Clinical Tandem Mass Spectrometry Analysis Method and System Based on Multidimensional Ion Exchange

Abstract

This 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. This invention significantly improves the relative concentration and resolution of low-abundance target analytes and reduces matrix interference by combining online SCX enrichment with multidimensional orthogonal separation (SCX-HILIC). Through intelligent triggering (dynamic window, noise threshold, intensity priority) and dynamic exclusion mass spectrometry acquisition strategies, the probability of effective qualitative identification of low-abundance ions is significantly improved. Finally, through a correction method combining isotope internal standard and retention time locking, quantitative distortion caused by pretreatment loss, matrix effects and instrument fluctuations is effectively overcome, thereby systematically solving the technical problems of qualitative omission and quantitative distortion faced in the analysis of low-abundance metabolites / peptides in clinical samples.

Description

Technical Field

[0001] This 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 Technology

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

[0003] Currently, over 90% of clinical laboratories use reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS / MS), which relies on hydrophobic separation mechanisms and exposes two major drawbacks in its application:

[0004] Separation failure of highly polar compounds: Positively charged / highly polar target compounds (such as creatinine acid and glutathione) are retained very weakly on RPLC columns and enter the mass spectrometer together with high-abundance matrices (such as sodium chloride and choline) that elute with the dead time, causing severe ion inhibition.

[0005] Co-elution leads to quantitative distortion: In complex samples, compounds with similar charge properties (such as arginine and lysophosphatidylcholine) are co-eluted in a one-dimensional gradient, and mass spectrometry cannot distinguish ions with the same m / z, triggering competitive suppression of the signal;

[0006] 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:

[0007] Lack of solvent orthogonality: Commonly used reversed-phase / reversed-phase combinations cannot separate differences in charge dimension; while when strong cation exchange (SCX) is connected in series with reversed-phase (RPLC), the high-salt eluent of SCX is incompatible with the organic phase of RPLC, resulting in column focusing failure and severe peak broadening;

[0008] Insufficient dynamic range coverage: Traditional data-dependent acquisition (DDA) uses a static m / z window for triggering, which only fragments high-abundance ions in the co-elution peak, ignoring low-abundance target analytes (<100 pg).

[0009] In summary, existing one-dimensional or online two-dimensional methods suffer from a dual shortcoming of "separation-detection" in the analysis of complex clinical samples:

[0010] Separation dimensions: Single-dimensional RPLCs do not retain strong polar substances well, and two-dimensional systems lack true physical orthogonal separation (charge × polarity), resulting in key low-abundance components being masked by the matrix;

[0011] Detection logic: Static triggering strategy cannot adapt to dynamic elution process, and competition among co-eluent ions leads to low mass spectrometry acquisition efficiency;

[0012] This ultimately leads to the omission of qualitative and quantitative information about low-abundance metabolites / peptides, severely hindering the translational application of clinical biomarkers. Summary of the Invention

[0013] This 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.

[0014] On the one hand, this invention provides a clinical tandem mass spectrometry analysis method based on multidimensional ion exchange, characterized by comprising the following steps:

[0015] Sample pretreatment: Add acidification reagent to clinical samples to remove high-abundance protein interference, retain target analytes, and inject the retained target analytes into an online SCX enrichment column for target analyte enrichment;

[0016] One-dimensional separation: The SCX enrichment column is eluted stepwise with five increasing ammonium salt concentrations, and the intensity of the elution peak is monitored by an ultraviolet detector. When the peak intensity decreases to the baseline level, the next concentration is switched for elution, and the target analyte eluted is directed to the HILIC column.

[0017] Two-dimensional separation: The eluted components after one-dimensional separation are guided to a HILIC column for further separation. The HILIC column achieves separation based on the polarity difference of the target analytes by changing the polarity of the mobile phase.

[0018] Mass spectrometry triggering mass spectrometry acquisition and processing: The eluted components after HILIC processing are acquired by mass spectrometry in the first stage to obtain the mass-to-charge ratio information of all ions. Then, based on the real-time baseline noise level, the mass-to-charge ratio range is divided into dynamic windows. When the signal intensity within the window exceeds 6 × baseline noise, the second stage mass spectrometry is triggered. If multiple ions exist in the same window, only the two ions with the highest intensity are triggered. At the same time, the triggered ions are added to a dynamic exclusion list, based on which the mass spectrometry detection data is obtained.

[0019] Quantitative correction: The mass spectrometry data are quantitatively corrected by a combination of isotope internal standard correction and retention time-locked standard.

[0020] This invention significantly improves the relative concentration and resolution of low-abundance target analytes and reduces matrix interference through online SCX enrichment combined with multidimensional orthogonal separation (SCX-HILIC). It also significantly increases the probability of effective qualitative identification of low-abundance ions through intelligent triggering (dynamic window, noise threshold, intensity priority) and dynamic exclusion mass spectrometry acquisition strategies. Finally, it effectively overcomes quantitative distortion caused by pretreatment losses, matrix effects, and instrument fluctuations through a correction method combining isotope internal standards and retention time locking. This systematically solves the technical challenges of qualitative omissions and quantitative distortions in the analysis of low-abundance metabolites / peptides in clinical samples.

[0021] Preferably, in the sample pretreatment, the target analyte in the SCX enrichment column is enriched using an elution buffer, and the conductivity change of the elution buffer is monitored in real time. When the conductivity change is lower than a preset threshold, the matrix removal is determined to be complete, and the enrichment process ends.

[0022] Preferably, in the one-dimensional separation, a weighted sum of the charge density of the target analyte and the ionic strength concentration of the eluent is introduced based on 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.

[0023] At each predetermined time, the peak height and peak area are calculated. Based on the calculated peak height and peak area, it is determined whether the target substance has been completely eluted. 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.

[0024] Preferably, in the two-dimensional separation, the mobile phase A in the HILIC analytical column is a high organic solvent and the mobile phase B is an aqueous phase. Starting from 100% flow towards A, linear gradient elution is performed until 80% mobile phase A is reached, and the duration is synchronized with the arrival time of the SCX elution peak.

[0025] Preferably, the slope of the linear gradient is dynamically adjusted, and the specific steps are as follows:

[0026] Initial gradient slope setting: Set an initial gradient slope based on the polarity range of the target compound;

[0027] Real-time peak arrival time monitoring: The arrival time of the elution peak is recorded in real time at the outlet of the HILIC column;

[0028] Calculate the time deviation: Based on the real-time peak arrival time monitoring results and the expected arrival time, calculate the time deviation;

[0029] Adjusting the gradient slope: Multiply the absolute value of the time deviation by the proportional coefficient to obtain the adjustment amount of the gradient slope; adjust the gradient slope based on the adjustment amount to obtain the adjusted gradient slope, wherein the gradient slope is decreased when the time deviation is greater than zero, and the gradient slope is increased when the time deviation is less than zero.

[0030] Preferably, the dynamic window division in the mass spectrometry-triggered mass spectrometry acquisition and processing includes the following steps:

[0031] Based on the scanning range of the first-level mass spectrometer, the mass-to-charge ratio range is divided into multiple initial windows, where the initial window is the ratio of the total mass-to-charge ratio range of the first-level mass spectrometer scan to the default window size.

[0032] 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 shrunk to obtain the updated window size; if the real-time noise level is less than the preset noise threshold, the default window is enlarged to obtain the updated window size. The noise threshold is obtained by multiplying the baseline noise level by a multiplier.

[0033] Preferably, a labeled isotope internal standard is added during the sample pretreatment stage;

[0034] At the end of the one-dimensional separation stage and before switching to the next ammonium salt concentration, the RT Lock standard is directly injected into the elution flow path of the SCX enrichment column through a solenoid valve. After mixing with the current salt concentration eluent, it enters the HILIC column. The injection point is located in the flow path between the SCX enrichment column and the HILIC column.

[0035] Preferably, the quantitative correction includes the following steps:

[0036] Data extraction and calibration of the dual internal standard system: Isotope internal standard ions were selected based on known mass-to-charge ratios, and their corresponding SCX concentrations and signal intensities at HILIC RT were obtained; Based on the fixed mass-to-charge ratio of the RT Lock standard, the measured retention time at the start of elution at each ammonium salt concentration was extracted.

[0037] Internal standard correction factor and retention time offset calculation: Based on the acquired data, the isotope internal standard correction factor and RT Lock retention time offset are calculated to form an isotope internal standard correction factor table and an RT Lock offset table;

[0038] Two-dimensional peak alignment of UV signal and RT Lock: Based on the elution end time of UV signal, the target signal is assigned to the corresponding SCX salt concentration window, the HILIC retention time is calibrated using the RT Lock offset corresponding to the salt concentration, and the signals of the same compound in different salt concentration windows are merged to form a two-dimensional coordinate table.

[0039] Fast-dimensional signal integration and quantitative integration: By comparing fragmented ions from secondary mass spectrometry with the spectral library, the mass-to-charge ratio of the target analyte and all its signal points in two-dimensional coordinates are confirmed. For all calibrated signal points of the same compound, the peak areas of the primary mass spectrometry are summed to eliminate the differences in extraction efficiency between samples and obtain the quantitative integration result of the target analyte.

[0040] On the other hand, the present invention provides a clinical tandem mass spectrometry analysis system based on multidimensional ion exchange, including an acidification reagent injection pump, a quantitative pump, an SCX enrichment pump, a gradient elution device, a flow path switching valve, a HILIC analysis column, and a tandem mass spectrometer.

[0041] 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 eluent; the metering pump is used to pump the isotope internal standard into the SCX enrichment column at a predetermined amount.

[0042] The target analyte obtained after passing through the SCX enrichment column is introduced into the gradient elution device to execute the gradient elution program. The intensity of the elution peak is monitored by a UV detection device. When the peak intensity decreases to the baseline level, the elution is switched to the next concentration. The elution peak that meets the intensity requirements and the RT lock standard are injected into the HILIC analysis column for processing through the flow path switching solenoid valve. The target analyte after processing by the HILIC analysis column is detected by the tandem mass spectrometer.

[0043] The beneficial effects of this invention include:

[0044] This invention significantly improves the relative concentration and resolution of low-abundance target analytes and reduces matrix interference through online SCX enrichment combined with multidimensional orthogonal separation (SCX-HILIC). It also significantly increases the probability of effective qualitative identification of low-abundance ions through intelligent triggering (dynamic window, noise threshold, intensity priority) and dynamic exclusion mass spectrometry acquisition strategies. Finally, it effectively overcomes quantitative distortion caused by pretreatment losses, matrix effects, and instrument fluctuations through a correction method combining isotope internal standards and retention time locking. This systematically solves the technical challenges of qualitative omissions and quantitative distortions in the analysis of low-abundance metabolites / peptides in clinical samples. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1This is an overall step diagram provided for an embodiment of the present invention.

[0047] Figure 2 This is a schematic diagram illustrating the specific steps of mass spectrometry-triggered mass spectrometry acquisition and processing provided in an embodiment of the present invention.

[0048] Figure 3 This is an overall system block diagram provided for an embodiment of the present invention. Detailed Implementation

[0049] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0050] Example 1

[0051] See Figure 1 As shown, the clinical tandem mass spectrometry analysis method based on multidimensional ion exchange includes the following steps:

[0052] S1. Sample pretreatment: Add acidification reagent to clinical samples to remove high-abundance protein interference, retain target analytes, and inject the retained target analytes into an online SCX enrichment column for target analyte enrichment;

[0053] Specifically, the following steps are included:

[0054] Sample pretreatment: After collection, clinical samples (such as plasma and urine) undergo preliminary treatment 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. Then, an acidification solution (such as an aqueous solution containing 0.1% formic acid) is added to the sample to adjust the pH value 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, which facilitates capture by the subsequent SCX enrichment column. Acidification can also inhibit enzyme activity and prevent further degradation of metabolites.

[0055] After acidification, the sample was filtered through a 0.22 μm filter membrane to remove particulate matter clogging the chromatographic column and unprecipitated proteins; the filtered sample was then subjected to an online SCX enrichment system.

[0056] Online SCX enrichment: The SCX enrichment column uses high-capacity, strong cation exchange packing material (such as sulfonic acid-functionalized silica gel). The SCX enrichment column operates in a high ionic strength (such as 500mM NH4HCO3) buffer, enabling efficient capture of positively charged targets (such as amino acids and peptides). The selection of the SCX enrichment column is based on its high selectivity for targets and high sample loading capacity, while effectively removing neutral or weakly polar interfering substances (such as phospholipids and salt ions).

[0057] After acidification, the sample is injected into the SCX enrichment column via an autosampler. In the high ionic strength eluent, the SCX enrichment column selectively captures positively charged target substances, while neutral or weakly polar interfering substances flow out with the eluent and are guided to the waste liquid. The conductivity of the eluent gradually decreases during matrix removal. When the conductivity drops to a preset threshold (e.g., 10% of the initial conductivity), matrix removal is considered complete, and the enrichment process ends. A 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 leads to a significant decrease in conductivity. Therefore, when the conductivity drops to the preset threshold, an enrichment completion signal is triggered, stopping the matrix rinsing process.

[0058] S2. One-dimensional separation: The SCX enrichment column is eluted stepwise with five increasing ammonium salt concentrations, and the intensity of the elution peak is monitored by an ultraviolet detector. When the peak intensity decreases to the baseline level, the next concentration is switched for elution.

[0059] In the one-dimensional separation of this step, the SCX enrichment column was eluted using five incremental ammonium salt concentrations (e.g., 50 mM, 100 mM, 150 mM, 200 mM, 500 mM), with each ammonium salt concentration corresponding to a specific charge density of the metabolite.

[0060] 50mM: Used for eluting single-charged amino acids;

[0061] 100mM and 150mM: used for eluting metabolites with moderate charge density;

[0062] 200mM and 500mM: used for eluting multi-charged peptides;

[0063] The elution buffer uses NH4HCO3 as a buffer to ensure a suitable pH value and avoid non-specific adsorption of the target analyte.

[0064] After each elution, the system 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 for each salt concentration is optimized based on the charge density and concentration of the target analyte to ensure complete elution and avoid residue. Therefore, to optimize the elution time within each salt concentration window, the following model is established to obtain the elution time for each salt concentration: ;

[0065] In the formula: Indicates the washout time; Indicates the charge density of the target object; Indicates the ionic strength concentration of the eluent; Indicates the influence coefficient of charge density; Indicates the concentration effect coefficient; Indicates baseline time;

[0066] After each salt pulse, an ultraviolet detector (214nm) monitors the elution peak in real time. When the peak height drops to 10% of the baseline, the system automatically switches to the next salt concentration. By monitoring the intensity and shape of the elution peak, the system ensures that the target analyte within each salt concentration window is completely eluted, avoiding residue and improving separation efficiency.

[0067] The logic for automatically switching to the next concentration is as follows:

[0068] Calculate peak height and peak area at each predetermined time point: Determine whether the target analyte has been completely eluted based on the calculated peak height and peak area, and determine whether to switch the salt concentration, based on the following conditions:

[0069] Peak height is below the threshold: ,in Indicates peak height; Indicates baseline signal strength;

[0070] Deviation between peak area and expected value: ,in: Indicates the allowable deviation range (5%); This represents the actual observed elution peak area; This represents the expected elution peak area, a value based on a combination of theoretical calculations and experimental experience.

[0071] Check if the peak height is below the threshold and the peak area deviation is within the allowable range. If the conditions are met, switch to the next salt concentration.

[0072] If the peak area is insufficient, the elution time should be dynamically adjusted. Indicates the adjustment time;

[0073] In this embodiment, one-dimensional separation employs a stepped ion intensity elution technique, which solves the problem of mass spectrometry signal suppression caused by co-elution of metabolites with similar charge properties in traditional gradient elution. By dividing the ion intensity of the eluent into multiple incremental discrete concentrations, each concentration corresponding to a component with a specific charge density, more efficient separation is achieved, co-elution phenomenon is reduced, and detection sensitivity and accuracy are improved.

[0074] S3. Two-dimensional separation: The eluted components after one-dimensional separation are guided to a HILIC column for further separation. The HILIC column achieves separation based on the polarity difference of the target analytes by changing the polarity of the mobile phase.

[0075] 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 decrease to 10% of the baseline, the solenoid valve is switched. The valve switch directs the current elution peak from the SCX enrichment column to the HILIC analysis column, ensuring that each SCX elution peak enters the HILIC column independently for further separation.

[0076] A highly polar stationary phase (such as amino or glycol-bonded silica gel) is used in the HILIC analytical column to enhance the retention of highly polar metabolites. Mobile phase A is a high organic solvent (such as 95% acetonitrile, 0.1% formic acid), and mobile phase B is an aqueous phase (such as 5% acetonitrile, 0.1% formic acid). Starting from 100% mobile phase A, linear gradient elution is performed to 80% mobile phase A (gradient program), with the duration synchronized with the arrival time of the SCX elution peak.

[0077] When the UV signal intensity of the SCX elution peak exceeds a preset threshold (e.g., 10 mAU), the HILIC gradient program is initiated. The HILIC gradient slope is dynamically adjusted based on the polarity differences of the target compounds to ensure sufficient separation of similar compounds. The steps for dynamically adjusting the HILIC gradient slope are as follows:

[0078] Initial gradient slope setting: Based on the polarity range of the target compound, set an initial gradient slope. For example, the initial gradient slope is 5% acetonitrile / min;

[0079] Real-time peak arrival time monitoring: The arrival time of the elution peak is recorded in real time at the HILIC column outlet. The expected arrival time is ;

[0080] Calculate the time deviation: Calculate the time deviation based on the real-time peak arrival time monitoring results. : ;

[0081] Adjust the gradient slope: If If the value is greater than 0, it indicates that the arrival time is later than expected, so the gradient slope should be reduced; if... A value less than 0 indicates that the actual arrival time is earlier than expected, thus increasing the gradient slope; the adjustment formula is as follows: ;

[0082] in: Indicates the adjusted gradient slope; The adjustment amount representing the gradient slope is determined based on the following formula: ;

[0083] In the formula: k represents the proportionality coefficient, which is determined based on experimental optimization (e.g., 0.5% acetonitrile / min·s);

[0084] The HILIC gradient procedure is updated in real time based on the adjusted gradient slope.

[0085] In this embodiment, the gradient slope is dynamically adjusted based on the real-time arrival time and peak width of the elution peaks to ensure that compounds with similar polarities are fully separated, reduce peak overlap, significantly improve the separation of low-abundance compounds in complex samples, and optimize the rate of change of the mobile phase gradient by dynamically adjusting the gradient slope, thereby shortening the overall analysis time.

[0086] Furthermore, in this embodiment, an appropriate amount of buffer solution (such as 10mM ammonium acetate) is added to the HILIC mobile phase to maintain pH stability and reduce matrix interference; and in this embodiment, an ultraviolet detector is configured at the HILIC column outlet to monitor the time point at which the elution peak reaches the mass spectrometer in real time.

[0087] In this embodiment, the polarity separation dimension of HILIC and the charge separation dimension of SCX are orthogonal to each other, which significantly improves the separation of low-abundance compounds in complex samples. Furthermore, by switching valves and starting the gradient program in real time, it is ensured that each SCX elution peak enters the HILIC column independently, avoiding peak mixing and signal overlap. In this embodiment, SCX separates charged molecules based on charge difference (one-dimensional), while HILIC separates strongly polar compounds based on polarity difference (two-dimensional). The two achieve physical orthogonal separation through valve switching, breaking through the solvent compatibility limitations of traditional two-dimensional LC (such as reversed phase / reverse phase).

[0088] S4. Mass Spectrometry Triggered Mass Spectrometry Acquisition and Processing: See [link / details] Figure 2 As shown, after HILIC separation, the mobile phase carries the separated components into the mass spectrometer. First, a rapid scan of the first-stage mass spectrometer (MS1) is performed. The main purpose of MS1 is to obtain the mass-to-charge ratio (m / z) information of all ions for subsequent analysis and to trigger tandem mass spectrometry (second-stage mass spectrometry, MS2).

[0089] Since the full scan of MS1 provides ion signals across the entire m / z range, baseline noise monitoring is performed based on this:

[0090] Blank region analysis: Regions without the target compound within the m / z range are selected as blank regions, and the signal intensity fluctuations in these regions are calculated. ;

[0091] In the formula: Indicates the noise level in the blank area; This represents the signal strength of the i-th data point in the blank area; This represents the average signal strength in the blank area; N represents the total number of data points in the blank area.

[0092] Adjacent Scan Difference: Compare the signal strength of the current scan with that of the previous scan, calculate the difference value to assess the noise level. ;

[0093] In the formula: The total signal strength is represented by M in the j-th scan; M represents the number of scans. This represents the total signal strength during the (j-1)th scan; Indicates the noise level of adjacent scans;

[0094] Sliding window method: Using a sliding window within the m / z range, calculate the standard deviation of the signal strength within each window, which is taken as the noise level of that region. ;

[0095] In the formula: This represents the signal strength of the l-th data point in the k-th sliding window; represents the average signal strength of the k-th sliding window; L represents the number of data points in a single sliding window; K represents the total number of sliding windows; This represents the noise level determined based on the sliding window method.

[0096] In this embodiment, the current noise level is obtained by weighted summation in three ways, or by selecting the maximum value of the noise level obtained in the three ways as the current noise level; while the baseline noise level is obtained based on the average of historical noise levels.

[0097] Initial window division: Based on the scan range of MS1 (e.g., 400-2000 m / z), the m / z range is divided into multiple initial windows: ;

[0098] In the formula: Indicates the initial number of windows in the partition; Indicates the default window size; This indicates the total m / z range of the MS1 scan; Indicates rounding up;

[0099] The default window size is dynamically adjusted based on the baseline noise level. ;

[0100] In the formula: This indicates the adjusted window size; Indicates the window scaling factor; Indicates the window scaling factor; The noise threshold is represented by a multiplier based on the baseline noise level. This represents the current noise level; based on the adjusted window size, it is substituted into the initial window partitioning formula to obtain the adjusted initial number of windows;

[0101] Signal intensity monitoring within the window: 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.

[0102] If the signal strength of multiple ions exceeds the trigger threshold within the same window, the multiple ions are sorted according to their signal strength, and only the two ions with the highest signal strength are triggered for MS2 acquisition. This can effectively avoid signal splitting and improve the efficiency and accuracy of data acquisition.

[0103] Ions that have been triggered for MS2 acquisition are recorded in a dynamic exclusion list. An exclusion time is set for each triggered ion. During the exclusion time, triggered ions will not be triggered for MS2 acquisition again. Ions outside the set exclusion time are removed from the exclusion list, and the exclusion list is updated in real time.

[0104] S5. Quantitative correction: The mass spectrometry detection data are quantitatively corrected by combining isotope internal standard correction with retention time-locked standard.

[0105] See Figure 3 As shown, in step S1, during the sample pretreatment stage, a labeled isotope internal standard (e.g., ...) is added. 13 C / 15 The internal standard (N-labeled analyte) has a different mass-to-charge ratio than the target analyte, and this property is used to detect it independently in the first-order mass spectrometry scan (MS1).

[0106] And inject RT Lock standard (retention time lock standard) to correct retention time drift of HILIC column, wherein the timing of injecting RT Lock standard is as follows:

[0107] Before switching to the next salt concentration, the RT Lock standard is directly injected into the elution flow path of the SCX enrichment column through the solenoid valve. After being mixed with the current salt concentration eluent, 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 one-dimensional separation stage and the beginning of the two-dimensional separation stage.

[0108] In step S4, MS1 will obtain the m / z values, signal intensity, HILIC retention time, and corresponding SCX salt concentration labels for all ions;

[0109] MS2 will produce fragment ion spectra triggered by the DIWT (sliding window) algorithm for qualitative matching of compounds;

[0110] Based on the data obtained above, quantitative correction is performed, and the specific steps are as follows:

[0111] Data extraction and calibration of the dual internal standard system:

[0112] Isotope internal standard ions are screened based on known m / z (e.g., target m / z+6), and their corresponding SCX concentrations and signal intensities (peak areas) at HILIC RT (representing the elution time of the target compound and RT Lock standard in a two-dimensional HILIC column) are obtained.

[0113] Based on the fixed m / z of the RT Lock standard (e.g., m / z 507.0 for ATP), extract the measured retention time at the start of elution at each salt concentration;

[0114] Based on the acquired data, the internal standard correction factor and RT Lock retention time offset are calculated:

[0115] ;

[0116] In the formula: Indicates the isotope internal standard correction factor; This indicates the amount of isotope internal standard theoretically added; This indicates the peak area of ​​the isotope internal standard in the primary mass spectrum. ;

[0117] In the formula: Indicates the retention time offset; Indicates the actual measured retention time of the RT Lock standard product; Indicates the theoretical retention time of the RT Lock standard product;

[0118] Based on the above, we obtained the isotope internal standard correction factor table (including salt concentration, RT, and correction factor) and the RT Lock offset table (including salt concentration, ...). );

[0119] Based on two-dimensional peak alignment between ultraviolet signals and RT Lock:

[0120] In the one-dimensional stage, the UV detector records the elution end time of each salt concentration pulse (e.g., the time point when the UV peak height reaches 10%), determining the SCX elution window for that salt concentration fraction. Therefore, in this step, based on the elution end time of the UV signal, the SCX salt concentration window corresponding to the target analyte signal value is assigned (e.g., if an ion is switched to the HILIC column by a valve during elution at an SCX 100mM salt concentration, its salt concentration label is 100mM). Then, for the target analyte at the corresponding salt concentration, the corresponding salt concentration is used... Value calibration HILIC retention time (e.g., for a target at an SCX 100 mM salt concentration, using the salt concentration corresponding to...) The value is used to calibrate the HILIC retention time, and the calibration formula is as follows: ;

[0121] In the formula: Indicates the original retention time; Indicates the retention time after calibration.

[0122] Based on this, the signals of the same compound in different salt concentration windows are combined (e.g., a peptide is eluted in both the SCX 150mM and 200mM windows due to partial protonation) to form the final two-dimensional coordinate table.

[0123] Cross-dimensional signal integration and quantitative integration: By comparing MS2 fragment ions with the spectral library, the m / z of the target analyte and all its signal points in two-dimensional coordinates were confirmed (e.g., a certain peptide showed elution signals at SCX 200mM salt concentration, HILIC RT 8min and SCX 500mM salt concentration, HILIC RT 15min).

[0124] For all calibrated signal points of the same compound, sum their MS1 peak areas as follows: In the formula: Indicates the total peak area of ​​the target object; This represents the peak area at the i-th signal point; This represents 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 strength.

[0125] Divide the total peak area of ​​the target analyte by the peak area of ​​the isotope internal standard to eliminate differences in extraction efficiency between samples and obtain the quantitative integral result of the target analyte: In the formula: Indicates the concentration of the target substance; Indicates the total peak area of ​​the isotope internal standard; This indicates the theoretical concentration of the isotope internal standard.

[0126] In this embodiment, an RT Lock standard (such as ATP) is injected before elution at each salt concentration, and the retention time drift (ΔRT) of the HILIC column is monitored in real time. After calibration, the retention time error is controlled within ±0.2 min, which solves the problem of peak misalignment caused by mobile phase fluctuations in two-dimensional separation and ensures the accuracy of peak alignment across samples.

[0127] In clinical sample processing, target analytes are easily lost due to protein precipitation, chromatographic separation, and other steps, leading to quantitative deviations. This protocol incorporates an isotope internal standard during the sample pretreatment stage. By calculating a correction factor, it can accurately compensate for losses of the target analyte during extraction, enrichment, and separation, greatly improving the accuracy of quantitative results and avoiding false negative or false positive results caused by sample processing losses.

[0128] Secondly, during two-dimensional separation, the retention time of the HILIC column is easily affected by factors such as changes in mobile phase composition and temperature fluctuations, leading to peak misalignment and affecting quantitative accuracy. In this embodiment, RT Lock standard is injected into the SCX column before each salt concentration switch, and calibration is performed by calculating the retention time offset. This effectively solves the peak misalignment problem in two-dimensional separation, ensuring accurate alignment of chromatographic peaks between different samples and laying the foundation for subsequent cross-dimensional signal integration.

[0129] Secondly, in clinical samples, low-abundance metabolites often have their signals masked or suppressed due to co-elution. This protocol integrates the signals of the same compound under different separation dimensions using calibration data provided by a dual internal standard system, combined with a two-dimensional coordinate table (SCX salt concentration and HILIC retention time); and performs internal standard normalization quantification; effectively avoiding signal loss caused by co-elution in single-dimensional separation, and improving the detection sensitivity of low-abundance compounds. Example

[0130] See 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 chamber, a gradient elution device, a flow path switching valve, a HILIC analysis column, and a tandem mass spectrometer.

[0131] 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 eluent; the metering pump is used to pump the isotope internal standard into the SCX enrichment column at a predetermined amount.

[0132] The target analyte obtained after passing through the SCX enrichment column is introduced into the gradient elution device to execute the gradient elution program. The intensity of the elution peak is monitored by a UV detection device. When the peak intensity decreases to the baseline level, the elution is switched to the next concentration. The elution peak that meets the intensity requirements and the RT lock standard are injected into the HILIC analysis column for processing through the flow path switching solenoid valve. The target analyte after processing by the HILIC analysis column is detected by the tandem mass spectrometer.

[0133] See Figure 3 As shown, the isotope internal standard is stored in an isotope internal standard storage pool and is pumped into the SCX enrichment column by a quantitative pump; the RT Lock standard is stored in an RT Lock standard storage pool and is introduced into the HILIC analysis column through a flow path switching solenoid valve; a peak detection sensor is set at the outlet of the HILIC analysis column to provide real-time feedback and drive gradient optimization, and the peak detection sensor also adopts an ultraviolet detector.

[0134] The HILIC analysis column is equipped with a peak detection sensor at the outlet to monitor the arrival time of the HILIC elution peak in real time, calculate the deviation from the expected time, and dynamically adjust the gradient slope based on the deviation.

[0135] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for clinical tandem mass spectrometric analysis based on multidimensional ion exchange, characterized in that, Comprising the following steps: Sample pretreatment: adding clinical samples into acidified reagent, removing high abundance protein interference, retaining target, and injecting the retained target into an online SCX enrichment column for target enrichment; Adding labeled isotope internal standards in the sample pretreatment stage; One-dimensional separation: stepwise elution of the SCX enrichment column using five increasing ammonium salt concentrations, and monitoring the intensity of the elution peak using a UV detector; when the peak intensity decreases to the baseline level, switching to the next concentration for elution, and directing the current eluted target to the HILIC column; at the end of the one-dimensional separation stage and before switching to the next ammonium salt concentration, injecting the RT Lock standard directly into the elution flow path of the SCX enrichment column through a solenoid valve, and mixing with the current salt concentration eluent to enter the HILIC column; the injection point is in the flow path between the SCX enrichment column and the HILIC column; Two-dimensional separation: directing the elution components after one-dimensional separation to the HILIC column for further separation, and realizing polarity difference separation of the target by changing the polarity of the mobile phase of the HILIC column; Mass spectrometry triggered mass spectrometry acquisition and processing: the components flowing out after HILIC processing are collected by mass spectrometry for mass-to-charge ratio information of all ions, and then the mass-to-charge ratio interval is divided into dynamic windows according to the real-time baseline noise level; when the signal intensity in a window exceeds 6 times the baseline noise, the secondary mass spectrometry is triggered; if there are multiple ions in the same window, only the top 2 ions with the highest intensity are triggered; at the same time, the triggered ions are added to a dynamic exclusion list, and mass spectrometry detection data is obtained based on this; Quantitative correction: using a combination of isotope internal standard correction and retention time lock standard to correct the mass spectrometry detection data; The quantitative correction comprises the following steps: Data extraction and calibration of the dual internal standard system: screening isotope internal standard ions according to known mass-to-charge ratios, and obtaining the signal intensity at the SCX concentration and HILIC RT corresponding to the isotope internal standard ions; extracting the measured retention time of the RT Lock standard at the beginning of elution of each ammonium salt concentration according to the fixed mass-to-charge ratio of the RT Lock standard; Internal standard correction factor and retention time offset calculation: calculating the isotope internal standard correction factor and the RT Lock retention time offset based on the obtained data, to form an isotope internal standard correction factor table and an RT Lock offset table; Two-dimensional peak alignment of UV signals and RT Lock: according to the elution end time of the UV signal, attributing the target signal to the corresponding SCX salt concentration window, using the RT Lock offset corresponding to the salt concentration to calibrate the HILIC retention time, and merging the signals of the same compound in different salt concentration windows to form a two-dimensional coordinate table; Cross-dimension signal integration and quantitative integration: confirming the mass-to-charge ratio of the target and all signal points of the target in the two-dimensional coordinate through comparison of the fragment ions of the secondary mass spectrometry with the spectral library, accumulating the peak area of the primary mass spectrometry of all calibrated signal points of the same compound to eliminate the extraction efficiency difference between samples, and obtaining the quantitative integration result of the target.

2. The multi-dimensional ion exchange based clinical tandem mass spectrometric method as claimed in claim 1, wherein, In the sample pretreatment, the target in the SCX enrichment column is enriched by using 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 completed, and the enrichment process is ended.

3. The multi-dimensional ion exchange based clinical tandem mass spectrometric method as claimed in claim 1, wherein, In the one-dimensional separation, on the basis of 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; At each predetermined time, the peak height and the peak area are calculated, and whether the target is completely eluted is judged according to the calculated peak height and peak area, if the peak height is lower than a preset threshold and the peak area deviation is within an allowable range, the next salt concentration is switched to; if the peak area is insufficient, the elution time is adjusted based on a preset adjustment time and the elution time obtained after optimization based on the elution optimization model.

4. The multi-dimensional ion exchange based clinical tandem mass spectrometric method as claimed in claim 1, wherein, In the two-dimensional separation, the mobile phase A in the HILIC analysis column is a high organic solvent, and the mobile phase B is an aqueous phase, starting from 100% mobile phase A, linear gradient elution to 80% mobile phase A, and the duration is synchronized with the SCX elution peak arrival time.

5. The multi-dimensional ion exchange based clinical tandem mass spectrometric method as claimed in claim 4, wherein, The slope of the linear gradient is dynamically adjusted, and the specific steps are as follows: Initial gradient slope setting: according to the polarity range of the target compound, an initial gradient slope is set; Real-time peak arrival time monitoring: the arrival time of the elution peak is recorded in real time at the outlet of the HILIC column; Calculate the time deviation: based on the real-time peak arrival time monitoring result and the expected arrival time, the time deviation is calculated; Adjust the gradient slope: multiply the absolute value of the time deviation by a proportionality coefficient to obtain the adjustment amount of the gradient slope; based on the adjustment amount, the adjusted gradient slope is obtained, wherein the gradient slope is decreased when the time deviation is greater than zero, and the gradient slope is increased when the time deviation is less than zero.

6. The multi-dimensional ion exchange based clinical tandem mass spectrometric method as claimed in claim 1, wherein, The dynamic window division in the mass spectrometry trigger mass spectrometry acquisition and processing includes the following steps: According to the scan range of the primary mass spectrum, the mass-to-charge ratio range is divided into multiple initial windows, wherein the initial window is the ratio of the total mass-to-charge ratio range of the primary mass spectrum scan to the default window size; Wherein the default window size is dynamically adjusted based on the baseline noise level, if the real-time noise level is greater than the preset noise threshold, the default window is reduced to obtain the updated window size; if the real-time noise level is less than the preset noise threshold, the default window is expanded to obtain the updated window size; wherein the noise threshold is obtained by multiplying the baseline noise level by a multiple coefficient.

7. A clinical tandem mass spectrometry system based on multidimensional ion exchange, characterized in that, A multi-dimensional ion exchange-based clinical tandem mass spectrometry analysis method for performing any one of claims 1 to 6, comprising an acidified reagent injection pump, a quantitative pump, an SCX enrichment column, a gradient elution device, a flow path switching valve, a HILIC analysis column, and a tandem mass spectrometer; The acidified reagent injection pump is used to pump the acidified clinical sample into the SCX enrichment column, wherein a conductivity sensor for detecting the conductivity of the eluent is installed in the enrichment column; the quantitative pump is used to pump the isotopic 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, a gradient elution program is performed, and the intensity of the elution peak is monitored by the ultraviolet detection device; when the peak intensity decreases to the baseline level, switching to the next concentration for elution; the elution peak meeting the intensity requirement and the RT lock standard are injected into the HILIC analysis column for processing through the flow path switching valve; and the target after passing through the HILIC analysis column is detected by the tandem mass spectrometer.

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