阅读说明：本技术 维生素d定量检测的样本前处理溶液及其使用方法 (Sample pretreatment solution for vitamin D quantitative detection and use method thereof ) 是由 王伟 于 2019-08-28 设计创作，主要内容包括：本发明公开了一种维生素D定量检测的样本前处理溶液,包括生理盐水、硫酸葡聚糖钠盐与β-异嗜性阻断剂,本发明使用的硫酸葡聚糖钠盐和β-异嗜性抗体在一定程度上能很好的使得待分析物25-羟基维生素D与结合蛋白充分分离,同时降低血清基质中非特异性的干扰作用,在样本的进一步分离纯化过程中能够很好的得以分离,经过处理的样本可以得到高质量的待分析物,提高了维生素D的定量检测的准确度。(The invention discloses a sample pretreatment solution for vitamin D quantitative detection, which comprises normal saline, dextran sulfate sodium salt and β -heterophilic blocking agent, wherein the dextran sulfate sodium salt and β -heterophilic antibody used in the invention can well separate 25-hydroxyvitamin D to be analyzed from binding protein to a certain extent, simultaneously reduce nonspecific interference effect in serum matrix, can be well separated in the further separation and purification process of the sample, the processed sample can obtain high-quality analyte, and the accuracy of the quantitative detection of vitamin D is improved.)

1. A sample pretreatment solution for quantitative vitamin D detection is characterized by comprising normal saline, dextran sulfate sodium salt and β -heterophilic blocking agent.

2. The sample pretreatment solution for vitamin D quantitative determination according to claim 1, wherein 80-100mg of dextran sulfate sodium salt and 1.5-2.0mg of β -heterophilic blocker are sequentially added to each ml of normal saline based on normal saline, and the mixture is uniformly mixed.

3. The sample pretreatment solution for vitamin D quantitative determination according to claim 1, wherein: the sodium chloride concentration of the physiological saline is 0.9%.

4. The method for using the sample pretreatment solution for vitamin D quantitative determination according to any one of claims 1 to 3, comprising the steps of:

s1, taking 100 mu L of the solution, taking 200 mu L of serum samples, mixing, uniformly mixing for 1min in a vortex mode, and standing for 5 min;

s2, putting the sample obtained in the S1 into a centrifuge, and centrifuging for 10min at 13000rpm and 4 ℃;

s3, centrifuging, taking supernatant, adding 200 mu L of ethanol, uniformly mixing for 1min in a vortex mode, and then standing for 5 min;

s4, centrifuging the sample obtained in the S3 for 10min at 13000rpm and 4 ℃;

s5, centrifuging, taking the supernatant, drying the supernatant in a new centrifuge tube by using nitrogen, adding a separation mobile phase, redissolving and uniformly mixing the supernatant and the separation mobile phase, and then using the mixture for LC-MS/MS detection.

Technical Field

The invention relates to the field of quantitative detection of vitamin D, in particular to a sample pretreatment solution for quantitative detection of vitamin D and a using method thereof.

Background

Vitamin D is a steroid hormone, including 2 types: vitamin D2 and vitamin D3. Is converted into 25-hydroxyvitamin D2[25-hydroxyvitamin D2, 25(OH) D2] and 25-hydroxyvitamin D3[25-hydroxyvitamin D3, 25(OH) D3] by absorption in the body, which are collectively called 25-hydroxyvitamin D [25-hydroxyvitamin D, 25(OH) D ]. 25(OH) D in the systemic circulation of the body is considered to be an important indicator of vitamin D levels in humans.

Vitamin D is an important microelement participating in the physiological functions of the organism, and the main physiological functions are participating in the calcium and phosphorus metabolism and the immune regulation of the organism. Patients with osteoporosis, osteomalacia, rickets, hyperparathyroidism, thyroid surgery, chronic diarrhea, the elderly with fractures or recent frequent falls, people in long-term bed with reduced sunlight or dark skin, patients with obesity, pregnancy and lactation, hormone therapy, malabsorption syndrome, liver failure, granuloma, chronic kidney disease and kidney transplantation, all belong to the high risk group with vitamin D deficiency. If there is a significant vitamin D deficiency, the vitamin D supplement can be supplemented. Because the individual difference of human body to the absorption of vitamin D supplements is very large, blind supplement may have potential risks, and detection before and after taking is necessary to avoid vitamin D poisoning caused by insufficient supplement or excessive supplement.

Detection methods used in clinical laboratories include competitive vitamin D protein binding assays (CPBA), immunoassays (enzyme linked immunosorbent, chemiluminescence, etc.), High Performance Liquid Chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The enzyme immunoassay method has been the most important method for measuring the concentration of 25(OH) D in serum, and the main defect of the enzyme immunoassay method is insufficient method specificity caused by cross reaction between antibodies. Chromatography allows for efficient separation of these analytes, and thus sufficient specificity. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has great advantages: high selectivity, specificity and sensitivity. This analytical technique has become increasingly widely used for the analysis of 25(OH) D in clinical laboratory tests.

The types of samples currently used for in vitro quantification of vitamin D in humans are: saliva samples, serum or plasma samples, Dried Blood Spots (DBS) samples, etc. The pretreatment modes of the sample comprise liquid-liquid extraction, solid-phase extraction, an extraction technology utilizing the specificity of the analyte and the like, and the treatment process mainly aims to effectively extract the analyte from the complex sample composition to obtain the high-quality analyte. Taking the clinical main type of sample to be tested (serum sample) as an example, the tight binding between 25(OH) D and binding protein in blood circulation, accurate quantification of vitamin D must first completely separate vitamin D from binding protein, and at the same time effectively control the nonspecific binding effect of other proteins in the matrix to the analyte 25(OH) D. Especially in some special serum samples, such as rheumatoid factor positive samples, the rheumatoid factor as a type of self antibody can interfere the test. Currently commercially available immunological methods have their own proprietary methods for the isolation of vitamin D and binding proteins. The latest detection method even comprises a special blocking agent, and can effectively reduce the interference of heterophile antibodies in a sample on a detection result. For LC-MS/MS, the binding protein in the sample is precipitated, the interference effect of the matrix is reduced, and the extraction of vitamin D by using an organic solvent is a necessary step for detection.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology is used as a standard detection means for small molecule biological analysis, and is now becoming one of important tools for quantitative analysis of macromolecular compounds (polypeptides, nucleotides and proteins) in biological matrixes. Most of the samples could not be directly analyzed by LC-MS/MS without pretreatment. The principle of sample pretreatment is the same regardless of the type of mass detector used for analysis. Two requirements of LC-MS/MS analysis are met: the liquid chromatography part is used for analyzing liquid samples, the samples entering the chromatography system should be liquid, and importantly, particles are not available, and proteins cannot be injected into the liquid chromatograph generally; the mass spectrometer section is used to generate gaseous ions and is tested under high vacuum conditions, and non-volatile acids, bases or salts cannot be introduced into the ion source to ensure good ionization efficiency. The matrix effect of matrix components on the target during ionization (i.e., interference effects: ion enhancement or ion suppression) is also of great interest in analysis. Matrix effects are often difficult to predict and if not handled properly, can lead to inaccurate quantitation. In most cases, the effect of certain matrix effects can be corrected using a stable isotope labeled internal standard (SIL-IS). How to improve the accuracy of detection and reduce the interference effect of the matrix, the pretreatment process of the sample plays a very critical role.

Disclosure of Invention

The invention aims to provide a sample pretreatment solution for vitamin D quantitative detection and a use method thereof, which can effectively reduce the interference effect of a matrix and improve the detection accuracy.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a sample pretreatment solution for quantitative vitamin D detection comprises normal saline, dextran sulfate sodium salt and β -heterophilic blocker.

Preferably, based on normal saline, 80-100mg of dextran sulfate sodium salt and 1.5-2.0mg of β -heterophilic blocking agent are added into each ml of normal saline in sequence and mixed evenly.

Preferably, the normal saline has a sodium chloride concentration of 0.9%.

The application method of the sample pretreatment solution for the vitamin D quantitative detection for reducing the matrix interference effect, which is provided by the invention, comprises the following steps of:

s1, taking 100 mu L of the solution, taking 200 mu L of serum samples, mixing, uniformly mixing for 1min in a vortex mode, and standing for 5 min;

s2, putting the sample obtained in the S1 into a centrifuge, and centrifuging for 10min at 13000rpm and 4 ℃;

s3, centrifuging, taking supernatant, adding 200 mu L of ethanol, uniformly mixing for 1min in a vortex mode, and then standing for 5 min;

s4, centrifuging the sample obtained in the S3 for 10min at 13000rpm and 4 ℃;

s5, centrifuging, taking the supernatant, drying the supernatant in a new centrifuge tube by using nitrogen, adding a separation mobile phase, redissolving and uniformly mixing the supernatant and the separation mobile phase, and then using the mixture for LC-MS/MS detection.

By adopting the technical scheme, the invention has the following beneficial effects:

the DSS (dextran sulfate sodium salt) and β -heterophilic antibody used in the invention can fully separate the 25-hydroxyvitamin D to be analyzed from the binding protein to a certain extent, simultaneously reduce the nonspecific interference effect in the serum matrix, can be well separated in the further separation and purification process of the sample, and the processed sample can obtain the high-quality analyte, thereby improving the accuracy of the quantitative detection of the vitamin D.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.