CN110068641A - The non-derived detection method of the HPLC-MS/MS of 25-OH Vintamin D2 and 25-hydroxyvitamin D3 - Google Patents
The non-derived detection method of the HPLC-MS/MS of 25-OH Vintamin D2 and 25-hydroxyvitamin D3 Download PDFInfo
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- CN110068641A CN110068641A CN201810059510.4A CN201810059510A CN110068641A CN 110068641 A CN110068641 A CN 110068641A CN 201810059510 A CN201810059510 A CN 201810059510A CN 110068641 A CN110068641 A CN 110068641A
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- G—PHYSICS
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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
- G01N30/88—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
- 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
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Abstract
The present invention provides the non-derived detection method of HPLC-MS/MS of a kind of 25-OH Vintamin D2 and 25-hydroxyvitamin D3.Specifically, detection kit of the invention includes (1) reference substance, (2) quality-control product, (3) Isotopic Internal Standard extracting solution, (4) precipitating reagent and (5) Mobile Phase Additives.Sample to be tested is detected using above-mentioned detection box and equipped with the high performance liquid chromatography mass spectrometer of two six-way valves, capture chromatographic column and analysis chromatographic column.This method used time is shorter, and detection efficiency is high.
Description
Technical field
The present invention relates to the technical field of 25-hydroxy-vitamin D detection, in particular to a kind of liquid chromatography mass is combined skill
The method of art detection 25-hydroxy-vitamin D.
Background technique
Vitamin D is sterol analog derivative, and ergosterol, vitamin D2 have anti-rachitic effect, also known as anti-rickets
Sick vitamin.Vitamin D family member includes vitamine D1, D2, D3, D4 and D5, and closer to healthy relationship is D2 (ergot
Calciferol) and D3 (gallbladder solidification alcohol).It is not only merely a kind of liposoluble vitamin, is substantially the one of a variety of physiological activity of tool
Kind hormone has extensive physiological metabolism activity.Vitamin D deficiency can cause the diseases such as rickets, tetany, osteomalacia,
But excessive vitamin D is taken in for a long time, blood vessel and organ calcification will be caused.Therefore in periodic detection human body vitamin D water
It is flat, for measurement body vitamin D supplementary result, and human health is maintained to have great importance.
Vitamin D can be generated by skin synthesis or food intake.Vitamin D half-life period about 3 hours, and with dimension
Raw element D binding protein is together；About 24 hours 1,25- (OH) 2 calciferols/D3 half-life period, content is extremely low in blood；25- hydroxyl
Base vitamin D is the marker being detected in blood, relatively stable because its half-life period is about 3 weeks, and is vitamin D in human body
Major storage form, by detection it may determine that the case where overall vitamin D.Vitamin D is the master for maintaining bone health
Want element.Childhood vitamin D there is a serious shortage of will lead to skeleton deformity, i.e. rickets.Vitamin D deficiency is secondary first shape
The common disease factor of other adenohypersthenia disease.25-hydroxy-vitamin D content is low also low with bone density related.With other clinical datas
In conjunction with testing result helps to determine bone metabolism.Discovered in recent years vitamin D and cancer, cardiovascular disease, diabetes etc. have
Correlation, enough internal vitamin D levels have certain prevention effect to cancer, multiple sclerosis, diabetes etc..
The crowd of vitamin D deficiency can improve internal vitamin D content by food, sunlight and vitamin replenisher,
Food source includes yolk, salmon, tuna, cod-liver oil, beef liver, margarine, Yoghourt, cheese etc.；What replenishers provided
Vitamin D includes two kinds of forms: D2 and D3.Two kinds of forms are all effective, each can guarantee sufficient vitamin D level.
But 2 and 3 be incoordinate.D3 is the form that human body generates, and some researches show that D3 quickly to improve on vitamin D level recently
Up to 3 times, and can remain more permanent.Replenishers amount depends on age and risk factor.The dietary standard of recommendation is 70
Year, old man was 600IU/ days, 71 and the above are 800IU/ days.Some researchers propose that vitamin D content height can bring a variety of
Health benefits, but will cause injury too much.According to United States Medicine research institute, more than 4000IU/ days, the risk of harm can increase
Add.
25 (OH) D2 and 25 (OH) D3 activity and therapeutic effect difference are big, and vitamine D3 should be used as preferred replenishers, but tie up
Raw element D2 is also indispensable, and separate detection is independent evaluation calciferol and vitamine D3 activity and function is inevitable requirement；Only
Detection 25-hydroxyvitamin D3 will lead to the horizontal inaccuracy of total 25-hydroxy-vitamin D, detect 25- hydroxyl D2/D3 ability respectively
The curative effect of correct assessment vitamin D level and vitamin D replacement therapy.Therefore, measurement 25-OH Vintamin D2/D3 can respectively
Directly to reflect the level of vitamin D Actual activity in organism, there is important clinical meaning and efficacy determination meaning.
Summary of the invention
It is an object of the present invention to provide a kind of 25-OH Vintamin D2s and 25-hydroxyvitamin D3 high-efficient liquid phase color
Compose the non-derived detection kit of mass spectrometry
It is a further object to provide a kind of 25-OH Vintamin D2s and 25-hydroxyvitamin D3 efficient liquid phase
The non-derived detection detection method of combined gas chromatography mass spectrometry.
The first aspect of the present invention provides a kind of 25-OH Vintamin D2 and 25-hydroxyvitamin D3 high-efficient liquid phase color
Compose the non-derived detection kit of mass spectrometry, comprising:
(1) reference substance,
(2) quality-control product；
(3) Isotopic Internal Standard extracting solution；
(4) precipitating reagent；
(5) Mobile Phase Additives；
Contain 25-OH Vintamin D2 and 25-hydroxyvitamin D3, Isotopic Internal Standard in the reference substance and quality-control product
Extracting solution is 25-hydroxyvitamin D3-d6.
In another preferred example, the concentration of the Isotopic Internal Standard extracting solution is 15ng/ml~25ng/ml.
In another preferred example, the concentration of the Isotopic Internal Standard extracting solution is 20ng/ml.
In another preferred example, the kit further includes mobile phase.
In another preferred example, the mobile phase includes mobile phase A and Mobile phase B.
In another preferred example, saturated monocarboxylic acid-aqueous solution that the mobile phase A is 0.28%~0.32%.
In another preferred example, saturated monocarboxylic acid-methanol solution that the Mobile phase B is 0.28%~0.32%.
In another preferred example, the precipitating reagent is acetonitrile.
In another preferred example, the Mobile Phase Additives are saturated monocarboxylic acid.
In another preferred example, the saturated monocarboxylic acid is selected from the group: formic acid, acetic acid, propionic acid, butyric acid or its group
It closes.
The second aspect of the present invention provides a kind of liquid phase fittings equipment, and the equipment includes: two six-way valves, capture
Chromatographic column and analysis chromatographic column；Wherein, two six-way valves are for connecting capture chromatographic column and analysis chromatographic column；Described
Capture chromatographic column is used for the acquisition of target compound and separates chaff interferent, the chromatography point of the analysis chromatographic column target compound
From.
In another preferred example, the filler of the analysis chromatographic column is octadecylsilane chemically bonded silica.
The third aspect of the present invention provides a kind of high performance liquid chromatography mass spectrometer comprising such as second party of the present invention
Liquid phase fittings equipment described in face.
The fourth aspect of the present invention provides a kind of 25-OH Vintamin D2 and 25-hydroxyvitamin D3 high-efficient liquid phase color
Compose the non-derived detection method of mass spectrometry, the method comprising steps of
(i) sample, precipitating reagent and Isotopic Internal Standard extracting solution are provided, the sample is blood serum sample to be detected, control
Product or quality-control product；
(ii) mixed processing is carried out to the sample and precipitating reagent and Isotopic Internal Standard extracting solution, mixed solution exists
It is cultivated 10~15 minutes at 2~8 DEG C；
(iii) solution carries out centrifugal treating after the cultivation obtained to step (ii), obtains supernatant；
(iv) in high performance liquid chromatography mass spectrometer as described in the third aspect of the present invention, supernatant is detected
Analysis.
In another preferred example, the reference substance is prepared by the method included the following steps: taking proper volume
The serum matrix managed is mixed with 4 μ l, 100 μ l, the 25-hydroxyvitamin D3 working solution that 200 μ l concentration are 1 μ g/ml respectively,
Up to 2~100ng/ml serum matrix standard curve sample, as reference substance.
In another preferred example, the quality-control product is prepared by the method included the following steps: taking proper volume
The serum matrix managed is mixed with 16 μ l, 40 μ l, the 25-hydroxyvitamin D3 working solution that 80 μ l concentration are 1 μ g/ml, i.e., respectively
Obtain the quality-control product of 8ng/ml (QC1), 20ng/ml (QC2), 40ng/ml (QC3).
In another preferred example, the centrifugal force in step (iii) is 15000g；And/or centrifugation time is 5 minutes.
In another preferred example, the HPLC-MS/MS system includes: two six-way valves, capture chromatographic column, analysis color
Compose column.
In another preferred example, precipitating reagent described in step (ii) is acetonitrile, and the Isotopic Internal Standard extracting solution is
Concentration is the 25-hydroxyvitamin D3-d6 solution of 15ng/ml~25ng/ml, and sample, precipitating reagent and the isotope
The volume ratio of internal standard extracting solution is 2~6 ︰, 0.5~2 ︰ 6~10.
In another preferred example, the volume ratio of the sample, precipitating reagent and Isotopic Internal Standard extracting solution is 4 ︰, 1 ︰ 8.
In another preferred example, the blood serum sample to be detected includes: human serum sample, cow's serum sample, rat serum
Final proof product, mice serum sample, rabbit anteserum, cow's serum.It should be understood that within the scope of the present invention, above-mentioned each technology of the invention
Can be combined with each other between feature and specifically described in below (e.g. embodiment) each technical characteristic, thus constitute it is new or
Preferred technical solution.Due to space limitations, I will not repeat them here.
Specific embodiment
The present inventor after extensive and in-depth study, by largely screening, develops a kind of 25- hydroxy vitamin for the first time
D2 and the non-derived detection kit of 25-hydroxyvitamin D3 high performance liquid chromatography mass spectrometry and detection method.Sample to be tested
Pre-treatment uses simple and effective precipitation of protein.Trapping column is acquired to analyte and separates chaff interferent, uses two six
Trapping column is connect by port valve with the performance liquid chromatographic column for being used for chromatographic isolation.Use atmospheric pressure chemical ionization (APCI) and deuterated
Inside being marked with ensures precision and robustness and minimizes ion inhibiting effect.This method is easy to operate, the used time is shorter, and can
To detect the concentration of 25-OH Vintamin D2 and 25-hydroxyvitamin D3 simultaneously in one test, and testing result is sensitive
Degree is high, and testing result accuracy is high.The present invention is completed on this basis.
As used herein, term " containing " or " including (including) " can be open, semi-enclosed and enclosed.It changes
Yan Zhi, the term also include " substantially by ... constitute " or " by ... constitute ".
Detection kit and detection method of the invention
Detection kit of the invention includes:
(1) reference substance；
(2) quality-control product；
(3) Isotopic Internal Standard extracting solution；
(4) precipitating reagent；
(5) Mobile Phase Additives；
Contain 25-OH Vintamin D2 and 25-hydroxyvitamin D3 in reference substance and quality-control product of the invention, in isotope
Mark extracting solution is 25-hydroxyvitamin D3-d6, and concentration is 15ng/ml~25ng/ml.
Reference substance is prepared by the method included the following steps: take the processed serum matrix of proper volume, respectively with
The 25-hydroxyvitamin D3 working solution of various concentration mixes, and obtains serum matrix standard curve sample, as reference substance.
The quality-control product is prepared by the method included the following steps: the processed serum matrix of proper volume is taken,
The quality-control product to get various concentration is mixed with the 25-hydroxyvitamin D3 working solution of various concentration respectively.
Precipitating reagent of the invention includes (but being not limited to) acetonitrile.
Mobile Phase Additives of the invention include (but being not limited to): formic acid, acetic acid, propionic acid, butyric acid, or combinations thereof.
Mobile phase of the invention includes mobile phase A and Mobile phase B, and the mobile phase A is that saturated monocarboxylic acid-is water-soluble
Liquid；The Mobile phase B is saturated monocarboxylic acid-methanol solution.
Detection method of the invention includes the following steps:
One, experimental implementation
(i) sample, precipitating reagent and Isotopic Internal Standard extracting solution are provided, the sample is blood serum sample to be detected, control
Product or quality-control product；
(ii) mixed processing is carried out to the sample and precipitating reagent and Isotopic Internal Standard extracting solution, mixed solution exists
It is cultivated 10~15 minutes at 2~8 DEG C；
(iii) solution carries out centrifugal treating after the cultivation obtained to step (ii), obtains supernatant；
(iv) supernatant is tested and analyzed in HPLC-MS/MS system.
Two six-way valves, capture chromatographic column, analysis chromatographic column etc. are specially provided in HPLC-MS/MS system of the invention
Accessory, for analyzing the concentration of 25-OH Vintamin D2 and 25-hydroxyvitamin D3.
Two, data are analyzed
Record chromatogram and detect sample 25-OH Vintamin D2,25-hydroxyvitamin D3 peak area and isotope in
Mark 25-hydroxyvitamin D3-d6 peak area.
The method for drafting of standard curve: using the mark concentration of 3 reference substances as abscissa (x), with the reality of 3 reference substances
The ratio for detecting peak area and respective internal standard peak area is ordinate (y), draws standard curve.
The fitting of calibration curve equation: mark concentration (x) is linearly returned with the peak area ratio (y) of 3 reference substances
Return.It can get regression equation: y=a+bx, wherein y is ordinate, and x is abscissa, and a is intercept, and b is slope.Detect sample knot
The calculating of fruit: the ratio of the practical peak area and internal standard peak area that will test sample substitutes into above-mentioned standard curvilinear equation, calculates inspection
The concentration of untested compound in test sample sheet.
Main advantages of the present invention include:
The present invention establishes 25-OH Vintamin D2 and 25-hydroxyvitamin D3 detection method, related examination in serum
Agent is few, and detecting step is simple, and accuracy is high, strong applicability, and one-time detection can obtain simultaneously 25-OH Vintamin D2 and
The concentration of 25-hydroxyvitamin D3 has good clinical value.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
Embodiment 1
Reference substance is prepared by the method included the following steps in the present embodiment: taking the processed serotonin of proper volume
Matter mixes respectively with 4 μ l, 100 μ l, the 25-hydroxyvitamin D3 working solution that 200 μ l concentration are 1 μ g/ml to get 2-100ng/
Ml serum matrix standard curve sample, as reference substance.
Quality-control product is prepared by the method included the following steps: take the processed serum matrix of proper volume, respectively with
16 μ l, 40 μ l, the 25-hydroxyvitamin D3 working solution that 80 μ l concentration are 1 μ g/ml mix to get 8ng/ml (QC1), 20ng/ml
(QC2), the quality-control product of 40ng/ml (QC3).
Contain 25-hydroxyvitamin D3-d6, concentration 50ng/ml in Isotopic Internal Standard extracting solution.Precipitating reagent is second
Nitrile, Mobile Phase Additives are formic acid.
One, experimental implementation
1. drawing 100 μ l samples/reference substance/quality-control product into 1.5ml centrifuge tube.
2. adding 25 μ l precipitating reagents.
3. adding 200 μ l internal standard extracting solutions, it is vortexed 60 seconds.
4. being cultivated 10 minutes at+4 DEG C.
5. being centrifuged 5 minutes in the case where centrifugal force is the revolving speed of 15000g.
6. shifting 200 μ l supernatants into 96 hole reaction plates.
7. 20 μ l supernatant of sample introduction is into HPLC-MS/MS system.
Two, liquid-phase condition
Mobile phase prepares
A phase: Mobile Phase Additives are added in aqueous solution in the ratio of 3:1000.
B phase: Mobile Phase Additives are added in methanol in the ratio of 3:1000.
Capture chromatographic column:
Analyze chromatographic column: octadecylsilane chemically bonded silica is filler or suitable person.
Chromatographic column column temperature: 25 DEG C.
Sample volume: 20 μ L.
Eluent gradient and flow velocity:
Two six-way valve settings:
Three, Mass Spectrometry Conditions
Multiple-reaction monitoring ion pair (MRM) and corresponding voltage parameter
Other mass spectrometry parameters
Ion source: atmospheric pressure chemical ionization (APCI) ion source.
Ionizing voltage: 5500V.
Auxiliary plus hot air temperature: 500 DEG C.
Atomization gas (GAS1): 55psi.
Gas curtain gas (CUR): 20psi.
Collision gas (CAD): 3psi.
Five, quantitative analysis
The method for drafting of standard curve: using the mark concentration of 3 reference substances as abscissa (x), with the reality of 3 reference substances
The ratio for detecting peak area and respective internal standard peak area is ordinate (y), draws standard curve.
The fitting of calibration curve equation: mark concentration (x) is linearly returned with the peak area ratio (y) of 3 reference substances
Return.It can get regression equation: y=a+bx, wherein y is ordinate, and x is abscissa, and a is intercept, and b is slope.Detect sample knot
The calculating of fruit: the ratio of the practical peak area and internal standard peak area that will test sample substitutes into above-mentioned standard curvilinear equation, calculates inspection
The concentration of 25-OH Vintamin D2 and 25-hydroxyvitamin D3 is respectively 4.22ng/mL and 23.32ng/mL in test sample sheet.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of 25-OH Vintamin D2 and the non-derived detection reagent of 25-hydroxyvitamin D3 high performance liquid chromatography mass spectrometry
Box characterized by comprising
(1) reference substance；
(2) quality-control product；
(3) Isotopic Internal Standard extracting solution；
(4) precipitating reagent；With
(5) Mobile Phase Additives；
Contain 25-OH Vintamin D2 and 25-hydroxyvitamin D3 in the reference substance and quality-control product, Isotopic Internal Standard extracts
Liquid is 25-hydroxyvitamin D3-d6.
2. kit as described in claim 1, which is characterized in that the concentration of the Isotopic Internal Standard extracting solution is 15ng/
Ml~25ng/ml.
3. kit as described in claim 1, which is characterized in that the precipitating reagent is acetonitrile.
4. kit as described in claim 1, which is characterized in that the Mobile Phase Additives are saturated monocarboxylic acid.
5. kit as described in claim 3, which is characterized in that the saturated monocarboxylic acid is selected from the group: formic acid, second
Acid, propionic acid, butyric acid, or combinations thereof.
6. a kind of liquid phase fittings equipment, which is characterized in that the equipment includes: two six-way valves, capture chromatographic column and analysis
Chromatographic column；Wherein, two six-way valves are for connecting capture chromatographic column and analysis chromatographic column；The capture chromatographic column is used
In target compound acquisition and separate chaff interferent, the chromatographic isolation of the analysis chromatographic column target compound.
7. a kind of high performance liquid chromatography mass spectrometer, which is characterized in that it includes liquid phase accessory as described in claim 6
Equipment.
8. a kind of 25-OH Vintamin D2 and the non-derived detection method of 25-hydroxyvitamin D3 high performance liquid chromatography mass spectrometry,
It is characterized in that, the method comprising steps of
(i) sample, precipitating reagent and Isotopic Internal Standard extracting solution be provided, the sample be blood serum sample to be detected, reference substance or
Quality-control product；
(ii) mixed processing is carried out to the sample and precipitating reagent and Isotopic Internal Standard extracting solution, mixed solution is 2~8
It is cultivated 10~15 minutes at DEG C；
(iii) solution carries out centrifugal treating after the cultivation obtained to step (ii), obtains supernatant；
(iv) in high performance liquid chromatography mass spectrometer as claimed in claim 7, supernatant is tested and analyzed.
9. detection method as described in claim 8, which is characterized in that precipitating reagent described in step (ii) is acetonitrile, institute
The Isotopic Internal Standard extracting solution stated is the 25-hydroxyvitamin D3-d6 solution that concentration is 15ng/ml~25ng/ml, and described
Sample, precipitating reagent and Isotopic Internal Standard extracting solution volume ratio be 2~6 ︰, 0.5~2 ︰ 6~10.
10. detection method as claimed in claim 9, which is characterized in that the blood serum sample to be detected includes: human serum sample
Product, cow's serum sample, rat blood serum sample, mice serum sample, rabbit anteserum, cow's serum.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110542735A (en) * | 2019-09-12 | 2019-12-06 | 贝知(上海)医疗科技有限公司 | method for high-throughput determination of multiple fat-soluble vitamins by ultra-high performance liquid mass spectrometry |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110542735A (en) * | 2019-09-12 | 2019-12-06 | 贝知(上海)医疗科技有限公司 | method for high-throughput determination of multiple fat-soluble vitamins by ultra-high performance liquid mass spectrometry |
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