CN105527364A - Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry - Google Patents
Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry Download PDFInfo
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Abstract
The invention provides a method for detecting 25-hydroxy-vitamin D through a high-sensitivity high-throughput ultra-performance liquid chromatography-tandem mass spectrometry device. The method comprises adding an internal standard substance of 25-hydroxy-vitamin D into a serum sample, adding a zinc sulfate solution and a methanol solution into the mixture so that protein deposition is realized, carrying out full mixing, adding a n-hexane solvent into the mixture, carrying out extraction, carrying out full mixing, carrying out centrifugation, taking the supernatant, carrying out nitrogen blowing drying, adding a compounded solution into the supernatant to obtain a sample to be detected, detecting the sample to be detected through an ultra-performance liquid chromatography-tandem quadrupole mass spectrometry device and carrying out quantitation through an internal standard curve method based on 25-hydroxy-vitamin D2 and/or 25-hydroxy-vitamin D3 quantitative and qualitative ion pair retention time as qualitative basis. The method has advantages of simple pre-treatment process, good specificity, good matrix interference resistance, short detection time, high throughput, high detection precision, high sensitivity and low cost.
Description
Technical field
The present invention relates to the technical field that vitamin D detects, particularly a kind of Ultra Performance Liquid Chromatography tandem mass spectrum detects the method for serum 25(OH)VD.
Background technology
Vitamin D is fat-soluble nonessential vitamin, it is steroid derivant, 25(OH)VD is that vitamin D transforms formation under the effect of hepatomicrosome 25 hydroxylase, because they are compared with other metabolic products of vitamin D, concentration is in blood 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.
Vitamin D is the most important thing is in vitamin D family member
2and vitamin D
3.Vitamin D
2be contained in plant food more, it be plant ergosterol through sunlight irradiate and synthesize.Vitamin D
3account for human body total amount 90%-95%, mainly synthesized under the ultraviolet irradiation of specific wavelength by the 7-DHC in skin, also obtain by food intake.Vitamin D has the metabolism of adjustment human body Serum Calcium Phosphorus, maintains the normal physiological effect of bone and muscle.Along with the further investigation for vitamin D finds, vitamin D deficiency ubiquity in crowd, and the generation of vitamin D deficiency and various diseases and develop closely related, angiocardiopathy and the metabolic syndrome diseases such as such as obesity, hypertension, diabetes, heart stalk.Therefore the mutual relationship of vitamin D deficiency and various diseases causes the extensive concern of domestic and international research institution.
Traditional 25(OH)VD detection method has radio immunoassay, euzymelinked immunosorbent assay (ELISA), chemoluminescence method, Electrochemiluminescince and high performance liquid chromatography etc.But mainly there are the following problems: the first, because 25(OH)VD is mainly combined with bindin of serum in human body, in human body, there is a large amount of high-affinity associated proteins, therefore detect and there is serious matrix interference.And traditional radioimmunology and luminescence method, effectively can not remove matrix interference; The second, existing detection technique pretreatment process is complicated, method poor specificity; 3rd, there is cross reaction in immunization sometimes, occurs false positive results, and luminescence method exists the problems such as luminescence efficiency is low simultaneously.Therefore immunization and luminescence method can not accurate quantitative analysis 25(OH)VDs simultaneously
2and 25(OH)VD
3content, the total content of 25(OH)VD accurately cannot be provided; 4th, the above-mentioned classic method whole testing process time is long, flux is low.
Therefore, the problem that Water demand those skilled in the art urgently solve is exactly: how to provide a kind of detection method being suitable for the 25(OH)VD of human serum.Compare traditional immunization method and liquid phase chromatography, liquid chromatography tandem mass spectrometry (LC-MS/MS) can solve the problem, and the current world generally believes that LC-MS/MS detects 25(OH)VD " goldstandard " in serum.But be somebody's turn to do " goldstandard " complex disposal process, sensitivity and precision need to be improved further.
Summary of the invention
The present invention is intended to solve the deficiencies in the prior art, provides a kind of detection method being applicable to the 25(OH)VD of human serum, effectively can remove matrix interference, detect fast.
In order to achieve the above object, technical scheme provided by the invention is:
The method that Ultra Performance Liquid Chromatography tandem mass spectrum detects serum 25(OH)VD comprises the steps:
(1) in blood serum sample, add the internal standard compound that volume is the 25(OH)VD of blood serum sample volume 1/15 to 2/15, add solution of zinc sulfate and methanol solution carries out protein precipitation; Normal hexane solvent extraction is added after abundant mixing; Fully mixing is rear centrifugal again, obtains supernatant and precipitation, dries up precipitation after pipetting supernatant with nitrogen, then add redissolution liquid, obtain testing sample; Described 25(OH)VD internal standard compound comprises
6d-25 hydroxy-vitamine D
2and/or
6d-25 hydroxy-vitamine D
3; Described redissolution liquid is that first alcohol and water is by 7:(2-4), the mixed liquor of the volume ratio mixing of preferred 7:3;
Wherein, the volume ratio of described blood serum sample, solution of zinc sulfate, methanol solution, normal hexane solvent is 1:(0.8-1.2): (1.8-2.2): (4-6), preferred 1:1:2:5; The volume ratio of described redissolution liquid and blood serum sample is 1:(1.8-2.2), preferred 1:2;
(2) testing sample Ultra Performance Liquid Chromatography QQ-TOF mass spectrometry instrument is detected.
Preferably, the described mass spectrum of step (2) adopts positive ion mode, and adopts the scan mode of multiple-reaction monitoring ion scan MRM;
In described positive ion mode, interior scalar quantity ion pair comprises
6d-25 hydroxy-vitamine D
2quota ion to
6d-25 hydroxy-vitamine D
3quota ion pair; Target quota ion is to comprising 25(OH)VD
2quota ion to and 25(OH)VD
3quota ion pair; The qualitative ion pair of target comprises 25(OH)VD
2qualitative ion pair and 25(OH)VD
3qualitative ion pair;
The mass-to-charge ratio condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair is:
6d-25 hydroxy-vitamine D
2the mass-to-charge ratio of the parent ion that quota ion is right is 419.3, and the mass-to-charge ratio of corresponding daughter ion is 83.1;
6d-25 hydroxy-vitamine D
3the mass-to-charge ratio of the parent ion that quota ion is right is 407.3, and the mass-to-charge ratio of corresponding daughter ion is 159.1;
The mass-to-charge ratio condition of the multiple-reaction monitoring ion scan MRM of target quota ion and the qualitative ion of target is:
25(OH)VD
2the mass-to-charge ratio of parent ion be 413.35, the mass-to-charge ratio of corresponding quota ion is 395.3, and the mass-to-charge ratio of qualitative ion is 83.1;
25(OH)VD
3the mass-to-charge ratio of parent ion be 401.35, the mass-to-charge ratio of corresponding quota ion is 383.3, and the mass-to-charge ratio of qualitative ion is 159.1;
Described liquid chromatography adopts gradient elution mode, and liquid phase chromatogram condition is:
Chromatographic column: ACQUITYUPLCBEHC18Column (2.1x50mm, 1.7 μm);
Chromatographic column column temperature: 45 DEG C;
Sample size: 20ul;
Flow velocity: 0.4ml/min;
Mobile phase: mobile phase A is the water containing 2mM ammonium acetate and 0.1% formic acid, Mobile phase B is the methyl alcohol containing 2mM ammonium acetate and 0.1% formic acid;
6d-25 hydroxy-vitamine D
3and 25(OH)VD
3retention time be 3.09min,
6d-25 hydroxy-vitamine D
2and 25(OH)VD
2retention time be 3.16min;
(3) with 25(OH)VD
2and 25(OH)VD
3retention time be qualitative foundation, judge 25(OH)VD
2and 25(OH)VD
3existence;
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 in blood serum sample
2and 25(OH)VD
3content.
In addition, the taper hole voltage of described reaction monitoring ion scan MRM, residence time, collision energy condition are as follows:
Wherein, * represents quota ion pair
The condition of described gradient elution is as follows:
In described mass spectrum, mass ions source dates is as follows:
Ionization source: electron spray ionisation ESI source;
Desolventizing temperature degree: 400 DEG C;
Desolventizing gas velocity: 900L/Hr;
Ion source temperature: 120 DEG C;
Capillary voltage: 2500V.
Preferably, condition centrifugal described in step (1) is: at room temperature, with the centrifugation 5-10min of 13000r/min.It is pass into the nitrogen of 45-55 DEG C until precipitation is dry that step (1) described nitrogen dries up precipitation.
Compared with prior art, beneficial effect of the present invention is:
The present invention's application Ultra Performance Liquid Chromatography tandem mass spectrometer detects, and the present invention can be simultaneously qualitative or quantitatively detect 25(OH)VD in human serum
2and 25(OH)VD
3;
The present invention carries out protein precipitation by solution of zinc sulfate co-production of methanol solution, and by normal hexane liquid-liquid extraction, nitrogen just can detect with Ultra Performance Liquid Chromatography QQ-TOF mass spectrometry instrument after drying up redissolution;
Pre-treatment of the present invention is simple, and effectively can remove matrix interference, and specificity, anti-matrix interference ability are strong;
The present invention's application Ultra Performance Liquid Chromatography tandem mass spectrometer detects, and detection time is short, and flux is high, and detection sensitivity, precision are high, with low cost.
In a word, the present invention adopts protein precipitation and liquid-liquid extraction method to obtain 25(OH)VD extract, utilizes Ultra Performance Liquid Chromatography tandem mass spectrometer to detect, isotopic dilution standard measure.Meet pre-treatment simple, effectively can remove matrix interference, accurate quantitative analysis detects 25(OH)VD simultaneously
2and 25(OH)VD
3, and the whole testing process time is short, and flux is high.Compare the document that the LC-MS/MS method reported at present detects 25(OH)VD, pre-treatment of the present invention is simpler, do not need to carry out the expensive instrument such as Solid phase extraction operation or on-line solid phase extraction instrument, sensitivity and precision higher, meet clinical detection requirement.In addition, compare conventional liquid-phase chromatographic column, the filler particles of ultra high efficiency chromatographic column is thinner, voltage endurance capability is stronger simultaneously, detection sensitivity is higher, can detect fast, shorten analysis time, therefore the present invention is under the prerequisite adopting LC-MS/MS method, optimize pretreatment process and instrument designing parameter, optimize the mobile phase in LC-MS/MS method and gradient condition simultaneously, and then improve peak shape and the response of chromatographic peak, the detection sensitivity of 25(OH)VD is significantly improved, is applicable to clinical expansion.
Accompanying drawing explanation
Fig. 1 is 25(OH)VD
2and 25(OH)VD
3quantitative limit (S/N=10).
Embodiment
Embodiment 1
The method that described Ultra Performance Liquid Chromatography tandem mass spectrometer detects 25(OH)VD comprises the steps: in fact
(1) pre-treatment:
In human serum sample, add the internal standard compound of 25(OH)VD, add solution of zinc sulfate and methanol solution carries out protein precipitation; Normal hexane solvent extraction is added after abundant mixing; Fully mixing is rear centrifugal again, then pipettes supernatant and carry out nitrogen to dry up, and adds redissolution liquid, obtains testing sample;
Wherein, the volume ratio of described human serum sample and described solution of zinc sulfate, methanol solution, n-hexane extraction solution is 1:1:2:5; Described supernatant is 1:1 with the volume ratio of corresponding original solution; The volume ratio of described redissolution liquid and human serum sample is 1:2.
25(OH)VD internal standard compound can comprise d
6-25(OH)VD
2and/or d
6-25(OH)VD
3.Namely d can be only included
6-25(OH)VD
2, also can only include d
6-25(OH)VD
3, also can comprise d simultaneously
6-25(OH)VD
2and d
6-25(OH)VD
3.
If only detect 25(OH)VD
2(25OHD
2), then only can add d
6-25(OH)VD
2; If only detect 25(OH)VD
3(25OHD
3), then only can add d
6-25(OH)VD
3;
If detect 25(OH)VD simultaneously
2and 25(OH)VD
3, then can add d simultaneously
6-25(OH)VD
2and d
6-25(OH)VD
3.
To the centrifugal condition adding the solution after normal hexane be: the centrifugation 5min of 13000r/min.
To the condition of the supernatant drying pipetted be: the nitrogen of logical 50 DEG C is until drying.
Described redissolution liquid is the potpourri of first alcohol and water, and wherein, the volume ratio of first alcohol and water is 70:30.
(2) detect;
Ultra Performance Liquid Chromatography QQ-TOF mass spectrometry instrument is adopted to detect to described testing sample;
Described detection adopts positive ion mode, and scan mode adopts multiple-reaction monitoring ion MRM;
MRM:MultipleReactionMonitor, refers to multiple-reaction monitoring ion scan;
Wherein, interior scalar quantity ion pair comprises d
6-25(OH)VD
2quota ion to and/or d
6-25(OH)VD
3quota ion pair;
Concrete, as detection d
6-25(OH)VD
2time, adopt d
6-25(OH)VD
2quota ion pair;
As detection d
6-25(OH)VD
3time, adopt d
6-25(OH)VD
3quota ion pair;
When detecting d simultaneously
6-25(OH)VD
2and d
6-25(OH)VD
3, adopt d
6-25(OH)VD
2quota ion to and d
6-25(OH)VD
3quota ion pair;
Target quota ion is to comprising 25(OH)VD
2quota ion to and/or 25(OH)VD
3quota ion pair; The qualitative ion pair of target comprises 25(OH)VD
2qualitative ion pair and/or 25(OH)VD
3qualitative ion pair;
Specifically, to 25(OH)VD
2when detecting, adopt 25(OH)VD
2quota ion to and 25(OH)VD
2qualitative ion pair;
To 25(OH)VD
3when detecting, adopt 25(OH)VD
3quota ion to and 25(OH)VD
3qualitative ion pair;
Simultaneously to 25(OH)VD
2and 25(OH)VD
3when detecting, adopt 25(OH)VD respectively
2quota ion to and 25(OH)VD
2qualitative ion pair and 25(OH)VD
3quota ion to and 25(OH)VD
3qualitative ion pair;
The condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair comprises:
6d-25 hydroxy-vitamine D
3the mass-to-charge ratio of the parent ion that quota ion is right is 407.3, and the mass-to-charge ratio of corresponding daughter ion is 159.1;
6d-25 hydroxy-vitamine D
2the mass-to-charge ratio of the parent ion that quota ion is right is 419.3, and the mass-to-charge ratio of corresponding daughter ion is 83.1;
The condition of the multiple-reaction monitoring ion scan MRM of target quota ion comprises:
25(OH)VD
3the mass-to-charge ratio of parent ion be 401.35, the mass-to-charge ratio of corresponding quota ion is 383.3, and the mass-to-charge ratio of qualitative ion is 159.1;
25(OH)VD
2the mass-to-charge ratio of parent ion be 413.35, the mass-to-charge ratio of corresponding quota ion is 395.3, and the matter/lotus ratio of qualitative ion is 83.1;
Wherein, described liquid chromatography adopts gradient elution mode, and liquid phase chromatogram condition comprises:
Chromatographic column: ACQUITYUPLCBEHC18Column (2.1x50mm, 1.7 μm);
Chromatographic column column temperature: 45 DEG C;
Sample size: 20ul;
Flow velocity: 0.4ml/min;
In gradient mode wash-out, mobile phase comprises: A mobile phase is the water containing 2mM ammonium acetate and 0.1% formic acid, and B mobile phase is the methyl alcohol containing 2mM ammonium acetate and 0.1% formic acid.
Adopt above-mentioned mobile phase, in gradient elution, 25(OH)VD
2retention time be 3.16min, 25(OH)VD
3retention time be 3.09min.
The condition of multiple-reaction monitoring ion scan MRM is as follows:
Wherein, * represents quota ion pair
Being appreciated that the condition of above-mentioned MRM is not only corresponding a kind of situation, can be detect 25(OH)VD
2, also can be detect 25(OH)VD
3, can also be detect 25(OH)VD simultaneously
2and 25(OH)VD
3.
Condition of gradient elution is as follows:
Mass ions source dates comprises:
Ionization source: electron spray ionisation ESI source;
Desolventizing temperature degree: 400 DEG C;
Desolventizing gas velocity: 900L/Hr;
Ion source temperature: 120 DEG C;
Capillary voltage: 2500V.
(3) qualitatively judge and quantitatively calculate:
According to 25(OH)VD
2and/or 25(OH)VD
3retention time be qualitative foundation, judge 25(OH)VD
2and/or 25(OH)VD
3existence;
Specifically, when detection 25(OH)VD
2time, according to 25(OH)VD
2retention time judge 25(OH)VD
2existence;
When detection 25(OH)VD
3time, according to 25(OH)VD
3retention time judge 25(OH)VD
3existence;
When detecting 25(OH)VD simultaneously
2and 25(OH)VD
3time, according to 25(OH)VD
2and 25(OH)VD
3retention time judge 25(OH)VD
2and 25(OH)VD
3existence.
With Ultra Performance Liquid Chromatography tandem mass spectrometry, sample is qualitatively judged, under same test conditions, in sample measured target material chromatographic peak quota ion to qualitative ion pair retention time with tie substance chromatographic peak quota ion in standard solution to consistent with the retention time of qualitative ion pair, then can there is corresponding target substance in judgement sample.
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 in human serum sample
2and/or 25(OH)VD
3content.
Specifically, when detection 25(OH)VD
2, 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 in human serum
2content.
When detection 25(OH)VD
3, 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 in human serum
3content.
When detecting 25(OH)VD simultaneously
2and 25(OH)VD
3time, 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 25(OH)VD in human serum
2and 25(OH)VD
3content.With 25(OH)VD
2and 25(OH)VD
3content sum as the content of 25(OH)VD.
Based on above-mentioned experiment condition, through test of many times checking, the RSD<9% of the withinday precision of the basic, normal, high concentration recovery testu of the invention process and day to day precision for three days on end.25(OH)VD
2and 25(OH)VD
3detection limit (S/N=3) be respectively 0.3ng/ml and 0.2ng/ml, 25(OH)VD
2and 25(OH)VD
3quantitative limit (S/N=10) be respectively 0.8ng/ml and 0.5ng/ml (see Fig. 1).Simple and effective pretreatment process, high resolving power and highly sensitive Liquid Chromatography-Tandem Mass Spectrometry instrument, the optimization of suitable liquid-phase condition and good mass spectrometry parameters, makes implementation data of the present invention be better than the data in literature reported.
Embodiment 2 understands the present invention better for making those skilled in the art, now provides an example so that the specific implementation process of the present invention for the detection method of 25(OH)VD to be described.
150ul human serum is pipetted in the 2ml centrifuge tube of cleaning, adds 10ul inner mark solution, mixing 10s, add 0.2M solution of zinc sulfate 150ul, mixing 10s, adds 300ul methyl alcohol, mixing 10s, adds 750ul normal hexane, mixing 30s, centrifugal (13000rpm, 5min), gets supernatant 600ul, 50 DEG C of nitrogen dry up, and finally redissolve with 75ul70% methanol aqueous solution, after vortex mixing, be transferred to glass-lined pipe, be loaded in liquid chromatography automatic sampler.
Sample is loaded into chromatographic column by liquid chromatography automatic sampler automatically, is then passed to mass spectrometer LC-MS/MS by mobile phase.LC-MS/MS adopts electron spray ionisation source (ESI) level Four bar mass analyzer of connecting to analyze as mass detector.Wherein, desolventizing temperature degree is 400 DEG C, and desolventizing gas velocity is 900L/Hr, and ion source temperature is 120 DEG C, and capillary voltage is 2500V.Adopt multiple-reaction monitoring ion MRM scanning, its condition is see table 1.
Table 1 reaction monitoring ion scan MRM condition
| Material | Ion pair | Taper hole voltage (V) | Residence time (S) | Collision energy (V) |
| 25OHD 2 | 413.35>395.3* | 24 | 0.05 | 10 |
| 25OHD 2 | 413.35>83.1 | 24 | 0.05 | 22 |
| 25OHD 3 | 401.35>383.3* | 24 | 0.05 | 10 |
| 25OHD 3 | 401.35>159.1 | 24 | 0.05 | 28 |
| d6-25OHD 2 | 419.35>83.1 | 24 | 0.05 | 22 |
| d6-25OHD 3 | 407.35>159.1 | 24 | 0.05 | 28 |
Wherein, * represents quota ion pair
The sample that 20ul purifies in advance is loaded in Ultra Performance Liquid Chromatography system by automatic sampler automatically, and liquid phase chromatogram condition is see table 2.Chromatographic column adopts ACQUITYUPLCBEHC18Column (2.1x50mm, 1.7 μm), A mobile phase is the water containing 2mM ammonium acetate and 0.1% formic acid, B mobile phase is the methyl alcohol containing 2mM ammonium acetate and 0.1% formic acid, wherein, chromatographic column column temperature is 45 DEG C, and sample size is 20ul, and flow velocity is 0.4ml/min.
Table 2 liquid phase chromatogram condition
| Step | Analysis time (min) | Flow velocity (ml/min) | A Phase Proportion (%) | B Phase Proportion (%) |
| 1 | 0.00 | 0.4 | 27 | 73 |
| 2 | 2.00 | 0.4 | 27 | 73 |
| 3 | 3.50 | 0.4 | 2 | 98 |
| 4 | 3.51 | 0.4 | 27 | 73 |
| 5 | 6.00 | 0.4 | 27 | 73 |
Sample enters mass spectrometer ion source or waste liquid after discharging column outlet with mobile phase under the effect of the pressure, is controlled to enter ionogenic sample channel by six-way valve.In ion gun, fluid sample is vaporized and ionizes as charged ion, and charged ion, under voltage and vacuum action, enters Q1, Q2 and Q3, and wherein, Q1 and Q3 is mass analyzer, only allows according to 25(OH)VD
2and 25(OH)VD
3mass-to-charge ratio select parent ion and daughter ion pass through, Q2 is collision cell, and parent ion collides with intert-gas atoms herein, produces specific fragmention.
Mass spectrometric first level Four bar (Q1) selection has 25(OH)VD
2, 25(OH)VD
3,
6d-25 hydroxy-vitamine D
3with
6d-25 hydroxy-vitamine D
2the 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
3passed through by selection with interior target fragmention, and other ions are removed.See table 3, illustrate the 25(OH)VD being used to Identification and determination.
The mass shift table of table 325 hydroxy-vitamine D
| Analyte | Parent ion (m/z) | Quota ion (m/z) | Qualitative ion (m/z) |
| 25OHD 3 | 401.35 | 383.3 | 159.1 |
| 25OHD 2 | 413.35 | 395.3 | 83.1 |
| 6d-25OHD 2 | 419.35 | 83.1 | - |
| 6d-25OH D 3 | 407.35 | 159.1 | - |
Along with ion and detecting device collide, the mass number captured is changed into the electronic impulse of digital signal by them.The data obtained are passed to computing machine, and collected ion signal intensity versus time is mapped by it, obtains mass spectrum total ion current figure.
The ratio analyzing thing and interior target peak area in serum standard is used to build interior mark typical curve, and then this curve is used to analyte concentration in calculation sample or quality control substance, the results are shown in Table 4;
Table 425 hydroxy-vitamine D
2and 25(OH)VD
3precision
Certain authoritative inspection center, the human serum 25OHD adopting this implementation method to carry out 1000 examples detects, and it is accurate that testing result display quality controls material result, and experimenter at least can detect 100 increment product every day, and detection efficiency is high.Illustrate that this method accurately and reliably, pre-treatment is simple, and analysis time is short.
Claims (7)
1. Ultra Performance Liquid Chromatography tandem mass spectrum detects the method for serum 25(OH)VD, and it is characterized in that, described method comprises the steps:
(1) in blood serum sample, add the internal standard compound that volume is the 25(OH)VD of blood serum sample volume 1/15 to 2/15, add solution of zinc sulfate and methanol solution carries out protein precipitation; Normal hexane solvent extraction is added after abundant mixing; Fully mixing is rear centrifugal again, obtains supernatant and precipitation, dries up precipitation after pipetting supernatant with nitrogen, then add redissolution liquid, obtain testing sample; Described 25(OH)VD internal standard compound comprises
6d-25 hydroxy-vitamine D
2and/or
6d-25 hydroxy-vitamine D
3; Described redissolution liquid is that first alcohol and water is by 7:(2-4) volume ratio mixing mixed liquor;
Wherein, the volume ratio of described blood serum sample, solution of zinc sulfate, methanol solution, normal hexane solvent is 1:(0.8-1.2): (1.8-2.2): (4-6); The volume ratio of described redissolution liquid and blood serum sample is 1:(1.8-2.2);
(2) testing sample Ultra Performance Liquid Chromatography QQ-TOF mass spectrometry instrument is detected.
2. the method for claim 1, is characterized in that, the described mass spectrum of step (2) adopts positive ion mode, and adopts the scan mode of multiple-reaction monitoring ion scan MRM;
In described positive ion mode, interior scalar quantity ion pair comprises
6d-25 hydroxy-vitamine D
2quota ion to and/or
6d-25 hydroxy-vitamine D
3quota ion pair; Target quota ion is to comprising 25(OH)VD
2quota ion to and/or 25(OH)VD
3quota ion pair; The qualitative ion pair of target comprises 25(OH)VD
2qualitative ion pair and/or 25(OH)VD
3qualitative ion pair;
The mass-to-charge ratio condition of the multiple-reaction monitoring ion scan MRM of interior scalar quantity ion pair is:
6d-25 hydroxy-vitamine D
2the mass-to-charge ratio of the parent ion that quota ion is right is 419.3, and the mass-to-charge ratio of corresponding daughter ion is 83.1;
6d-25 hydroxy-vitamine D
3the mass-to-charge ratio of the parent ion that quota ion is right is 407.3, and the mass-to-charge ratio of corresponding daughter ion is 159.1;
The mass-to-charge ratio condition of the multiple-reaction monitoring ion scan MRM of target quota ion and the qualitative ion of target is:
25(OH)VD
2the mass-to-charge ratio of parent ion be 413.35, the mass-to-charge ratio of corresponding quota ion is 395.3, and the mass-to-charge ratio of qualitative ion is 83.1;
25(OH)VD
3the mass-to-charge ratio of parent ion be 401.35, the mass-to-charge ratio of corresponding quota ion is 383.3, and the mass-to-charge ratio of qualitative ion is 159.1;
Described liquid chromatography adopts gradient elution mode, and liquid phase chromatogram condition is:
Chromatographic column: ACQUITYUPLCBEHC18Column (2.1x50mm, 1.7 μm);
Chromatographic column column temperature: 45 DEG C;
Sample size: 20ul;
Flow velocity: 0.4ml/min;
Mobile phase: mobile phase A is the water containing 2mM ammonium acetate and 0.1% formic acid, Mobile phase B is the methyl alcohol containing 2mM ammonium acetate and 0.1% formic acid;
6d-25 hydroxy-vitamine D
3and 25(OH)VD
3retention time be 3.09min,
6d-25 hydroxy-vitamine D
2and 25(OH)VD
2retention time be 3.16min;
(3) with 25(OH)VD
2and 25(OH)VD
3retention time be qualitative foundation, judge 25(OH)VD
2and 25(OH)VD
3existence;
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 in blood serum sample
2and 25(OH)VD
3content.
3. method as claimed in claim 2, it is characterized in that, the taper hole voltage of described reaction monitoring ion scan MRM, residence time, collision energy condition are as follows:
。
4. method according to claim 2, the condition of described gradient elution is as follows:
。
5. method as claimed in claim 2, it is characterized in that, in described mass spectrum, mass ions source dates is as follows:
Ionization source: electron spray ionisation ESI source;
Desolventizing temperature degree: 400 DEG C;
Desolventizing gas velocity: 900L/Hr;
Ion source temperature: 120 DEG C;
Capillary voltage: 2500V.
6. the method for claim 1, is characterized in that, condition centrifugal described in step (1) is: at room temperature, with the centrifugation 5-10min of 13000r/min.
7. the method for claim 1, is characterized in that, it is pass into the nitrogen of 45-55 DEG C until precipitation is dry that step (1) described nitrogen dries up precipitation.
Priority Applications (1)
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| CN106442838A (en) * | 2016-10-19 | 2017-02-22 | 杭州佰辰医学检验所有限公司 | Isotope dilution ultra-high performance liquid chromatography-mass spectrometry method for measuring vitamin B1 in serum |
| 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 |
| CN108020625A (en) * | 2017-12-01 | 2018-05-11 | 上海宝藤生物医药科技股份有限公司 | The detection method and threatened abortion diagnostic kit of anandamide and 2- arachidonic glycerol contents in blood plasma |
| CN108107133A (en) * | 2018-01-17 | 2018-06-01 | 中国医学科学院北京协和医院 | Liquid Chromatography-Tandem Mass Spectrometry measures the method for vitamin A and E in serum |
| CN108593790A (en) * | 2018-04-10 | 2018-09-28 | 中国医学科学院北京协和医院 | Detect serum 24,25 simultaneously（OH）The method of 2D and 25OHD |
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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 |
| CN110487943A (en) * | 2019-08-28 | 2019-11-22 | 杭州佰辰医学检验所有限公司 | A kind of method of liposoluble vitamin in detection blood sample |
| CN110715996A (en) * | 2018-07-12 | 2020-01-21 | 中国科学院生态环境研究中心 | Method for full-automatic online detection of free-state small molecular compound in serum |
| CN111830146A (en) * | 2020-05-27 | 2020-10-27 | 江苏豪思睦可生物科技有限公司 | LC-MS/MS high-throughput detection method and kit for 25-hydroxy vitamin D in dried blood tablets |
| CN111983071A (en) * | 2020-08-13 | 2020-11-24 | 上海交通大学医学院附属上海儿童医学中心 | Mass spectrum detection method for vitamin D in peripheral trace blood |
| CN114674943A (en) * | 2022-03-03 | 2022-06-28 | 中国计量科学研究院 | Detection of 25(OH) D in serum2And 25(OH) D3Liquid chromatography-mass spectrometry tandem detection method |
| CN114778727A (en) * | 2022-04-19 | 2022-07-22 | 天津国科医工科技发展有限公司 | Method for detecting fat-soluble vitamins in cryopreserved breast milk |
| CN115078559A (en) * | 2022-03-24 | 2022-09-20 | 杭州佰辰医学检验所有限公司 | Rapid vitamin D detection method based on single quadrupole mass spectrometry, kit and application |
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| CN106442838A (en) * | 2016-10-19 | 2017-02-22 | 杭州佰辰医学检验所有限公司 | Isotope dilution ultra-high performance liquid chromatography-mass spectrometry method for measuring vitamin B1 in serum |
| CN106442838B (en) * | 2016-10-19 | 2018-07-10 | 杭州佰辰医学检验所有限公司 | Vitamin B in a kind of measure serum1Isotopic dilution ultra performance liquid chromatography mass spectrometry combination method |
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| CN108107133B (en) * | 2018-01-17 | 2020-08-21 | 中国医学科学院北京协和医院 | Method for measuring vitamin A and vitamin E in serum by liquid chromatography tandem mass spectrometry |
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| CN108593790B (en) * | 2018-04-10 | 2020-11-13 | 中国医学科学院北京协和医院 | Method for simultaneously detecting 24,25(OH)2D and 25OHD of serum |
| CN108802221A (en) * | 2018-06-12 | 2018-11-13 | 杭州度安医学检验实验室有限公司 | The Sparklet testing method of vitamin A and vitamin E in peripheral blood |
| CN108645942A (en) * | 2018-06-20 | 2018-10-12 | 杭州凯莱谱精准医疗检测技术有限公司 | The high performance liquid chromatography tandem mass spectrum detection method of 25-hydroxy-vitamin D in serum |
| CN110715996A (en) * | 2018-07-12 | 2020-01-21 | 中国科学院生态环境研究中心 | Method for full-automatic online detection of free-state small molecular compound in serum |
| CN110715996B (en) * | 2018-07-12 | 2022-04-12 | 中国科学院生态环境研究中心 | Method for full-automatic online detection of free-state small molecular compound in serum |
| 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 |
| CN110487943A (en) * | 2019-08-28 | 2019-11-22 | 杭州佰辰医学检验所有限公司 | A kind of method of liposoluble vitamin in detection blood sample |
| CN111830146A (en) * | 2020-05-27 | 2020-10-27 | 江苏豪思睦可生物科技有限公司 | LC-MS/MS high-throughput detection method and kit for 25-hydroxy vitamin D in dried blood tablets |
| CN111830146B (en) * | 2020-05-27 | 2023-09-01 | 江苏豪思睦可生物科技有限公司 | LC-MS/MS high-throughput detection method and kit for 25-hydroxyvitamin D in dried blood slices |
| CN111983071A (en) * | 2020-08-13 | 2020-11-24 | 上海交通大学医学院附属上海儿童医学中心 | Mass spectrum detection method for vitamin D in peripheral trace blood |
| CN114674943A (en) * | 2022-03-03 | 2022-06-28 | 中国计量科学研究院 | Detection of 25(OH) D in serum2And 25(OH) D3Liquid chromatography-mass spectrometry tandem detection method |
| CN115078559A (en) * | 2022-03-24 | 2022-09-20 | 杭州佰辰医学检验所有限公司 | Rapid vitamin D detection method based on single quadrupole mass spectrometry, kit and application |
| CN114778727A (en) * | 2022-04-19 | 2022-07-22 | 天津国科医工科技发展有限公司 | Method for detecting fat-soluble vitamins in cryopreserved breast milk |
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