A kit for pre-treatment of a dihydroxy vitamin d metabolite and a method for pre-treating a sample

Abstract

The application provides a double-hydroxy vitamin D metabolite pretreatment kit and a sample pretreatment method, and belongs to the technical field of in vitro diagnostic reagents. The double-hydroxy vitamin D metabolite pretreatment kit provided by the application comprises a magnetic bead working solution, the magnetic bead working solution comprises hydrophilic-lipophilic balance magnetic beads with a surface modified benzofuran ring system; a sample processing solution, the sample processing solution is a zinc sulfate heptahydrate solution; a leaching solution, the leaching solution is a solution containing fatty acid methyl ester ethoxylate and acetic acid; an eluent, the eluent is an acetonitrile-isopropyl alcohol mixed solution containing 2,6-di-tert-butyl-p-cresol; and a sample diluent, the sample diluent is a solution containing acetic acid. The kit provided by the application has the advantages of good stability, low detection limit and simultaneous detection of multiple target objects.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, and more specifically, to a pretreatment kit and sample pretreatment method for dihydroxyvitamin D metabolites. Background Technology

[0002] Vitamin D exists in the human body mainly in two forms: vitamin D3 and vitamin D2. Vitamin D undergoes two hydroxylation steps in the body to be converted into biologically active 1,25-dihydroxyvitamin D (1,25-DHVD), which plays a central role in physiological processes such as calcium and phosphorus metabolism, immune regulation, and cell differentiation and proliferation. Clinically, the level of 1,25-DHVD is often measured to understand the level of active vitamin D in metabolic bone diseases. Furthermore, 24,25-dihydroxyvitamin D (24,25-DHVD), as the main metabolic clearance product of 25-OH vitamin D, has serum concentrations that are of significant reference value for assessing vitamin D metabolic status and diagnosing certain bone and kidney diseases.

[0003] However, due to the extremely low concentrations of dihydroxy metabolites such as 1,25-DHVD and 24,25-DHVD in serum, and the lack of easily ionized groups in their molecular structures, effective quantitative detection of dihydroxy vitamin D metabolites has become a recognized challenge in the industry. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is the "gold standard" for detecting trace substances. In the LC-MS / MS quantitative detection of dihydroxy vitamin D metabolites, the pretreatment steps of most methods are also very complex. Existing pretreatment methods include derivatization, liquid-liquid extraction, solid-phase extraction, and antibody immunomagnetic bead enrichment. However, derivatization typically employs PTA... Reagents such as D or DAPTAD have drawbacks such as strict reaction conditions, long processing time, uneven derivatization, and the need for quenching, and they are also prone to contaminating the mass spectrometry ion source. Liquid-liquid extraction requires the use of large amounts of toxic organic solvents such as n-hexane and ethyl acetate, and involves cumbersome steps such as extraction, centrifugation, nitrogen blowing, and reconstitution, which are difficult to automate and have poor reproducibility. Solid-phase extraction involves multiple steps such as activation, equilibration, sample loading, washing, and elution, which are time-consuming and dependent on positive or negative pressure equipment. Improper flow rate control can easily lead to large deviations in results. Although antibody immunomagnetic bead enrichment has the advantage of high selectivity, the preparation cost of antibodies, especially high-quality antibodies against small molecule haptens, is extremely high, the stability is poor, the batch-to-batch variability is large, and the incubation time is long.

[0004] Therefore, developing a pretreatment method that is simple, rapid, requires no derivatization, is low in cost, and can simultaneously achieve highly sensitive detection of multiple dihydroxyvitamin D metabolites is an urgent need in current clinical diagnosis. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to overcome the shortcomings of the existing technology in the pretreatment of dihydroxyvitamin D metabolites, such as low throughput, low recovery rate, cumbersome operation, and difficulty in meeting the needs of high throughput for clinical automation and the stability requirements of trace detection.

[0006] To address the aforementioned technical problems, the first aspect of this invention provides a pretreatment kit for dihydroxyvitamin D metabolites, the pretreatment kit comprising:

[0007] The magnetic bead working solution contains magnetic beads that are hydrophilic-lipophilic balanced magnetic beads with a surface-modified benzofuran ring system.

[0008] The sample processing solution is a zinc sulfate heptahydrate solution.

[0009] The rinsing solution is a solution containing fatty acid methyl ester ethoxylate and acetic acid;

[0010] The eluent is an acetonitrile-isopropanol mixed solution containing 2,6-di-tert-butyl-p-cresol;

[0011] The sample diluent is a solution containing acetic acid.

[0012] In the technical solution provided by this invention, the magnetic bead working fluid uses hydrophilic and lipophilic balanced magnetic beads coated with a benzofuran ring system. These beads have a styrene-based polymer backbone, providing a lipophilic / hydrophobic cross-linked structure. The benzofuran ring structure exhibits strong hydrophobicity and the ability to generate π-π interactions. Through surface modification, an electron-rich aromatic heterocyclic benzofuran ring system is introduced. Its rigid planar structure has a larger conjugated area and electron cloud density, resulting in stronger directional π-π stacking and hydrogen bonding. N-vinylpyrrolidone provides hydrophilic groups, giving the material surface a certain degree of hydrophilicity and reducing non-specific adsorption of proteins, etc. Dihydroxyvitamin D metabolites such as 1,25-DHVD and 24,25-DHVD possess sterol rings. The benzofuran ring-coated hydrophilic-lipophilic balanced magnetic beads exhibit a strong hydrophobic effect with substances containing sterol rings, thanks to the synergistic effect of the benzene and benzofuran rings. This effect is particularly enhanced in aqueous buffers containing organic solvents. The conjugated diene structure in the sterol ring forms π-π stacking with the benzene and benzofuran rings on the magnetic surface. Furthermore, the two hydroxyl groups (C1α / C24 and C25 positions) of 1,25-DHVD and 24,25-DHVD can form hydrogen bonds with the hydrophilic groups on the magnetic surface, enhancing specific molecular recognition and selective adsorption. This allows the benzofuran ring-coated hydrophilic-lipophilic balanced magnetic beads to simultaneously extract trace amounts of 1,25-DHVD and 24,25-DHVD from serum. By employing magnetic bead extraction technology in conjunction with a fully automated extraction instrument, four dihydroxyvitamin D metabolites can be extracted simultaneously in a single sample pretreatment.

[0013] Preferably, the content of the surface-modified benzofuran ring-based hydrophilic-lipophilic balanced magnetic beads in the working solution is 0.05~2g / mL.

[0014] This invention optimizes the content of magnetic beads to effectively balance their adsorption capacity and selectivity, ensuring that all four target substances extracted are efficiently captured. It avoids competitive adsorption of non-target substances such as lipids and proteins, reduces matrix interference, and at the same time, the appropriate content can effectively avoid magnetic bead residue of target analytes caused by magnetic bead saturation, thereby improving sample recovery rate.

[0015] Preferably, in the working solution of the magnetic beads, the dispersion medium of the magnetic beads is an aqueous solution of glycerol, wherein the volume ratio of glycerol to water is 2:3.

[0016] By balancing the volume ratio of glycerol to water, the magnetic beads can be prevented from settling in a short time, ensuring that they are in a uniformly dispersed state. This allows for more accurate addition of the magnetic beads. In contrast, traditional water-based or alcohol-based magnetic bead suspensions tend to settle rapidly during automated sample addition and incubation, resulting in uneven bead amounts between wells or batches, which severely affects the repeatability of the recovery rate.

[0017] Preferably, in the sample processing solution, the concentration of zinc sulfate heptahydrate is 0.01~0.05M, and the solvent of the sample processing solution is a first methanol aqueous solution, wherein the volume ratio of methanol to water in the first methanol aqueous solution is 1:(1~2).

[0018] In the technical solution provided by this invention, the zinc ions of zinc sulfate heptahydrate in the sample processing solution can complex or salt out with the phosphate ions of phospholipids in serum and certain specific proteins, so that the specific binding sites on the surface of the magnetic beads can more efficiently focus on capturing the target analytes; methanol and water avoid the extensive co-precipitation of lipid substances caused by excessive organic solvents; compared with traditional crude precipitation methods (such as methanol or acetonitrile), the method of this invention significantly reduces the co-precipitation of hydrophobic lipid impurities while achieving similar protein precipitation efficiency, thereby avoiding the loss of target analytes due to entrainment.

[0019] Preferably, in the eluent, the volume fraction of the fatty acid methyl ester ethoxylate is 0.01‰~0.05‰, the volume fraction of the acetic acid is 0.05%~0.2%, and the solvent of the eluent is a second methanol aqueous solution, wherein the volume ratio of methanol to water in the second methanol aqueous solution is 1:(5~10).

[0020] In the elution solution provided by this invention, methanol-water can remove hydrophobic impurities such as lipids and proteins; fatty acid methyl ester ethoxylate is used to reduce the interfacial tension between the analyte and the magnetic bead surface to improve the dispersibility and stability of the magnetic beads, thereby increasing dispersibility and reducing the magnetic bead residue of the target analyte; acetic acid is used to remove drug metabolites, acidic lipids and other "moderately polar" or "weakly acidic" coexisting interfering substances.

[0021] Preferably, in the eluent, the concentration of 2,6-di-tert-butyl-p-cresol is 0.1~1 mg / mL, and the volume ratio of acetonitrile to isopropanol is (1~3):1.

[0022] In the technical solution provided by this invention, the role of the eluent is to elute the substances captured and adsorbed by the magnetic beads. Acetonitrile is the eluent for dihydroxyvitamin D metabolites, and isopropanol is used to ensure that the target analytes adsorbed by the magnetic beads are fully released, thereby improving the recovery rate and sensitivity and reducing the magnetic bead residue of the target analytes. 2,6-Di-tert-butyl-p-cresol can make the dihydroxyvitamin D metabolites more stable during the elution process and reduce the oxidative loss of the target analytes.

[0023] Preferably, the sample diluent contains 0.05% to 0.2% acetic acid by volume, and the solvent of the sample diluent is a third methanol aqueous solution, wherein the volume ratio of methanol to water in the third methanol aqueous solution is 1:(5 to 20).

[0024] In the technical solution provided by this invention, acetic acid in the sample diluent is used to maintain the pH of the sample diluent, keeping the test solution in an acidic environment. The sample diluent provided by this invention is compatible with the LC-MS / MS mobile phase for dihydroxyvitamin D metabolites and has the advantages of high sensitivity and symmetrical peak shape. Substances eluted by diluting the eluent with the sample diluent provided by this invention can be directly injected, reducing the oxidative loss of 1,25-DHVD and 24,25-DHVD, and shortening the entire sample pretreatment time to 10 minutes. In traditional methods, after elution, the eluent needs to be further purged with nitrogen, incubated with derivatization reagents, quenched, and then analyzed, with a total time of >1 hour. Compared with traditional methods for processing target analytes, the sample pretreatment time is improved by more than 500%.

[0025] Preferably, the dihydroxyvitamin D metabolite pretreatment kit further includes:

[0026] The calibrators include 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, and 24,25(OH)2D2;

[0027] Quality control materials, including 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, and 24,25(OH)2D2;

[0028] Isotope internal standards, including 1,25(OH)2D3-IS, 1,25(OH)2D2-IS, 24,25(OH)2D3-IS and 24,25(OH)2D2-IS.

[0029] The second aspect of the present invention provides a sample pretreatment method for dihydroxyvitamin D metabolites, specifically, the sample is pretreated using the dihydroxyvitamin D metabolite pretreatment kit described in the first aspect.

[0030] Preferably, the sample preprocessing method includes the following steps:

[0031] S1. Sample loading: Take the sample to be tested, add the isotope internal standard, sample processing solution and magnetic bead working solution in sequence, mix evenly and then perform magnetic separation, retaining the magnetic beads;

[0032] S2, rinsing: Add rinsing solution to the magnetic beads retained in step S1, mix well, and then perform magnetic separation to retain the magnetic beads;

[0033] S3, Elution: Add eluent to the magnetic beads retained in step S2, mix well, perform magnetic separation, and collect the eluent;

[0034] S4. Dilution: Add sample diluent to the eluent collected in step S3, mix well, and obtain the test solution. The pretreatment is now complete.

[0035] Based on the pretreatment kit for dihydroxyvitamin D metabolites provided in the first aspect, if the four dihydroxyvitamin D metabolites extracted in one sample pretreatment are processed by an LC-MS / MS system, the effect of simultaneously extracting and detecting four dihydroxyvitamin D substances in one sample pretreatment can be achieved. This achieves the goals of high throughput, high recovery rate, simple operation, and simplified equipment, which can effectively meet the needs of high-throughput automation in clinical practice.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. This invention employs hydrophilic and lipophilic balanced magnetic beads with a benzofuran ring system on their surface. The benzofuran ring system has a rigid planar structure, providing a larger conjugated area and electron cloud density, resulting in stronger directional π-π stacking and hydrogen bonding. This allows for specific molecular recognition and selective adsorption of the steroidal ring structure in dihydroxyvitamin D metabolites. Simultaneously, the hydrophilic and lipophilic skeleton provides excellent retention capacity. The synergistic effect of these two components enables the simultaneous and efficient extraction of trace amounts of 1,25-DHVD and 24,25-DHVD from serum, significantly enhancing the specific adsorption and enrichment capacity for trace targets, effectively avoiding competitive adsorption of non-target substances, and reducing matrix interference.

[0038] 2. This invention introduces a glycerol-water suspension system into the magnetic bead working solution. By increasing the viscosity of the system with a specific proportion of glycerol, the magnetic beads do not settle over a longer period of time and remain in a uniformly dispersed state. This design completely solves the industry problem that traditional water-based or alcohol-based magnetic bead suspensions are prone to rapid sedimentation during sample loading and incubation in automated instruments, resulting in uneven sample loading between wells or batches. This significantly improves the repeatability and consistency of extraction recovery.

[0039] 3. This invention breaks away from the cumbersome process of traditional detection. The eluted solution is directly added to a sample diluent containing a specific proportion of acetic acid, mixed, and then directly injected for analysis. This method completely eliminates the time-consuming steps required in traditional LC-MS / MS detection, such as nitrogen blowing concentration, incubation with derivatization reagents, and derivatization quenching. This not only avoids column contamination caused by incomplete quenching of derivatization reagents and significantly reduces the oxidation and entrainment loss of trace substances during complex processing, but also shortens the entire sample pretreatment time to within 10 minutes, improving efficiency by more than 500% compared to traditional methods.

[0040] 4. The reagent kit provided by this invention has an extremely low limit of quantitation, a wide linear range, and a stable recovery rate, meeting stringent clinical diagnostic requirements;

[0041] 5. The reagent kit provided by this invention uses magnetic bead extraction technology combined with a fully automated extractor. The entire sample pretreatment process can be completed automatically in a single device without manual shaking, centrifugation, or solution transfer. The programmed extraction process completely eliminates errors caused by manual operation and does not rely on complicated laboratory auxiliary equipment such as positive and negative pressure devices, centrifuges, vortex mixers, nitrogen cylinders, and nitrogen blowing devices. This significantly reduces equipment procurement costs and laboratory space occupation, making it extremely beneficial for conducting high-throughput, standardized routine testing in clinical testing centers. Attached Figure Description

[0042] Figure 1 This is the chromatogram of the isotopic internal standard 1,25(OH)2D3-IS in Example 2 of the present invention;

[0043] Figure 2 This is the chromatogram of 1,25(OH)2D3 in the sample to be tested in Example 2 of the present invention;

[0044] Figure 3 This is the chromatogram of the isotopic internal standard 1,25(OH)2D2-IS in Example 2 of the present invention;

[0045] Figure 4 This is the chromatogram of 1,25(OH)2D2 in the sample to be tested in Example 2 of the present invention;

[0046] Figure 5 This is the chromatogram of the isotopic internal standard 24,25(OH)2D3-IS in Example 2 of the present invention;

[0047] Figure 6 This is the chromatogram of 24,25(OH)2D3 in the sample to be tested in Example 2 of the present invention;

[0048] Figure 7 This is the chromatogram of the isotopic internal standard 24,25(OH)2D2-IS in Example 2 of the present invention;

[0049] Figure 8 This is the chromatogram of 24,25(OH)2D2 in the sample to be tested in Example 2 of the present invention;

[0050] Figure 9 This is the standard curve of 1,25(OH)2D3 in Example 2 of the present invention;

[0051] Figure 10 This is the standard curve of 1,25(OH)2D2 in Example 2 of the present invention;

[0052] Figure 11 This is the standard curve of 24,25(OH)2D3 in Example 2 of the present invention;

[0053] Figure 12 This is the standard curve of 24,25(OH)2D2 in Example 2 of the present invention. Detailed Implementation

[0054] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0055] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0056] As described in the background section, current methods for detecting dihydroxyvitamin D metabolites suffer from drawbacks such as complex pretreatment steps and low stability. Therefore, this invention provides a dihydroxyvitamin D metabolite pretreatment kit, which specifically includes:

[0057] The magnetic bead working solution contains magnetic beads that are hydrophilic-lipophilic balanced magnetic beads with a surface-modified benzofuran ring system.

[0058] The sample processing solution is a zinc sulfate heptahydrate solution.

[0059] The rinsing solution is a solution containing fatty acid methyl ester ethoxylate and acetic acid;

[0060] The eluent is an acetonitrile-isopropanol mixed solution containing 2,6-di-tert-butyl-p-cresol;

[0061] The sample diluent is a solution containing acetic acid.

[0062] In the magnetic bead working solution of the above embodiments, the content of the surface-modified benzofuran ring system hydrophilic-lipophilic balanced magnetic beads is 0.05~2g / mL.

[0063] In the magnetic bead working solution of the above embodiments, the dispersion medium of the magnetic beads is an aqueous glycerol solution, wherein the volume ratio of glycerol to water is 2:3.

[0064] In the sample processing solution of the above embodiments, the concentration of zinc sulfate heptahydrate is 0.01~0.05M, and the solvent of the sample processing solution is a first methanol aqueous solution, wherein the volume ratio of methanol to water in the first methanol aqueous solution is 1:(1~2).

[0065] In the rinsing solution of the above embodiments, the volume fraction of the fatty acid methyl ester ethoxylate is 0.01‰~0.05‰, the volume fraction of the acetic acid is 0.05%~0.2%, and the solvent of the rinsing solution is a second methanol aqueous solution, wherein the volume ratio of methanol to water in the second methanol aqueous solution is 1:(5~10).

[0066] In the eluent of the above embodiments, the concentration of 2,6-di-tert-butyl-p-cresol is 0.1~1 mg / mL, and the volume ratio of acetonitrile to isopropanol is (1~3):1.

[0067] In the sample diluent of the above embodiments, the volume fraction of acetic acid is 0.05%~0.2%, and the solvent of the sample diluent is a third methanol aqueous solution, wherein the volume ratio of methanol to water in the third methanol aqueous solution is 1:(5~20).

[0068] More specifically, the dihydroxyvitamin D metabolite pretreatment kit provided in the specific embodiments of the present invention further includes:

[0069] The calibrators include a mixed solution of 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, and 24,25(OH)2D2;

[0070] Quality control material, wherein the quality control material comprises a mixed solution of 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, and 24,25(OH)2D2;

[0071] Isotope internal standards, including 1,25(OH)2D3-IS, 1,25(OH)2D2-IS, 24,25(OH)2D3-IS and a mixed solution of 24,25(OH)2D2-IS.

[0072] More specifically, in the above embodiments, the concentration of calibrator 1,25(OH)2D2 is preferably 10~700 pg / mL, the concentration of calibrator 1,25(OH)2D3 is preferably 10~700 pg / mL, the concentration of calibrator 24,25(OH)2D3 is preferably 0.1~20 ng / mL, and the concentration of calibrator 24,25(OH)2D2 is preferably 0.1~20 ng / mL.

[0073] More specifically, in the above embodiments, the calibrator can be a solution containing a single target substance or a mixed solution containing multiple target substances.

[0074] More specifically, in the above embodiments, the calibrator is prepared using bovine serum albumin containing sodium ascorbate.

[0075] More specifically, in the above embodiments, the concentration of each calibrator is preferably 3 to 10.

[0076] More specifically, in the above embodiments, the concentration of quality control 1,25(OH)2D2 is preferably 30~120 pg / mL, the concentration of quality control 1,25(OH)2D3 is preferably 30~120 pg / mL, the concentration of quality control 24,25(OH)2D3 is preferably 1~5 ng / mL, and the concentration of quality control 24,25(OH)2D2 is preferably 1~5 ng / mL.

[0077] More specifically, in the above embodiments, the quality control product can be a solution containing a single target substance or a mixed solution containing multiple target substances.

[0078] More specifically, in the above embodiments, the quality control material is prepared using human serum containing sodium ascorbate.

[0079] More specifically, in the above embodiments, the concentration of each quality control product is at least two.

[0080] In the above embodiments, the preferred isotopic internal standard is a mixed solution of 1,25(OH)2D3-IS, 1,25(OH)2D2-IS, 24,25(OH)2D3-IS and 24,25(OH)2D2-IS.

[0081] More specifically, in the above embodiments, the concentration of 1,25(OH)2D3-IS is preferably 300~500 pg / mL, the concentration of 1,25(OH)2D2-IS is preferably 300~500 pg / mL, the concentration of 24,25(OH)2D3-IS is preferably 1~5 ng / mL, and the concentration of 24,25(OH)2D2-IS is preferably 1~5 ng / mL.

[0082] A specific embodiment of the present invention also provides a method for pretreating samples using the aforementioned dihydroxyvitamin D metabolite pretreatment kit, which specifically includes the following steps:

[0083] S1. Sample loading: Take the sample to be tested, add the isotope internal standard, sample processing solution and magnetic bead working solution in sequence, mix evenly and then perform magnetic separation, retaining the magnetic beads;

[0084] S2, rinsing: Add rinsing solution to the magnetic beads retained in step S1, mix well, and then perform magnetic separation to retain the magnetic beads;

[0085] S3, Elution: Add eluent to the magnetic beads retained in step S2, mix well, perform magnetic separation, and collect the eluent;

[0086] S4. Dilution: Add sample diluent to the eluent collected in step S3, mix well, and obtain the test solution. The pretreatment is now complete.

[0087] The kit provided by the specific embodiments of the present invention can simultaneously extract four dihydroxyvitamin D metabolites, achieving rapid and efficient extraction of dihydroxyvitamin D metabolites.

[0088] The technical solutions of the present invention are further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. In some cases, terms with a conventional understanding are limited herein for clarification or ease of reference, and such limitations should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0089] Example 1

[0090] reagent kit composition

[0091] In this embodiment, the pretreatment kit for dihydroxyvitamin D metabolites includes the following components: calibrator, quality control, isotope internal standard, magnetic bead working solution, sample processing solution, rinsing solution, elution solution, sample dilution solution, mobile phase (aqueous phase), and mobile phase (organic phase). The calibrator, quality control, isotope internal standard, and magnetic bead working solution are stored at 2-8°C, while the remaining components can be stored at room temperature.

[0092] The calibrators were prepared as follows: 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3 and 24,25(OH)2D2 standards were prepared into mixed solutions of 6 different concentration levels using bovine serum albumin containing 0.3% sodium ascorbate. The specific concentrations are shown in Table 1.

[0093] Table 1

[0094]

[0095] The preparation method of the quality control product is as follows: human serum was collected, mixed, and 0.3% sodium ascorbate was added. Then, 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3 and 24,25(OH)2D2 standard solutions were added to prepare two serum solutions with different concentration levels. After aliquoting, the solutions were lyophilized and the lyophilized powder was assigned a value. In this embodiment, the concentration of the quality control product is shown in Table 2.

[0096] Table 2

[0097]

[0098] The preparation method of the isotope internal standard is as follows: 1,25(OH)2D3-IS, 1,25(OH)2D2-IS, 24,25(OH)2D3-IS and 24,25(OH)2D2-IS standards are prepared into a mixed solution using an acetonitrile solution containing 0.5 mg / mL BHT (2,6-di-tert-butyl-p-cresol). In this embodiment, the concentration of the isotope internal standard is shown in Table 3.

[0099] Table 3

[0100]

[0101] The magnetic bead working solution is prepared as follows: Take 3 mL of water, 2 mL of glycerol, and 500 mg of hydrophilic and lipophilic magnetic beads with a benzofuran ring system on the surface to prepare the magnetic bead working solution.

[0102] The sample processing solution is prepared as follows: Take 0.575 g of zinc sulfate heptahydrate, 50 mL of methanol, and 50 mL of ultrapure water to prepare the sample processing solution.

[0103] The eluent is prepared as follows: Take 2 μL of fatty acid methyl ester ethoxylate, 50 μL of acetic acid, 10 mL of methanol, and 90 mL of ultrapure water to prepare the eluent.

[0104] The eluent is prepared as follows: Take 50 mL of acetonitrile, 50 mL of isopropanol, and 50 mg of 2,6-di-tert-butyl-p-cresol to prepare the eluent.

[0105] The sample diluent was prepared as follows: Take 0.575 g of zinc sulfate heptahydrate, 50 mL of methanol, and 50 mL of ultrapure water to prepare the sample diluent.

[0106] The preparation method of the mobile phase (aqueous phase) is as follows: Take 150 μL of acetic acid and 500 mL of ultrapure water, filter and degas the mixture to prepare the mobile phase (aqueous phase).

[0107] The preparation method of the mobile phase (organic phase) is as follows: Take 300 μL of acetic acid and 1000 mL of methanol, filter and degas them to prepare the mobile phase (organic phase).

[0108] More specifically, in this embodiment, the components of the kit are shown in Table 4.

[0109] Table 4

[0110]

[0111] Example 2

[0112] Sample pretreatment and LC-MS / MS detection

[0113] In this embodiment, the kit provided in Example 1 was used to process the sample to extract dihydroxyvitamin D metabolites. All steps were performed in a fully automated extractor, and the specific steps are as follows:

[0114] Take 500 μL of the sample to be tested, and add 200 μL of isotope internal standard, 200 μL of sample processing solution, and 20 μL of magnetic bead working solution sequentially. Mix at 1000 rpm for 180 s to separate the magnetic beads and solution. Then, add 200 μL of eluent to the separated magnetic beads, mix at 1000 rpm for 10 s to separate the magnetic beads and solution. Next, add 50 μL of elution buffer to the separated magnetic beads, mix at 1000 rpm for 30 s to separate the magnetic beads and solution. Finally, add 30 μL of sample diluent to the separated solution, mix at 1000 rpm for 30 s to obtain the test solution for LC-MS / MS detection. Specifically, the extraction program of the fully automated extractor is shown in Table 5.

[0115] Table 5

[0116]

[0117] In this embodiment, LC-MS / MS was used to detect the test solution, and the mass spectrometry source parameters are shown in Table 6.

[0118] Table 6

[0119]

[0120] The list of target compounds and isotope internal standards is shown in Table 7.

[0121] Table 7

[0122]

[0123] The chromatographic conditions for liquid chromatography are shown in Table 8.

[0124] Table 8

[0125]

[0126] The liquid phase gradient is shown in Table 9.

[0127] Table 9

[0128]

[0129] The liquid chromatography-tandem mass spectrometry system analysis software automatically plots the calibration curve based on the calibration results. The ratio of the labeled concentration of the calibrator to the concentration of the internal standard is used as the abscissa (x), and the ratio of the actual measured peak area of ​​the calibrator to the peak area of ​​its respective internal standard is used as the ordinate (y). Linear regression is performed using the least squares method to automatically plot the standard curve, and the regression equation can be obtained as: y=ax+b, where y is the ordinate, x is the abscissa, a is the slope, b is the intercept, and the r (correlation coefficient) value is calculated.

[0130] Using the sample pretreatment method and LC-MS / MS detection method described above, four dihydroxyvitamin D metabolites were extracted and detected in a single step. The chromatograms of the isotope internal standards and sample detection results for the four dihydroxyvitamin D metabolites are shown below. Figures 1-8 For the standard curves of the four dihydroxyvitamin D metabolites, please refer to [link / reference needed]. Figures 9-12 .

[0131] in, Figure 1 The internal standard for isotopes 1,25(OH)2D3-IS (1,25(OH)2D3- 13 Chromatogram of C5), Figure 2 The chromatogram of 1,25(OH)2D3 in the sample to be tested is shown. Figure 3 The internal standard for isotopes is 1,25(OH)₂D₂-IS (1,25(OH)₂D₂- 13 Chromatogram of C5); Figure 4 The chromatogram of 1,25(OH)2D2 in the sample to be tested is shown. Figure 5 The internal standard for isotopes 24,25(OH)2D3-IS (24,25(OH)2D3- 13 Chromatogram of C5); Figure 6 The chromatogram of 24,25(OH)2D3 in the sample to be tested is shown. Figure 7 The chromatogram of the isotopic internal standard 24,25(OH)2D2-IS (24,25(OH)2D2-d3); Figure 8 The chromatogram of 24,25(OH)2D2 in the sample to be tested is shown.

[0132] Figure 9 The standard curve for 1,25(OH)2D3; Figure 10 The standard curve for 1,25(OH)2D2; Figure 11 The standard curve for 24,25(OH)2D3; Figure 12 The standard curve for 24,25(OH)2D2.

[0133] The linear relationships of the four dihydroxyvitamin D metabolites were analyzed. For 1,25(OH)₂D₃ and 1,25(OH)₂D₂ in the range of 15–600 pg / mL, and for 2,4,25(OH)₂D₃ and 2,4,25(OH)₂D₂ in the range of 0.3–12 ng / mL, the linear correlation coefficient (r) should be no less than 0.9900. The validation results of the standard curves for the four dihydroxyvitamin D metabolites are shown in Table 10.

[0134] Table 10

[0135]

[0136] As shown in Table 10, the four dihydroxyvitamin D metabolites exhibit good linearity within the linear concentration range. The kit provided in this embodiment has a wide linear range and a lower limit of quantitation, which is higher than the linear range of current technologies and meets the needs of clinical diagnosis.

[0137] Example 3

[0138] Reagent kit performance validation

[0139] In this embodiment, the performance verification of the reagent kit specifically includes accuracy, precision, limit of quantitation, and accuracy of quality control materials.

[0140] Specifically, the accuracy of the test results is expressed as recovery rate (%). According to the industry consensus on in vitro diagnostic reagents, the recovery rate should be between 85.0% and 115.0%.

[0141] Specifically, the accuracy determination method is as follows: within the linear range of the kit, standard solutions of low, medium and high concentrations are added to serum to prepare spiked samples of low, medium and high concentrations respectively, and the theoretical spiked concentration of each analyte at each concentration is calculated.

[0142] The method for preparing low-concentration spiked samples is as follows: Take 80 μL of low-concentration standard solution, add 2920 μL of serum sample, vortex at speed 10 for 20 min to prepare low-concentration spiked samples.

[0143] The preparation method for medium-concentration spiked samples is as follows: Take 80 μL of medium-concentration standard solution, add 2920 μL of serum sample, vortex at speed 10 for 20 min to prepare medium-concentration spiked samples;

[0144] The method for preparing high-concentration spiked samples is as follows: Take 100 μL of high-concentration standard solution, add 2900 μL of serum sample, vortex at speed 10 for 20 min to prepare high-concentration spiked samples.

[0145] The test results are shown in Table 11.

[0146] Table 11

[0147]

[0148] As shown in Table 11, for the kit provided in Example 1, the spiked recoveries of all analytes were between 85.0% and 115.0%, which met the kit quality standards.

[0149] Specifically, for precision, the main focus is on repeatability and batch-to-batch precision. For repeatability, the coefficient of variation (CV) should not exceed 10.0%; for batch-to-batch precision, the coefficient of variation (CV) should not exceed 10.0%.

[0150] In this embodiment, three different batches of reagent kits were used to test quality control samples at two concentration levels, low and high. Ten replicates were performed for each batch and each concentration. Intra-batch, inter-batch, and total CV were calculated. The test results are shown in Table 12.

[0151] Table 12

[0152]

[0153] As shown in Table 12, for the kit provided by this invention, the intra-batch and inter-batch CVs of all analytes are less than 10%, and the precision meets the kit quality standards.

[0154] Specifically, regarding the limits of quantitation (LOQ), based on the detection results of Example 2, the LQ for 1,25(OH)₂D₃ and 1,25(OH)₂D₂ should be 15 pg / mL, and the LQ for 24,25(OH)₂D₃ and 24,25(OH)₂D₂ should be 0.3 ng / mL. For samples with concentrations of 1,25(OH)₂D₃ and 1,25(OH)₂D₂ within the range of (15.0 ± 3.0) pg / mL, and concentrations of 24,25(OH)₂D₃ and 24,25(OH)₂D₂ within the range of (0.30 ± 0.06) ng / mL, the relative deviation between the measured values ​​and the theoretical values ​​should not exceed ±15.0%, and the coefficient of variation (CV) should not be greater than 20.0%.

[0155] The specific detection results for the limit of quantitation are shown in Table 13.

[0156] Table 13

[0157]

[0158] As shown in Table 13, the relative deviation of the quantitation limits for all analytes was less than 15%, and the coefficient of variation (CV) was less than 20%, indicating that the quantitation limits met the quality standards of the kit.

[0159] Specifically, to assess the accuracy of the quality control products, one set of our quality control products was tested using the matching reagent kit. Three tests were repeated for each concentration, and the average value was calculated. The relative deviation should not exceed ±20.0%. The specific test results are shown in Table 14.

[0160] Table 14

[0161]

[0162] As shown in Table 14, the relative deviation between the detected values ​​and the target values ​​of all analytical material controls is less than 20%, and the accuracy of the quality control products meets the quality standards of the kit.

[0163] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A pretreatment kit for dihydroxyvitamin D metabolites, characterized in that, include: The magnetic bead working solution contains magnetic beads that are hydrophilic-lipophilic balanced magnetic beads with a surface-modified benzofuran ring system. The sample processing solution is a zinc sulfate heptahydrate solution. The rinsing solution is a solution containing fatty acid methyl ester ethoxylate and acetic acid; The eluent is an acetonitrile-isopropanol mixed solution containing 2,6-di-tert-butyl-p-cresol; The sample diluent is a solution containing acetic acid.

2. The pretreatment kit for dihydroxyvitamin D metabolites as described in claim 1, characterized in that, In the magnetic bead working solution, the content of the surface-modified benzofuran ring system hydrophilic-lipophilic balanced magnetic beads is 0.05~2g / mL.

3. The pretreatment kit for dihydroxyvitamin D metabolites as described in claim 1, characterized in that, In the working solution of the magnetic beads, the dispersion medium of the magnetic beads is an aqueous solution of glycerol, wherein the volume ratio of glycerol to water is 2:

3.

4. The pretreatment kit for dihydroxyvitamin D metabolites as described in claim 1, characterized in that, In the sample processing solution, the concentration of zinc sulfate heptahydrate is 0.01~0.05M, and the solvent of the sample processing solution is a first methanol aqueous solution, in which the volume ratio of methanol to water is 1:(1~2).

5. The pretreatment kit for dihydroxyvitamin D metabolites as described in claim 1, characterized in that, In the eluent, the volume fraction of the fatty acid methyl ester ethoxylate is 0.01‰~0.05‰, the volume fraction of the acetic acid is 0.05%~0.2%, and the solvent of the eluent is a second methanol aqueous solution, wherein the volume ratio of methanol to water in the second methanol aqueous solution is 1:(5~10).

6. The pretreatment kit for dihydroxyvitamin D metabolites as described in claim 1, characterized in that, In the eluent, the concentration of 2,6-di-tert-butyl-p-cresol is 0.1~1 mg / mL, and the volume ratio of acetonitrile to isopropanol is (1~3):

1.

7. The pretreatment kit for dihydroxyvitamin D metabolites as described in claim 1, characterized in that, The sample diluent contains acetic acid at a volume fraction of 0.05% to 0.2%, and the solvent is a third methanol aqueous solution with a volume ratio of methanol to water of 1:(5 to 20).

8. The pretreatment kit for dihydroxyvitamin D metabolites as described in claim 1, characterized in that, The dihydroxyvitamin D metabolite pretreatment kit also includes: The calibrators include 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, and 24,25(OH)2D2; Quality control materials, including 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, and 24,25(OH)2D2; Isotope internal standards, including 1,25(OH)2D3-IS, 1,25(OH)2D2-IS, 24,25(OH)2D3-IS, and 24,25(OH)2D2-IS.

9. A sample pretreatment method for dihydroxyvitamin D metabolites, characterized in that, The sample was pretreated using the dihydroxyvitamin D metabolite pretreatment kit described in any one of claims 1 to 8.

10. The sample pretreatment method as described in claim 9, characterized in that, Includes the following steps: S1. Sample loading: Take the sample to be tested, add the isotope internal standard, sample processing solution and magnetic bead working solution in sequence, mix evenly and then perform magnetic separation, retaining the magnetic beads; S2, rinsing: Add rinsing solution to the magnetic beads retained in step S1, mix well, and then perform magnetic separation to retain the magnetic beads; S3, Elution: Add eluent to the magnetic beads retained in step S2, mix well, perform magnetic separation, and collect the eluent; S4. Dilution: Add sample diluent to the eluent collected in step S3, mix well, and obtain the test solution. The pretreatment is now complete.

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