Method for determining 25-hydroxyvitamin D in serum by two-dimensional column switching high performance liquid chromatography

Abstract

The invention belongs to the technical field of analysis and detection, and particularly provides a method for determining 25-hydroxyvitamin D in serum by two-dimensional column switching high performance liquid chromatography. The method comprises the following steps: preparing a protein precipitant; adding a protein precipitant into the serum to be detected to obtain a mixed sample solution; centrifuging the mixed sample liquid to obtain supernate; preparing a mixed working solution containing 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3; detecting the mixed working solution by adopting a two-dimensional column switching high performance liquid chromatography to obtain a calibration curve; and detecting the supernate by adopting a two-dimensional column switching high performance liquid chromatography, and obtaining the content of the 25-hydroxyvitamin D in the serum to be detected according to the calibration curve. According to the method provided by the invention, the contents of the 25-hydroxy vitamin D2 and the 25-hydroxy vitamin D3 in the serum can be accurately, respectively and simultaneously determined.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for determining 25-hydroxyvitamin D in serum by using a two-dimensional column switching high performance liquid chromatography method. Background Art

[0002] Multidimensional liquid chromatography is an important direction of liquid chromatography development. It connects multiple chromatographic systems with different separation mechanisms, improves the separation capacity of the chromatographic system, and meets the separation requirements of complex samples. Among the multidimensional chromatographic separation methods, two-dimensional chromatographic separation is the most commonly used. Two-dimensional liquid chromatography coupling technology is usually a liquid chromatography technology that combines two chromatographic columns with different separation mechanisms but are independent of each other in series. The connection between different chromatographic columns, the sharing of multiple chromatographic columns and detectors, and the change of the flow direction of the mobile phase are achieved through switching valves. Relying on switching valves, the change of mobile phase and the switching of chromatographic modes can be flexibly achieved, also known as column switching technology. It can transfer several or all components separated by the first-dimensional chromatographic column to the second dimension for further analysis, and process and integrate the results of the two data analysis. Compared with ordinary one-dimensional liquid chromatography, two-dimensional liquid chromatography can provide multiple peak capacity, and through the automated operation of online coupling, it greatly simplifies the sample pretreatment process, which is particularly suitable for the analysis of trace samples and complex samples.

[0003] According to whether the transferred components enter the second dimension directly, it can be divided into two modes: offline and online. Offline operation is to manually collect the components separated by the previous chromatographic column and then inject them into the second chromatographic column for analysis. Online operation is to directly switch the target components in the first-dimensional elution fraction to the second-dimensional system or use a collection switching device to collect the first-dimensional components for secondary analysis. According to the switching mode, it can be divided into two modes: partial and overall switching, including simple heart cutting, multi-component heart cutting and full two-dimensional analysis.

[0004] The main function of vitamin D is to promote the absorption of calcium and phosphorus in the intestine, thereby maintaining the balance of blood calcium and phosphorus. This is essential for the growth and development of bones and the health of teeth. In addition, vitamin D can also promote the reabsorption of calcium and phosphorus by the renal tubules, further ensuring the balance of calcium and phosphorus in the body. Therefore, vitamin D has a significant effect on preventing bone diseases such as rickets and osteoporosis. In addition to its effects on bones, vitamin D also plays an important role in the immune system. It can regulate the proliferation and differentiation of immune cells and enhance the activity of immune cells, thereby improving the body's immunity. Studies have shown that adequate intake of vitamin D helps reduce the risk of infectious diseases. In addition, vitamin D is also closely related to the occurrence and development of a variety of diseases. Studies have found that vitamin D deficiency is associated with an increased risk of a variety of diseases such as cardiovascular disease, diabetes, and autoimmune diseases. However, the main storage form of vitamin D in the human body is 25-hydroxyvitamin D, which accounts for more than 95% of the total. Therefore, 25-hydroxyvitamin D is recognized as the best indicator for measuring vitamin D nutritional status.

[0005] At present, the main detection methods of 25-hydroxyvitamin D are immunoassay and chromatography. Among them, the immunoassay is divided into radioimmunoassay, enzyme-linked immunosorbent assay, and chemiluminescent immunoassay. These immunoassays have common disadvantages: the determination is easily affected by factors such as matrix and antibody, which can easily lead to low accuracy of the determination results, and generally cannot distinguish 25-hydroxyvitamin D2 from 25-hydroxyvitamin D3, the detection range is narrow, there are large deviations in intra-batch and inter-batch repeatability and precision, and antibodies that specifically bind to 25-hydroxyvitamin D have different degrees of reaction with other vitamin hydroxylates, which affects the determination results. Chromatography is divided into high-performance liquid chromatography and high-performance liquid chromatography-tandem mass spectrometry. High-performance liquid chromatography can separate 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, and complete the determination of both at the same time. However, since the content of 25-hydroxyvitamin D2 in the blood is much lower than that of 25-hydroxyvitamin D3, the sensitivity of high-performance liquid chromatography cannot meet the clinical detection needs of 25-hydroxyvitamin D2. The equipment cost of high-performance liquid chromatography-tandem mass spectrometry is relatively high, and the economic value is not high.

[0006] Under the premise that the sensitivity of high performance liquid chromatography cannot meet the clinical detection needs of 25-hydroxyvitamin D2, a low-cost, highly sensitive and effective method for detecting 25-hydroxyvitamin D in serum is urgently needed. Summary of the invention

[0007] In order to solve the problems in the prior art, the purpose of the present invention is to provide a method for determining 25-hydroxyvitamin D in serum by two-dimensional column switching high performance liquid chromatography, which can save time and has high sensitivity and low reagent and consumables costs, and the initial investment in instruments and equipment is also relatively low.

[0008] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0009] According to a first aspect of the present invention, a method for determining 25-hydroxyvitamin D in serum by two-dimensional column switching high performance liquid chromatography is provided, the method comprising the following steps:

[0010] Step 1: preparing protein precipitant;

[0011] Step 2: adding the protein precipitant to the serum to be tested to obtain a mixed sample solution;

[0012] Step 3: centrifuging the mixed sample solution to obtain a supernatant;

[0013] Step 4: preparing a mixed working solution containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3;

[0014] Step 5: using two-dimensional column switching high performance liquid chromatography to detect the mixed working solution prepared in step 4 to obtain a calibration curve;

[0015] Step 6: Detecting the supernatant by using the two-dimensional column switching high performance liquid chromatography, and obtaining the content of 25-hydroxyvitamin D in the serum to be detected according to the detection result and the calibration curve obtained in step 5;

[0016] Wherein, in the two-dimensional column switching high performance liquid chromatography in step 5 and step 6, the one-dimensional column is a C8 chromatographic column and the two-dimensional column is a C18 chromatographic column;

[0017] The first-dimensional mobile phase is a mixed solution of methanol and potassium dihydrogen phosphate aqueous solution; the volume of the potassium dihydrogen phosphate aqueous solution accounts for more than 5% of the volume of the first-dimensional mobile phase, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 15-25mmol / L; the second-dimensional mobile phase is a mixed solution of methanol and potassium dihydrogen phosphate aqueous solution; the volume of the potassium dihydrogen phosphate aqueous solution accounts for more than 5% of the volume of the second-dimensional mobile phase, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 15-25mmol / L.

[0018] In one aspect of the present invention, in step 1, the protein precipitant is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1:8-10.

[0019] In one aspect of the present invention, in step 1, the protein precipitant is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1:9.

[0020] In one aspect of the present invention, in step 2, the volume ratio of the serum to be detected and the protein precipitant is 1:2-4.

[0021] In one aspect of the present invention, in step 2, the volume ratio of the serum to be detected and the protein precipitant is 1:2.5.

[0022] In one aspect of the present invention, in step 3, the rotation speed of the centrifugation process is 5000-15000 rpm, and the centrifugation time is 5-15 min.

[0023] In one aspect of the present invention, the first-dimensional mobile phase is a mixed solution of methanol and potassium dihydrogen phosphate aqueous solution; the volume of the potassium dihydrogen phosphate aqueous solution accounts for 10% or more of the volume of the first-dimensional mobile phase, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 20 mmol / L.

[0024] In one aspect of the present invention, the second-dimensional mobile phase is a mixed solution of methanol and potassium dihydrogen phosphate aqueous solution; the volume of the potassium dihydrogen phosphate aqueous solution accounts for 5% or more of the volume of the second-dimensional mobile phase, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 20 mmol / L.

[0025] In one aspect of the present invention, the two-dimensional column switching high performance liquid chromatography is implemented by a two-dimensional high performance liquid chromatography system.

[0026] In one aspect of the present invention, the two-dimensional high performance liquid chromatography system comprises a one-dimensional chromatographic column, a one-dimensional infusion pump, a two-dimensional chromatographic column, a two-dimensional infusion pump, an automatic sampler, an ultraviolet detector and a six-way valve; wherein the one-dimensional infusion pump is connected to the automatic sampler and the one-dimensional chromatographic column to form a flow path, and the two-dimensional infusion pump is connected to the ultraviolet detector and the two-dimensional chromatographic column to form another flow path; the two flow paths are respectively connected to two positions of the six-way valve.

[0027] In one aspect of the present invention, the calibration curve of 25-hydroxyvitamin D2 is Y=3.05×10 -1 X+5.04, r≥0.9990; r is the linear correlation coefficient; Y is the peak area of ​​the corresponding peak in the chromatogram, in mAU·sec; X is the concentration of 25-hydroxyvitamin D2, in ng / mL.

[0028] In one aspect of the present invention, the calibration curve of 25-hydroxyvitamin D3 is Y=3.71×10 -1 X+3.75, r≥0.9990; r is the linear correlation coefficient; Y is the peak area of ​​the corresponding peak in the chromatogram, in mAU·sec; X is the concentration of 25-hydroxyvitamin D3, in ng / mL.

[0029] In one aspect of the present invention, in step 5 and step 6, the detection conditions of the two-dimensional column switching high performance liquid chromatography are: the quantitative detection wavelength is 260-270nm, the qualitative detection wavelength is 260-270nm, and the column temperature is 30-45°C.

[0030] The beneficial effects of the present invention are:

[0031] (1) The method for determining 25-hydroxyvitamin D in serum by two-dimensional column switching high performance liquid chromatography provided by the present invention can accurately and simultaneously determine the contents of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum.

[0032] (2) Compared with mass spectrometry, the two-dimensional column switching high performance liquid chromatography provided by the present invention requires lower cost of instruments and equipment, and lower cost of reagents and consumables.

[0033] (3) The two-dimensional column switching high performance liquid chromatography method provided by the present invention does not need to repeatedly measure the calibration curve, further saving time and reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the overall structure of the two-dimensional high performance liquid chromatography system of the present invention.

[0035] Figure 2 It is a schematic diagram of the connection mode of the two-dimensional high performance liquid chromatography system of the present invention.

[0036] Figure 3 It is the chromatogram of the repeatability test of Example 1 of the present invention.

[0037] Figure 4 This is the calibration curve test chromatogram of Example 1 of the present invention.

[0038] Figure 5 It is the chromatogram of the test of comparative example 1 of the present invention.

[0039] Figure 6 It is the chromatogram of the test of comparative example 2 of the present invention.

[0040] Figure 7 It is the chromatogram of the test of comparative example 3 of the present invention. DETAILED DESCRIPTION

[0041] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they should also fall within the scope of the present application.

[0042] The present invention implements a method for determining 25-hydroxyvitamin D in serum by two-dimensional column switching high performance liquid chromatography through a two-dimensional high performance liquid chromatography system; Figure 1 and Figure 2 As shown in the figure, the two-dimensional high performance liquid chromatography system includes a one-dimensional chromatographic column 13, a one-dimensional infusion pump 11, a two-dimensional chromatographic column 14, a two-dimensional infusion pump 12, an automatic sampler 31, an ultraviolet detector 41 and a six-way valve 32; the one-dimensional chromatographic column 13 and the two-dimensional infusion pump 12 are located in a column oven 51, and the system also includes a solvent tray 61 for placing the test solvent and a waste liquid barrel 62 for placing the test waste liquid.

[0043] like Figure 2 As shown, the six-way valve 32 includes six positions 1-6. In the first state, position 1 of the six-way valve is connected to position 6, the two-dimensional infusion pump 12 is connected to position 1 of the six-way valve 32, and position 6 is connected to the two-dimensional chromatographic column 14 and the ultraviolet detector 41 in sequence; the one-dimensional infusion pump 11 is connected to the automatic sampler 31 and then connected to position 3 of the six-way valve 32, position 3 of the six-way valve 32 is connected to position 2, position 2 is connected to the inlet end of the one-dimensional chromatographic column 13, the outlet end of the one-dimensional chromatographic column 13 is connected to position 5 of the six-way valve 32, position 5 of the six-way valve 32 is connected to position 4, and position 4 is connected to the waste liquid bucket 62; the state at this time is as shown in FIG. Figure 2 As shown; when the six-way valve 32 changes from the first state to the second state, the No. 1 position of the six-way valve 32 is connected to the No. 2 position; the two-dimensional infusion pump 12 is connected to the No. 1 position of the six-way valve 32, the No. 2 position is connected to the inlet end of the one-dimensional chromatographic column 13, the outlet end of the one-dimensional chromatographic column 13 is connected to the No. 5 position of the six-way valve 32, the No. 5 position is connected to the No. 6 position, and the No. 6 position is sequentially connected to the two-dimensional chromatographic column 14 and the ultraviolet detector 41. In the present invention, after the sample is concentrated by the one-dimensional chromatographic column 13, the six-way valve 32 is used to switch the column from the first state to the second state, and the concentrated sample is transferred from the one-dimensional chromatographic column 13 to the two-dimensional chromatographic column 14 for further separation, and finally the detection is completed.

[0044] In the present invention, the 25-hydroxyvitamin D2 stock solution and the 25-hydroxyvitamin D3 stock solution can be obtained by the following steps:

[0045] Accurately weigh 10 mg of 25-hydroxyvitamin D3 and transfer it to a 10 mL volumetric flask. Add methanol until the 25-hydroxyvitamin D3 is completely dissolved, then add methanol to make the volume up to 10 mL to obtain a 1.0 mg / mL 25-hydroxyvitamin D3 stock solution, which is stored at -20°C.

[0046] Accurately weigh 10 mg of 25-hydroxyvitamin D2 and transfer it to a 10 mL volumetric flask. Add methanol until 25-hydroxyvitamin D2 is completely dissolved, then add methanol to make up to 10 mL to obtain a 1.0 mg / mL 25-hydroxyvitamin D2 stock solution, which should be stored at -20°C.

[0047] In the present invention, the calibration curve can be obtained by the following steps:

[0048] Take 20 μL of the above 1.0 mg / mL 25-hydroxyvitamin D2 stock solution, 20 μL of the 1.0 mg / mL 25-hydroxyvitamin D3 stock solution, and then add 960 μL of newborn calf serum to prepare a mixed working solution 1.

[0049] Take 100 μL of mixed working solution 1 and add 900 μL of newborn calf serum to prepare mixed working solution 2.

[0050] Take 160 μL of mixed working solution 2 and add 840 μL of newborn calf serum to prepare calibrator point 6.

[0051] Take 500 μL of calibrator point 6 and add 500 μL of newborn calf serum to prepare calibrator point 5.

[0052] Take 500 μL of calibrator point 5 and add 500 μL of newborn calf serum to prepare calibrator point 4.

[0053] Take 500 μL of calibrator point 4 and add 500 μL of newborn calf serum to prepare calibrator point 3.

[0054] Take 500 μL of calibrator point 3 and add 500 μL of newborn calf serum to prepare calibrator point 2.

[0055] Take 500 μL of calibrator point 2 and add 500 μL of newborn calf serum to prepare calibrator point 1.

[0056] For the above calibration point 1 to calibration point 6, the relationship between concentration and peak area is constructed to obtain a calibration curve.

[0057] In the present invention, the method for determining 25-hydroxyvitamin D in serum by two-dimensional column switching high performance liquid chromatography using a two-dimensional high performance liquid chromatography system comprises the following:

[0058] Step 1: preparing a protein precipitant; the protein precipitant comprises methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1:9;

[0059] Step 2: Add 1000 μL of protein precipitant to 400 μL of serum to be tested to obtain a mixed sample solution;

[0060] Step 3: Centrifuge the mixed sample solution at 10000 rpm for 10 min to obtain a supernatant;

[0061] Step 4: preparing a 25-hydroxyvitamin D2 stock solution and a 25-hydroxyvitamin D3 stock solution; and using the 25-hydroxyvitamin D2 stock solution and the 25-hydroxyvitamin D3 stock solution to sequentially prepare a mixed working solution 1 and a mixed working solution 2;

[0062] Step 5: Using two-dimensional column switching high performance liquid chromatography, the calibration sample points 1-6 obtained by diluting the mixed working solution 2 prepared in step 4 are tested to obtain a calibration curve;

[0063] Step 6: 400 μL of the supernatant was tested by the two-dimensional column switching high performance liquid chromatography, and the content of 25-hydroxyvitamin D in the serum to be tested was obtained according to the test results and the calibration curve obtained in step 5. According to the test results of the collected chromatogram analysis, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were completely separated without interference, and the peak shape was symmetrical.

[0064] The detection conditions of the above-mentioned two-dimensional column switching high performance liquid chromatography are: quantitative detection wavelength 262nm, qualitative detection wavelength 265nm, column temperature 40°C; the first-dimensional chromatographic column adopts a reversed-phase C8 silica gel chromatographic column with a specification of 5μm, ID4.6×50mm; the second-dimensional chromatographic column adopts a reversed-phase C18 silica gel chromatographic column with a specification of 5μm, ID4.6×150mm.

[0065] The experimental conditions of the aforementioned two-dimensional column switching high performance liquid chromatography also include: the first-dimensional mobile phase is a mixed solution formed by 20 mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 20 mmol / L potassium dihydrogen phosphate aqueous solution accounts for ≥10%; the second-dimensional mobile phase is a mixed solution formed by 20 mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 20 mmol / L potassium dihydrogen phosphate aqueous solution accounts for ≥5%.

[0066] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0067] Example:

[0068] Embodiment 1:

[0069] Embodiment 1 comprises the following steps:

[0070] 1) Take 400 μL of serum and add it to a centrifuge tube, add 1000 μL of protein precipitant and mix for 3 minutes to obtain a mixed sample solution, wherein the protein precipitant is a mixed organic phase solution of methanol:acetonitrile=1:9 (v / v).

[0071] 2) Place the centrifuge tube in a small centrifuge and centrifuge at 10000 rpm for 10 min. Take 1000 μL of the supernatant and place it in a sample injection bottle to obtain the sample to be tested.

[0072] 3) Accurately weigh 10 mg of 25-hydroxyvitamin D3 and transfer it to a 10 mL volumetric flask, add methanol until 25-hydroxyvitamin D3 is completely dissolved, then add methanol to make up to 10 mL to obtain a 1.0 mg / mL 25-hydroxyvitamin D3 stock solution, which is stored at -20°C. Accurately weigh 10 mg of 25-hydroxyvitamin D2 and transfer it to a 10 mL volumetric flask, add methanol until 25-hydroxyvitamin D2 is completely dissolved, then add methanol to make up to 10 mL to obtain a 1.0 mg / mL 25-hydroxyvitamin D2 stock solution, which is stored at -20°C.

[0073] 4) Take 20 μL of the above 1.0 mg / mL 25-hydroxyvitamin D2 stock solution, 20 μL of the 1.0 mg / mL 25-hydroxyvitamin D3 stock solution, and add 960 μL of newborn calf serum to prepare mixed working solution 1. Take 100 μL of mixed working solution 1, add 900 μL of newborn calf serum to prepare mixed working solution 2. Take 160 μL of mixed working solution 2, add 840 μL of newborn calf serum to prepare calibration point No. 6. Take 500 μL of calibration point No. 6, add 500 μL of newborn calf serum to prepare calibration point No. 5. Take 500 μL of calibration point No. 5, add 500 μL of newborn calf serum to prepare calibration point No. 4. Take 500 μL of calibration point No. 4, add 500 μL of newborn calf serum to prepare calibration point No. 3. Take 500 μL of calibration point 3 and add 500 μL of newborn calf serum to obtain calibration point 2. Take 500 μL of calibration point 2 and add 500 μL of newborn calf serum to obtain calibration point 1. For the above calibration points 1 to 6, construct the relationship between concentration and peak area, such as Figure 4 As shown, the calibration curve is obtained through the test data. Figure 4 It can be seen that 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 are completely separated, there is no interference, and the peak shape is symmetrical.

[0074] The calibration curve is obtained from the data in Table 1 and Table 2 below:

[0075] Table 1

[0076]

[0077] Table 2

[0078]

[0079] 5) Using the two-dimensional liquid phase center cutting mode, 400μL of the sample to be tested was injected into the two-dimensional liquid chromatography system for detection; wherein the first-dimensional mobile phase was a mixed solution formed by 20mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 20mmol / L potassium dihydrogen phosphate aqueous solution accounted for 10%; the second-dimensional mobile phase was a mixed solution formed by 20mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 20mmol / L potassium dihydrogen phosphate aqueous solution accounted for 5%. The detection conditions were: the quantitative detection wavelength was 262nm, the qualitative detection wavelength was 265nm, and the column temperature was 40℃; the first-dimensional chromatographic column used a reversed-phase C8 silica gel chromatographic column (SupersilC8), with a specification of 5μm and ID4.6×50mm; the second-dimensional chromatographic column used a reversed-phase C18 silica gel chromatographic column (Superesil ODS-B), with a specification of 5μm and ID4.6×150mm.

[0080] The samples were tested 7 times to test the repeatability of the method provided by the present invention; the results are as follows Figure 3 As shown in Tables 3 and 4 below:

[0081] Table 3

[0082]

[0083] Table 4

[0084]

[0085] From Tables 3 and 4, and Figure 3 It can be seen that the method provided by the present disclosure has good repeatability.

[0086] Comparative Example 1:

[0087] The steps of Comparative Example 1 are the same as those of Example 1, except that a mixed organic phase solution of methanol:acetonitrile = 9:1 (v / v) is used in Comparative Example 1. The test results of 25-hydroxyvitamin D in the same serum sample in Comparative Example 1 and Example 1 are as follows: Figure 5 shown.

[0088] Comparative Example 2:

[0089] The steps of Comparative Example 2 are the same as those of Example 1, except that the one-dimensional chromatographic column used in Comparative Example 2 is Supersil C8 5μm, ID4.6×150mm, and the two-dimensional chromatographic column is Superesil ODS-B 5μm, ID4.6×250mm; which are used to replace the one-dimensional chromatographic column Supersil C8 5μm, ID4.6×50mm and the two-dimensional chromatographic column Superesil ODS-B 5μm, ID4.6×150mm used in Example 1. The test results of Comparative Example 2 and Example 1 on 25-hydroxyvitamin D in the same serum sample are as follows: Figure 6 shown.

[0090] Comparative Example 3:

[0091] The steps of Comparative Example 3 are the same as those of Example 1, except that the first-dimensional mobile phase used in Comparative Example 3 is a mixed solution of 10mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 10mmol / L potassium dihydrogen phosphate aqueous solution accounts for 10%; the second-dimensional mobile phase is a mixed solution of 10mmol / L potassium dihydrogen phosphate aqueous solution and methanol, wherein the 10mmol / L potassium dihydrogen phosphate aqueous solution accounts for 5%. Comparative Example 3 and Example 1 test the 25-hydroxyvitamin D in the same serum sample as shown in Figure 1. Figure 7 shown.

[0092] from Figures 5 to 7 It can be seen that comparative examples 1-3 cannot completely separate 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, and there is interference between the peaks.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining 25-hydroxyvitamin D in serum by two-dimensional column switching high performance liquid chromatography, characterized in that: The method comprises the following steps: Step 1: preparing protein precipitant; Step 2: adding the protein precipitant to the serum to be tested to obtain a mixed sample solution; Step 3: centrifuging the mixed sample solution to obtain a supernatant; Step 4: preparing a mixed working solution containing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3; Step 5: using two-dimensional column switching high performance liquid chromatography to detect the mixed working solution prepared in step 4 to obtain a calibration curve; Step 6: Detecting the supernatant by using the two-dimensional column switching high performance liquid chromatography, and obtaining the content of 25-hydroxyvitamin D in the serum to be detected according to the detection result and the calibration curve obtained in step 5; Wherein, in the two-dimensional column switching high performance liquid chromatography in step 5 and step 6, the one-dimensional column is a C8 chromatographic column and the two-dimensional column is a C18 chromatographic column; The first-dimensional mobile phase is a mixed solution of methanol and potassium dihydrogen phosphate aqueous solution; the volume of the potassium dihydrogen phosphate aqueous solution accounts for more than 5% of the volume of the first-dimensional mobile phase, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 15-25mmol / L; the second-dimensional mobile phase is a mixed solution of methanol and potassium dihydrogen phosphate aqueous solution; the volume of the potassium dihydrogen phosphate aqueous solution accounts for more than 5% of the volume of the second-dimensional mobile phase, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 15-25mmol / L.

2. The method according to claim 1, characterized in that In step 1, the protein precipitant is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1:8-10.

3. The method according to claim 1, characterized in that In step 2, the volume ratio of the serum to be detected and the protein precipitant is 1:2-4.

4. The method according to claim 1, characterized in that In step 3, the rotation speed of the centrifugation process is 5000-15000 rpm, and the centrifugation time is 5-15 min.

5. The method according to claim 1, characterized in that The first-dimensional mobile phase is a mixed solution of methanol and potassium dihydrogen phosphate aqueous solution; the volume of the potassium dihydrogen phosphate aqueous solution accounts for 10% or more of the volume of the first-dimensional mobile phase, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 20mmol / L.

6. The method according to claim 1, characterized in that The second-dimensional mobile phase is a mixed solution of methanol and potassium dihydrogen phosphate aqueous solution; the volume of the potassium dihydrogen phosphate aqueous solution accounts for 5% or more of the volume of the second-dimensional mobile phase, and the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 20 mmol / L.

7. The method according to claim 1, characterized in that The two-dimensional column switching high performance liquid chromatography method is implemented by a two-dimensional high performance liquid chromatography system.

8. The method according to claim 1, characterized in that The calibration curve of 25-hydroxyvitamin D2 is Y=3.05×10 -1 X+5.04, r≥0.9990; r is the linear correlation coefficient; Y is the peak area of ​​the corresponding peak in the chromatogram, in mAU·sec; X is the concentration of 25-hydroxyvitamin D2, in ng / mL.

9. The method according to claim 1, characterized in that: The calibration curve of 25-hydroxyvitamin D3 is Y=3.71×10 -1 X+3.75, r≥0.9990; r is the linear correlation coefficient; Y is the peak area of ​​the corresponding peak in the chromatogram, in mAU·sec; X is the concentration of 25-hydroxyvitamin D3, in ng / mL.

10. The method according to claim 1, characterized in that In step 5 and step 6, the detection conditions of the two-dimensional column switching high performance liquid chromatography are: the detection wavelength for quantitative detection is 260-270 nm, the detection wavelength for qualitative detection is 260-270 nm, and the column temperature is 30-45°C.

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