Liquid chromatography-tandem mass spectrometry detection method for metabolites in metabolic pathway of vitamin D in serum

Abstract

The invention provides a liquid chromatography-tandem mass spectrometry detection method for metabolites in a metabolic pathway of vitamin D in serum. Agarose gel antibody immune particles or magnetic bead antibodies are adopted for immune enrichment pretreatment, mass spectrum conditions in liquid chromatogram tandem mass spectrum detection are improved, an APCI ion source is adopted, the temperature of the ion source is optimized, alternative unconventional mobile phase additives are provided, and the signal-to-noise ratio of mass spectrum signals of a low-response instrument is increased. According to the method, the lower quantitative limit of 1, 25-dihydroxy vitamin D is as low as 8 pg / mL, so that synchronous detection of 1, 25-dihydroxy vitamin D with extremely low content, 25-dihydroxy vitamin D with relatively high content and vitamin D metabolic pathway related metabolites including 24, 25-dihydroxy vitamin D is realized; a mobile phase, an eluent, a deriving agent and the like are subjected to adaptive optimization, and a four-dimensional liquid chromatography-tandem mass spectrometry detection method is combined, so that the flux is higher, and a detection scheme with high availability is provided for clinical detection.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and specifically relates to a liquid chromatography tandem mass spectrometry detection method for metabolites in serum vitamin D metabolic pathway. Background Art

[0002] Vitamin D is a steroid derivative and a fat-soluble vitamin. Vitamin D is mainly synthesized by human skin after exposure to ultraviolet light, and a small amount is ingested from food or supplements. Vitamin D not only affects calcium and phosphorus metabolism, but also has a wide range of physiological effects. It is an essential substance for maintaining human health, cell growth and development, and is closely related to many diseases. There are two forms of vitamin D in the human body, vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D is converted into 25-hydroxyvitamin D (25(OH)D) by hydroxylation in the liver and then converted into active 1,25-dihydroxyvitamin D (1,25-DHVD) in the kidneys. Clinically, the level of active vitamin D in metabolic bone disease is measured by measuring the level of 1,25-DHVD. Under certain physiological conditions, such as hypercalcemia or hyperphosphatemia, 25(OH)D is converted into 24,25-dihydroxyvitamin D (24,25-DHVD) by enzymes (mainly 24-hydroxylase, CYP24A1). 24,25-DHVD plays an auxiliary role in regulating calcium absorption and phosphorus metabolism, and can reduce the synthesis of 1,25-DHVD through a negative feedback mechanism to help maintain homeostasis. Although the biological activity of 24,25-DHVD is not as strong as that of 1,25-DHVD, it can play a role in maintaining bone health, immune regulation, and the regulation of cell proliferation and differentiation.

[0003] The level of 1,25-dihydroxyvitamin D in serum is extremely low (normal range is about 18-78 pg / mL), the half-life is short (1-20 hours), and its content is regulated by parathyroid hormone (PTH), calcium and phosphorus content, making its accurate quantification very challenging. Current methods for detecting 1,25-dihydroxyvitamin D include ELISA, radioimmunoassay (RIA), high performance liquid chromatography (HPLC), and liquid chromatography tandem mass spectrometry (LC-MS / MS). Immunoassays cannot distinguish between 1,25-dihydroxyvitamin D2 (1,25-DHVD2) and 1,25-dihydroxyvitamin D3 (1,25-DHVD3), and the test results are easily interfered by isomers such as 24,25-dihydroxyvitamin D and 4,25-dihydroxyvitamin D (24,25-DHVD and 4,25-DHVD), and are also easily affected by vitamin D binding proteins, resulting in poor accuracy and specificity of the test results. There are also some reports on liquid chromatography-tandem mass spectrometry methods in the literature, but because the serum content of 1,25-dihydroxyvitamin D is extremely low and the ionization efficiency is poor, the pretreatment steps of most detection methods are very complicated, such as the need for immunoenrichment, SPE extraction or derivatization, which greatly increases the complexity of daily operations in clinical laboratories.

[0004] In addition, studies have shown that CYP24A1 plays an important role in the metabolic balance and regulation of vitamin D. The active metabolite 1,25-DHVD and the inactive metabolite 24,25-DHVD are dependent on 25(OH)D: when the serum 25(OH)D concentration is lower than 20 ng / mL, the concentration ratio of 25(OH)D to 24,25-DHVD increases, and the inactive metabolite 24,25-DHVD is downregulated by CYP24A1 to facilitate the production of the active metabolite 1,25-DHVD; when the 25(OH)D concentration is high, CYP24A1 regulation tends to produce more inactive metabolites 24,25-DHVD to reduce the production of active metabolites 1,25-DHVD, thereby preventing toxicity. Therefore, determination of serum inactive metabolite 24,25-DHVD concentration helps identify patients with CYP24A1 loss-of-function mutations, especially for patients with renal calcification or kidney stones. If an elevated concentration ratio of 25(OH)D to 24,25-DHVD is detected, vitamin D supplementation should be prohibited to prevent hypercalcemia and stone formation.

[0005] So far, there is no feasible technical solution with better effect for the simultaneous detection of serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D. The existing technology focuses on the detection of 25-hydroxyvitamin D, such as patent CN113063871B, but the content of 25-hydroxyvitamin D is nearly a thousand times that of 1,25-dihydroxyvitamin D, and its detection is relatively easy. The content of 1,25-dihydroxyvitamin D is extremely low, contains isomer interferences, and has a strong matrix effect, which is more difficult to detect. The detection scheme for 25-hydroxyvitamin D cannot serve as a reference for the detection of 1,25-dihydroxyvitamin D. There are also detection methods for 24,25-dihydroxyvitamin D, such as patent CN110058028B, but combined with antibody selectivity, its detection scheme has no reference for the detection of 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D. At present, the best technical solution for 1,25-dihydroxyvitamin D in China reported in the literature is to use SPE extraction and enrichment, but its lower limit of quantification can only reach 80 pg / mL (the lower limits of quantification of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 are both 40 pg / mL, and the two indicators need to be added to determine the total content, so the lower limit of quantification of 1,25-dihydroxyvitamin D is 40+40=80 pg / mL), but the clinical reference range of human serum samples is 18-78 pg / mL. Therefore, the lower limit of quantification of this method is already higher than the upper limit of the normal reference range, and it cannot fully meet the clinical sensitivity requirements for the detection of 1,25-dihydroxyvitamin D content. It cannot perform differential diagnosis on patients with 1,25-dihydroxyvitamin D deficiency, nor can it effectively indicate patients with abnormal renal function due to CYP24A1 deficiency.

[0006] Therefore, there is an urgent need to find a highly sensitive, highly accurate and high-throughput solution for the simultaneous detection of serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D to meet the needs of clinical testing. Summary of the invention

[0007] To solve the above problems, the present invention provides a simple and efficient sample pretreatment method based on agarose bead antibody / streptavidin magnetic bead antibody. When combined with liquid chromatography tandem mass spectrometry (LC-MS / MS), the detection time of six indicators of a single-injection sample is 4 minutes, or when combined with a multi-channel high-performance liquid chromatography tandem mass spectrometry detection (TLX4-MS / MS) scheme, the detection time of a single-injection sample can be shortened to 1.5 minutes. Within one hour, six indicators such as 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D and three additive indicators, as well as the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D in 40 human serum samples can be accurately and efficiently detected, a total of ten clinical indicators, with the quantitative lower limit of 25-hydroxyvitamin D3 being 1 ng / mL, the quantitative lower limit of 25-hydroxyvitamin D2 being 0.4 ng / mL, and the quantitative lower limit of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 being 8. pg / mL, the lower limit of quantification of 24,25-dihydroxyvitamin D3 is 0.1 ng / mL, and the lower limit of quantification of 24,25-dihydroxyvitamin D2 is 0.05 ng / mL.

[0008] On the one hand, a method for detecting metabolites related to the serum vitamin D metabolic pathway by liquid chromatography tandem mass spectrometry is provided, the method comprising a pretreatment method, the pretreatment method adopting an antibody immune particle extraction method or a magnetic bead antibody extraction method, and the metabolites related to the vitamin D metabolic pathway include any one or more of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and 24,25-dihydroxyvitamin D.

[0009] The present invention optimizes the pretreatment of samples and selects an antibody immune particle extraction method or a magnetic bead antibody extraction method. The content of 1,25-dihydroxyvitamin D in serum is negligible. Agarose bead antibodies or streptavidin-coupled magnetic bead antibodies are used for high-specific enrichment and extraction, which can effectively improve the detection sensitivity, reduce the matrix effect, alleviate the interference of structural analogs such as isomers, and broaden the quantitative lower limit; at the same time, it can effectively take into account the extraction of relatively high content 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D, thereby achieving effective extraction of key metabolites in the vitamin D metabolic pathway. First, the antibody extraction method can specifically identify and capture the target molecule, thereby reducing interfering substances in the sample, reducing matrix interference of liquid phase co-elution components, improving the signal-to-noise ratio of LC-MS / MS detection and reducing the detection limit; second, agarose gel-coupled immune antibodies can use 96-well filter plates to separate and wash samples; and the streptavidin-coupled magnetic bead antibody extraction method can use an automated magnetic bead extractor to quickly separate and wash samples, thereby shortening the sample pretreatment time and steps and achieving standardization of the sample pretreatment process. The current best technology for 1,25-dihydroxyvitamin D is to use SPE plate solid phase extraction, but the detection sensitivity is only 40 pg / mL. Using the antibody extraction method of the present invention, the detection sensitivity can reach 8pg / mL, and it can take into account the simultaneous extraction of key active and inactive metabolites in the vitamin D metabolic pathway, effectively improving the detection throughput.

[0010] The pretreatment of serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D in the present invention adopts an antibody immune particle extraction method or an antibody magnetic bead extraction method, which is groundbreaking and creative: vitamin D is a steroid derivative, and its chemical properties determine that once the position of the hydroxyl group changes, its protein binding site will change almost completely. For the key active and inactive metabolites of the vitamin D metabolic pathway, the present invention screens out specific antibodies based on their structures and chemical properties, and uses agarose beads to couple to antibody solutions (immune particle suspensions) or streptavidin magnetic beads to couple to magnetic bead antibodies, so as to achieve specific synchronous immune extraction of serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D, and then distinguishes the antibodies from interfering components by filtration (agarose bead antibody solution) or magnetism (magnetic bead antibody), which is the basis for improving sensitivity and reducing the limit of quantification.

[0011] Furthermore, the pretreatment method also includes derivatization, and the derivatization uses 4-phenyl-1,2,4-triazine-3,5-dione as a derivatization reagent.

[0012] In some ways, through derivatization reactions, the chemical and physical properties of the analyte can be changed to make it easier to be detected by the detector, thereby improving the detection sensitivity.

[0013] Furthermore, the 25-hydroxyvitamin D includes 25-hydroxyvitamin D3 and / or 25-hydroxyvitamin D2; the 1,25-dihydroxyvitamin D includes 1,25-dihydroxyvitamin D3 and / or 1,25-dihydroxyvitamin D2; and the 24,25-dihydroxyvitamin D includes 24,25-dihydroxyvitamin D3 and / or 24,25-dihydroxyvitamin D2.

[0014] In some ways, the content of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 in the vitamin D metabolic pathway metabolites is very low in serum. The current detection technology can only detect 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 with a quantitative lower limit of only 80 pg / mL, while the clinical reference range of human serum samples is 18-78 pg / mL, so it cannot meet clinical requirements. The detection method provided by the present invention can quantitatively detect 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 with a lower limit of 8 pg / mL, which can meet clinical needs. Although the methods for detecting 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, 24,25-dihydroxyvitamin D3, and 24,25-dihydroxyvitamin D2 provided by other commercially available kits can meet clinical requirements, the detection accuracy is not high and is controlled within ±15%, while the accuracy of the detection method provided by the present invention can be controlled within ±5%, which greatly improves the accuracy of the detection.

[0015] Furthermore, the antibody immune particle extraction method uses agarose beads coupled to antibodies for extraction, and the magnetic bead antibody extraction method uses magnetic beads coupled to antibodies for extraction; in the pretreatment method, the agarose bead coupled antibodies are agarose beads coupled to anti-vitamin D mouse monoclonal antibodies, and the magnetic bead antibodies are streptavidin coupled to anti-vitamin D mouse monoclonal antibodies.

[0016] Compared with other existing sample pretreatment methods, the sample pretreatment method of agarose beads coupled to vitamin D mouse monoclonal antibody and the sample pretreatment method of streptavidin magnetic beads coupled to mouse monoclonal antibody provided by the present invention have the advantages of being able to achieve high-efficiency specific adsorption and extraction of target analytes in biological samples by using antibodies, while separating the antibodies bound to the analytes from potential interfering components in the sample by means of physical separation, fully reducing the matrix effect of the biological sample, improving the ionization efficiency and sensitivity in the detection process, and fully meeting the sensitivity and accuracy requirements of clinical detection. Other sample pretreatment methods cannot meet the sensitivity monitoring requirements of the present invention, especially for 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 with extremely low levels in serum. The antibody immune particle method is compared with the magnetic bead antibody extraction method. In terms of manipulation, agarose beads need to be collected and separated by centrifugation or small column sieve plates, which is relatively cumbersome to operate. However, due to the characteristics of magnetic materials, magnetic beads can be manipulated and collected by external magnetic force, which is more convenient and quick to operate. In terms of operating efficiency, magnetic beads have higher operating efficiency than agarose beads. The magnetism of magnetic beads can make them completely concentrated on the magnetic rod position, and they can be separated and extracted through the automated extraction process of the magnetic bead instrument, avoiding the weightlessness problem that may be caused by centrifugation. Agarose beads are easily dispersed by gravity during centrifugation, while the operation is more complicated and costly when separated by small column sieve plates. In terms of repeatability, the automated extraction of magnetic beads has better reusability and can be washed and separated multiple times by adding and removing magnetic force, while agarose beads often need multiple centrifugation or small column elution to separate the supernatant and residue in the experiment, which is easy to cause loss and scouring. Therefore, streptavidin magnetic beads coupled to mouse monoclonal antibodies can make the test results more accurate.

[0017] Preferably, the sample pre-treatment method is streptavidin magnetic beads coupled to mouse monoclonal antibodies.

[0018] Furthermore, the mass spectrometry ion source of the detection method adopts an APCI ion source; the temperature of the mass spectrometry ion source of the detection method is 350-450° C., which may vary between different instruments.

[0019] The present invention has been proved through research that the selection of ion source has a very important influence on the detection of 1,25-dihydroxyvitamin D, while for several other analytes, the influence is not so obvious.

[0020] The method provided by the present invention also conducted a large number of tests on mass spectrometry parameters, thereby finding a suitable adjustment scheme and selecting the APCI ion source. The APCI ion source is more suitable for detecting 1,25-dihydroxyvitamin D than the ESI ion source. Under the APCI source conditions, the target analyte with a relatively low boiling point can be selectively vaporized by adjusting the ion source temperature to produce clean gas-phase charged ions, thereby reducing the dirtiness of the triple quadrupole of the mass spectrometer, thereby improving the sensitivity and accuracy of this low-concentration metabolite detection. In the ESI source, the ionization process is more susceptible to interference from a variety of substances such as phospholipids in serum samples. During the process of Coulomb explosion, these interfering substances such as phospholipids will compete with 1,25-dihydroxyvitamin D, inhibit the ionization of the target analyte, and cause the signal to be reduced. The charged particles of the APCI source can use gas phase reactions to eliminate the influence of substances such as phospholipids that are not easy to vaporize, and enhance the signal-to-noise ratio of 1,25-dihydroxyvitamin D detection by effectively reducing baseline interference.

[0021] The ion source temperature of the mass spectrometer, especially the APCI source temperature, will also affect the detection sensitivity of 1,25-dihydroxyvitamin D, because the ion source temperature will affect the efficiency of gasification of each component in the injection solution. Selecting a suitable APCI ion source temperature can reduce the gasification efficiency of interfering components without affecting the gasification ionization efficiency of the target analyte, which can effectively reduce the influence of the matrix effect and improve the detection sensitivity. After repeated tests and screening, we found that when using the APCI source, 350-450 ° C is a moderate ion source temperature, which can avoid the temperature being not high enough and causing the gasification efficiency of 1,25-dihydroxyvitamin D derivatives to be insufficient, and can also effectively reduce the gasification efficiency of interfering components, effectively improve the signal-to-noise ratio of target detection, thereby improving the detection sensitivity. Therefore, the present invention selects 350-450 ° C as the ion source temperature.

[0022] Preferably, the mass spectrometry ion source temperature of the detection method is 350-450°C.

[0023] Further preferably, the mass spectrometry ion source temperature of the detection method of the Calibra-CalQuantS instrument is about 375°C, which may vary between different instrument models.

[0024] Furthermore, the mobile phase A of the liquid chromatography tandem mass spectrometry is water, and the mobile phase B is methanol; the mobile phase additives of phase A and phase B are formic acid or organic amines; the pretreatment method also includes eluting and eluting steps, the eluent is formic acid water, and the eluent is ethanol, acetonitrile or a mixed solution of ethanol and acetonitrile; the organic amine includes any one or more of methylamine, ethylamine, and triethylamine.

[0025] Furthermore, the liquid chromatography tandem mass spectrometry detection method may be a single-channel or multi-channel liquid chromatography tandem mass spectrometry detection method.

[0026] In some embodiments, the liquid chromatography tandem mass spectrometry is a four-dimensional (four-channel) liquid chromatography tandem mass spectrometry detection method.

[0027] A liquid chromatography-tandem mass spectrometry detection method was established, and the detection time for a single sample of 6 indicators was 4 minutes; at the same time, a four-dimensional liquid chromatography-tandem mass spectrometry detection method using four-dimensional liquid phase was established, and the detection time for a single sample was 1.5 minutes. The detection efficiency is high and it is easy to promote clinically.

[0028] Furthermore, a kit for detecting metabolites related to the serum vitamin D metabolic pathway by liquid chromatography tandem mass spectrometry is provided, the kit comprising a sample diluent, a washing agent, an eluent, a derivatizing agent, an internal standard solution, an agarose bead-coupled antibody solution or a streptavidin-coupled magnetic bead antibody solution; the antibody selected is an anti-vitamin D mouse monoclonal antibody; and the metabolites related to the vitamin D metabolic pathway include any one or more of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and 24,25-dihydroxyvitamin D.

[0029] Furthermore, the eluent is formic acid water; the eluent is ethanol, acetonitrile or a mixed solution of ethanol and acetonitrile; and the derivatizing agent is 4-phenyl-1,2,4-triazine-3,5-dione.

[0030] Furthermore, an APCI ion source is used to improve the sensitivity of liquid chromatography tandem mass spectrometry detection of serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D, wherein the APCI ion source temperature is 350-450°C; the mobile phase additive can be formic acid or ethylamine; and ethylamine is used as the mobile phase additive.

[0031] The beneficial effects of the present invention are:

[0032] 1. The pretreatment schemes of antibody immune particle extraction method and antibody magnetic bead extraction method were provided for the simultaneous detection of serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D. A sample pretreatment extraction method with high specificity, high extraction efficiency and simple operation was developed, which significantly improved the sensitivity of liquid chromatography tandem mass spectrometry detection. The lower limits of quantification of 25-hydroxyvitamin D3 were 1 ng / mL, the lower limits of quantification of 25-hydroxyvitamin D2 were 0.4 ng / mL, the lower limits of quantification of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 were 8 pg / mL, the lower limits of quantification of 24,25-dihydroxyvitamin D3 were 0.1 ng / mL, and the lower limits of quantification of 24,25-dihydroxyvitamin D2 were 0.05 ng / mL, providing a highly usable detection scheme for clinical detection;

[0033] 2. The mass spectrometry conditions in liquid chromatography-tandem mass spectrometry detection were improved, and specific settings were made for the specific purpose of detecting 25-hydroxyvitamin D and its metabolites. The APCI ion source was used and the ion source temperature was optimized. This scheme significantly improved the signal-to-noise ratio of mass spectrometry detection, making it easy to detect 1,25-dihydroxyvitamin D, which was originally weak and difficult to detect; the mobile phase additives were optimized to increase the response of low-content 25-hydroxyvitamin D2 and its metabolites to meet the sensitivity requirements of clinical sample detection; when organic amines were used as unconventional mobile phase additives, the response of low-content 25-hydroxyvitamin D2, 1,25-DHVD2 and 24,25-DHVD2 and their metabolites could be increased by more than 5 times;

[0034] 3. The mobile phase, eluent, derivatization agent, etc. have been optimized for adaptability, and a four-dimensional liquid chromatography-tandem mass spectrometry detection method using a four-channel liquid phase has been added, making the overall detection scheme more optimized, convenient to use, and with high detection accuracy, high sensitivity, and high throughput;

[0035] 4. A kit for detecting serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D by liquid chromatography-tandem mass spectrometry is provided. The kit is combined with a liquid chromatography-tandem mass spectrometer to realize standardized operations for the detection of 25-hydroxyvitamin D, metabolites 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D, and standardize and normalize the detection of 25-hydroxyvitamin D and its metabolic pathways, benefiting a large number of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the ion current spectrum of two indicators (1,25-dihydroxyvitamin D) in the liquid chromatography tandem mass spectrometry detection process;

[0037] Figure 2It is the ion current spectrum of two indicators (24,25-dihydroxyvitamin D) in the liquid chromatography tandem mass spectrometry detection process;

[0038] Figure 3 It is the ion current spectrum of two indicators (25-hydroxyvitamin D) during the liquid chromatography tandem mass spectrometry detection process;

[0039] Figure 4 The ion current spectrum of 24,25-DHVD2 during the liquid chromatography-tandem mass spectrometry detection process (there are isomers, and the figure shows the peaks of the substances that are correctly assigned);

[0040] Figure 5 This is the chromatogram of 1,25-Dihydroxy Vitamin D3 by four-channel liquid chromatography tandem mass spectrometry;

[0041] Figure 6 This is the chromatogram of 1,25-Dihydroxy Vitamin D2 by four-channel liquid chromatography tandem mass spectrometry;

[0042] Figure 7 This is the response spectrum of the LLOQ of 1,25-Dihydroxy Vitamin D3 in the ESI ion source;

[0043] Figure 8 This is the response spectrum of the LLOQ of 1,25-Dihydroxy Vitamin D3 in the APCI ion source;

[0044] Fig. 9 This is the response spectrum of 1,25-Dihydroxy Vitamin D3 in serum sample in ESI ion source;

[0045] Fig.10 This is the response spectrum of 1,25-Dihydroxy Vitamin D3 in serum sample in APCI ion source;

[0046] Fig.11 Mass spectra at different ion source temperatures (from left to right, the tested ion source temperatures are 250 ℃ and 300 ℃ respectively);

[0047] Fig.12 Mass spectra at different ion source temperatures (from left to right, the tested ion source temperatures are 325 °C and 350 °C respectively);

[0048] Fig.13 Mass spectra at different ion source temperatures (from left to right, the tested ion source temperatures are 375 °C and 400 °C respectively);

[0049] Fig.14Mass spectra at different ion source temperatures (from left to right, the tested ion source temperatures are 450 ℃ and 500 ℃ respectively). DETAILED DESCRIPTION

[0050] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. It should be pointed out that the following embodiments are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. All features disclosed in the embodiments of the present invention, or all steps in the methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0051] Example 1: Detection method

[0052] 1. Solution preparation

[0053] The solution preparations involved in this application are shown in Tables 1 to 5.

[0054] (1) Prepare a mixed working solution: Prepare the standard substances 1,25-DHVD3, 1,25-DHVD2, 24,25-DHVD3, 24,25-DHVD2, 25-OH VD3, and 25-OH VD2 into a mixed working solution as shown in Table 1.

[0055] Table 1. Preparation of mixed working solution

[0056]

[0057] (2) Prepare standard solution: Use the above mixed working solution and ethanol to prepare calibrant and quality control working solutions 50*S1-50*S6, as shown in Table 2 below.

[0058] Table 2. Preparation and concentration of calibrator and quality control working solutions

[0059]

[0060] Take 20 μL of the six 50X calibrators and quality control working solutions 50*S1-50*S6 in Table 2 above and mix them with 980 μL of standard curve matrix to prepare six standard solutions SD1-SD6 for preparing the standard curve. The configuration volume of the standard solution and the concentration of each component are shown in Table 3 below.

[0061] Table 3. Preparation of standard solution for calibration curve

[0062]

[0063] (3) Preparation of quality control products: Take the three calibration products 50*S2, 50*S5, and 50*S6 and the quality control product working solution configured in Table 2 above and mix them with the quality control matrix to prepare L1, L2, and L3 quality control products. The configuration of L1, L2, and L3 quality control products and the content of their components are shown in Table 4 below.

[0064] Table 4. Preparation of quality control products

[0065]

[0066] (4) Preparation of internal standard solution: 1,25-DHVD3-IS, 1,25-DHVD2-IS, 24,25-DHVD3-IS, 24,25-DHVD2-IS, 25-OHVD3-IS and 25-OHVD2-IS were respectively prepared into primary stock solutions. The six primary stock solutions were then prepared into secondary stock solutions. Different volumes of the six secondary stock solutions were respectively taken and added with 70% methanol to prepare internal standard solutions. The specific configurations of the primary stock solutions, secondary stock solutions and internal standard solutions are shown in Table 5.

[0067] Table 5. Preparation and concentration of internal standard solution

[0068]

[0069] 2. Sample pretreatment

[0070] Sample requirements: Use yellow or red cap blood collection tubes to collect blood, and separate serum of no less than 1 mL. It can be stored at room temperature or refrigerated for seven days, and can be frozen for long-term storage.

[0071] 2.1 Antibody immune particle extraction method:

[0072] (1) Take 200 μL of agarose beads and 200 μL of antibody (Aimejie, anti-1,25(OH)2VD mouse monoclonal antibody), add to 1 mL 96-well filter plate, add 100 μL of polyethylene glycol, 200 μL of blocking agent (containing 10 w / v% bovine serum albumin, 10 w / v% casein, 5 w / v% glycine and 5 w / v% glutamate), incubate at room temperature with shaking for 1 hour, wash three times with PBS buffer, and obtain 400 μL of agarose bead-coupled antibody solution;

[0073] (2) Take 400 μL of the sample to be tested to a 1 mL 96-well filter plate, add 50 μL of internal standard solution and 20 μL of agarose bead-coupled antibody solution, place in a centrifuge, centrifuge at 1000 rpm for 1 minute at room temperature, and vortex mix at 800 rpm for 1 hour;

[0074] (3) Use positive pressure to push the liquid down the filter plate; rinse the 96-well filter plate with the eluent, repeat twice; press dry after the second rinse;

[0075] (4) Elute with 200 μL of eluent, collect the eluate in a 96-well plate, and blow dry with nitrogen gas;

[0076] (5) Add 50 μL of derivatization agent to the 96-well collection plate, derivatize at 37 °C for 30 min, quench with aqueous solution and analyze on the analyzer.

[0077] The agarose beads were coupled with 24,25-(OH)2VD mouse monoclonal antibody (purchased from Amygen) and 25-OHVD mouse monoclonal antibody (purchased from Amygen) respectively using the same method as above, and the samples were pre-treated.

[0078] 2.2 Magnetic bead antibody extraction method:

[0079] (1) Take 500 μL of magnetic beads (Dynabeads™ MyOne™ Streptavidin T1 magnetic beads (Cat. No. 65601) from Thermo Fisher) and 500 μL of antibody (Aimejie, anti-1,25(OH)2VD mouse monoclonal antibody, which has been biotinylated in advance), add 250 μL of polyethylene glycol and 500 μL of blocking agent (containing 10 w / v% bovine serum albumin, 10 w / v% casein, 5 w / v% glycine and 5 w / v% glutamate), incubate at room temperature with shaking for 1 hour, wash three times with PBS buffer under magnetic stand adsorption conditions, and re-dissolve to obtain 1000 μL of magnetic bead antibody solution for standby use;

[0080] (2) Take 400 μL of the sample to be tested to a 96-well plate, add 50 μL of internal standard solution and 50 μL of magnetic bead antibody solution in sequence, and vortex to mix to obtain the diluted sample;

[0081] (3) Place the 96-well plate in a magnetic bead extractor, mix for 10 minutes, and absorb the magnet for 30 seconds;

[0082] (4) The magnetic bead extractor moves the magnetic bar into the elution plate 1 and performs elution step 1, with a mixing time of 60 seconds and magnetic attraction for 30 seconds;

[0083] (5) The magnetic bead extractor moves the magnetic bar into the elution plate 2 and performs the elution step 2, with a mixing time of 60 seconds and magnetic attraction for 30 seconds;

[0084] (6) The magnetic bead extractor moves the magnetic bar into the elution plate and performs the elution step with a mixing time of 150 seconds and magnetic attraction for 30 seconds;

[0085] (7) Dry the elution plate with nitrogen; add 50 μL of derivatization agent to each well, derivatize at 37 °C for 30 minutes, quench with aqueous solution and analyze on the analyzer.

[0086] The streptavidin magnetic beads were coupled to the pre-biotinylated 24,25-(OH)2VD mouse monoclonal antibody (purchased from Aimejie) and 25-OHVD mouse monoclonal antibody (purchased from Aimejie) respectively using the same method as above, and the samples were pre-treated.

[0087] The derivatizing agent in the methods described in 2.1 and 2.2 is 4-phenyl-1,2,4-triazine-3,5-dione (PTAD).

[0088] The above two pretreatment methods were used to detect the quality control products L1-L3 in Table 4 respectively. It was found that the use of the magnetic bead antibody extraction method made the quality control products detected more accurate and with a smaller coefficient of variation. The relative deviation could be controlled within ±5%, while the antibody immune particle method had a large coefficient of variation for the quality control products and a relative deviation within ±10%. Preferably, the magnetic bead antibody extraction method was selected as the sample pretreatment method. Compared with the magnetic bead antibody extraction method, the antibody immune particle method has the following advantages: in terms of manipulation, agarose beads need to be collected and separated by centrifugation, which is relatively cumbersome, while magnetic beads can be manipulated and collected by external magnetic force due to the characteristics of their magnetic materials, which is more convenient and quicker to operate; in terms of precipitation efficiency, magnetic beads have higher precipitation efficiency than agarose beads. The magnetism of magnetic beads can make them completely concentrated on the magnetic rod, avoiding the weightlessness problem that may be caused by centrifugation, while agarose beads are easily dispersed by gravity during centrifugation; in terms of repeatability, magnetic beads have good reusability and can be washed and separated multiple times by adding and removing magnetic force, while agarose beads often require multiple centrifugation to separate the supernatant and residue in experiments, which is easy to cause loss and scouring.

[0089] In summary, the magnetic bead antibody extraction method is preferred.

[0090] 3. Comparison of detection methods

[0091] The method of extracting antibodies with magnetic beads was used for pretreatment, and the samples were detected by liquid chromatography tandem mass spectrometry system and four-channel liquid chromatography tandem mass spectrometry system.

[0092] (1) Liquid chromatography tandem mass spectrometry system: CalQμant-S

[0093] Mobile phase A: 0.1% ethylamine in water; Mobile phase B: 0.1% ethylamine in methanol; Injection volume: 30 μL; Flow rate: 0.5 mL / min; Chromatographic column: Disigns Column 002 (C18 Column 3.0 × 30 mm); Column temperature: 40 °C;

[0094] Gradient elution parameters are shown in Table 6:

[0095] Table 6. Gradient elution parameters

[0096]

[0097] Retention time (min):

[0098] 1,25-Dihydroxy Vitamin D3: 1.96;

[0099] 1,25-Dihydroxy Vitamin D2: 2.07;

[0100] 24,25-Dihydroxy Vitamin D3: 1.02;

[0101] 24,25-Dihydroxy Vitamin D2: 1.47;

[0102] 25-Hydroxy Vitamin D3: 2.21;

[0103] 25-Hydroxy Vitamin D2: 2.25.

[0104] The specific ion selection chromatograms are shown in Figure 1-Figure 4 , the retention time will vary with different detection times and should be within ±0.1 min.

[0105] Mass spectrometry conditions: APCI source, positive ion mode; spray voltage: 5500 V; temperature: 375 o C; atomizing gas: 50 psi; auxiliary heating gas: 50 psi;

[0106] Curtain gas: 20 psi; multiple reaction monitoring (MRM) scanning mode.

[0107] The mass spectrometry parameters are shown in Table 7:

[0108] Table 7. Mass spectrometry parameters

[0109]

[0110] The internal standard method was used to determine the concentrations of various components of the six standard solutions SD1-SD6 in Table 3, and a standard curve was established. The linear relationship verification record is shown in Table 8. The measurement units of 1,25-DHVD3 and 1,25-DHVD2 are pg / mL, and the others are ng / mL.

[0111] Table 8. Standard curve results of liquid chromatography tandem mass spectrometry system

[0112]

[0113] According to the data analysis in Table 8, the linear correlation coefficients of the six components can all reach above 0.99. It can be seen that the linear relationship of the detection of each indicator is good within the concentration range of the standard curve.

[0114] The quantitative lower limit and linear range were obtained by using the standard curve of the above detection, as shown in Table 9 below.

[0115] Table 9. Detection limit and linear range of liquid chromatography tandem mass spectrometry system

[0116]

[0117] To determine the detection precision and accuracy of the above-mentioned lower limit of quantitation, the standard solution SD1 in Table 3 was tested 6 times, and the standard deviation, coefficient of variation and relative deviation were calculated, as shown in Table 10 below.

[0118] Table 10. Precision and accuracy verification of lower limit of quantitation (LLOQ)

[0119]

[0120] According to the analysis of the data results in Tables 9 and 10, the lower limit of quantification of 1,25-DHVD3 can reach 8 pg / mL, the lower limit of quantification of 1,25-DHVD2 can reach 8 pg / mL, the lower limit of quantification of 24,25-DHVD3 can reach 0.1 ng / mL, the lower limit of quantification of 24,25-DHVD2 can reach 0.05 ng / mL, the lower limit of quantification of 25-OHVD3 can reach 1 ng / mL, and the lower limit of quantification of 25-OHVD2 can reach 0.4 ng / mL. The quantitative lower limit determination results of the above six substances have good precision and accuracy.

[0121] The internal standard method was used to measure the concentrations of the quality control products L1 to L3 in Table 4 to evaluate the accuracy of the kit provided by the present invention. Each sample was tested three times and the average value was calculated. The test results are shown in Table 11 below.

[0122] Table 11. Test results of quality control products

[0123]

[0124] According to the data analysis in Table 11, the relative deviation between the detection value and the theoretical value of each test object is within ±5%, indicating that the kit provided by the present invention has high accuracy in detecting six substances at the same time.

[0125] (2) Four-channel liquid chromatography tandem mass spectrometry system

[0126] Four-channel liquid phase system: Transcend II TLX-4; mass spectrometer: Thermo TSQ Altis; mobile phase A: 0.1% ethylamine in water; mobile phase B: 0.1% ethylamine in methanol; injection volume: 40 μL; flow rate: 0.7 mL / min; switching valve cut-in time for mass spectrometer: Start: 0.9 min; Duration: 1.5 min; software version: Thermo Xcalibur 4.1.50.

[0127] The gradient elution parameters are shown in Table 12 below.

[0128] Table 12. Gradient elution parameters

[0129]

[0130] Retention time:

[0131] 1,25-Dihydroxy Vitamin D3: 1.06;

[0132] 1,25-Dihydroxy Vitamin D2: 1.17;

[0133] 24,25-Dihydroxy Vitamin D3: 0.12;

[0134] 24,25-Dihydroxy Vitamin D2: 0.57;

[0135] 25-Hydroxy Vitamin D3: 1.31;

[0136] 25-Hydroxy Vitamin D2: 1.35.

[0137] The specific ion selection chromatograms of 1,25-Dihydroxy Vitamin D3 and 1,25-Dihydroxy Vitamin D2 are shown in Figure 5-Figure 6 , the retention time should be within ± 0.1 min.

[0138] Mass spectrometry conditions: ion source: APCI; sheath gas: 45; auxiliary gas: 10; purge gas: 2; ion transfer tube temperature ( o C): 300; Atomizing gas temperature ( o C): 375; Q1 resolution (FWHM): 0.7; Q3 resolution (FWHM): 1.2; Collision gas (mTorr): 2.

[0139] The mass spectrometry parameters are shown in Table 13 below:

[0140] Table 13. Mass spectrometry parameters

[0141]

[0142] The internal standard method was used to determine the concentrations of the various components of the six standard solutions SD1-SD6 in Table 3, and a standard curve was established, as shown in Table 14 below. The measurement units of 1,25-DHVD3 and 1,25-DHVD2 were pg / ml, and the others were ng / ml.

[0143] Table 14. Results of four-dimensional liquid phase tandem mass spectrometry standard curve

[0144]

[0145] According to the data analysis in Table 14, the linear correlation coefficients of the six components can all reach above 0.99. It can be seen that the linear relationship of the detection of each indicator is good within the standard curve concentration range.

[0146] The internal standard method was used to determine the concentrations of the quality control products L1-L3 in Table 4 to evaluate the accuracy of the kit provided by the present invention. Each sample was tested three times and the average value was calculated. The test results are shown in Table 15 below.

[0147] Table 15. Test results of quality control products

[0148]

[0149] According to the data analysis in Table 15, the relative deviation between the detection value of each test object and the theoretical value is within ±5%, indicating that the kit provided by the present invention has high accuracy in simultaneously detecting six substances.

[0150] In summary, after the sample is treated with the method provided by the present invention, whether it is detected by a single-channel liquid chromatography tandem mass spectrometry detection method or a multi-channel liquid chromatography tandem mass spectrometry detection method, the linear range and accuracy of the detected sample are very high.

[0151] Example 2: Comparison of different sample pretreatment methods and different kit detection methods

[0152] Among the six vitamin D metabolic pathway metabolites, 1,25-DHVD3, 1,25-DHVD2, 24,25-DHVD3, 24,25-DHVD2, 25-OHVD3, and 25-OHVD2, the contents of 1,25-DHVD3 and 1,25-DHVD2 in serum are much lower than those of the other four components and are difficult to detect. Therefore, the detection sensitivity and anti-interference ability of the present invention are evaluated by detecting the contents of 1,25-DHVD3 and 1,25-DHVD2.

[0153] This example compares the sensitivity and accuracy of different kits. In addition to setting up two groups of samples extracted by agarose beads coupled to anti-DHVD mouse monoclonal antibodies and streptavidin magnetic beads coupled to anti-DHVD mouse monoclonal antibodies provided by the present invention and then used for detection with the kit provided by the present invention, the 1,25-(OH)2-vitamin D3 / D2 ImmuTube LC-MS / MS Extraktionskit (Cat. No. KM1100) kit from Immun Diagnostik and the AB Sciex Amplifex Diene Kit SPE kit were purchased for comparison based on literature information. The 1,25-(OH)2-vitamin D3 / D2 ImmuTubeLC-MS / MS Extraktionskit kit is set up with two methods of derivatization and non-derivatization to detect samples; the AB SciexAmplifex Diene Kit SPE kit is an SPE derivatization method, so the SPE pretreatment non-derivatization method reported in the literature is also used for comparison. The SPE non-derivatization method pretreatment used the method in the literature (J Mass Spectrom. 2022 Jan; 57(1): e4792. doi: 10.1002 / jms.4792) to enrich, extract and detect 1,25-DHVD in the sample.

[0154] Set the following six groups of sample pretreatment methods:

[0155] Group 1: ImmuTube LC-MS / MS Extraktionskit non-derivatized method;

[0156] Group 2: ImmuTube LC-MS / MS Extraktionskit derivatization method;

[0157] Group 3: SPE non-derivatized method (addition of acetyl lithium) combined with the detection method described in the above paper;

[0158] Group 4: AB Sciex Amplifex Diene Kit SPE kit derivatization method;

[0159] The fifth group: agarose beads coupled to anti-DHVD mouse monoclonal antibody combined with the kit provided by the present invention (derivation method);

[0160] Group 6: Streptavidin-coupled anti-DHVD mouse monoclonal antibody combined with the kit provided by the present invention (derivation method).

[0161] The sample pretreatment methods of the first and second groups refer to the instructions of the ImmuTube LC-MS / MS Extraktionskit kit, with the difference that a derivatizing agent is added to the samples of the first group during pretreatment, while no derivatizing agent is added to the samples of the second group. The sample detection method refers to the liquid chromatography tandem mass spectrometry detection in Example 1.

[0162] The sample pretreatment and sample detection methods of the third group refer to the above paper, specifically: take 200 μL of the sample to be tested to a 96-well plate, add 50 μL of internal standard solution and 200 μL of methanol in turn, vortex mix for 5 seconds, and then add 0.5 mL of 0.1 M zinc sulfate in n-hexane / ethyl acetate solution (n-hexane: ethyl acetate = 8:2). The mixed solution was vortexed at 1200 rpm for 5 minutes and then centrifuged at 1200 g for 10 minutes. After centrifugation, the supernatant was transferred to a 96-well plate and dried with nitrogen at 37 degrees. After drying, 100 μL of 50% methanol aqueous solution was added to the 96-well plate for re-dissolution. The re-solution was tested on the machine, and the injection volume was 50 μL.

[0163] The sample pretreatment method of the fourth group was referred to the AB Sciex Amplifex Diene Kit SPE kit instructions, specifically: 0.5 mL of serum was diluted with 0.4 mL of water, and 20 μL of 2500 pg / mL DHVD2-d6 and DHVD3-d6 internal standards were added. The samples were centrifuged at 2000 g for 10 min. Two-stage solid phase extraction was performed using the filter cartridges included in the kit: C1000 (first stage; diatomaceous earth) and S500 (second stage; silica). 900 μL of sample was added to the C1000 column, gravity flowed for 10 min, DHVD was eluted onto the S500 cartridge with diisopropyl ether (1 mL*4), and then washed twice with 4.5 mL of 4% isopropanol in hexane solution, and then washed twice with 6 mL of 6% isopropanol in hexane solution. DHVD was eluted from the S500 cartridge with 4.5 mL of 30% isopropanol in n-hexane solution, and the eluate was completely dried by centrifugal evaporation. The amplified diene reagent (derivative agent) was prepared by diluting the prepared diluent 1:2, adding 50 μL of the diluted reagent to each sample and mixing by oscillation for 30 min. After 30 min, the derivatization reaction was quenched with 50 μL of water, and the sample was transferred to the injection bottle for LC-MS / MS analysis. The sample detection parameters were detected by liquid chromatography tandem mass spectrometry as described in Example 1.

[0164] The sample pretreatment methods and sample detection methods of the fifth and sixth groups refer to the two sample pretreatment methods and detection by liquid chromatography-tandem mass spectrometry in Example 1.

[0165] The samples to be tested treated by the above six different sample pretreatment methods are selected from the quality control products in Example 1, and the sensitivity under the six pretreatment and detection methods is calculated. The results are shown in Table 16. The quantitative lower limits of 1,25-DHVD3 and 1,25-DHVD2 are similar to the linear range trends. The quantitative lower limit detection result of 1,25-DHVD2 is taken as an example.

[0166] Table 16. Quantitative lower limit and linear range of different sample pretreatment methods

[0167]

[0168] According to the data analysis in Table 16, the quantitative lower limit and linear range of 1,25-DHVD2 detected after the samples were treated by the derivatization method (the second, fourth, fifth, and sixth groups) and the non-derivative method (the first and third groups) were compared. The quantitative lower limit of 1,25-DHVD2 can be significantly reduced and the linear range of detection can be expanded after the samples are treated by the derivatization method (the second, fourth, fifth, and sixth groups). This shows that the derivatization method can improve the sensitivity of detection; the quantitative lower limit and linear range of 1,25-DHVD2 detected after the samples were treated by the four derivatization methods (the second, fourth, fifth, and sixth groups) were compared. The sample pretreatment method provided by the present invention combined with the kit provided by the present invention can make the quantitative lower limit of 1,25-DHVD2 lower, reaching 8 pg / mL, indicating that the kit provided by the present invention has higher detection sensitivity and wider linear range than the commercially available ImmuTube LC-MS / MS Extraktionskit and AB Sciex Amplifex Diene Kit. The speculated reason is that during the sample pretreatment process, after the agarose beads coupled to the anti-DHVD mouse monoclonal antibody adsorb the analyte, the interfering components can be removed by filtration. After the streptavidin-coupled anti-DHVD mouse monoclonal antibody adsorbs the analyte, the interfering components can be removed by magnetic selection in an automated manner on a magnetic bead extractor, thereby improving the detection sensitivity.

[0169] Comparing the sample pretreatment methods of the fifth group and the sixth group, the detection accuracy of the same quality control product was higher when the streptavidin-coupled anti-DHVD mouse monoclonal antibody was used to detect multiple times, the coefficient of variation was smaller, and the relative deviation could be controlled within ±5%, while the agarose bead-coupled anti-DHVD mouse monoclonal antibody was used to detect multiple times the same quality control product, and the coefficient of variation was large and the relative deviation was within ±10%. Preferably, the selection of streptavidin-coupled anti-DHVD mouse monoclonal antibody in combination with the kit provided by the present invention to detect samples can further improve the detection sensitivity of 1,25-dihydroxyvitamin D.

[0170] The above six sample pretreatment methods were used to treat three normal human serum samples, and then the concentrations of 1,25-DHVD3 and 1,25-DHVD2 were detected. The detection method was referred to Example 1. Each sample was tested three times and the average value was taken. The test results are shown in Table 17 below.

[0171] Table 17. Comparison of test results of samples with different pre-treatment methods

[0172]

[0173] According to the data analysis in Table 17, the 1,25-DHVD3 concentration detected by the third group of SPE non-derivatization methods (adding acetyl lithium) in combination with the detection method described in the above paper and the fourth group of AB Sciex Amplifex Diene Kit SPE kit derivatization methods exceeded the upper limit of the reference range for normal people, resulting in false positive results. There are many isomers of 1,25-dihydroxyvitamin D in biological samples. The reason why the SPE method is prone to false positive results is speculated to be the relatively weak impurity removal ability; when the analysis time of liquid chromatography-tandem mass spectrometry is extended to more than 15 minutes, interference peaks that are not completely baseline separated can be observed in the samples pre-treated by SPE. In the process of using specific antibodies for sample pre-treatment, the antibody selectively extracts the target analyte, which can effectively filter out isomers that are difficult to separate in the sample. According to literature reports, it is speculated that the potential endogenous interfering substance with a high content in the SPE extraction process is 4β,25-DHVD; this interfering component will not be bound by the antibody during the antibody enrichment process. The fifth and sixth groups of methods can more accurately detect the concentration of 1,25-DHVD3, and the sixth group of methods is the most preferred.

[0174] In summary, the kit provided by the present invention uses magnetic bead-coupled Aimejie anti-1,25 (OH) 2VD mouse monoclonal antibody, which can effectively separate interfering components while achieving specific extraction of 1,25-DHVD in serum samples, and has higher sensitivity and accuracy, and is preferred.

[0175] Example 3: Mass spectrometry ion source ionization mode, ion source temperature

[0176] During the research process of the present invention, it was found that the detection signal-to-noise ratio of the APCI ion source is much higher than that of the ESI ion source reported in the literature. Therefore, APCI and ESI are respectively selected as ion sources in this embodiment. The other parameters are the same as those in Example 1. The magnetic bead antibody extraction method and liquid chromatography tandem mass spectrometry are selected for detection. The detection spectrum of 1,25-DHVD3 is shown in FIG. Figure 7-10 .like Figure 7 As shown in the two figures, the LLOQ response of 1,25-DHVD3 in the ESI ion source, the detection signal-to-noise ratio is about 5; Figure 8 As shown in the two figures, the response of the LLOQ of 1,25-DHVD3 detected in the APCI ion source, the detection signal-to-noise ratio is about 25; Fig. 9 As shown in the two figures, the response of 1,25-DHVD3 in serum samples to the ESI source, the detection signal-to-noise ratio is about 8; Fig.10 As shown in the two figures, the response of 1,25-DHVD3 in serum samples detected by APCI source, and the detection signal-to-noise ratio is about 40.

[0177] The results show that when using ESI source for detection, the baseline peak response is high and the signal-to-noise ratio of the analyte is low; when using APCI source for detection, the baseline peak response is low, the signal-to-noise ratio is high, and the detection effect is better. Figure 7-Figure 8 The ESI ion source and Figure 9-10 The LLOQ of the APCI source and the actual serum sample detection spectrum show that the signal-to-noise ratio of APCI detection is about 5 times that of the ESI source detection. The active metabolites of 25-hydroxyvitamin D, 1,25-DHVD3 and 1,25-DHVD2, are extremely low in serum samples, and the mass spectrometry signals are relatively weak. The selection of the APCI source reduces baseline noise and improves the signal-to-noise ratio of the analytes, which is especially suitable for the detection of 1,25-DHVD3 and 1,25-DHVD2, and can significantly improve the detection sensitivity; while for the other analytes, the ion source does not have such a significant effect on them. In order to improve the detection sensitivity of 1,25-DHVD3 and 1,25-DHVD2, it is necessary to use the APCI ion source.

[0178] The present invention found in the research process that the temperature of the ion source has a great influence on the detection sensitivity of 1,25-DHVD. Therefore, in this embodiment, the ion source temperature of the mass spectrometer is adjusted respectively. The other parameters are the same as those in Example 1. The magnetic bead antibody extraction method and the ordinary liquid chromatography tandem mass spectrometry are selected for detection. The detection spectrum of 1,25-DHVD3 is shown in FIG. Figure 11-Figure 14 . Fig.11 From left to right, the tested ion source temperatures are 250 °C and 300 °C; Fig.12 From left to right, the ion source temperatures tested are 325 °C and 350 °C; Fig.13 From left to right, the tested ion source temperatures are 375 °C and 400 °C; Fig.14 From left to right, the tested ion source temperatures are 450 ℃ and 500 ℃ respectively.

[0179] The results show that the response of the target analyte increases first and then decreases with the increase of temperature. After 1,25-DHVD produces gas-phase ions in the ion source, it is easy to remove one molecule of water, and can further remove two molecules of water. This process is greatly affected by temperature. Among them, the parent ion that removes one molecule of water has a higher sensitivity and is selected for quantitative detection. When the ion source temperature is 250°C, the ionization efficiency and response of the target analyte are low, which will have a greater impact on the detection; when the ion source temperature is equal to or higher than 450°C, the parent ion may be further dehydrated or cracked, resulting in a decrease in signal. When the ion source temperature is 350-450°C, the detection effect is good and the sensitivity is high. The ion source temperature of 375°C is preferred, and the sensitivity is the highest at this time.

[0180] In parallel testing of multiple mass spectrometers, it was found that the optimal ion source temperature conditions vary between instruments and need to be set separately for different instruments, but the optimal temperature range for ionization efficiency and signal-to-noise ratio is 350-450 °C.

[0181] Example 4: Screening and Optimization of Derivatization Agents and Optimization of Derivatization Conditions

[0182] In the research process of the present invention, in order to improve the detection sensitivity of trace amounts of 1,25-DHVD3 and 1,25-DHVD2 in LC-MS / MS, the ionization efficiency of the target analyte was improved by derivatization reaction to achieve a lower detection limit. Therefore, different derivatization agents were investigated in this example, and the following types of derivatization agents were used for comparison:

[0183] (1) Amplifex™ Diene Reagent used in the aforementioned AB Sciex Amplifex Diene Kit SPE kit;

[0184] (2) Cookson-type reagents: PTAD (4-phenyl-1,2,4-triazolin-3,5-dione), MBOTAD (4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazolin-3,5-dione, DMEQTAD (4-[2-(6,7dimethoxy-4-methyl-3-oxo-3,4-dihydroqμinoxalyl)ethyl]-1,2,4-triazolin-3,5-dione), MTAD (4-methyl-1,2,4-triazolin-3,5-dione) and 4-carboxyphenyl-TAD, etc. Among the 4-substituted 1,2,4-triazolin-3,5-dione Cookson reagents, PTAD has the highest cost-effectiveness and is most commonly used in the derivatization reaction of diene compounds. Therefore, PTAD is selected as the derivatization and component in the kit detection scheme;

[0185] (3) The 2-nitrosopyridine reported in the literature has a high derivatization reaction efficiency, but the derivatized product has four stereoisomers, which will affect the liquid phase separation and mass spectrometry quantification process.

[0186] Compared with PTAD, the derivatization activity of Amplifex™ Diene Reagent is too high, resulting in extremely harsh storage and transportation conditions. It needs to be transported under anhydrous conditions using dry ice at -70 ℃ or below, and the storage conditions are -70 ℃ or below. If it is used as the derivatization agent component of the DHVD kit, the storage and transportation conditions of the entire kit will be limited to below -70 ℃, greatly increasing the storage and transportation costs of the kit and limiting its stability in use: it is not recommended to reuse the derivatization agent after opening the bottle. If the sample volume of the testing laboratory is not large enough, a kit may need to match multiple bottles of derivatization agents, which will further increase the testing cost. In addition, the use of such a highly active and low-stability reagent in the production process will pose great challenges to the operating requirements of production personnel and the quality control of product components, and may even affect its quality.

[0187] 2-Nitrosopyridine can rapidly and efficiently react with conjugated dienes in the structure of 25-hydroxyvitamin D and its metabolites through Diels-Alder addition reaction, and the reaction product has a high ionization efficiency, which is helpful for improving the detection sensitivity. However, the reason for limiting its wide application is that each vitamin D-derived product has four stereoisomers. Under the premise of pursuing high-throughput detection, it is difficult for the liquid phase separation system to achieve complete baseline separation of the four stereoisomers in a short separation time. At the same time, the four stereoisomers have different polarities and will show different separation effects. In addition, 25-hydroxyvitamin D and its metabolites themselves have many isomers, which will increase the number of peaks in the detection process by four times, making it difficult to identify and attribute the specificity of each indicator, which can easily lead to incorrect attribution in the quantitative process. After comprehensive comparison and consideration, it is not recommended to use it.

[0188] After 1,25-DHVD was extracted by the magnetic bead antibody extraction method described in Example 1, liquid chromatography tandem mass spectrometry was used for detection. The difference was that different derivatization agents were used. The quantitative lower limit and linear range results are shown in Table 18. The quantitative lower limit and linear range trends of 1,25-DHVD3 and 1,25-DHVD2 are similar. The quantitative lower limit detection result of 1,25-DHVD2 is taken as an example.

[0189] Table 18. Quantitative limit and linear range under different derivatization agents

[0190]

[0191] According to the data analysis in Table 18, PTAD as a derivatizing agent can reduce the detection limit of 1,25-dihydroxyvitamin D2 to 8 pg / mL, with a larger linear range. The detection limits of other derivatizing agents are all higher than 8 pg / mL.

[0192] Therefore, after comprehensively considering the reaction activity, kit cost, stability, convenience of data processing, and challenges in quality control, the cost-effective triazolinedione compound PTAD was selected as the derivatization reagent used in this study. After using PTAD as a derivatization reagent, the detection signal-to-noise ratio increased by more than 2 times, and the detection LLOQ could be reduced from 20 pg / mL without the addition of a derivatization agent to 8 pg / mL.

[0193] When PTAD is used as a derivatizing agent, the commonly used derivatization condition in the literature is heating derivatization at 50-55 °C in an acetonitrile solution. However, in actual operation, especially in the process of using a 96-well plate to derivatize a large number of samples, it is found that during the heating process of derivatization, acetonitrile will easily volatilize, and the silicone pad covering the 96-well plate will be pushed open by the acetonitrile vapor; even if the silicone pad on the 96-well plate is reinforced with sticky paper, it will still be pushed open, and cross-contamination between wells is prone to occur; when the silicone pad is enabled and the sticky paper is used to seal the plate, there will also be a miscellaneous peak of 1,25-dihydroxyvitamin D. The present invention optimizes the derivatization temperature, and the test temperatures are room temperature, 30 °C, 37 °C, 50 °C and 55 °C, the derivatization time is 30 minutes, and after the magnetic bead antibody extraction method described in Example 1 is used to extract 1,25-DHVD, liquid chromatography tandem mass spectrometry is used for detection, and the detection signal-to-noise ratio results are shown in Table 19 below.

[0194] Table 19. Signal-to-noise ratio results of different derivative temperature detection

[0195]

[0196] According to the data analysis in Table 19, under various derivatization temperature conditions, there were no significant differences in the derivatization efficiency and low-concentration signal-to-noise ratio of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2. However, under the derivatization condition of a lower temperature, the silicone cover pad of the 96-well plate would not be pushed open, effectively avoiding cross-contamination between samples. Therefore, the present invention and the kit method recommend the use of a derivatization condition of 37°C.

[0197] Example 5: Screening and Optimization of Mobile Phase and Additives

[0198] In order to improve the detection sensitivity of trace 1,25-DHVD in LCMS, according to its chemical properties, adding formic acid can effectively increase its separation, but the mobile phase needs to be optimized. The difference between this embodiment and embodiment 1 is that multiple sets of mobile phase schemes are set, as follows:

[0199] (1) Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol;

[0200] (2) Mobile phase A: 2 mM ammonium acetate + 0.1% formic acid in water; Mobile phase B: 2 mM ammonium acetate + 0.1% formic acid in water;

[0201] (3) Mobile phase A: 2 mM ammonium fluoride + 0.1% formic acid in water; mobile phase B: 2 mM ammonium fluoride + 0.1% formic acid in methanol (100% methanol containing 0.1% formic acid);

[0202] (4) Mobile phase A: 2 mM ammonium fluoride in water; Mobile phase B: 2 mM ammonium fluoride in methanol (100% methanol containing 2 mM ammonium fluoride);

[0203] (5) Mobile phase A: 0.1% methylamine in water; Mobile phase B: 0.1% methylamine in methanol (100% methanol contains 0.1% methylamine);

[0204] (6) Mobile phase A: 0.1% ethylamine in water; Mobile phase B: 0.1% ethylamine in methanol (100% methanol containing 0.1% ethylamine);

[0205] (7) Mobile phase A: 0.1% triethylamine in water; Mobile phase B: 0.1% triethylamine in methanol (100% methanol containing 0.1% triethylamine);

[0206] (8) Mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile (100% acetonitrile containing 0.1% formic acid).

[0207] The concentration of each component in the quality control product L1 was detected by LCMSMS according to the above eight groups of schemes, and the LLOQ signal-to-noise ratio was calculated based on the test results. The results are shown in Table 20 below.

[0208] Table 20. Detection signal-to-noise ratio of LLOQ under different mobile phases

[0209]

[0210] According to the analysis in Table 20, by comparing the LLOQ signal-to-noise ratio results of different mobile phases, the signal-to-noise ratios of the six components detected by the sixth group of mobile phases were the highest, indicating that the sixth group of schemes (mobile phase A: 0.1% ethylamine water; mobile phase B: 0.1% ethylamine methanol) had the highest response of each indicator and was most conducive to the detection of 1,25-DHVD with the best sensitivity; except for the sixth group of schemes, the signal-to-noise ratios of the six components detected by the first, fifth and seventh groups of mobile phases were higher than those of the remaining four groups, and the sensitivity was better. Generally speaking, the signal-to-noise ratio of the liquid chromatograph should be above 5:1 to ensure the accuracy and reliability of the data, so the first group and the 5-7 groups of mobile phases are preferred. However, in order to simultaneously detect 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 24,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D2 with high sensitivity, especially to improve the detection sensitivity of 1,25-dihydroxyvitamin D, the sixth group of mobile phase schemes is the most preferred.

[0211] Example 6: Screening and Optimization of Eluents and Eluents

[0212] Optimization of eluent: In the process of using antibodies to extract 1,25-DHVD in samples, the components of the eluent were optimized and compared according to the sensitivity of LLOQ. The magnetic bead antibody extraction method described in Example 1 was used to extract 1,25-DHVD3 and 1,25-DHVD2. Different eluents were added during the extraction process, and liquid chromatography-tandem mass spectrometry was used for detection. Different eluents and detection quantitative lower limits and linear ranges are shown in Table 21 below. The quantitative lower limits of 1,25-DHVD3 and 1,25-DHVD2 have similar trends to the linear range. The quantitative lower limit detection results of 1,25-DHVD2 are taken as an example.

[0213] Table 21. Screening and optimization of eluents

[0214]

[0215] According to the data analysis in Table 21, when water containing an organic solvent is used as the eluent, it has a certain elution ability for 1,25-DHVD, resulting in the removal of part of 1,25-DHVD in the sample and reduced sensitivity. When water, 0.2% formic acid water and 0.2% ammonia water are used as eluents, the sensitivity is not affected. Preferably, water and 0.2% formic acid water are selected, and combined with the antibacterial requirements of the reagent for long-term storage, 0.2% formic acid water is finally locked in as the eluent (0.2% formic acid water).

[0216] Optimization of eluent: In the process of using antibodies to extract 1,25-DHVD and other vitamin D metabolites in the sample, the components of the eluent were optimized. The magnetic bead antibody extraction method described in Example 1 was used to extract 1,25-DHVD, and liquid chromatography tandem mass spectrometry was used for detection. The magnetic bead antibody extraction method used different eluents during the extraction process and the nitrogen drying time after elution was different. Different eluents and detection sensitivity and linear range are shown in Table 22 below. The quantitative lower limit of 1,25-DHVD3 and 1,25-DHVD2 have similar trends to the linear range. The quantitative lower limit detection result of 1,25-DHVD2 is taken as an example.

[0217] Table 22. Screening and optimization of eluents

[0218]

[0219] According to the data analysis in Table 22, comparing the nitrogen drying time and sensitivity test results required after elution with different eluents, the nitrogen drying time after elution with ethanol is the shortest, and the sensitivity for detecting 1,25-DHVD is the highest. Therefore, the combination of ethanol as the eluent and 0.2% formic acid water as the eluent is the optimal combination.

[0220] Example 7: Clinical Example

[0221] A child in a children's hospital presented with hypercalcemia. The hospital's test found that the test result of 25-hydroxyvitamin D3 exceeded 150 ng / mL (the upper limit of the detection range), reaching the toxic concentration of the normal population. The doctor suspected that the child might have a vitamin D metabolic pathway disorder or a deficiency of kidney-related metabolic enzymes.

[0222] After the serum samples of the children were sent to the laboratory, the magnetic bead antibody extraction method described in Example 1 was used to extract 25-hydroxyvitamin D and its metabolites in the patient serum samples, and liquid chromatography-tandem mass spectrometry was used for detection. The detection results of each indicator are shown in Table 23 below.

[0223] Table 23. Detection results of 25-hydroxyvitamin D and its metabolites in patient serum samples

[0224]

[0225] According to the analysis of the test results in Table 23 above, the patient's 25-hydroxyvitamin D content is high, reaching the toxic concentration of the normal population; the 1,25-dihydroxyvitamin D content is high, exceeding the upper limit of the reference range of the normal population; the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D = 163.2 / 2.04 = 80. According to the reference range data in the literature, when the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D is less than 25, the result is normal; when the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D exceeds 25 but is less than 80, the patient may have a heterozygous variation of 24-hydroxylase (CYP24A1); when the ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D exceeds 80, the patient may have a biallelic variation or deletion of 24-hydroxylase.

[0226] Based on the test results of the method of the present invention, the doctor conducted a genetic test on the child and confirmed that the child had a CYP24A1 mutation. After targeted treatment, the child's blood calcium and phosphorus concentrations returned to normal.

[0227] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting metabolites related to serum vitamin D metabolic pathway by liquid chromatography tandem mass spectrometry, characterized in that: The method includes a pretreatment method, which adopts an antibody immune particle extraction method or a magnetic bead antibody extraction method. The vitamin D metabolic pathway-related metabolites include 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D.

2. The method according to claim 1, characterized in that: The 25-hydroxyvitamin D includes 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2; the 1,25-dihydroxyvitamin D includes 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2; and the 24,25-dihydroxyvitamin D includes 24,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D2.

3. The method according to claim 2, characterized in that: The pretreatment method adopts a magnetic bead antibody extraction method; the magnetic bead antibody is a streptavidin-coupled anti-vitamin D mouse monoclonal antibody.

4. The method according to claim 3, characterized in that: The mass spectrometry ion source of the detection method adopts an APCI ion source; the temperature of the mass spectrometry ion source of the detection method is 350-450° C., which may vary between different instruments.

5. The method according to claim 4, characterized in that: The pretreatment method also includes derivatization, and the derivatization uses 4-phenyl-1,2,4-triazolidine-3,5-dione as a derivatization reagent.

6. The method according to claim 5, characterized in that: The mobile phase A of the liquid chromatography tandem mass spectrometry is ethylamine water; the mobile phase B is ethylamine methanol; the pretreatment method also includes eluting and eluting steps, the eluent is formic acid water, and the eluent is ethanol, acetonitrile or a mixed solution of ethanol and acetonitrile.

7. The method according to claim 6, characterized in that: The liquid chromatography tandem mass spectrometry detection method can be a single-channel or multi-channel liquid chromatography tandem mass spectrometry detection method.

8. A kit for detecting metabolites related to serum vitamin D metabolic pathway by liquid chromatography tandem mass spectrometry, characterized in that: The kit comprises a sample diluent, a washing agent, an eluting agent, a derivatizing agent, an internal standard solution, an agarose bead-coupled antibody solution or a streptavidin-coupled magnetic bead antibody solution; the antibodies comprise an anti-25-hydroxyvitamin D mouse monoclonal antibody, an anti-1,25-dihydroxyvitamin D mouse monoclonal antibody and an anti-24,25-dihydroxyvitamin D mouse monoclonal antibody.

9. The kit according to claim 8, characterized in that: The eluent is formic acid water; the eluent is ethanol, acetonitrile or a mixed solution of ethanol and acetonitrile; and the derivatizing agent is 4-phenyl-1,2,4-triazine-3,5-dione.

10. Use of an APCI ion source in improving the detection sensitivity of simultaneous detection of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D in serum by liquid chromatography-tandem mass spectrometry, characterized in that: The APCI ion source temperature is 350-450°C.

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