CN115356408A - Method for detecting 25-hydroxy vitamin D in serum - Google Patents
Method for detecting 25-hydroxy vitamin D in serum Download PDFInfo
- Publication number
- CN115356408A CN115356408A CN202210895724.1A CN202210895724A CN115356408A CN 115356408 A CN115356408 A CN 115356408A CN 202210895724 A CN202210895724 A CN 202210895724A CN 115356408 A CN115356408 A CN 115356408A
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- detecting
- detection method
- serum
- liquid chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002966 serum Anatomy 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 22
- JWUBBDSIWDLEOM-XHQRYOPUSA-N (3e)-3-[(2e)-2-[1-(6-hydroxy-6-methylheptan-2-yl)-7a-methyl-2,3,3a,5,6,7-hexahydro-1h-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol Chemical compound C1CCC2(C)C(C(CCCC(C)(C)O)C)CCC2\C1=C\C=C1/CC(O)CCC1=C JWUBBDSIWDLEOM-XHQRYOPUSA-N 0.000 title claims abstract description 18
- 235000021318 Calcifediol Nutrition 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 36
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 8
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims abstract description 5
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000003550 marker Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 5
- 239000012491 analyte Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000011159 matrix material Substances 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 239000013076 target substance Substances 0.000 claims description 3
- 238000004807 desolvation Methods 0.000 claims 1
- 239000007921 spray Substances 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 description 10
- 150000002500 ions Chemical class 0.000 description 6
- 239000003643 water by type Substances 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 241000282412 Homo Species 0.000 description 2
- 229930003316 Vitamin D Natural products 0.000 description 2
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 235000019166 vitamin D Nutrition 0.000 description 2
- 239000011710 vitamin D Substances 0.000 description 2
- 150000003710 vitamin D derivatives Chemical class 0.000 description 2
- 229940046008 vitamin d Drugs 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- WRLFSJXJGJBFJQ-WPUCQFJDSA-N Calcifediol monohydrate Chemical compound O.C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C\C=C1\C[C@@H](O)CCC1=C WRLFSJXJGJBFJQ-WPUCQFJDSA-N 0.000 description 1
- UCTLRSWJYQTBFZ-UHFFFAOYSA-N Dehydrocholesterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCCC(C)C)CCC33)C)C3=CC=C21 UCTLRSWJYQTBFZ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8877—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample optical isomers
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Library & Information Science (AREA)
- Engineering & Computer Science (AREA)
- Dispersion Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of chemical detection, and discloses a method for detecting 25-hydroxy vitamin D in serum. The detection method adopts liquid chromatography tandem mass spectrometry for detection, and the conditions of the liquid chromatography comprise: the chromatographic column is UPLC ACQUITY TM HSS PFP column; the mobile phase A is 0.1-0.5% formic acid water solution, and the mobile phase B is methanol. The method has strong specificity and high accuracy, and is suitable for detecting 25-hydroxy vitamin D in serum samples of people of all ages.
Description
Technical Field
The invention belongs to the technical field of chemical detection, and particularly relates to a method for detecting 25-hydroxy vitamin D in serum.
Background
Vitamin D (hereinafter VD) is a fat-soluble steroid derivative, is a vitamin essential to human bodies and mainly comprises two forms of VD2 and VD 3. VD can be converted in humans by sunlight or ingested from food, for example, 7-dehydrocholesterol stored under the skin can be converted to VD3 by ultraviolet irradiation in sunlight, while VD2 can be supplemented by food. VD2 and VD3 in human bodies are metabolized into 25-OH VD2 and 25-OH VD3 through livers, the concentration of 25-OH VD2 and 25-OH VD3 in blood is high, the main existing form of VD in the human bodies is obtained, the stability is good, the half life period of circulation in the human bodies is about 2-3 weeks, and therefore the 25-OH VD2 and 25-OH VD3 in the blood become the most reliable indexes for evaluating the nutrition state of the VD in the human bodies.
However, the epimer of 25-OH VD3, 3-epi 25-OH VD3, is also present in humans, accounting for approximately 4% of the total 25-OH VD, and is present in lower amounts in adults, but in very high concentrations in newborns, accounting for approximately 60% of the total 25-OH VD. 3-epi 25-OH VD3 has no biological activity, and if the substance cannot be effectively separated in the detection process, the detection result of the 25-OH VD3 has a higher error.
The traditional vitamin D detection method comprises a radioimmunoassay, a competitive protein binding method, a high performance liquid chromatography and the like, wherein the radioimmunoassay and the competitive protein binding method have the defects of complex pretreatment, long analysis time, low flux and poor method specificity, and the content of 25-OH VD2 and 25-OH VD3 cannot be accurately quantified simultaneously. At present, the LC-MS/MS is universally considered as the 'gold standard' for detecting 25-hydroxy vitamin D in serum internationally, however, the currently used method still cannot effectively separate epimers, and the detection result has deviation.
Therefore, the invention hopes to establish a method for detecting 25 hydroxy vitamin D in human serum, which is simple and convenient to operate, high in specificity and good in sensitivity.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a method for detecting 25 hydroxyvitamin D in serum, which has strong specificity and high accuracy and is suitable for detecting 25 hydroxyvitamin D in serum samples of people of all ages.
The invention provides a method for detecting 25-hydroxy vitamin D in serum, which adopts liquid chromatography tandem mass spectrometry (LC-MS/MS) to detect, wherein the conditions of the liquid chromatography comprise: the chromatographic column is UPLC ACQUITY TM HSS PFP column; the mobile phase A is 0.1-0.5% formic acid water solution, and the mobile phase B is methanol.
The invention provides that the mobile phase system is combined with a specific chromatographic column type, so that the effective separation of 25-OH VD2, 25-OH VD3 and 3-epi 25-OH VD3 can be realized, and the detection accuracy of 25-hydroxy vitamin D in serum is improved.
Preferably, the size of the column is 2.1mm x 50mm,1.8 μm.
Preferably, the liquid chromatography is performed by gradient elution.
More preferably, the conditions of the gradient elution are:
the volume fraction of the mobile phase B is changed from 70% to 95% in 0-4 min;
4-4.01min, the volume fraction of the mobile phase B is changed from 95% to 70%;
4.01-4.5min, and the volume fraction of the mobile phase B is 70%.
Preferably, the conditions of the liquid chromatography further comprise: the flow rate is 0.4mL/min; the column temperature is 40 ℃; the sample amount is 2-10 μ L.
Preferably, the conditions of the mass spectrum include: ESI source positive ion mode, multi-reaction monitoring scanning mode; spraying voltage: 1.5-2.5kV; the temperature of the solvent removal: 450-550 ℃; the flow rate of the atomizer is 800-1000L/h; ion source temperature: 120-150 ℃; taper hole air flow velocity: 55-65L/hr.
Preferably, the detection parameters of the mass spectrum are as follows:
| target analyte | Q1(Da) | Q3(Da) | CV(V) | CE(V) |
| 25-OH VD2 | 413.3 | 113.1 | 20 | 10 |
| 25-OH VD3 | 383.3 | 257.2 | 20 | 15 |
| 25-OH VD2_IS | 416.4 | 113.1 | 20 | 10 |
| 25-OH VD3_IS | 386.3 | 257.2 | 20 | 15 |
Preferably, the detection method further comprises:
preparing a calibrator solution containing target substances with different concentration gradients by taking BSA as a matrix, adding an isotope marker for pretreatment, and detecting by using LC-MS/MS; taking the concentration of the target in the calibrator solution as an X axis, and taking the peak area ratio of the target to the corresponding isotope marker in the calibrator solution as a Y axis, and drawing to obtain a standard working curve;
adding an isotope marker into a serum sample to be detected, pretreating, detecting by using LC-MS/MS, and obtaining the content of 25 hydroxyvitamin D in the serum sample to be detected according to a standard working curve.
Compared with the prior art, the invention has the following beneficial effects:
(1) The detection method provided by the invention has strong specificity and high accuracy, can realize the separation of 25-OH VD3 and epimer 3-epi 25-OH VD3 thereof, and is suitable for the detection of 25-hydroxy vitamin D in serum samples of people of all ages;
(2) The detection method provided by the invention is simple to operate, strong in operability, short in analysis time and capable of realizing high-throughput detection.
Drawings
FIG. 1 shows the results of the serum samples of children in example 2;
FIG. 2 is a graph showing the results of detection in control 1 in example 3;
FIG. 3 is a graph showing the results of detection in control group 2 of example 3;
FIG. 4 is the test results of the experimental group in example 3.
Detailed Description
In order to make the technical solutions of the present invention more clearly apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
Example 1
The embodiment provides a method for detecting 25 hydroxy vitamin D in serum, which adopts liquid chromatography tandem mass spectrometry (LC-MS/MS) to detect; wherein,
the liquid chromatography conditions were:
the chromatographic column is UPLC ACQUITY TM HSS PFP column(2.1mm×50mm,1.8μm,Waters)。
The liquid chromatography tandem mass spectrometry (LC-MS/MS) detection instrument is a Waters UPLC Xevo TQ-S Micro IVD.
Mobile phase a was 0.1% aqueous formic acid and mobile phase B was methanol.
Gradient elution is adopted, and the conditions of the gradient elution are as follows:
the volume fraction of the mobile phase B is changed from 70% to 95% in 0-4 min;
4-4.01min, the volume fraction of the mobile phase B is changed from 95% to 70%;
4.01-4.5min, and the volume fraction of the mobile phase B is 70%.
The flow rate is 0.4mL/min; the column temperature was 40 ℃; the amount of sample was 5. Mu.L.
The mass spectrum conditions are as follows:
under an ESI source positive ion mode, a multi-reaction monitoring scanning mode (MRM) is adopted; spraying voltage: 2.0kV; the temperature of the solvent removal: 500 ℃; the flow rate of the atomizer is 800L/h; ion source temperature: 150 ℃; taper hole air flow velocity: 65L/hr;
parameters of parent ions Q1, daughter ions Q3, declustering voltage CV and collision energy voltage CE of target analytes (25-OH VD2, 25-OH VD3 and isotopic markers thereof) are shown in Table 1.
TABLE 1 Mass spectrometric parameters
| Target analyte | Q1(Da) | Q3(Da) | CV(V) | CE(V) |
| 25-OH VD2 | 413.3 | 113.1 | 20 | 10 |
| 25-OH VD3 | 383.3 | 257.2 | 20 | 15 |
| 25-OH VD2_IS | 416.4 | 113.1 | 20 | 10 |
| 25-OH VD3_IS | 386.3 | 257.2 | 20 | 15 |
Mass spectrometry acquisition and analysis were performed using MassLynx V4.2 software.
The specific detection steps are as follows:
1. pretreatment of a sample to be detected: transferring 100 mu L of serum sample to be detected, adding 400 mu L of acetonitrile containing an internal standard (isotope label), and vortexing for 2min; centrifuging at 4 deg.C for 5min, and collecting 100 μ L supernatant; adding 20 mu L of ultrapure water, uniformly mixing, and performing LC-MS/MS detection;
2. drawing a standard curve: preparing a calibrator solution containing target substances with different concentration gradients by using 5-percent BSA as a matrix, performing the same pretreatment as the serum sample, and performing LC-MS/MS detection; taking the concentration of the target in the calibrator solution as an X axis, and taking the peak area ratio of the target in the calibrator solution to the corresponding isotope marker as a Y axis, and drawing to obtain a standard working curve;
3. and calculating to obtain the concentrations of 25-OH-VD2 and 25-OH-VD3 in the serum sample to be detected according to the standard working curve and the LC-MS/MS detection result of the serum sample to be detected.
Example 2
By adopting the detection method in the embodiment 1 to detect the child serum sample, as can be seen from fig. 1, the detection method in the embodiment 1 can realize effective separation of 25-OH VD3 and epimer 3-epi 25-OH VD3 thereof, thereby improving the detection accuracy of 25-hydroxy vitamin D.
Example 3: column type comparison
In order to compare the influence of the chromatographic column type on the separation effect of 25-OH VD3 and the epimer 3-epi 25-OH VD3 thereof, a serum sample containing 3-epi 25-OH VD3 is taken as an experimental object, the detection method of example 1 is basically adopted, and the following 3 chromatographic columns are respectively used for testing the sample, and the single-standard peak time of 3-epi 25-OH VD3 is detected simultaneously, so that the separation condition of 25-OH VD3 and the epimer 3-epi 25-OH VD3 thereof is determined.
Control group 1: BEH C18 (2.1 mm. Times.50mm, 1.7 μm, waters);
control group 2: BEH Phenyl column (2.1 mm. Times.50mm, 1.7 μm, waters);
experimental groups: HSS PFP column (2.1 mm. Times.50mm, 1.8 μm, waters);
the results in FIGS. 2-4 show that the peak-off times for 25-OH VD3 and its epimer 3-epi 25-OH VD3 in control 1 and control 2 were consistent and separation of 25-OH VD3 from its epimer 3-epi 25-OH VD3 could not be achieved; and the experimental group can achieve good separation effect. The above results show that the type of column plays an important role in the separation of 25-OH VD3 from its epimer, 3-epi 25-OH VD3, and that good separation is only achieved with a specific column type in combination with the flow in example 1.
Example 4: performance evaluation of detection methods
1. Linear regression equation and linear correlation coefficient
The method described in example 1 was used to establish a working curve, the results are shown in table 2, the target analyte standard curve has good linearity, the correlation coefficient is above 0.99, and the quantitative requirements are met.
TABLE 2 Linear regression equation and Linear correlation coefficient
2. Quantitative NIST-SRM972a accuracy assessment
The detection method of example 1 is adopted to carry out quantitative detection on SRM972a (NIST in America) frozen human serum and compare with a target value, and the results of Table 3 show that the accuracy of 25-OH VD2 and 25-OH VD3 in Level3 is 100.83 percent and 93.38 percent respectively; the accuracy of 25-OH VD3 in Level4 is 93.03% (if the Level4 contains 26.0ng/mL of 3-epi 25-OH VD3, the quantitative result is high if the separation cannot be carried out), and the result shows that the accuracy is high by adopting the method in the example 1.
TABLE 3 quantitative results for NIST samples
3. Quantitative normal human serum sample accuracy evaluation
The detection method of the embodiment 1 is adopted to evaluate the low, medium and high spiked recovery rates of the serum of the normal person, and the results of the table 4 show that the spiked recovery rate is between 89.22% and 96.39%, and can meet the quantitative requirement.
TABLE 4 recovery of normal human serum spiked
4. Investigation of precision
The normal human serum is taken to carry out the labeling of low, medium and high concentrations, 6 samples in each group are parallel and are continuously processed for three days, the intra-day precision and the inter-day precision are calculated, and the results are shown in Table 5.
TABLE 5 precision within day and day
As can be seen from the performance verification results, the method provided in example 1 has good accuracy and precision, strong specificity, and is suitable for detecting 25 hydroxyvitamin D in serum samples of people of all ages.
The embodiments of the present application have been described in detail with reference to the drawings, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
Claims (8)
1. The method for detecting 25 hydroxy vitamin D in serum is characterized by adopting liquid chromatography tandem mass spectrometry to detect, wherein the conditions of the liquid chromatography comprise: the chromatographic column is UPLC ACQUITY TM HSS PFP column; the mobile phase A is 0.1-0.5% formic acid water solution, and the mobile phase B is methanol.
2. The method of claim 1, wherein the size of the column is 2.1mm x 50mm,1.8 μm.
3. The detection method according to claim 1, wherein the liquid chromatography employs a gradient elution method.
4. The detection method according to claim 3, wherein the conditions of the gradient elution are:
the volume fraction of the mobile phase B is changed from 70% to 95% in 0-4 min;
4-4.01min, the volume fraction of the mobile phase B is changed from 95% to 70%;
4.01-4.5min, and the volume fraction of the mobile phase B is 70%.
5. The detection method according to claim 1, wherein the conditions of the liquid chromatography further comprise: the flow rate is 0.4mL/min; the column temperature was 40 ℃; the sample amount is 2-10 μ L.
6. The detection method according to claim 1, wherein the conditions of the mass spectrum comprise: ESI source positive ion mode, multi-reaction monitoring scanning mode; spray voltage: 1.5-2.5kV; the temperature of the desolvation: 450-550 ℃; the flow rate of the atomizer is 800-1000L/h; ion source temperature: 120-150 ℃; taper hole air flow rate: 55-65L/hr.
7. The method of claim 1, wherein the detection parameters of the mass spectrum are as follows:
8. The detection method according to claim 1, characterized in that the detection method further comprises:
preparing a calibrator solution containing target substances with different concentration gradients by taking BSA as a matrix, adding an isotope marker for pretreatment, and detecting by using LC-MS/MS; taking the concentration of the target in the calibrator solution as an X axis, and taking the peak area ratio of the target to the corresponding isotope marker in the calibrator solution as a Y axis, and drawing to obtain a standard working curve;
adding an isotope marker into a serum sample to be detected, pretreating, detecting by using LC-MS/MS, and obtaining the content of 25 hydroxyvitamin D in the serum sample to be detected according to a standard working curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210895724.1A CN115356408A (en) | 2022-07-27 | 2022-07-27 | Method for detecting 25-hydroxy vitamin D in serum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210895724.1A CN115356408A (en) | 2022-07-27 | 2022-07-27 | Method for detecting 25-hydroxy vitamin D in serum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115356408A true CN115356408A (en) | 2022-11-18 |
Family
ID=84031379
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210895724.1A Pending CN115356408A (en) | 2022-07-27 | 2022-07-27 | Method for detecting 25-hydroxy vitamin D in serum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115356408A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106526026A (en) * | 2016-11-10 | 2017-03-22 | 广州博厚医疗技术有限公司 | Method for detection of 25-hydroxyvitamin D in serum |
| CN111208248A (en) * | 2020-01-14 | 2020-05-29 | 南京品生医学检验实验室有限公司 | Method for detecting 5 fat-soluble vitamins in serum by ultra-high performance liquid chromatography tandem mass spectrometry |
| US20210088486A1 (en) * | 2018-01-29 | 2021-03-25 | Dh Technologies Development Pte. Ltd. | Methods and systems for detection of vitamin d metabolites |
| CN112557539A (en) * | 2020-12-02 | 2021-03-26 | 成都和合医学检验所有限公司 | Method for simultaneously determining vitamin A, vitamin E and 25-hydroxyvitamin D in blood plasma |
-
2022
- 2022-07-27 CN CN202210895724.1A patent/CN115356408A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106526026A (en) * | 2016-11-10 | 2017-03-22 | 广州博厚医疗技术有限公司 | Method for detection of 25-hydroxyvitamin D in serum |
| US20210088486A1 (en) * | 2018-01-29 | 2021-03-25 | Dh Technologies Development Pte. Ltd. | Methods and systems for detection of vitamin d metabolites |
| CN111208248A (en) * | 2020-01-14 | 2020-05-29 | 南京品生医学检验实验室有限公司 | Method for detecting 5 fat-soluble vitamins in serum by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN112557539A (en) * | 2020-12-02 | 2021-03-26 | 成都和合医学检验所有限公司 | Method for simultaneously determining vitamin A, vitamin E and 25-hydroxyvitamin D in blood plasma |
Non-Patent Citations (4)
| Title |
|---|
| JOHANNES M.W. VAN DEN OUWELAND: "Overestimation of 25-hydroxyvitamin D3 by increased ionisationefficiency of 3-epi-25-hydroxyvitamin D3 in LC–MS/MS methods notseparating both metabolites as determined by an LC–MS/MS methodfor separate quantification of 25-hydroxyvitamin D3,3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in huma", JOURNAL OF CHROMATOGRAPHY B, vol. 967 * |
| YANG YIFEI ET AL.: "High-throughput measurement of 25-hydroxyvitamin D by LC–MS/MS with separation of the C3-epimer interference for pediatric populations", CLINICA CHIMICA ACTA, vol. 454, pages 102 - 106, XP029400062, DOI: 10.1016/j.cca.2016.01.004 * |
| 尹丹阳 等: "超高效液相色谱-串联质谱法测定血清中25-羟基维生素D和维生素K1", 卫生研究, vol. 49, no. 4, pages 597 - 602 * |
| 王睿;付志成;胡贻椿;毛德倩;杨晓光;陈竞;杨丽琛;: "超高效液相色谱――串联质谱法同时测定血清中维生素A、维生素E", 中国食物与营养, no. 09 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Du et al. | Simultaneous determination of 10 β2-agonists in swine urine using liquid chromatography–tandem mass spectrometry and multi-walled carbon nanotubes as a reversed dispersive solid phase extraction sorbent | |
| CN110487943B (en) | Method for detecting fat-soluble vitamins in blood sample | |
| Wang et al. | Solid phase microextraction combined with thermal-desorption electrospray ionization mass spectrometry for high-throughput pharmacokinetics assays | |
| BRPI0819605B1 (en) | methods for detecting dihydroxyvitamin d metabolites by mass spectrometry | |
| CN113125601B (en) | Method for simultaneously detecting concentrations of 4 fat-soluble vitamins in serum | |
| CN106841427B (en) | A kind of tandem mass spectrum kit detecting PKU and CAH | |
| Doerge et al. | Detection and confirmation of β-agonists in bovine retina using LC/APCI-MS | |
| CA3058642A1 (en) | Methods for quantitation of insulin and c-peptide | |
| Hart et al. | Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry | |
| CN113063871A (en) | Metabolic small molecule detection method and detection system | |
| DE112014001433T5 (en) | Method for determining derivatized analytes in a separated biological fluid | |
| Şentürk et al. | Analytical methods for determination of selective serotonin reuptake inhibitor antidepressants | |
| CN112666273A (en) | Method for detecting concentration of methotrexate substances in erythrocytes | |
| Hoff et al. | Use of capillary electrophoresis with laser-induced fluorescence detection to screen and liquid chromatography–tandem mass spectrometry to confirm sulfonamide residues: Validation according to European Union 2002/657/EC | |
| CN113607854A (en) | Method and detection kit for simultaneously detecting multiple vitamins | |
| CN111239303B (en) | Method for simultaneously determining concentrations of ticagrelor, active metabolites thereof and endogenous adenosine in human plasma by liquid chromatography-mass spectrometry | |
| CN115356408A (en) | Method for detecting 25-hydroxy vitamin D in serum | |
| CN112362765B (en) | Solid-phase extraction and detection method and kit for doxepin and metabolite N-nor doxepin thereof | |
| CN113311080A (en) | Joint detection method for multiple vitamin D metabolites, detection kit and application thereof | |
| CN111896645A (en) | Kit for detecting mycophenolic acid and metabolites thereof in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN115078559B (en) | Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application | |
| Tang et al. | Rapid determination of biogenic amines in ossotide injections by microfluidic chip-mass spectrometry platform: Optimization of microfluidic chip derivatization using response surface methodology | |
| CN114563504B (en) | Method and kit for determining content of free aldosterone in blood plasma | |
| CN117554536B (en) | Simultaneous analysis method for 15 thyroid hormones in environmental water sample | |
| KR20060123270A (en) | Method for the determination of 25-hydroxycholecalciferol in feed |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |