Clinical tandem mass spectrometry method and system based on multi-dimensional ion exchange

Abstract

The invention belongs to the technical field of clinical mass spectrometry, and particularly relates to a clinical tandem mass spectrometry analysis method and system based on multi-dimensional ion exchange, the relative concentration and the separation degree of a low-abundance target object are remarkably improved through combination of SCX online enrichment and multi-dimensional orthogonal separation (SCX-HILIC), and matrix interference is reduced; through an intelligent triggering (dynamic window, noise threshold and intensity priority) and dynamic exclusion mass spectrum acquisition strategy, the probability that low-abundance ions are effectively qualitative is remarkably improved; finally, quantitative distortion caused by pretreatment loss, matrix effect and instrument fluctuation is effectively overcome through a correction method combining an isotope internal standard and retention time locking, so that the technical problems of qualitative omission and quantitative distortion faced by low-abundance metabolite / peptide fragment analysis in a clinical sample are systematically solved.

Description

Technical Field

[0001] The present application belongs to the field of clinical mass spectrometry technology, and more specifically, relates to a clinical tandem mass spectrometry analysis method and system based on multidimensional ion exchange. Background Art

[0002] With the advancement of precision medicine, the accurate detection of low-abundance metabolites and peptides (such as early disease markers and drug metabolites) has become a core challenge for clinical mass spectrometry. These molecules are often overwhelmed by the high-abundance matrix (salt ions, phospholipids, and proteins) in complex biological samples (such as plasma and urine). Their qualitative and quantitative accuracy directly determines the reliability of disease screening. In areas such as cardiovascular disease and early cancer screening, detection sensitivity for highly polar / charged small molecules (such as amino acid derivatives and phosphorylated peptides) must reach the ng / mL level, placing extremely high demands on the separation technology's resolution and resistance to matrix interference.

[0003] Currently, over 90% of clinical laboratories use reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS / MS), which relies on a hydrophobic separation mechanism and has two key drawbacks: Failure to separate highly polar compounds: Positively charged / highly polar targets (such as creatinine and glutathione) are very weakly retained on the RPLC column and enter the mass spectrometer together with the high-abundance matrix (such as sodium chloride and choline) that co-elutes during the dead time, causing severe ion suppression. Co-elution leads to quantitative distortion: Compounds with similar charge properties (such as arginine and lysophosphatidylcholine) in complex samples co-elute in a single-dimensional gradient. The mass spectrometer cannot distinguish ions with the same m / z, triggering competitive inhibition of the signal. To improve separation capabilities, the academic community has developed online two-dimensional chromatography (such as RPLC-RPLC and SCX-RPLC), but fundamental bottlenecks still exist: Lack of solvent orthogonality: Commonly used reversed-phase / reversed-phase combinations cannot separate differences in charge dimensions. When strong cation exchange (SCX) is coupled with reversed-phase LC (RPLC), the high-salt eluent of SCX is incompatible with the organic phase of RPLC, resulting in failure of on-column focusing and severe peak broadening. Insufficient dynamic range coverage: Traditional data-dependent acquisition (DDA) uses a static m / z window trigger, which only fragments high-abundance ions in co-eluting peaks, while low-abundance targets (<100 pg) are ignored.

[0004] In summary, existing single-dimensional or online two-dimensional methods have the following shortcomings in the analysis of complex clinical samples: separation and detection: Separation dimension: Single-dimensional RPLC has insufficient retention of highly polar compounds, and the two-dimensional system lacks true physical orthogonal separation (charge × polarity), resulting in the masking of key low-abundance components by the matrix; Detection logic: The static trigger strategy cannot adapt to the dynamic elution process, and competition between co-eluting ions leads to low mass spectrometry acquisition efficiency; This ultimately leads to qualitative omissions and quantitative distortions of low-abundance metabolites / peptides, severely restricting the translational application of clinical biomarker research. Summary of the Invention

[0005] The present invention provides a clinical tandem mass spectrometry analysis method and system based on multidimensional ion exchange, which aims to solve the current technical problems of qualitative omission and quantitative distortion of low-abundance metabolites / peptides.

[0006] On the one hand, the present invention provides a clinical tandem mass spectrometry analysis method based on multidimensional ion exchange, characterized in that it comprises the following steps: Sample pretreatment: Acidification reagent is added to clinical samples to remove high-abundance protein interference and retain the target. The retained target is injected into the online SCX enrichment column for target enrichment; One-dimensional separation: The SCX enrichment column is eluted stepwise using five increasing ammonium salt concentrations, and the intensity of the elution peak is monitored using a UV detector. When the peak intensity drops to the baseline level, the elution is switched to the next concentration and the currently eluted target is directed to the HILIC column. Two-dimensional separation: The eluted components after one-dimensional separation are directed to the HILIC column for further separation. The HILIC column achieves polarity-differential separation of the target compounds by changing the polarity of the mobile phase. Mass spectrometry triggers mass spectrometry acquisition and processing: The components eluted after HILIC treatment are collected by the mass spectrometer for primary mass spectrometry to obtain the mass-to-charge ratio information of all ions. The mass-to-charge ratio range is then divided into dynamic windows based on the real-time baseline noise level. When the signal intensity within the window exceeds 6× the baseline noise, the secondary mass spectrometer is triggered. If multiple ions exist in the same window, only the top two ions with the highest intensity are triggered. At the same time, the triggered ions are added to the dynamic exclusion list, based on which the mass spectrometry detection data is obtained. Quantitative calibration: The mass spectrometry data were quantitatively calibrated using a combination of isotope internal standard calibration and retention time locked standards.

[0007] The present invention significantly improves the relative concentration and separation degree of low-abundance targets and reduces matrix interference through SCX online enrichment combined with multidimensional orthogonal separation (SCX-HILIC); significantly improves the probability of low-abundance ions being effectively qualitatively identified through intelligent triggering (dynamic window, noise threshold, intensity priority) and dynamic exclusion of mass spectrometry acquisition strategies; finally, through a correction method combining isotope internal standards and retention time locking, effectively overcomes the quantitative distortion caused by pretreatment losses, matrix effects and instrument fluctuations, thereby systematically solving the technical problems of qualitative omissions and quantitative distortion faced in the analysis of low-abundance metabolites / peptides in clinical samples.

[0008] Preferably, in the sample pretreatment, the target in the SCX enrichment column is enriched with an eluent, and the conductivity change of the eluent is monitored in real time. When the conductivity change is lower than a preset threshold, it is determined that the matrix removal is complete and the enrichment process is finished.

[0009] Preferably, in the one-dimensional separation, a weighted sum of the charge density of the target and the ionic strength concentration of the eluent is introduced on the basis of the baseline time to establish an elution time optimization model, and the elution time for each ammonium salt concentration is obtained based on the established elution time optimization model; The peak height and peak area are calculated at each predetermined time, and whether the target is completely eluted is determined based on the calculated peak height and peak area. If the peak height is lower than the preset threshold and the peak area deviation is within the allowable range, the next salt concentration is switched; if the peak area is insufficient, the elution time is adjusted based on the preset adjustment time and the elution time obtained after optimization based on the elution optimization model.

[0010] Preferably, in the two-dimensional separation, the mobile phase A in the HILIC analytical column is a high organic solvent, the mobile phase B is an aqueous phase, and the linear gradient elution starts from 100% mobile phase A to 80% mobile phase A, and the duration is synchronized with the arrival time of the SCX elution peak.

[0011] Preferably, the slope of the linear gradient is dynamically adjusted, and the specific steps are as follows: Initial gradient slope setting: Set an initial gradient slope according to the polarity range of the target compound; Real-time peak arrival time monitoring: At the outlet of the HILIC column, the arrival time of the elution peak is recorded in real time; Calculate time deviation: Calculate time deviation based on the real-time peak arrival time monitoring results and the expected arrival time; Adjusting the gradient slope: multiplying the absolute value of the time deviation by the proportional coefficient to obtain an adjustment amount of the gradient slope; adjusting the gradient slope based on the adjustment amount to obtain an adjusted gradient slope, wherein the gradient slope is reduced when the time deviation is greater than zero, and the gradient slope is increased when the time deviation is less than zero.

[0012] Preferably, the mass spectrometry triggered mass spectrometry acquisition and dynamic window division in processing includes the following steps: According to the scanning range of the primary mass spectrometer, the mass-to-charge ratio range is divided into a plurality of initial windows, wherein the initial window is the ratio of the total mass-to-charge ratio range of the primary mass spectrometer scan to the default window size; The default window size is dynamically adjusted based on the baseline noise level. If the real-time noise level is greater than a preset noise threshold, the default window is reduced to obtain an updated window size; if the real-time noise level is less than the preset noise threshold, the default window is expanded to obtain an updated window size; wherein the noise threshold is obtained based on the baseline noise level multiplied by a multiplication coefficient.

[0013] Preferably, a labeled isotope internal standard is added during the sample pretreatment stage; At the end of the first-dimensional separation stage and before switching to the next ammonium salt concentration, the RT Lock standard is injected directly into the elution flow path of the SCX enrichment column through the solenoid valve. After mixing with the eluent of the current salt concentration, it enters the HILIC column. The injection point is located in the flow path between the SCX enrichment column and the HILIC column.

[0014] Preferably, the quantitative calibration comprises the following steps: Data extraction and calibration of the dual internal standard system: Isotopic internal standard ions are screened based on known mass-to-charge ratios to obtain their corresponding SCX concentrations and signal intensities at the HILIC RT. Based on the fixed mass-to-charge ratio of the RT Lock standard, the measured retention time at the beginning of elution at each ammonium salt concentration is extracted. Internal standard correction factor and retention time offset calculation: Calculate the isotope internal standard correction factor and RT Lock retention time offset based on the acquired data to form the isotope internal standard correction factor table and RT Lock offset table; Two-dimensional peak alignment of UV signal and RT Lock: According to the end time of UV signal elution, the target signal is assigned to the corresponding SCX salt concentration window. The RT Lock offset corresponding to the salt concentration is used to calibrate the HILIC retention time. The signals of the same compound in different salt concentration windows are combined to form a two-dimensional coordinate table. Fast dimensional signal integration and quantitative integration: By comparing the fragmented ions of the secondary mass spectrometer with the spectral library, the mass-to-charge ratio of the target and all its signal points in the two-dimensional coordinates are confirmed. For all calibrated signal points of the same compound, the primary mass spectrum peak area is accumulated to eliminate the difference in extraction efficiency between samples and obtain the quantitative integration result of the target.

[0015] On the other hand, the present invention provides a clinical tandem mass spectrometry analysis system based on multidimensional ion exchange, comprising an acidifying reagent injection pump, a quantitative pump, an SCX enrichment housing, a gradient elution device, a flow path switching valve, a HILIC analysis column, and a tandem mass spectrometer; The acidification reagent injection pump is used to pump the acidified clinical sample into the SCX enrichment column, wherein the enrichment column is equipped with a conductivity sensor for detecting the conductivity of the eluate; the dosing pump is used to pump the isotope internal standard into the SCX enrichment column according to a predetermined amount; The target obtained after passing through the SCX enrichment column is introduced into the gradient elution device, and the gradient elution program is executed. The intensity of the elution peak is monitored by the ultraviolet detection device. When the peak intensity decreases to the baseline level, it is switched to the next concentration for elution. The elution peak that meets the intensity requirements and the RT lock standard are injected into the HILIC analytical column through the flow path switching solenoid valve for processing. The target after processing by the HILIC analytical column is detected by the tandem mass spectrometer.

[0016] The beneficial effects of the present invention include: The present invention significantly improves the relative concentration and separation degree of low-abundance targets and reduces matrix interference through SCX online enrichment combined with multidimensional orthogonal separation (SCX-HILIC); significantly improves the probability of low-abundance ions being effectively qualitatively identified through intelligent triggering (dynamic window, noise threshold, intensity priority) and dynamic exclusion of mass spectrometry acquisition strategies; finally, through a correction method combining isotope internal standards and retention time locking, effectively overcomes the quantitative distortion caused by pretreatment losses, matrix effects and instrument fluctuations, thereby systematically solving the technical problems of qualitative omissions and quantitative distortion faced in the analysis of low-abundance metabolites / peptides in clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0018] Figure 1 This is a flowchart of the overall steps provided by an embodiment of the present invention.

[0019] Figure 2 Schematic diagram of the specific steps of mass spectrometry triggered mass spectrometry acquisition and processing provided in an embodiment of the present invention.

[0020] Figure 3 This is a block diagram of the overall system provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0021] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, this application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0022] Example 1 See also Figure 1As shown, the clinical tandem mass spectrometry analysis method based on multidimensional ion exchange includes the following steps: S1. Sample pretreatment: Acidification reagent is added to clinical samples to remove high-abundance protein interference and retain the target. The retained target is then injected into an online SCX enrichment column for target enrichment. The specific steps include: Sample pretreatment: After clinical samples (such as plasma and urine) are collected, they undergo preliminary processing to prevent sample degradation or contamination. Plasma samples need to be centrifuged to remove cell debris, and urine samples need to be diluted to reduce the concentration of organic matter. An acidifying solution (such as an aqueous solution containing 0.1% formic acid) is then added to the sample to adjust the pH value of the sample to 2.0-3.0. The purpose of acidification is to precipitate proteins while retaining target metabolites (such as amino acids and peptides) in a positively charged form to facilitate subsequent capture on the SCX enrichment column. Acidification can inhibit enzyme activity and prevent further degradation of metabolites. After acidification, the sample was filtered through a 0.22 μm filter membrane to remove particulate matter and unprecipitated proteins that blocked the chromatographic column; the filtered sample was subjected to an online SCX enrichment system; Online SCX enrichment: SCX enrichment columns utilize high-capacity, strong cation exchange fillers (e.g., sulfonic acid-functionalized silica gel). These columns operate in high-ionic-strength buffers (e.g., 500 mM NH4HCO3), enabling efficient capture of positively charged targets (e.g., amino acids, peptides). SCX enrichment columns are selected for their high selectivity for target species and high sample loading capacity, while also effectively removing neutral or weakly polar interferences (e.g., phospholipids, salt ions). The acidified sample is injected into the SCX enrichment column through an automatic sampler. In the high ionic strength eluent, the SCX enrichment column selectively captures the positively charged target, while neutral or weakly polar interferences flow out with the eluent and are directed to the waste liquid. The conductivity of the eluent gradually decreases during the matrix removal process. When the conductivity drops to a preset threshold (such as 10% of the initial conductivity), the matrix removal is determined to be complete and the enrichment process is terminated. The conductivity sensor is installed at the outlet of the SCX enrichment column to monitor the conductivity changes of the eluent in real time. The elution of high-abundance matrix will cause a significant decrease in conductivity. Therefore, when the conductivity drops to the preset threshold, the enrichment completion signal is triggered and the matrix flushing process is stopped.

[0023] S2. 1D separation: The SCX enrichment column was eluted stepwise using five increasing ammonium salt concentrations. The intensity of the elution peak was monitored using a UV detector. When the peak intensity dropped to the baseline level, the elution was switched to the next concentration. In this step of one-dimensional separation, five increasing ammonium salt concentrations (e.g., 50mM, 100mM, 150mM, 200mM, 500mM) are used to distribute the elution from the SCX enrichment column. Each ammonium salt concentration corresponds to a metabolite with a specific charge density: 50mM: used to elute singly charged amino acids; 100mM and 150mM: used to elute metabolites with medium charge density; 200mM and 500mM: used to elute multiply charged peptides; The eluent uses NH4HCO3 as a buffer to ensure a moderate pH value and avoid nonspecific adsorption of the target; After each elution is completed, it automatically switches to the next salt concentration. By controlling the ionic strength of the eluent, it ensures that only specific targets are eluted within each salt concentration window. The elution time of each salt concentration is optimized according to the charge density and concentration of the target to ensure that the target is completely eluted and avoid residues. Therefore, in order to optimize the elution time within each salt concentration window, the following model is established to obtain the elution time for each salt concentration: ; Where: Indicates elution time; represents the charge density of the target; Indicates the ionic strength concentration of the eluent; represents the charge density influence coefficient; represents the concentration influence coefficient; represents baseline time; After each salt pulse, the UV detector (214 nm) monitors the elution peak in real time. When the peak height drops to 10% of the baseline, it automatically switches to the next salt concentration. By monitoring the intensity and shape of the elution peak, it ensures that the target within each salt concentration window is completely eluted, avoiding residue and improving separation efficiency. The judgment logic for automatically switching to the next concentration is as follows: Calculate the peak height and peak area at each predetermined time: Determine whether the target is completely eluted based on the calculated peak height and peak area, and decide whether to switch the salt concentration. The judgment conditions are as follows: Peak height below threshold: ,in Indicates peak height; represents the baseline signal intensity; Deviation of peak area from expected value: ,in: Indicates the allowable deviation range (5%); represents the actual observed elution peak area; It represents the expected elution peak area, which is a value based on a combination of theoretical calculation and experimental experience; Check whether the peak height is below the threshold and the peak area deviation is within the allowable range. If so, switch to the next salt concentration; If the peak area is insufficient, the elution time is adjusted dynamically. Indicates adjustment time; In this embodiment, one-dimensional separation uses a stepped ionic strength elution technique to solve the problem of mass spectrometry signal suppression caused by co-elution of metabolites with similar charge properties in traditional gradient elution. By dividing the ionic strength of the eluent into multiple increasing discrete concentrations, each concentration corresponds to a component with a specific charge density, more efficient separation is achieved, co-elution is reduced, and detection sensitivity and accuracy are improved.

[0024] S3. 2D Separation: The eluted fractions from the first-dimensional separation are directed to a HILIC column for further separation. The HILIC column achieves polarity-dependent separation of the target compounds by varying the polarity of the mobile phase. In this embodiment, when the peak height of a one-dimensional SCX elution peak (such as an amino acid or peptide) is detected by the UV detector to be reduced to 10% of the baseline, the solenoid valve is triggered to switch. The valve switching directs the current elution peak from the SCX enrichment column to the HILIC analytical column, ensuring that each SCX elution peak independently enters the HILIC column for further separation; A highly polar stationary phase (e.g., amino- or diol-bonded silica) is used in the HILIC column to enhance the retention of highly polar metabolites. Mobile phase A is a highly organic solvent (e.g., 95% acetonitrile, 0.1% formic acid), and mobile phase B is an aqueous phase (e.g., 5% acetonitrile, 0.1% formic acid). A linear gradient elution is performed starting from 100% mobile phase A to 80% mobile phase A (gradient program), with the duration synchronized with the arrival time of the SCX elution peak. When the UV signal intensity of the SCX elution peak exceeds a preset threshold (e.g., 10 mAU), the HILIC gradient program is started. The HILIC gradient slope is dynamically adjusted based on the polarity differences of the target compounds to ensure adequate separation of similar compounds. The steps for dynamically adjusting the HILIC gradient slope are as follows: Initial gradient slope setting: Set an initial gradient slope according to the polarity range of the target compound , for example, the initial gradient slope is 5% acetonitrile / min; Real-time peak arrival time monitoring: At the outlet of the HILIC column, the arrival time of the elution peak is recorded in real time , where the expected arrival time is ; Calculate time deviation: Calculate time deviation based on real-time peak arrival time monitoring results : ; Adjust the gradient slope: If If it is greater than 0, it means the arrival time is later than expected, then the gradient slope is reduced; if If it is less than 0, it means that the actual arrival time is earlier than expected, and the gradient slope is increased; the adjustment formula is as follows: ; in: represents the adjusted gradient slope; Indicates the adjustment amount of the gradient slope, which is determined based on the following formula: ; Where: k represents the proportionality coefficient, which is determined based on experimental optimization (e.g., 0.5% acetonitrile / min·s); The HILIC gradient program is updated in real time based on the adjusted gradient slope.

[0025] In this embodiment, the gradient slope is dynamically adjusted based on the real-time arrival time and peak width of the elution peak to ensure that compounds with similar polarity are fully separated, reduce peak overlap, and significantly improve the separation of low-abundance compounds in complex samples. In addition, by dynamically adjusting the gradient slope, the gradient change rate of the mobile phase is optimized, thereby shortening the overall analysis time.

[0026] Furthermore, in this embodiment, an appropriate amount of buffer (e.g., 10 mM ammonium acetate) is added to the HILIC mobile phase to maintain pH stability and reduce matrix interference. In this embodiment, a UV detector is configured at the outlet of the HILIC column to monitor in real time the time point when the elution peak reaches the mass spectrometer.

[0027] In this example, the polarity separation dimension of HILIC and the charge separation dimension of SCX are mutually orthogonal, significantly improving the resolution of low-abundance compounds in complex samples. Furthermore, through real-time valve switching and gradient program activation, each SCX elution peak enters the HILIC column independently, avoiding peak mixing and signal overlap. Furthermore, in this example, SCX separates charged molecules based on charge differences (one-dimensional), while HILIC separates highly polar compounds based on polarity differences (two-dimensional). Both achieve physical orthogonal separation through valve switching, overcoming the solvent compatibility limitations of traditional two-dimensional LC (such as reversed-phase / reversed-phase).

[0028] S4. Mass spectrometry triggering and mass spectrometry acquisition and processing: see Figure 2 As shown in the figure, after the HILIC separation is completed, the mobile phase carries the separated components into the mass spectrometer, and first performs a rapid scan of the primary mass spectrometer (MS1). The main purpose of MS1 is to obtain the mass-to-charge ratio (m / z) information of all ions for subsequent analysis and triggering of tandem mass spectrometry (secondary mass spectrometry, MS2). Since the full scan of MS1 provides ion signals across the entire m / z range, baseline noise monitoring is performed based on this: Blank area analysis: Select an area without target compounds within the m / z range as a blank area and calculate the signal intensity fluctuation in this area: ; Where: Indicates the noise level in the blank area; Represents the signal strength of the i-th data point in the blank area; represents the mean signal strength of the blank area; N represents the total number of data points in the blank area; Adjacent Scan Difference: Compares the signal strength of the current scan with the previous scan and calculates the difference to assess the noise level: ; Where: represents the total signal intensity of the jth scan; M represents the number of scans; represents the total signal intensity of the j-1th scan; represents the noise level of adjacent scans; Sliding window method: Use a sliding window within the m / z range and calculate the standard deviation of the signal intensity within each window as the noise level in that area: ; Where: represents the signal strength of the lth data point in the kth sliding window; represents the average signal strength of the kth sliding window; L represents the number of data points in a single sliding window; K represents the total number of sliding windows; represents the noise level determined based on the sliding window method; In this embodiment, the current noise level is obtained by weighted summing the three methods, or selecting the maximum value of the noise levels obtained by the three methods as the current noise level; and the baseline noise level is obtained based on the average value of the historical noise levels; Initial window division: Based on the MS1 scanning range (e.g., 400-2000 m / z), the m / z range is divided into multiple initial windows: ; Where: Indicates the number of initial windows divided; Indicates the default window size; represents the total m / z range of the MS1 scan; Indicates rounding up; The default window size is dynamically adjusted based on the baseline noise level. ; Where: Indicates the adjusted window size; Indicates the window reduction factor; Indicates the window expansion factor; represents the noise threshold, which is obtained by multiplying the baseline noise level by a multiplication factor; Indicates the current noise level; based on the adjusted window size, it is brought into the initial window division formula to obtain the adjusted initial window number; In-window signal intensity monitoring: The ion signal intensity within each dynamic window is monitored in real time. When the signal intensity within the window exceeds the set trigger threshold, MS2 acquisition is triggered. The trigger threshold is 6 times the baseline noise level to ensure that meaningful signals are captured without introducing excessive noise. If there are multiple ions with signal intensities exceeding the trigger threshold in the same window, the multiple ions are sorted according to signal intensities, and only the first two ions with the highest signal intensities are triggered for MS2 acquisition. This can effectively avoid signal splitting and improve the efficiency and accuracy of data acquisition. The ion information that has been triggered for MS2 acquisition is recorded in the dynamic exclusion list, and an exclusion time is set for each triggered ion. During the exclusion time, the triggered ions will not be triggered again for MS2 acquisition. According to the set exclusion time, the ions outside the exclusion time are removed from the exclusion list, and the exclusion list is updated in real time.

[0029] S5. Quantitative calibration: quantitative calibration of the mass spectrometry data using an isotope internal standard calibration combined with a retention time locked standard; See also Figure 3 As shown, in step S1, during the sample pre-treatment stage, a labeled isotope internal standard (e.g. 13 C / 15 N marker); the internal standard has a different mass-to-charge ratio from the target compound, and this characteristic is utilized to allow it to be independently detected in the first scan (MS1) of the mass spectrometer; Inject RT Lock standard (retention time locking standard) to correct the retention time drift of the HILIC column. The timing of injecting RT Lock standard is as follows: Before switching to the next salt concentration, the RT Lock standard is injected directly into the elution flow path of the SCX enrichment column through the solenoid valve. After mixing with the eluent of the current salt concentration, it enters the HILIC column. The injection point is located in the flow path between the SCX enrichment column and the HILIC column, which is the end of the first-dimensional separation stage and the beginning of the second-dimensional separation stage. In step S4, MS1 will obtain the m / z values, signal intensities, HILIC retention times, and corresponding SCX salt concentration labels of all ions; MS2 will obtain fragment ion spectra triggered by the DIWT (sliding window) algorithm for compound qualitative matching; Quantitative calibration is performed based on the data obtained above. The specific steps are as follows: Data extraction and calibration of the dual internal standard system: Isotopic internal standard ions are screened based on known m / z values ​​(e.g., target compound m / z+6), and the corresponding SCX concentration and signal intensity (peak area) at the HILIC RT (indicating the elution time of the target compound and RT Lock standard on the 2D HILIC column) are obtained. Based on the fixed m / z of the RT Lock standard (e.g., m / z 507.0 for ATP), extract its measured retention time at the beginning of elution at each salt concentration; Calculate the internal standard correction factor and RT Lock retention time shift based on the acquired data: ; Where: represents the isotope internal standard correction factor; Indicates the theoretical amount of isotope internal standard added; It represents the peak area of ​​the isotope internal standard in the primary mass spectrum; ; Where: Indicates retention time offset; Indicates the measured retention time of the RT Lock standard; Indicates the theoretical retention time of RT Lock standard; Based on the above, we can get the isotope internal standard correction factor table (salt concentration, RT and correction factor) and RT Lock offset table (salt concentration, ); 2D peak alignment based on UV signal and RT Lock: In the one-dimensional stage, the UV detector will record the elution end time of each salt concentration pulse (such as the time point when the UV peak height reaches 10%) to determine the SCX elution window of the salt concentration component; therefore, in this step, the SCX salt concentration window corresponding to the target signal attribution value will be assigned according to the elution end time of the UV signal (for example, if an ion is switched to the HILIC column by the valve during the elution of SCX 100mM salt concentration, its salt concentration label will be 100mM); then, for the target at the corresponding salt concentration, the SCX salt concentration corresponding to the target signal will be assigned according to the elution end time of the UV signal. Calibrate the HILIC retention time using the values ​​of the target compound at 100 mM salt concentration in SCX (e.g., the target compound at 100 mM salt concentration should be calibrated using the retention time corresponding to that salt concentration). The HILIC retention time is calibrated using the following formula: ; Where: represents the original retention time; Indicates the calibrated retention time.

[0030] Based on this, the signals of the same compound in different salt concentration windows are merged (for example, a peptide segment is eluted in both SCX150mM and 200mM windows due to partial protonation) to form the final two-dimensional coordinate table.

[0031] Cross-dimensional signal integration and quantitative integration: By comparing MS2 fragment ions with the spectral library, the m / z of the target compound and all signal points in the two-dimensional coordinates are confirmed (for example, a peptide segment has elution signals at SCX 200mM salt concentration, HILIC RT 8min and SCX 500mM salt concentration, HILIC RT 15min); For all calibrated signal points of the same compound, the MS1 peak areas are accumulated according to the following formula: Where: Represents the total peak area of ​​the target compound; represents the peak area of ​​the i-th signal point; Indicates the isotope internal standard correction factor corresponding to the i-th signal point, which is dynamically calculated based on the RT Lock offset and the internal standard signal intensity; Divide the total peak area of ​​the target compound by the peak area of ​​the isotope internal standard to eliminate the difference in extraction efficiency between samples and obtain the quantitative integration result of the target compound: Where: Indicates the concentration of the target substance; represents the total peak area of ​​the isotope internal standard; represents the theoretical concentration of the isotope internal standard.

[0032] In this example, an RT Lock standard (e.g., ATP) was injected before elution at each salt concentration, and the HILIC column retention time drift (ΔRT) was monitored in real time. After calibration, the retention time error was controlled within ±0.2 min, addressing the peak misalignment problem caused by mobile phase fluctuations in two-dimensional separations and ensuring the accuracy of cross-sample peak alignment.

[0033] During the clinical sample processing process, the target object is easily lost due to steps such as protein precipitation and chromatographic separation, resulting in quantitative deviation. It has the same chemical properties as the target object, with only a difference in mass-to-charge ratio, and can go through the entire sample processing process synchronously with the target object. This solution adds an isotope internal standard during the sample pre-processing stage; by calculating the correction factor, it can accurately compensate for the loss of the target object during the extraction, enrichment and separation processes, greatly improving the accuracy of the quantitative results and avoiding false negative or false positive results due to sample processing losses.

[0034] Secondly, during two-dimensional separations, the retention time of the HILIC column is susceptible to drift due to factors such as changes in mobile phase composition and temperature fluctuations, resulting in chromatographic peak misalignment and affecting quantitative accuracy. In this example, the RT Lock standard was injected into the SCX column before each salt concentration switch, and calibration was performed by calculating the retention time offset. This effectively solved the peak misalignment problem in two-dimensional separations, ensured the precise alignment of chromatographic peaks between different samples, and laid the foundation for subsequent cross-dimensional signal integration.

[0035] Secondly, in clinical samples, low-abundance metabolites often have their signals masked or suppressed due to coelution. This protocol uses calibration data provided by a dual internal standard system, combined with a two-dimensional coordinate table (SCX salt concentration and HILIC retention time), to integrate the signals of the same compound across different separation dimensions. This protocol also performs internal standard-normalized quantification. This effectively avoids signal loss due to coelution in single-dimensional separations and improves detection sensitivity for low-abundance compounds. Example

[0036] See also Figure 3 As shown, a clinical tandem mass spectrometry analysis system based on multidimensional ion exchange includes an acidification reagent injection pump, a quantitative pump, an SCX enrichment housing, a gradient elution device, a flow path switching valve, a HILIC analytical column, and a tandem mass spectrometer; The acidification reagent injection pump is used to pump the acidified clinical sample into the SCX enrichment column, wherein the enrichment column is equipped with a conductivity sensor for detecting the conductivity of the eluate; the dosing pump is used to pump the isotope internal standard into the SCX enrichment column according to a predetermined amount; The target obtained after passing through the SCX enrichment column is introduced into the gradient elution device, and the gradient elution program is executed. The intensity of the elution peak is monitored by the ultraviolet detection device. When the peak intensity decreases to the baseline level, it is switched to the next concentration for elution. The elution peak that meets the intensity requirements and the RT lock standard are injected into the HILIC analytical column through the flow path switching solenoid valve for processing. The target after processing by the HILIC analytical column is detected by the tandem mass spectrometer.

[0037] See also Figure 3 As shown, the isotopic internal standard is stored in an isotopic internal standard storage pool and pumped into the SCX enrichment column by a quantitative pump; the RT Lock standard is stored in the RT Lock standard storage pool and introduced into the HILIC analytical column through a flow path switching solenoid valve; a peak detection sensor is provided at the outlet of the HILIC analytical column to provide real-time feedback and drive gradient optimization, and the peak detection sensor also uses a UV detector; A peak detection sensor is provided at the outlet of the HILIC analytical column to monitor the arrival time of the HILIC elution peak in real time, calculate its deviation from the expected time, and dynamically adjust the gradient slope based on the deviation.

[0038] The above is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A clinical tandem mass spectrometry analysis method based on multidimensional ion exchange, characterized in that: The following steps are involved: Sample pretreatment: Acidification reagent is added to clinical samples to remove high-abundance protein interference and retain the target. The retained target is injected into the online SCX enrichment column for target enrichment; One-dimensional separation: The SCX enrichment column is eluted stepwise using five increasing ammonium salt concentrations, and the intensity of the elution peak is monitored using a UV detector. When the peak intensity drops to the baseline level, the elution is switched to the next concentration and the currently eluted target is directed to the HILIC column. Two-dimensional separation: The eluted components after one-dimensional separation are directed to the HILIC column for further separation. The HILIC column achieves polarity-differential separation of the target compounds by changing the polarity of the mobile phase. Mass spectrometry triggers mass spectrometry acquisition and processing: The components eluted after HILIC treatment are collected by the mass spectrometer for primary mass spectrometry to obtain the mass-to-charge ratio information of all ions. The mass-to-charge ratio range is then divided into dynamic windows based on the real-time baseline noise level. When the signal intensity within the window exceeds 6× the baseline noise, the secondary mass spectrometer is triggered. If multiple ions exist in the same window, only the top two ions with the highest intensity are triggered. At the same time, the triggered ions are added to the dynamic exclusion list, based on which the mass spectrometry detection data is obtained. Quantitative calibration: The mass spectrometry data were quantitatively calibrated using a combination of isotope internal standard calibration and retention time locked standards.

2. The clinical tandem mass spectrometry analysis method based on multidimensional ion exchange according to claim 1, characterized in that: In the sample pretreatment, the target in the SCX enrichment column is enriched with an eluent, and the conductivity change of the eluent is monitored in real time. When the conductivity change is lower than a preset threshold, it is determined that the matrix removal is complete and the enrichment process is finished.

3. The clinical tandem mass spectrometry analysis method based on multidimensional ion exchange according to claim 1, characterized in that: In the one-dimensional separation, based on the baseline time, a weighted sum value based on the charge density of the target and the ionic strength concentration of the eluent is introduced to establish an elution time optimization model, and the elution time of each ammonium salt concentration is obtained based on the established elution time optimization model; The peak height and peak area are calculated at each predetermined time, and whether the target is completely eluted is determined based on the calculated peak height and peak area. If the peak height is lower than the preset threshold and the peak area deviation is within the allowable range, the next salt concentration is switched; if the peak area is insufficient, the elution time is adjusted based on the preset adjustment time and the elution time obtained after optimization based on the elution optimization model.

4. The clinical tandem mass spectrometry analysis method based on multidimensional ion exchange according to claim 1, characterized in that: In the two-dimensional separation, mobile phase A in the HILIC analytical column is a high organic solvent, and mobile phase B is an aqueous phase. Starting from 100% mobile phase A, a linear gradient elution is performed to 80% mobile phase A, and the duration is synchronized with the arrival time of the SCX elution peak.

5. The clinical tandem mass spectrometry analysis method based on multidimensional ion exchange according to claim 4, characterized in that: The slope of the linear gradient is dynamically adjusted, and the specific steps are as follows: Initial gradient slope setting: Set an initial gradient slope according to the polarity range of the target compound; Real-time peak arrival time monitoring: At the outlet of the HILIC column, the arrival time of the elution peak is recorded in real time; Calculate time deviation: Calculate time deviation based on the real-time peak arrival time monitoring results and the expected arrival time; Adjusting the gradient slope: multiplying the absolute value of the time deviation by the proportional coefficient to obtain an adjustment amount of the gradient slope; adjusting the gradient slope based on the adjustment amount to obtain an adjusted gradient slope, wherein the gradient slope is reduced when the time deviation is greater than zero, and the gradient slope is increased when the time deviation is less than zero.

6. The clinical tandem mass spectrometry analysis method based on multidimensional ion exchange according to claim 1, characterized in that: The dynamic window division in the mass spectrometry triggered mass spectrometry acquisition and processing includes the following steps: According to the scanning range of the primary mass spectrometer, the mass-to-charge ratio range is divided into a plurality of initial windows, wherein the initial window is the ratio of the total mass-to-charge ratio range of the primary mass spectrometer scan to the default window size; The default window size is dynamically adjusted based on the baseline noise level. If the real-time noise level is greater than a preset noise threshold, the default window is reduced to obtain an updated window size; if the real-time noise level is less than the preset noise threshold, the default window is expanded to obtain an updated window size; wherein the noise threshold is obtained based on the baseline noise level multiplied by a multiplication coefficient.

7. The clinical tandem mass spectrometry analysis method based on multidimensional ion exchange according to claim 1, characterized in that: adding a labeled isotope internal standard during the sample pretreatment stage; At the end of the first-dimensional separation stage and before switching to the next ammonium salt concentration, the RT Lock standard is injected directly into the elution flow path of the SCX enrichment column through the solenoid valve. After mixing with the eluent of the current salt concentration, it enters the HILIC column. The injection point is located in the flow path between the SCX enrichment column and the HILIC column.

8. The clinical tandem mass spectrometry analysis method based on multidimensional ion exchange according to claim 1, characterized in that: The quantitative calibration comprises the following steps: Data extraction and calibration of the dual internal standard system: Isotopic internal standard ions are screened based on known mass-to-charge ratios to obtain their corresponding SCX concentrations and signal intensities at the HILIC RT. Based on the fixed mass-to-charge ratio of the RT Lock standard, the measured retention time at the beginning of elution at each ammonium salt concentration is extracted. Internal standard correction factor and retention time offset calculation: Calculate the isotope internal standard correction factor and RTLock retention time offset based on the acquired data to form the isotope internal standard correction factor table and the RT Lock offset table; Two-dimensional peak alignment of UV signal and RT Lock: According to the end time of UV signal elution, the target signal is assigned to the corresponding SCX salt concentration window. The RT Lock offset corresponding to the salt concentration is used to calibrate the HILIC retention time. The signals of the same compound in different salt concentration windows are combined to form a two-dimensional coordinate table. Fast dimensional signal integration and quantitative integration: By comparing the fragmented ions of the secondary mass spectrometer with the spectral library, the mass-to-charge ratio of the target and all its signal points in the two-dimensional coordinates are confirmed. For all calibrated signal points of the same compound, the primary mass spectrum peak area is accumulated to eliminate the difference in extraction efficiency between samples and obtain the quantitative integration result of the target.

9. A clinical tandem mass spectrometry analysis system based on multidimensional ion exchange, characterized in that: Including acidification reagent injection pump, quantitative pump, SCX enrichment unit, gradient elution device, flow path switching valve, HILIC analytical column and tandem mass spectrometer; The acidification reagent injection pump is used to pump the acidified clinical sample into the SCX enrichment column, wherein the enrichment column is equipped with a conductivity sensor for detecting the conductivity of the eluate; the dosing pump is used to pump the isotope internal standard into the SCX enrichment column according to a predetermined amount; The target obtained after passing through the SCX enrichment column is introduced into the gradient elution device, and the gradient elution program is executed. The intensity of the elution peak is monitored by the ultraviolet detection device. When the peak intensity decreases to the baseline level, it is switched to the next concentration for elution. The elution peak that meets the intensity requirements and the RT lock standard are injected into the HILIC analytical column through the flow path switching solenoid valve for processing. The target after processing by the HILIC analytical column is detected by the tandem mass spectrometer.

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