A multi-component combined detection method, device and application for synchronous analysis of bone metabolism markers

Abstract

The application discloses a multi-component joint detection method, device and application for synchronous analysis of bone metabolism markers. The multi-component joint detection method comprises the following steps: pretreating a serum sample, and simultaneously extracting small molecule sterols, short peptides and protein characteristic peptide segments; adopting a reverse phase chromatography system to perform chromatographic separation on the extracts, and realizing synchronous elution of lipid-soluble substances and polar substances through a variable gradient elution program; and adopting a segmented multi-reaction monitoring mode to perform mass spectrometry detection, and simultaneously detecting high-abundance indexes and low-abundance indexes in bone metabolism markers in a same sampling period. The application realizes synchronous quantitative detection of five bone metabolism markers, solves technical problems such as fragmentation, large concentration span, incompatible chromatographic behavior and isomer interference in the prior art, and improves detection efficiency by more than 400%, and reduces detection cost by 50%-70%.

Description

Technical Field

[0001] This invention belongs to the field of clinical medical testing technology, and relates to a multi-component detection method, device and application for simultaneous analysis of bone metabolism markers. Background Technology

[0002] Bone metabolism markers are key indicators reflecting bone turnover status, assessing osteoporosis treatment efficacy, and monitoring metabolic bone diseases. The clinically recognized "five bone metabolism markers" include: 25-hydroxyvitamin D (25-OH VD), parathyroid hormone (PTH), osteocalcin (OC), type I collagen N-terminal elongated peptide (PINP), and β-collagen specific sequence (β-CTX).

[0003] Currently, clinical laboratories mainly use chemiluminescent immunoassay (CLIA) for detection. Although the immunoassay is simple to operate, it has the following significant drawbacks: (1) Limited specificity: The immunoassay has difficulty distinguishing structurally similar metabolites (such as isomers of VD3) and is easily affected by cross-reactions of endogenous antibodies, PTH oxidized fragments, or inactive degradation products in the sample, leading to deviations in the test results. (2) Fragmented detection: The five bone metabolism items cover everything from highly polar small molecules (β-CTX) to hydrophobic lipid-soluble components (25-OH VD), and then to long peptides / proteins with huge molecular weights (PTH, PINP). Due to the wide range of physicochemical properties, existing technologies usually require these indicators to be split into multiple independent platforms or extracted and tested multiple times, which seriously affects the time to market (TAT) of clinical reports. (3) Bottlenecks in existing mass spectrometry technology: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the development direction of clinical testing due to its high specificity and wide linear range, but it still faces the following bottlenecks when realizing "multi-component joint detection": Contradiction between concentration and signal range: 25-OH VD has a high concentration in serum (ng / mL level), while PTH is at an extremely low abundance (pg / mL level). How to ensure that high abundance indicators do not cause detector saturation within the same injection cycle, while ensuring that low abundance indicators have a sufficient signal-to-noise ratio (S / N), is a recognized problem in the industry. Incompatibility of chromatographic elution behavior: Small molecule sterols require strong organic phase elution, while large molecule peptides or characteristic peptides after enzymatic digestion often require precise gradient switching to achieve good peak separation. Existing chromatographic methods often suffer from one problem at the expense of the other, resulting in peak tailing or overlap during simultaneous analysis of multiple components. Complexity of pretreatment: Existing multi-detection schemes are usually lengthy, and there are conflicts in the requirements of different indicators for pretreatment reagents (such as reducing agents and protease inhibitors). Currently, there is a lack of a method that can achieve simultaneous and accurate quantification of the above five heterogeneity indicators in a single injection using a unified pretreatment process.

[0004] Therefore, developing a simultaneous analysis system capable of covering characteristic peptides of steroids, small molecule peptides, and medium- to large molecule proteins has significant clinical value. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a multi-component assay method, apparatus, and application for simultaneous analysis of bone metabolism biomarkers. The primary objective is to overcome the fragmented nature of existing bone metabolism biomarker detection technologies and provide a simultaneous analysis method and apparatus capable of covering steroids (25-OH VD), small molecule peptides (β-CTX), and characteristic peptides of medium- and large molecule proteins (PTH, OC, PINP). By constructing a unified chromatographic-mass spectrometric analysis window, simultaneous detection of five bone metabolism indicators can be achieved in a single injection cycle, significantly improving the efficiency of clinical laboratories.

[0006] Another objective of this invention is to resolve the discrepancy of concentration ranges exceeding 1000 times between different indicators. By optimizing multiple reaction monitoring (MRM) parameters, segmented triggering techniques, and ion pair selection in mass spectrometry, it ensures that high concentrations of 25-OH VD do not lead to detector saturation, while simultaneously guaranteeing that extremely low abundances of PTH (pg / mL level) have sufficient signal-to-noise ratio (S / N), thus achieving accurate quantification across the entire range.

[0007] The purpose of this invention is to leverage the high resolution of mass spectrometry to address the cross-reactivity issue inherent in immunoassays. By screening for highly specific characteristic amino acid sequences, it effectively distinguishes active PTH from inactive fragments, and utilizes chromatographic separation techniques to eliminate interference from the C3- epimer of 25-OH VD3. Monitoring of oxidized sites is introduced into the detection system to ensure that the detection results reflect the true bioactive concentration in the sample.

[0008] The industrial application objective of this invention is to construct a high-throughput standardized clinical mass spectrometry device. Through an optimized chromatographic gradient elution strategy, the target analyte is effectively separated from interfering substances such as serum endogenous phospholipids, reducing the influence of matrix effects on quantification. This ensures that the device maintains a stable response value and an extremely low contamination rate even after processing hundreds of clinical samples continuously, meeting the actual needs of high-throughput and low-cost maintenance in clinical settings.

[0009] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a multi-component assay method for simultaneous analysis of bone metabolism markers. The multi-component assay method includes: pretreatment of serum samples to simultaneously extract small molecule sterols, short peptides, and characteristic protein peptides; chromatographic separation of the extracts using a reversed-phase chromatography system, achieving simultaneous elution of lipid-soluble and polar substances through a variable gradient elution program; and mass spectrometry detection using a segmented multiple reaction monitoring (MRM) mode to simultaneously detect high-abundance and low-abundance indicators of bone metabolism markers within the same injection cycle. The time-segment monitoring logic diagram for the simultaneous detection of five bone metabolism markers in this invention is shown below. Figure 1 As shown.

[0010] This invention employs a "reverse integration" pretreatment technique for cross-dimensional target analytes, breaking away from the traditional mass spectrometry approach of "precipitating proteins first, then using the supernatant to detect small molecules," and resolving the mutual incompatibility issue between extraction conditions for large protein molecules and small sterol molecules. It utilizes an "in-situ enzymatic digestion-simultaneous extraction" strategy. Before removing the matrix protein, a controlled slightly alkaline environment (pH 7.5-8.5) is used to induce unfolding of large proteins (PTH, OC, PINP), and a highly efficient trypsin is added to rapidly convert them into stable characteristic peptides. Subsequently, a high proportion of organic phase is introduced, and while precipitating the matrix protein, polarity switching is used to achieve the dissociation of small molecule 25-OH VD from the binding protein (DBP) and the simultaneous dissolution of peptides. This strategy ensures that five indicators of different magnitudes and polarities reach thermodynamic equilibrium in a single extract, with significantly higher recovery consistency than stepwise processing.

[0011] This invention employs a "concentration stabilization" and segmented mass spectrometry acquisition control logic to resolve signal conflicts between high-abundance substances at the ng / mL level and low-abundance substances at the pg / mL level within the same injection cycle. Dynamic gain adjustment includes: setting time window switching in the mass spectrometry monitoring software, employing a low residence time (<15 ms) and subsensitive ion pairs for the 25-OH VD window to artificially avoid detector saturation. Signal gain enhancement includes: for the PTH window, employing a long residence time (>150 ms) and superimposed energy gradient optimization in the collision chamber (Q2) to maximize ion transport efficiency of pg / mL characteristic peptides. This "high-low pressure balancing" technology enables the dynamic linear range of full-item detection to span four orders of magnitude, achieving precise quantification within the same standard curve framework.

[0012] This invention employs a multi-site monitoring and oxidation correction algorithm for characteristic peptides to solve the problem of inaccurate quantification of indicators such as PTH and OC caused by in vitro oxidation or degradation. Through experimentation, 2-3 characteristic peptides with high specificity and containing oxidation-sensitive sites (such as Met8 and Met18) are identified. The "reduced" and "oxidized" ion pairs of PTH are simultaneously monitored in mass spectrometry. Using the conversion formula established in this invention, the final concentration is corrected in real time based on the oxidation ratio. This ensures that the detection results reflect the total amount of biologically active indicators in the sample, completely overcoming the technical blind spot of immunoassays in distinguishing oxidized and degraded components.

[0013] This invention employs a "precise isomer stripping" technique under a complex chromatographic gradient to overcome the chromatographic separation bottleneck of 25-OH VD3 and its epimer (C3-Epi). A "gentle slope-steep slope" switching elution gradient was developed. After the polar peptide elution is complete, by precisely controlling the growth slope of the organic phase (growth rate controlled at 2.5%-3.5% per minute), baseline separation of VD3 and C3-Epi (resolution R>1.5) was achieved within 5 minutes. Combined with the dipole interaction of the polar embedded C18 stationary phase, complete removal of interfering isomers was achieved without prolonging the chromatographic run time, ensuring the "gold standard" nature of the detection results.

[0014] Preferably, the pretreatment includes: taking a serum sample and adding a composite stabilizing solution containing an isotope-labeled internal standard group; adding trypsin for enzymatic hydrolysis to convert macromolecular proteins into characteristic signal peptides; adding an extraction solvent to simultaneously precipitate proteins and release small molecule targets; centrifuging to collect the supernatant, which is then concentrated and reconstituted.

[0015] Preferably, the composite stabilizer contains any one or a combination of at least two of tris(2-carboxyethyl)phosphine (TCEP), 4-aminoethylbenzenesulfonyl fluoride (AEBSF), or ethylenediaminetetraacetic acid (EDTA).

[0016] Preferably, the enzymatic hydrolysis is performed at a temperature of 37°C-42°C (e.g., 37°C, 40°C or 42°C) for a time of 30-60 min (e.g., 30 min, 50 min or 60 min).

[0017] Preferably, the extraction solvent comprises a solution containing methanol or acetonitrile.

[0018] Preferably, the reversed-phase chromatography system uses a C18 column with embedded polar groups; mobile phase A is an aqueous solution containing 0.05%-0.2% (e.g., 0.05%, 0.1% or 0.2%) formic acid and 1-5 mmol / L (e.g., 1 mmol / L, 3 mmol / L or 5 mmol / L) ammonium acetate, and mobile phase B is a mixed solution of acetonitrile and methanol containing 0.05%-0.2% (e.g., 0.05%, 0.1% or 0.2%) formic acid.

[0019] Preferably, the variable gradient elution procedure includes the following steps: (1) 0-2.0 min: Maintain 3%-8% (e.g., 3%, 5% or 8%) of organic phase, and elute highly polar peptides first; (2) 2.0-5.0 min: linearly increase the organic phase to 92%-97% (e.g., 92%, 95% or 97%), and elute hydrophobic small molecule targets and isomers; (3) 5.0-7.0 min: Maintain 92%-97% organic phase to clean the column cavity and remove matrix interferences such as serum phospholipids.

[0020] Preferably, the mass spectrometry detection using a segmented multiple reaction monitoring mode includes setting three monitoring windows: a polarity window, a sensitivity core window, and a high abundance window. The polar window has a time range of 0-2.5 min and a residence time of 80-120 ms, and is used to monitor β-collagen special sequences and type I collagen amino-terminal elongated peptides, with corresponding characteristic ion pairs of 709→110 and 642→227, respectively. The sensitivity core window has a time range of 2.5-5.5 min, a residence time of 120-200 ms, a declustering potential of 40-70 V, and a collision energy of 15-30 eV. It is used to monitor parathyroid hormone and osteocalcin, and their corresponding characteristic ion pairs are 853→317 and 521→189, respectively. The high abundance window has a time range of 5.5-8.5 min, a residence time of 5-20 ms, a declustering potential of 30-50 V, and a collision energy of 10-20 eV. It is used to monitor 25-hydroxyvitamin D, and its corresponding characteristic ion pair is 401→179.

[0021] In this invention, the polarity window preferentially elutes short polar peptides (specific β-collagen sequences and extended amino-terminal peptides of type I collagen), the sensitivity core window acquires low-abundance pg / mL level indicators (parathyroid hormone and osteocalcin) with high sensitivity, and the high-abundance window is used for the detection of high-abundance indicators (25-hydroxyvitamin D) at the ng / mL level to prevent detector saturation.

[0022] Preferably, the bone metabolism markers include a combination of at least two of the following: 25-hydroxyvitamin D, parathyroid hormone, osteocalcin, type I collagen N-terminal elongated peptide, or a specific sequence of β-collagen.

[0023] Preferably, the method further includes the step of monitoring and correcting the oxidation state of parathyroid hormone: simultaneously monitoring the ion pairs of the reduced state characteristic peptide and the oxidized state characteristic peptide of parathyroid hormone, and performing real-time back-calculation correction of the final concentration based on the oxidation ratio.

[0024] Preferably, the calculation formula for the back-calculation correction is as shown in equation (1): Ctotal = (Areared + Areaox × α) / AreaIS Formula (1); Wherein, α is the oxidation response correction factor, Ctotal is the total activity concentration of parathyroid hormone, Areared is the mass spectrum peak area of ​​the characteristic peptide of reduced parathyroid hormone, Areaox is the mass spectrum peak area of ​​the characteristic peptide of oxidized parathyroid hormone, and AreaIS is the mass spectrum peak area of ​​the isotope-labeled internal standard.

[0025] Preferably, the method further includes a step of chromatographic separation of 25-hydroxyvitamin D3 and its C3-epimer: baseline separation of the two is performed by controlling the organic phase growth slope at 2.5%-3.5% per minute within a high abundance window.

[0026] Secondly, the present invention provides a multi-component detection device for simultaneous analysis of bone metabolism markers, the multi-component detection device comprising: a pretreatment module, a chromatographic separation module, and a mass spectrometry detection module; The preprocessing module is used to perform the following: preprocessing the serum sample and extracting small molecule sterols, short peptides and characteristic peptides of proteins. The chromatographic separation module is used to perform the following: chromatographic separation of the extract using a reversed-phase chromatography system, and simultaneous elution of lipid-soluble and polar substances through a variable gradient elution program; The mass spectrometry detection module is used to perform the following: mass spectrometry detection using a segmented multiple reaction monitoring mode, simultaneously detecting high-abundance and low-abundance indicators in bone metabolism markers within the same injection cycle; the concentration of the high-abundance indicator in serum is at the ng / mL level, and the concentration of the low-abundance indicator in serum is at the pg / mL level.

[0027] Preferably, the pretreatment module includes: a composite stabilizing solution addition unit, a trypsin hydrolysis unit, an organic solvent extraction unit, and a concentration and reconstitution unit.

[0028] Preferably, the chromatographic separation module uses a C18 column with embedded polar groups; mobile phase A is an aqueous solution containing 0.05%-0.2% formic acid and 1-5 mmol / L ammonium acetate, and mobile phase B is a mixed solution of acetonitrile and methanol containing 0.05%-0.2% formic acid.

[0029] Preferably, the mass spectrometry detection module includes: a polarity window, a sensitivity core window, and a high abundance window; the polarity window is used to monitor specific sequences of β-collagen and characteristic peptide segments of type I collagen N-terminal elongated peptides, with a residence time of 80-120 ms; the sensitivity core window is used to monitor characteristic peptide segments of parathyroid hormone and osteocalcin, with a residence time of 120-200 ms; and the high abundance window is used to monitor 25-hydroxyvitamin D, with a residence time of 5-20 ms.

[0030] Preferably, the mass spectrometry detection module further includes an oxidation correction unit, which is used to simultaneously monitor the reduced and oxidized characteristic peptides of parathyroid hormone and perform real-time back-calculation correction of the concentration based on the oxidation ratio.

[0031] Preferably, the calculation formula for the back-calculation correction is as shown in equation (1): Ctotal = (Areared + Areaox × α) / AreaIS Formula (1); Wherein, α is the oxidation response correction factor, Ctotal is the total activity concentration of parathyroid hormone, Areared is the mass spectrum peak area of ​​the characteristic peptide of reduced parathyroid hormone, Areaox is the mass spectrum peak area of ​​the characteristic peptide of oxidized parathyroid hormone, and AreaIS is the mass spectrum peak area of ​​the isotope-labeled internal standard.

[0032] Preferably, the bone metabolism markers include a combination of at least two of the following: 25-hydroxyvitamin D, parathyroid hormone, osteocalcin, type I collagen N-terminal elongated peptide, or a specific sequence of β-collagen.

[0033] Thirdly, the present invention provides the application of the multi-component detection method described in the first aspect or the multi-component detection device described in the second aspect in the preparation of diagnostic reagents or kits for bone metabolic diseases.

[0034] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses a "one-needle" multi-indicator testing technology to simultaneously complete the testing of five indicators within 7-10 minutes, increasing the single-machine throughput of clinical laboratories by more than 400% and shortening the time from sample entry to the issuance of a complete report by 60%; (2) This invention successfully achieved simultaneous and accurate monitoring of ng / mL (25-OHVD) and pg / mL (PTH) indicators through segmented mass spectrometry acquisition and signal gain modulation technology. The linear correlation coefficient (R) of each indicator was also achieved. 2 All values ​​were greater than 0.99, and the dynamic linear range of the detection spanned four orders of magnitude. Even when patients experienced extreme pathological fluctuations in their indicators (such as severe osteoporosis or hyperparathyroidism), the system could still maintain an extremely wide detection range without the need for secondary dilution or retesting of the samples, thus ensuring the continuity and reliability of the results. (3) The present invention achieves baseline separation of 25-OH VD3 and its epimer (C3-Epi) with a resolution R>1.5; by monitoring the specific characteristic peptides of PTH, the cross-reaction of inactive fragments commonly found in the immunoassay method is eliminated. (4) This invention is the first to propose a real-time monitoring and correction algorithm for PTH oxidation sites. When a sample undergoes slight oxidation changes, it can automatically calculate and restore the true active PTH concentration, and the correction deviation can reach 15%-30%. (5) Significantly reduced individual testing costs. Compared to the cumulative cost of individual testing, the overall testing cost after implementing this invention is reduced by approximately 50%-70%. Attached Figure Description

[0035] Figure 1 This is a time-period monitoring logic diagram for the simultaneous detection of five bone metabolism parameters.

[0036] Figure 2 To achieve chromatographic separation of 25-OH VD3 and C3-Epi epimers through gradient optimization.

[0037] Figure 3 This is a mass spectrometry scan of PTH and its characteristic oxidized peptides.

[0038] Figure 4 This is a superimposed standard curve of five bone metabolism parameters across multiple orders of magnitude concentration ranges.

[0039] Figure 5 The Bland-Altman plot shows the consistency between "before correction" and "after correction" of the oxidation correction algorithm in clinical samples. Detailed Implementation

[0040] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] The term LC-MS / MS stands for Liquid Chromatography-Tandem Mass Spectrometry.

[0043] The term RSD stands for Relative Standard Deviation.

[0044] Example 1 Multi-component detection method and linear validation for simultaneous analysis of bone metabolism markers.

[0045] 1. Pre-processing flow (1) Sample pretreatment: Take 100 μL of human serum and add 20 μL of internal standard containing isotope-labeled groups (the isotope-labeled internal standard groups include: 25-hydroxyvitamin D-D6 (25-OH VD-D6), parathyroid hormone characteristic peptide - 13 C6 15 N2 (PTH-IS), characteristic peptide of parathyroid hormone oxidized state - 13 C6 15 N2 (PTH-ox-IS), osteocalcin characteristic peptides - 13 C6 15 N2 (OC-IS), a characteristic peptide segment of type I collagen N-terminal elongated peptide - 13 C6 15 N2 (PINP-IS), β-collagen special sequence characteristic peptide - 13 C6 15 N2 (β-CTX-IS); the composite internal standard working solution contains 25-OH VD-D6 at a concentration of 200 ng / mL, and PTH-IS, PTH-ox-IS, OC-IS, PINP-IS, and β-CTX-IS at a concentration of 100 ng / mL;) and a composite stabilizing solution (2.5 mmol / L LTCEP, 0.5 mg / mL AEBSF, 2.0 mg / mL EDTA-2K, 0.05% (v / v) Tween-20, 50 mmol / L HEPES, pH 6.8); (2) Enzymatic hydrolysis: Add trypsin solution and hydrolyze at 37°C for 45 min to convert macromolecular proteins (PTH, OC, PINP) into characteristic signal peptides with specific mass numbers; (3) Liquid-liquid extraction / protein precipitation: Add 300 μL of acetonitrile (containing 0.1% formic acid) to simultaneously precipitate protein and release 25-OH VD; (4) Concentration and redissolution: Vortex centrifuge, take the supernatant, blow dry with nitrogen, redissolve with acetonitrile solution containing 0.1% formic acid, and then analyze.

[0046] 2. Cross-dimensional chromatographic separation A highly compatible reversed-phase chromatography system is used to achieve simultaneous elution of lipid-soluble and polar substances.

[0047] Chromatographic column: A C18 column (C18-PA) with intercalated polar groups is used to enhance the retention of polar peptides (β-CTX).

[0048] Mobile phase system: Mobile phase A is a 0.1% formic acid aqueous solution (containing 2 mmol / L ammonium acetate), and mobile phase B is a 0.1% formic acid acetonitrile / methanol mixed solution.

[0049] Variable gradient elution procedure: 0-2.0 min: Maintain a low proportion of organic phase (5% B) to allow highly polar β-CTX and peptides to elute first.

[0050] 2.0-5.0 min: linearly increase the organic phase to 95% B, elute the strongly hydrophobic 25-OH VD and isomers.

[0051] 5.0-7.0 min: Maintain 97% organic phase B to clean the column cavity and remove stubborn matrix such as serum phospholipids.

[0052] 3. Segmented mass spectrometry monitoring Polarity window: time range 0-2.5 min, maintain mobile phase 5% B, isocratic elution, monitor indicators β-CTX and PINP, residence time 100 ms; Sensitivity core window: time range 2.5-5.5 min, linear increase of mobile phase to 75% B, linear gradient (23.3% / min), monitoring indicators PTH, OC, residence time 150 ms; High abundance window: time range 5.5-8.5 min, with a slow gradient to increase the mobile phase to 85% B, slow gradient (3% / min), monitoring indicators 25-OH VD, C3-Epi, residence time 10 ms.

[0053] Column cavity purification and matrix removal: time range 8.5-10 min, maintain mobile phase 95% B, isocratic rinsing.

[0054] Column equilibration: time range 10-12 min, linearly reduce mobile phase to 5% B, linear backflushing.

[0055] 4. Selection of characteristic ion pairs PTH activity assay: A specific sequence characteristic peptide (SEQ ID NO.1: SVSEIQLMHNLGK) (corresponding to amino acid sequences 7-20 of PTH) was monitored. This peptide contains a methionine oxidation site (Met8). Simultaneously, its oxidation state (+16 Da) ion pair was monitored, and the final concentration was corrected in real-time based on the oxidation ratio. Figure 3 The calculation formula for back-calculation correction is shown in equation (1): Ctotal = (Areared + Areaox × α) / AreaIS Formula (1); Wherein, α is the oxidation response correction factor, Ctotal is the total activity concentration of parathyroid hormone, Areared is the mass spectrum peak area of ​​the characteristic peptide of reduced parathyroid hormone, Areaox is the mass spectrum peak area of ​​the characteristic peptide of oxidized parathyroid hormone, and AreaIS is the mass spectrum peak area of ​​the isotope-labeled internal standard.

[0056] OC integrity detection: Monitoring the amino acid sequence of human osteocalcin (OC) C-terminal specific pancreatic enzyme peptide (SEQ ID NO.2: GFQEAYRRFYGPV) corresponding to amino acid positions 37-49; this sequence is located in the hydrophobic region of the OC C-terminus, is a characteristic sequence unique to full-length OC and missing from the truncated form, and can specifically characterize the integrity of OC molecules, avoiding interference from N-terminal degradation fragments.

[0057] 25-OH VD isomer exclusion: Using specific characteristic fragment ions combined with chromatographic retention time, 25-OHVD3 and C3-Epi-25-OH VD3 can be distinguished.

[0058] 5. Experimental Results Linearity: 25-OH VD showed R in the range of 1-150 ng / mL. 2 >0.99; PTH in the range of 5-2000 pg / mL R 2 >0.99; OC in the range of 5-1000 ng / mL R 2 >0.99; PINP in the range of 10-500 ng / mL R 2 >0.99; β-CTX in the range of 0.1-10 ng / mL R 2 >0.99 ( Figure 4 ).

[0059] Accuracy: The recovery rate of quality control samples for each indicator is between 90% and 110%.

[0060] The above results demonstrate that the present invention has successfully achieved "detection of coexistence of high and low concentrations". The device and method of the present invention have excellent full-range index coverage and detection accuracy.

[0061] C3-Epi interference exclusion and isomer separation detection: Elution gradient: In the high abundance window, the organic phase was slowly increased from 75% B to 85% B at a slope of 3% per minute. Results: 25-OH VD3 and C3-Epi-25-OH VD3 were baseline separated with a resolution R=1.65, and the detection results were not affected by isomer interference.

[0062] Example 2 Validation of the PTH oxidation correction algorithm in clinical samples.

[0063] Twenty clinical serum samples were collected and left at room temperature for 5 hours. The reduced (Mred) and oxidized (Mox) peptides of parathyroid hormone (PTH) were simultaneously monitored using the method described in Example 1 of this invention. Concentration was calculated using the formula Ctotal = (Areared + Areaox × α) / AreaIS (where α is the oxidation response correction factor, Ctotal is the total active concentration of parathyroid hormone, Areared is the mass spectrum peak area of ​​the characteristic reduced PTH peptide, Areaox is the mass spectrum peak area of ​​the characteristic oxidized PTH peptide, and AreaIS is the mass spectrum peak area of ​​the isotope-labeled internal standard). Results: The deviation between the detection results of the corrected method of this invention and those of immediately frozen samples was reduced to within 4%. Traditional detection method (uncorrected): The results were approximately 22% lower than those of immediately frozen samples. Figure 5 ).

[0064] The above results indicate that by monitoring oxidation sites and correcting the algorithm, this invention can significantly reduce the risk of false low values ​​in clinical samples due to improper preprocessing.

[0065] Comparative Example 1 This comparative example uses conventional LC-MS / MS single-gradient mass spectrometry to eliminate C3-Epi interference and verify isomer separation, as detailed below: 1. Chromatographic conditions Chromatographic column: Conventional C18 column (ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 µm, 3 mm x 100 mm).

[0066] Mobile phase: A: 0.1% formic acid aqueous solution; B: 0.1% Formic acid acetonitrile (without methanol addition).

[0067] 2. Elution gradient (single linear, no window segmentation): 0-1.0 min: 10% B isotherm; 1.0-6.0 min: 10% B linearly increases to 90% B (slope ≈ 16% / min); 6.0-8.0 min: Rinse with 90% B solution; 8.0-10.0 min: Return to 10% B equilibrium.

[0068] 3. Mass spectrometry monitoring The process is unsegmented, with a uniform residence time of 50 ms throughout, and β-CTX, PINP, PTH, OC, and 25-OH VD ion pairs are collected simultaneously; there is no monitoring of PTH oxidation state, and no dedicated separation procedure for 25-OH VD isomers.

[0069] 4. Experimental Results like Figure 2 As shown, the chromatographic peaks of 25-OH VD3 and C3-Epi-25-OH VD3 completely overlapped, with a resolution R < 0.8, resulting in an artificially high detection value of 25-OH VD3 by 12%-18%. At the same time, the peaks of PTH and OC peptides showed severe tailing and significant matrix interference, making it impossible to achieve simultaneous and accurate quantification of the five indicators.

[0070] The above results demonstrate that the method of the present invention has excellent specificity and significant advantages in eliminating isomer interference, especially for the stripping of vitamin D isomers.

[0071] In summary, this invention enables simultaneous quantitative detection of five biomarkers of bone metabolism, solving technical problems in existing technologies such as fragmentation, large concentration range, incompatible chromatographic behavior, and interference from isomers. It improves detection efficiency by more than 400% and reduces detection costs by 50%-70%.

[0072] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A multi-component assay method for simultaneous analysis of bone metabolism markers, characterized in that, The multi-component assay method includes: pretreatment of serum samples to extract small molecule sterols, short peptides, and characteristic protein peptides; chromatographic separation of the extracts using a reversed-phase chromatography system, achieving simultaneous elution of lipid-soluble and polar substances through a variable gradient elution program; and mass spectrometry detection using a segmented multiple reaction monitoring mode to simultaneously detect high-abundance and low-abundance indicators of bone metabolism markers within the same injection cycle; wherein the concentration of the high-abundance indicator in serum is at the ng / mL level, and the concentration of the low-abundance indicator in serum is at the pg / mL level.

2. The multi-component joint detection method according to claim 1, characterized in that, The pretreatment includes: taking a serum sample and adding a composite stabilizing solution containing isotope-labeled internal standard groups; adding trypsin for enzymatic hydrolysis to convert large molecular proteins into characteristic signal peptides; adding an extraction solvent to simultaneously precipitate proteins and release small molecular target substances; centrifuging to collect the supernatant, which is then concentrated and reconstituted. Preferably, the composite stabilizer contains any one or a combination of at least two of tris(2-carboxyethyl)phosphine (TCEP), 4-aminoethylbenzenesulfonyl fluoride (AEBSF), or ethylenediaminetetraacetic acid (EDTA). Preferably, the enzymatic hydrolysis is performed at a temperature of 37°C-42°C for 30-60 minutes. Preferably, the extraction solvent comprises a solution containing methanol or acetonitrile.

3. The multi-component joint detection method according to claim 1 or 2, characterized in that, The reversed-phase chromatography system uses a C18 column with embedded polar groups; mobile phase A is an aqueous solution containing 0.05%-0.2% formic acid and 1-5 mmol / L ammonium acetate, and mobile phase B is a mixed solution of acetonitrile and methanol containing 0.05%-0.2% formic acid.

4. The multi-component joint detection method according to any one of claims 1-3, characterized in that, The variable gradient elution procedure includes the following steps: (1) 0-2.0 min: Maintain 3%-8% organic phase, and elute highly polar peptides first; (2) 2.0-5.0 min: linearly increase the organic phase to 92%-97% to elute hydrophobic small molecule target compounds and isomers; (3) 5.0-7.0 min: Maintain 92%-97% organic phase to clean the column cavity and remove matrix interferences such as serum phospholipids.

5. The multi-component joint detection method according to any one of claims 1-4, characterized in that, The mass spectrometry detection using the segmented multiple reaction monitoring mode includes setting three monitoring windows: a polarity window, a sensitivity core window, and a high abundance window. The polar window has a time range of 0-2.5 min and a residence time of 80-120 ms, and is used to monitor β-collagen special sequences and type I collagen amino-terminal elongated peptides, with corresponding characteristic ion pairs of 709→110 and 642→227, respectively. The sensitivity core window has a time range of 2.5-5.5 min, a dwell time of 120-200 ms, a declustering potential of 40-70 V, and a collision energy of 15-30 eV. It is used to monitor parathyroid hormone and osteocalcin, and their corresponding characteristic ion pairs are 853→317 and 521→189, respectively. The high abundance window has a time range of 5.5-8.5 min, a residence time of 5-20 ms, a declustering potential of 30-50 V, and a collision energy of 10-20 eV. It is used to monitor 25-hydroxyvitamin D, and its corresponding characteristic ion pair is 401→179.

6. The multi-component joint detection method according to any one of claims 1-5, characterized in that, The bone metabolism markers include a combination of at least two of the following: 25-hydroxyvitamin D, parathyroid hormone, osteocalcin, type I collagen N-terminal elongated peptide, or a specific sequence of β-collagen.

7. The multi-component joint detection method according to any one of claims 1-6, characterized in that, The method further includes the step of monitoring and correcting the oxidation state of parathyroid hormone: simultaneously monitoring the ion pairs of the reduced state characteristic peptide and the oxidized state characteristic peptide of parathyroid hormone, and performing real-time back-calculation correction of the final concentration based on the oxidation ratio. Preferably, the calculation formula for the back-calculation correction is as shown in equation (1): Ctotal = (Areared + Areaox × α) / AreaIS Formula (1); Wherein, α is the oxidation response correction factor, Ctotal is the total activity concentration of parathyroid hormone, Areared is the mass spectrum peak area of ​​the characteristic peptide of reduced parathyroid hormone, Areaox is the mass spectrum peak area of ​​the characteristic peptide of oxidized parathyroid hormone, and AreaIS is the mass spectrum peak area of ​​the isotope-labeled internal standard. Preferably, the method further includes a step of chromatographic separation of 25-hydroxyvitamin D3 and its C3-epimer: baseline separation of the two is performed by controlling the organic phase growth slope at 2.5%-3.5% per minute within a high abundance window.

8. A multi-component detection device for simultaneous analysis of bone metabolism markers, characterized in that, The multi-component detection device includes: a pretreatment module, a chromatographic separation module, and a mass spectrometry detection module; The preprocessing module is used to perform the following: preprocessing the serum sample and extracting small molecule sterols, short peptides and characteristic peptides of proteins. The chromatographic separation module is used to perform the following: chromatographic separation of the extract using a reversed-phase chromatography system, and simultaneous elution of lipid-soluble and polar substances through a variable gradient elution program; The mass spectrometry detection module is used to perform the following: mass spectrometry detection using a segmented multiple reaction monitoring mode, simultaneously detecting high-abundance and low-abundance indicators in bone metabolism markers within the same injection cycle; the concentration of the high-abundance indicator in serum is at the ng / mL level, and the concentration of the low-abundance indicator in serum is at the pg / mL level.

9. The multi-component joint inspection device according to claim 8, characterized in that, The pretreatment module includes: a composite stabilizing solution addition unit, a trypsin hydrolysis unit, an organic solvent extraction unit, and a concentration and reconstitution unit; Preferably, the chromatographic separation module uses a C18 column with embedded polar groups; mobile phase A is an aqueous solution containing 0.05%-0.2% formic acid and 1-5 mmol / L ammonium acetate, and mobile phase B is a mixed solution of acetonitrile and methanol containing 0.05%-0.2% formic acid; Preferably, the mass spectrometry detection module includes: a polarity window, a sensitivity core window, and a high abundance window; the polarity window is used to monitor specific sequences of β-collagen and characteristic peptide segments of type I collagen N-terminal extended peptides, with a residence time of 80-120 ms; the sensitivity core window is used to monitor characteristic peptide segments of parathyroid hormone and osteocalcin, with a residence time of 120-200 ms; the high abundance window is used to monitor 25-hydroxyvitamin D, with a residence time of 5-20 ms; Preferably, the mass spectrometry detection module further includes an oxidation correction unit, which is used to simultaneously monitor the reduced and oxidized characteristic peptides of parathyroid hormone and perform real-time back-calculation correction of the concentration based on the oxidation ratio. Preferably, the calculation formula for the back-calculation correction is as shown in equation (1): Ctotal = (Areared + Areaox × α) / AreaIS Formula (1); Wherein, α is the oxidation response correction factor, Ctotal is the total activity concentration of parathyroid hormone, Areared is the mass spectrum peak area of ​​the characteristic peptide of reduced parathyroid hormone, Areaox is the mass spectrum peak area of ​​the characteristic peptide of oxidized parathyroid hormone, and AreaIS is the mass spectrum peak area of ​​the isotope-labeled internal standard. Preferably, the bone metabolism markers include a combination of at least two of the following: 25-hydroxyvitamin D, parathyroid hormone, osteocalcin, type I collagen N-terminal elongated peptide, or a specific sequence of β-collagen.

10. The use of the multi-component detection method according to any one of claims 1-7 or the multi-component detection device according to claims 8 and 9 in the preparation of diagnostic reagents or kits for bone metabolic diseases.

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