CN115236253B

CN115236253B - Kit for detecting 25-hydroxy vitamin D in serum and application thereof

Info

Publication number: CN115236253B
Application number: CN202210866413.2A
Authority: CN
Inventors: 栗琳; 李小侠; 张新星; 吴政晖; 应洪波; 王倩倩; 秦于杰; 周立; 丁亮
Original assignee: Hunan Haosi Biotechnology Co ltd; Jiangsu Haosi Muke Biotechnology Co ltd; Beijing Haosi Biotechnology Co ltd
Current assignee: Hunan Haosi Biotechnology Co ltd; Jiangsu Haosi Muke Biotechnology Co ltd; Beijing Haosi Biotechnology Co ltd
Priority date: 2022-07-22
Filing date: 2022-07-22
Publication date: 2023-08-08
Anticipated expiration: 2042-07-22
Also published as: CN115236253A

Abstract

The invention provides a kit for detecting 25-hydroxy vitamin D in serum and application thereof, and relates to the technical field of vitamin D detection. The kit is used for detecting 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in serum by adopting a high performance liquid chromatography, takes buffer solution containing bovine serum albumin as diluent of a calibrator, further contains precipitant and sample extract for pretreatment of a sample, and takes laurylbenzophenone in the sample extract as an internal standard, thereby meeting the requirements of related regulations on linearity, repeatability, batch-to-batch difference, accuracy, stability and the like of the kit and having an important role in monitoring human vitamin D.

Description

Kit for detecting 25-hydroxy vitamin D in serum and application thereof

Technical Field

The invention relates to the technical field of vitamin D detection, in particular to a kit for detecting 25-hydroxy vitamin D in serum and application thereof.

Background

Vitamins are a kind of small molecular organic matters which are necessary for maintaining normal physiological functions of organisms and specific metabolic reactions in cells, are essential nutrient substances necessary for human health, and play an important role in regulating the metabolic processes of substances. Vitamin D belongs to fat-soluble vitamins, is an important vitamin for regulating bone metabolism, and can lead to rickets due to vitamin D deficiency of infants and osteoporosis due to adult deficiency. When the body ingests and accumulates excessive vitamin D, the negative feedback regulation of vitamin D in the body is deregulated, which can lead to hypercalcemia and a series of adverse reactions such as nausea, vomiting, constipation, pancreatitis, acute kidney injury, etc. The half-life of the 25-hydroxy vitamin D is longer, the in vivo existing form is stable, the concentration is higher, and the 25-hydroxy vitamin D is a marker for monitoring the in vivo vitamin D nutrition level. 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 are the main existence forms of 25-hydroxy vitamin D in blood circulation, and can be used as detection indexes of vitamin D, so that the content of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in human bodies is monitored, and the evaluation of the condition of human vitamin D is facilitated. There is no high performance liquid chromatography kit for diagnosing vitamin D in human serum in the current market.

In view of this, the present invention has been made.

Disclosure of Invention

The first aim of the invention is to provide a kit for detecting 25-hydroxy vitamin D in serum, so as to solve the problem of lack of an accurate and efficient vitamin D high performance liquid chromatography kit in the prior art.

A second object of the present invention is to provide the use of the above-described kit for the detection of 25-hydroxyvitamin D in serum for non-diagnostic and therapeutic purposes.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention there is provided a kit for detecting 25-hydroxyvitamin D in serum, the 25-hydroxyvitamin D comprising 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3;

the kit comprises a calibrator for high performance liquid chromatography, wherein the calibrator is a mixed solution containing 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3; a diluent for diluting the calibrator; and a precipitant and a sample extract for pre-treating the sample;

the diluent is a buffer solution containing bovine serum albumin;

the precipitant is an aqueous solution of sulfate or nitrate;

the sample extract is an organic solution containing laurylbenzophenone.

Preferably, the diluent is a PBS buffer containing bovine serum albumin.

Preferably, the diluent is PBS buffer containing 1-5% bovine serum albumin;

preferably, the diluent is PBS buffer containing 1% bovine serum albumin.

Preferably, the precipitant is an aqueous solution of zinc sulfate or an aqueous solution of silver nitrate;

preferably, the precipitant is zinc sulfate aqueous solution with the concentration of 0.01-0.2 mol/L;

preferably, the precipitant is an aqueous solution of zinc sulfate at a concentration of 0.2 mol/L.

Preferably, the solvent of the sample extract is ethyl acetate or n-hexane;

preferably, the sample extract is ethyl acetate solution of laurylbenzophenone with the concentration of 800-1200 ng/mL;

preferably, the sample extract is an ethyl acetate solution of laurylbenzophenone at a concentration of 1000ng/mL.

Preferably, the calibration standard comprises a calibration standard S1, a calibration standard S2, a calibration standard S3, a calibration standard S4, a calibration standard S5 and a calibration standard S6; preparing each calibrator according to the target concentration, and then carrying out higher-level calibrator assignment to obtain the marked concentration of the calibrator; the target concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the calibrators S1-S6 were, in order, 70, 100, 200, 500, 800 and 1000ng/mL.

Preferably, the kit further comprises a quality control.

Preferably, the quality control product comprises a low-value quality control product and a high-value quality control product; preparing each quality control product according to the target concentration, and then carrying out higher-level calibration product assignment to obtain the marked concentration of the quality control product; the target concentrations of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the low-value quality control product and the high-value quality control product are 200ng/mL and 800ng/mL in sequence.

Preferably, the kit comprises calibrator S1-S6, low-value quality control product, high-value quality control product, diluent, precipitant and sample extract;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S1 is 70ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S2 is 100ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S3 is 200ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S4 is 500ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S5 is 800ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S6 is 1000ng/mL, and methanol is used as a solvent;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the low-value quality control product is 200ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the high-value quality control product is 800ng/mL, and methanol is used as a solvent;

the diluent is PBS buffer solution containing 1% bovine serum albumin according to mass percent; the precipitant is zinc sulfate water solution with the concentration of 0.2 mol/L; the sample extract was an ethyl acetate solution of laurylbenzophenone at a concentration of 1000ng/mL.

According to another aspect of the present invention there is also provided the use of the above kit for the detection of 25-hydroxyvitamin D in serum for non-diagnostic and therapeutic purposes, said detection comprising subjecting a pre-treated sample to high performance liquid chromatography for the analysis of 25-hydroxyvitamin D in serum; the pretreatment includes precipitation and extraction of the sample.

Compared with the prior art, the invention has the following beneficial effects:

aiming at the blank of the high performance liquid chromatography kit for diagnosing the vitamin D in the human serum in the market, the invention researches a high performance liquid chromatography detection kit capable of accurately and efficiently evaluating the 25-hydroxy vitamin D2 and the 25-hydroxy vitamin D3 in the serum.

The kit provided by the invention adopts the buffer solution containing bovine serum albumin as a substitute matrix of a serum sample, and has the advantages of easiness in acquisition, low cost, easiness in quality control and the like. The kit provided by the invention further comprises a precipitant and a sample extract for pretreatment of the sample. The precipitant is sulfate aqueous solution for precipitating protein in serum, and 25-hydroxy vitamin D in serum can be rapidly extracted by adding sample extract and simple vibration extraction. The sample extract is an organic solution containing laurylbenzophenone, which can rapidly extract 25-hydroxy vitamin D in the sample after precipitation treatment, and the laurylbenzophenone in the extract is used as an internal standard substance in high performance liquid chromatography. The commonly used internal standard for testing the 25-hydroxy vitamin D is an isotope internal standard, but the kit adopts a liquid chromatograph, rather than a mass spectrometer, and the liquid chromatograph can not achieve the separation effect on the isotope internal standard, so that laurylbenzophenone is used as the internal standard, and the kit has the advantages that: 1. the nature of the material is similar to that of the target; 2. the method can eliminate endogenous interference, has certain stability under the same pretreatment and chromatographic conditions, and has the retention time close to but not overlapped with that of 25-hydroxy vitamin D, so that the method can be used as an internal standard, and errors caused by changes of operating conditions and the like are eliminated to a certain extent.

The kit for detecting 25-hydroxy vitamin D in serum provided by the invention can accurately and efficiently evaluate the contents of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in serum, can meet the requirements of related regulations on linearity, repeatability, batch difference, accuracy, stability and the like of the kit, and has an important role in monitoring human vitamin D.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a chromatogram of an HPLC analysis performed by an isocratic procedure using the kit of the present invention in effect example 10;

FIG. 2 is a chromatogram of HPLC analysis performed by gradient procedure 1 using the kit of the present invention in effect example 10;

fig. 3 and 4 show chromatograms of the high performance liquid chromatography analysis using the kit according to the present invention in the effect example 10 according to the gradient program 2.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

According to one aspect of the present invention there is provided a kit for detecting 25-hydroxyvitamin D in serum, the 25-hydroxyvitamin D comprising 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

The kit provided by the invention comprises a precipitant and a sample extract for pretreatment of a sample, a calibrator for high performance liquid chromatography, and optionally a quality control product for quality control of detection results, and a diluent for diluting the calibrator and the quality control product.

The kit provided by the invention is used for carrying out pretreatment on a sample and a sample extract, wherein the precipitant is an aqueous solution of sulfate or an aqueous solution of nitrate; the sulfate is optionally zinc sulfate and the nitrate is optionally silver nitrate.

In some alternative embodiments, the precipitant is an aqueous solution of zinc sulfate at a concentration of 0.01 to 0.2mol/L, such as, but not limited to, an aqueous solution of zinc sulfate at a concentration of 0.01, 0.05, 0.1, or 0.2mol/L, preferably 0.2 mol/L.

The sample extract is an organic solution containing laurylbenzophenone, preferably n-hexane or ethyl acetate, and in some alternative embodiments, the sample extract is an ethyl acetate solution of laurylbenzophenone at a concentration of 800 to 1200ng/mL, such as, but not limited to, 800, 900, 1000, 1100 or 1200ng/mL, preferably 1000ng/mL.

The protein in the serum is precipitated by adding the sulfate aqueous solution, and the 25-hydroxy vitamin D in the serum can be rapidly extracted by adding the organic solvent and simple extraction and shaking. The pretreatment reagent can achieve the effect of rapidly extracting 25-hydroxy vitamin D in serum, and the pretreatment method is simple and rapid.

The sample extract is an organic solution containing laurylbenzophenone, which can rapidly extract 25-hydroxy vitamin D in the sample after precipitation treatment, and the laurylbenzophenone in the extract is used as an internal standard substance in high performance liquid chromatography. The property of the laurylbenzophenone is similar to that of the 25-hydroxy vitamin D, the laurylbenzophenone is selected as an internal standard, so that endogenous interference can be eliminated, and the laurylbenzophenone has certain stability under the same pretreatment and chromatographic conditions, is close to but not overlapped with the retention time of the 25-hydroxy vitamin D, thus being used as an internal standard, and eliminating errors caused by the change of operating conditions and the like to a certain extent.

When the high performance liquid chromatography is used for analyzing a sample, a standard curve is firstly required to be established, wherein the standard curve is a function relation between the content of a substance to be detected in a calibrator with known concentration and the characteristic value of a chromatogram thereof, for example, a function relation between the content and the peak area or the peak height. And then, the characteristic value of the chromatogram of the sample to be detected is put into the constructed standard curve, so that the content of the substance to be detected in the sample to be detected can be obtained. Therefore, the kit provided by the invention also contains a calibrator for constructing a standard curve. The calibrator provided by the invention is a mixed solution containing 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3.

The kit provided by the invention also contains a diluent, the diluent is used for diluting the calibrator, and when the kit also contains a quality control product, the diluent can also be used for diluting the quality control product. The dilution is a buffer containing bovine serum albumin, preferably a PBS buffer containing bovine serum albumin, more preferably a PBS buffer containing 1-5% bovine serum albumin, and the concentration may be, for example, but not limited to, 1%, 2%, 3%, 4% or 5%. The diluent is used as a substitute matrix of a serum sample, and has the advantages of easy acquisition, low cost, easy quality control and the like.

Six concentration points are preferably arranged on the calibrator in the kit, wherein the calibration points comprise a calibrator S1, a calibrator S2, a calibrator S3, a calibrator S4, a calibrator S5 and a calibrator S6, each calibrator is prepared according to target concentration, and then the marked concentration of the calibrator is obtained through higher-level calibrator assignment. The target concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the calibrators S1-S6 were, in order, 70, 100, 200, 500, 800 and 1000ng/mL.

The term "target concentration" in the present invention refers to an expected value for a target concentration of a calibrator at the time of preparing the calibrator, not an actual concentration of the calibrator. In actual operation, the calibration material is prepared by errors caused by factors such as operation, instruments, containers and the like, so that the actual concentration of the calibration material deviates from the theoretical concentration of the pre-prepared calibration material, namely, the target concentration. In practice, a calibration standard for establishing a standard curve is usually corrected and assigned by using a calibration standard of a higher level, and the marked concentration of the calibration standard assigned by the calibration standard of the higher level may deviate from the value of the target concentration, which is acceptable in the field. Therefore, when preparing a calibrator based on the target concentration, the resulting calibrator is within the scope of the present invention, even if not strictly conforming to the target concentration, within an acceptable error in the art.

The kit provided by the invention preferably further comprises quality control products for controlling the quality of the detection results, and in order to evaluate the detection results more comprehensively, the quality control products preferably comprise low-value quality control products and high-value quality control products. The target concentrations of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the low-value quality control product and the high-value quality control product are 200ng/mL and 800ng/mL in sequence. The meaning of the quality control target concentration refers to the description of the calibration target concentration, and is not described herein.

In a preferred embodiment, the kit for detecting 25-hydroxyvitamin D in serum consists of the following reagents: calibrators S1-S6, low-value quality control products, high-value quality control products, diluent, precipitant and sample extract.

The target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S1 is 70ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S2 is 100ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S3 is 200ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S4 is 500ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S5 is 800ng/mL, and methanol is used as a solvent; the target concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in calibrator S6 were 1000ng/mL with methanol as solvent.

The target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the low-value quality control product is 200ng/mL, and methanol is used as a solvent; the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the high-value quality control product is 800ng/mL, and methanol is used as a solvent.

The kit for detecting 25-hydroxy vitamin D in serum provided by the invention can meet the requirements of related regulations on linearity, repeatability, inter-batch difference, accuracy, stability and the like of the kit, and has an important effect on monitoring human vitamin D. The linear range of the 25-hydroxy vitamin D2 of the kit is 7-100 ng/mL, the linear range of the 25-hydroxy vitamin D3 is 7-100 ng/mL, and the correlation coefficient r is more than or equal to 0.990; the Coefficient of Variation (CV) of the repeatability of the low-value quality control product is less than or equal to 20 percent, and the Coefficient of Variation (CV) of the repeatability of the high-value quality control product is less than or equal to 15 percent; the relative difference (R) between batches of the low-value quality control product is less than or equal to 20 percent, and the relative difference (R) between batches of the high-value quality control product is less than or equal to 15 percent; the relative deviation (B) of the accuracy is less than or equal to +/-15 percent.

According to another object of the present invention, there is also provided the use of the above kit for the detection of 25-hydroxyvitamin D in serum for non-diagnostic and therapeutic purposes, said detection comprising subjecting a pre-treated sample to high performance liquid chromatography for the analysis of 25-hydroxyvitamin D in serum; the pretreatment includes precipitation and extraction of the sample. The kit is applied to detecting 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in serum, and has the advantages of high detection accuracy, good stability, simple operation, short detection time and the like.

The technical solution and advantageous effects of the present invention are further described below in connection with preferred embodiments. 1. Reagent, standard substance, quality control substance and important consumable material information

TABLE 1

2. Instrument for measuring and controlling the intensity of light

2.1 analytical instrument

TABLE 2

Instrument name	Model number	Branding	Instrument numbering
				Liquid chromatograph	LC 2300	Harmony instrument	MY20201216-A005

2.2 other instruments

TABLE 3 Table 3

Instrument name	Model number	Branding
			Vortex mixing instrument	G560E	VORT
High-speed refrigerated centrifuge	D3024R	Dalong Xingzhuang (Chinese character) wound
			Nitrogen blowing instrument	NV96-G-8	Agela
96-well plate oscillator	MB100-4A	Hangzhou ao Sheng
			100 mu L pipette	10-100μL	eppendorf
200 mu L pipettor	20-200μL	eppendorf
			1000 mu L pipettor	100-1000μL	eppendorf

Example 1 kit

The kit comprises the following components:

the kit provided in this example comprises the following components:

TABLE 4 Table 4

(II) preparation of reagents

1. Work calibrator and quality control product preparation

1.1 Preparation of 25-hydroxy vitamin D intermediate

Precisely transferring appropriate amount of 25-hydroxy vitamin D2 and D3 standard substances, placing into a proper centrifuge tube, adding methanol, and mixing.

Table 5 intermediate liquid formulation

1.2 And preparing 25-vitamin D working calibrators G1-G6 and working quality control products GQCL-GQCH, wherein the working calibrators and the working quality control products are respectively high-grade quality control products of the calibrators and the quality control products in the kit and are used for assigning values to the calibrators and the quality control products in the kit.

The calibrator G1 to G6 and the working quality control GQCL to GQCH were prepared by diluting the intermediate liquid with methanol, respectively, and the dilution ratio is shown in the following table (note that the sampling amounts of the intermediate liquid are different).

TABLE 6 working calibrants G1-G6 and working quality control GQCL-GQCH formulations

2. Product calibrator and quality control product preparation

2.1 25-hydroxyvitamin D2 (SSC-D2) and 25-hydroxyvitamin D3 (SSC-D3) stock solutions are prepared by taking 1mg/mL of 25-hydroxyvitamin D2/D3 stock solution as an example:

the purchased 25-hydroxy vitamin D2 (BePure, MU-0005-1 mg) has accurate quality, 1mL (accurately removed) methanol is directly added without weighing, ultrasound is carried out until the 25-hydroxy vitamin D2 is completely dissolved, 1.00mg/mL 25-hydroxy vitamin D2 stock solution (SSC-D2) is obtained, the stock solution is transferred into a 1.5mL brown vial, and the stock solution is labeled and stored at the temperature of minus 20 ℃;

the purchased 25-hydroxyvitamin D3 (BePure, MU-0010-1 mg) has accurate quality, 1mL (accurately removed) of methanol is directly added without weighing, ultrasound is performed until the 25-hydroxyvitamin D3 is completely dissolved, 1.00mg/mL of 25-hydroxyvitamin D3 stock solution (SSC-D3) is obtained, the stock solution is transferred into a 1.5mL brown vial, labeled, and stored at-20 ℃.

2.2 preparation of calibrator S6, see Table below

TABLE 7

2.3 preparation of calibration materials S1 to S5

TABLE 8

2.4 quality control preparation

TABLE 9

3. Sample extract preparation

Accurately weighing 20mg of laurylbenzophenone, converting the purity of standard substances and the carried crystal water or salt during actual weighing, transferring into a 50.00mL volumetric flask, adding a certain amount of methanol, covering a bottle stopper to prevent leakage, reversing upside down until the laurylbenzophenone is completely dissolved, adding methanol to fix volume to 50.00mL to obtain 400 mug/mL laurylbenzophenone stock solution (ISSC), transferring into a 60mL brown glass bottle, labeling, and preserving at-20 ℃.

Taking 5.6L sample extract as an example, accurately transferring 7mL of ISSC to a container (about 2.8L scale points are determined in advance), fixing the volume of HPLC-grade ethyl acetate to the scale, reversing the top and bottom for 15 times to thoroughly mix uniformly, preparing a sample extract, repeating the steps twice to obtain 5.6L sample extract, wherein the laurylbenzophenone content is 1000ng/mL, and the preparation method is referred to in the following table.

Table 10

4. Preparation of dilution

Take the amount of diluent to produce a 60-box kit product as an example.

(1) 190mL of 10 XPBS was measured in a 2.5L glass vessel, and 1710mL of pure water was added thereto to mix the mixture uniformly, thereby obtaining 1 XPBS.

(2) 18.48g BSA was weighed into a 2.5L glass container, 1848mL of 1 XPBS was weighed into the 2.5L glass container using a cartridge, the lid was closed, and the mixture was shaken up and down until the BSA was completely dissolved.

5. Preparation of precipitant

Taking the amount of precipitant for producing 60 kit products as an example, weighing 69.012g of zinc sulfate, placing into a 2L glass container, measuring 1.2L of ultrapure water by using a measuring cylinder, placing into the 2L glass container, covering a bottle cap, and oscillating up and down until the zinc sulfate is completely dissolved.

Method for using kit

1. The sample treatment method comprises the following steps:

1) Adding a calibrator and a quality control product solution: precisely transferring 30 mu L of calibrator and quality control product solution, and respectively adding into 2mL centrifuge tubes;

2) Adding a diluent: precisely transferring 270 mu L of diluent, and respectively adding into the corresponding centrifugal tubes;

3) Serum samples were added: precisely transferring 300 mu L of serum sample, and respectively adding into 2mL centrifuge tubes;

4) Adding a precipitating agent: precisely transferring 150 mu L of precipitant, respectively adding into the corresponding 2mL centrifuge tubes, and slightly shaking for a few seconds;

5) Adding a sample extract: precisely transferring 800 mu L of sample extract, and respectively adding into the corresponding 2mL centrifuge tubes;

6) Oscillating: covering a centrifugal tube cover, placing on a 2mL vortex mixer, and sufficiently vibrating at 3000rpm for 15min;

7) And (3) centrifuging: centrifuge at 10000rpm for 10min at 4 ℃. Taking 600 mu L of supernatant and placing the supernatant into a 96-well U-shaped plate;

8) Nitrogen blowing: placing the 96-well plate in the step 7 into a nitrogen blowing instrument, and blowing nitrogen at 60 ℃ to dry;

9) And (3) re-dissolving: 80. Mu.L (acetonitrile/water: 8:2) was removed and added to each well used;

10 Oscillating): covering a 96-well plate pad, placing in a 96-well plate mixing instrument, and sufficiently vibrating at 1000rpm for 10min;

11 Detecting: the 96-well plate was placed in LC for detection.

2. High performance liquid chromatography conditions

2.1 chromatographic conditions:

TABLE 11

2.2 elution procedure

Table 12

Time (min)	Flow rate mL/min	Mobile phase a	Mobile phase B
				0	1	20	80
3	1	23	77
				4	1	40	60
5	1	37	63
				15	1	33	67
17	1	5	95
				20	1	5	95
22	1	20	80

Effect example 1 kit appearance

1. Appearance of

1) The verification method comprises the following steps: 1 bottle of each calibrator in the kit is randomly taken and visually observed under natural light to correct vision.

2) Acceptance criteria: the package is complete and has no damage; the characters and the contents of the minimum packaging label are clear and accurate, the seal is tight, and no leakage exists; the components should be clear, free of sediment, particles or flocs.

2. Experimental results:

(a) The components of the kit are complete and have no damage.

(b) The characters and contents of the minimum packaging label should be clear and accurate, the seal is tight, and no leakage exists.

(c) The liquid component should be clear, transparent, free of sediment, particles or flocs.

3. Conclusion: the package is complete and has no damage; the characters and the contents of the minimum packaging label are clear and accurate, the seal is tight, and no leakage exists; the components are clear, have no sediment, particles or floccules, and meet the acceptance criteria.

Effect example 2 kit amount

1. Filling amount

1) The verification method comprises the following steps: the kit 1 was randomly taken and the net content of each liquid component was measured using a universal measuring tool.

2) Acceptance criteria: the net content of each liquid reagent in the kit should be not less than the standard value.

2. Test results:

TABLE 13

3. Conclusion: the net content of each liquid reagent in the kit is not less than the standard value, and meets the acceptance standard.

Effect example 3 Linear Range of kit

1. Linear range

1) The verification method comprises the following steps: and (3) treating the solutions of the calibrator S1-S6 of the product to be tested according to a sample treatment method, and repeating the test for 3 times for each concentration. The correlation coefficient r of the linear regression can be calculated by referring to a formula, and the correlation coefficient r is more than or equal to 0.990.

r: linear regression correlation coefficient

xi: concentration of S1 to S6

yi: peak area ratio average value of calibrator and internal standard in corresponding concentration solution

2) Acceptance criteria: the correlation coefficient r of the linear regression of the 25-hydroxy vitamin D2 and the 25-hydroxy vitamin D3 is more than or equal to 0.990.

2. Experimental results:

linear range: vitamin D2/D3:7 ng/mL-100 ng/mL

TABLE 14

3. Conclusion: the correlation coefficient r of the linear regression of the 25-hydroxy vitamin D2 and the 25-hydroxy vitamin D3 is more than or equal to 0.990, and the acceptance standard is met.

Effect example 4 kit reproducibility

1. Repeatability of

1) The verification method comprises the following steps: under the condition of repeatability, the quality control product is tested by using the kit according to the test method, and the test is repeated for 10 times. The Coefficient of Variation (CV) of repeatability can be calculated by referring to a formula, the CV of the low-value quality control product is less than or equal to 20%, and the CV of the high-value quality control product is less than or equal to 15%.

CV: coefficient of variation of repeatability

Average of 10 measurements

S: standard deviation of 10 measurements

2) Acceptance criteria: the coefficient of variation CV of the low-value quality control product is less than or equal to 20 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent.

2. Experimental results:

TABLE 15

3. Conclusion: the coefficient of variation CV of the low-value quality control product is less than or equal to 20 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent, thereby meeting the acceptance standard.

Effect example 5 kit batch to batch differences

1. Difference between batches

1) The verification method comprises the following steps: quality control was tested according to the test method using three different lot number kits and repeated 3 times. The relative range (R) between batches can be calculated by referring to a formula, the relative range R between batches of a low-value quality control product is less than or equal to 20 percent, and the relative range R between batches of a high-value quality control product is less than or equal to 15 percent.

R: relative differences between batches;

average of 3 measurements per batch; />Maximum value of (2); />Is the minimum value of (a); />And 3, detecting the average value in batches.

2) Acceptance criteria: the relative range R between batches of the low-value quality control product is less than or equal to 20 percent, and the relative range R between batches of the high-value quality control product is less than or equal to 15 percent.

2. Experimental results

Table 16

3. Conclusion: the relative range R between batches of the low-value quality control product is less than or equal to 20 percent, and the relative range R between batches of the high-value quality control product is less than or equal to 15 percent, thereby meeting the acceptance standard.

Effect example 6 kit accuracy

1. Accuracy of

1) The verification method comprises the following steps: the standard reference substance SRM2972a was tested using the kit according to the test method of example 1, and the measurement was repeated 3 times. The relative deviation (B) of the accuracy can be calculated by referring to a formula, and the relative deviation B is less than or equal to +/-15 percent.

B＝(M-T)/T×100％；

B: a relative deviation; m: testing the average value of the results; t: the values are indicated.

2) Acceptance criteria: the relative deviation B of the accuracy is less than or equal to +/-15 percent.

2. Experimental results:

TABLE 17

3. Conclusion: the relative deviation B of the accuracy is less than or equal to +/-15 percent, and meets the acceptance standard.

Effect example 7 stability of the kit during the expiration date

1. Stability of expiration date

1) The verification method comprises the following steps: and (3) taking the kit which is stored in a dark place at the normal storage temperature (-18+/-8 ℃) for 1 month beyond the effective period (6 months), and detecting according to the effect examples 1-4 and 6, wherein the result meets the corresponding requirements.

2) Acceptance criteria: the package is complete and has no damage; the characters and the contents of the minimum packaging label are clear and accurate, the seal is tight, and no leakage exists; the components should be clear without precipitation, particles or flocs; the net content of each liquid reagent in the kit should be not less than the standard value; the correlation coefficient r of the linear regression of the 25-hydroxy vitamin D2 and the 25-hydroxy vitamin D3 is more than or equal to 0.990; the coefficient of variation CV of the low-value quality control product is less than or equal to 20 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent; the relative deviation B of the accuracy is less than or equal to +/-15 percent.

2. Experimental results

1) Appearance of

(a) The components of the kit are complete and have no damage.

2) Filling amount

TABLE 18

Filling amount	Actual measurement	standard	Determination of
				S1	390μL	≥360μL	Qualified product
S2	390μL	≥360μL	Qualified product
				S3	390μL	≥360μL	Qualified product
S4	390μL	≥360μL	Qualified product
				S5	390μL	≥360μL	Qualified product
S6	390μL	≥360μL	Qualified product
				QCL	390μL	≥360μL	Qualified product
QCH	390μL	≥360μL	Qualified product
				Sample extract	83mL	≥80mL	Qualified product
Precipitant	18mL	≥15mL	Qualified product
				Dilution liquid	28mL	≥26mL	Qualified product

3) Linear range: vitamin D2/D3, 7 ng/mL-100 ng/mL

TABLE 19

4) Repeatability of

Table 20

5) Accuracy of

Table 21

3. Conclusion: the package is complete and has no damage; the characters and the contents of the minimum packaging label are clear and accurate, the seal is tight, and no leakage exists; the components are clear, and no sediment, particles or floccules exist; the net content of each liquid reagent in the kit is not less than the standard value; the correlation coefficient r of the linear regression of the 25-hydroxy vitamin D2 and the 25-hydroxy vitamin D3 is more than or equal to 0.990; the coefficient of variation CV of the low-value quality control product is less than or equal to 20 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent; the relative deviation B of the accuracy is less than or equal to +/-15 percent, and meets the acceptance standard.

Effect example 8 kit calibrator

1. Appearance of

3) Experimental results:

(a) The components of each calibrator should be packaged completely without damage.

(b) The characters and the contents of the packing label of each calibrator component should be clear and accurate, the sealing is tight, and no leakage exists.

(c) The components of each calibrator should be clear without sediment, particles or floc.

4) Conclusion: the package is complete and has no damage; the characters and the contents of the minimum packaging label are clear and accurate, the seal is tight, and no leakage exists; the components should be clear, free of sediment, particles or flocs, meeting the acceptance criteria.

2. Filling amount

1) The verification method comprises the following steps: 1 bottle of calibration product in the kit is randomly taken, and the volume is detected by using a universal measuring tool.

2) Acceptance criteria: the net content of the calibrator in the kit should be not less than the standard value.

3) Experimental results

Table 22

Filling amount	Actual measurement	standard	Determination of
				S1	390μL	≥360μL	Qualified product
S2	390μL	≥360μL	Qualified product
				S3	390μL	≥360μL	Qualified product
S4	390μL	≥360μL	Qualified product
				S5	390μL	≥360μL	Qualified product
S6	390μL	≥360μL	Qualified product

4) Conclusion: the net content of the calibrator in the kit is not less than the standard value, and meets the acceptance standard.

3. Accuracy of

1) The verification method comprises the following steps: and calibrating by using the working calibrator according to the detection method, testing each calibrator of the kit, and repeatedly detecting for 3 times. The relative deviation (B) of the accuracy can be calculated by referring to a formula, and the relative deviation B is less than or equal to +/-15 percent.

B＝(M-T)/T×100％

3) Experimental results

Table 23

4) Conclusion: the relative deviation B of the accuracy is less than or equal to +/-15 percent, and meets the acceptance standard.

4. Uniformity of

1) The verification method comprises the following steps: 10 bottles of calibrator of the same lot were tested according to the test method, and the average of 10 measurements was calculated according to formulas (1) and (2)And standard deviation (S1). The test was repeated 10 times again for 1 bottle in the lot number, and the average value +.>And standard deviation (S2). And (3) calculating a Coefficient of Variation (CV) according to the formula (3) and the formula (4), wherein the coefficient of variation CV of the S1-S3 calibrator is less than or equal to 20%, and the coefficient of variation CV of the S4-S6 calibrator is less than or equal to 15%. />

Let cv=0 when S1 < S2.

2) Acceptance criteria: the variation coefficient CV of the calibration products of S1-S3 is less than or equal to 20 percent, and the variation coefficient CV of the calibration products of S4-S6 is less than or equal to 15 percent.

3) Experimental results:

table 24

Table 25

/>

4) Conclusion: the variation coefficient CV of the calibration products of S1-S3 is less than or equal to 20 percent, the variation coefficient CV of the calibration products of S4-S6 is less than or equal to 15 percent, and the acceptance standard is met.

5. Stability of

a) Stability of opening bottle

1) The verification method comprises the following steps: after unsealing, the kit calibrator is stored in a dark place at the normal storage temperature (-18 ℃ +/-8 ℃) for more than 1 week after the bottle opening validity period (15 days), and is detected according to the 2 nd part (loading amount), the 3 rd part (accuracy) and the 4 th part (uniformity) of the effect example 8, and the result meets the corresponding requirements.

2) Acceptance criteria: the net content of the calibrator in the kit is not less than the standard value; the relative deviation B of the accuracy is less than or equal to +/-15 percent; the variation coefficient CV of the calibration products of S1-S3 is less than or equal to 20 percent, and the variation coefficient CV of the calibration products of S4-S6 is less than or equal to 15 percent.

3) Experimental results:

a) Filling amount

Table 26

B) Accuracy of

Table 27

/>

C) Uniformity of

Table 28

Table 29

/>

4) Conclusion: the net content of the calibrator in the kit is not less than the standard value; the relative deviation B of the accuracy of the calibrator is less than or equal to +/-15 percent; the variation coefficient CV of the calibration products of S1-S3 is less than or equal to 20 percent, the variation coefficient CV of the calibration products of S4-S6 is less than or equal to 15 percent, and the acceptance standard is met.

b) Stability of expiration date

1) The verification method comprises the following steps: and (3) taking a kit calibrator which is stored in a dark place at the normal storage temperature (-18+/-8 ℃) for more than 1 month after the effective period (6 months), and detecting according to the 1 st part (appearance) to the 4 th part (uniformity) of the effect example 8, wherein the result meets the corresponding requirement.

2) Acceptance criteria: the package is complete and has no damage; the characters and the contents of the minimum packaging label are clear and accurate, the seal is tight, and no leakage exists; the components should be clear without precipitation, particles or flocs; the net content of the calibration product in the kit should be not less than the standard value; the relative deviation B of the accuracy is less than or equal to +/-15 percent; the variation coefficient CV of the calibration products of S1-S3 is less than or equal to 20 percent, and the variation coefficient CV of the calibration products of S4-S6 is less than or equal to 15 percent.

3) Experimental results:

a) Appearance:

B) The filling amount is as follows:

table 30

C) Accuracy:

table 31

D) Uniformity of

Table 32

/>

Table 33

4) Conclusion: the calibrator is completely packaged and is not damaged; the characters and the contents of the minimum packaging label are clear and accurate, the seal is tight, and no leakage exists; the components are clear, and no sediment, particles or floccules exist; the net content of the calibrator in the kit is not less than the standard value; the relative deviation B of the accuracy is less than or equal to +/-15 percent; the variation coefficient CV of the calibration products of S1-S3 is less than or equal to 20 percent, the variation coefficient CV of the calibration products of S4-S6 is less than or equal to 15 percent, and the acceptance standard is met.

Effect example 9 quality control product of kit

1. Appearance of

1) The verification method comprises the following steps: and randomly taking 1 bottle of quality control products in the kit, and visually observing under natural light to correct vision.

3) Experimental results:

(a) The components of each quality control product are completely packaged without damage.

(b) The characters and the contents of the packaging label of each quality control product component should be clear and accurate, the sealing is tight, and no leakage exists.

(c) The quality control components are clear and have no sediment, particles or floccules.

2. The filling amount is as follows:

1) The verification method comprises the following steps: and randomly taking 1 bottle of each quality control product in the kit, and detecting the volume by using a universal measuring tool.

2) Acceptance criteria: the net content of the quality control product in the kit should be not less than the standard value.

3) Experimental results:

watch 34

Filling amount	Actual measurement	standard	Determination of
				QCL	390μL	≥360μL	Qualified product
QCH	390μL	≥360μL	Qualified product

4) Conclusion: the net content of the quality control product in the kit is not less than the standard value, and meets the acceptance standard.

3. The expected results are:

1) The verification method comprises the following steps: and (3) operating according to a detection method, calibrating by using a product calibrator, testing each quality control product in the kit, and repeating the test for 3 times. The detection result of the quality control product is within the acceptable range of the quality control product.

2) Acceptance criteria: the detection result of the quality control product is within the acceptable range of the quality control product.

3) Experimental results:

table 35

4) Conclusion: the detection result of the quality control product is within the acceptable range of the quality control product, and meets the acceptance standard.

4. Uniformity of

1) The verification method comprises the following steps: and testing 10 bottles of quality control products in the same batch of kits according to a detection method, wherein the quality control products with each concentration level are randomly numbered 1-10, and the test is repeated for 3 times per bottle.

The 3 tests were performed in the following order: 1. 3, 5, 7, 9, 2, 4, 6, 8, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9.

Recording the detection result, and calculating F, S according to the formulas (5-14) _bb 、S _r And CV (CV) _{Bottle room} 。

/>

SS _{In the bottle} ＝SS _{Sum total} -SS _{Bottle room} … … … … … … … … … … … … … … formula (7)

CV: coefficient of variation; SS: variance; v (V): degree of freedom; MS: mean square; f: f, checking a value; n is n ₀ : the number of effective measurements; s is(s) _bb : standard deviation between bottles; s is(s) _r : standard deviation of repeatability; x: measuring or calculating the result;total average.

When F is less than or equal to 1, s is used as _r Instead of s _bb Calculation of CV _{Bottle room} The result should meet the corresponding requirements.

When F is less than or equal to F0.05 (v 1, v 2), the test result shows that the uniformity among bottles has no significant difference, and the result CV is calculated _{Bottle room} The result should meet the corresponding requirements.

When F is greater than F0.05 (v 1, v 2), s _bb When the delta is less than or equal to 0.3 delta, the uniformity among bottles is considered to be good, and the CV is calculated _{Bottle room} The result should meet the corresponding requirements.

When F is greater than F0.05 (v 1, v 2), s _bb At > 0.3 delta, the uniformity between bottles was considered poor, and the corresponding requirements were not met.

Note that: delta is the target standard deviation.

3) Experimental results:

table 36

F0.05 =2.39, qcl:0.3δ=0.15×target=0.15×20=0.3

Table 37

/>

F0.05 =2.39, qch:0.3δ= 0.15×target value=0.15×80=1.2

4) Conclusion: the coefficient of variation CV of the low-value quality control product is less than or equal to 20 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent, thereby meeting the acceptance standard.

5. Stability of

1) The verification method comprises the following steps: after unsealing, taking quality control products of the kit which are preserved in dark at the normal storage temperature (-18+/-8 ℃) for more than the effective period of bottle opening (15 days), respectively taking 3 quality control products, and repeatedly measuring each quality control product for 2 times. The difference significance test can be performed with reference to the formula, and the difference should not be significant.

The average value of the measurement at the end of the bottle opening stabilization period; />The measurement average value of the newly opened bottle; n is n ₁ : the number of times of newly opening the bottle; n is n ₂ : the number of times of measurement at the end of the stable period of opening the bottle; s is(s) ₁ : measuring standard deviation of a newly opened bottle; s is(s) ₂ : standard deviation was measured at the end of the open vial stabilization period.

The degree of freedom when t < significance level a (a=0.05) is the critical value ta (n) of (n1+n2-2) ₁ +n ₂ -2) there is no significant difference between the two averages.

2) Acceptance criteria: t < significance level a (a=0.05) degrees of freedom (n) ₁ +n ₂ -2) critical value ta (n) ₁ +n ₂ -2) there is no significant difference between the two averages.

3) Experimental results:

table 38

/>

4) Conclusion: t < significance level a (a=0.05) degrees of freedom (n) ₁ +n ₂ -2) critical value ta (n) ₁ +n ₂ -2) meeting the acceptance criteria.

b) Validity period stability:

1) The verification method comprises the following steps: and (3) carrying out statistical treatment on the stability research data, and carrying out trend significance test by referring to a formula, wherein the trend is not significant.

t _0.05，n-2 =tinv (0.05, n-2) … … … … … … … … … … … … … … … … equation (18)

When |b1| < t0.05, n-2·s (b 1), the trend is not significant, otherwise the trend is significant.

2) Acceptance criteria: ib1| < t0.05, n-2 s (b 1).

3) Experimental results:

QCL

table 39

Table 40

/>

QCH

Table 41

Table 42

4) Conclusion: and b1 is less than t0.05, n-2.s (b 1) meets the acceptance criterion.

Effect example 10 liquid chromatography condition optimization Process

1. Fig. 1 is acetonitrile: and (3) a spectrogram of the human serum sample when water (9:1) is subjected to isocratic test, wherein a green spectrogram is a standard sample, and a red spectrogram and a blue spectrogram are two human serum samples respectively. From the spectrogram, when the human serum sample is tested isocratically, the interfering substance in the human matrix overlaps with the VD2, so that the content of the VD2 in the human body cannot be accurately quantified.

2. Liquid chromatography was performed with the following liquid gradient procedure 1, and the results are shown in fig. 2, wherein blue is a standard spectrum and red is a human serum spectrum.

Table 43

Time (min)	Flow rate mL/min	Mobile phase a	Mobile phase B
				0	1	20	80
3	1	25	75
				5	1	30	70
13	1	30	70
				15	1	5	95
17	1	5	95
				19	1	20	80

3. Liquid chromatography was performed using the following liquid gradient procedure 2, and the results are shown in fig. 3 and 4, wherein red is a standard spectrum and blue is a human serum spectrum in fig. 3 and 4.

Table 44

Through testing different human serum samples, after continuously optimizing the gradient program, from the spectrograms, the liquid phase gradient program 2 can effectively separate VD3 and VD2, and no interferents near the VD3 and the VD2 influence the quantitative test.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. Use of a kit for the detection of 25-hydroxyvitamin D in serum for non-diagnostic and therapeutic purposes, characterized in that said 25-hydroxyvitamin D comprises 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3;

the kit comprises a calibrator for high performance liquid chromatography, wherein the calibrator is a mixed solution containing 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3; a diluent for diluting the calibrator and a precipitant and a sample extract for pre-treating the sample;

the diluent is a buffer solution containing bovine serum albumin;

the precipitant is an aqueous solution of sulfate or nitrate;

the sample extract is ethyl acetate solution containing laurylbenzophenone;

the detection comprises the steps of analyzing 25-hydroxy vitamin D in serum by adopting a high performance liquid chromatography on a pretreated sample; the pretreatment comprises precipitation and extraction of a sample;

the conditions for the analysis by high performance liquid chromatography include:

the high performance liquid chromatography adopts a chromatographic column: c18, specification 5 μm 4.6X105 mm;

mobile phase a was 0.07% formic acid in water by volume percent; mobile phase B was 0.07% acetonitrile formate solution;

the gradient elution procedure was:

time 0min, mobile phase A20%, mobile phase B80%;

3 min, mobile phase A23%, mobile phase B77%;

time 4 min, mobile phase A40%, mobile phase B60%;

time 5min, mobile phase A37%, mobile phase B63%;

time 15min, mobile phase A33%, mobile phase B67%;

time 17 min, mobile phase A5%, mobile phase B95%;

time 20 min, mobile phase A5%, mobile phase B95%;

time 22 min, mobile phase A20%, mobile phase B80%.

2. The use according to claim 1, wherein the diluent is a PBS buffer containing bovine serum albumin.

3. The use according to claim 2, wherein the diluent is a PBS buffer containing 1-5% bovine serum albumin by mass percent.

4. The use according to claim 3, wherein the dilution is a PBS buffer containing 1% bovine serum albumin in mass percent.

5. Use according to claim 1, characterized in that the precipitant is an aqueous solution of zinc sulphate or an aqueous solution of silver nitrate.

6. The use according to claim 5, wherein the precipitant is an aqueous solution of zinc sulfate having a concentration of 0.01 to 0.2 mol/L.

7. The use according to claim 6, wherein the precipitant is an aqueous zinc sulphate solution having a concentration of 0.2 mol/L.

8. The use according to claim 1, wherein the sample extract is an ethyl acetate solution of laurylbenzophenone at a concentration of 800-1200 ng/mL.

9. The use according to claim 8, wherein the sample extract is an ethyl acetate solution of laurylbenzophenone at a concentration of 1000ng/mL.

10. The use according to claim 1, wherein the calibration standard comprises calibration standard S1, calibration standard S2, calibration standard S3, calibration standard S4, calibration standard S5 and calibration standard S6; preparing each calibrator according to the target concentration, and then carrying out higher-level calibrator assignment to obtain the marked concentration of the calibrator; the target concentrations of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S1-S6 are sequentially 70, 100, 200, 500, 800 and 1000ng/mL.

11. The use of claim 1, wherein the kit further comprises a quality control.

12. The use of claim 11, wherein the quality control comprises a low value quality control and a high value quality control; preparing each quality control product according to the target concentration, and then carrying out higher-level calibration product assignment to obtain the marked concentration of the quality control product; the target concentrations of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the low-value quality control product and the high-value quality control product are 200ng/mL and 800ng/mL in sequence.

13. The use according to any one of claims 1 to 12, wherein the kit comprises calibrator S1 to S6, low value quality control, high value quality control, diluent, precipitant and sample extract;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S1 is 70ng/mL, and methanol is used as a solvent;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S2 is 100ng/mL, and methanol is used as a solvent;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S3 is 200ng/mL, and methanol is used as a solvent;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S4 is 500ng/mL, and methanol is used as a solvent;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S5 is 800ng/mL, and methanol is used as a solvent;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the calibrator S6 is 1000ng/mL, and methanol is used as a solvent;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the low-value quality control product is 200ng/mL, and methanol is used as a solvent;

the target concentration of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in the high-value quality control product is 800ng/mL, and methanol is used as a solvent;

the diluent is PBS buffer solution containing 1% bovine serum albumin according to mass percent;

the precipitant is zinc sulfate water solution with the concentration of 0.2 mol/L;

the sample extract was an ethyl acetate solution of laurylbenzophenone at a concentration of 1000ng/mL.