CN108490096A - The detection method of 25(OH)VD in human serum - Google Patents
The detection method of 25(OH)VD in human serum Download PDFInfo
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Abstract
Include the steps that sample pre-treatments and sample detection the present invention relates to a kind of detection method of 25(OH)VD in serum, specially：Take human serum sample, the methanol solution containing 25(OH)VD internal standard compound is added, then zinc sulfate solution is added into the human serum sample and methanol carries out albumen precipitation, n-hexane solvent is added after mixing well to be extracted, it is centrifuged after mixing well again, it is added after taking supernatant and being dried and redissolves liquid, obtain sample to be tested；The sample to be tested is detected with ultra performance liquid chromatography series connection quadrupole mass spectrometer again.The detection method pre-treatment of the present invention is simple, and high sensitivity, specific, anti-matrix interference ability is strong, and detection time is short, and required sample size is few, and flux is high.
Description
Technical field
The present invention relates to analysis technical fields, more particularly to a kind of detection method of 25(OH)VD in human serum.
Background technology
Vitamin D is a kind of liposoluble vitamin, is sterol analog derivative, is a kind of necessary to maintain human health
Compound.Vitamin D includes 5 kinds of compounds, and closely related with health is calciferol and vitamine D3.Calciferol and dimension
Monoxidase (25 hydroxyl oxidizing ferment) oxidations of the raw element D3 through liver forms 25(OH)VD 2 and 25(OH)VD 3.25
The concentration of hydroxy-vitamine D in blood is high, cycle period is long, therefore becomes concentration indicator of the vitamin D in human body, is
The evaluation index of body vitamin D nutrition conditions.
It is now recognized that vitamin D is also a kind of steroid hormone, most important member is VD2 in vitamin D family member
(ergocalciferol) and VD3 (Vitamin D3).Calciferol is contained in plant food more, it is passed through by the ergosterol of plant
Sunlight and synthesize.Vitamine D3 accounts for body vitamin D total amount 90%-95%, mainly by the 7- dehydrogenation courages in skin
Sterol synthesizes under the ultraviolet irradiation of specific wavelength, can also be obtained by food intake.Vitamin D, which has, adjusts human body
Serum Calcium Phosphorus is metabolized, and maintains the effect of the normal physiological of bone and muscle.With the further investigation discovery for vitamin D, crowd
Middle vitamin D deficiency generally existing, and the occurrence and development of vitamin D deficiency and various diseases are closely related, such as it is fat, high
The diseases such as blood pressure, diabetes, heart infarction, in recent years, many researchs have been found that body vitamin D level and tumour, autoimmune
There are relevant risk factors for disease, angiocardiopathy, metabolic syndrome etc..Therefore the phase of vitamin D deficiency and various diseases
Mutual relation causes the extensive concern of domestic and international research institution.
The correlative study of vitamin D deficiency is necessarily required to develop the accurate detection method to vitamin D in blood.
The world generally believes that LC-MS/MS is 25(OH)VD " goldstandard " in detection serum at present, but related field is developed
The method that 25(OH)VD is detected based on high performance liquid chromatography tandem mass spectrum instrument, generally use C18 columns as chromatographic column,
Its sensitivity is relatively low, and sample size needed for experiment is larger every time.
Invention content
Based on this, the present invention provides a kind of detection method of 25(OH)VD in human serum, the detection spirits of this method
Sensitivity is high, and sample requirement is few.
Specific technical solution is as follows：
The detection method of 25(OH)VD, includes the following steps in a kind of human serum：
Sample pre-treatments：Take human serum sample, the methanol solution containing 25(OH)VD internal standard compound be added, then to
Zinc sulfate solution and methanol are added in the human serum sample and carries out albumen precipitation, be added after mixing well n-hexane solvent into
Row extraction, then centrifuged after mixing well, it is added after taking supernatant and being dried and redissolves liquid, obtain sample to be tested；
Sample detection：The sample to be tested is detected with ultra performance liquid chromatography series connection quadrupole mass spectrometer,
Wherein, the condition of ultra performance liquid chromatography includes：
Chromatographic column：WatersACQUITY UPLC HSS T3Column,2.1×50mm,1.8μm；
Chromatogram column temperature：37℃-43℃；
Sample size：18-22uL；
Flow velocity：0.42-0.48mL/min；
Mobile phase：Mobile phase A is the aqueous solution containing 1.8-2.2mM ammonium acetates and 0.09-0.11v/v% formic acid, flowing
Phase B is the methanol solution containing 1.8-2.2mM ammonium acetates and 0..09-0.11v/v% formic acid, gradient elution；
The 25(OH)VD is 25(OH)VD 2 and/or 25(OH)VD 3,25 hydroxy vitamin
D internal standard compounds are 3d-25 hydroxy vitamin D2s and/or 6d-25 hydroxycholecalciferols.
In specific detection process, if only detecting 25(OH)VD 2,3d-25 hydroxy vitamins can be only added
D2 is as internal standard compound；If only detecting 25(OH)VD 3,6d-25 hydroxycholecalciferols can be only added as internal standard
Object；If to detect 25(OH)VD 2 and 25(OH)VD 3 simultaneously, 3d-25 hydroxy vitamins can be added simultaneously
D2 and 6d-25 hydroxycholecalciferols are internal standard compound.
In wherein some embodiments, the condition of ultra performance liquid chromatography includes：
Chromatographic column：WatersACQUITY UPLC HSS T3Column,2.1×50mm,1.8μm；
Chromatogram column temperature：40℃；
Sample size：20uL；
Flow velocity：0.45mL/min；
Mobile phase：Mobile phase A is the aqueous solution containing 2mM ammonium acetates and 0.1% formic acid, and Mobile phase B is to contain 2mM acetic acid
The methanol solution of ammonium and 0.1% formic acid, gradient elution.
In wherein some embodiments, the condition of the gradient elution is as follows：
In wherein some embodiments, the ultra performance liquid chromatography of the ultra performance liquid chromatography series connection quadrupole mass spectrometer
Acquisition window is 2.30-2.70min.
In wherein some embodiments, Mass Spectrometry Conditions include：
In wherein some embodiments, the Mass Spectrometry Conditions further include：The detection ion pair of the 25(OH)VD 2
It is 413.3>395.2 and 413.3>83.1, wherein 413.3>395.2 be quota ion pair；The inspection of the 25(OH)VD 3
Measured ion is to being 401.3>383.2 and 401.3>159.1 wherein 401.3>383.2 be quota ion pair；The 3d-25 hydroxyls
The quantitative detection ion pair of calciferol is 416.35>358.3；The quantitative detection ion pair of the 6d-25 hydroxycholecalciferols
It is 407.35>159.1.
The detection method of the present invention, target quota ion pair includes the quota ion pair of 25(OH)VD 2,25 hydroxyls
The quota ion pair of base vitamine D3；Target qualitative ion pair includes the qualitative ion pair and 25 hydroxyls dimension of 25(OH)VD 2
The qualitative ion pair of raw element D3.Specifically, when being detected to 25(OH)VD 2, using determining for 25(OH)VD 2
Measure ion pair and qualitative ion pair；When being detected to 25(OH)VD 3, using the quota ion of 25(OH)VD 3
Pair and qualitative ion pair.Wherein, the condition of the multiple-reaction monitoring ion scan of target quota ion pair includes：25 hydroxy vitamins
The mass-to-charge ratio of the parent ion of D2 is 413.3, and the mass-to-charge ratio of corresponding daughter ion is 395.2；The parent ion of 25(OH)VD 3
Mass-to-charge ratio be 401.3, the mass-to-charge ratio of corresponding daughter ion is 383.2.The multiple-reaction monitoring ion of target qualitative ion pair is swept
The condition retouched includes：The mass-to-charge ratio of the parent ion of 25(OH)VD 2 is 413.3, and the mass-to-charge ratio of corresponding daughter ion is
83.1；The mass-to-charge ratio of the parent ion of 25(OH)VD 3 is 401.3, and the mass-to-charge ratio of corresponding daughter ion is 159.1.
Correspondingly, the quota ion pair of internal standard compound includes the quota ion pair and 6d-25 hydroxyls of 3d-25 hydroxy vitamin D2s
The quota ion pair of vitamine D3.Specifically, when detecting 3d-25 hydroxy vitamin D2s, it is fixed using 3d-25 hydroxy vitamin D2s
Measure ion pair；When detecting 6d-25 hydroxycholecalciferols, using 6d-25 hydroxycholecalciferol quota ion pairs；It is detected when simultaneously
When 3d-25 hydroxy vitamin D2s and 6d-25 hydroxycholecalciferols, using 3d-25 hydroxy vitamin D2s and 6d-25 hydroxy vitamins
D3 quota ion pairs.Wherein, the condition of the multiple-reaction monitoring scanning of internal standard quota ion pair includes：3d-25 hydroxy vitamin D2s
The mass-to-charge ratio of parent ion be 416.35, the mass-to-charge ratio of corresponding daughter ion is 358.3；6d-25 hydroxycholecalciferols it is female from
The mass-to-charge ratio of son is 407.35, and the mass-to-charge ratio of corresponding daughter ion is 159.1.
In wherein some embodiments, the corresponding orifice potential of detection ion pair and collision energy are as follows：
Above table is not only to correspond to a kind of situation, can be detection 25(OH)VD 2, can also be detection 25
Hydroxycholecalciferol can also be while detect 25(OH)VD 2 and 25(OH)VD 3.
In wherein some embodiments, according to 25(OH)VD 2 and/or 25(OH)VD 3 retention time with
And relative ion abundance is than judging to whether there is 25(OH)VD 2 and/or 25 hydroxy vitamins in the human serum sample
D3, the relative ion abundance is than the peak area of the qualitative ion pair for each object to be measured object and the peak area of quota ion pair
Ratio, the object to be measured object include 25(OH)VD 2 and/or 25(OH)VD 3.
In wherein some embodiments, a concentration of 248- of the methanol solution containing 25(OH)VD internal standard compound
252ng/mL。
In wherein some embodiments, the human serum sample and the methanol containing 25(OH)VD internal standard compound
The volume ratio of solution is 1：0.15-0.25.
In wherein some embodiments, a concentration of 0.15-0.25mol/L of the zinc sulfate solution.
In wherein some embodiments, the human serum sample and the solution of zinc sulfate, the body of methanol, n-hexane solvent
Product is than being 1：1：1.8-2.2：8-12.
In wherein some embodiments, the volume ratio of the supernatant and n-hexane solvent is 1：1.1-1.3.
It is described to redissolve the mixture that liquid is first alcohol and water, the volume ratio of the first alcohol and water in wherein some embodiments
For 63-67：33-37.
In wherein some embodiments, the liquid and the volume ratio of the human serum sample of redissolving is 2-3：2.
In wherein some embodiments, the condition of the centrifugation includes：At room temperature, with 12000-14000rpm/min's
Speed centrifuges 4-6min；
In wherein some embodiments, the condition of the drying includes：It is passed through 45 DEG C~55 DEG C of nitrogen drying.
The detection method of 25(OH)VD has the following advantages and beneficial effect in the human serum of the present invention：
The present invention provides a kind of detection methods of the 25(OH)VD in human serum based on UPLC-MS/MS, should
Method pre-treatment is simple, and WatersACQUITYUPLC HSS T3Column is selected to be detected in conjunction with specific chromatography as chromatographic column
Condition, larger improves instrument detection signal strength, improves the sensitivity of method, reduces the sample size needed for experiment,
50uL human serum samples are only needed to can be obtained accurate testing result, and can be simultaneously to 25(OH)VD 2 and 25 hydroxyls
Vitamine D3 carries out accurate quantitative analysis.Compared with the conventional method, detection method of the invention has detection sensitivity height, accuracy
Good, required sample size is few, and detection time is short, and flux is high, high specificity, the advantages that anti-matrix interference ability is strong.
Further, the condition of chromatography and Mass Spectrometry Conditions and pre-treatment is further optimized, it can be into one
Step improves sensitivity and the accuracy of detection.Inventor also found, use mass spectrometric qualitative function (relative ion abundance ratio)
25(OH)VD 2 and 25(OH)VD 3 can be carried out more accurately judging, high specificity is qualitative using two kinds
Basis for estimation excludes co-elute substance and elutes the interference to test substance in identical retention time, improves the accuracy of detection.
Description of the drawings
Fig. 1 is the detection chromatogram of the human serum sample of embodiment 1；
Fig. 2 is the 25(OH)VD 2 and its internal standard compound and 25(OH)VD 3 of embodiment 1 and mixing for internal standard compound
The detection chromatogram of standardization working solution；
Fig. 3 is the detection mass spectrogram of the human serum sample of embodiment 1；
Fig. 4 is the 25(OH)VD 2 and its internal standard compound and 25(OH)VD 3 of embodiment 1 and its mixing for internal standard compound
The detection mass spectrogram of standardization working solution；
Fig. 5 is the canonical plotting of the 25(OH)VD 2 and 25(OH)VD 3 of embodiment 1.
Specific implementation mode
The detection method of the present invention is further described in detail below in conjunction with specific embodiment.
Embodiment 1
The detection method tool of 25(OH)VD, which is stopped, in human serum provided in this embodiment includes the following steps：
(1) sample pre-treatments
It takes 50uL human serums to be poured into clean centrifuge tube, 25 hydroxy vitamins of a concentration of 250ng/mL of 10uL is added
The methanol solution of D internal standard compounds, whirlpool mixing 10s, adds the zinc sulfate solution of the 0.2mol/L of 50uL and the chromatography of 100uL
Pure methanol, whirlpool mixing 10s add 500uL n-hexane solvents and are extracted to precipitate protein in human serum, whirlpool mixing
After 30s, 5min is centrifuged in centrifuge 13000rpm/min, supernatant 400uL is then taken to be transferred in clean centrifuge tube,
It is dried up with nitrogen under the conditions of 50 DEG C, the methanol aqueous solution for being eventually adding 75uL 65% (volumetric concentration) is redissolved, and whirlpool is mixed
To get sample to be tested after even.
(2) sample detection
Sample to be tested is transferred on maximum returnable bottle, then is installed into liquid chromatogram autosampler, liquid chromatogram is certainly
Sample is loaded into ultra performance liquid chromatography series connection quadrupole mass spectrometer analyzer (UPLC-MS/MS) to this by dynamic injector automatically
Sample to be tested is detected.
Autosampler automatically by the sample to be tested that 20uL has been purified in advance be loaded into ultra performance liquid chromatography system into
Row sample detaches, and the condition of ultra performance liquid chromatography is referring to table 1.The ultra high efficiency liquid of the ultra performance liquid chromatography tandem mass spectrometer
Phase chromatography acquisition window is 2.30-2.70min.Chromatographic column using Waters ACQUITY UPLC HSS T3Column, 2.1 ×
50mm,1.8μm；Chromatographic column column temperature is 40 DEG C.
1. ultra performance liquid chromatography condition of table
UPLC-MS/MS uses the Waters Xevo TQD tandem mass spectrometers with electron spray ionisation source (ESI) as inspection
It surveys device and carries out analysis detection.Wherein, capillary voltage 2KV, ion source temperature are 150 DEG C, and desolvation temperature is 350 DEG C,
Taper hole gas velocity is 50L/Hr, Desolvention gas velocity 1000L/Hr, and desolventizing gas, taper hole gas are high pure nitrogen；Collision gas
For high-purity argon gas.It is scanned using multiple-reaction monitoring ion scan pattern MRM, (monitoring ion pair is (qualitative and quantitative for condition
Ion pair), orifice potential and impact energy) referring to table 2.
Table 2
After sample is detected with mobile phase discharge column outlet, enter mass spectrometer ion source or useless under the effect of the pressure
Liquid by six-way valve control into the sample channel of ion source, and enters switching time.Fluid sample is vaporized simultaneously in ion source
And ionization is charged ion, charged ion is under voltage and vacuum action, into Q1, Q2 and Q3, wherein Q1 and Q3 is quality mistake
Filter only allows to be tieed up according to 25(OH)VD 2,25(OH)VD 3,3d-25 hydroxy vitamin D2s and 6d-25 hydroxyls to give birth to
The parent ion and daughter ion of the mass-to-charge ratio selection of plain D3 pass through, and Q2 is collision cell, and parent ion is touched with intert-gas atoms here
It hits, generates specific fragment ion.
Mass spectrometric first quadrupole (Q1) selection is with 25(OH)VD 2,25(OH)VD 3,3d-25 hydroxyls
The ion of the specific mass-to-charge ratio m/z of calciferol and 6d-25 hydroxycholecalciferols, with these m/z than ion be allowed into
Enter Q2, the fragment ion that Q2 is generated enters Q3, wherein only 25(OH)VD 2,25(OH)VD 3,3d-25 hydroxyls
The fragment ion of calciferol and 6d-25 hydroxycholecalciferols is selected to pass through, and other ions are removed.Be used for differentiate and
The mass-to-charge ratio of quantitative 25(OH)VD is referring to above-mentioned table 2.
As ion and detector collide, the number of ions captured is converted to the electronic impulse of digital signal by they.Institute
The data of acquisition are passed to computer, and collected number of ions is plotted against time to get mass spectrogram.
(3) Analysis of test results
(3.1) it is qualitatively judged using retention time and relative ion abundance ratio.
Retention time and 25(OH)VD 2 and 25 hydroxyls according to 25(OH)VD 2 and 25(OH)VD 3
The relative ion abundance ratio of base vitamine D3, judges the presence of 25(OH)VD 2 and 25(OH)VD 3.Specifically,
When detecting 25(OH)VD 2, according to 25(OH)VD 2 relative retention time and 25(OH)VD 2 it is opposite
Presence of the abundance of ions than judging 25(OH)VD 2；When detecting 25(OH)VD 3, according to 25(OH)VD 3
Relative retention time and 25(OH)VD 3 presence of the relative ion abundance than judging 25(OH)VD 3；When simultaneously
When detecting both objects, D3 relative retention times and 25(OH)VD 2 and D3 according to 2 sum of 25(OH)VD
Presence of the relative ion abundance than judging 25(OH)VD 2 and D3.
Sample is qualitatively judged with ultra performance liquid chromatography tandem mass spectrometer, under the conditions of same detection, in sample
Measured target substance chromatographic peak retention time and tie substance chromatographic peak retention time in standard solution are almost the same；Sample to be tested
Chromatogram selected in detection ion pair relative ion abundance than the relative ion abundance with suitable concentration standard solution
The deviation of ratio is no more than preset rules range, then may determine that there are corresponding target substances in sample.Wherein, 25 hydroxyl
The preset range of calciferol is：0.57 ± 20%；The preset range of 25(OH)VD 3 is：0.24 ± 25%.Wherein, institute
State relative ion abundance than the peak area of the qualitative ion pair for 25(OH)VD 2 or 25(OH)VD 3 with it is quantitative from
The ratio of the peak area of son pair.
In the present embodiment, the chromatogram of human serum sample is as shown in Figure 1,25(OH)VD 2 and 25(OH)VD 3
Hybrid standard working solution (standard working solution：Pipette 2 standard of the 25(OH)VD storage of suitable a concentration of 5 μ g/mL
3 standard reserving solution of 25(OH)VD of standby liquid and a concentration of 10 μ g/mL, with methanol dilution to aimed concn；Internal standard work is molten
Liquid：Pipette the 6d-25 hydroxyls of the 3d-25 hydroxy vitamin D2s standard reserving solution and a concentration of 1 μ g/mL of suitable a concentration of 1 μ g/mL
Base vitamine D3 standard reserving solution, with methanol dilution to aimed concn；25(OH)VD 2 in the hybrid standard working solution
Final concentration with 25(OH)VD 3 is 25ng/mL, the end of 3d-25 hydroxy vitamin D2s and 6d-25 hydroxycholecalciferols
Concentration is 50ng/mL) chromatogram it is as shown in Figure 2.From the result of Fig. 1 and Fig. 2：25 under the testing conditions of the present embodiment
The retention time of hydroxy vitamin D2 is 2.54min；The retention time of 25(OH)VD 3 is 2.48min；The leted others have a look at blood of Fig. 1
The relative ion abundance of 25(OH)VD 2 is 25 hydroxy vitamins in standard working solution shown in 0.555, Fig. 2 in final proof product
The relative ion abundance of D2 is 0.624；The relative ion abundance of 25(OH)VD 3 is in human serum sample shown in Fig. 1
The relative ion abundance of 25(OH)VD 3 is 0.226 in standard working solution shown in 0.237, Fig. 2.From the knot of Fig. 1 and Fig. 2
Fruit can determine whether that there are 25(OH)VDs 2 and 25(OH)VD 3 in the human serum sample of the present embodiment.
In the present embodiment, the detection mass spectrogram of human serum sample is as shown in figure 3,25(OH)VD 2 and the dimension life of 25 hydroxyls
Hybrid standard working solution (the standard working solution of plain D3：The 25(OH)VD 2 for pipetting suitable a concentration of 5 μ g/mL is marked
3 standard reserving solution of 25(OH)VD of quasi- storing solution and a concentration of 10 μ g/mL, with methanol dilution to aimed concn；Internal standard work
Make solution：Pipette the 6d- of the 3d-25 hydroxy vitamin D2s standard reserving solution and a concentration of 1 μ g/mL of suitable a concentration of 1 μ g/mL
3 standard reserving solution of 25(OH)VD, with methanol dilution to aimed concn, 25 hydroxyls dimension is given birth in the hybrid standard working solution
The final concentration of 25ng/mL of plain D2 and 25(OH)VD 3,3d-25 hydroxy vitamin D2s and 6d-25 hydroxycholecalciferols
Final concentration of 50ng/mL) detection mass spectrogram it is as shown in Figure 4.From the result of Fig. 3 and Fig. 4：Human serum sample shown in Fig. 3
In the mass-to-charge ratio that detects be respectively：25(OH)VD 3 (m/z=401.29), 6d-25 hydroxycholecalciferols (m/z=
407.35), 25(OH)VD 2 (m/z=413.29), 3d-25 hydroxy vitamin D2s (m/z=416.35)；The indicating of Fig. 4 institutes
The mass-to-charge ratio detected in quasi- working solution is respectively：25(OH)VD 3 (m/z=401.29), 6d-25 hydroxy vitamins
D3 (m/z=407.35), 25(OH)VD 2 (m/z=413.29), 3d-25 hydroxy vitamin D2s (m/z=416.35).From
The result of Fig. 3 and Fig. 4 can determine whether that there are 25(OH)VDs 2 and 25(OH)VD 3 in the human serum sample of the present embodiment
Detection signal.
(3.2) quantitative calculating is carried out using calibration curve method.
Peak area ratio according to 25(OH)VD 2 and 25(OH)VD 3 with corresponding internal standard compound on standard curve
Value calculates the content of the 25(OH)VD 2 and 25(OH)VD 3 in human serum sample.Specifically, when 25 hydroxyls of detection
When base calciferol, the peak area ratio according to 25(OH)VD 2 with corresponding internal standard compound on standard curve calculates people's blood
The content of 25(OH)VD 2 in final proof product.When detecting 25(OH)VD 3, according to 25(OH)VD 3 with it is right
Peak area ratio of the internal standard compound on standard curve is answered, the content of the 25(OH)VD 3 in human serum sample is calculated.When same
When detection 25(OH)VD 2 and when 25(OH)VD 3, according to 25(OH)VD 2 and 25(OH)VD 3 with it is right
Peak area ratio of the internal standard compound on standard curve is answered, the 25(OH)VD 2 in human serum sample and the dimension life of 25 hydroxyls are calculated
The content of plain D3.Content of the sum of the content using 25(OH)VD 2 and 25(OH)VD 3 as 25(OH)VD.
The structure of (3.2.1) standard curve
The preparation of standard working solution：Pipette suitable a concentration of 5 μ g/mL 2 standard reserving solution of 25(OH)VD and
3 Standard Stock solutions of 25(OH)VD of a concentration of 10 μ g/mL, with methanol dilution to aimed concn.
The preparation of internal standard working solution：Pipette the 3d-25 hydroxy vitamin D2 standard inventories of suitable a concentration of 1 μ g/mL
The 6d-25 hydroxycholecalciferol standard reserving solutions of liquid and a concentration of 1 μ g/mL, with methanol dilution to aimed concn.
The drafting of standard curve：Standard working solution and internal standard working solution are added separately in bare substrate, with this
The method of embodiment step (1) and step (2) carries out sample pre-treatments, upper machine testing.Each concentration point waits in standard working curve
The final concentration of analyte is respectively：1ng/mL, 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 75ng/mL, 100ng/mL,
Interior target is final concentration of：50ng/mL.
By the ratio of the peak area of the analysans and internal standard compound of each concentration point in standard working curve for building standard
Curve, with a concentration of x-axis of analysans, passes through using the ratio of analysans and the detection peak area of internal standard compound as y-axis
The software TargetLynx of Waters companies analyzes detection signal, standard curve is built, then in terms of the standard curve
Calculate the concentration of analyte in sample to be tested or quality control substance.Its canonical plotting is as shown in Figure 5.
(3.2.2) is calculated and result
Standard curve：
25(OH)VD 2：Y=0.135947*x+0.0689148, r2=0.998132；
25(OH)VD 3：Y=0.237172*x+0.050634, r2=0.998298.
The testing result of the present embodiment：
The peak area that 25(OH)VD 2 can be obtained from Fig. 1 results is the peak face of 1298.491,3d-25 hydroxy vitamin D2s
Product is 1543.691, i.e. the ratio of the peak area of 25(OH)VD 2 and internal standard peak area is 2.103, and it is public to substitute into standard curve
Formula calculates to obtain a concentration of 15.0ng/mL of 25(OH)VD 2 in the blood serum sample；25 hydroxy vitamins can be obtained from Fig. 1 results
The peak area of D3 is that the peak area of 3853.525,6d-25 hydroxycholecalciferols is 2829.498, the i.e. peak of 25(OH)VD 3
The ratio of area and internal standard peak area is 3.405, substitutes into calibration curve formula and calculates to obtain 25 hydroxy vitamins in the blood serum sample
A concentration of 14.1ng/mL of D3.
It based on the experiment condition of embodiment 1, is verified by test of many times, the precision RSD of the detection method<15%, inspection
Survey time average out to 5min.
2 methodology validation of embodiment
1, linear
Verification method：
The preparation of standard working solution：Pipette suitable a concentration of 5 μ g/mL 2 standard reserving solution of 25(OH)VD and
3 Standard Stock solutions of 25(OH)VD of a concentration of 10 μ g/mL, with methanol dilution to aimed concn.
The preparation of internal standard working solution：Pipette the 3d-25 hydroxy vitamin D2 standard inventories of suitable a concentration of 1 μ g/mL
The 6d-25 hydroxycholecalciferol standard reserving solutions of liquid and a concentration of 1 μ g/mL, with methanol dilution to aimed concn.
The drafting of standard curve：Standard working solution and internal standard working solution are added separately in bare substrate, then
Sample pre-treatments are carried out, upper machine testing, experiment condition is the same as embodiment 1.The analysans of each concentration point in standard working curve
Final concentration is respectively：1ng/mL、5ng/mL、10ng/mL、25ng/mL、50ng/mL、75ng/mL、100ng/mL.Interior target is whole
It is a concentration of：50ng/mL.
By the ratio of the peak area of the analysans and internal standard compound of each concentration point in standard working curve for building standard
Curve, with a concentration of x-axis of analysans, passes through using the ratio of analysans and the detection peak area of internal standard compound as y-axis
The software TargetLynx of Waters companies analyzes detection signal, builds standard curve and calculates the phase of the standard curve
Relationship number r2。
It based on the experiment condition of embodiment 1, is repeatedly tested, test every time all rebuilds standard curve, with standard
The correlation coefficient r of curve2Evaluation criterion curve it is linear.
Verification result：
Standard curve 1：
25(OH)VD 2：Y=0.180168*x+0.049236；r2=0.998373；
25(OH)VD 3：Y=0.253746*x+0.0857472；r2=0.998901.
Standard curve 2：
25(OH)VD 2：Y=0.174125*x+0.0766472；r2=0.998638；
25(OH)VD 3：Y=0.257958*x+0.0820327；r2=0.998653.
Standard curve 3：
25(OH)VD 2：Y=0.173406*x+0.0387316；r2=0.999395；
25(OH)VD 3：Y=0.265267*x+0.00307133；r2=0.999587.
Standard curve 4：
25(OH)VD 2：Y=0.169681*x+0.0278474；r2=0.999811；
25(OH)VD 3：Y=0.239016*x+0.0530473；r2=0.999266.
The related coefficient that can be seen that the standard curve of test structure every time from the above experimental data is all higher than 0.99, full
Sufficient requirement of experiment.
2, withinrun precision and batch interior accuracy test：
Verification method：
The preparation of standard working solution：Pipette suitable a concentration of 5 μ g/mL 2 standard reserving solution of 25(OH)VD and
3 Standard Stock solutions of 25(OH)VD of a concentration of 10 μ g/mL, with methanol dilution to aimed concn.
The preparation of internal standard working solution：Pipette the 3d-25 hydroxy vitamin D2 standard inventories of suitable a concentration of 1 μ g/mL
The 6d-25 hydroxycholecalciferol standard reserving solutions of liquid and a concentration of 1 μ g/mL, with methanol dilution to aimed concn.
The preparation of recovery of standard addition test sample：Standard working solution and internal standard working solution are added separately to blank base
The final concentration that analysans is obtained in matter is respectively：2ng/mL, 40ng/mL, 80ng/mL, interior target are final concentration of：50ng/mL
Test sample, then carry out sample pre-treatments, upper machine testing, experiment condition is the same as embodiment 1.Simultaneously with the method for embodiment 1
Build standard curve.
Detection signal is analyzed by the software TargetLynx of Waters companies, mark-on is returned using standard curve
Yield test sample is calculated, and obtains the concentration of test sample.
Based on the experiment condition of embodiment 1, in same batch, while to the recovery of standard addition test sample of three concentration
It is tested, the recovery of standard addition test sample parallel testing of each concentration 6 times, by the recovery of standard addition for calculating test result
And the coefficient of variation, evaluate withinrun precision and batch interior accuracy of the detection method of the present invention.
Calculation formula：
Formula 1,
Formula 2,
Formula 3,
Formula 4,
(Indicate average value, unit ng/mL；∑ c indicates the sum of each parallel testing sample result；N indicates parallel testing
Quantity；csIndicate the setting concentration of recovery of standard addition sample, i.e. final concentration；SD indicates standard deviation, unit ng/mL；CV
Indicate the coefficient of variation, unit %)
Verification result：
The recovery of standard addition and coefficient of variation test result of low concentration point (2ng/mL)：
| Serial number | 25(OH)VD 2 | 25(OH)VD 3 |
| 1 | 1.8043 | 1.7673 |
| 2 | 2.1657 | 1.7794 |
| 3 | 1.8469 | 1.8233 |
| 4 | 1.9244 | 2.0126 |
| 5 | 1.6458 | 2.0261 |
| 6 | 1.7593 | 1.8619 |
| Average value (ng/mL) | 1.86 | 1.88 |
| Recovery of standard addition (%) | 92.89 | 93.92 |
| SD(ng/mL) | 0.18 | 0.11 |
| CV (%) | 9.53 | 6.08 |
The recovery of standard addition and coefficient of variation test result of middle concentration point (40ng/mL)：
| Serial number | 25(OH)VD 2 | 25(OH)VD 3 |
| 1 | 38.226 | 38.5492 |
| 2 | 38.0587 | 37.6959 |
| 3 | 38.7729 | 39.1063 |
| 4 | 39.0163 | 39.0362 |
| 5 | 39.0074 | 38.4376 |
| 6 | 37.6504 | 37.737 |
| Average value (ng/mL) | 38.46 | 38.43 |
| Recovery of standard addition (%) | 96.14 | 96.07 |
| SD(ng/mL) | 0.56 | 0.61 |
| CV (%) | 1.46 | 1.59 |
The recovery of standard addition and coefficient of variation test result of high concentration spot (80ng/mL)：
As can be seen from the above data, the recovery of standard addition of low concentration, middle concentration and high concentration spot 100 ± 15% with
Interior, the coefficient of variation is within 15%, it was demonstrated that the withinrun precision of detection method of the invention and batch interior accuracy are good.
3, betweenrun precision and batch between accuracy test：
Verification method：
The preparation of standard working solution：Pipette suitable a concentration of 5 μ g/mL 2 standard reserving solution of 25(OH)VD and
3 Standard Stock solutions of 25(OH)VD of a concentration of 10 μ g/mL, with methanol dilution to aimed concn.
The preparation of internal standard working solution：Pipette the 3d-25 hydroxy vitamin D2 standard inventories of suitable a concentration of 1 μ g/mL
The 6d-25 hydroxycholecalciferol standard reserving solutions of liquid and a concentration of 1 μ g/mL, with methanol dilution to aimed concn.
The preparation of recovery of standard addition test sample：Standard working solution and internal standard working solution are added separately to blank base
The final concentration that analysans is obtained in matter is respectively：2ng/mL, 40ng/mL, 80ng/mL, interior target are final concentration of：50ng/mL
Test sample, then carry out sample pre-treatments, upper machine testing, experiment condition is the same as embodiment 1.Simultaneously with the method for embodiment 1
Build standard curve.
Detection signal is analyzed by the software TargetLynx of Waters companies, mark-on is returned using standard curve
Yield test sample is calculated, and obtains the concentration of test sample.
Based on the experiment condition of embodiment 1, the test of 1 batch is carried out daily in for three days on end, each batch is right respectively
The recovery of standard addition test sample parallel testing of each concentration twice, calculates recovery of standard addition and the change of 3 batch experimental results
Different coefficient, accuracy between evaluating the betweenrun precision of the detection method of the present invention and criticizing.
Calculation formula：
Formula 1,
Formula 2,
Formula 3,
Formula 4,
(Indicate average value, unit ng/mL；∑ c indicates the sum of each parallel testing sample result；N indicates parallel testing
Quantity；csIndicate the setting concentration of recovery of standard addition sample, i.e. final concentration；SD indicates standard deviation, unit ng/mL；CV
Indicate the coefficient of variation, unit %)
Verification result：
The recovery of standard addition and coefficient of variation test result of low concentration point (2ng/mL)：
| Batch | 25(OH)VD 2 | 25(OH)VD 3 |
| 1 | 1.8937 | 1.7689 |
| 1 | 1.8359 | 1.885 |
| 2 | 2.0084 | 2.1198 |
| 2 | 2.0667 | 2.3212 |
| 3 | 1.7741 | 2.053 |
| 3 | 1.8833 | 2.0443 |
| Average value (ng/mL) | 1.91 | 2.03 |
| Recovery of standard addition (%) | 95.52 | 101.60 |
| SD(ng/mL) | 0.11 | 0.19 |
| CV (%) | 5.69 | 9.41 |
The recovery of standard addition and coefficient of variation test result of middle concentration point (40ng/mL)：
The recovery of standard addition and coefficient of variation test result of high concentration spot (80ng/mL)：
| Batch | 25(OH)VD 2 | 25(OH)VD 3 |
| 1 | 78.6563 | 78.0705 |
| 1 | 78.7858 | 78.9077 |
| 2 | 79.4095 | 93.9998 |
| 3 | 79.3335 | 86.11 |
| 3 | 81.1337 | 84.1695 |
| Average value (ng/mL) | 79.46 | 84.25 |
| Recovery of standard addition (%) | 99.33 | 105.31 |
| SD(ng/mL) | 0.99 | 6.43 |
| CV (%) | 1.25 | 7.63 |
As can be seen from the above data, the recovery of standard addition of low concentration, middle concentration and high concentration spot 100 ± 15% with
Interior, the coefficient of variation is within 15%, it was demonstrated that the betweenrun precision of detection method of the invention and batch between accuracy it is good.
4, lower limit of quantitation
Verification method：
The preparation of standard working solution：Pipette suitable a concentration of 5 μ g/mL 2 standard reserving solution of 25(OH)VD and
3 Standard Stock solutions of 25(OH)VD of a concentration of 10 μ g/mL, with methanol dilution to aimed concn.
The preparation of internal standard working solution：Pipette the 3d-25 hydroxy vitamin D2 standard inventories of suitable a concentration of 1 μ g/mL
The 6d-25 hydroxycholecalciferol standard reserving solutions of liquid and a concentration of 1 μ g/mL, with methanol dilution to aimed concn.
The preparation of recovery of standard addition test sample：Standard working solution and internal standard working solution are added separately to blank base
The final concentration of of analysans is obtained in matter：1ng/mL, interior target are final concentration of：Then the test sample of 50ng/mL carries out sample
Product pre-treatment, upper machine testing, experiment condition is the same as embodiment 1.Simultaneously standard curve is built with the method for embodiment 1.
Detection signal is analyzed by the software TargetLynx of Waters companies, mark-on is returned using standard curve
Yield test sample is calculated, and obtains the concentration of sample to be tested.
Based on the experiment condition of embodiment 1, continuously above-mentioned recovery of standard addition test sample is tested 6 times, calculates 6 experiments
As a result recovery of standard addition and the coefficient of variation evaluate lower limit of quantitation.
| Serial number | 25(OH)VD 2 | 25(OH)VD 3 |
| 1 | 0.9012 | 0.7629 |
| 2 | 0.8036 | 0.8482 |
| 3 | 0.7995 | 0.8516 |
| 4 | 0.8943 | 0.8613 |
| 5 | 1.0178 | 0.8194 |
| 6 | 0.9174 | 0.8355 |
| Average value (ng/mL) | 0.89 | 0.83 |
| Recovery of standard addition (%) | 88.90 | 82.98 |
| SD(ng/mL) | 0.08 | 0.04 |
| CV (%) | 9.12 | 4.32 |
As can be seen from the above data, within 100 ± 20%, the coefficient of variation exists the recovery of standard addition of lower limit of quantitation
Within 20%, it was demonstrated that the lower limit of quantitation of detection method of the invention can meet requirement of experiment, high sensitivity.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. the detection method of 25(OH)VD in a kind of human serum, which is characterized in that include the following steps：
Sample pre-treatments：Human serum sample is taken, the methanol solution containing 25(OH)VD internal standard compound is added, then to described
Zinc sulfate solution is added in human serum sample and methanol carries out albumen precipitation, n-hexane solvent is added after mixing well and is extracted
It takes, then is centrifuged after mixing well, be added after taking supernatant and being dried and redissolve liquid, obtain sample to be tested；
Sample detection：The sample to be tested is detected with ultra performance liquid chromatography series connection quadrupole mass spectrometer,
Wherein, the condition of ultra performance liquid chromatography includes：
Chromatographic column：Waters ACQUITY UPLC HSS T3 Column,2.1×50mm,1.8μm；
Chromatogram column temperature：37℃-43℃；
Sample size：18-22uL；
Flow velocity：0.42-0.48mL/min；
Mobile phase：Mobile phase A is the aqueous solution containing 1.8-2.2mM ammonium acetates and 0.09-0.11v/v% formic acid, and Mobile phase B is
Methanol solution containing 1.8-2.2mM ammonium acetates and 0..09-0.11v/v% formic acid, gradient elution；
The 25(OH)VD is 25(OH)VD 2 and/or 25(OH)VD 3, in the 25(OH)VD
It is 3d-25 hydroxy vitamin D2s and/or 6d-25 hydroxycholecalciferols to mark object.
2. the detection method of 25(OH)VD in human serum according to claim 1, which is characterized in that ultra high efficiency liquid
The condition of phase chromatography includes：
Chromatographic column：Waters ACQUITY UPLC HSS T3 Column,2.1×50mm,1.8μm；
Chromatogram column temperature：40℃；
Sample size：20uL；
Flow velocity：0.45mL/min；
Mobile phase：Mobile phase A is the aqueous solution containing 2mM ammonium acetates and 0.1% formic acid, Mobile phase B be containing 2mM ammonium acetates and
The methanol solution of 0.1% formic acid, gradient elution.
3. the detection method of 25(OH)VD in human serum according to claim 1, which is characterized in that the gradient
The condition of elution is as follows：
4. the detection method of 25(OH)VD in human serum according to claim 1, which is characterized in that the superelevation
The ultra performance liquid chromatography acquisition window of effect liquid phase chromatogram series connection quadrupole mass spectrometer is 2.30-2.70min.
5. according to the detection method of 25(OH)VD in claim 1-4 any one of them human serums, which is characterized in that
Mass Spectrometry Conditions include：
6. the detection method of 25(OH)VD in human serum according to claim 5, which is characterized in that the mass spectrum
Condition further includes：The detection ion pair of the 25(OH)VD 2 is 413.3>395.2 and 413.3>83.1, wherein 413.3>
395.2 be quota ion pair；The detection ion pair of the 25(OH)VD 3 is 401.3>383.2 and 401.3>159.1,
In 401.3>383.2 be quota ion pair；The quantitative detection ion pair of the 3d-25 hydroxy vitamin D2s is 416.35>
358.3；The quantitative detection ion pair of the 6d-25 hydroxycholecalciferols is 407.35>159.1.
7. the detection method of 25(OH)VD in human serum according to claim 6, which is characterized in that detection ion
It is as follows to corresponding orifice potential and collision energy：
8. according to the detection method of 25(OH)VD in claim 1-4 any one of them human serums, which is characterized in that
According to the retention time and relative ion abundance of 25(OH)VD 2 and/or 25(OH)VD 3 than judging people's blood
It whether there is 25(OH)VD 2 and/or 25(OH)VD 3 in final proof product, the relative ion abundance ratio is each to be measured
The ratio of the peak area of the qualitative ion pair of object and the peak area of quota ion pair, the object to be measured object include 25 hydroxyls
Calciferol and/or 25(OH)VD 3.
9. according to the detection method of 25(OH)VD in claim 1-4 any one of them human serums, which is characterized in that
A concentration of 248-252ng/mL of the methanol solution containing 25(OH)VD internal standard compound, and/or, the zinc sulfate water
A concentration of 0.15-0.25mol/L of solution；And/or
Described to redissolve the mixture that liquid is first alcohol and water, the volume ratio of the first alcohol and water is 63-67：33-37.
10. the detection method of 25(OH)VD in human serum according to claim 9, which is characterized in that people's blood
The volume ratio of final proof product and the methanol solution containing 25(OH)VD internal standard compound is 1：0.15-0.25；And/or
The human serum sample and the volume ratio of the solution of zinc sulfate, methanol, n-hexane solvent are 1：1：1.8-2.2：8-12；
And/or
The volume ratio of the supernatant and n-hexane solvent is 1：1.1-1.3；And/or
The liquid and the volume ratio of the human serum sample of redissolving is 2-3：2.
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| CN109358148A (en) * | 2018-09-20 | 2019-02-19 | 杭州和合医学检验实验室有限公司 | The method of inspection of vitamin D in blood |
| 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 |
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