CN103308621B - The method of high flux Liquid Chromatography tandem mass spectrography detection 25(OH)VD - Google Patents
The method of high flux Liquid Chromatography tandem mass spectrography detection 25(OH)VD Download PDFInfo
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Abstract
The method embodiments providing a kind of high flux Liquid Chromatography tandem mass spectrography detection 25(OH)VD, the acetonitrile solution added containing 25(OH)VD internal standard substance to human serum sample carries out albumen precipitation；Fully add n-hexane extraction solvent after mixing；It is centrifuged after fully mixing again, then pipettes supernatant and carry out dried, adding double solvents, obtain testing sample；Testing sample high flux liquid chromatography tandem level Four bar mass spectrograph is detected；With 25(OH)VD2And/or 25(OH)VD3Relative retention time and the abundance ratio of quota ion pair of detection be qualitative foundation, adopt Internal standard curve method quantitative.Embodiment of the present invention pre-treatment is simple, and specificity, anti-matrix interference ability are strong；The detection time is short, and flux is high, and detection precision is high, with low cost.<!--1-->
Description
Technical field
The present invention relates to the technical field of vitamin D detection, particularly to a kind of method that high flux Liquid Chromatography tandem mass spectrography detects 25(OH)VD.
Background technology
Vitamin D is steroid derivant, 25(OH)VD is vitamin D product after liver mono-oxygenase 25 hydroxyl oxidize oxydasis, owing to it is compared with other metabolite of vitamin D, concentration in blood is high and cycle period is long, and become the good indicator of vitamin D bulk concentration, it is the evaluation index of body vitamin D nutriture.
And most important member is vitamin D2 and vitamin D3 in vitamin D family member.Vitamin D2 is contained in plant food more, and it is to be irradiated through sunlight by the ergosterol of plant and synthesize.Vitamin D3 can be synthesized by human body skin self, it is possible to passes through food intake.Vitamin D has important physiological role in adjustment human serum calcium, phosphorus level.
It is of equal value that vitamin D2 and D3 are considered as biology for a long time, but nearest report shows that the vitamin D of both forms is likely to be of difference (Armasetc, (2004) J.Clin.Endocrinol.Metab.89:5387-5391) on biological activity and bioavailability.
Traditional 25(OH)VD detection method has radioimmunology, competition protein binding method, high performance liquid chromatography etc., but it is primarily present following problem: the first, owing to 25(OH)VD is mainly combined with bindin of serum DBP in human body, and in human body, there is substantial amounts of high-affinity associated proteins, therefore there is serious matrix interference in detection.And traditional radioimmunology and competition protein binding method, it is impossible to effectively dispel matrix interference；Second, existing detection technique, complex pretreatment, analysis time are long, method poor specificity；3rd, it is impossible to accurate quantitative analysis 25(OH)VD simultaneously2And 25(OH)VD3Content, it is impossible to provide the content of 25(OH)VD accurately；4th, whole detection process time length, flux are low.
Therefore, need the technical problem that those skilled in the art urgently solve exactly: how can provide the detection method of a kind of 25(OH)VD suitable in human serum, pre-treatment is simple, and can effectively dispel matrix interference, it is possible to the 25(OH)VD of accurate quantitative analysis detection simultaneously2And 25(OH)VD3, and whole detection process time is short, flux is high.
Summary of the invention
Embodiment of the present invention technical problem to be solved is to provide the detection method of a kind of 25(OH)VD suitable in human serum, it is possible to effectively removes matrix interference, quickly detects.
In order to solve the problems referred to above, the method that the invention discloses a kind of high flux liquid chromatography tandem mass spectrometry detection 25(OH)VD, including:
(1), the acetonitrile solution added containing 25(OH)VD internal standard substance to human serum sample carries out albumen precipitation；Fully add n-hexane extraction solvent after mixing；Fully centrifugal after mixing, then pipette supernatant and carry out dried, adding double solvents, obtain testing sample；
Wherein, described human serum sample is 1:2:4 with the volume ratio of described acetonitrile solution, n-hexane extraction solution；Described supernatant is 1:2 with the volume ratio of corresponding original solution；The volume ratio of described redissolution liquid and human serum sample is 1:2；
(2), described testing sample liquid chromatography tandem level Four bar mass spectrograph is detected；
Described detection adopts positive ion mode, and scan mode adopts multiple-reaction monitoring ion scan MRM；
Wherein, target quota ion is to including 25(OH)VD2Quota ion pair, and 25(OH)VD3Quota ion pair；The condition of the multiple-reaction monitoring ion scan MRM of target quota ion includes:
25(OH)VD2The matter/lotus ratio of parent ion be 412.7～413.7, the matter/lotus ratio of corresponding daughter ion is 394.8～395.8；
25(OH)VD3The matter/lotus ratio of parent ion be 400.7～401.7, the matter/lotus ratio of corresponding daughter ion is 382.9～383.9；
Wherein, described liquid chromatograph adopts gradient mode eluting, and liquid phase chromatogram condition includes:
Chromatographic column:2.7um2.1mm×50C18；
Chromatographic column column temperature: 55 DEG C；
Sample size: 20ul；
Flow velocity: 0.9ml/min；
Mobile phase: containing the methanol of 0.1% formic acid, and, containing the water of 0.1% formic acid；
25(OH)VD3Retention time be 2.60min, and/or, 25(OH)VD2Retention time be 2.80min；
(3), according to 25(OH)VD2And/or 25(OH)VD3Relative retention time, and, 25(OH)VD2Quota ion to and/or 25(OH)VD3The abundance ratio of quota ion pair, it is judged that 25(OH)VD2And/or 25(OH)VD3Existence；
According to 25(OH)VD2And/or 25(OH)VD3With corresponding internal standard substance peak area ratio on interior mark standard curve, calculate the 25(OH)VD in human serum sample2And/or 25(OH)VD3Content.
It is preferred that described 25(OH)VD internal standard substance includes6D-25 hydroxy-vitamine D2And/or6D-25 hydroxy-vitamine D3。
It is preferred that6D-25 hydroxy-vitamine D2Retention time be 2.61min,6D-25 hydroxy-vitamine D3Retention time be 2.81；
Interior scalar quantity ion pair includes6D-25 hydroxy-vitamine D2Quota ion pair, and/or,6D-25 hydroxy-vitamine D3Quota ion pair；
The condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair includes:
6D-25 hydroxy-vitamine D2The matter of the parent ion of quota ion pair/lotus ratio is 419.4, and the matter/lotus ratio of corresponding daughter ion is 401.3；
6D-25 hydroxy-vitamine D3The matter of the parent ion of quota ion pair/lotus ratio is 407.2, the 389.4 of corresponding daughter ion.
It is preferred that the condition of described multiple-reaction monitoring ion scan MRM also includes:
It is preferred that the condition of described gradient elution includes:
| Time (min) | A Phase Proportion (%) | B Phase Proportion (%) |
| 0 | 30 | 70 |
| 2.3 | 20 | 80 |
| 2.9 | 20 | 80 |
| 3.0 | 5 | 95 |
| 3.5 | 5 | 95 |
| 3.55 | 30 | 70 |
| 5.0 | 30 | 70 |
Wherein, A phase is the water containing 0.1% formic acid, and B phase is the methanol containing 0.1% formic acid.
It is preferred that described high flux Liquid Chromatography-Tandem Mass Spectrometry instrument includes two set high flux liquid chromatographic systems, the liquid phase chromatogram condition of described two set high flux liquid chromatographic systems is the same；First set liquid phase acquisition window is 2.0min-3.0min；Second set liquid phase acquisition window is 2.0min-3.0min.
It is preferred that described mass ions source dates includes:
Ionization source: electron spray ionisation ESI source；
Gas curtain gas CUR:20psi；
Add steam Gs1:45psi；
Auxiliary adds steam Gs2:45psi；
Add hot air temperature: 500 DEG C；
Collision gas: high pure nitrogen, 6psi；
Electron spray pin voltage: 5500V.
It is preferred that described centrifugal condition includes: at room temperature, with the centrifugation 10min of 10000r/min.
It is preferred that described dry condition includes: the nitrogen of logical 55 DEG C～60 DEG C is until drying.
It is preferred that the mixture that described double solvents is first alcohol and water, wherein, the volume ratio of described first alcohol and water is 50:50.
Compared with background technology, the embodiment of the present invention has the advantage that
Embodiment of the present invention application high flux liquid chromatography tandem level Four bar mass spectrograph detects, and compared with background technology, the embodiment of the present invention simultaneously qualitative or detection by quantitative can go out the 25(OH)VD in human serum2And 25(OH)VD3。
The embodiment of the present invention carries out protein precipitation, normal hexane liquid-liquid extraction by acetonitrile, just can detect with liquid chromatography tandem level Four bar mass spectrograph after redissolution, and pre-treatment is simple, and can effectively remove matrix interference, and specificity, anti-matrix interference ability are strong.
The embodiment of the present invention adopts high flux Liquid Chromatography-Tandem Mass Spectrometry instrument to detect, and the detection time is short, and flux is high, and detection precision is high, with low cost.
Accompanying drawing explanation
Fig. 1 is the flow chart of steps of the embodiment of the method for a kind of high flux Liquid Chromatography tandem mass spectrography detection 25(OH)VD of the embodiment of the present invention.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, below in conjunction with detailed description of the invention, the present invention is further detailed explanation.
Referring to Fig. 1, it is shown that the flow chart of steps of the detection method embodiment of a kind of 25(OH)VD of the embodiment of the present invention, specifically may include steps of:
Step 101, pre-treatment；
The acetonitrile solution added containing 25(OH)VD internal standard substance to human serum sample carries out albumen precipitation；Fully add n-hexane extraction solvent after mixing；Fully after mixing, it is centrifuged and then pipettes supernatant and carry out dried, add double solvents, obtain testing sample；
Wherein, described human serum sample is 1:2:4 with the volume ratio of described acetonitrile solution, n-hexane extraction solution；Described supernatant is 1:2 with the volume ratio of corresponding original solution；The volume ratio of described redissolution liquid and human serum sample is 1:2；
555 in a preferred exemplary of the embodiment of the present invention, and 25(OH)VD internal standard substance can include6D-25 hydroxy-vitamine D2And/or6D-25 hydroxy-vitamine D3.Namely can only include6D-25 hydroxy-vitamine D2, it is possible to only include6D-25 hydroxy-vitamine D3, it is also possible to include simultaneously6D-25 hydroxy-vitamine D2With6D-25 hydroxy-vitamine D3。
In implementing, if only detecting 25(OH)VD2(25OHD2), then can only add6D-25 hydroxy-vitamine D2(6D-25OHD2)；If only detecting 25(OH)VD3(25OHD3), then can only add6D-25 hydroxy-vitamine D3(6D-25OHD3)；If to detect 25(OH)VD simultaneously2And 25(OH)VD3, then can add simultaneously6D-25 hydroxy-vitamine D2With6D-25 hydroxy-vitamine D3。
In a preferred exemplary of the embodiment of the present invention, the centrifugal condition of the solution after adding n-hexane be may include that at room temperature, with the centrifugation 10min of 10000r/min.
In a preferred exemplary of the embodiment of the present invention, the condition that the supernatant pipetted is dried may include that the nitrogen of logical 55 DEG C～60 DEG C until drying.
In a preferred exemplary of the embodiment of the present invention, double solvents can be the mixture of methanol and water, and wherein, the volume ratio of described first alcohol and water can be 50:50.
Step 102, detection；
Described testing sample liquid chromatography tandem level Four bar mass spectrograph is detected；
Described detection adopts positive ion mode, and scan mode adopts multiple-reaction monitoring ion scan MRM；
MRM:MultiReactionMonitor, refers to multiple-reaction monitoring ion scan.
Wherein, target quota ion is to including 25(OH)VD2Quota ion pair, and/or, 25(OH)VD3Quota ion pair.
Specifically, to 25(OH)VD2When detecting, adopt 25(OH)VD2Quota ion pair；
To 25(OH)VD3When detecting, adopt 25(OH)VD3Quota ion pair；
Simultaneously to 25(OH)VD2And 25(OH)VD3When detecting, adopt 25(OH)VD2Quota ion to and 25(OH)VD3Quota ion pair.
The condition of the multiple-reaction monitoring ion scan MRM of target quota ion includes:
25(OH)VD2The matter/lotus ratio of parent ion be 412.7～413.7, the matter/lotus ratio of corresponding daughter ion is 394.8～395.8；
25(OH)VD3The matter/lotus ratio of parent ion be 400.7～401.7, the matter/lotus ratio of corresponding daughter ion is 382.9～383.9；
Wherein, described liquid chromatograph adopts gradient mode eluting, and liquid phase chromatogram condition includes:
Chromatographic column:2.7um2.1mm×50C18；
Chromatographic column column temperature: 55 DEG C；
Sample size: 20ul；
Flow velocity: 0.9ml/min；
In the embodiment of the present invention, in gradient mode eluting, mobile phase may include that the methanol containing 0.1% formic acid, and, containing the water of 0.1% formic acid；
Adopt above-mentioned mobile phase, in gradient elution, 25(OH)VD2Retention time be 2.60min；And/or, 25(OH)VD3Retention time be 2.80min；
In a preferred exemplary of the embodiment of the present invention, interior scalar quantity ion pair includes6D-25 hydroxy-vitamine D2Quota ion pair, and/or,6D-25 hydroxy-vitamine D3Quota ion pair；
Concrete, work as detection6D-25 hydroxy-vitamine D2Time, adopt6D-25 hydroxy-vitamine D2Quota ion pair；
Work as detection6D-25 hydroxy-vitamine D3Time, adopt6D-25 hydroxy-vitamine D3Quota ion pair；
Detect when simultaneously6D-25 hydroxy-vitamine D2With6D-25 hydroxy-vitamine D3Time, adopt6D-25 hydroxy-vitamine D2Quota ion to6D-25 hydroxy-vitamine D3Quota ion pair.
The condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair includes:
6D-25 hydroxy-vitamine D2The matter of the parent ion of quota ion pair/lotus ratio is 419.4, and the matter/lotus ratio of corresponding daughter ion is 401.3；
6D-25 hydroxy-vitamine D3The matter of the parent ion of quota ion pair/lotus ratio is 407.2, the 389.4 of corresponding daughter ion；
In a preferred exemplary of the embodiment of the present invention, the condition of multiple-reaction monitoring ion scan MRM also includes:
It is appreciated that above table is not a corresponding a kind of situation, it is possible to be detection 25(OH)VD2, it is also possible to it is detection 25(OH)VD3, it is also possible to it is detect 25(OH)VD simultaneously2And 25(OH)VD3。
In a preferred exemplary of the embodiment of the present invention, gradient elution includes:
A phase is the water containing 0.1% formic acid, and B phase is the methanol containing 0.1% formic acid
| Time (min) | A Phase Proportion (%) | B Phase Proportion (%) |
| 0 | 30 | 70 |
| 2.3 | 20 | 80 |
| 2.9 | 20 | 80 |
| 3.0 | 5 | 95 |
| 3.5 | 5 | 95 |
| 3.55 | 30 | 70 |
| 5.0 | 30 | 70 |
In a preferred exemplary of the embodiment of the present invention, high flux Liquid Chromatography-Tandem Mass Spectrometry instrument can include two set high flux liquid chromatographic systems, and the liquid phase chromatogram condition of described two set high flux liquid chromatographic systems is the same；First set liquid phase acquisition window is 2.0min-3.0min；Second set liquid phase acquisition window is 2.0min-3.0min.
In a preferred exemplary of the embodiment of the present invention, mass ions source dates includes:
Ionization source: electron spray ionisation ESI source；
Gas curtain gas CUR:20psi；
Add steam Gs1:45psi；
Auxiliary adds steam Gs2:45psi；
Add hot air temperature: 500 DEG C；
Collision gas: high pure nitrogen, 6psi；
Electron spray pin voltage: 5500V.
Step 103, qualitatively judges and quantitative Analysis.
According to 25(OH)VD2And/or 25(OH)VD3Relative retention time, and, 25(OH)VD2Quota ion to and/or 25(OH)VD3The abundance ratio of quota ion pair, it is judged that 25(OH)VD2And/or 25(OH)VD3Existence；
Specifically, when detection 25(OH)VD2Time, according to 25(OH)VD2Relative retention time and 25(OH)VD2The abundance ratio of quota ion pair judges 25(OH)VD2Existence；
When detection 25(OH)VD3Time, according to 25(OH)VD3Relative retention time and 25(OH)VD3The abundance ratio of quota ion pair judges 25(OH)VD3Existence；
25(OH)VD is detected when simultaneously2And 25(OH)VD3Time, according to 25(OH)VD2And 25(OH)VD3Relative retention time, and, 25(OH)VD2Quota ion to and 25(OH)VD3The abundance ratio of quota ion pair, it is judged that 25(OH)VD2And 25(OH)VD3Existence.
With Liquid Chromatography-Tandem Mass Spectrometry, sample being qualitatively judged, under same test conditions, in sample, measured target material chromatographic peak retention time is consistent with tie substance chromatographic peak retention time in standard solution；The relative abundance of the detection ion pair selected in sample chromatogram figure less than preset rules scope, then may determine that in sample the target substance that there is correspondence than the deviation with the ion relative abundance ratio of suitable concentration standard solution.
According to 25(OH)VD2And/or 25(OH)VD3With corresponding internal standard substance peak area ratio on interior mark curve, calculate the 25(OH)VD in human serum sample2And/or 25(OH)VD3Content.
Specifically, when detection 25(OH)VD2Time, according to 25(OH)VD2With corresponding internal standard substance peak area ratio on interior mark standard curve, calculate the 25(OH)VD in human serum sample2Content.
When detection 25(OH)VD3Time, according to 25(OH)VD3With corresponding internal standard substance peak area ratio on interior mark standard curve, calculate the 25(OH)VD in human serum sample3Content.
25(OH)VD is detected when simultaneously2And 25(OH)VD3Time, according to 25(OH)VD2And 25(OH)VD3With corresponding internal standard substance peak area ratio on interior mark standard curve, calculate the 25(OH)VD in human serum sample2And 25(OH)VD3Content.
In embodiments of the present invention, with 25(OH)VD2And 25(OH)VD3Content sum as the content of 25(OH)VD.
Based on above-mentioned experiment condition, verifying through test of many times, < 10%, detection time average is 2.5min to embodiment of the present invention precision RSD.
For making those skilled in the art be more fully understood that the present invention, an example presented below illustrate the embodiment of the present invention for 25(OH)VD detection method implement process.
200ul human serum is poured into cleaning centrifuge tube in, add 400ul contain internal standard substance (6D-25 hydroxy-vitamine D3) trifluoroacetic acid aqueous solution, carry out albumen precipitation, after whirlpool mixing 30s, add 800ul normal hexane and carry out liquid-liquid extraction, after whirlpool mixing 30s, in centrifuge 10000r/min centrifugation 10min, after take in the supernatant 700ul centrifuge tube being transferred to cleaning, under 55 DEG C of conditions, nitrogen dries up, finally redissolve with 100ul50% methanol-water, after whirlpool mixing, it is transferred on 96 orifice plates, is loaded in liquid chromatograph automatic sampler.
Sample is loaded into Liquid Chromatography-Tandem Mass Spectrometry instrument analyzer LC-MS/MS by liquid chromatograph automatic sampler automatically.LC-MS/MS adopts the AppliedBiochemistryAPI4000plus Tandem Mass Spectrometry Analysis instrument with electron spray ionisation source (ESI) to be analyzed as detector.Wherein, gas curtain gas CUR is 20psi, and adding steam Gs1 is 45psi, and it is 45psi that auxiliary adds steam Gs2, and adding hot air temperature is 500 DEG C, and collision gas is the high pure nitrogen of 6psi, and electron spray pin voltage is 5500V.Adopting reaction detection ion scan MRM scanning, its condition is referring to table 1.
Table 1. reaction detection ion scan MRM condition
The sample that 20ul purifies in advance is loaded in S1 and S2 high flux liquid chromatographic system by automatic sampler automatically, and liquid phase chromatogram condition S1 and S2 is identical, and liquid phase chromatogram condition is referring to form 2.First set liquid phase acquisition window is 2.0min-3.0min；Second set liquid phase acquisition window is 2.0min-3.0min.Chromatographic column adopts Aglientporoshell1202.7um2.1mm × 50C18 chromatographic column, Mobile phase B is the Chromatographic Pure Methanol containing 0.1% formic acid, and mobile phase A is the ultra-pure water containing 0.1% formic acid.Wherein, chromatographic column column temperature is 55 DEG C, and flow velocity is 0.9ml/min.
Table 2. liquid phase chromatogram condition
| Step | Analysis time (min) | Flow velocity (μ L/min) | Mobile phase A % | Mobile phase B % |
| 1 | 0.00 | 900 | 30.0 | 70.0 |
| 2 | 2.30 | 900 | 20.0 | 80.0 |
| 3 | 2.90 | 900 | 20.0 | 80.0 |
| 4 | 3.00 | 900 | 5.0 | 95.0 |
| 5 | 3.50 | 900 | 5.0 | 95.0 |
| 6 | 3.55 | 900 | 30.0 | 70.0 |
| 7 | 5.00 | 900 | 30.0 | 70.0 |
After sample discharges column outlet with mobile phase, enter mass spectrometer ion source or waste liquid under the effect of the pressure, six-way valve control to enter ionogenic sample channel, and enter switching time.In ion source, fluid sample is vaporized and ionizes as charged molecule, and charged molecule, under voltage and vacuum action, enters Q1, Q2 and Q3, and wherein, Q1 and Q3 is mass filter, only allows according to 25(OH)VD2And 25(OH)VD3Mass-to-charge ratio select parent ion and daughter ion pass through, Q2 is collision cell, parent ion herein with intert-gas atoms collide, produce specific fragment ion.
Mass spectrometric first quadrupole (Q1) selects have 25(OH)VD2, 25(OH)VD3With6D-25 hydroxy-vitamine D3The ion of the specific mass-to-charge ratio m/z of (interior mark), has the ion of these m/z ratios and is allowed to enter the fragment ion that Q2, Q2 produce and enters into Q3, wherein only have 25(OH)VD2, 25(OH)VD3It is chosen with interior target fragment ion and passes through, and other ion is removed.Referring to table 3, it is shown that be used to differentiate and quantitative 25(OH)VD.
The mass shift table of table 3.25 hydroxy-vitamine D
| Analyte | Q1 parent ion (m/z) | Q3 daughter ion (m/z) |
| 25OHD3 | 401.2 | 383.4 |
| 25OHD2 | 413.2 | 395.3 |
| 6d-25OHD3 | 407.2 | 389.4 |
Along with ion and detector collision, the number of ions captured is changed into the electronic impulse of digital signal by them.The data obtained are passed to computer, and collected number of ions is plotted against time by it, obtain quality chromatography spectrogram.
In serum standard, the ratio of analyte and interior target peak area is used to build interior mark standard curve, and then this curve is used to calculate analyte concentration in sample or quality control substance.
Certain authority inspection center, adopts the present embodiment method to carry out the human serum 25OHD detection of 1000 examples, and testing result shows that between the method S1 and S2 system, result there was no significant difference, and detection Quality Control material result is accurately, < 10%, detection efficiency is high for precision RSD.Illustrating that this method is accurately and reliably, efficiency is high.
The method of a kind of high flux Liquid Chromatography tandem mass spectrography detection the 25(OH)VD above embodiment of the present invention provided, it is described in detail, principle and the embodiment of the embodiment of the present invention are set forth by specific case used herein, and the explanation of above example is only intended to help to understand method and the core concept thereof of the embodiment of the present invention；Simultaneously for one of ordinary skill in the art, according to the thought of the embodiment of the present invention, all will change in specific embodiments and applications, in sum, this specification content should not be construed as limitation of the present invention.
Claims (8)
1. the method for a high flux liquid chromatography tandem mass spectrometry detection 25(OH)VD, it is characterised in that including:
(1), the acetonitrile solution added containing 25(OH)VD internal standard substance to human serum sample carries out albumen precipitation；Fully add n-hexane extraction solvent after mixing；Fully centrifugal after mixing, then pipette supernatant and carry out dried, adding double solvents, obtain testing sample；
Wherein, described human serum sample is 1:2:4 with the volume ratio of described acetonitrile solution, n-hexane extraction solution；Described supernatant is 1:2 with the volume ratio of corresponding original solution；The volume ratio of described redissolution liquid and human serum sample is 1:2；
(2), described testing sample liquid chromatography tandem level Four bar mass spectrograph is detected；
Described detection adopts positive ion mode, and scan mode adopts multiple-reaction monitoring ion scan MRM；
Wherein, target quota ion is to including 25(OH)VD2Quota ion pair, and 25(OH)VD3Quota ion pair；The condition of the multiple-reaction monitoring ion scan MRM of target quota ion includes:
25(OH)VD2The matter/lotus ratio of parent ion be 412.7～413.7, the matter/lotus ratio of corresponding daughter ion is 394.8～395.8；
25(OH)VD3The matter/lotus ratio of parent ion be 400.7～401.7, the matter/lotus ratio of corresponding daughter ion is 382.9～383.9；
Wherein, described liquid chromatograph adopts gradient mode eluting, and liquid phase chromatogram condition includes:
Chromatographic column:2.7μm2.1mm×50C18；
Chromatographic column column temperature: 55 DEG C；
Sample size: 20 μ 1；
Flow velocity: 0.9m1/min；
Mobile phase: containing the methanol of 0.1% formic acid, and, containing the water of 0.1% formic acid；
25(OH)VD3Retention time be 2.60min, and/or, 25(OH)VD2Retention time be 2.80min；
(3), according to 25(OH)VD2And/or 25(OH)VD3Relative retention time, and, 25(OH)VD2Quota ion to and/or 25(OH)VD3The abundance ratio of quota ion pair, it is judged that 25(OH)VD2And/or 25(OH)VD3Existence；
According to 25(OH)VD2And/or 25(OH)VD3With corresponding internal standard substance peak area ratio on interior mark standard curve, calculate the 25(OH)VD in human serum sample2And/or 25(OH)VD3Content；
Wherein, described 25(OH)VD internal standard substance includes6D-25 hydroxy-vitamine D2And/or6D-25 hydroxy-vitamine D3；
The condition of described gradient elution includes:
Wherein, A phase is the water containing 0.1% formic acid, and B phase is the methanol containing 0.1% formic acid.
2. method according to claim 1, it is characterised in that 6d-25 hydroxy-vitamine D2Retention time be 2.61min,6D-25 hydroxy-vitamine D3Retention time be 2.81；
Interior scalar quantity ion pair includes6D-25 hydroxy-vitamine D2Quota ion pair, and/or,6D-25 hydroxy-vitamine D3Quota ion pair；
The condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair includes:
6D-25 hydroxy-vitamine D2The matter of the parent ion of quota ion pair/lotus ratio is 419.4, and the matter/lotus ratio of corresponding daughter ion is 401.3；
6D-25 hydroxy-vitamine D3The matter of the parent ion of quota ion pair/lotus ratio is 407.2, the 389.4 of corresponding daughter ion.
3. method according to claim 1 and 2, it is characterised in that the condition of described multiple-reaction monitoring ion scan MRM also includes:
4. method according to claim 1, it is characterized in that, described high flux Liquid Chromatography-Tandem Mass Spectrometry instrument includes two set high flux liquid chromatographic systems, and the liquid phase chromatogram condition of described two set high flux liquid chromatographic systems is the same: first set liquid phase acquisition window is 2.0min-3.0min；Second set liquid phase acquisition window is 2.0min-3.0min.
5. method according to claim 1, it is characterised in that described mass ions source dates includes:
Ionization source: electron spray ionisation ESI source；
Gas curtain gas CUR:20psi；
Add steam Gs1:45psi:
Auxiliary adds steam Gs2:45psi；
Add hot air temperature: 500 DEG C；
Collision gas: high pure nitrogen, 6psi；
Electron spray pin voltage: 5500V.
6. method according to claim 1, it is characterised in that described centrifugal condition includes: at room temperature, with the centrifugation 10min of 10000r/min.
7. method according to claim 1, it is characterised in that described dry condition includes: the nitrogen of logical 55 DEG C～60 DEG C is until drying.
8. method according to claim 1, it is characterised in that described double solvents is the mixture of first alcohol and water, wherein, the volume ratio of described first alcohol and water is 50:50.
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| CN105527364A (en) * | 2015-08-26 | 2016-04-27 | 袁洪 | Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry |
| CN105158394B (en) * | 2015-09-14 | 2016-04-27 | 济南英盛生物技术有限公司 | A kind of method simultaneously detecting multiple liposoluble vitamin in blood sample |
| CN105651901A (en) * | 2016-04-11 | 2016-06-08 | 北京洛奇临床检验所股份有限公司 | Liquid chromatogram tandem mass spectrum detection method for 25-hydroxy vitamin D in dry blood sample and kit |
| CN105911160A (en) * | 2016-04-11 | 2016-08-31 | 北京洛奇临床检验所股份有限公司 | Liquid chromatography-tandem mass spectrometry detection method of 25-hydroxyvitamin D in serum or blood plasma, and kit thereof |
| CN107621499A (en) * | 2016-07-14 | 2018-01-23 | 上海可力梅塔生物医药科技有限公司 | 25 hydroxy vitamin D2s and 25 hydroxycholecalciferol high performance liquid chromatography MS detection kits |
| CN106526026B (en) * | 2016-11-10 | 2019-01-08 | 博厚健康科技股份有限公司 | The detection method of 25-hydroxy-vitamin D in serum |
| CN108344821A (en) * | 2017-01-25 | 2018-07-31 | 上海可力梅塔生物医药科技有限公司 | The liquid phase Tandem Mass Spectrometry Analysis method of vitamin A in biological sample |
| CN108362759A (en) * | 2017-05-10 | 2018-08-03 | 中国科学院过程工程研究所 | A kind of oxygen-containing acid group monitoring system |
| CN107478761A (en) * | 2017-09-21 | 2017-12-15 | 岛津企业管理（中国）有限公司 | 25 hydroxy-vitamine D in serum2/D3The parallel liquid chromatography mass combination detection method of content of dual flow path |
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| CN109212091A (en) * | 2018-10-24 | 2019-01-15 | 天津国科医工科技发展有限公司 | 25-hydroxy-vitamin D Liquid Chromatography-Tandem Mass Spectrometry combination detection method and kit in serum |
| CN109212090A (en) * | 2018-10-24 | 2019-01-15 | 天津国科医工科技发展有限公司 | A kind of method and kit based on 25-hydroxy-vitamin D in liquid chromatography tandem mass spectrometry detection serum |
| CN109781916A (en) * | 2018-11-05 | 2019-05-21 | 苏州帕诺米克生物医药科技有限公司 | Target the quantitative automatic analysis method of metabolism group and device, electronic equipment |
| CN109900821A (en) * | 2019-02-27 | 2019-06-18 | 武汉康圣达医学检验所有限公司 | Method and kit a kind of while that detect a variety of liposoluble vitamins in blood |
| CN110441537B (en) * | 2019-08-23 | 2022-09-13 | 北京丹大生物技术有限公司 | Method for measuring content of 25-hydroxy vitamin D |
| CN110487943B (en) * | 2019-08-28 | 2021-01-15 | 杭州佰辰医学检验所有限公司 | Method for detecting fat-soluble vitamins in blood sample |
| CN110726799A (en) * | 2019-12-02 | 2020-01-24 | 成都和合医学检验所有限公司 | Method for simultaneously detecting 25 hydroxy-vitamin D3 and 25 hydroxy-vitamin D2 contents in blood |
| CN111198237A (en) * | 2020-01-15 | 2020-05-26 | 苏州康吉诊断试剂有限公司 | Extraction method and detection method of vitamin D metabolite-25 (OH) D3 in serum |
| CN114441674A (en) * | 2022-01-17 | 2022-05-06 | 天津国科医工科技发展有限公司 | Trace detection method and kit for vitamin D |
| CN117630233A (en) * | 2024-01-26 | 2024-03-01 | 北京豪思生物科技股份有限公司 | Method for detecting 25-hydroxy vitamin D in whole blood |
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