CN115356408A - Method for detecting 25-hydroxy vitamin D in serum - Google Patents

Abstract

The invention belongs to the technical field of chemical detection and discloses a detection method for 25-hydroxyvitamin D in serum. The detection method adopts liquid chromatography tandem mass spectrometry for detection, and the conditions of the liquid chromatography include: the chromatographic column is UPLC ACQUITY ^TM HSS PFP column; the mobile phase A is 0.1%-0.5% formic acid aqueous solution, and the mobile phase B is methanol. The method has strong specificity and high accuracy, and is suitable for the detection of 25-hydroxyvitamin D in serum samples of people of all ages.

Description

A method for detecting 25-hydroxyvitamin D in serum

technical field

The invention belongs to the technical field of chemical detection, in particular to a method for detecting 25-hydroxyvitamin D in serum.

Background technique

Vitamin D (hereinafter referred to as VD) is a fat-soluble steroid derivative and is an essential vitamin for the human body, mainly including two forms of VD2 and VD3. VD in the human body can be transformed by sunlight or taken from food. For example, 7-dehydrocholesterol stored under the skin can be converted into VD3 after being irradiated by ultraviolet rays in the sun, and VD2 can be supplemented by food. The VD2 and VD3 in the human body will be metabolized into 25-OH VD2 and 25-OH VD3 by the liver. The concentration of 25-OH VD2 and 25-OH VD3 in the blood is high, which is the main form of VD in the body, and it is stable and has a half-life in the body. It is about 2-3 weeks, so 25-OHVD2 and 25-OH VD3 in the blood have become the most reliable indicators for evaluating the nutritional status of human VD.

However, there is also an epimer of 25-OH VD3 in the human body - 3-epi 25-OH VD3, which accounts for about 4% of the total 25-OH VD, and its content is low in adults, but in newborns The concentration in the body is extremely high, accounting for about 60% of the total concentration of 25-OH VD. 3-epi 25-OH VD3 has no biological activity. If the substance cannot be effectively separated during detection, it will cause errors in the detection results of 25-OHVD3.

Traditional vitamin D detection methods include radioimmunoassay, competitive protein binding method, high performance liquid chromatography, etc. Among them, radioimmunoassay and competitive protein binding method have the disadvantages of complicated pretreatment, long analysis time, low throughput, and poor method specificity , and cannot accurately quantify the content of 25-OH VD2 and 25-OH VD3 at the same time. At present, LC-MS/MS is generally considered to be the "gold standard" for the detection of 25-hydroxyvitamin D in serum. However, the current methods still cannot effectively separate the epimers, and the detection results are biased.

Therefore, the present invention hopes to establish a method for detecting 25-hydroxyvitamin D in human serum with simple operation, high specificity and good sensitivity.

Contents of the invention

The present invention aims to solve at least one of the technical problems in the above-mentioned prior art. Therefore, the present invention proposes a method for detecting 25-hydroxyvitamin D in serum, which has strong specificity and high accuracy, and is suitable for detecting 25-hydroxyvitamin D in serum samples of people of all age groups.

The invention provides a method for detecting 25-hydroxyvitamin D in serum, which is detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). The conditions of the liquid chromatography include: the chromatographic column is UPLC ACQUITY ^TM HSS PFPcolumn; The mobile phase A is 0.1%-0.5% formic acid aqueous solution, and the mobile phase B is methanol.

The present invention proposes that the effective separation of 25-OH VD2, 25-OH VD3 and 3-epi 25-OH VD3 can be achieved by combining the above-mentioned mobile phase system with a specific chromatographic column type, thereby improving the detection accuracy of 25-hydroxyvitamin D in serum sex.

Preferably, the size of the chromatographic column is 2.1mm×50mm, 1.8μm.

Preferably, the liquid chromatography adopts a gradient elution method.

More preferably, the conditions of the gradient elution are:

0-4min, the volume fraction of mobile phase B changes from 70% to 95%;

4-4.01min, the volume fraction of mobile phase B changes from 95% to 70%;

4.01-4.5min, the volume fraction of mobile phase B is 70%.

Preferably, the conditions of the liquid chromatography further include: a flow rate of 0.4 mL/min; a column temperature of 40° C.; and an injection volume of 2-10 μL.

Preferably, the conditions of the mass spectrometer include: ESI source positive ion mode, multiple reaction monitoring scan mode; spray voltage: 1.5-2.5kV; desolvation temperature: 450-550°C; nebulizer flow rate 800-1000L/h; Source temperature: 120-150°C; cone gas flow rate: 55-65L/hr.

Preferably, the detection parameters of the mass spectrum are as follows:

target analyte Q1(Da) Q3(Da) CV(V) CE(V) 25-OH VD2 413.3 113.1 20 10 25-OH VD3 383.3 257.2 20 15 25-OH VD2_IS 416.4 113.1 20 10 25-OH VD3_IS 386.3 257.2 20 15

Preferably, the detection method also includes:

Using BSA as the matrix, prepare calibrator solutions containing target substances with different concentration gradients, add isotope labels for pretreatment, and use LC-MS/MS for detection; take the concentration of target substances in the calibrator solution as the X-axis, and take the concentration of the calibrator The peak area ratio of the target substance and its corresponding isotope label in the solution is the Y axis, and the standard working curve is drawn;

Add isotope markers to the serum samples to be tested, perform pretreatment and detect with LC-MS/MS, and obtain the content of 25-hydroxyvitamin D in the serum samples to be tested according to the standard working curve.

Compared with the prior art, the beneficial effects of the present invention are as follows:

(1) The detection method proposed by the present invention has strong specificity and high accuracy, can realize the separation of 25-OH VD3 and its epimer 3-epi 25-OH VD3, and is suitable for 25-OH VD3 in serum samples of people of all ages. Hydroxyvitamin D detection;

(2) The detection method proposed by the present invention is simple in operation, strong in operability, short in analysis time, and can realize high-throughput detection.

Description of drawings

Fig. 1 is the detection result of children's serum sample in embodiment 2;

Fig. 2 is the detection result of control group 1 in embodiment 3;

Fig. 3 is the detection result of control group 2 in embodiment 3;

Fig. 4 is the detection result of the experimental group in embodiment 3.

Detailed ways

In order to make those skilled in the art understand the technical solution of the present invention more clearly, the following examples are listed for illustration. It should be pointed out that the following examples are only preferred embodiments of the present invention, and do not constitute a limitation to the scope of protection required by the present invention. Any modifications, substitutions and combinations made under the spirit and principles of the present invention are all included in the protection scope of the present invention.

Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels, or can be obtained by existing known methods.

Example 1

This embodiment provides a method for detecting 25-hydroxyvitamin D in serum, which is detected by liquid chromatography tandem mass spectrometry (LC-MS/MS); wherein,

The liquid chromatography conditions are:

The chromatographic column is UPLC ACQUITY ^TM HSS PFP column (2.1mm×50mm, 1.8μm, Waters).

The liquid chromatography tandem mass spectrometry (LC-MS/MS) detection instrument is Waters UPLC Xevo TQ-S Micro IVD.

The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was methanol.

Adopt gradient elution, the condition of gradient elution is:

0-4min, the volume fraction of mobile phase B changes from 70% to 95%;

4-4.01min, the volume fraction of mobile phase B changes from 95% to 70%;

4.01-4.5min, the volume fraction of mobile phase B is 70%.

The flow rate was 0.4 mL/min; the column temperature was 40°C; the injection volume was 5 μL.

The mass spectrometry conditions are:

In the positive ion mode of the ESI source, the multiple reaction monitoring scanning mode (MRM) is adopted; the spray voltage: 2.0kV; the solvent removal temperature: 500°C; the nebulizer flow rate is 800L/h; the ion source temperature: 150°C; the cone gas flow rate: 65L/hr;

The parameters of the target analytes (25-OH VD2, 25-OH VD3 and their isotope labels) such as precursor ion Q1, product ion Q3, declustering voltage CV, and collision energy voltage CE are shown in Table 1.

Table 1 Mass spectrometry detection parameters

target analyte Q1(Da) Q3(Da) CV(V) CE(V) 25-OH VD2 413.3 113.1 20 10 25-OH VD3 383.3 257.2 20 15 25-OH VD2_IS 416.4 113.1 20 10 25-OH VD3_IS 386.3 257.2 20 15

Mass spectra were collected and analyzed using MassLynx V4.2 software.

The specific detection steps are:

1. Pretreatment of the sample to be tested: pipette 100 μL of the serum sample to be tested, add 400 μL of acetonitrile containing an internal standard (isotope label), vortex for 2 minutes; centrifuge at 4°C for 5 minutes, take 100 μL of the supernatant; add 20 μL of ultrapure Water, mixed for LC-MS/MS detection;

2. Standard curve drawing: 5% BSA was used as the matrix to prepare calibrator solutions containing target substances with different concentration gradients, and after the same pretreatment as serum samples, LC-MS/MS detection was performed; the target substances in the calibrator solution were The concentration of the concentration is the X-axis, and the peak area ratio of the target substance and its corresponding isotope label in the calibrator solution is the Y-axis, and the standard working curve is drawn;

3. Calculate the concentrations of 25-OH-VD2 and 25-OH-VD3 in the serum samples to be tested according to the standard working curve and the LC-MS/MS detection results of the serum samples to be tested.

Example 2

The detection method in Example 1 was used to detect children's serum samples. As can be seen from Figure 1, the detection method in Example 1 can be used to achieve 25-OH VD3 and its epimer 3-epi 25-OH VD3 Effective separation, thereby improving the detection accuracy of 25 hydroxyvitamin D.

Embodiment 3: Comparison of chromatographic column types

In order to compare the impact of the chromatographic column type on the separation effect of 25-OH VD3 and its epimer 3-epi 25-OH VD3, the serum sample containing 3-epi 25-OH VD3 was used as the experimental object, basically using Example 1 Using the following three kinds of chromatographic columns to test the samples, and simultaneously detect the single-marked peak time of 3-epi 25-OH VD3 to determine 25-OH VD3 and its epimer 3-epi 25-OH VD3 of separation.

Control group 1: BEH C18 (2.1mm×50mm, 1.7μm, Waters);

Control group 2: BEH Phenyl column (2.1mm×50mm, 1.7μm, Waters);

Experimental group: HSS PFP column (2.1mm×50mm, 1.8μm, Waters);

The results in Figure 2-4 show that the peak time of 25-OH VD3 and its epimer 3-epi 25-OHVD3 in control group 1 and control group 2 is consistent, and the peak time of 25-OH VD3 and its epimer cannot be achieved The separation of 3-epi 25-OH VD3; while the experimental group can achieve a good separation effect. The above results show that the type of chromatographic column plays an important role in the separation of 25-OH VD3 and its epimer 3-epi 25-OH VD3, and only a specific chromatographic column type combined with the mobile phase in Example 1 can play a role Good separation effect.

Embodiment 4: Performance evaluation of detection method

1. Linear regression equation and linear correlation coefficient

The working curve was established by the method described in Example 1, and the results are shown in Table 2. The linearity of the target analyte standard curve is good, and the correlation coefficient is above 0.99, which meets the quantitative requirements.

Table 2 Linear regression equation and linear correlation coefficient

2. Quantitative American NIST-SRM972a accuracy evaluation

Adopt the detection method of embodiment 1 to carry out quantitative detection to SRM972a (U.S. NIST) freezing human serum, and compare with target value, the result of table 3 shows that the accuracy of 25-OH VD2 and 25-OH VD3 in Level3 is 100.83% respectively , 93.38%; the accuracy of 25-OH VD3 in Level4 is 93.03% (contain 26.0ng/mL 3-epi 25-OHVD3 in Level4, if can not separate, can cause quantitative result on the high side), this result shows that adopt embodiment 1 method has high accuracy.

Table 3 Quantitative results of NIST samples

3. Evaluation of the accuracy of quantitative normal human serum samples

The detection method in Example 1 was used to evaluate the standard-added recoveries of low, medium and high levels of normal human serum. The results in Table 4 show that the standard-added recoveries were between 89.22% and 96.39%, which could meet the quantitative requirements.

Table 4 normal human serum standard recovery rate

4. Inspection of precision

Serum from normal people was spiked at three concentrations of low, medium and high. Six parallel samples in each group were processed for three consecutive days. The intra-day precision and inter-day precision were calculated. The results are shown in Table 5.

Table 5 Intra-day precision

From the above performance verification results, it can be seen that the method proposed in Example 1 has good accuracy and precision, and strong specificity, and is suitable for the detection of 25-hydroxyvitamin D in serum samples of people of all ages.

The embodiments of the present application have been described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned embodiments, and within the scope of knowledge of those of ordinary skill in the art, various modifications can be made without departing from the purpose of the present application. Variety. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other under the condition of no conflict.

Claims (8)

1. The method for detecting 25 hydroxy vitamin D in serum is characterized by adopting liquid chromatography tandem mass spectrometry to detect, wherein the conditions of the liquid chromatography comprise: the chromatographic column is UPLC ACQUITY ^TM HSS PFP column; the mobile phase A is 0.1-0.5% formic acid water solution, and the mobile phase B is methanol.

2. The method of claim 1, wherein the size of the column is 2.1mm x 50mm,1.8 μm.

3. The detection method according to claim 1, wherein the liquid chromatography employs a gradient elution method.

4. The detection method according to claim 3, wherein the conditions of the gradient elution are:

the volume fraction of the mobile phase B is changed from 70% to 95% in 0-4 min;

4-4.01min, the volume fraction of the mobile phase B is changed from 95% to 70%;

4.01-4.5min, and the volume fraction of the mobile phase B is 70%.

5. The detection method according to claim 1, wherein the conditions of the liquid chromatography further comprise: the flow rate is 0.4mL/min; the column temperature was 40 ℃; the sample amount is 2-10 μ L.

6. The detection method according to claim 1, wherein the conditions of the mass spectrum comprise: ESI source positive ion mode, multi-reaction monitoring scanning mode; spray voltage: 1.5-2.5kV; the temperature of the desolvation: 450-550 ℃; the flow rate of the atomizer is 800-1000L/h; ion source temperature: 120-150 ℃; taper hole air flow rate: 55-65L/hr.

7. The method of claim 1, wherein the detection parameters of the mass spectrum are as follows:

target analyte Q1(Da) Q3(Da) CV(V) CE(V) 25-OH VD2 413.3 113.1 20 10 25-OH VD3 383.3 257.2 20 15 25-OH VD2_IS 416.4 113.1 20 10 25-OH VD3_IS 386.3 257.2 20 15

8. The detection method according to claim 1, characterized in that the detection method further comprises:

preparing a calibrator solution containing target substances with different concentration gradients by taking BSA as a matrix, adding an isotope marker for pretreatment, and detecting by using LC-MS/MS; taking the concentration of the target in the calibrator solution as an X axis, and taking the peak area ratio of the target to the corresponding isotope marker in the calibrator solution as a Y axis, and drawing to obtain a standard working curve;

adding an isotope marker into a serum sample to be detected, pretreating, detecting by using LC-MS/MS, and obtaining the content of 25 hydroxyvitamin D in the serum sample to be detected according to a standard working curve.

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