CN109212091A - 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination detection method and kit in serum - Google Patents
25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination detection method and kit in serum Download PDFInfo
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The present invention provides 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination detection method and kits in a kind of serum, 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum, comprising the following steps: (1) takes blood serum sample in sample cell；(2) precipitating reagent containing internal standard mixed liquor is added into each sample, is vortexed and mixes；(3) extractant is added into each sample, is vortexed and is mixed；(4) sample takes supernatant in sample feeding pipe after standing；(5) it is added after being dried with nitrogen at room temperature and redissolves liquid, it is detected after vortex using liquid chromatography tandem mass spectrometry, and obtains the content of 25-hydroxy-vitamin D in test serum by comparing the internal standard peak area of 25-hydroxy-vitamin D and 25-hydroxy-vitamin D.25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum of the present invention, can further improve accuracy in detection, precision and shorten the pre-treatment time.
Description
Technical field
The invention belongs to 25-hydroxy-vitamin D detection technique fields, can detect 25- hydroxyl simultaneously more particularly, to one kind
Base vitamin D2And 25-hydroxy-vitamin D3Method and kit.
Background technique
Vitamin D has the function of classics for adjusting alcium and phosphor metabolization and influences the non-classical effects such as cell proliferation and differentiation, is to maintain
A kind of essential steroid hormone of human health.It can lead to children rachitis and adult human cartilage when the shortage of vitamin D
Disease.In recent years, in-depth study show vitamin D deficiency will increase suffer from tumour, cardiovascular disease, spontaneous immunity disease and
The risk of the common multiple illness such as mental disease.For the importance of human health and current Chinese are directed to based on vitamin D
The relatively low status of group's vitamin D level, it is necessary to popularized in Chinese population vitamin D level detection and to key population into
Row screening.25-hydroxy-vitamin D (is divided into 25-hydroxy-vitamin D2And 25-hydroxy-vitamin D3) level is defined as vitamin D
The functional parameter of nutritional status has become the optimal parameter of measurement vitamin D generally acknowledged in the world.
The competitive protein binding method (CPBA) of method of 25 (OH) D contents of currently used detection, radioimmunology
(RIA), enzyme linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), high performance liquid chromatography (HPLC) etc..
Some methods are time-consuming, step is lengthy and jumbled, are unable to satisfy clinical easy, high-throughput requirement；Some methods have radioactive pollution
Potential risk has damage to experimenter and environment；Some methods are easy that cross reaction occurs with other non-targeted compounds,
Cause method specificity insufficient, HPLC can distinguish 25 (OH) D relative to immunization2With 25 (OH) D3, but 25 (OH) D2It is dense
Degree is too low, and liquid chromatography detection limit does not reach requirement.
With the development of detection technique and precision, HPLC is gradually replaced by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
Generation.LC-MS/MS is 25 (OH) D detection methods the most commonly used in chromatography, and sample uses liquid chromatogram to separate after processing,
Detector is tandem mass spectrometer, under multiple-reaction monitoring scan pattern, can detect 25 (OH) D simultaneously2With 25 (OH) D3, have non-
Often high sensitivity, specificity and accuracy, by it is internationally recognized be goldstandard that 25 (OH) D are detected.Internal vitamin D concentrations
Accurate quantitative analysis is the important indicator for diagnosing vitamin D deficiency related disease diagnosing and treating feedback effects.
Existing based on most of detection method associated with liquid chromatogram and tandem mass spectrum is laboratory from construction method, is lacked
Standardization.The processing method of the reagent, standard items and the sample that are used due to different laboratories is different, often results in different experiments room
Between testing result difference it is big, and can also consume a large amount of time in preceding processing.Therefore, using include calibration object, extraction
It is the important channel for solving the problems, such as this that the kit of reagent needed for reagent, quality-control product etc. detect, which carries out detection,.
Summary of the invention
In view of this, the present invention is directed to propose a kind of 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination inspection in serum
Survey method handles the time before further shortening under the premise of high accuracy, precision to overcome the deficiencies of existing technologies,
Improve working efficiency.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum, comprising the following steps:
(1) take blood serum sample in sample cell；
(2) precipitating reagent containing internal standard mixed liquor is added into each sample, is vortexed and mixes；
(3) extractant is added into each sample, is vortexed and is mixed；
(4) sample takes supernatant in sample feeding pipe after standing；
(5) it is added after being dried with nitrogen at room temperature and redissolves liquid, detected after vortex using liquid chromatography tandem mass spectrometry, and
25- hydroxyl dimension life in test serum is obtained by comparing the internal standard peak area of 25-hydroxy-vitamin D and 25-hydroxy-vitamin D
The content of plain D.It should be noted that the liquid chromatography tandem mass spectrometry in the present invention is combined using Liquid Chromatography-Tandem Mass Spectrometry
Instrument.
Preferably, in step (2), internal standard mixed liquor includes 25-hydroxy-vitamin D2Internal standard and 25-hydroxy-vitamin D3It is interior
Mark.
Preferably, in step (1), the selection amount of serum is 90-200 μ L；It is heavy containing internal standard mixed liquor in step (2)
The dosage of shallow lake agent is 300-600 μ L, and wherein the volume ratio of internal standard mixed liquor and precipitating reagent is 1:(10-30), vortex mixing time
For 30-90s；In step (3), the dosage of extractant is 800-1000 μ L, and vortex mixing time is 60-120s；In step (4),
Sample time of repose is 30-90s, and the supernatant volume that sample feeding pipe is added is 750-900 μ L；In step (5), the dosage of liquid is redissolved
For 100-200 μ L, vortex time 30-60s；It is further preferred that the selection amount of serum is 100 μ L in step (1)；Step
(2) in, the dosage of the precipitating reagent containing internal standard mixed liquor is 310 μ L, and wherein the volume ratio of internal standard mixed liquor and precipitating reagent is 1:
(10-30), vortex mixing time are 60s；In step (3), the dosage of extractant is 900 μ L, and vortex mixing time is 120s；Step
Suddenly in (4), sample time of repose is 60s, and the supernatant volume that sample feeding pipe is added is 800 μ L；In step (5), the use of liquid is redissolved
Amount is 100 μ L, vortex time 30s.
Preferably, in step (2), precipitating reagent is the mixed solution of methanol and acetonitrile 1:1 mixing by volume；Step (3)
In, extractant is HPLC grades of n-hexanes；In step (5), redissolve liquid be 40-60v/v% methanol aqueous solution, wash needle liquid be 50~
The methanol aqueous solution of 80v/v%；Preferably, in step (5), the methanol aqueous solution that liquid is 50v/v% is redissolved, washing needle liquid is 50v/
The methanol aqueous solution of v%.It should be noted that the effect for washing needle liquid is the fixed front and back mobile phase, auxiliary measuring, cleaning of balancing side
Probe and avoid influence of the preceding sample to rear sample measures.
Preferably, in step (5), chromatographic condition when liquid chromatography tandem mass spectrometry detects is as follows: using gradient elution
Program, the aqueous formic acid of 0.05~0.3v/v% is as mobile phase A, and the formic acid methanol solution of 0.05~0.3v/v% is as stream
Dynamic phase B；Preferably, mobile phase A is using the aqueous formic acid of 0.1v/v% in gradient elution program, and Mobile phase B is using 0.1v/
The formic acid methanol solution of v%.
Preferably, in step (5), the parameter of gradient elution is as follows:
| Time | The volume ratio of mobile phase A | The volume ratio of Mobile phase B |
| 0-2min | 25→2 | 75→98 |
| 2-5min | 98 | 98 |
| 5-5.5min | 2→25 | 98→75 |
| 5.5-7min | 25 | 75 |
Preferably, the chromatographic condition in step (5), when liquid chromatography tandem mass spectrometry detects further include: chromatographic column: C18
2.1 × 100mm, 3 μm；Chromatographic parameter: flow rate of mobile phase 0.4-1.0mL/min, 35-60 DEG C of column temperature, detection time 4-10min,
Sample volume 10-50 μ L；Preferably, flow rate of mobile phase 0.5mL/min, 40 DEG C of column temperature；Detection time 7min, 30 μ L of sample volume.
Preferably, in step (5), Mass Spectrometry Conditions when liquid chromatography tandem mass spectrometry detects include:
。
Preferably, in step (5), Mass Spectrometry Conditions when liquid chromatography tandem mass spectrometry detects further include following ion source ginseng
Number: ionization source is the source ESI, and using positive ion mode, other parameters are as follows:
Preferably, other parameters are as follows:
| Gas curtain gas CUR (psi) | 16 |
| Sprayer GS1 (psi) | 20 |
| Auxiliary heating gas GS2 (psi) | 50 |
| Temperature TEM (DEG C) | 500 |
| Interface heats Ihe | ON |
| Collision gas CAD (psi) | 4 |
It is a kind of for 25-hydroxy-vitamin D liquid phase color in serum as described above another object of the present invention is to propose
The kit of tandem mass spectrum combination detection method is composed, to be used for 25-hydroxy-vitamin D liquid chromatography tandem in above-mentioned serum
Mass spectrometry detection method detects 25-hydroxy-vitamin D in serum.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
A kind of examination for 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination detection method in serum as described above
Agent box, including following solution:
(1) calibration object: 25 (OH) D of 5-40ng/mL2With 25 (OH) D of 5-120ng/mL3；
(2) quality-control product: 25 (OH) D of 5.4-23.5ng/mL2With 25 (OH) D of 4.8-64.6ng/mL3；
(3) internal standard mixed liquor: the 25-hydroxy-vitamin D of 200ng/mL2The 25-hydroxy-vitamin D of internal standard and 500ng/mL3
Internal standard；
(4) precipitating reagent: the mixed solution of methanol and acetonitrile 1:1 mixing by volume；
(5) extractant: HPLC grades of n-hexanes；
(6) liquid: the methanol aqueous solution of 40~60v/v% is redissolved；Preferably, redissolving liquid is methanol and water 1:1 by volume
Mixed solution；
(7) mobile phase: mobile phase A uses the aqueous formic acid of 0.05~0.3v/v%, Mobile phase B using 0.05~
The formic acid methanol solution of 0.3v/v%；Preferably, mobile phase A uses the aqueous formic acid of 0.1v/v%, and Mobile phase B uses
The formic acid methanol solution of 0.1v/v%；
(8) needle liquid: the methanol aqueous solution of 50~80v/v% is washed；Preferably, the methanol aqueous solution that needle liquid is 50v/v% is washed.
Compared with the existing technology, 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination inspection in serum of the present invention
Survey method has the advantage that
(1) 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum of the present invention, is trying
Under the modification of agent box component and pretreatment mode, it is no longer necessary to individually add micro internal standard, avoid experimental error；No longer need
It is centrifuged the operating procedure of this lengthy and jumbled time-consuming, substantially reduces the time required for pre-treatment, and simplifies and entirely detected
Journey, while also ensuring the high accuracy, high precision and high detection flux of detection.
(2) 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum of the present invention, passes through
25-hydroxy-vitamin D molecular cleavage at the daughter ion of different mass-to-charge ratioes, is scanned the daughter ion quantity of different mass-to-charge ratioes by voltage,
With reference to target concentration in known 25-hydroxy-vitamin D, 25-hydroxy-vitamin D concentration to be measured is calculated, using side of the invention
Method and kit detect 25-hydroxy-vitamin D, and preci-sion and accuracy is higher.
(3) 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum of the present invention, can be from
25-hydroxy-vitamin D is extracted in serum, 25-hydroxy-vitamin D in sample is quantified using tandem mass spectrometer,
25-hydroxy-vitamin D level in human body can be evaluated and be monitored by analysis, to be carried out to vitamin related disease
Diagnostic and therapeutic effects assessment, detection cycle significantly shorten.
It is described a kind of for 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination detection method in serum as described above
Kit and above-mentioned serum in 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination detection method compared with the existing technology
Possessed advantage is identical, and details are not described herein.
Detailed description of the invention
Fig. 1 is 1 standard chromatogram of embodiment；
Fig. 2 is that embodiment 1 surveys sample chromatogram figure；
Fig. 3 is embodiment 125- hydroxy-vitamine D2Working curve diagram；
Fig. 4 is embodiment 125- hydroxy-vitamine D3Working curve diagram.
Specific embodiment
In addition to being defined, technical term used in following embodiment has universal with those skilled in the art of the invention
The identical meanings of understanding.Test reagent used in following embodiment is unless otherwise specified conventional biochemical reagent；It is described
Experimental method is unless otherwise specified conventional method.
Below with reference to examples and drawings, the present invention will be described in detail.
Embodiment 1
The present embodiment is that 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum:
One, major experimental step:
(1) prepare internal standard working solution: the 25- hydroxyl that chemical (TRC) company provides being studied using Toronto and ties up life
Plain D2-d6, the 25-hydroxy-vitamin D of Medical Isotopes company offer3-d6；According to when in time experimental specimen amount calculating
Mark the amount of standard working solution.Above-mentioned internal standard is mixed to get high concentration internal standard mixed liquor, in use, with methanol dilution high concentration
Internal standard mixed liquor mixes well and internal standard working solution can be obtained.
The specific preparation method of above-mentioned internal standard mixed liquor are as follows:
1) according to different packing specifications, two kinds of internal standard stock solutions is first diluted to 20 μ g/mL, are denoted as ISS D respectively2-d6
And ISS D3-d6；
2) 100 μ L ISS D are taken2-d6, add 900 μ L methanol, obtaining concentration is 2 μ g/mL, is denoted as ISS1 D2-d6；Take 250 μ
L ISS D3-d6, add 750 μ L methanol, obtaining concentration is 5 μ g/mL, is denoted as ISS1 D3-d6；
3) the 2 μ g/mL ISS1D of 500 μ L are taken2-d6, the 5 μ g/mL ISS1D of 500 μ L3-d6, add the methanol of 4mL, obtain interior
Mark mixed liquor, i.e., 25 (OH) D2-d6Concentration is 200ng/mL, 25 (OH) D3-d6Concentration is 500ng/mL, is denoted as ISW.
(2) analytic process:
1, take 100 μ L blood serum samples in sample cell；
2,310 μ L precipitating reagents containging interior traget are added into each sample, is vortexed and mixes 60s；
3,900 μ L extractants are added into each sample, are vortexed and are mixed 120s；
4, sample takes 800 μ L supernatants in sample feeding pipe after standing 60s；
5,100 μ L are added after being dried with nitrogen at room temperature and redissolve liquid, sample detection after vortex 30s.
In above-mentioned analytic process: internal standard mixed liquor includes 25-hydroxy-vitamin D2Internal standard and 25-hydroxy-vitamin D3Internal standard；
Precipitating reagent is the mixed solution of methanol and acetonitrile 1:1 mixing by volume；Extractant is HPLC grades of n-hexanes；Redissolution liquid is 50v/
The methanol aqueous solution of v%；Wash the methanol aqueous solution that needle liquid is 50v/v%.
(3) result calculates:
In tandem mass spectrum system software can will test result by comparing detectable substance and it is corresponding in target peak area come into
Row calculates, and provides the concentration of detectable substance and generates data report.
(4) points for attention:
1, -20 DEG C of refrigerators should be immediately placed in after isotopic standard product are finished to save；
If 2, reagent is poured out from reagent bottle, can not be used again.
Two, main agents:
1, in detection method of the invention, the ratio of water, formic acid and methanol can be adjusted in respective range in mobile phase, together
When the adjustment of relevant parameter must be carried out on mass spectrograph, the accuracy of vitamin content result detected in this way and precision are not
It is impacted；
2, all reagents select chromatography rank；
3, the ultrapure water of wet concentration tri-distilled water or water purification machine filtering used, resistance>=18M Ω or conductance<5us/ are configured
Cm, pH value are 7.0 ± 0.2.
Three, mass spectrometry parameters:
It is referred in the setting of tandem mass spectrum instrument AB Sciex company API4000 and is shown in Table 1 with Mass Spectrometry Conditions:
1 mass spectrum relative parameters setting of table
It should be noted that above-mentioned parameter parent ion is fixed, for different machine or same brand machine mass spectrometry parameters
When variation, daughter ion is possible to change.
Wherein, chromatographic condition is as follows:
Chromatographic column: C18 2.1 × 100mm, 3 μm
Chromatographic parameter: flow rate of mobile phase 0.5mL/min, 40 DEG C of column temperature, detection time 7min, 30 μ L of sample volume；
Using gradient elution mode, elution parameters are shown in Table 2:
2 condition of gradient elution of table
Mobile phase A uses the aqueous formic acid of 0.1v/v% in above-mentioned gradient elution program, and Mobile phase B uses 0.1v/v%
Formic acid methanol solution.
Mass Spectrometer Method condition is as follows:
Source parameters: the source ESI, positive ion mode
3 Mass Spectrometer Method condition of table
| Gas curtain gas CUR (psi) | 16 |
| Sprayer GS1 (psi) | 20 |
| Auxiliary heating gas GS2 (psi) | 50 |
| Temperature TEM (DEG C) | 500 |
| Interface heats Ihe | ON |
| Collision gas CAD | 4 |
It should be noted that the differences such as precision of machine, the above parameter only supplies since every machine is when installing debugging
With reference to specific tuning parameter does a little change by engineer.
The resulting mark product chromatogram of method through this embodiment as shown in Figure 1, it is shown in Fig. 2 through this embodiment
The resulting actual sample chromatogram of method, abscissa represent the time, and ordinate represents ionic strength, and the figure shows different detections
Appearance situation of the object in specific time can be seen that the peak type of detectable substance is in normal distribution curve figure by Fig. 1, Fig. 2, and
It can obviously be distinguished with chaff interferent, and in the chromatogram of Fig. 2 actual sample when the mass chromatography peak reservation of measured target substance
Between it is consistent with the mass chromatography peak retention time of tie substance in Fig. 1 mark product chromatogram, and then may determine that detectable substance appearance reaches
To testing requirements.
The working curve diagram of the resulting detectable substance of method through this embodiment as shown in Figure 3, Figure 4, abscissa represent
Detectable substance concentration, ordinate represent the peak area of detectable substance, it can be seen that are in good linear relationship, 25 (OH) D2Linear pass
System is y=0.0522x+0.0235, linear coefficient r=0.9983；25(OH)D3Linear relationship y=0.0296x+0.0154,
Linear coefficient r=0.9996.Testing concentration can be calculated by the linear relationship in the figure.It is computed, in actual sample, 25
(OH)D2Concentration be 11.34ng/mL；25(OH)D3Concentration be 9.75ng/mL.
Embodiment 2
The present embodiment is the kit of 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination detection in serum:
The kit of this implementation can detect 25 (OH) D simultaneously2With 25 (OH) D3。
The kit of the present embodiment includes: calibration object, quality-control product, internal standard mixed liquor, precipitating reagent, extractant, redissolves liquid, stream
Dynamic phase, is specifically shown in Table 4.It include internal standard product in internal standard mixed liquor, internal standard product study chemical (TRC) company using Toronto
The 25-hydroxy-vitamin D of offer2-d6, the 25-hydroxy-vitamin D of Medical Isotopes company offer3-d6。
The composition of 4 kit of table
(1) use of the precipitating reagent containing internal standard mixed liquor:
Internal standard in kit is mixed into precipitating reagent, and the mixing that is vortexed.
(2) formula that mobile phase uses:
Mobile phase A uses the aqueous formic acid of 0.1v/v%, and Mobile phase B uses the formic acid methanol solution of 0.1v/v%.
(3) needle liquid: the methanol aqueous solution of 50v/v% is washed.
The kit of the domestic common tandem mass spectrometer (American AB company) of the method cooperation of embodiment 1 and embodiment 2,
It can be by once testing while detecting (OH) D 25 in serum2With 25 (OH) D3, 25 (OH) D2With 25 (OH) D3Detection batch in not
Precision and batch between imprecision be shown in Table 5,25 (OH) D2With 25 (OH) D3The sample recovery rate of detection is shown in Table 6.It can by table 5 and table 6
See, testing result has very high accuracy and accuracy.
Table 5 25 (OH) D2With 25 (OH) D3Detection batch in imprecision and batch between imprecision
Table 6 25 (OH) D2With 25 (OH) D3The sample recovery rate of detection
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum, it is characterised in that: including following step
It is rapid:
(1) take blood serum sample in sample cell；
(2) precipitating reagent containing internal standard mixed liquor is added into each sample, is vortexed and mixes；
(3) extractant is added into each sample, is vortexed and is mixed；
(4) sample takes supernatant in sample feeding pipe after standing；
(5) it is added after being dried with nitrogen at room temperature and redissolves liquid, detected after vortex using liquid chromatography tandem mass spectrometry, and pass through
Compare the internal standard peak area of 25-hydroxy-vitamin D and 25-hydroxy-vitamin D to obtain 25-hydroxy-vitamin D in test serum
Content.
2. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum according to claim 1,
Be characterized in that: in step (2), internal standard mixed liquor includes 25-hydroxy-vitamin D2Internal standard and 25-hydroxy-vitamin D3Internal standard.
3. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum according to claim 1,
Be characterized in that: in step (1), the selection amount of serum is 90-200 μ L；In step (2), the precipitating reagent containing internal standard mixed liquor
Dosage is 300-600 μ L, and wherein the volume ratio of internal standard mixed liquor and precipitating reagent is 1:(10-30), vortex mixing time is 30-
90s；In step (3), the dosage of extractant is 800-1000 μ L, and vortex mixing time is 60-120s；In step (4), sample is quiet
Setting the time is 30-90s, and the supernatant volume that sample feeding pipe is added is 750-900 μ L；In step (5), the dosage for redissolving liquid is 100-
200 μ L, vortex time 30-60s；Preferably, in step (1), the selection amount of serum is 100 μ L；In step (2), containing interior
The dosage for marking the precipitating reagent of mixed liquor is 310 μ L, and wherein the volume ratio of internal standard mixed liquor and precipitating reagent is 1:(10-30), it is vortexed
Mixing time is 60s；In step (3), the dosage of extractant is 900 μ L, and vortex mixing time is 120s；In step (4), sample
Time of repose is 60s, and the supernatant volume that sample feeding pipe is added is 800 μ L；In step (5), the dosage for redissolving liquid is 100 μ L, whirlpool
The rotation time is 30s.
4. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum according to claim 1,
Be characterized in that: in step (2), precipitating reagent is the mixed solution of methanol and acetonitrile 1:1 mixing by volume；In step (3), extraction
Agent is HPLC grades of n-hexanes；In step (5), the methanol aqueous solution that liquid is 40-60v/v% is redissolved, washing needle liquid is 50~80v/v%
Methanol aqueous solution；Preferably, in step (5), the methanol aqueous solution that liquid is 50v/v% is redissolved, washes the first that needle liquid is 50v/v%
Alcohol solution.
5. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum according to claim 1,
Be characterized in that: in step (5), chromatographic condition when liquid chromatography tandem mass spectrometry detects is as follows: gradient elution program is used,
The aqueous formic acid of 0.05~0.3v/v% is as mobile phase
The formic acid methanol solution of A, 0.05~0.3v/v% are as Mobile phase B；Preferably, mobile phase A is adopted in gradient elution program
With the aqueous formic acid of 0.1v/v%, Mobile phase B uses the formic acid methanol solution of 0.1v/v%.
6. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum according to claim 1,
Be characterized in that: in step (5), the parameter of gradient elution is as follows:
7. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum according to claim 1,
It is characterized in that: the chromatographic condition in step (5), when liquid chromatography tandem mass spectrometry detects further include: chromatographic column: C18 2.1 ×
100mm, 3 μm；Chromatographic parameter: flow rate of mobile phase 0.4-1.0mL/min, 35-60 DEG C of column temperature, detection time 4-10min, sample volume
10-50μL；Preferably, flow rate of mobile phase 0.5mL/min, 40 DEG C of column temperature；Detection time 7min, 30 μ L of sample volume.
8. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum according to claim 1,
Be characterized in that: in step (5), Mass Spectrometry Conditions when liquid chromatography tandem mass spectrometry detects include:
。
9. 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry is combined detection method in serum according to claim 1,
Be characterized in that: in step (5), Mass Spectrometry Conditions when liquid chromatography tandem mass spectrometry detects further include following source parameters: electricity
It is the source ESI from source, using positive ion mode, other parameters are as follows:
Preferably, other parameters are as follows:
。
10. one kind is for 25-hydroxy-vitamin D liquid chromatography tandem matter in serum as claimed in any one of claims 1 to 9
The kit of spectrum combination detection method, it is characterised in that: including following solution:
(1) calibration object: 25 (OH) D of 5-40ng/mL2With 25 (OH) D of 5-120ng/mL3；
(2) quality-control product: 25 (OH) D of 5.4-23.5ng/mL2With 25 (OH) D of 4.8-64.6ng/mL3；
(3) internal standard mixed liquor: the 25-hydroxy-vitamin D of 200ng/mL2The 25-hydroxy-vitamin D of internal standard and 500ng/mL3It is interior
Mark；
(4) precipitating reagent: the mixed solution of methanol and acetonitrile 1:1 mixing by volume；
(5) extractant: HPLC grades of n-hexanes；
(6) liquid: the methanol aqueous solution of 40~60v/v% is redissolved；Preferably, redissolving liquid is the mixed of methanol and water 1:1 by volume
Close solution；
(7) mobile phase: mobile phase A uses the aqueous formic acid of 0.05~0.3v/v%, and Mobile phase B uses 0.05~0.3v/v%
Formic acid methanol solution；Preferably, mobile phase A uses the aqueous formic acid of 0.1v/v%, and Mobile phase B uses the first of 0.1v/v%
Sour methanol solution；
(8) needle liquid: the methanol aqueous solution of 50~80v/v% is washed；Preferably, the methanol aqueous solution that needle liquid is 50v/v% is washed.
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