Mass spectrum separation and detection method for acyl carnitine isomeride

Abstract

The invention belongs to the technical field of mass spectrometric detection, and relates to a mass spectrometric separation and detection method for acyl carnitine isomers. According to the method, HILIC chromatographic separation is adopted, a separation mechanism based on hydrophilicity / polarity difference is utilized, the phenomenon of co-elution of the isomers is reduced, a foundation is laid for separation, then parent ion and characteristic daughter ion maps of all the isomers are obtained through mass spectrometry, daughter ion fragment difference generated by structural difference of different isomers is accurately captured, and the separation accuracy of the isomers is improved. The method comprises the following steps: establishing a multi-MRM channel based on characteristic daughter ions, and combining peak area calculation with standard curve comparison, so that even if partial co-elution exists, accurate quantification of each isomeride can be realized through a specific signal of the characteristic daughter ions; quantitative distortion caused by signal superposition in a traditional method is avoided, meanwhile, dependence on high-resolution mass spectrum, special chromatographic columns or complex derivatization is not needed, and the core problem that the isomeride is difficult to accurately distinguish and quantify is solved.

Description

Technical Field

[0001] The present application belongs to the field of mass spectrometry detection technology, and more specifically, relates to a mass spectrometry separation and detection method for acylcarnitine isomers. Background Art

[0002] Acylcarnitines (ACs) are key intermediates in fatty acid β-oxidation and energy metabolism, and are widely present in human blood, urine, and other biological samples. Their types and concentrations are closely associated with various inherited metabolic diseases, such as organic acidemias and fatty acid oxidation disorders, making them important biomarkers for clinical diagnosis and screening. For example, elevated isobutyrylcarnitine suggests methylmalonic acidemia (MMA), while elevated butyrylcarnitine may be associated with short-chain acyl-CoA dehydrogenase deficiency (SCAD). Accurately distinguishing between these two is crucial for disease classification and treatment decisions.

[0003] Currently, the detection of acylcarnitines primarily relies on liquid chromatography-tandem mass spectrometry (LC-MS / MS), particularly reversed-phase liquid chromatography-tandem mass spectrometry (RP-LC-MS / MS) coupled with multiple reaction monitoring (MRM) mode. This technology, with its advantages of high sensitivity, high specificity, and high throughput, has become a mainstream detection method in clinical laboratories. Its basic principle is to separate acylcarnitines in a sample using a reversed-phase column (based on hydrophobicity differences), and then detect the specific transitions (MRM transitions) between the parent ion and characteristic product ions using mass spectrometry to achieve qualitative and quantitative analysis.

[0004] However, there are a large number of isomers in the acylcarnitine family (such as C4:0-carnitine and isobutyrylcarnitine, C5:0-carnitine and isovalerylcarnitine, 2-methylbutyrylcarnitine, etc.); these isomers have the same molecular weight and similar chemical structure, and only differ in the branching position or length of the acyl chain.

[0005] Therefore, due to the minimal difference in hydrophobicity between isomers, it is difficult for reversed-phase columns to achieve effective separation through hydrophobic interactions, resulting in the co-elution of multiple isomers within the same retention time window; the co-eluting isomers have the same parent ion (for example, the parent ions of C4 isomers are all m / z 260.1) and share some characteristic product ions (such as the m / z 85.0 fragment of the carnitine skeleton); therefore, the signal detected by the MRM channel is the sum of multiple isomers and cannot distinguish the true concentration of a single isomer, which may lead to "false positive" or "false negative" results (for example, the increase in isobutyrylcarnitine is mistakenly judged as abnormal butyrylcarnitine). Summary of the Invention

[0006] The present invention provides a mass spectrometry separation and detection method for acylcarnitine isomers, which aims to solve the current technical problems of widespread co-elution and quantitative distortion.

[0007] The mass spectrometry separation and detection method of acylcarnitine isomers comprises the following steps: S1. Extract a sample from plasma or dried blood spot extract, precipitate protein using methanol, and remove the precipitate by centrifugation to obtain a supernatant; S2. Separate the supernatant using HILIC chromatography and adjust the chromatographic separation conditions; S3. For each peak after chromatographic separation, perform parent ion scanning using a mass spectrometer, select the parent ion of each acylcarnitine isomer, and record its mass to obtain parent ion information for each acylcarnitine isomer; perform collision-induced dissociation on the selected parent ion to obtain product ions, scan the generated product ions in a mass spectrometer, compare the product ion spectra of different isomers, extract characteristic fragment ions, and generate a characteristic product ion spectrum for each isomer; S4. Based on the characteristic product ion spectrum of each isomer, construct multiple MRM channels and perform quantification by calculating the area of ​​each channel. The obtained data are compared with the standard curve to obtain the accurate concentration of each isomer.

[0008] The present invention adopts HILIC chromatography separation and utilizes its separation mechanism based on hydrophilicity / polarity differences to make up for the separation deficiencies of traditional reverse chromatography caused by small hydrophobicity differences, reduce the co-elution phenomenon of isomers, and lay the foundation for separation. Secondly, the parent ion and characteristic daughter ion spectrum of each isomer is obtained through mass spectrometry analysis, accurately capturing the differences in daughter ion fragments caused by structural differences of different isomers, providing a molecular surface characteristic basis for distinguishing co-eluting components, and then establishing multiple MRM channels based on the characteristic daughter ions. Combined with peak area calculation and comparison with the standard curve, even if there is partial co-elution, the specific signals of the characteristic daughter ions can be used to achieve accurate quantification of each isomer, avoiding the quantitative distortion caused by signal superposition in traditional methods. At the same time, there is no need to rely on high-resolution mass spectrometry, special chromatographic columns or complex derivatization. While ensuring high throughput and practicality, it solves the core problem of the difficulty in accurately distinguishing and quantifying isomers.

[0009] Preferably, in step S1, after extracting the sample, a stable isotope-labeled internal standard of known concentration needs to be added to the sample.

[0010] Preferably, the step S3 further comprises treating the pure standard solution of the isomers, comprising the following steps: Use the pure product of each target isomer, dissolve and dilute it with methanol to prepare a single pure product standard solution with a concentration of 1-10μM; A predetermined volume of pure standard solution was injected into the HILIC chromatograph and separated using the same analytical conditions as for the supernatant analysis. When the pure standard solution was eluted with the mobile phase to the mass spectrometer detector, the Q1 scan full mode of the triple quadrupole mass spectrometer was used to record the change in ion signal intensity with mass-to-charge ratio. The quasi-molecular ion peak with the highest abundance was screened from the scan results and recorded as the parent ion of the pure product, and the corresponding retention time of the parent ion was recorded. Then, the predetermined extracted pure standard solution is injected into the HILIC chromatograph and separated using the analytical conditions exactly the same as those for the supernatant analysis. When the pure standard solution is eluted with the mobile phase to the mass spectrometer detector, the product ion scan mode is turned on based on the determined parent ion and retention time. Then, a gradient scan is performed within the collision energy range of 15-45 eV. The product ions generated by the parent ion fragmentation at different collision energies and their relative abundances are recorded to obtain a CID mass spectrum. Compare the CID mass spectra of the same group of isomers and screen the high-abundance product ions shared by all isomers as common ions; then analyze the abundance differences of product ions between isomers to screen out specific product ions.

[0011] Preferably, in step S3, the supernatant is subjected to HILIC chromatography separation under the same processing conditions as the pure product labeled solution, and then product ion scanning is performed within the determined retention time window and under the conditions of the determined parent ion; the product ion type and abundance of the target substance in the supernatant are obtained.

[0012] Preferably, the multiple MRM channels include a universal quantitative channel, a linear characteristic channel and a branched characteristic channel; The universal quantitative channel is used to monitor the total signal of all target acylcarnitines as a quantitative benchmark to reflect the total concentration of C4 acylcarnitines; The linear characteristic channel is used to monitor the signal that specifically responds to linear acylcarnitine; The branched-chain characteristic channel is used to monitor the specific response branched-chain acylcarnitine.

[0013] Preferably, step S4 includes collision energy optimization, comprising the following steps: For each channel, a collision energy gradient scan was performed in the range of 15–45 eV with a step size of 5 eV. The signal intensity of the product ions at different collision energies was recorded, and the collision energy value with the strongest signal and the least interference was selected.

[0014] Preferably, obtaining the accurate concentration of each isomer comprises the following steps: Based on the optimized collision energy, the signals of all MRM channels of the unknown sample are collected synchronously. The single acquisition time of each channel is the same and the automatic gain control is activated for signal acquisition. Based on the determined retention time, the chromatographic peaks of the three channels were aligned, and the peak area of ​​each channel was calculated using the automatic integration function. Baseline interference or false peaks were eliminated through manual correction to obtain the integrated peak area data; Calculate the linear characteristic ratio and branched characteristic ratio of the unknown sample based on the integrated peak area data; Using a mixed standard of known proportions and based on optimized collision energy, the signals of the MRM channels were synchronously collected, and the linear characteristic ratio and branched characteristic ratio were calculated. A standard curve corresponding to the linear characteristic ratio / and a standard curve corresponding to the branched characteristic ratio were established respectively; the linear characteristic ratio and branched characteristic ratio of the unknown sample were substituted into the corresponding standard curve to obtain the acylcarnitine content of the unknown sample.

[0015] The beneficial effects of the present invention include: The present invention adopts HILIC chromatography separation and utilizes its separation mechanism based on hydrophilicity / polarity differences to make up for the separation deficiencies of traditional reverse chromatography caused by small hydrophobicity differences, reduce the co-elution phenomenon of isomers, and lay the foundation for separation. Secondly, the parent ion and characteristic daughter ion spectrum of each isomer is obtained through mass spectrometry analysis, accurately capturing the differences in daughter ion fragments caused by structural differences of different isomers, providing a molecular surface characteristic basis for distinguishing co-eluting components, and then establishing multiple MRM channels based on the characteristic daughter ions. Combined with peak area calculation and comparison with the standard curve, even if there is partial co-elution, the specific signals of the characteristic daughter ions can be used to achieve accurate quantification of each isomer, avoiding the quantitative distortion caused by signal superposition in traditional methods. At the same time, there is no need to rely on high-resolution mass spectrometry, special chromatographic columns or complex derivatization. While ensuring high throughput and practicality, it solves the core problem of the difficulty in accurately distinguishing and quantifying isomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Figure 1 This is a flowchart of the overall steps provided by an embodiment of the present invention.

[0018] Figure 2 This is a schematic diagram of the specific steps of step S4 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0019] 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.

[0020] See also Figure 1 As shown, the preferred embodiment of the present invention is further described; The mass spectrometry separation and detection method of acylcarnitine isomers comprises the following steps: S1. Extract a sample from plasma or dried blood spot extract, precipitate protein using methanol, and remove the precipitate by centrifugation to obtain a supernatant; In this example, a plasma or dried blood spot sample (50 μL) was transferred to a centrifuge tube. A methanol solution containing a specific concentration of a stable isotope internal standard, such as d3-acetylcarnitine (d3-C2), d3-propionylcarnitine (d3-C3), d3-butyrylcarnitine (d3-C4:0), or d3-isovalerylcarnitine (d3-C5), was added to the sample tube. The volume of the methanol solution was 5 times the sample volume to ensure sufficient protein precipitation efficiency while maintaining the organic phase ratio of the final extract, compatibility with the injection conditions of subsequent HILIC chromatography, and minimizing solvent effects. It should be noted that the role of the isotope internal standard is baseline correction, and how to use an isotope internal standard for baseline correction is a conventional technique in the art. Therefore, this will not be described in detail in the present invention as it is not the focus of the present invention.

[0021] Use a vortex mixer to vigorously and thoroughly vortex the contents of the centrifuge tube to ensure that the internal standard is in uniform contact with the target analyte in the sample to obtain a mixed solution; The mixture is then placed in a pre-cooled (4°C) or room temperature microcentrifuge and centrifuged at high speed (13,000-14,000 rpm) for 8-15 minutes to form a dense precipitate and highly clarify the supernatant. The supernatant is then aspirated using a micropipette and transferred to an injection vial for subsequent HILIC chromatography analysis. In this example, a high proportion of methanol was used as the precipitant, resulting in a high organic phase content (compatible with methanol and acetonitrile) in the final supernatant, which perfectly matches the initial high organic phase (80-95% B) mobile phase conditions of the HILIC column. This reduces peak broadening or deformation caused by solvent effects, facilitating the optimization and reproducibility of the chromatographic separation effect in step S2.

[0022] S2. Separate the supernatant using HILIC chromatography and adjust the chromatographic separation conditions; In this example, hydrophilic interaction chromatography (HIC) mode was used to maximize separation of target acylcarnitine isomer pairs (particularly linear and branched isomers, such as C4:0-carnitine vs. isobutyrylcarnitine; C5:0-carnitine vs. isovalerylcarnitine vs. 2-methylbutyrylcarnitine). This laid the foundation for subsequent accurate quantification based on the ratio of characteristic product ions. Even with some coelution, the optimized chromatographic conditions ensured good peak shape and stable retention times. Adjusting the deep chromatography separation conditions includes: In this example, a HILIC column with a strongly hydrophilic surface functional group is used, such as an amide column (Waters Acquity UPLC BEH Amide (e.g., 1.7 μm, 2.1 × 100 / 150 mm)), which has good retention and separation effects on carnitine compounds. A zwitterion column (Merck SeQuant ZIC-HILIC (e.g., 3.5 μm 1.7 μm, 2.1 × 100 / 150 mm)) has a unique zwitterion layer, providing good selectivity. Mobile phase design employed 10 mM ammonium formate aqueous solution + 0.1% (v / v) formic acid in phase A (aqueous phase). Ammonium formate provided buffering capacity (pH ~3.5) to maintain stable protonation of the target, optimizing its retention and peak shape on the HILIC column. Formic acid enhanced protonation efficiency and improved ionization response in ESI+ mode. Phase B (organic phase) employed acetonitrile + 0.1% (v / v) formic acid. Acetonitrile, the primary organic solvent in HILIC mode, leveraged its strong elution power and low viscosity to achieve efficient separation. For the optimization of the elution program, a gradient elution program was adopted in this embodiment, as follows: Initial starting section: 95% B (acetonitrile + 0.1% formic acid) / 5% A (10 mM ammonium formate + 0.1% formic acid aqueous solution); in the initial state, maintain 95% B at the beginning, with a relatively high organic phase ratio; ensure that the target compound is effectively enriched and focused on the chromatographic column, while similar weak polar impurities are quickly eluted to avoid affecting subsequent separation, thereby ensuring that the target acylcarnitine and its isomers can stably enter the column and reduce baseline fluctuations.

[0023] Maintain a high organic phase: 95% B / 5% A, time 0.0-2.0 min; maintain a high organic phase ratio for 2 minutes to allow impurities and weak polar components to be washed out as early as possible, ensuring effective separation and accumulation of the target, and avoiding premature elution of isomers, thereby increasing the operability of the separation; Key separation stage: Linear decrease from 95% B -> 60% B, 5% A -> 40% A, time 2.0-10.0 min; starting from two minutes, the proportion of organic phase B is gradually reduced until 10 minutes. By linearly decreasing the organic phase concentration, the mobile phase is effectively increased, promoting the separation of acylcarnitines and their isomers. As the proportion of phase B gradually decreases, the more hydrophilic acylcarnitine isomers are gradually eluted; During the critical separation phase, gradient changes directly affect the resolution between isomers. Subtle differences in molecular polarity can affect their hydrophilic interactions on the stationary phase, thus affecting the separation effect. Therefore, based on the sample data, a slow linear gradient was used between 2.0 and 6 minutes, and an accelerated gradient change was used between 6 and 10 minutes to improve the resolution of isomers with large differences. The specific steps for adjusting the slope in gradient elution are as follows: The gradient slope is calculated based on the known 95% B at the beginning of the critical separation, 60% B at the end, and a 10-min time period: Where: represents the change in the proportion of solvent B; Indicates time changes; represents the concentration of solvent B at the starting point of the gradient; represents the concentration of solvent B at the end point of the gradient; Indicates the time when the gradient ends; Indicates the time when the gradient ends; Based on the above, we know that the concentration of solvent B needs to be reduced at a rate of 3.5% per minute within 10 minutes, that is, the rate of gradient descent is Based on the above formula, we can adjust the rate of decrease of phase B in the range of 2.0 - 10.0 min (such as extending it to 12 or 15 min), or use a nonlinear gradient (such as a convex gradient) to maximize the resolution (Rs) between isomer pairs (such as C4 and C5 isomers).

[0024] Isocratic section: 60% B -> 95% B, 40% A -> 5% A, time 10.0 - 10.5 min. During this section, the high organic phase ratio is quickly restored for 0.5 min to ensure that residual impurities are completely eluted. It is important to note that this section no longer participates in the separation of the target compound, but is used for cleaning and column recovery to ensure stability for the next injection. Equilibration section: 95% B / 5% A, time 10.5 - 15.0 min, under high organic phase conditions, restores column stability, reduces column efficiency decay, and ensures consistency of retention time and peak shape in each analysis.

[0025] In this example, by optimizing the gradient elution program, acylcarnitine isomers, especially linear and branched isomers, can be effectively separated, and the stability of the peak shape and good separation degree are guaranteed. According to the experimental data feedback, the gradient change rate and the slope of the separation section can be flexibly adjusted to further improve the separation effect and ensure the efficiency and accuracy of the chromatographic analysis.

[0026] Column temperature optimization: The column temperature was selected between 30°C and 40°C, and the effects of different column temperatures on the separation and peak shape of key isomers were tested. The stable temperature with the best separation effect was selected. In this example, 36°C was selected.

[0027] Flow rate optimization: Select a flow rate of 0.3 mL / min - 0.5 mL / min (for a 2.1 mm inner diameter column). Test the effects of different flow rates on the resolution of key isomers, analysis time, and system pressure under the selected chromatographic column, gradient, and column temperature. Select a flow rate that balances analytical efficiency while ensuring adequate resolution.

[0028] S3. For each peak after chromatographic separation, perform parent ion scanning using a mass spectrometer, select the parent ion of each acylcarnitine isomer, and record its mass to obtain parent ion information for each acylcarnitine isomer; perform collision-induced dissociation on the selected parent ion to obtain product ions, scan the generated product ions in a mass spectrometer, compare the product ion spectra of different isomers, extract characteristic fragment ions, and generate a characteristic product ion spectrum for each isomer; The step S3 further includes processing the pure standard solution of isomers, including the following steps: Use the pure product of each target isomer, dissolve and dilute it with methanol to prepare a single pure product standard solution with a concentration of 1-10μM; A predetermined volume of pure standard solution is injected into the HILIC chromatograph and separated using analytical conditions identical to those used for supernatant analysis (i.e., the conditions set in step S2). When the pure standard solution is eluted with the mobile phase into the mass spectrometer detector, the Q1 scan full mode of the triple quadrupole mass spectrometer is used to record the change in ion signal intensity as a function of mass-to-charge ratio. The quasi-molecular ion peak with the highest abundance is screened from the scan results and recorded as the parent ion of the pure product, and the corresponding retention time of the parent ion is recorded. Then, the predetermined extracted pure standard solution was injected into the HILIC chromatograph and separated using the analytical conditions exactly the same as those for the supernatant analysis. When the pure standard solution was eluted with the mobile phase to the mass spectrometer detector, the product ion scan mode was turned on based on the determined parent ion and retention time. A gradient scan was performed in the collision energy range of 15-45 eV. The product ions generated by the fragmentation of the parent ion at different collision energies and their relative abundances were recorded to obtain a CID mass spectrum. Compare the CID mass spectra of the same group of isomers and screen the high-abundance product ions shared by all isomers as common ions; then analyze the abundance differences of product ions between isomers to screen out specific product ions.

[0029] In step S3, the supernatant is subjected to HILIC chromatography under the same processing conditions as the pure product labeled solution (i.e., the conditions set in step 2), and then product ion scanning is performed within a determined retention time window and under determined parent ion conditions to obtain the product ion type and abundance of the target substance in the supernatant.

[0030] S4. Based on the characteristic product ion spectrum of each isomer, construct multiple MRM channels and perform quantification by calculating the area of ​​each channel. The obtained data are compared with the standard curve to obtain the accurate concentration of each isomer.

[0031] See also Figure 2 As shown, the multiple MRM channels include a universal quantitative channel, a linear characteristic channel, and a branched characteristic channel; The universal quantitative channel is used to monitor the total signal of all target acylcarnitines, serving as a quantitative benchmark to reflect the total concentration of C4 acylcarnitines; parent ion: m / z 260.1 ([M+H]⁺ shared by C4 isomers), product ion: m / z 85.0 (carnitine characteristic fragment shared by all acylcarnitines); The linear characteristic channel is used to monitor the signal that specifically responds to linear acylcarnitines, reflecting the signal of butyrylcarnitine, with a parent ion of m / z 260.1 (consistent with the universal channel) and a daughter ion of m / z 71.0 (a high-abundance characteristic fragment of linear C4 acylcarnitine). The branched characteristic channel is used to monitor the signal of isobutyrylcarnitine, which specifically responds to branched acylcarnitine. The parent ion is m / z 260.1 (consistent with the universal channel) and the daughter ion is m / z 99.0 (a high-abundance characteristic fragment of branched C4 acylcarnitine). The step S4 includes collision energy optimization, comprising the following steps: For each channel, a collision energy gradient scan was performed in the range of 15-45 eV with a step size of 5 eV. The signal intensity of the product ions at different collision energies was recorded, and the collision energy value with the strongest signal and the least interference was selected. The optimization results are as follows: Universal quantitative channel (m / z 260.1→85.0): optimal CE = 20 eV; Linear characteristic channel (m / z 260.1→71.0): optimal CE=25 eV; Branched chain characteristic channel (m / z 260.1→99.0): optimal CE=30 eV.

[0032] As a possible implementation of this embodiment, obtaining the accurate concentration of each isomer includes the following steps: Based on the optimized collision energy, the signals of all MRM channels of the unknown sample are collected synchronously. The single acquisition time of each channel is the same and the automatic gain control is activated for signal acquisition. Based on the determined retention time, the chromatographic peaks of the three channels were aligned, and the peak area of ​​each channel was calculated using the automatic integration function. Baseline interference or false peaks were eliminated through manual correction to obtain the integrated peak area data; The linear characteristic ratio and branched characteristic ratio of the unknown sample are calculated based on the integrated peak area data; the specific expressions are as follows: ; Where: represents the proportion of straight chain features; Represents the chromatographic peak area of ​​the linear characteristic channel; represents the chromatographic peak area of ​​the universal quantitative channel and the total C4 signal; ; Where: represents the branch characteristic ratio; Represents the chromatographic peak area of ​​the branched characteristic channel.

[0033] The characteristic ratio is then converted to actual concentration using a standard curve. Based on the peak area of ​​the universal quantitative channel and the standard curve of the total C4 standard, the total concentration of butyrylcarnitine and isobutyrylcarnitine is obtained: ; Where: represents the total C4 acylcarnitine concentration; Represents the fitting function of the total C4 standard curve (such as the linear equation y=ax+b); represents the universal channel peak area; Substitute the linear characteristic ratio and branched characteristic ratio of the unknown sample into the corresponding standard curve to obtain the concentration of butyrylcarnitine and isobutyrylcarnitine in the unknown sample; the specific expression is as follows: ; Where: Represents the proportion of linear features of unknown samples; represents the inverse function of the butyrylcarnitine standard curve; represents the absolute concentration of butyrylcarnitine in the unknown sample; ; Where: represents the absolute concentration of isobutyrylcarnitine in the unknown sample; represents the proportion of branched features in the unknown sample; Represents the inverse function of the isobutyrylcarnitine standard curve.

[0034] In this example, the mutual exclusivity of the fragmentation paths of the linear (71.0) and branched (99.0) characteristic product ions was utilized to establish independent quantitative channels, completely avoiding the separation bottleneck caused by similar physical and chemical properties.

[0035] The above are only preferred embodiments of the present application and are 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 method for separating and detecting acylcarnitine isomers by mass spectrometry, characterized in that: The following steps are involved: S1. Extract a sample from plasma or dried blood spot extract, precipitate protein using methanol, and remove the precipitate by centrifugation to obtain a supernatant; S2. Based on the obtained supernatant, separation is performed using HILIC chromatography, and the chromatographic separation conditions are adjusted; S3. For each peak after chromatographic separation, perform parent ion scanning using a mass spectrometer, select the parent ion of each acylcarnitine isomer, and record its mass to obtain parent ion information for each acylcarnitine isomer; perform collision-induced dissociation on the selected parent ion to obtain product ions, scan the generated product ions in a mass spectrometer, compare the product ion spectra of different isomers, extract characteristic fragment ions, and generate a characteristic product ion spectrum for each isomer; S4. Based on the characteristic product ion spectrum of each isomer, construct multiple MRM channels and perform quantification by calculating the area of ​​each channel. The obtained data are compared with the standard curve to obtain the accurate concentration of each isomer.

2. The method for mass spectrometric separation and detection of acylcarnitine isomers according to claim 1, wherein: In step S1, after extracting the sample, a stable isotope-labeled internal standard of known concentration needs to be added to the sample.

3. The method for mass spectrometric separation and detection of acylcarnitine isomers according to claim 1, wherein: The step S3 further includes processing the pure standard solution of isomers, including the following steps: Use the pure product of each target isomer, dissolve and dilute it with methanol to prepare a single pure product standard solution with a concentration of 1-10μM; A predetermined volume of pure standard solution was injected into the HILIC chromatograph and separated using the same analytical conditions as for the supernatant analysis. When the pure standard solution was eluted with the mobile phase to the mass spectrometer detector, the Q1 scan full mode of the triple quadrupole mass spectrometer was used to record the change in ion signal intensity with mass-to-charge ratio. The quasi-molecular ion peak with the highest abundance was screened from the scan results and recorded as the parent ion of the pure product, and the corresponding retention time of the parent ion was recorded. Then, the predetermined extracted pure standard solution is injected into the HILIC chromatograph and separated using the analytical conditions exactly the same as those for the supernatant analysis. When the pure standard solution is eluted with the mobile phase to the mass spectrometer detector, the product ion scan mode is turned on based on the determined parent ion and retention time. Then, a gradient scan is performed within the collision energy range of 15-45 eV. The product ions generated by the parent ion fragmentation at different collision energies and their relative abundances are recorded to obtain a CID mass spectrum. Compare the CID mass spectra of the same group of isomers and screen the high-abundance product ions shared by all isomers as common ions; then analyze the abundance differences of product ions between isomers to screen out specific product ions.

4. The method for mass spectrometric separation and detection of acylcarnitine isomers according to claim 3, characterized in that: In step S3, the supernatant is subjected to HILIC chromatography under the same treatment conditions as the pure product labeled solution, and then product ion scanning is performed within the determined retention time window and under the conditions of the determined parent ion; Obtain the product ion type and abundance of the target compound in the supernatant.

5. The method for mass spectrometric separation and detection of acylcarnitine isomers according to claim 1, wherein: The multiple MRM channels include a universal quantitative channel, a linear characteristic channel and a branched characteristic channel; The universal quantitative channel is used to monitor the total signal of all target acylcarnitines as a quantitative benchmark to reflect the total concentration of C4 acylcarnitines; The linear characteristic channel is used to monitor the signal that specifically responds to linear acylcarnitine; The branched-chain characteristic channel is used to monitor the specific response branched-chain acylcarnitine.

6. The method for mass spectrometric separation and detection of acylcarnitine isomers according to claim 5, wherein: The step S4 includes collision energy optimization, including the following steps: For each channel, a collision energy gradient scan was performed in the range of 15–45 eV with a step size of 5 eV. The signal intensity of the product ions at different collision energies was recorded, and the collision energy value with the strongest signal and the least interference was selected.

7. The method for mass spectrometric separation and detection of acylcarnitine isomers according to claim 6, wherein: Obtaining the accurate concentration of each isomer involves the following steps: Based on the optimized collision energy, the signals of all MRM channels of the unknown sample are collected synchronously. The single acquisition time of each channel is the same and the automatic gain control is activated for signal acquisition. Based on the determined retention time, the chromatographic peaks of the three channels were aligned, and the peak area of ​​each channel was calculated using the automatic integration function. Baseline interference or false peaks were eliminated through manual correction to obtain the integrated peak area data; Calculate the linear characteristic ratio and branched characteristic ratio of the unknown sample based on the integrated peak area data; Using a mixed standard of known proportions and based on optimized collision energy, the signals of the MRM channels were synchronously collected, and the linear characteristic ratio and branched characteristic ratio were calculated. A standard curve corresponding to the linear characteristic ratio / and a standard curve corresponding to the branched characteristic ratio were established respectively; the linear characteristic ratio and branched characteristic ratio of the unknown sample were substituted into the corresponding standard curve to obtain the acylcarnitine content of the unknown sample.

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