it is dissolved, constant volume, is shaken up to get vitamine D3 stock solution with dehydrated alcohol, absorbance is measured using ultraviolet specrophotometer,
Measuring concentration is 6.4091 μ g/ml.The preparation of farnoquinone stock solution: precision weighs farnoquinone standard items 10.30mg and is placed in
It in 100ml brown volumetric flask, is dissolved, constant volume, is shaken up to get farnoquinone stock solution, using ultraviolet spectrometry light with ethyl acetate
Degree meter measurement absorbance, measuring concentration is 100 μ g/ml.Vitamine D3 stock solution and farnoquinone stock solution are placed in -18 DEG C
It is stored for future use in refrigerator.
Embodiment 2
The preparation of the present embodiment progress standard working solution.
Precision draws vitamine D3 stock solution 10.00ml, farnoquinone stock solution 2.50ml is placed in 25ml brown volumetric flask
In, dehydrated alcohol is added and is settled to scale, shakes up to get standard working solution, it is accurate respectively to draw 1 μ l of working solution, 2 μ l, 5
μ l, 8 μ l, 10 μ l, in following liquid phase chromatogram condition: chromatographic column be 250 × 4.6mm of Agilent C18,5 μm；Chromatographic column column temperature
It is 40 DEG C；Detection wavelength is 264nm；Mobile phase includes mobile phase A and Mobile phase B, and mobile phase A is 95% methanol-water solution, stream
Dynamic phase B is isopropanol；Flow rate of mobile phase is 1mL/min；Using gradient elution, 0.01~20.00min, mobile phase A and mobile phase
The volume ratio of B is 98:2；The volume ratio of 20.01~38.00min, mobile phase A and Mobile phase B is 50:50；38.01~
The volume ratio of 42.00min, mobile phase A and Mobile phase B is 0:100；The body of 42.01~45.00min mobile phase A and Mobile phase B
Product is than being 50:50；The volume ratio of 45.01~53.00min, mobile phase A and Mobile phase B is 98:2, carries out analysis measurement, is drawn
Liquid chromatogram standard working curve, vitamine D3 standard working curve and farnoquinone standard working curve are please respectively refering to Fig. 3
And Fig. 4.Wherein, the sample volume of liquid chromatographic detection is 20 μ l.
Embodiment 3
Precision weighs determinand 1.1000g (1g~3g), is placed in 10ml (or 25ml) brown volumetric flask, be added just oneself
Alkane: in the mixed solution of dehydrated alcohol (20:80), it is placed in progress 30min ultrasonic extraction in 60 DEG C of Ultrasound Instruments, is taken out, holding chamber
Temperature, with n-hexane: the mixed solution of dehydrated alcohol (20:80) is settled to scale, shakes up 0.45 μm of organic filter membrane, obtains for examination
Product solution, the sample volume for carrying out liquid chromatographic detection is 20 μ l (10 μ l of μ l~30), and liquid phase chromatogram condition condition is the same as 2 liquid of embodiment
Phase chromatographic condition.
The content of vitamine D3 or farnoquinone in determinand is calculated according to formula X=V × C/M × K.
In formula: X is vitamine D3 or farnoquinone content in determinand, and unit is μ g/；
C is the concentration of vitamine D3 or farnoquinone in test solution, and unit is μ g/mL；
M is the quality of determinand, unit g；
V is the diluted volume of determinand, unit mL.
K is unit conversion coefficient.
As a result Tables 1 and 2 is please referred to, respectively using the method for the present invention detection determinand vitamine D3 and farnoquinone
Data.
Table 1 detects the data of determinand vitamine D3 using the method for the present invention
Table 2 detects the data of determinand farnoquinone using the method for the present invention
Comparative example 1
The measurement of vitamine D3 is carried out according to the 4th method of GB 5009.82-2016, comprising the following steps:
(1) vitamine D3 standard solution is prepared
It accurately weighs vitamine D3 standard items 3.5mg chromatographically pure dehydrated alcohol and dissolves and be settled to 500mL, obtain dimension life
Plain D3 Standard Stock solutions, are sealed in -20 DEG C of refrigerators, and validity period 3 months.100 μ L vitamine D3 standard inventories are molten
Liquid is settled to scale in the brown volumetric flask of 10mL, with dehydrated alcohol, mixes, obtains vitamine D3 standard solution, use respectively
1cm quartz cuvette, using dehydrated alcohol as blank reference, by formula CVitamine D3It is molten that=A × 10000/E calculates vitamine D3 standard
The concentration of liquid.
In formula: CVitamine D3For the concentration of vitamine D3 standard solution, unit is μ g/mL；
A is the average UV light absorption value of vitamine D3 standard solution；
10000 be conversion coefficient；
E is that vitamine D3 1% compares chroma brightness coefficient.
(2) processing of determinand
Saponification: weigh 1.0g determinand (accurately to 0.01g) through homogenization in 150mL boiling flask, add
1.0g ascorbic acid and 0.1g BHT are mixed, and 30mL dehydrated alcohol is added, and 10mL~20mL potassium hydroxide solution, Bian Jia is added
Side shaking is saponified 30min in 80 DEG C of reflux in constant temperature blender with magnetic force after mixing, is cooled to room temperature immediately with cold water after saponification,
Obtain saponification liquor.
It extracts: saponification liquor is transferred in the separatory funnel of 250mL with 30mL water, 50mL petroleum ether, oscillation extraction is added
Lower layer's solution is transferred in the separatory funnel of another 250mL by 5min, and the petroleum ether that 50mL is added extracts again, merges ether layer.
Washing: using about 150mL water washing ether layer, be repeated 3 times, until ether layer, which is washed till neutrality, (detects lower layer with pH test paper
Solution ph), remove lower layer's water phase.
Concentration: the ether layer after washing is filtered in 250mL rotary evaporation bottle or nitrogen concentration tube through anhydrous sodium sulfate (3g),
Separatory funnel and anhydrous sodium sulfate 2 times are rinsed with 15mL petroleum ether, is incorporated in evaporative flask, and connect in rotary evaporator or gas
On body concentrating instrument, vacuum distillation or air-flow concentration remove evaporative flask, nitrogen is blown to when ether in bottle is left 2mL in 40 DEG C of water-baths
It is dry, it is settled to 10mL with n-hexane, 0.22 μm of organic system membrane filtration is prepared for partly preparing Normal-phase HPLC system half,
Purify prepare liquid.
The condition of reversed-phase liquid chromatography detection is as follows:
Chromatographic column: C18 column, column length 250mm, column internal diameter 4.6mm, 5 μm of partial size or the chromatographic column for having equal performance；
Mobile phase: methanol+water=95+5；
Flow velocity: 1mL/min；
Detection wavelength: 264nm；
Column temperature: 35 DEG C ± 1 DEG C；
According to formula XVitamine D3=ρ × VVitamine D3× f × 100/m calculates the content of vitamine D3 in determinand, as a result please join
Read table 3.
In formula: XVitamine D3For the content of vitamine D3 in determinand, unit is μ g/；
ρ is the concentration of vitamine D3 in the determinand being calculated according to standard working curve, and unit is μ g/mL；
VVitamine D3For n-hexane constant volume, unit mL；
F is the extension rate of prepare liquid dilution；
100 be the conversion coefficient that amount is calculated in determinand with every 100 grams；
M is the sample weighting amount of determinand, and unit is gram g.
Table 3 detects the data of determinand vitamine D3 using the 4th method of GB 5009.82-2016
Comparative example 2
The measurement of farnoquinone is carried out referring to TCJC-SOP/B-589-2016, comprising the following steps:
(1) farnoquinone standard solution is prepared
It weighs farnoquinone standard items 5mg and is accurate to 0.01mg in 50mL volumetric flask, add ethyl acetate ultrasonic dissolution, it is fixed
Hold, shake up, cross 0.45 μm filter membrane to get.
(2) processing of determinand
It takes content to be uniformly mixed 20 determinands and weighs about 1.5g and be placed in 25ml volumetric flask, acetic acid second is added
Ester, 35 DEG C of ultrasound (power 500W, frequency 50kHz) processing are taken out, are let cool to extracting completely (15min), and constant volume shakes up, warp
0.45 μm of membrane filtration carries out liquid chromatographic detection to get prepare liquid.
The condition of liquid chromatographic detection is as follows:
Chromatographic column: using the strong conjunction silica gel of octadecylsilane as filler (column length 25cm, internal diameter 4.6mm, 5 μm of partial size) or together
Etc. performances chromatographic column；
Column temperature: 40 DEG C；
Detection wavelength: 270nm；
Mobile phase: 50% mobile phase A: methanol: 0.1% acetic acid-aqueous solution=95:5
50% Mobile phase B: isopropanol
Flow velocity: 1.0ml/min；
According to formula XFarnoquinone=VFarnoquinone×CFarnoquinone/MFarnoquinone×KFarnoquinoneThe content of vitamine D3 in determinand is calculated,
As a result table 4 is please referred to.
In formula: XFarnoquinoneFor farnoquinone content in determinand, unit is μ g/；
CFarnoquinoneFor the concentration of farnoquinone in test solution, unit is μ g/mL；
MFarnoquinoneFor the quality of determinand, unit g；
VFarnoquinoneFor the diluted volume of prepare liquid, mL.
KFarnoquinoneFor unit conversion coefficient.
Data of the table 4 referring to TCJC-SOP/B-589-2016 detection determinand farnoquinone
Embodiment 4
The embodiment carries out the data that embodiment 3 detects the data of vitamine D3 and comparative example 1 detects vitamine D3 pair
Than the data that embodiment 3 detects the data of farnoquinone and comparative example 2 detects vitamine D3 are compared.As a result it please refers to
Table 5 and table 6.The result shows that detection method is scientific and effective to the assay of farnoquinone and vitamine D3, it can be to soup
Minister is good for the purpose that the content of farnoquinone and vitamine D3 in calcium Vitamin D Vitamin K soft capsule plays quality control again.
5 embodiment 3 of table and comparative example 1 detect the data comparison of vitamine D3
6 embodiment 3 of table and comparative example 2 detect the data comparison of farnoquinone
Embodiment 5
The present embodiment carries out methodology validation.
(1) specificity is tested
Referring to Fig. 2, pressing the liquid chromatogram of 2 liquid phase chromatogram condition of embodiment measurement for working solution in the embodiment of the present invention
Figure, Fig. 2 show to contain vitamine D3 peak and farnoquinone in working solution.Sample is not weighed, handles blank by 3 method of embodiment
Solution measures blank solution by 2 liquid phase chromatogram condition of embodiment, as a result as shown in Figure 1, compareing with vitamine D3 and farnoquinone
The appearance time of product solution compares, the results showed that blank solution equal nothing at the appearance time of vitamine D3 and farnoquinone
Absorption peak shows that blank is substantially noiseless to the measurement result at vitamine D3 peak and farnoquinone.
(2) range of linearity
The test data of embodiment 2 is respectively as shown in table 7 and table 8, i.e. the test data of vitamine D3 and farnoquinone.With
Peak area is abscissa, and concentration is ordinate, is drawn respectively according to the test data of table 7 and table 8 and obtains vitamine D3 standard work
Make curve and farnoquinone standard working curve, i.e. Fig. 3 and Fig. 4.The result shows that the related coefficient of vitamine D3 and farnoquinone
R2 is respectively 0.9999206,0.9999592, and the method for the present invention measures vitamine D3 in 0.12818 μ g/ml to 1.28182 of concentration
μ g/ml, farnoquinone are good linear to presenting between 5.13970 μ g/ml in 0.51397 μ g/ml of concentration, meet GB/
The requirement of T27404-2008 " Good Laboratory control specification " (GB/T27404-2008 requires coefficient R >=0.99).
The test data of 7 vitamine D3 of table
The test data of 8 farnoquinone of table
(3) detection limit and quantitative limit
The detection limit DL and quantitative limit QL of analysis method are calculated by signal-to-noise ratio (S/N).DL is defined as corresponding when S/N=3
Concentration to be analyzed, QL are defined as corresponding concentration to be analyzed when S/N=10.
1) detection limit
When signal-to-noise ratio (S/N) be 3 when, the detection limit of vitamine D3 and farnoquinone be respectively 0.01156 μ g/ml,
0.02685 μ g/ml, when the amount of weighing is 1.1g, vitamine D3 and farnoquinone detection limit are respectively in detection method
10.5072μg/100g、0.2455mg/kg。
2) quantitative limit
When signal-to-noise ratio (S/N) be 10 when, the quantitative limit of vitamine D3 and farnoquinone be respectively 0.03853 μ g/ml,
0.09000 μ g/ml, when the amount of weighing is 1.1g, the quantitative limit of vitamine D3 and farnoquinone difference in detection method
For 35.0273 μ g/100g, 0.8182mg/kg.
(4) precision test
6 parts of determinands are weighed, determinand is handled by 3 method of embodiment, detects vitamine D3 and farnoquinone in determinand
Content, and calculate its RSD (%), the data difference of vitamine D3 and farnoquinone is as shown in Table 9 and Table 10.The result shows that inspection
The RSD for surveying 6 parts of determinand vitamine D3s and farnoquinone content is respectively 3.6%, 3.3%, and detection method has preferably
Precision, meeting the requirement of GB/T27404-2008 " Good Laboratory control specification ", (GB/T27404-2008 requires RSD
(%)≤7.5%).
The data of vitamine D3 in 9 precision test of table
The data of farnoquinone in 10 precision test of table
(5) stability test
By vitamine D3, farnoquinone test solution respectively at room temperature place 0h, 1h, 4h, 8h, 12h, for 24 hours after, press
5.1 conditions measure concentration, calculate its RSD (%).The data of vitamine D3 and farnoquinone are respectively such as table 11 in stability test
With shown in table 12.The result shows that by test solution respectively at room temperature place 0h, 1h, 4h, 8h, 12h, for 24 hours after, detection for examination
The RSD (%) of vitamine D3 and farnoquinone is respectively 1.5%, 4.3% in product solution, shows vitamin in test solution
D3, farnoquinone at room temperature for 24 hours in have good stability.
The data of vitamine D3 in 11 stability test of table
The data of farnoquinone in 12 stability test of table
(6) accuracy test (rate of recovery)
Mark-on: precision weighs about 1.1g determinand, and vitamine D3 and farnoquinone content are respectively as follows: 5.0 μ g/ in determinand
G, 16 μ g/g) 9 parts, it is divided into 3 groups, every group 3 parts, each group is accurate respectively to be added vitamine D3 stock solution (concentration is 6.4091 μ g/
ml)0.60ml,0.80ml,1.00ml；It is successively accurate respectively again that farnoquinone stock solution (concentration 0.0102794mg/ is added
Ml) 1.10ml, 1.60ml, 1.90ml obtain mark-on sample.Again by the determinand after 3 method of embodiment processing mark-on.
Measure additional amount=mark-on sample measured amount-determinand measured amount
The rate of recovery (%)=measure additional amount/theoretical addition amount × 100%
The data of vitamine D3 and farnoquinone are respectively as shown in table 13 and table 14 in accuracy test.
The experimental results showed that the average recovery rate of vitamine D3 and farnoquinone is respectively 97.66%, 99.06%, relatively
Standard deviation (RSD) is respectively 1.7%, 1.3%, meets the requirement of GB/T27404-2008 " Good Laboratory control specification "
(it is 95~105% that GB/T27404-2008, which requires the rate of recovery).Also, the accuracy rate of vitamine D3 detection is improved, and mentions
High 2.8% (rate of recovery of the 4th method vitamine D3 of GB5009.82-2016 is 95%, and the rate of recovery of detection method reaches
97.66%)
The data of vitamine D3 in 13 accuracy test of table
The data of farnoquinone in 14 accuracy test of table
The above result shows that the present invention detects the specificity test of the method for vitamin D and vitamin K, linear model simultaneously
It encloses, precision test, stability test, accuracy test, detection limit and quantitative limit meet the " laboratory GB/T27404-2008
Quality control specifications " requirement, show that content assaying method of the present invention is scientific and effective, can to soup minister again be good for calcium vitamin D dimension life
Vitamine D3 and farnoquinone content play the purpose of quality control in plain K soft capsule.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method for detecting vitamin D and vitamin K simultaneously, which comprises the following steps:
A) extraction of vitamine D3 and farnoquinone is carried out to determinand using the mixed solution of n-hexane and dehydrated alcohol,
B) detection that the determinand vitamine D3 and farnoquinone are carried out using liquid chromatography, obtains Liquid Chromatography data,
According to the liquid chromatogram standard working curve of vitamine D3 and farnoquinone, the determinand vitamine D3 and farnoquinone are obtained
Content.
2. the method according to claim 1, wherein the determinand is calcium Vitamin D Vitamin K soft capsule.
3. the method according to claim 1, wherein in the mixed solution of the n-hexane and dehydrated alcohol just oneself
The volume ratio of pure and mild dehydrated alcohol is (10~20): (80~90).
4. the method according to claim 1, wherein vitamine D3 in the liquid chromatogram standard working curve
Concentration is 0.128 μ of μ g/ml~1.28 g/ml；
The concentration of farnoquinone is 0.513 μ of μ g/ml~5.13 g/ml in the liquid chromatogram standard working curve.
5. the method according to claim 1, wherein the condition of the liquid chromatography are as follows:
Chromatographic column is C18 chromatographic column；
Chromatographic column column temperature is 30 DEG C~50 DEG C；
Detection wavelength is 264nm；
Mobile phase includes mobile phase A and Mobile phase B, and the mobile phase A is 95% methanol-water solution, and the Mobile phase B is isopropyl
Alcohol；
Flow rate of mobile phase is 0.5mL/min~1.5mL/min.
6. according to the method described in claim 5, it is characterized in that, the length of the C18 chromatographic column is 250nm, the C18 color
The internal diameter for composing column is 4.6mm, and the packing material size of the C18 chromatographic column is 5 μm.
7. according to the method described in claim 5, it is characterized in that, the liquid chromatography uses gradient elution.
8. the method according to the description of claim 7 is characterized in that in the gradient elution, 0.01~20.00min, the stream
The volume ratio of dynamic phase A and the Mobile phase B is 98:2；20.01~38.00min, the body of the mobile phase A and the Mobile phase B
Product is than being 50:50；The volume ratio of 38.01~42.00min, the mobile phase A and the Mobile phase B is 0:100；42.01~
The volume ratio of 45.00min, the mobile phase A and the Mobile phase B is 50:50；45.01~53.00min, the mobile phase A
Volume ratio with the Mobile phase B is 98:2.
9. the method according to claim 1, wherein described be extracted as ultrasonic extraction.
10. according to the method described in claim 9, it is characterized in that, the frequency of the ultrasonic extraction is 20KHz~60KHz；
The temperature of the ultrasonic extraction is 55 DEG C~65 DEG C；
The time of the ultrasonic extraction is 25min~35min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811535984.8A CN109613277B (en) | 2018-12-14 | 2018-12-14 | Method for simultaneously detecting vitamin D and vitamin K |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811535984.8A CN109613277B (en) | 2018-12-14 | 2018-12-14 | Method for simultaneously detecting vitamin D and vitamin K |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109613277A true CN109613277A (en) | 2019-04-12 |
| CN109613277B CN109613277B (en) | 2022-02-18 |
Family
ID=66010049
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811535984.8A Active CN109613277B (en) | 2018-12-14 | 2018-12-14 | Method for simultaneously detecting vitamin D and vitamin K |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109613277B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110057933A (en) * | 2019-04-29 | 2019-07-26 | 杭州民生健康药业有限公司 | Vitamin K in one kind of multiple vitamin and minerals compound preparations2Detection method |
| CN111574348A (en) * | 2020-06-15 | 2020-08-25 | 浙江诚意药业股份有限公司 | Purification process of vitamin K1 |
| CN115308338A (en) * | 2022-08-24 | 2022-11-08 | 哈药集团技术中心 | Method for detecting vitamin D3 related substances in vitamin D3 drops |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130236979A1 (en) * | 2005-04-06 | 2013-09-12 | Quest Diagnostics Investments Incorporated | Methods for detecting vitamin d metabolites by mass spectrometry |
| CN104502501A (en) * | 2014-12-26 | 2015-04-08 | 江苏艾兰得营养品有限公司 | Chromatographic method for rapidly measuring content of vitamin D3 |
| CN106153796A (en) * | 2015-04-17 | 2016-11-23 | 西藏卫信康医药股份有限公司 | The content analysis detection method of 12 kinds of compound vitamines of injection |
| CN106383186A (en) * | 2016-08-31 | 2017-02-08 | 重庆方通动物药业有限公司 | High performance liquid chromatographic analysis method for simultaneous determination of content of 14 kinds of vitamins |
| CN106404947A (en) * | 2016-08-31 | 2017-02-15 | 陈大为 | Method for determination of fat-soluble vitamins by using semi-automatic sample treatment liquid chromatographic technology |
| CN107688068A (en) * | 2017-11-02 | 2018-02-13 | 威海百合生物技术股份有限公司 | A kind of detection method of farnoquinone content |
-
2018
- 2018-12-14 CN CN201811535984.8A patent/CN109613277B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130236979A1 (en) * | 2005-04-06 | 2013-09-12 | Quest Diagnostics Investments Incorporated | Methods for detecting vitamin d metabolites by mass spectrometry |
| CN104502501A (en) * | 2014-12-26 | 2015-04-08 | 江苏艾兰得营养品有限公司 | Chromatographic method for rapidly measuring content of vitamin D3 |
| CN106153796A (en) * | 2015-04-17 | 2016-11-23 | 西藏卫信康医药股份有限公司 | The content analysis detection method of 12 kinds of compound vitamines of injection |
| CN106383186A (en) * | 2016-08-31 | 2017-02-08 | 重庆方通动物药业有限公司 | High performance liquid chromatographic analysis method for simultaneous determination of content of 14 kinds of vitamins |
| CN106404947A (en) * | 2016-08-31 | 2017-02-15 | 陈大为 | Method for determination of fat-soluble vitamins by using semi-automatic sample treatment liquid chromatographic technology |
| CN107688068A (en) * | 2017-11-02 | 2018-02-13 | 威海百合生物技术股份有限公司 | A kind of detection method of farnoquinone content |
Non-Patent Citations (6)
| Title |
|---|
| KARA 等: "Seasonal variation of vitamin and sterol content of chironomidae larvae", 《PAKISTAN JOURNAL OF BIOLOGICAL SCIENCES》 * |
| PARLAK, AKIF E 等: "The determination of the effect of some 1,3,4 thiadiazole derivatives on biochemical content (Fatty Acids, Sterols, Lipophilic Vitamins) in rat liver", 《CELLULAR AND MOLECULAR BIOLOGY》 * |
| 周围等: "超高效合相色谱法同时测定复合维生素片中11种脂溶性维生素及其衍生物", 《分析化学》 * |
| 曾俊芬等: "HPLC法测定复合维生素注射液(13种)中4种脂溶性维生素的含量", 《中国药师》 * |
| 王聪等: "液相色谱-质谱联用法测定保健食品中9种脂溶性维生素", 《食品安全质量检测学报》 * |
| 陈玉花等: "高效液相色谱法在多种维生素药物检测中的应用", 《黑龙江医药》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110057933A (en) * | 2019-04-29 | 2019-07-26 | 杭州民生健康药业有限公司 | Vitamin K in one kind of multiple vitamin and minerals compound preparations2Detection method |
| CN110057933B (en) * | 2019-04-29 | 2022-08-30 | 杭州民生健康药业股份有限公司 | Method for detecting vitamin K2 in multivitamin mineral compound preparation |
| CN111574348A (en) * | 2020-06-15 | 2020-08-25 | 浙江诚意药业股份有限公司 | Purification process of vitamin K1 |
| CN111574348B (en) * | 2020-06-15 | 2022-10-25 | 浙江诚意药业股份有限公司 | Vitamin K1 purification process |
| CN115308338A (en) * | 2022-08-24 | 2022-11-08 | 哈药集团技术中心 | Method for detecting vitamin D3 related substances in vitamin D3 drops |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109613277B (en) | 2022-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109613277A (en) | Method that is a kind of while detecting vitamin D and vitamin K | |
| CN109283274A (en) | A kind of multicomponent content assaying method of Hedan tablet | |
| CN110068633A (en) | The liquid chromatography detecting method of caution structure substance in a kind of brufen | |
| CN101718690B (en) | Method for measuring content of ellagic acid substance in raspberry | |
| CN105486776B (en) | The gas chromatography analysis method of Metoclopramide | |
| CN107199012A (en) | A kind of magnetic fullerene nanomaterial and its application in SPE | |
| CN106198801A (en) | A kind of vitamin D3the detection method of content | |
| CN107037152B (en) | The detection method of carminic acid content in a kind of gelatine capsule | |
| CN106706835B (en) | A kind of quality determining method of trollflower effervescent tablet | |
| CN107703239A (en) | A kind of assay method of soyabean protein powder vitamine D3 | |
| CN115436544B (en) | Preparation method of vitamin D test solution and detection method of vitamin D in vitamin product | |
| CN111579684B (en) | Method for measuring content of total capsaicin in capsule wall material of capsule | |
| CN104849381B (en) | The method simultaneously measuring 7 kinds of Radix Astragalis based on high performance liquid chromatography-charged aerosol detectors method | |
| CN103513000B (en) | Method for carrying out identification and content measurement on nymphaea candida presl | |
| CN106153795A (en) | Measure chenodeoxycholic acid crude drug content and the method having related substance thereof | |
| CN110007033A (en) | Synchronous detection qinghaosu, dihydroartemisinine, Artesunate content method | |
| CN105758977A (en) | Method for detecting quality of Lycium ruthenicum Murr fruit or Lycium ruthenicum Murr fruit extracts | |
| CN108918721A (en) | A kind of measuring method of tussilago Content of Chlorogenic Acid | |
| CN109085258A (en) | A kind of quality determining method of Ficus lyrata | |
| CN101169395A (en) | Cosmetic product hydrocortisone high efficiency liquid chromatography detection method | |
| CN108205033A (en) | A kind of method of solanine and black nightshade polysaccharide in synchronous extracting and developing, measure black nightshade Chinese olive | |
| CN106596783A (en) | Method for determining content of garlicin content in garlicin tablets | |
| CN102253157A (en) | Method for detecting components of corsvenor momordica fruit soaking liquid in white spirit | |
| CN107290468B (en) | A kind of method of betel nut alkali content in LC-MS quantitative detection children's indigestion tablet | |
| CN105136939B (en) | A kind of method that use HPLC methods determine citric acid content in mulberries medicinal material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |