Method for detecting 25-hydroxy vitamin D and 1, 25-dihydroxy vitamin D and application thereof

Abstract

The invention relates to a method for detecting 25-hydroxy vitamin D and 1, 25-dihydroxy vitamin D and application thereof. The method comprises the following steps: pretreating a sample to be detected, carrying out derivatization reaction, and detecting by adopting ultra-high performance liquid chromatography-tandem mass spectrometry, the reagent for the derivatization reaction comprises 4-phenyl-1, 2, 3-triazoline-3, 5-diketone and benzylamine, and the reagent for the derivatization reaction comprises 4-phenyl-1, 2, 3-triazoline-3, 5-diketone and benzylamine. According to the invention, 4-phenyl-1, 2, 3-triazoline-3, 5-diketone and benzylamine are adopted for combined derivatization, and a nitrogen-containing group is introduced through imine bond formation, so that the ESI < + > ionization efficiency is remarkably improved, and the signal intensity is improved by 5-10 times compared with silanization.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D and an application thereof. Background Art

[0002] Vitamin D is an essential fat-soluble vitamin for humans. In addition to regulating calcium and phosphorus metabolism and maintaining bone health, it also participates in tissue cell differentiation, proliferation, and immune regulation. Vitamin D is converted into metabolites in the liver, 25-hydroxyvitamin D1 and 1,25-dihydroxyvitamin D. These metabolites reflect vitamin D levels in the body and are important indicators for assessing vitamin D levels. They are crucial for the diagnosis of osteoporosis, kidney disease, and endocrine diseases. Current detection methods include immunoassays, competitive protein binding assays, or high-performance liquid chromatography. Immunoassays rely on antibody specificity and are susceptible to interference from structural analogs (e.g., vitamin D2 / D3 cross-reactivity). They also have low sensitivity (detection limits are typically in the ng range) and are unable to distinguish between 25-OH D2 and 25-OH D3, or between 1,25(OH)2D2 and 1,25(OH)2D3.

[0003] In existing mass spectrometry, vitamin D metabolites are often detected using silanization or pre-column fluorescence derivatization. Silylation derivatization reagents are susceptible to hydrolysis, resulting in instability in the derivatized products and the need for strict control of reaction conditions. Fluorescence derivatization has limited sensitivity and requires specific wavelength excitation, making it unsuitable for samples with complex matrices. Direct mass spectrometry detection without derivatization results in low ion response, particularly for dihydroxy compounds, and poor signal-to-noise ratios, making it difficult to meet clinical trace detection requirements.

[0004] Currently available technologies have limited detection limits for low-concentration metabolites that meet clinical needs. Immunoassays cannot distinguish between D2 and D3 isomers, chromatographic methods require long columns for separation, and are time-consuming. Existing derivatization methods require multiple steps (such as silanization, which requires pyridine catalysis and high temperatures), which can easily introduce errors and are unsuitable for batch sample testing. Therefore, a rapid, efficient, and highly sensitive method for detecting vitamin D metabolites is urgently needed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D and its application. In the present invention, the test sample 4-phenyl-1,2,3-triazolin-3,5-dione (PTAD) and benzylamine are jointly derivatized to significantly improve the specificity of detection, shorten the detection time, and can be used for batch detection.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, wherein the sample to be tested is subjected to a derivatization reaction and then detected by ultra-high performance liquid chromatography tandem mass spectrometry; the reagents for the derivatization reaction include 4-phenyl-1,2,3-triazolin-3,5-dione and benzylamine.

[0008] In this invention, a new derivatization strategy is designed for the detection of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. 4-phenyl-1,2,3-triazolin-3,5-dione and benzylamine are used for combined derivatization. Nitrogen-containing groups are introduced through imine bond formation, which significantly improves the ESI + The ionization efficiency is 5-10 times higher than that of silanization. The traditional silanization reaction takes 2 hours, while the method of the present invention uses two reagents to synergistically complete the hydroxyl derivatization in one step, shortening the reaction time to 10 minutes.

[0009] The detection method of the present invention can effectively separate vitamin D2 / D3 isomers, with a separation degree greater than 1.5 and a retention time difference greater than 0.3 minutes.

[0010] Preferably, the derivatization reaction comprises adding 4-phenyl-1,2,3-triazolin-3,5-dione and benzylamine to the sample to be tested, incubating, blowing dry with nitrogen, and re-dissolving with a solvent to prepare a test sample.

[0011] Preferably, the concentration of 4-phenyl-1,2,3-triazolin-3,5-dione is 50-100 mM, for example, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM.

[0012] Preferably, the concentration of benzylamine is 50-100 mM, for example, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM.

[0013] Preferably, the volume ratio of 4-phenyl-1,2,3-triazolin-3,5-dione to benzylamine is 1:(0.5-2). Said (0.5-2) may be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8 or 2.

[0014] Preferably, the incubation temperature is 55-65°C, and the incubation time is 5-15 minutes, for example, 55°C, 56°C, 58°C, 60°C, 62°C, 64°C, or 65°C. The 5-15 minutes can be, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes.

[0015] Preferably, the solvent includes mobile phase A and mobile phase B of the ultra-high performance liquid chromatography tandem mass spectrometry.

[0016] Preferably, the volume ratio of mobile phase A to mobile phase B in the solvent is 1:(0.5-2). Said (0.5-2) can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8 or 2.

[0017] Preferably, the pretreatment comprises adding an internal standard to the sample to be tested, precipitating the protein with a precipitant, mixing and then centrifuging, and taking the supernatant and drying it with nitrogen.

[0018] Preferably, the internal standard includes deuterated 25-hydroxyvitamin D2, deuterated 25-hydroxyvitamin D3, deuterated 1,25-dihydroxyvitamin D2 and deuterated 1,25-dihydroxyvitamin D3.

[0019] The present invention adopts deuterated internal standard for correction, synchronously corrects derivatization efficiency and matrix effect, the recovery rate reaches 90%-110%, and the intra-batch variation coefficient is less than 5%.

[0020] Preferably, the concentration of deuterated 25-hydroxyvitamin D2 is 5-15 ng / mL, the concentration of deuterated 25-hydroxyvitamin D3 is 5-15 ng / mL, the concentration of deuterated 1,25-dihydroxyvitamin D2 is 5-15 ng / mL, and the concentration of deuterated 1,25-dihydroxyvitamin D3 is 5-15 ng / mL. The concentration of deuterated 25-hydroxyvitamin D2 is 5-15 ng / mL, for example, it can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL. The concentration of the deuterated 25-hydroxyvitamin D3 is 5-15 ng / mL, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, or 15 ng / mL. The concentration of the deuterated 1,25-dihydroxyvitamin D2 is 5-15 ng / mL, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, or 15 ng / mL. The concentration of the deuterated 1,25-dihydroxyvitamin D3 is 5-15 ng / mL, for example, it can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL, etc.

[0021] Preferably, the volume ratio of the sample to be tested, the internal standard, and the precipitant is (5-15):1:(30-50). The (5-15) may be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The (30-50) may be, for example, 30, 35, 40, 45, or 50.

[0022] Preferably, the sample to be tested includes serum and / or blood serum.

[0023] Preferably, the precipitant comprises methanol and / or acetonitrile.

[0024] Preferably, the centrifugation speed is 10,000-15,000 rpm, and the time is 5-15 min. The 10,000-15,000 rpm may be, for example, 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, or 15,000 rpm. The 5-15 min may be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min.

[0025] Preferably, the chromatographic column of the ultra-high performance liquid chromatography comprises a C18 column (BEH C18, 1.7 um, 2.1 mm*100 mm);

[0026] Preferably, the mobile phase A of the ultra-high performance liquid chromatography is a 0.05-0.3% formic acid aqueous solution, and the mobile phase B is a 0.05-0.3% formic acid acetonitrile solution. The 0.05-0.3% can be, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.25% or 0.3%.

[0027] Preferably, the flow rate of the ultra-high performance liquid chromatography is 0.1-0.5 mL / min, for example, 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min.

[0028] Preferably, the column temperature of the ultra-high performance liquid chromatography is 35-45° C. For example, it can be 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C. or 45° C.

[0029] Preferably, the elution procedure of the ultra-high performance liquid chromatography is:

[0030] 0-1 min, (3-8)% mobile phase B;

[0031] 1-1.5 min, (3-8)% average rate change to (92-98)% mobile phase B;

[0032] 2.5-3 min, (92-98)% mobile phase B;

[0033] 3-3.1 min, (92-98)% average rate change to (3-8)% mobile phase B;

[0034] 3.1-4 min, (3-8)% mobile phase B.

[0035] The (3-8)% may be, for example, 3%, 4%, 5%, 6%, 7% or 8%, etc. The (92-98)% may be, for example, 92%, 93%, 94%, 95%, 96%, 97% or 98%, etc.

[0036] Preferably, the method further comprises preparing a standard curve.

[0037] Preferably, the standard curve concentration range of 25-hydroxyvitamin D2 is 0.01-100 ng / mL, for example, 0.01 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL or 100 ng / mL.

[0038] Preferably, the standard curve concentration range of 25-hydroxyvitamin D3 is 0.01-100 ng / mL, for example, 0.01 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL or 100 ng / mL.

[0039] Preferably, the standard curve concentration range of 1,25-dihydroxyvitamin D2 is 0.5-200 pg / mL, for example, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 150 pg / mL or 200 pg / mL.

[0040] Preferably, the standard curve concentration range of 1,25-dihydroxyvitamin D2 is 0.5-200 pg / mL, for example, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 150 pg / mL or 200 pg / mL.

[0041] In a second aspect, the present invention provides an application of the method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to the first aspect in detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

[0042] Compared with the prior art, the present invention has at least the following beneficial effects:

[0043] 1. The present invention adopts PTAD-benzylamine combined derivatization to introduce nitrogen-containing groups through imine bond formation, which significantly improves ESI + The ionization efficiency significantly improves the detection signal intensity and enhances the detection accuracy. The dual reagents work together to complete the hydroxyl derivatization in one step, greatly shortening the detection time and can be used for batch detection.

[0044] 2. The method of the present invention can effectively detect the D2 / D3 isomers of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, using multiple reaction monitoring (MRM) to simultaneously detect four targets and the internal standard. A single run takes only 3-5 minutes. In addition, deuterated internal standard correction is used to simultaneously correct for derivatization efficiency and matrix effects, significantly reducing the intra-batch coefficient of variation. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the TIC spectrum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0046] Figure 2 This is the TIC spectrum of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3.

[0047] Figure 3 The mass spectra of 25-hydroxyvitamin D3 before and after derivatization.

[0048] Figure 4 The mass spectra of 1,25-dihydroxyvitamin D3 before and after derivatization.

[0049] Figure 5 This is the standard curve of 1,25-dihydroxyvitamin D2.

[0050] Figure 6 This is the standard curve of 1,25-dihydroxyvitamin D3.

[0051] Figure 7 This is the standard curve of 25-hydroxyvitamin D2.

[0052] Figure 8 This is the standard curve of 25-hydroxyvitamin D3. DETAILED DESCRIPTION

[0053] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0054] Example 1

[0055] This example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D

[0056] To 100 μL of blank serum, add 10 ng / mL 25-hydroxyvitamin D2, 10 ng / mL 25-hydroxyvitamin D3, 10 ng / mL 1,25-dihydroxyvitamin D2, and 10 ng / mL 1,25-dihydroxyvitamin D3, then add 10 μL of deuterated 25-hydroxyvitamin D (25-OH D2-d3; 25-OH D3-d3) and deuterated 1,25-dihydroxyvitamin D (1,25-(OH)2D2-d3; 1,25-(OH)2D3-d3) (mixed internal standard, a total of 10 μL), add 400 μL of methanol to precipitate protein, vortex for 1 min, centrifuge at 13,000 rpm for 10 min, and take the supernatant and blow dry with nitrogen.

[0057] Add 50 μL of 50 mM 4-phenyl-1,2,3-triazolin-3,5-dione and 50 μL of 100 mM benzylamine to the dried residue, incubate at 60°C for 10 min, and blow dry with nitrogen. Reconstitute with mobile phase A and B in a 1:1 volume ratio, and inject for detection.

[0058] The detection conditions for ultra-high performance liquid chromatography were as follows: BEH C18 column, 1.7 μm, 2.1 mm x 100 mm; flow rate, 0.3 mL / min; column temperature, 40°C; mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in acetonitrile. The elution procedure for ultra-high performance liquid chromatography was as follows:

[0059] 0-1 min, 5% mobile phase B;

[0060] 1-2.5 min, 5% average rate change to 95% mobile phase B;

[0061] 2.5-3 min, 95% mobile phase B;

[0062] 3-3.1 min, 95% average rate change to 5% mobile phase B;

[0063] 3.1-4 min, 5% mobile phase B.

[0064] The mass spectrometry conditions were: spray voltage 4500 V, sheath gas pressure 40 psi, booster pressure 15 psi, ion transfer tube temperature 300°C, inlet voltage 8 V, and collision outlet voltage 10 V. The ion parameters in the mass spectrum are shown in Table 1.

[0065] Table 1

[0066]

[0067]

[0068] Example 2

[0069] In this example, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were detected by adding 15 ng / mL 25-hydroxyvitamin D2, 15 ng / mL 25-hydroxyvitamin D3, 15 ng / mL 1,25-dihydroxyvitamin D2, and 15 ng / mL 1,25-dihydroxyvitamin D3 to 150 μL of blank serum, and then adding 10 μL of deuterated 25-hydroxyvitamin D (25-OH D2-d3; 25-OH D3-d3) and deuterated 1,25-dihydroxyvitamin D (1,25-(OH)2D2-d3; 1,25-(OH)2D3-d3) (mixed internal standard, a total of 10 μL), adding 500 μL of methanol to precipitate protein, vortexing for 1 min, centrifuging at 15,000 rpm for 5 min, and taking the supernatant and blowing it dry with nitrogen.

[0070] 100 μL of 100 mM 4-phenyl-1,2,3-triazolin-3,5-dione and 50 μL of 50 mM benzylamine were added to the dry residue, and the mixture was reconstituted with a mobile phase A and B solvent with a volume ratio of 1:0.5, and injected for detection.

[0071] Ultra-high performance liquid chromatography (UPLC) detection conditions were as follows: C18 column (BEH C18, 1.7 μm, 2.1 mm x 100 mm); flow rate 0.1 mL / min; column temperature 45°C; mobile phase A 0.3% formic acid in water; mobile phase B 0.05% formic acid in acetonitrile. The UPLC elution procedure was:

[0072] 0-1 min, 5% mobile phase B;

[0073] 1-2.5 min, 5% average rate change to 95% mobile phase B;

[0074] 2.5-3 min, 95% mobile phase B;

[0075] 3-3.1 min, 95% average rate change to 5% mobile phase B;

[0076] 3.1-4 min, 5% mobile phase B.

[0077] The mass spectrometry conditions were the same as those in Example 1.

[0078] Example 3

[0079] This example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D

[0080] To 50 μL of blank serum, add 5 ng / mL 25-hydroxyvitamin D2, XX ng / mL 25-hydroxyvitamin D3, 5 ng / mL 1,25-dihydroxyvitamin D2, and XX ng / mL 1,25-dihydroxyvitamin D3. Then, add 10 μL of deuterated 25-hydroxyvitamin D (25-OH D2-d3; 25-OH D3-d3) and deuterated 1,25-dihydroxyvitamin D (1,25-(OH)2D2-d3; 1,25-(OH)2D3-d3) (mixed internal standard, total 10 μL). Add 300 μL of methanol to precipitate protein, vortex for 1 min, centrifuge at 10,000 rpm for 15 min, and remove the supernatant and blow dry with nitrogen.

[0081] Add 50 μL of 50 mM 4-phenyl-1,2,3-triazolin-3,5-dione and 100 μL of 50 mM benzylamine to the dried residue. Reconstitute with mobile phase A and B in a 1:2 volume ratio and inject for detection.

[0082] The detection conditions of ultra-high performance liquid chromatography were as follows: C18 column; flow rate 0.5 mL / min, column temperature 35°C, mobile phase A 0.05% formic acid in water, mobile phase B 0.3% formic acid in acetonitrile. The elution procedure of ultra-high performance liquid chromatography was:

[0083] 0-1 min, 5% mobile phase B;

[0084] 1-2.5 min, 5% average rate change to 95% mobile phase B;

[0085] 2.5-3 min, 95% mobile phase B;

[0086] 3-3.1 min, 95% average rate change to 5% mobile phase B;

[0087] 3.1-4 min, 5% mobile phase B.

[0088] The mass spectrometry conditions were the same as those in Example 1.

[0089] Example 4

[0090] This example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The only difference from Example 1 is that the concentration of 4-phenyl-1,2,3-triazolin-3,5-dione is 25 mM, and the rest is consistent with Example 1.

[0091] Example 5

[0092] This example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The only difference from Example 1 is that the concentration of benzylamine is 200 mM, and the rest is consistent with Example 1.

[0093] Example 6

[0094] This example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The only difference from Example 1 is that the ultra-high performance liquid chromatography elution procedure used in this example is:

[0095] 0-1 min, 5% mobile phase B;

[0096] 1-4 min, 5% average rate change to 95% mobile phase B;

[0097] 4-5 min, 95% mobile phase B;

[0098] 5-5.1 min, 95% average rate change to 5% mobile phase B;

[0099] 5.1-6min, 5% mobile phase B. The rest is the same as in Example 1

[0100] Example 7

[0101] This example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The only difference between this example and Example 1 is that mobile phase A is a methanol solution and mobile phase B is a formic acid solution. The rest is the same as Example 1.

[0102] Example 8

[0103] This example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The only difference from Example 1 is that the re-dissolution solvent is adjusted to acetonitrile, and the rest is the same as Example 1.

[0104] Example 9

[0105] This example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The only difference from Example 1 is that no internal standard is added. The rest is the same as Example 1.

[0106] Comparative Example 1

[0107] This comparative example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The only difference between this comparative example and Example 1 is that benzylamine is not added, and the rest is the same as Example 1.

[0108] Comparative Example 2

[0109] This comparative example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The only difference between this comparative example and Example 1 is that 4-phenyl-1,2,3-triazolin-3,5-dione is not added, and the rest is consistent with Example 1.

[0110] Comparative Example 3

[0111] This comparative example detects 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. The difference between this comparative example and Example 1 is that this example adopts traditional silanization detection. The specific steps are as follows:

[0112] Sample pretreatment was consistent with Example 1. 10 ng / mL 25-hydroxyvitamin D2, XX ng / mL 25-hydroxyvitamin D3, 10 ng / mL 1,25-dihydroxyvitamin D2, and XX ng / mL 1,25-dihydroxyvitamin D3 were added to 100 μL of blank serum. 10 μL of deuterated 25-hydroxyvitamin D (25-OH D2-d3; 25-OH D3-d3) and deuterated 1,25-dihydroxyvitamin D (1,25-(OH)2D2-d3; 1,25-(OH)2D3-d3) were then added. 400 μL of methanol was added to precipitate protein, the sample was vortexed for 1 min, and the sample was centrifuged at 13,000 rpm for 10 min. The supernatant was taken and blown dry with nitrogen.

[0113] After silylation derivatization, 50 μL of pyridine (as a catalyst) and 50 μL of BSTFA (containing 1% TMCS) were added to the dried residue and incubated at 70°C for 2 hours. Excess reagents were removed by nitrogen purge. For reconstitution and injection, the residue was reconstituted in 100 μL of acetonitrile (non-mobile phase mixed solvent). The sample was injected and analyzed by UPLC-MS / MS, using the same chromatographic and mass spectrometric conditions as in Example 1.

[0114] Test Example 1

[0115] This test example detects the mass spectrum intensity

[0116] This test example adopts the method of the above embodiment and comparative example to detect and detect the mass spectrometry signal intensity. The specific results are shown in Table 2. Table 2 is a comparison table of mass spectrometry signal intensity after derivatization (peak area × 10 6 ).like Figure 1 As shown, [M+H] + The ionic strength is the highest, which is increased by 8 times and 12 times compared with single reagent derivatization.

[0117] Table 2

[0118]

[0119]

[0120] (1) The results of Examples 1-3 show that the PTAD-benzylamine combined derivatization introduces nitrogen-containing groups through imine bonds, significantly improving ESI + Ionization efficiency. Test results are as follows Figure 1 and Figure 2 As shown, the signal intensity of 25-OH D2 TIC is 5.87×106 The signal intensity of 25-OH D3 TIC was 6.08×10 6 The signal intensity of 1,25-(OH)2D2 in TIC was 8.33×10 4 The signal intensity of 1,25-(OH)2D3 TIC was 4.94×10 4 .

[0121] pass Figure 3 and Figure 4 Compared with the baseline without derivatization, the signal intensity of 25-OH D3 before derivatization was 2.88×10 5 The signal intensity after derivatization was 3.53×10 6 The signal intensity after derivatization increased by about 12 times. The signal intensity before 1,25-(OH)2D3 derivatization was 7.2×10 4 The signal intensity before 1,25-(OH)2D3 derivatization was 5.68×10 5 After derivatization, the signal intensity increased by about 8 times.

[0122] (2) By comparing Example 1 with Examples 4-5, it can be seen that when the concentrations of PTAD and benzylamine are outside the range of the present invention, the detected signal intensity decreases by 60-70%.

[0123] (3) By comparing Example 1 with Examples 6-7, it can be seen that the use of other elution procedures leads to insufficient separation and a 40% reduction in signal intensity. The use of other mobile phases will cause the signal intensity to plummet by 85% due to an imbalance in solvent polarity.

[0124] (4) Comparison of Example 1 with Examples 8-9 shows that the signal intensity decreased by 80% when reconstituted with pure acetonitrile due to the incompatibility of the solvent with the mobile phase. Without the use of an internal standard, the signal fluctuation increased due to uncorrected matrix effects, with the CV% rising to 15%.

[0125] (5) By comparing Example 1 with Comparative Examples 1-2, it can be seen that using only PTAD or benzylamine will result in a signal intensity of only 15-20% of that of the dual reagent, proving that the combination of PTAD and benzylamine can significantly improve the detection signal intensity.

[0126] (6) By comparing Example 1 with Comparative Example 3, it can be seen that the traditional silanization method has low derivatization efficiency and poor stability, and the signal intensity is the lowest, only 10% of that in Example 1. The traditional silanization method requires the addition of a catalyst (pyridine) and the reaction time is as long as 2 hours (the present invention only requires 10 minutes), which is not suitable for batch samples. Figure 1 In contrast, the signal intensity of 25-OHD3 after silanization is only 10% of that of the PTAD-benzylamine method.

[0127] Test Example 2

[0128] This test case verifies the recovery and precision

[0129] 1 ng / mL 25-OH VD3 and 0.1 pg / mL 1,25-(OH)2D3 were added to blank human serum. Each experiment was repeated 6 times. The samples were processed according to the methods of Examples 1-9 and Comparative Examples 1-3, respectively.

[0130] Preparation of standard curve: The concentration gradient of 25-OH VD3 was 0.01 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL. The concentration gradient of 25-OH VD2 was 0.01 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL. The concentration gradient of 1,25-(OH)2D3 was 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, and 200 pg / mL. The concentration gradient of the standard curve of 1,25-(OH)2D2 was 0.5pg / mL, 1pg / mL, 5pg / mL, 10pg / mL, 50pg / mL, 100pg / mL and 200pg / mL. A linear regression was performed using the peak area (y) versus the concentration (x) with a weighting factor of 1 / x. The standard curves of 25-OH VD3 and 1,25-(OH)2D3 are shown in the figure below. Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown in the figure, the linear range of the 25-OH VD3 standard curve is 0.01-100 ng / mL, r 2 is 0.9993, the linear range of 1,25-(OH)2D3 is 0.5-200 pg / mL, r 2 The linear range of the 25-OH VD2 standard curve was 0.01-100 ng / mL, r 2 is 0.9991, the linear range of 1,25-(OH)2D2 is 0.5-200 pg / mL, r 2 The recovery rate and coefficient of variation were calculated using the formula: Recovery rate (%) = (spike concentration / measured concentration) × 100%. The specific results are as follows: Table 3 compares the recovery rate and precision of 25-OH VD3, and Table 4 compares the recovery rate and precision of 1,25-(OH)2D3.

[0131] Table 3

[0132] Group Mean observed concentration (ng / mL) Standard deviation Recovery (%) CV (%) Example 1 0.952 ±0.031 95.2 3.1 Example 2 0.938 ±0.035 93.8 3.7 Example 3 0.945 ±0.038 94.5 4.0 Example 4 0.810 ±0.068 81.0 8.4 Example 5 0.825 ±0.072 82.5 8.7 Example 6 0.880 ±0.050 88.0 5.7 Example 7 0.650 ±0.085 65.0 13.1 Example 8 0.720 ±0.078 72.0 10.8 Example 9 0.920 ±0.110 92.0 12.0 Comparative Example 1 0.750 ±0.090 75.0 12.0 Comparative Example 2 0.680 ±0.095 68.0 14.0 Comparative Example 3 0.580 ±0.105 58.0 18.1

[0133] Table 4

[0134]

[0135]

[0136] The results show that:

[0137] (1) By comparing Examples 1-3, it can be seen that within the scope of the present invention, the recovery rate is 93.8%-98.7%, and the precision CV is <4%, which is the best effect. This shows that the synergistic derivatization of PTAD and benzylamine improves the ionization efficiency and the deuterated internal standard effectively corrects the matrix effect.

[0138] (2) By comparing Example 1 with Examples 4-5, it can be seen that when the reagent concentration is outside the range, the recovery rate will drop to 81%-83.5% due to incomplete derivatization.

[0139] (3) By comparing Example 1 with Examples 7-8, it can be seen that worse mobile phase and solvent will lead to a decrease in chromatographic separation effect, causing the recovery rate to plummet to 62%-70%.

[0140] (4) By comparing Example 1 with Example 9, it can be seen that the lack of internal standard correction causes the coefficient of variation to increase to 12%-13.3%, significantly reducing the accuracy and precision of the detection.

[0141] (5) Comparison of Example 1 and Comparative Example 3 shows that the conventional silanization method significantly reduces the recovery rate and increases the coefficient of variation to the highest level due to the unstable derivatization efficiency. Furthermore, the matrix effect is corrected due to the unstable derivatization efficiency and the lack of an internal standard.

[0142] Test Example 3

[0143] Comparison of clinical sample testing in this test case

[0144] Thirty clinical serum samples (containing various vitamin D levels, such as deficiency, sufficiency, and excess) were tested. Two experiments were conducted: an experimental group and a control group. The experimental group was tested using the method in Example 1, while the control group was tested using ELISA kits (25-OH Vitamin D Assay Kit: Manufacturer: DIAsource Immunoassays SA, Catalog No.: KAP1971, Detection Limit: 25-OH D > 1 ng / mL; 1,25-(OH)2 Vitamin D Assay Kit: Manufacturer: Immunodiagnostic Systems (IDS), Catalog No.: AC-57F1, Detection Range: 5–200 pg / mL). 25-OH D2, 25-OH D3, 1,25-(OH)2D2, and 1,25-(OH)2D3 were simultaneously tested using the above method. The specific test results are shown in Table 5.

[0145] Table 5

[0146]

[0147] From the above results we can see that:

[0148] The ELISA detected only 73.3% of 25-OH D samples (missing 8 samples with concentrations <1 ng / mL), while the method in Example 1 detected all samples (minimum 25-OH D2 level 5.2 ng / mL). The ELISA did not detect 1,25-(OH)2D at all (due to insufficient detection limit), while the method in Example 1 detected all samples (minimum 1,25-(OH)2D2 level 3.5 pg / mL).

[0149] The method of Example 1 can achieve baseline separation, such as Figure 3 As shown, although the retention time difference between 25-OH D2 and D3 is minimal, they can be fully distinguished using channels with different molecular weights. However, ELISA cannot differentiate D2 and D3 due to antibody cross-reactivity. Antibodies in ELISA methods can cross-react with vitamin D binding protein (VDBP), leading to false positives and inability to detect trace amounts of 1,25-(OH)2VD (clinically required concentrations are typically 20–60 pg / mL). The method of the present invention, however, uses specific MRM channels to avoid interference (such as 25-OH VD2), and internal standard correction reduces matrix effects.

[0150] In summary, in the present invention, 4-phenyl-1,2,3-triazolin-3,5-dione and benzylamine are used for combined derivatization to introduce nitrogen-containing groups through imine bond formation, which significantly improves the ESI+ ionization efficiency and increases the signal intensity by 5-10 times compared to silanization.

[0151] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, characterized in that: The method comprises: performing a derivatization reaction on a sample to be tested, and then detecting the sample by ultra-high performance liquid chromatography tandem mass spectrometry; The reagents for the derivatization reaction include 4-phenyl-1,2,3-triazolin-3,5-dione and benzylamine.

2. The method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to claim 1, characterized in that: The derivatization reaction includes: adding 4-phenyl-1,2,3-triazolin-3,5-dione and benzylamine to a sample to be tested, incubating, blowing dry with nitrogen, and re-dissolving with a solvent to prepare a test sample.

3. The method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to claim 2, wherein: The concentration of the 4-phenyl-1,2,3-triazolin-3,5-dione is 50-100 mM; Preferably, the concentration of benzylamine is 50-100 mM; Preferably, the volume ratio of 4-phenyl-1,2,3-triazolin-3,5-dione and benzylamine is 1:(0.5-2); Preferably, the incubation temperature is 55-65° C. and the incubation time is 5-15 min.

4. The method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to claim 2 or 3, characterized in that: The solvent includes mobile phase A and mobile phase B of the ultra-high performance liquid chromatography tandem mass spectrometry; Preferably, the volume ratio of mobile phase A to mobile phase B in the solvent is 1:(0.5-2).

5. The method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to any one of claims 1 to 4, characterized in that: The derivatization reaction also includes a step of pre-treating the sample to be tested; Preferably, the pretreatment comprises adding an internal standard to the sample to be tested, precipitating the protein with a precipitant, mixing and then centrifuging, and taking the supernatant and drying it with nitrogen.

6. The method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to claim 5, characterized in that: The internal standards include deuterated 25-hydroxyvitamin D2, deuterated 25-hydroxyvitamin D3, deuterated 1,25-dihydroxyvitamin D2 and deuterated 1,25-dihydroxyvitamin D3; Preferably, the concentration of deuterated 25-hydroxyvitamin D2 is 5-15 ng / mL, the concentration of deuterated 25-hydroxyvitamin D3 is 5-15 ng / mL, the concentration of deuterated 1,25-dihydroxyvitamin D2 is 5-15 ng / mL, and the concentration of deuterated 1,25-dihydroxyvitamin D3 is 5-15 ng / mL; Preferably, the volume ratio of the sample to be tested, the internal standard and the precipitant is (5-15):1:(30-50); Preferably, the sample to be tested includes serum and / or blood serum; Preferably, the precipitant comprises methanol and / or acetonitrile; Preferably, the centrifugal speed is 10000-15000 rpm, and the time is 5-15 min.

7. The method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to any one of claims 1 to 6, characterized in that: The chromatographic column of the ultra-high performance liquid chromatography comprises a C18 column; Preferably, the mobile phase A of the ultra-high performance liquid chromatography is 0.05-0.3% formic acid aqueous solution, and the mobile phase B is 0.05-0.3% formic acid acetonitrile solution; Preferably, the flow rate of the ultra-high performance liquid chromatography is 0.1-0.5 mL / min; Preferably, the column temperature of the ultra-high performance liquid chromatography is 35-45°C.

8. The method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to any one of claims 1 to 7, characterized in that: The elution procedure of the ultra-high performance liquid chromatography is: 0-1 min, (3-8)% mobile phase B; 1-2.5 min, (3-8)% average rate change to (92-98)% mobile phase B; 2.5-3 min, (92-98)% mobile phase B; 3-3.1 min, (92-98)% average rate change to (3-8)% mobile phase B; 3.1-4 min, (3-8)% mobile phase B.

9. The method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to any one of claims 1 to 8, characterized in that: The method further comprises preparing a standard curve; Preferably, the standard curve concentration range of 25-hydroxyvitamin D2 is 0.01-100 ng / mL; Preferably, the standard curve concentration range of 25-hydroxyvitamin D3 is 0.01-100 ng / mL; Preferably, the standard curve concentration range of 1,25-dihydroxyvitamin D2 is 0.5-200 pg / mL; Preferably, the concentration range of the standard curve of 1,25-dihydroxyvitamin D2 is 0.5-200 pg / mL.

10. Use of the method for detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D according to any one of claims 1 to 9 in detecting 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D.

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