CN111474255A - Method for detecting 25-hydroxy vitamin D based on dried blood sample - Google Patents
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Abstract
The invention provides a method for detecting 25 hydroxyvitamin D based on a dried blood slice sample, which combines the analysis of the dried blood slice sample with high performance liquid chromatography-mass spectrometry, so that the sample dosage is reduced, only the dried blood slice sample with the total blood volume of about 12L is needed, compared with the analysis method in the prior art, the analysis method provided by the invention has obvious advantages in the aspects of analysis flow, time length and sensitivity, and the 25 hydroxyvitamin D analysis method with low sample consumption, accuracy, sensitivity and quickness is established.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for detecting 25-hydroxy vitamin D based on a dried blood sample.
Background
Vitamin D (vitamind) is a sterol derivative with rickets resistant function, also called as rickets resistant vitamin, which is a fat soluble vitamin, deficiency of vitamin D can cause rickets, tetany, osteomalacia and other diseases, but long-term intake of excessive vitamin D can cause calcification of blood vessels and organs. Therefore, the regular detection of the vitamin D level in the human body has important significance for measuring the vitamin D supplement effect of the human body and maintaining the health of the human body.
The main components of vitamin D are two compounds, vitamin D2 and D3, which are metabolized in the liver to form 25-OHD and in the kidney to form 1,25(OH) 2D. In blood, vitamin D is mainly present in the form of 25-OHD, accounting for about 95% of the total vitamin D, and has the longest half-life and very stable properties. At present, 25-OHD is considered to be the best index for measuring the level of available vitamin D in vivo in most of the world.
The immunological method has the problems of antibody cross reaction and inconsistent recognition of 25-OHD2 and 25-OHD3, and the detection accuracy is in question. L CMS/MS analysis technology has high specificity, is considered to detect the gold standard of 25-OHD, and reports about a method for simultaneously detecting 25-OHD2 and 25-OHD3 by using L CMS/MS.
Disclosure of Invention
Based on the problems in the prior art, the invention aims to provide a method for detecting 25 hydroxyvitamin D based on a dried blood slice sample, which combines the dried blood slice sample with high performance liquid chromatography-mass spectrometry analysis, so that the sample dosage is reduced, only the dried blood slice sample with the total blood volume of about 12 mu L is needed, compared with the analysis method in the prior art, the analysis method provided by the invention has obvious advantages in the aspects of analysis flow, time length and sensitivity, and the 25 hydroxyvitamin D analysis method with low sample consumption, accuracy, sensitivity and quickness is established.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for detecting 25-hydroxy vitamin D based on a dried blood sample comprises the following steps:
step S1, preparing a standard working solution and a quality control working solution, respectively weighing a 25-hydroxyvitamin D2 standard substance and a 25-hydroxyvitamin D3 standard substance, dissolving the standard substance solutions by using methanol, mixing the two standard substance solutions into a standard substance mixed solution mother solution, wherein the concentration ratio of the two standard substance solutions in the standard substance mixed solution mother solution is 25-hydroxyvitamin D2: 25-hydroxyvitamin D3 ═ 1: 4; diluting the mother liquor of the standard substance mixed liquor into a series of standard substance mixed liquor with concentration gradient by using methanol to obtain standard working liquor; diluting the mother liquor of the mixed solution of the standard product into a solution with quality control concentration by using methanol to obtain a working solution with quality control;
step S2 whole blood matrix preparation, taking anticoagulated whole blood, centrifuging to remove upper plasma, and carrying out multiple redissolution cleaning on the precipitate by using Phosphate Buffered Saline (PBS), wherein the volume ratio of blood cells is as follows: bovine Serum Albumin (BSA) solution 55: adding 7% Bovine Serum Albumin (BSA) solution into 45%, and uniformly mixing for later use;
s3 preparing a standard curve and a quality control sample of the dried blood sheet, taking a plurality of parts of the whole blood matrix processed in the step S2, respectively adding standard working solutions with series concentrations to prepare a series of standard curve solutions, uniformly mixing, then respectively taking blood drops from the series of standard curve solutions to the blood filter paper sheet, and airing for later use;
taking a plurality of parts of the whole blood substrate treated in the step S2, respectively adding a quality control working solution to prepare a quality control solution, uniformly mixing, respectively taking blood from the quality control solution to drop on a blood filter paper sheet, and airing for later use;
step S4, preprocessing a sample, namely, punching out the part with the blood sample from each blood filter paper sheet obtained in the step S3, respectively placing the blood sample into methanol containing an internal standard substance, swirling, and then using ultrasonic wave to assist extraction; drying the extract, adding 4-phenyl-1, 2, 4-triazoline-3, 5-dione (PTAD) solution, mixing uniformly, reacting for 20-40min, adding ethanol to terminate the reaction, drying, and adding 70% methanol to redissolve to obtain a sample solution to be analyzed for later use;
and step S5, respectively carrying out high performance liquid chromatography-mass spectrometry on each sample solution to be analyzed obtained in the step S4.
According to the scheme, the concentrations of the standard working solution and the quality control working solution in each series are as follows:
the standard working solution with 6 concentrations forms the standard working solution with a series of concentrations.
According to the above protocol, the anticoagulated whole blood in step S2 is EDTA-K2Anticoagulated whole blood; the centrifugation condition is 3500r/min centrifugation for 15 min; the repeated redissolution cleaning of the sediment by using Phosphate Buffered Saline (PBS) is to add 1x Phosphate Buffered Saline (PBS) with the volume of 3 times of that of the blood cells, mix the mixture evenly, centrifuge the mixture for 15min at 3500r/min and repeatedly clean the mixture for 3 to 5 times; the pH of the 1 Xphosphate buffered saline (PBS) was 7.4.
According to the scheme, the concentrations of 25-hydroxyvitamin D2 in the series of standard curve solutions in the step S3 are respectively 0.5, 1.0, 2.5, 5.0, 25 and 50ng/m L, the concentrations of 25-hydroxyvitamin D3 are respectively 2.0, 4.0, 10, 20, 100 and 200ng/m L, and the concentrations of 25-hydroxyvitamin D2 in the quality control solution in the step S3 are respectively 1.5, 10 and 40ng/m L, and the concentrations of 25-hydroxyvitamin D3 are respectively 6, 40 and 160ng/m L.
According to the scheme, the methanol inner standard products in the step S4 are 25-hydroxyvitamin D2-D3 and 25-hydroxyvitamin D3-D3, the concentration of the methanol inner standard products is 20ng/m L, the vortex time is 5S, the ultrasonic auxiliary treatment time is 30min, the ultrasonic frequency is 40kHz, and the drying is drying by using nitrogen.
According to the above scheme, the chromatographic conditions in step S5 are: the column temperature is 45 ℃; the relevant liquid phase gradients are as follows:
| time (min) | Flow rate (m L/min) | Mobile phase A | Mobile phase B |
| 0.00 | 0.6 | 30 | 70 |
| 0.60 | 0.6 | 1 | 99 |
| 1.80 | 0.6 | 1 | 99 |
| 1.81 | 0.6 | 30 | 70 |
| 3.00 | 0.6 | 30 | 70 |
Phase A of the mobile phase was 10mM ammonium formate in water containing 0.1% FA; phase B was a 10mM ammonium formate solution in methanol containing 0.1% FA.
According to the above scheme, the mass spectrum conditions in step S5 are: adopting ESI ion source in positive ion mode, multiple reaction monitoring scanning (MRM);
the ion pairs (m/z) are respectively:
| metabolites | Q1(m/z) | Q3(m/z) |
| 25-hydroxyvitamin D2 | 570.4 | 298.2 |
| 25-hydroxyvitamin D3 | 558.4 | 298.2 |
| 25-hydroxyvitamin D2-D3 | 573.4 | 301.2 |
| 25-hydroxyvitamin D3-D3 | 561.4 | 301.2 |
Source parameter, air curtain gas (CUR): 25; collision gas (CAD): 7;
compound parameters: the declustering voltage (DP) is 40; the impact energy (CE) is 20; the inlet voltage (EP) is 10; the collision cell exit voltage (CXP) is 15.
The invention has the beneficial effects that:
1) the related reagents are few in types and only comprise methanol, ethanol, acetonitrile and 4-phenyl-1, 2, 4-triazoline-3, 5-diketone (PTAD) solution; the analysis time is greatly shortened, namely the chromatographic analysis time is shortened to 3min, and the total time of ultrasonic extraction and derivatization involved in the pretreatment step is 1 h.
2) The selection range of the internal standards is widened, the double internal standards are adopted, the accuracy of the analysis result is improved, and the method is good in accuracy, high in sensitivity and strong in practicability.
3) The sampling method of the Dry Blood Sheet (DBS) is not limited by regions and time, and the Dry Blood Sheet (DBS) sample is convenient to store and has good biological safety.
Drawings
FIG. 1 is a standard graph of 25-OHD2 and 25-OHD3 in the examples;
FIG. 2 is a LL OQ (0.5ng/m L) chromatogram of 25-OHD2 in example and a chromatogram of internal standard 25-OHD2-d 3.
FIG. 3 is a chromatogram of LL OQ (2.0ng/m L) and of an internal standard 25-OHD3-d3 of example 25-OHD 3.
Detailed Description
The technical solution of the present invention is described below with reference to the accompanying drawings and examples.
A method for detecting 25-hydroxy vitamin D based on a dried blood sample comprises the following steps:
step S1, preparing a standard working solution and a quality control working solution, respectively weighing a 25-hydroxyvitamin D2 standard substance and a 25-hydroxyvitamin D3 standard substance, respectively dissolving and preparing a 25-hydroxyvitamin D2 standard substance with a concentration of 50 mug/m L and a 25-hydroxyvitamin D3 standard substance with a concentration of 100 mug/m L by using methanol, mixing the two standard substance solutions into a standard mixed solution mother solution according to a volume ratio of 1: 2, wherein the concentration ratio of the two standard substances in the standard mixed solution mother solution is 25-hydroxyvitamin D2: 25-hydroxyvitamin D3 ═ 1: 4, diluting the standard mixed solution mother solution into a series of standard mixed solution with a concentration gradient by using methanol to obtain a standard working solution, and diluting the standard mixed solution mother solution into a quality control concentration solution by using methanol to obtain a quality control working solution;
the concentrations of the standard working solution and the quality control working solution in each series are as follows:
the standard working solution with 6 concentrations forms the standard working solution with a series of concentrations.
Step S2 Whole blood matrix preparation, EDTA-K2Anticoagulating whole blood, centrifuging at 3500r/min for 15min, removing upper plasma, adding 1x phosphate buffer salt solution (pH 7.4) with volume 3 times of blood cells, mixing, centrifuging at 3500r/min for 15min, repeatedly adding phosphate buffer salt solution, centrifuging and cleaning for 5 timesAnd then the volume ratio of blood cells: bovine serum albumin solution 55: adding 7% bovine serum albumin solution into 45, and mixing uniformly to obtain the whole blood matrix for later use.
Step S3 preparation of a standard curve and a quality control sample of the dried blood sheet, taking six parts of the whole blood matrix processed in the step S2, adding standard working solution with different concentrations of 4 mu L into each part of 76 mu L, namely adding standard working solution with one concentration into one part of the whole blood matrix to prepare a series of standard curve solutions with six concentrations, wherein the concentrations of 25-hydroxyvitamin D2 in the series of standard curve solutions are respectively 0.5, 1.0, 2.5, 5.0, 25 and 50ng/m L, and the concentrations of 25-hydroxyvitamin D3 in the series of standard curve solutions are respectively 2.0, 4.0, 10, 20, 100 and 200ng/m L, mixing uniformly, and then respectively taking 75 mu L blood drops from the series of standard curve solutions to the blood filter paper to be dried for later use;
and taking three parts of the whole blood matrix treated in the step S2, adding quality control working solutions with different concentrations of 4 mu L into each part of 76 mu L parts of the whole blood matrix, namely adding one part of the whole blood matrix into one part of the quality control working solution with one concentration to prepare three concentrations of quality control solutions, wherein the concentrations of 25-hydroxyvitamin D2 in the quality control solutions are respectively 1.5, 10 and 40ng/m L, and the concentrations of 25-hydroxyvitamin D3 in the quality control solutions are respectively 6, 40 and 160ng/m L, uniformly mixing, taking out 75 mu L blood drops from the quality control solutions, dripping the blood drops on blood filter paper sheets, and airing for later use.
Step S4, preprocessing the sample, punching out the part with the blood sample from each blood filter paper sheet obtained in step S3 by a puncher with the diameter of 6mm, respectively placing the parts in a 2m L centrifuge tube, adding 500 mu L methanol containing internal standard substances, wherein the internal standard substances are 25-hydroxyvitamin D2-D3 and 25-hydroxyvitamin D3-D3, the concentration of the internal standard substances is 20ng/m L, whirling for 5S, performing ultrasonic treatment at 40kHz for 30min for auxiliary extraction, transferring the extract liquid 450 mu L into a new 1.5m L centrifuge tube, drying the centrifuge tube by using nitrogen, adding 100 mu L4-phenyl-1, 2, 4-triazoline-3, 5-dione solution, uniformly mixing, reacting for 30min, adding 20 mu L ethanol for stopping the reaction, drying the reaction by using nitrogen, adding 60 mu L of 70% methanol for redissolution, obtaining the sample solution to be analyzed, and transferring the sample solution to a 96 pore plate to be loaded on a computer.
Step S5, performing hplc-ms analysis on each sample solution to be analyzed obtained in step S4, wherein the chromatographic conditions in step S5 are as follows: the column temperature is 45 ℃; the relevant liquid phase gradients are as follows:
phase A of the mobile phase was 10mM ammonium formate in water containing 0.1% FA; phase B was 10mM ammonium formate in methanol with 0.1% FA and the chromatographic results are shown in FIGS. 2 and 3.
The mass spectrum conditions were: adopting ESI ion source in positive ion mode, multi-reaction detecting and scanning;
the ion pairs are respectively:
| metabolites | Q1(m/z) | Q3(m/z) |
| 25-hydroxyvitamin D2 | 570.4 | 298.2 |
| 25-hydroxyvitamin D3 | 558.4 | 298.2 |
| 25-hydroxyvitamin D2-D3 | 573.4 | 301.2 |
| 25-hydroxyvitamin D3-D3 | 561.4 | 301.2 |
Source parameters, gas curtain gas: 25; collision gas: 7;
compound parameters: the declustering voltage is 40; the collision energy was 20; the inlet voltage is 10; the collision cell exit voltage is 15.
The results of FIG. 1 show that 25-OHD2 has good linearity in the range of 0.5-50ng/m L and 25-OHD3 has good linearity in the range of 2.0-200ng/m L.
The above embodiments are only used for illustrating but not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.
Claims (7)
1. A method for detecting 25-hydroxy vitamin D based on a dried blood sample is characterized by comprising the following steps:
step S1, preparing a standard working solution and a quality control working solution, respectively weighing a 25-hydroxyvitamin D2 standard substance and a 25-hydroxyvitamin D3 standard substance, dissolving the standard substance solutions by using methanol, mixing the two standard substance solutions into a standard substance mixed solution mother solution, wherein the concentration ratio of the two standard substance solutions in the standard substance mixed solution mother solution is 25-hydroxyvitamin D2: 25-hydroxyvitamin D3 ═ 1: 4; diluting the mother liquor of the standard substance mixed liquor into a series of standard substance mixed liquor with concentration gradient by using methanol to obtain standard working liquor; diluting the mother liquor of the mixed solution of the standard product into a solution with quality control concentration by using methanol to obtain a working solution with quality control;
step S2 whole blood matrix preparation, taking anticoagulated whole blood, centrifuging to remove upper plasma, and carrying out multiple redissolution cleaning on the precipitate by using phosphate buffered saline solution, wherein the volume ratio of blood cells is as follows: bovine serum albumin solution 55: adding 7% bovine serum albumin solution into 45, and mixing uniformly for later use;
s3 preparing a standard curve and a quality control sample of the dried blood sheet, taking a plurality of parts of the whole blood matrix processed in the step S2, respectively adding standard working solutions with series concentrations to prepare a series of standard curve solutions, uniformly mixing, then respectively taking blood drops from the series of standard curve solutions to the blood filter paper sheet, and airing for later use;
taking a plurality of parts of the whole blood substrate treated in the step S2, respectively adding a quality control working solution to prepare a quality control solution, uniformly mixing, respectively taking blood from the quality control solution to drop on a blood filter paper sheet, and airing for later use;
step S4, preprocessing a sample, namely, punching out the part with the blood sample from each blood filter paper sheet obtained in the step S3, respectively placing the blood sample into methanol containing an internal standard substance, swirling, and then using ultrasonic wave to assist extraction; drying the extract, adding 4-phenyl-1, 2, 4-triazoline-3, 5-diketone solution, uniformly mixing, reacting for 20-40min, adding ethanol to terminate the reaction, drying, and adding 70% methanol to redissolve to obtain a sample solution to be analyzed for later use;
and step S5, respectively carrying out high performance liquid chromatography-mass spectrometry on each sample solution to be analyzed obtained in the step S4.
2. The method for detecting 25 hydroxyvitamin D based on dried blood sample as claimed in claim 1, wherein the concentrations of the standard working solution and the quality control working solution are as follows:
the standard working solution with 6 concentrations forms the standard working solution with a series of concentrations.
3. The dry blood sample-based detection of 25 hydroxyl groups of claim 1The method of vitamin D, wherein the anticoagulated whole blood in step S2 is EDTA-K2Anticoagulated whole blood; the centrifugation condition is 3500r/min centrifugation for 15 min; the repeated redissolution cleaning of the sediment by using phosphate buffered saline solution is to add 1x phosphate buffered saline solution with the volume of 3 times of blood cells, mix the mixture evenly, centrifuge the mixture for 15min at 3500r/min and repeatedly clean the mixture for 3 to 5 times; the pH of the 1 Xphosphate buffered saline solution is 7.4.
4. The method for detecting 25 hydroxyvitamin D based on dry blood slice sample as claimed in claim 1, wherein the concentrations of 25-hydroxyvitamin D2 in the series of standard curve solutions in step S3 are respectively 0.5, 1.0, 2.5, 5.0, 25, 50ng/m L: 25-hydroxyvitamin D3 are respectively 2.0, 4.0, 10, 20, 100, 200ng/m L, and the concentrations of 25-hydroxyvitamin D2 in the quality control solution in step S3 are respectively 1.5, 10, 40ng/m L: 25-hydroxyvitamin D3 are respectively 6, 40, 160ng/m L.
5. The method for detecting 25 hydroxyvitamin D based on dried blood sample as claimed in claim 1, wherein the methanol in step S4 has internal standard of 25-hydroxyvitamin D2-D3 and 25-hydroxyvitamin D3-D3, concentration of 20ng/m L, vortex time of 5S, ultrasonic wave auxiliary treatment time of 30min, ultrasonic wave frequency of 40kHz, and drying by blowing with nitrogen.
6. The method for detecting 25 hydroxyvitamin D based on dried blood sample as claimed in claim 1, wherein the chromatographic conditions in step S5 are: the column temperature is 45 ℃; the relevant liquid phase gradients are as follows:
Phase A of the mobile phase was 10mM ammonium formate in water containing 0.1% FA; phase B was a 10mM ammonium formate solution in methanol containing 0.1% FA.
7. The method for detecting 25 hydroxyvitamin D based on dried blood sample as claimed in claim 1, wherein the mass spectrometric conditions in step S5 are: adopting ESI ion source in positive ion mode, multi-reaction detecting and scanning;
the ion pairs are respectively:
Source parameters, gas curtain gas: 25; collision gas: 7;
compound parameters: the declustering voltage is 40; the collision energy was 20; the inlet voltage is 10; the collision cell exit voltage is 15.
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Cited By (2)
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| CN115343379A (en) * | 2022-04-20 | 2022-11-15 | 杭州谱聚医疗科技有限公司 | Preparation method of blank blood matrix and standard substance and detection method of vitamin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111879876A (en) * | 2020-08-07 | 2020-11-03 | 苏州新波生物技术有限公司 | Rapid and sensitive analysis method for 25 hydroxyvitamin D based on immune mass spectrometry technology |
| CN115343379A (en) * | 2022-04-20 | 2022-11-15 | 杭州谱聚医疗科技有限公司 | Preparation method of blank blood matrix and standard substance and detection method of vitamin |
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