CN103308621A - Method for detecting 25(hydroxyl)vitamin D by using high-pass liquid chromatography-tandem mass spectrometry - Google Patents
Method for detecting 25(hydroxyl)vitamin D by using high-pass liquid chromatography-tandem mass spectrometry Download PDFInfo
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Abstract
The embodiment of the invention provides a method for detecting 25(hydroxyl)vitamin D by using a high-pass liquid chromatography-tandem mass spectrometry. The method comprises the following steps of: adding an acetonitrile solution containing an internal standard substance of the 25(hydroxyl)vitamin D into a human serum sample to carry out protein precipitation; sufficiently and uniformly mixing the solution, and then, adding an n-hexane extracting solvent; sufficiently and uniformly mixing the solution, then centrifuging the solution, movably taking a supernatant and drying the supernatant, and adding a complex solution to obtain a sample to be detected; detecting the sample to be detected by using a high-pass liquid chromatography-tandem quadrupole mass spectrometer; and quantifying according to the relative retention time of 25(hydroxyl)vitamin D2 and/or 25(hydroxyl)vitamin D3 and the detected abundance ratio of quantitative ion pairs by using an internal standard curve method. According to the embodiment of the invention, the method has the advantages of simplicity in pretreatment, strong specificity and matrix interference resistance, short detection time, high pass, high detection precision and low cost.
Description
Technical field
The present invention relates to the technical field that vitamin D detects, particularly a kind of high flux liquid phase chromatography tandem mass spectrometry detects the method for 25(OH)VD.
Background technology
Vitamin D is the steroid derivant, 25(OH)VD is the product of vitamin D behind liver MO-25 hydroxyl oxidize oxydasis, because it is compared with other metabolic product of vitamin D, concentration in blood is high and cycle period is long, and become the good indicator of vitamin D bulk concentration, be the evaluation index of body vitamin D nutrition condition.
And most important member is calciferol and cholecalciferol among the vitamin D family member.Calciferol is contained in the plant food more, and it is that ergosterol by plant is through solar radiation and synthetic.Cholecalciferol can be synthetic by human body skin self, also can be by food intake.Vitamin D has important physiological role in adjustment human serum calcium, phosphorus level.
Calciferol and D3 are considered to the biology equivalence for a long time, yet nearest report shows the vitamin D of these two kinds of forms and may have difference (Armas etc, (2004) J.Clin.Endocrinol.Metab.89:5387-5391) on biologically active and bioavailability.
Traditional 25(OH)VD detection method has radioimmunology, competition protein combination method, high performance liquid chromatography etc., but mainly there are the following problems: the first, mainly be combined with bindin of serum DBP in human body owing to 25(OH)VD, and exist a large amount of high-affinities in conjunction with albumen in the human body, therefore there is serious matrix interference in detection.And traditional radioimmunology and competition protein combination method can not effectively be dispelled matrix interference; The second, existing detection technique, complex pretreatment, analysis time are long, the method poor specificity; The 3rd, can not while accurate quantitative analysis 25(OH)VD
2And 25(OH)VD
3Content, can't provide the accurately content of 25(OH)VD; The 4th, the whole testing process time is long, flux is low.
Therefore, need the urgent technical matters that solves of those skilled in the art to be exactly: how a kind of detection method that is applicable to the 25(OH)VD of human serum can be provided, pre-treatment is simple, and can effectively dispel matrix interference, and accurate quantitative analysis detects 25(OH)VD simultaneously
2And 25(OH)VD
3, and the whole testing process time is short, flux is high.
Summary of the invention
Embodiment of the invention technical matters to be solved provides a kind of detection method that is applicable to the 25(OH)VD of human serum, can effectively remove matrix interference, fast detecting.
In order to address the above problem, the invention discloses the method that a kind of high flux Liquid Chromatography-Tandem Mass Spectrometry method detects 25(OH)VD, comprising:
(1), adds the acetonitrile solution that contains the 25(OH)VD internal standard compound to the human serum sample and carry out albumen precipitation; Fully add the n-hexane extraction solvent behind the mixing; Fully centrifugal behind the mixing, after then pipetting supernatant and carrying out drying, add double solvents, obtain testing sample;
Wherein, the volume ratio of described human serum sample and described acetonitrile solution, n-hexane extraction solution is 1:2:4; Described supernatant is 1:2 with the volume ratio of corresponding original solution; Described redissolution liquid and human serum sample's volume ratio is 1:2;
(2), described testing sample is detected with liquid chromatography series connection level Four bar mass spectrometer;
Positive ion mode is adopted in described detection, and scan mode adopts multiple-reaction monitoring ion scan MRM;
Wherein, the target quota ion is to comprising 25(OH)VD
2Quota ion pair, and 25(OH)VD
3Quota ion pair; The condition of the multiple-reaction monitoring ion scan MRM of target quota ion comprises:
25(OH)VD
2Parent ion matter/the lotus ratio is 412.7～413.7, corresponding daughter ion matter/the lotus ratio is 394.8～395.8;
25(OH)VD
3Parent ion matter/the lotus ratio is 400.7～401.7, corresponding daughter ion matter/the lotus ratio is 382.9～383.9;
Wherein, described liquid chromatography adopts the gradient mode wash-out, and liquid phase chromatogram condition comprises:
Chromatographic column:
2.7um2.1mm * 50C18;
Chromatographic column column temperature: 55 ℃;
Sample size: 20ul;
Flow velocity: 0.9ml/min;
Mobile phase: contain the methyl alcohol of 0.1% formic acid, and, contain the water of 0.1% formic acid;
25(OH)VD
3Retention time be 2.60min, and/or, 25(OH)VD
2Retention time be 2.80min;
(3), according to 25(OH)VD
2And/or 25(OH)VD
3Relative retention time, and, 25(OH)VD
2Quota ion to and/or 25(OH)VD
3The abundance ratio that quota ion is right is judged 25(OH)VD
2And/or 25(OH)VD
3Existence;
According to 25(OH)VD
2And/or 25(OH)VD
3With the peak area ratio of corresponding internal standard compound on interior mark typical curve, calculate the 25(OH)VD among the human serum sample
2And/or 25(OH)VD
3Content.
Preferably, described 25(OH)VD internal standard compound comprises
6The d-25 hydroxy-vitamine D
2And/or
6The d-25 hydroxy-vitamine D
3
Preferably,
6The d-25 hydroxy-vitamine D
2Retention time be 2.61min,
6The d-25 hydroxy-vitamine D
3Retention time be 2.81;
Interior scalar quantity ion pair comprises
6The d-25 hydroxy-vitamine D
2Quota ion pair, and/or,
6The d-25 hydroxy-vitamine D
3Quota ion pair;
The condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair comprises:
6The d-25 hydroxy-vitamine D
2The matter of the parent ion that quota ion is right/lotus ratio is 419.4, corresponding daughter ion matter/the lotus ratio is 401.3;
6The d-25 hydroxy-vitamine D
3The matter of the parent ion that quota ion is right/lotus ratio is 407.2,389.4 of corresponding daughter ion.
Preferably, the condition of described multiple-reaction monitoring ion scan MRM also comprises:
Preferably, the condition of described gradient elution comprises:
| Time (min) | A phase ratio (%) | B phase ratio (%) |
| 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, for containing the water of 0.1% formic acid, B is mutually for containing the methyl alcohol of 0.1% formic acid mutually for A.
Preferably, described high flux Liquid Chromatography-Tandem Mass Spectrometry instrument comprises two cover high flux liquid chromatographic systems, and the liquid phase chromatogram condition of described two cover high flux liquid chromatographic systems is the same; First set liquid phase acquisition window is 2.0min-3.0min; The second cover liquid phase acquisition window is 2.0min-3.0min.
Preferably, described mass ion source parameter comprises:
Ionization source: electron spray ionisation ESI source;
Gas curtain gas CUR:20psi;
Heat air Gs1:45psi;
Auxiliary heating gas Gs2:45psi;
Heat air temperature: 500 ℃;
Collision gas: high pure nitrogen, 6psi;
Electron spray pin voltage: 5500V.
Preferably, described centrifugal condition comprises: at room temperature, and with the centrifugal 10min of the speed of 10000r/min.
Preferably, the condition of described drying comprises: logical 55 ℃～60 ℃ nitrogen is until drying.
Preferably, described double solvents is the potpourri of first alcohol and water, and wherein, the volume ratio of described first alcohol and water is 50:50.
Compare with background technology, the embodiment of the invention has the following advantages:
The embodiment of the invention is used high flux liquid chromatography series connection level Four bar mass spectrometer and is detected, and compares with background technology, and the embodiment of the invention is can be simultaneously qualitative or quantitatively detect 25(OH)VD in the human serum
2And 25(OH)VD
3
The embodiment of the invention is carried out protein precipitation, normal hexane liquid-liquid extraction by acetonitrile, just can detect with the liquid chromatography level Four bar mass spectrometer of connecting after the redissolution, pre-treatment is simple, and can effectively remove matrix interference, and specificity, anti-matrix interference ability are strong.
The embodiment of the invention adopts high flux Liquid Chromatography-Tandem Mass Spectrometry instrument to detect, and detection time is short, and flux is high, detects precision high, with low cost.
Description of drawings
Fig. 1 is the flow chart of steps that a kind of high flux liquid phase chromatography tandem mass spectrometry of the embodiment of the invention detects the embodiment of the method for 25(OH)VD.
Embodiment
For above-mentioned purpose of the present invention, feature and advantage can be become apparent more, the present invention is further detailed explanation below in conjunction with embodiment.
Referring to Fig. 1, show the flow chart of steps of detection method embodiment of a kind of 25(OH)VD of the embodiment of the invention, specifically can comprise the steps:
Add the acetonitrile solution that contains the 25(OH)VD internal standard compound to the human serum sample and carry out albumen precipitation; Fully add the n-hexane extraction solvent behind the mixing; Fully behind the mixing, centrifugal then pipette supernatant and carry out drying after, add double solvents, obtain testing sample;
Wherein, the volume ratio of described human serum sample and described acetonitrile solution, n-hexane extraction solution is 1:2:4; Described supernatant is 1:2 with the volume ratio of corresponding original solution; Described redissolution liquid and human serum sample's volume ratio is 1:2;
555 in a preferred exemplary of the embodiment of the invention, and the 25(OH)VD internal standard compound can comprise
6The d-25 hydroxy-vitamine D
2And/or
6The d-25 hydroxy-vitamine D
3Namely can include only
6The d-25 hydroxy-vitamine D
2, can include only
6The d-25 hydroxy-vitamine D
3, also can comprise simultaneously
6The d-25 hydroxy-vitamine D
2With
6The d-25 hydroxy-vitamine D
3
In specific implementation, if only detect 25(OH)VD
2(25OHD2), then can only add
6The d-25 hydroxy-vitamine D
2(
6D-25OHD2); If only detect 25(OH)VD
3(25OHD3), then can only add
6The d-25 hydroxy-vitamine D
3(
6D-25OHD3); If detect simultaneously 25(OH)VD
2And 25(OH)VD
3, then can add simultaneously
6The d-25 hydroxy-vitamine D
2With
6The d-25 hydroxy-vitamine D
3
In a preferred exemplary of the embodiment of the invention, can comprise the centrifugal condition that adds the solution behind the normal hexane: at room temperature, with the centrifugal 10min of the speed of 10000r/min.
In a preferred exemplary of the embodiment of the invention, can comprise the condition of the supernatant drying that pipettes: logical 55 ℃～60 ℃ nitrogen is until drying.
In a preferred exemplary of the embodiment of the invention, double solvents can be the potpourri of methyl alcohol and water, and wherein, the volume ratio of described first alcohol and water can be 50:50.
Step 102 detects;
Described testing sample is detected with liquid chromatography series connection level Four bar mass spectrometer;
Positive ion mode is adopted in described detection, and scan mode adopts multiple-reaction monitoring ion scan MRM;
MRM:Multi Reaction Monitor refers to the multiple-reaction monitoring ion scan.
Wherein, the target quota ion is to comprising 25(OH)VD
2Quota ion pair, and/or, 25(OH)VD
3Quota ion pair.
Particularly, to 25(OH)VD
2When detecting, adopt 25(OH)VD
2Quota ion pair;
To 25(OH)VD
3When detecting, adopt 25(OH)VD
3Quota ion pair;
Simultaneously to 25(OH)VD
2And 25(OH)VD
3When detecting, adopt 25(OH)VD
2Quota ion to and 25(OH)VD
3Quota ion pair.
The condition of the multiple-reaction monitoring ion scan MRM of target quota ion comprises:
25(OH)VD
2Parent ion matter/the lotus ratio is 412.7～413.7, corresponding daughter ion matter/the lotus ratio is 394.8～395.8;
25(OH)VD
3Parent ion matter/the lotus ratio is 400.7～401.7, corresponding daughter ion matter/the lotus ratio is 382.9～383.9;
Wherein, described liquid chromatography adopts the gradient mode wash-out, and liquid phase chromatogram condition comprises:
Chromatographic column:
2.7um2.1mm * 50C18;
Chromatographic column column temperature: 55 ℃;
Sample size: 20ul;
Flow velocity: 0.9ml/min;
In the embodiment of the invention, mobile phase can comprise in the gradient mode wash-out: contain the methyl alcohol of 0.1% formic acid, and, contain the water of 0.1% formic acid;
Adopt above-mentioned mobile phase, in gradient elution, 25(OH)VD
2Retention time be 2.60min; And/or, 25(OH)VD
3Retention time be 2.80min;
In a preferred exemplary of the embodiment of the invention, interior scalar quantity ion pair comprises
6The d-25 hydroxy-vitamine D
2Quota ion pair, and/or,
6The d-25 hydroxy-vitamine D
3Quota ion pair;
Concrete, work as detection
6The d-25 hydroxy-vitamine D
2The time, adopt
6The d-25 hydroxy-vitamine D
2Quota ion pair;
Work as detection
6The d-25 hydroxy-vitamine D
3The time, adopt
6The d-25 hydroxy-vitamine D
3Quota ion pair;
When detecting simultaneously
6The d-25 hydroxy-vitamine D
2With
6The d-25 hydroxy-vitamine D
3The time, adopt
6The d-25 hydroxy-vitamine D
2Quota ion to
6The d-25 hydroxy-vitamine D
3Quota ion pair.
The condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair comprises:
6The d-25 hydroxy-vitamine D
2The matter of the parent ion that quota ion is right/lotus ratio is 419.4, corresponding daughter ion matter/the lotus ratio is 401.3;
6The d-25 hydroxy-vitamine D
3The matter of the parent ion that quota ion is right/lotus ratio is 407.2,389.4 of corresponding daughter ion;
In a preferred exemplary of the embodiment of the invention, the condition of multiple-reaction monitoring ion scan MRM also comprises:
Be appreciated that above table is not corresponding a kind of situation only, can be to detect 25(OH)VD
2, also can be to detect 25(OH)VD
3, can also be to detect simultaneously 25(OH)VD
2And 25(OH)VD
3
In a preferred exemplary of the embodiment of the invention, gradient elution comprises:
For containing the water of 0.1% formic acid, B is mutually for containing the methyl alcohol of 0.1% formic acid mutually for A
| Time (min) | A phase ratio (%) | B phase ratio (%) |
| 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 invention, high flux Liquid Chromatography-Tandem Mass Spectrometry instrument can comprise two cover high flux liquid chromatographic systems, and the liquid phase chromatogram condition of described two cover high flux liquid chromatographic systems is the same; First set liquid phase acquisition window is 2.0min-3.0min; The second cover liquid phase acquisition window is 2.0min-3.0min.
In a preferred exemplary of the embodiment of the invention, the mass ion source parameter comprises:
Ionization source: electron spray ionisation ESI source;
Gas curtain gas CUR:20psi;
Heat air Gs1:45psi;
Auxiliary heating gas Gs2:45psi;
Heat air temperature: 500 ℃;
Collision gas: high pure nitrogen, 6psi;
Electron spray pin voltage: 5500V.
Step 103 qualitatively judges and quantitatively calculates.
According to 25(OH)VD
2And/or 25(OH)VD
3Relative retention time, and, 25(OH)VD
2Quota ion to and/or 25(OH)VD
3The abundance ratio that quota ion is right is judged 25(OH)VD
2And/or 25(OH)VD
3Existence;
Particularly, when detecting 25(OH)VD
2The time, according to 25(OH)VD
2Relative retention time and 25(OH)VD
2The right abundance ratio of quota ion is judged 25(OH)VD
2Existence;
When detecting 25(OH)VD
3The time, according to 25(OH)VD
3Relative retention time and 25(OH)VD
3The right abundance ratio of quota ion is judged 25(OH)VD
3Existence;
Detect 25(OH)VD when simultaneously
2And 25(OH)VD
3The time, according to 25(OH)VD
2And 25(OH)VD
3Relative retention time, and, 25(OH)VD
2Quota ion to and 25(OH)VD
3The abundance ratio that quota ion is right is judged 25(OH)VD
2And 25(OH)VD
3Existence.
With Liquid Chromatography-Tandem Mass Spectrometry sample is qualitatively judged, under same test conditions, measured target material chromatographic peak retention time is consistent with tie substance chromatographic peak retention time in the standard solution in the sample; Then can there be corresponding target substance in the relative abundance of selected detection ion pair in the judgement sample than with the Ion Phase of suitable concentration standard solution the deviation of abundance ratio being no more than default specialized range among the sample chromatogram figure.
According to 25(OH)VD
2And/or 25(OH)VD
3With the peak area ratio of corresponding internal standard compound on interior mark curve, calculate the 25(OH)VD among the human serum sample
2And/or 25(OH)VD
3Content.
Particularly, when detecting 25(OH)VD
2The time, according to 25(OH)VD
2With the peak area ratio of corresponding internal standard compound on interior mark typical curve, calculate the 25(OH)VD among the human serum sample
2Content.
When detecting 25(OH)VD
3The time, according to 25(OH)VD
3With the peak area ratio of corresponding internal standard compound on interior mark typical curve, calculate the 25(OH)VD among the human serum sample
3Content.
Detect 25(OH)VD when simultaneously
2And 25(OH)VD
3The time, according to 25(OH)VD
2And 25(OH)VD
3With the peak area ratio of corresponding internal standard compound on interior mark typical curve, calculate the 25(OH)VD among the human serum sample
2And 25(OH)VD
3Content.
In embodiments of the present invention, with 25(OH)VD
2And 25(OH)VD
3The content sum as the content of 25(OH)VD.
Based on above-mentioned experiment condition, through test of many times checking, embodiment of the invention precision RSD＜10%, detection time average out to 2.5min.
For making those skilled in the art understand better the present invention, below provide an example to illustrate that the embodiment of the invention is used for the specific implementation process of the detection method of 25(OH)VD.
The 200ul human serum is poured in the clean centrifuge tube, add 400ul contain internal standard compound (
6The d-25 hydroxy-vitamine D
3) trifluoroacetic acid aqueous solution, carry out albumen precipitation, behind the whirlpool mixing 30s, add the 800ul normal hexane and carry out liquid-liquid extraction, behind the whirlpool mixing 30s, in hydro-extractor 10000r/min centrifuging 10min, after get supernatant 700ul and be transferred in the clean centrifuge tube, nitrogen dries up under 55 ℃ of conditions, redissolve with the 100ul50% methanol-water at last, behind the whirlpool mixing, be transferred on 96 orifice plates, be loaded in the liquid chromatography automatic sampler.
The liquid chromatography automatic sampler is loaded into sample Liquid Chromatography-Tandem Mass Spectrometry instrument analyzer LC-MS/MS automatically.LC-MS/MS adopts the Applied Biochemistry API4000plus Tandem Mass Spectrometry Analysis instrument with electron spray ionisation source (ESI) to analyze as detecting device.Wherein, gas curtain gas CUR is 20psi, and heat air Gs1 is 45psi, and auxiliary heating gas Gs2 is 45psi, and the heat air temperature is 500 ℃, and collision gas is the high pure nitrogen of 6psi, and electron spray pin voltage is 5500V.Adopt reaction detection ion scan MRM scanning, its condition is referring to table 1.
Table 1. reaction detection ion scan MRM condition
The sample that automatic sampler purifies 20ul automatically in advance is loaded in S1 and the S2 high flux liquid chromatographic system, and liquid phase chromatogram condition S1 is identical with S2, and liquid phase chromatogram condition is referring to form 2.First set liquid phase acquisition window is 2.0min-3.0min; The second cover liquid phase acquisition window is 2.0min-3.0min.Chromatographic column adopting Aglient poroshell120
2.7um2.1mm * 50C18 chromatographic column, Mobile phase B are the Chromatographic Pure Methanol that contains 0.1% formic acid, mobile phase A is the ultrapure water that contains 0.1% formic acid.Wherein, the chromatographic column column temperature is 55 ℃, 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 |
Sample enters mass spectrometer ion source or waste liquid after discharging the chromatographic column outlet with mobile phase under the effect of pressure, enter ionogenic sample channel by six-way valve control, and enter switching time.Fluid sample is vaporized and ionizes and is charged molecule in ion gun, and charged molecule enters Q1, Q2 and Q3 under voltage and vacuum action, and wherein, Q1 and Q3 are mass filter, only allows according to 25(OH)VD
2And 25(OH)VD
3Mass-to-charge ratio parent ion and the daughter ion selected pass through, Q2 is collision cell, parent ion herein with the intert-gas atoms collision, produce specific fragmention.
Mass spectrometric first four utmost point (Q1) selects to have 25(OH)VD
2, 25(OH)VD
3With
6The d-25 hydroxy-vitamine D
3The ion of the specific mass-to-charge ratio m/z of (interior mark), the ion with these m/z ratios is allowed to enter Q2, and the fragmention that Q2 produces enters into Q3, wherein only has 25(OH)VD
2, 25(OH)VD
3Pass through with interior target fragmention is selected, and other ion is removed.Referring to table 3, show the 25(OH)VD that is used to Identification and determination.
The quality indicator 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 detecting device collision, they change into the number of ions that captures the electronic impulse of digital signal.The data that obtain are passed to computing machine, and it is mapped collected number of ions to the time, namely get quality chromatography spectrogram.
The ratio of analyte and interior target peak area is used to make up interior mark typical curve in the serum reference material, and then this curve is used to analyte concentration in calculation sample or the quality control substance.
Certain authoritative inspection center, the human serum 25OHD that adopts the present embodiment method to carry out 1000 examples detects, and testing result shows between the method S1 and S2 system as a result there was no significant difference, detects that Quality Control material result is accurate, precision RSD＜10%, and detection efficiency is high.This method is described accurately and reliably, efficient is high.
Above a kind of high flux liquid phase chromatography tandem mass spectrometry that the embodiment of the invention is provided detects the method for 25(OH)VD, be described in detail, used specific case herein principle and the embodiment of the embodiment of the invention are set forth, the explanation of above embodiment just is used for helping to understand method and the core concept thereof of the embodiment of the invention; Simultaneously, for one of ordinary skill in the art, the thought according to the embodiment of the invention all will change in specific embodiments and applications, and in sum, this description should not be construed as limitation of the present invention.
Claims (10)
1. the method for a high flux Liquid Chromatography-Tandem Mass Spectrometry method detection 25(OH)VD is characterized in that, comprising:
(1), adds the acetonitrile solution that contains the 25(OH)VD internal standard compound to the human serum sample and carry out albumen precipitation; Fully add the n-hexane extraction solvent behind the mixing; Fully centrifugal behind the mixing, after then pipetting supernatant and carrying out drying, add double solvents, obtain testing sample;
Wherein, the volume ratio of described human serum sample and described acetonitrile solution, n-hexane extraction solution is 1:2:4; Described supernatant is 1:2 with the volume ratio of corresponding original solution; Described redissolution liquid and human serum sample's volume ratio is 1:2;
(2), described testing sample is detected with liquid chromatography series connection level Four bar mass spectrometer;
Positive ion mode is adopted in described detection, and scan mode adopts multiple-reaction monitoring ion scan MRM;
Wherein, the target quota ion is to comprising 25(OH)VD
2Quota ion pair, and 25(OH)VD
3Quota ion pair; The condition of the multiple-reaction monitoring ion scan MRM of target quota ion comprises:
25(OH)VD
2Parent ion matter/the lotus ratio is 412.7～413.7, corresponding daughter ion matter/the lotus ratio is 394.8～395.8;
25(OH)VD
3Parent ion matter/the lotus ratio is 400.7～401.7, corresponding daughter ion matter/the lotus ratio is 382.9～383.9;
Wherein, described liquid chromatography adopts the gradient mode wash-out, and liquid phase chromatogram condition comprises:
Chromatographic column:
2.7um2.1mm * 50C18;
Chromatographic column column temperature: 55 ℃;
Sample size: 20ul;
Flow velocity: 0.9ml/min;
Mobile phase: contain the methyl alcohol of 0.1% formic acid, and, contain the water of 0.1% formic acid;
25(OH)VD
3Retention time be 2.60min, and/or, 25(OH)VD
2Retention time be 2.80min;
(3), according to 25(OH)VD
2And/or 25(OH)VD
3Relative retention time, and, 25(OH)VD
2Quota ion to and/or 25(OH)VD
3The abundance ratio that quota ion is right is judged 25(OH)VD
2And/or 25(OH)VD
3Existence;
According to 25(OH)VD
2And/or 25(OH)VD
3With the peak area ratio of corresponding internal standard compound on interior mark typical curve, calculate the 25(OH)VD among the human serum sample
2And/or 25(OH)VD
3Content.
2. method according to claim 1 is characterized in that, described 25(OH)VD internal standard compound comprises
6The d-25 hydroxy-vitamine D
2And/or
6The d-25 hydroxy-vitamine D
3
3. method according to claim 2 is characterized in that,
6The d-25 hydroxy-vitamine D
2Retention time be 2.61min,
6The d-25 hydroxy-vitamine D
3Retention time be 2.81;
Interior scalar quantity ion pair comprises
6The d-25 hydroxy-vitamine D
2Quota ion pair, and/or,
6The d-25 hydroxy-vitamine D
3Quota ion pair;
The condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair comprises:
6The d-25 hydroxy-vitamine D
2The matter of the parent ion that quota ion is right/lotus ratio is 419.4, corresponding daughter ion matter/the lotus ratio is 401.3;
6The d-25 hydroxy-vitamine D
3The matter of the parent ion that quota ion is right/lotus ratio is 407.2,389.4 of corresponding daughter ion.
4. according to claim 1 and 2 or 3 described methods, it is characterized in that, the condition of described multiple-reaction monitoring ion scan MRM also comprises:
5. method according to claim 1 is characterized in that, the condition of described gradient elution comprises:
Wherein, for containing the water of 0.1% formic acid, B is mutually for containing the methyl alcohol of 0.1% formic acid mutually for A.
6. according to claim 1 or 5 method, it is characterized in that, described high flux Liquid Chromatography-Tandem Mass Spectrometry instrument comprises two cover high flux liquid chromatographic systems, and the liquid phase chromatogram condition of described two cover high flux liquid chromatographic systems is the same; First set liquid phase acquisition window is 2.0min-3.0min; The second cover liquid phase acquisition window is 2.0min-3.0min.
7. method according to claim 1 is characterized in that, described mass ion source parameter comprises:
Ionization source: electron spray ionisation ESI source;
Gas curtain gas CUR:20psi;
Heat air Gs1:45psi;
Auxiliary heating gas Gs2:45psi;
Heat air temperature: 500 ℃;
Collision gas: high pure nitrogen, 6psi;
Electron spray pin voltage: 5500V.
8. method according to claim 1 is characterized in that, described centrifugal condition comprises: at room temperature, and with the centrifugal 10min of the speed of 10000r/min.
9. method according to claim 1 is characterized in that, the condition of described drying comprises: logical 55 ℃～60 ℃ nitrogen is until drying.
10. method according to claim 1 is characterized in that, described double solvents is the potpourri of first alcohol and water, and wherein, the volume ratio of described first alcohol and water is 50:50.
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