CN115980211A - Kit and method for quantitatively detecting 25-hydroxyvitamin D and application thereof - Google Patents
Kit and method for quantitatively detecting 25-hydroxyvitamin D and application thereof Download PDFInfo
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Abstract
The invention provides a kit and a method for quantitatively detecting 25-hydroxyvitamin D and application thereof, and relates to the technical field of biological detection. The invention provides a kit for quantitatively detecting 25-hydroxyvitamin D, wherein a sample extracting solution of the kit is a methanol acetonitrile solution containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 isotope internal standards, the stable isotope internal standards are used for eliminating the influence of matrix effect, the simultaneous determination of various trace sample types is realized, and the 25-hydroxyvitamin D in dry blood discs/whole blood/serum/plasma can be directly and efficiently extracted. According to tests, only two pieces of 3/6mm blood spots, 20 muL whole blood, 10 muL serum or 10 muL plasma are needed for detection by the kit, the using amount of samples is small, the types are multiple, the kit is suitable for detection screening of multiple sampling methods and multiple crowds, and multiple sample types can be detected by one kit at the same time.
Description
Field of the invention
The invention relates to the technical field of biological detection, in particular to a kit and a method for quantitatively detecting 25-hydroxyvitamin D and application thereof.
Background of the invention
Vitamin D is a fat-soluble vitamin, a compound essential for maintaining human health. Vitamin D comprises 5 compounds, and those closely related to health are vitamin D2 and vitamin D3. Vitamin D2 and vitamin D3 are oxidized by liver monooxygenase (25-hydroxyoxidase) to form 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, which are further metabolized in the kidney to form 1, 25-dihydroxyvitamin D. In the blood, vitamin D is mainly present in the form of 25-hydroxyvitamin D, which accounts for about 95% of the total amount of vitamin D, and has the longest half-life and very stable properties. Therefore, 25-hydroxyvitamin D is an indicator of the concentration of vitamin D in the human body and is an index for evaluating the nutritional status of vitamin D in the human body.
At present, methods for detecting 25-hydroxyvitamin D in a human body mainly include an electrochemiluminescence immunoassay method, a radioimmunoassay, a high performance liquid chromatography method, a liquid chromatography-tandem mass spectrometry method and the like. The immunization method generally has the defects of low specificity, sensitivity to matrix effect, incapability of distinguishing 25-hydroxyvitamin D2 from 25-hydroxyvitamin D3 and the like. The high performance liquid chromatography also has the defects of complex pretreatment, low sensitivity, easy interference and the like. The liquid chromatography tandem mass spectrometry has the advantages of high selectivity, specificity, sensitivity and the like, is more and more widely used for clinical examination, and is a currently accepted gold standard method for quantitative detection of 25-hydroxyvitamin D. At present, the blood sample types for detecting the concentration of 25-hydroxyvitamin D by adopting a liquid chromatography-tandem mass spectrometry method comprise dried blood slices, whole blood, serum, plasma and the like, however, no kit exists so far, which can simultaneously and quantitatively detect the dried blood slices and trace amounts of 25-hydroxyvitamin D in the whole blood, the serum and the plasma, and different kits need to be purchased for different types of sample detection, so that the detection cost is increased, and the detection efficiency is also reduced. Therefore, there is an urgent need to develop a detection kit and method for quantitatively detecting 25-hydroxyvitamin D in dry blood slices and various types of blood samples such as trace whole blood, serum, plasma and the like, so that the disadvantages of the existing kit and method can be overcome.
Summary of the invention
In view of the above, the present invention provides a kit for quantitatively detecting 25-hydroxyvitamin D, which can simultaneously and quantitatively detect the concentration of 25-hydroxyvitamin D in dry blood slices and trace amounts of whole blood, serum and plasma.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a kit for quantitatively detecting 25-hydroxyvitamin D, which comprises a calibrator, a quality control product, a sample extracting solution, a derivative solution, a stop solution, a redissolution and a mobile phase additive; the sample extracting solution is a methanol acetonitrile solution containing 25-hydroxyvitamin D2-D6 and 25-hydroxyvitamin D3-D3, and the volume ratio of methanol to acetonitrile is (1 to 9.5): 1.
preferably, the calibrator is a series of dry blood tablets with standard concentrations, which are respectively S0: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 0ng/mL, S1: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 2ng/mL, S2: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 12ng/mL, S3: both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 20ng/mL, S4: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 40ng/mL, S5: both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 120ng/mL, S6: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 200ng/mL.
More preferably, the preparation method of the calibrator comprises the following steps:
preparing a 5-percent BSA buffer solution containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, said 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations each being 0, 4, 24, 40, 80, 240, 400ng/mL, respectively;
treating whole blood to obtain red blood cells free of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3; and (3) mixing the red blood cells with BSA buffer solutions with different concentrations in an equal volume, dripping the mixture on a filter paper sheet, and airing to obtain a series of dry blood sheet calibrators with standard concentrations.
Preferably, the quality control product is three dry blood slices with different concentrations, which are QC1: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 10ng/mL, QC2: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 20ng/mL, QC3: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were each 40ng/mL.
More preferably, the preparation method of the quality control product comprises the following steps:
detecting the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the whole blood sample, diluting the whole blood sample to the concentration corresponding to QC1 by using 5% BSA buffer solution, adding the diluted whole blood sample to the concentrations corresponding to QC2 and QC3, dropping the whole blood sample on a filter paper sheet and airing the whole blood sample to obtain the dry blood sheet quality control products with three different concentrations.
Preferably, the derivatizing agent solution is a 0.2mg/mL solution of PTDA ethyl acetate; the stop solution is absolute ethyl alcohol; the complex solution is 0.1 percent formic acid-80 percent methanol water solution; the mobile phase additive is 50% formic acid aqueous solution.
The invention also provides a method for quantitatively detecting 25-hydroxyvitamin D, which comprises the following steps:
mixing the calibrator dry blood slices and the quality control dry blood slices with a sample to be detected, adding a sample extracting solution, shaking and centrifuging, mixing the obtained supernatant with a derivatization agent solution for derivatization reaction, then sequentially adding a stop solution and a redissolution, filtering the obtained solution, and performing detection analysis to calculate the concentration of 25-hydroxyvitamin D in the sample to be detected.
Preferably, the detection analysis adopts a liquid chromatography tandem mass spectrum, and the mass spectrum conditions are as follows: a positive ion mode, wherein the scanning mode is multi-reaction monitoring ion scanning; in the positive ion mode, the target quantitative ion pair comprises: 25-hydroxy vitamin D2 derivative 570.4 → 298.1, 25-hydroxy vitamin D2-D6 derivative 576.4 → 298.1, 25-hydroxy vitamin D3 derivative 558.4 → 298.1, 25-hydroxy vitamin D3-D3 derivative 561.4 → 301.2.
More preferably, the chromatographic column of the liquid chromatogram is C18, the mobile phase A of the liquid chromatogram is water containing 0.1-0.3% of formic acid, and the mobile phase B is methanol containing 0.1-0.3% of formic acid; the flow rate of the liquid chromatogram is 0.2-1.0mL/min, the column temperature is 30-45 ℃, and the sample injection volume is 20-40 muL.
The invention also provides application of the kit or the method in quantitative detection of 25-hydroxyvitamin D in trace whole blood, serum, plasma and/or dried blood slices.
The invention provides a kit for quantitatively detecting 25-hydroxyvitamin D, wherein a sample extracting solution of the kit is a methanol acetonitrile solution containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 isotope internal standards, the influence of a matrix effect is eliminated by using the stable isotope internal standards, the simultaneous determination of various trace sample types is realized, and the 25-hydroxyvitamin D in a dry blood paper sheet/whole blood/serum/plasma can be directly and efficiently extracted; the pretreatment adopts a 96-pore plate, so that the automation level and the analysis flux are improved; the liquid chromatography-tandem mass spectrometry is used for simultaneously detecting the 25-hydroxyvitamin D2 and the 25-hydroxyvitamin D3, the ionization efficiency of an analyte is enhanced through derivatization reaction, the detection sensitivity is further improved, and the sample dosage is reduced. According to tests, only two 3/6mm blood spots, 20 muL whole blood, 10 muL serum samples or 10 muL plasma are needed for detection by the kit, the sample usage amount is small, the sample types are multiple, the kit is suitable for detection screening of multiple sampling methods and multiple crowds, particularly newborns and children, and multiple sample types can be detected by one kit.
Drawings
FIG. 1 is a chromatogram of 25-hydroxyvitamin D2 calibrator S5.
FIG. 2 is a chromatogram of 25-hydroxyvitamin D3 calibrator S5.
FIG. 3 is a chromatogram of 25-hydroxyvitamin D2 calibrator S0.
FIG. 4 is a chromatogram of 25-hydroxyvitamin D3 calibrator S0
FIG. 5 is a chromatogram of a sample of a 25-hydroxyvitamin D2 dried blood paper sheet.
FIG. 6 is a chromatogram of a sample of 25-hydroxyvitamin D3 dried blood paper.
FIG. 7 is a chromatogram of a 25-hydroxyvitamin D2 whole blood sample.
FIG. 8 is a chromatogram of a 25-hydroxyvitamin D3 whole blood sample.
FIG. 9 is a chromatogram of a 25-hydroxyvitamin D2 plasma sample.
FIG. 10 is a chromatogram of a 25-hydroxyvitamin D3 plasma sample.
FIG. 11 is a chromatogram of a 25-hydroxyvitamin D2 serum sample.
FIG. 12 is a chromatogram of a 25-hydroxyvitamin D3 serum sample.
FIG. 13 is a chromatogram of 25-hydroxyvitamin D2 with a lower limit of quantitation of 2 ng/mL.
FIG. 14 is a chromatogram of 25-hydroxyvitamin D3 with a lower limit of quantitation of 2 ng/mL.
FIG. 15 is a standard curve of 25-hydroxyvitamin D2 in the range of 2ng/mL to 200ng/mL.
FIG. 16 is a graph of the standard curve of 25-hydroxyvitamin D3 in the range of 2ng/mL to 200ng/mL.
Detailed description of the preferred embodiments
The invention provides a kit for quantitatively detecting 25-hydroxyvitamin D, which comprises a calibrator, a quality control product, a sample extracting solution, a derivative solution, a stop solution, a redissolution and a mobile phase additive; the sample extracting solution is a methanol acetonitrile solution containing 25-hydroxyvitamin D2-D6 and 25-hydroxyvitamin D3-D3, and the volume ratio of methanol to acetonitrile is (1 to 9.5): 1.
in the invention, the concentration of 25-hydroxyvitamin D2-D6 in the sample extracting solution is preferably 10 to 100ng/mL, and more preferably 20ng/mL; the concentration of the 25-hydroxyvitamin D3-D3 is preferably 10 to 100ng/mL, and more preferably 20ng/mL. The sample extracting solution adopts methanol acetonitrile solution containing 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 isotope internal standards, and the stable isotope internal standards are used for eliminating the influence of matrix effect, so that the simultaneous determination of various trace sample types is realized, and the 25-hydroxy vitamin D in the dry blood paper sheet/whole blood/serum/plasma can be directly and efficiently extracted, thereby simultaneously realizing the detection of the 25-hydroxy vitamin D in various sample types such as dry blood sheets and trace whole blood, serum, plasma and the like.
In the invention, the calibrator is a series of dry blood tablets with standard concentration, which are respectively S0: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 0ng/mL, S1: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 2ng/mL, S2: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 12ng/mL, S3: both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 20ng/mL, S4: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 40ng/mL, S5: both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 120ng/mL, S6: both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 200ng/mL. In the present invention, the preparation method of the calibrator preferably comprises the following steps: preparing a 5-percent BSA buffer solution containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, said 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations each being 0, 4, 24, 40, 80, 240, 400ng/mL, respectively; processing whole blood to obtain red blood cells free of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3; and (3) mixing the red blood cells with BSA buffer solutions with different concentrations in an equal volume, dripping the mixture on a filter paper sheet, and airing to obtain a series of dry blood sheet calibrators with standard concentrations. In the invention, the mixed liquid dripped on the filter paper sheet is preferably 50 to 100 muL, and more preferably 75 muL.
In the invention, the quality control products are three dry blood slices with different concentrations, which are QC1: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 10ng/mL, QC2: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 20ng/mL, QC3: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were each 40ng/mL. In the present invention, the preparation method of the quality control material preferably comprises the following steps: detecting the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the whole blood sample, diluting the whole blood sample to the concentration corresponding to QC1 by using 5% BSA buffer solution, adding the diluted whole blood sample to the concentrations corresponding to QC2 and QC3, dropping the whole blood sample on a filter paper sheet and airing the whole blood sample to obtain the dry blood sheet quality control products with three different concentrations. In the present invention, the 5-percent BSA buffer solution is preferably a 5-percent BSA buffer solution containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the buffer solution being 0, 4, 24, 40, 80, 240, 400ng/mL, respectively. In the invention, the mixed liquid dripped on the filter paper sheet is preferably 50 to 100 muL, and more preferably 75 muL.
In the present invention, the derivatizing agent solution is preferably a 0.2mg/mL solution of ethyl PTDA (4-phenyl-1, 2, 4-triazoline-3, 5-dione) acetate; the stop solution is preferably absolute ethyl alcohol; the compound solution is preferably 0.1% formic acid-80% methanol aqueous solution; the mobile phase additive is preferably 50% aqueous formic acid.
In the invention, the kit preferably further comprises consumables, and the consumables preferably comprise a 96-well plate, a 96-well filter plate, a 96-well conical bottom collection plate, a 96-well plate silica gel cover and an operation instruction.
The invention also provides a method for quantitatively detecting 25-hydroxyvitamin D, which comprises the following steps:
mixing the calibrator dry blood slices and the quality control dry blood slices with a sample to be detected, adding a sample extracting solution, shaking and centrifuging, mixing the obtained supernatant with a derivatization agent solution for derivatization reaction, then sequentially adding a stop solution and a redissolution, filtering the obtained solution, and performing detection analysis to calculate the concentration of 25-hydroxyvitamin D in the sample to be detected.
In the present invention, the calibrator, the quality controller, the sample extracting solution, the derivatizing agent solution, the complex solution, the mobile phase additive and the consumable material are preferably equilibrated to room temperature before use. In the invention, the preferred number of the calibrator dried blood tablets and the preferred number of the quality control dried blood tablets are 2. In the present invention, the sample to be tested is preferably a dried blood sheet, whole blood, serum or plasma. When the sample to be detected is a dried blood slice, the addition amount of the sample to be detected is preferably 2 blood spots of 3 mm; when the sample to be detected is whole blood, the addition amount of the sample to be detected is preferably 20 muL; when the sample to be detected is serum, the addition amount of the sample to be detected is preferably 10 muL, and when the sample to be detected is plasma, the addition amount of the sample to be detected is preferably 10 muL. In the invention, the addition amount of the sample extracting solution is preferably 150-300 muL, the addition amount of the derivative solution is preferably 50-250 muL, the addition amount of the stop solution is preferably 10-50 muL, and the addition amount of the complex solution is preferably 50-150 muL. In the invention, the time of the derivatization reaction is preferably 20 to 60min, and more preferably 30min; the filtration is preferably performed in a 96-well filter plate.
In the present invention, the detection analysis is preferably liquid chromatography tandem mass spectrometry, and the mass spectrometry conditions are preferably: in the positive ion mode, the scanning mode is multi-reaction monitoring ion scanning; in the positive ion mode, the target quantitative ion pair comprises: 25-hydroxy vitamin D2 derivative 570.4 → 298.1, 25-hydroxy vitamin D2-D6 derivative 576.4 → 298.1, 25-hydroxy vitamin D3 derivative 558.4 → 298.1, 25-hydroxy vitamin D3-D3 derivative 561.4 → 301.2. The chromatographic column of the liquid chromatography is preferably C18, and the conditions of the liquid chromatography are preferably as follows: the mobile phase A is water containing 0.1-0.3% of formic acid, and the mobile phase B is methanol containing 0.1-0.3% of formic acid; the flow rate of the liquid chromatogram is 0.2-1.0mL/min, the column temperature is 30-45 ℃, and the sample injection volume is 20-40 muL. In the present invention, the method for calculating the concentration of 25-hydroxyvitamin D in the sample to be tested is preferably: and (3) calculating the concentration of the 25-hydroxyvitamin D in the sample or the quality control product by adopting a standard curve constructed by the peak area ratio of the 25-hydroxyvitamin D in the calibrator and the internal standard thereof and the theoretical concentration.
The invention also provides application of the kit or the method in quantitative detection of 25-hydroxyvitamin D in trace whole blood, serum, plasma and/or dried blood slices.
The invention provides a kit capable of quantitatively detecting 25-hydroxyvitamin D in dry blood slices and trace whole blood, serum and plasma at the same time, only two 3/6mm blood spots, 20 mu L whole blood, 10 mu L serum samples or 10 mu L plasma are needed for detection, the sample usage amount is small, the sample types are multiple, the kit is suitable for detection and screening of various sampling methods and various crowds, particularly newborns and children, the multiple sample types can be simultaneously detected by one kit, the detection efficiency is greatly improved, and the detection cost is reduced.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
In the following examples, unless otherwise specified, all methods are conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The detection indexes of the kit for quantitatively detecting 25-hydroxyvitamin D provided by the invention comprise: 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 25-hydroxyvitamin D (the sum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3).
The composition of a kit for quantitative determination of 25-hydroxyvitamin D provided in this example is shown in Table 1.
TABLE 1 kit composition for quantitative determination of 25-hydroxyvitamin D
The calibrator in table 1 is a dried blood tablet, S0: 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 0ng/mL, S1: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 2ng/mL, S2: 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 12ng/mL, S3: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 20ng/mL, S4: 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 40ng/mL, S5: 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 120ng/mL, S6: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 200ng/mL.
The preparation method of the calibrator comprises the following steps: preparing a series of 5-percent BSA buffer solutions containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 at concentrations of 0, 2, 12, 20, 40, 120 and 200ng/mL respectively, treating whole blood to obtain erythrocytes without 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, mixing the erythrocytes with the BSA buffer solutions containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 at different concentrations in an equal volume to obtain whole blood calibrators containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 at different concentrations, dripping 75 mu L of the whole blood calibrators onto a filter paper sheet, and naturally drying to obtain a series of dried blood sheet calibrators.
The quality control substances in table 1 are three dry blood slices with different concentrations, namely low, medium and high, which are QC1: concentration of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 was 10ng/mL, QC2: concentration of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 was 20ng/mL, QC3: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 40ng/mL.
The preparation method of the quality control product comprises the following steps: detecting the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the whole blood sample, diluting the whole blood sample to the concentration corresponding to QC1 by using 5% BSA buffer solution, adding the diluted whole blood sample to the concentrations corresponding to QC2 and QC3 to obtain a low, medium and high concentration whole blood quality control product, then taking 75 mu L of the whole blood quality control product to drip on a filter paper sheet, and naturally drying the whole blood quality control product.
The sample extract in table 1 is a methanolic acetonitrile solution containing 25-hydroxyvitamin D2-D6 and 25-hydroxyvitamin D3-D6 at a concentration of 30ng/mL, the volume ratio of methanol to acetonitrile being 90:10; the sample extract derivatizing agent solution was 0.2mg/mL of a solution of PTDA (4-phenyl-1, 2, 4-triazoline-3, 5-dione) in ethyl acetate; the stop solution is absolute ethyl alcohol; the compound solution is 0.1 percent formic acid-80 percent methanol water solution; the mobile phase additive is 50% formic acid aqueous solution.
Example 2
Method for detecting 25-hydroxyvitamin D using kit for quantitative detection of 25-hydroxyvitamin D of example 1
The method mainly comprises the following steps:
1. sample pretreatment
(1) Taking out the calibrator, the quality control material, the sample extracting solution, the derivating agent solution, the complex solution and the mobile phase additive which are all described in the embodiment 1, and balancing to room temperature;
(2) Respectively taking 2 calibrators, 2 quality control products and a proper amount of samples and adding the calibrators, the quality control products and the samples into a 96-well plate (the dry blood sample comprises two 3/6mm blood spots, the whole blood sample comprises 20 mu L, the serum sample comprises 10 mu L and the plasma sample comprises 10 mu L);
(3) Adding 200 μ L of sample extractive solution, and shaking at room temperature for 30min;
(4) Taking all the supernatant to a new 96-well plate, and blowing nitrogen to dry at room temperature;
(5) Adding 100 mu L of derivatization agent solution, and performing derivatization reaction for 30min at room temperature;
(6) Adding 20 mu L of stop solution, standing for 10 min, and blowing the reaction solution to be dry at room temperature by nitrogen;
(7) Adding 80 μ L of the complex solution, and shaking at room temperature for 5 min;
(8) The whole solution was transferred to a 96-well filter plate, covered with a silica gel cap, and centrifuged at room temperature for 10 min (3000 r/min), the solution was collected in a 96-well conical bottom collection plate, covered with a silica gel cap. Thus obtaining the calibration product, the quality control product and the sample to be processed.
2. Liquid chromatography tandem mass spectrometry detection
(1) Preparation on machine
Preparing a mobile phase, adding 2 mL of mobile phase additive into 1000 mL of deionized water, adding 2 mL of mobile phase additive into 1000 mL of methanol, uniformly mixing, and filtering; the mobile phase of the apparatus was replaced with the mobile phase prepared above, and a Thermo C18 column (50 mm. Times.2.1 mm, 2.6 μm) was connected to the column; and transferring the 96-hole conical bottom sample plate to be detected to an LC-MS/MS sample introduction system, and balancing an instrument by adopting a corresponding chromatographic mass spectrometry method for detection.
(2) Chromatographic process
Ultra-high performance liquid chromatography: shimadzu LC-30 liquid phase system.
A chromatographic column: thermo C18 column (50 mm. Times.2.1 mm, 2.6 μm);
column temperature: 40 ℃;
sample injector temperature: 10 ℃;
sample injection volume: 10 mu L of the solution;
mobile phase: phase A is 0.1% formic acid water, phase B is methanol containing 0.1% formic acid;
the flow rate is 0.4 mL/min;
the elution gradient is shown in table 2:
TABLE 2 liquid chromatography elution gradient
Under the chromatographic conditions, the retention time of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 is 1.99 min and 1.97 min respectively.
(3) Mass spectrometry method
A tandem mass spectrometer with an electrospray ion source was used as the detector for analysis. Wherein the pressure of the air curtain is 20.0psi, the pressure of the heating air is 50psi, the pressure of the auxiliary heating air is 50psi, the temperature of the heating air is 500 ℃, the collision air is high-purity nitrogen, and the pressure is 6 psi; the voltage of the electrospray needle is 5500V. And a positive ion mode is adopted, and a scanning mode adopts multi-reaction monitoring ion scanning. After flowing out of the analytical column along with the mobile phase, the substance to be detected enters the ion source of the mass spectrometer under the action of pressure, and the substance enters the sample channel of the ion source under the control of the six-way valve. In the ion source, a liquid sample is vaporized and ionized into charged molecules, and the charged molecules enter Q1, Q2 and Q3 under the action of voltage and vacuum, wherein Q1 and Q3 are mass filters, only a parent ion and a daughter ion selected according to the mass-to-charge ratio of 25-hydroxyvitamin D and a derivative product of an internal standard substance are allowed to pass through, and Q2 is a collision unit, and the parent ion collides with an inert gas atom to generate a specific fragment ion. The first quadrupole (Q1) of the mass spectrometer selects parent ions with a particular mass-to-charge ratio m/z of 25 hydroxyvitamin D and its internal standard derivatisation products, the parent ions with these m/z ratios being admitted to Q2, the fragment ions generated by Q2 being admitted to Q3, wherein the fragment ions (daughter ions) of 25 hydroxyvitamin D and its internal standard derivatisation products are selected to pass through, while the other ions are removed. The parameters are detailed in table 3.
TABLE 3 Multi-reaction monitoring of ion Scan parameters
As the ions collide with the detector, they convert the number of ions captured into an electronic pulse of digital signal. The data obtained is passed to a computer which plots the number of ions collected against time, resulting in a total ion flow graph (TIC graph) (as shown in figures 1 and 2).
As can be seen from the figures 1-4 and 13, the 25-hydroxy vitamin D2 has symmetrical peak shape and no interference, has good peak height and peak shape when the content is 2.0 ng/mL, has more than 10 times of signal-to-noise ratio, and the content value of 2.0 ng/mL can be quantified; as can be seen from the figures 1-4 and 14, the 25-hydroxy vitamin D3 has symmetrical peak shape and no interference, has good peak height and peak shape when the content is 2.0 ng/mL, has more than 10 times of signal-to-noise ratio, can be quantified in the content value of 2.0 ng/mL, and has high sensitivity.
A standard curve is established by adopting an internal standard method, as shown in figure 15, figure 16 and table 4, the linear correlation of 25-hydroxyvitamin D2 in the linear range of 2 ng/mL-200 ng/mL and the linear correlation of 25-hydroxyvitamin D3 in the linear range of 2 ng/mL-200 ng/mL are good, and the correlation coefficients r are respectively 0.99548 and 0.99762.
TABLE 4 results of standard curves
| Compound (I) | Linear equation of equations | Correlation coefficient |
| 25 hydroxy vitamin D2 | y=6.28924×10 -4 x+0.00123 | 0.99548 |
| 25-hydroxy vitamin D3 | y=0.00677x+0.00963 | 0.99762 |
Blood samples of volunteers are collected, dry blood slices, trace whole blood, trace serum and trace plasma are respectively prepared and are parallelly measured for three times, and the results are shown in Table 5, wherein the detected concentration of 25-hydroxyvitamin D2 is 0.5101 ng/mL, the relative standard deviation is 4.37%, the detected concentration of 25-hydroxyvitamin D3 is 13.28 ng/mL, the relative standard deviation is 5.73%, the repeatability is reliable, and the detection error is small.
TABLE 5 results of reproducibility measurement
| Sample numbering | 25-Hydroxyvitamin D2 (ng/mL) | 25-Hydroxyvitamin D3 (ng/mL) |
| Dried |
0.5028 | 13.20 |
| Dried |
0.4936 | 12.79 |
| Dried blood tablet 3 | 0.5322 | 11.92 |
| |
0.5037 | 13.05 |
| Micro |
0.4969 | 13.54 |
| Micro whole blood 3 | 0.5464 | 14.26 |
| Minute amount of |
0.5415 | 12.36 |
| Micro-serum 2 | 0.4981 | 13.78 |
| Micro-serum 3 | 0.4745 | 14.41 |
| Micro amount of |
0.5292 | 13.81 |
| Micro amount of |
0.4911 | 12.62 |
| Micro amount of plasma 3 | 0.5115 | 13.58 |
| RSD% | 4.37% | 5.73% |
The kit can quantitatively detect the concentration of 25-hydroxyvitamin D in dry blood slices and trace whole blood, serum and plasma at the same time, realizes that one kit can detect various sample types at the same time, and greatly improves the detection efficiency; the kit provided by the invention reduces the dosage of the sample, and has the advantages of high detection sensitivity, good repeatability and good application prospect.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (9)
1. The kit for quantitatively detecting 25-hydroxyvitamin D is characterized by comprising a calibrator, a quality control material, a sample extracting solution, a derivatizing agent solution, a stop solution, a redissolution and a mobile phase additive; the sample extracting solution is a methanol acetonitrile solution containing 25-hydroxy vitamin D2-D6 and 25-hydroxy vitamin D3-D3, and the volume ratio of methanol to acetonitrile is (1 to 9.5): 1; the derivatizing agent solution is a 0.2mg/mL PTDA ethyl acetate solution; the stop solution is absolute ethyl alcohol; the complex solution is 0.1 percent formic acid-80 percent methanol water solution; the mobile phase additive is 50% formic acid water solution.
2. The kit of claim 1, wherein the calibrator is a series of standard concentrations of dried blood, S0: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 0ng/mL, S1: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 2ng/mL, S2: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 12ng/mL, S3: both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 20ng/mL, S4: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 40ng/mL, S5: both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations were 120ng/mL, S6: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 200ng/mL.
3. The kit according to claim 2, wherein the method for preparing the calibrator comprises the steps of:
preparing a 5-percent BSA buffer solution containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, said 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations each being 0, 4, 24, 40, 80, 240, 400ng/mL, respectively;
treating whole blood to obtain red blood cells free of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3; and (3) mixing the red blood cells with BSA buffer solutions with different concentrations in equal volume, dripping the mixture on a filter paper sheet, and airing to obtain a series of dry blood sheet calibrators with standard concentrations.
4. The kit according to claim 1, wherein the quality control product is three dried blood slices with different concentrations, which are QC1: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 10ng/mL, QC2: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were both 20ng/mL, QC3: the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 40ng/mL each.
5. The kit according to claim 4, wherein the preparation method of the quality control product comprises the following steps:
detecting the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the whole blood sample, diluting the whole blood sample to the concentration corresponding to QC1 by using 5% BSA buffer solution, adding the diluted whole blood sample to the concentrations corresponding to QC2 and QC3, dropping the whole blood sample on a filter paper sheet and airing the whole blood sample to obtain the dry blood sheet quality control products with three different concentrations.
6. A method for quantitatively detecting 25-hydroxyvitamin D, comprising the steps of:
mixing the calibrator dry blood slices and the quality control dry blood slices with a sample to be detected, adding a sample extracting solution, shaking and centrifuging, mixing the obtained supernatant with a derivatization agent solution for derivatization reaction, then sequentially adding a stop solution and a redissolution, filtering the obtained solution, and performing detection analysis to calculate the concentration of 25-hydroxyvitamin D in the sample to be detected.
7. The method of claim 6, wherein the detection analysis employs liquid chromatography tandem mass spectrometry under the following conditions: a positive ion mode, wherein the scanning mode is multi-reaction monitoring ion scanning; in the positive ion mode, the target quantitative ion pair comprises: a 25-hydroxyvitamin D2 derivative 570.4 → 298.1, 25-hydroxyvitamin D2-D6 derivative 576.4 → 298.1, 25-hydroxyvitamin D3 derivative 558.4 → 298.1, 25-hydroxyvitamin D3-D3 derivative 561.4 → 301.2.
8. The method according to claim 7, wherein the chromatographic column of the liquid chromatography is C18, the mobile phase A of the liquid chromatography is water containing 0.1-0.3% formic acid, the mobile phase B is methanol containing 0.1-0.3% formic acid; the flow rate of the liquid chromatogram is 0.2-1.0mL/min, the column temperature is 30-45 ℃, and the sample injection volume is 20-40 muL.
9. Use of a kit according to any one of claims 1 to 5 or a method according to any one of claims 6 to 8 for the quantitative determination of 25-hydroxyvitamin D in trace amounts of whole blood, serum, plasma and/or dried blood.
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