Method and kit for simultaneously analyzing acyl coenzyme A with different lengths based on LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) system

Abstract

The invention provides a method and a kit for simultaneously analyzing acyl coenzyme A with different lengths based on an LC-MS / MS. The method belongs to the technical field of biological analysis, and comprises the following steps: (1) obtaining standard curves of Acyl-CoAs standard substances with different lengths; and (2) treating a to-be-detected sample, detecting by adopting an LC-MS / MS system, determining the type of the acyl coenzyme A, and calculating the content of different types of acyl coenzyme A by utilizing the standard curve in the step (1). According to the method, the chromatographic peak tailing effect is reduced by utilizing a phenyl-hexyl column, the elution capacity for Acyl-CoAs with different properties is improved by gradually increasing the content of triethylamine in the chromatography, the coverage range for Acyl-CoAs is expanded, chromatographic separation and quantitative detection of acyl coenzyme A from a short chain to an ultra-long chain can be realized within 20 minutes, and the method is suitable for industrial production. The method has good precision and accuracy and a wide linear range.

Description

Technical Field

[0001] The invention belongs to the field of biological analysis, and particularly relates to a method and a kit for simultaneously analyzing acyl-CoA of different lengths based on an LC-MS / MS system. Background Art

[0002] Acyl-CoA is a thiol compound of fatty acid and coenzyme A (RCO-CoA), an active metabolic intermediate in the synthesis and decomposition of fatty acids, which produces fatty acids and coenzyme A when hydrolyzed. In addition, ATP is consumed during the synthesis of fatty acids and coenzyme A. Therefore, they are involved in the metabolism of proteins, carbohydrates, ketone bodies and lipids, as well as processes such as cell signaling and epigenetic regulation. They exist in multiple metabolic pathways. The relative distribution of cellular acyl-CoA represents the fingerprint of cell metabolism and can provide information on the activity of metabolic or signaling pathways, including epigenetic regulation. Therefore, the analysis of acyl-CoA has become the main focus of current metabolomics and epigenomics.

[0003] High performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been used to analyze a large number of acyl-CoAs, but their acyl chains vary greatly, and acyl-CoA is a very heterogeneous group of metabolites, with hydrophobic and hydrophilic neutral or charged substances with different carbon chain lengths, saturation and branching levels. Due to the difference in material polarity, the existing quantitative detection methods for acyl-CoA have a very limited detection coverage from short-chain acyl-CoA (carbon chain length less than 6) to long-chain acyl-CoA (carbon chain length greater than 12). In addition, there are also orders of magnitude differences in their concentrations. The endogenous level of acyl-CoA is low, it is highly unstable in aqueous solution, extremely easy to hydrolyze, and there are problems such as low recovery rate and low signal intensity, which is not conducive to sample preparation. On the other hand, the detection of acyl-CoA using LC-MS / MS systems will result in severe peak tailing, signal degradation and poor detection limits. Therefore, it is difficult to analyze the entire acyl-CoA metabolome with a single analytical method.

[0004] In order to cover the full range of acyl-CoA. In reverse phase liquid chromatography (RPLC), short-chain to medium-chain Acyl-CoAs are suitable for slightly acidic mobile phases, while medium-chain to long-chain Acyl-CoAs are more suitable for alkaline mobile phases. Previously, there was a method to use a quaternary pump liquid chromatography-mass spectrometry (LC-MS) system to cover short-chain, medium-chain and long-chain Acyl-CoAs. However, due to the need for multi-liquid chromatography system operation and secondary separation, this method introduces complexity and increases analysis time, which in turn affects high-throughput analysis. Therefore, finding a simple, effective and wide-ranging method to analyze Acyl-CoAs remains a major challenge. Summary of the invention

[0005] The object of the present invention is to provide a simple, effective and wide-ranging method for analyzing Acyl-CoAs.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for simultaneously analyzing acyl-CoA of different lengths based on an LC-MS / MS system, comprising the following steps:

[0008] (1) preparing working solutions of Acyl-CoAs standards of different lengths, injecting the solutions, and obtaining standard curves of Acyl-CoAs standards of different lengths;

[0009] (2) adding extract A to the sample to be tested, homogenizing and centrifuging, taking the supernatant, and detecting it using an LC-MS / MS system to determine the type of acyl-CoA in the sample to be tested, and using the standard curve of step (1) to calculate the content of different types of acyl-CoA;

[0010] The chromatographic column in the LC-MS / MS system is a phenyl-hexyl chromatographic column.

[0011] Preferably, the Acyl-CoAs standard comprises:

[0012] CoA0:0, CoA2:0, CoA3:0, COA3-keto10:0, CoA3O2, COA3-keto16:0, CoA4:0, CoA6:0, CoA8:0, CoA10:0, CoA12:0, CoA14:0, CoA16:0, CoA17:0, CoA18:0, CoA18:1, CoA20:0, CoA22:0, CoA22:6, CoA24:0 and CoA-SUC.

[0013] Preferably, the concentration of the working solution of the Acyl-CoAs standard in step (1) is 90 to 110 ng / mL.

[0014] Preferably, the extract A in step (2) is composed of methanol, acetonitrile and water in a volume ratio of 2-4:2-4:1-2, and the total addition amount is 8-12 μL / mg of the sample to be tested.

[0015] Preferably, the centrifugation conditions in step (2) are 13000-15000 g, 3-5° C., and 8-12 min.

[0016] Preferably, in steps (1) and (2), the mass spectrometer adopts a multiple reaction ion detection mode, a positive ion mode for collection, and an electrospray ion source; capillary temperature: 310-330°C; spray voltage: 3.6-4.0 kV; maximum spray current: 80-120 μA; sheath gas flow rate: 28-32 L / min; auxiliary gas flow rate: 4-6 L / min; probe heating temperature: 280-320°C.

[0017] Preferably, in steps (1) and (2), mobile phase A is water containing 4.5-5.5 mM ammonium bicarbonate;

[0018] The mobile phase B comprises acetonitrile, methanol and water in a mass ratio of 7.5-8.5:1.5-2.5:1, wherein the mobile phase B also contains 4.5-5.5 mM ammonium bicarbonate and 0.08-0.12% triethylamine;

[0019] Preferably, during the chromatographic gradient elution process, the mobile phase A is 95-97% within the elution time of 0-10 min, 50-55% at the elution time of 10-11 min, adjusted to 28-32% at the elution time of 14-16 min, and 8-12% at the elution time of 18-19.5 min;

[0020] The mobile phase B is 3-5% during the elution time of 0-10 minutes, adjusted to 45-50% when the elution time reaches 10-11 minutes, adjusted to 68-72% when the elution time reaches 14-16 minutes, and adjusted to 88-92% when the elution time reaches 18-19.5 minutes;

[0021] The elution flow rate is 0.28~0.32mL / min.

[0022] The present invention provides a kit for simultaneously analyzing acyl-CoAs of different lengths based on an LC-MS / MS system, comprising the following reagents: Acyl-CoAs standard solutions of different lengths, an extract A, a reagent A and a reagent B;

[0023] The reagent A is ammonium bicarbonate solid powder, which is prepared into a 4.5-5.5 mM ammonium bicarbonate solution with water as mobile phase A;

[0024] The reagent B is used as mobile phase B, which is prepared by mixing acetonitrile, methanol and water in a mass ratio of 7.5-8.5:1.5-2.5:1, and then adding ammonium bicarbonate and triethylamine, wherein the final concentration of ammonium bicarbonate in the reagent B is 4.5-5.5 mM, and the volume fraction of triethylamine is 0.08-0.12%.

[0025] The present invention provides a method and a kit for simultaneously analyzing acyl-CoAs of different lengths based on an LC-MS / MS system. The present invention utilizes a phenyl-hexyl column to reduce the retention effect of a conventional reverse chromatographic column (such as C18) on Acyl-CoAs, thereby reducing the tailing effect of the chromatographic peak; the present invention gradually increases the content of triethylamine in the chromatogram to improve the elution capacity of Acyl-CoAs of different properties, thereby expanding the coverage of Acyl-CoAs by the present method; the present invention dynamically adjusts the solution ratio and elution gradient in the mobile phase, so that the present invention can not only realize the chromatographic separation and quantitative detection of acyl-CoAs from short chain (CoA0:0) to ultra-long chain (CoA24:0) under one chromatographic condition, but also can simultaneously detect multiple Acyl-CoAs (covering short chain, medium chain, long chain and even ultra-long chain Acyl-CoAs of C24) within 20 minutes. The drawback that short-chain and long-chain acyl-CoAs cannot be covered simultaneously in the past is solved, which greatly saves time and economic costs in the analysis process and reduces the phenomenon of decreased activity of biological samples caused by batch analysis.

[0026] The present invention is also verified by using the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" given by the Chinese Pharmacopoeia, which verifies that the quantitative detection method provided by the present invention has good precision, accuracy and a wide linear range. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the overall diagram of the liquid phase separation of acyl-CoA standards;

[0028] Figure 2 This is a single-label diagram of the liquid phase separation of some acyl-CoA standards;

[0029] Figure 3 This is a single-label diagram of the liquid phase separation of some acyl-CoA standards;

[0030] Figure 4 This is a single-label diagram of the liquid phase separation of some acyl-CoA standards;

[0031] Figure 5 is the separation effect of C18 column on Acyl-CoAs;

[0032] Figure 6 The separation effect of phenylhexyl column on Acyl-CoAs

[0033] Figure 7 The separation effect of mobile phase group 1 on Acyl-CoAs;

[0034] Figure 8 The separation effect of mobile phase group 2 on Acyl-CoAs;

[0035] Fig. 9is the concentration of Acyl-CoAs in kidney tissues of mice in different groups. DETAILED DESCRIPTION

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] 1. Sample processing

[0039] After C57BL / 6 mice were killed by cervical dislocation, 25 mg of kidney tissue was collected and added with extract A (composed of methanol, acetonitrile and water in a volume ratio of 2:2:1) at a ratio of 10 μL / mg. After homogenization, the mixture was centrifuged at 14000g and 4°C for 10 min. After centrifugation, the supernatant was collected and stored at -80°C until it was loaded onto the machine.

[0040] 2. Preparation of Acyl-CoAs Standard Stock Solution and Working Solution

[0041] Accurately weigh the acyl-CoA standard (see Table 2), dissolve it in anhydrous methanol and prepare a 1.0 mg / mL standard stock solution for subsequent experiments, and store it in a -20°C refrigerator.

[0042] Accurately pipette the above 1.0 mg / mL standard stock solution and dilute it with anhydrous methanol to obtain a working solution concentration of 100 ng / mL; simultaneously prepare a standard mixture with a concentration of 100 ng / mL. Store them in a -20°C refrigerator.

[0043] 3. Mass spectrometry parameters

[0044] The mass spectrometer was operated in multiple reaction ion detection mode, with positive ion mode acquisition and electrospray ionization (ESI) source;

[0045] Capillary temperature: 320℃;

[0046] Spray Voltage: 3.8 kV;

[0047] Maximum spray current (Max Spray Current): 100μA;

[0048] Sheath Gas Flow: 30L / min;

[0049] Aux Gas Flow: 5L / min;

[0050] Probe Heater Temp: 300°C.

[0051] 4. Chromatographic parameters

[0052] Chromatographic column: ACQUITY Premier CSH Phenyl-Hexyl Column 2.5 μm, 2.1 x 100 mm, 1 / pk;

[0053] Mobile phase: Phase A, water (containing 5 mM ammonium bicarbonate);

[0054] Phase B: acetonitrile: methanol: water = 8:2:1 (containing 5 mM ammonium bicarbonate, 0.1% triethylamine);

[0055] The injection volume was 3 μL;

[0056] The elution gradient is shown in Table 1.

[0057] Table 1 Elution gradient

[0058] Time (min) Phase A (%) Phase B (%) Flow rate (mL / min) 0.00 97.00％ 3.00％ 0.300 1.00 97.00％ 3.00％ 0.300 1.50 95.70％ 4.30％ 0.300 10.00 95.00％ 5.00％ 0.300 11.00 55.00％ 45.00％ 0.300 14.00 30.00％ 70.00％ 0.300 16.00 30.00％ 70.00％ 0.300 18.00 10.00％ 90.00％ 0.300 19.50 10.00％ 90.00％ 0.300 20.00 97.00％ 3.00％ 0.300

[0059] 5. Data processing

[0060] Agilent OpenLab software was used for data acquisition, and Agilent MassHunter software was used for qualitative and quantitative analysis.

[0061] 6. Mass spectrometry optimization results

[0062] The 21 acyl-CoAs standards in the "Preparation of Acyl-CoAs Standard Stock Solution and Working Solution" were prepared into a mixed standard solution, and the standard precursor ions and product ions were extracted in the Agilent MassHunter software analysis using the "mass spectrometry parameters" and "chromatographic parameters" conditions mentioned above. After extraction, the overall liquid phase separation diagram of the acyl-CoAs standard was obtained ( Figure 1 ) and single plot (sorted by molecular weight of parent ions) ( Figure 2 to Figure 4 ), the Acyl-CoAs standard mass spectrum parameter optimization information table is shown in Table 2.

[0063] Table 2 Acyl-CoAs standard mass spectrum parameter optimization information table

[0064]

[0065]

[0066] Depend on Figure 1 , 2It can be seen that this method has a high separation ability for short-chain to ultra-long-chain acyl-CoA standards and is suitable for the detection and analysis of acyl-CoA.

[0067] 7. Method Validation

[0068] Linearity and limit of quantitation

[0069] During the method validation, three standard curves prepared in parallel were evaluated. Linearity was evaluated by plotting the peak area ratio (Y) of each analyte against the theoretical concentration (X) (Table 3). This time, the least squares method was selected for linear regression (weighting factor 1 / X) to evaluate linearity. The relative error (RE%) between the actual concentration and the theoretical concentration of the calibration standard should not exceed ±15% of its labeled value (±20% in the lower limit of quantitation concentration sample). Each batch of standard curve samples contains at least six valid concentration samples and at least 75% of the calibration standard samples in each batch should meet the specified standards.

[0070] The Acyl-CoAs standard curve equation, linear correlation coefficient and weight are shown in Table 3.

[0071] Table 3 Acyl-CoAs standard curve equation, linear correlation coefficient and weight

[0072]

[0073] Acyl-CoAs detection precision and accuracy

[0074] To verify the precision and accuracy, three batches of samples were injected and measured on two consecutive days. Each batch of samples included double blank samples, linear calibration standards, low concentration quality control samples (LQC), medium concentration quality control samples (MQC) and high concentration quality control samples (HQC). Each quality control sample of different concentrations was prepared in parallel with six copies for measurement. The precision (RSD%) of the concentration of each quality control sample was used to evaluate the precision. For each quality control level, the coefficient of variation (CV) should not exceed ±15%, and for samples of LQC concentration, it should not exceed ±20%. The measurement results are shown in Table 4;

[0075] Table 4 Intra-batch and inter-batch precision accuracy of Acyl-CoAs

[0076]

[0077]

[0078]

[0079] As shown in Table 4, the precision and accuracy of the method were evaluated by at least 3 analytical batches and at least two days, with three concentrations of LQC, MQC and HQC for each batch, and at least 5 measured values ​​for each concentration. The intra-batch and inter-batch precision of this method meets the HQC, the relative standard deviation (RSD, %) of MQC does not exceed 15%, and the RSD of LQC does not exceed 20%. The results show that the mean accuracy is generally within the range of ±15% of the labeled value of the quality control sample, and the intra-batch and inter-batch accuracy of LQC is within the range of ±20% of the labeled value. After the above verification, this method has good precision and accuracy.

[0080] Extraction recovery and matrix effect of Acyl-CoAs in biological matrices

[0081] The investigation of matrix effect is mainly carried out by calculating the matrix factor of each analyte and internal standard. The matrix factor is the ratio of the peak area of ​​the analyte measured by mixing 6 portions of extracted blank matrix with the working solution to the peak area of ​​the pure standard solution at the corresponding concentration (n-6). The matrix factor is further standardized by dividing the matrix factor of the analyte by the matrix factor of the internal standard. The matrix factor normalized by the internal standard is used as the investigation index of the matrix effect. The coefficient of variation of the matrix factor after internal standard normalization must be within 15% and should be determined at three quality control concentrations. The investigation of extraction recovery rate is carried out by comparing the average peak area of ​​the analyte in LQC, MQC and HQC (n=6) and the ratio of the extraction blank matrix of the corresponding analyte concentration to calculate the recovery rate. The results are shown in Table 5

[0082] Table 5 Extraction recovery and matrix effect of Acyl-CoAs in kidney tissue

[0083]

[0084]

[0085] As shown in Table 5, we used 6 batches of kidney tissue blank matrices from different donors as experimental objects for tissue samples, and investigated the matrix effect and extraction recovery of kidney tissue at three concentration points of HQC, MQC, and LQC. The mean values ​​of matrix effects in mouse kidney tissue at different concentration points ranged from 0.78 to 1.19, and the average extraction recovery in kidney tissue ranged from 0.83 to 1.19. The matrix effect indicates that the sample suppresses or enhances the ions of the analytes to be measured. When applied to real samples, the corresponding IS is used to reduce matrix interference. However, in this verification process, the RSD basically meets the requirements of LQC less than 20%, MQC, and HQC less than 15%, which can indicate that the detection method and extraction method have reliable stability and repeatability.

[0086] Example 2 Effect of different chromatographic conditions on Acyl-CoAs separation conditions

[0087] 1) Chromatographic column

[0088] In order to explore the influence of different chromatographic columns on the separation effect, phenylhexyl column and C18 column were selected for the separation of Acyl-CoAs. Figure 5 ) and phenylhexyl column ( Figure 6 ) have the following separation effects on Acyl-CoAs: Figure 5 and Figure 6 As shown:

[0089] like Figure 5 and Figure 6 As shown in the figure, the peak shape of the same concentration of compounds has been significantly improved under the same other conditions, which shows that the phenylhexyl column is more suitable for the separation of Acyl-CoAs than the C18 column used in the past. The reason is that although the C18 column and the phenylhexyl column are both reverse chromatographic columns, the C18 column is modified by an octadecyl group and has a better hydrophobic effect, which is suitable for the separation of non-polar and medium-polar compounds. It has a good separation effect on acidic, basic and amphoteric compounds, and is especially suitable for the analysis of compounds such as long-chain fatty acids and drug components. The phenyl column is modified by a phenyl group, has high stability and good separation efficiency, and is suitable for the analysis of a variety of compounds. The phenyl group provides a strong hydrophobic effect, which is conducive to the separation of non-polar and medium-polar substances, and is especially suitable for the separation of aliphatic compounds and natural products.

[0090] 2) Mobile phase

[0091] Choose different compositions of mobile phases for testing;

[0092] Group 1 (separation results as Figure 7 ):

[0093] Phase A, H O containing 1 mM ammonium acetate and 0.05% formic acid;

[0094] Phase B, acetonitrile contains 1 mM ammonium acetate and 0.05% triethylamine.

[0095] Group 2 (separation results as Figure 8 ):

[0096] Phase A, H O containing 1 mM ammonium acetate;

[0097] Phase B, acetonitrile contained 1 mM ammonium acetate and 0.05% triethylamine, and the rest of the experimental conditions were the same.

[0098] like Figure 7 and Figure 8As shown in the figure, when the pH of the mobile phase increases, the separation efficiency of Acyl-CoAs can be effectively improved. The reason is that triethylamine is an organic base that can effectively adjust the pH value of the eluent. In the column experiment, the appropriate pH value helps to improve the separation effect between the target substance and the impurities. By adding triethylamine, the pH of the eluent can be accurately adjusted to achieve the best separation conditions. Adding triethylamine to the mobile phase is mainly used to solve the tailing phenomenon of basic compounds. Compounds with polar groups such as -COOH, -NH2, -NHR, and -NR2 are prone to tailing. Triethylamine combines with silanol groups to hinder its binding with compounds, thereby reducing the occurrence of tailing.

[0099] In summary, we found that selecting a suitable chromatographic column and adjusting the pH value of the eluent can effectively improve the separation effect of Acyl-CoAs.

[0100] Example 3: Cell experiment

[0101] Animal experiments and sample collection

[0102] C57BL / 6 male mice of the same batch, 6 weeks old and weighing about 20g, were selected as research subjects and purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., with production license number: SCXK (Su) 2020-0009 and use license number SYXK (Shanghai) 2019-0020. They were kept in the SPF-level animal laboratory mouse room of Shanghai University School of Medicine, with an indoor temperature of 25°C and a humidity of 60%. The mouse room was illuminated for 12 hours in a cycle, and food and water were available freely. Grouping and drug administration began after one week of environmental adaptation. Animal ethics were reviewed by the Science and Technology Ethics Committee of Shanghai University, in accordance with ethical principles and relevant national regulations, with approval number ECSHU2024-108.

[0103] The animals were randomly divided into a blank solvent control group, a 0.2 mg / kg aristolochic acid I (AAI) administration group, and a 20 mg / kg aristolochic acid I (AAI) administration group, with 12 animals in each group. The drugs were administered for 5 consecutive days, and the animals were killed by dislocation of the neck 2 days after drug withdrawal, and the heart, liver, spleen, lung, kidney and other tissues were obtained.

[0104] In this experiment, 25 mg of kidney tissue was taken, and extract A was added at a ratio of 10 uL / mg. After homogenization, it was centrifuged at 14000g and 4°C for 10 min. After centrifugation, the supernatant was taken and stored at -80°C until it was loaded onto the machine. The LC-MS / MS system was used for detection, and the mass spectrometry parameters, chromatographic parameters and data processing methods were the same as those in Example 1. The types of acyl-CoA in kidney tissue were determined, and the contents of different types of acyl-CoA were calculated using the Acyl-CoAs standard curve equation provided in Table 3. The results are as follows: Fig. 9 shown.

[0105] Depend on Fig. 9 It can be seen that a total of 14 acyl-CoA were quantified in the renal tissues of mice administered with aristolochic acid, of which 11 showed a significant change trend with the change of administration concentration.

[0106] From the above quantitative analysis results, it can be seen that the detection method provided in the present application can be effectively applied in animal kidney tissue and express the changing trend of acyl-CoA of different concentrations and different chain lengths under different administration concentrations, and has good analytical ability.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously analyzing acyl-CoA of different lengths based on an LC-MS / MS system, characterized in that: The following steps are involved: (1) preparing working solutions of Acyl-CoAs standards of different lengths, injecting the solutions, and obtaining standard curves of Acyl-CoAs standards of different lengths; (2) adding extract A to the sample to be tested, homogenizing and centrifuging, taking the supernatant, and detecting it using an LC-MS / MS system to determine the type of acyl-CoA in the sample to be tested, and using the standard curve of step (1) to calculate the content of different types of acyl-CoA; The chromatographic column in the LC-MS / MS system is a phenyl-hexyl chromatographic column.

2. The method according to claim 1, characterized in that: The Acyl-CoAs standard includes: CoA0:0, CoA2:0, CoA3:0, COA3-keto10:0, CoA3O2, COA3-keto16:0, CoA4:0, CoA6:0, CoA8:0, CoA10:0, CoA12:0, CoA14:0, CoA16:0, CoA17:0, CoA18:0, CoA18:1, CoA20:0, CoA22:0, CoA22:6, CoA24:0 and CoA-SUC.

3. The method according to claim 2, characterized in that: The concentration of the working solution of the Acyl-CoAs standard in step (1) is 90 to 110 ng / mL.

4. The method according to claim 3, characterized in that: The extract A in step (2) is composed of methanol, acetonitrile and water in a volume ratio of 2-4:2-4:1-2, and the total addition amount is 8-12 μL / mg of the sample to be tested.

5. The method according to claim 4, characterized in that: The centrifugal conditions in step (2) are 13000-15000 g, 3-5° C., and 8-12 min.

6. The method according to claim 5, characterized in that In steps (1) and (2), the mass spectrometer adopts multiple reaction ion detection mode, positive ion mode acquisition, and electrospray ion source; capillary temperature: 310-330°C; spray voltage: 3.6-4.0 kV; maximum spray current: 80-120 μA; sheath gas flow rate: 28-32 L / min; auxiliary gas flow rate: 4-6 L / min; probe heating temperature: 280-320°C.

7. The method according to claim 6, characterized in that: In steps (1) and (2), mobile phase A is water containing 4.5-5.5 mM ammonium bicarbonate; The mobile phase B comprises acetonitrile, methanol and water in a mass ratio of 7.5-8.5:1.5-2.5:1, wherein the mobile phase B also contains 4.5-5.5 mM ammonium bicarbonate and 0.08-0.12% triethylamine.

8. The method according to claim 7, characterized in that: During the chromatographic gradient elution process, the mobile phase A is 95-97% within the elution time of 0-10 minutes, 50-55% at the elution time of 10-11 minutes, adjusted to 28-32% at the elution time of 14-16 minutes, and 8-12% at the elution time of 18-19.5 minutes; The mobile phase B is 3-5% during the elution time of 0-10 minutes, adjusted to 45-50% when the elution time reaches 10-11 minutes, adjusted to 68-72% when the elution time reaches 14-16 minutes, and adjusted to 88-92% when the elution time reaches 18-19.5 minutes; The elution flow rate is 0.28~0.32mL / min.

9. A kit for simultaneously analyzing acyl-CoA of different lengths based on an LC-MS / MS system, characterized in that: The kit includes the following reagents: Acyl-CoAs standard solutions of different lengths, extracting solution A, reagent A and reagent B; The reagent A is ammonium bicarbonate solid powder, which is prepared into a 4.5-5.5 mM ammonium bicarbonate solution with water as mobile phase A; The reagent B is used as mobile phase B, which is prepared by mixing acetonitrile, methanol and water in a mass ratio of 7.5-8.5:1.5-2.5:1, and then adding ammonium bicarbonate and triethylamine, wherein the final concentration of ammonium bicarbonate in the reagent B is 4.5-5.5 mM, and the volume fraction of triethylamine is 0.08-0.12%.

Images