A method for simultaneous analysis of common free oxidized lipids and their precursor compounds in human milk or milk powder samples

Abstract

The application relates to a method for simultaneously analyzing common free oxidized lipids and precursor compounds in breast milk or milk powder samples, and belongs to the technical field of biological separation and analysis. The method for simultaneously analyzing the application comprises the following steps: 1, adding methanol to the sample, vortexing and then standing; 2, centrifuging to collect the supernatant, and then adding formic acid-water solution and mixing; 3, taking an OasisPRiMEHLB (1cc, 30mg) solid-phase extraction column for pretreatment, respectively using formic acid-water solution and methanol-water solution containing formic acid to rinse the column, finally using acetonitrile / methanol to elute, and then centrifuging the eluent to obtain the supernatant after standing overnight; and 4, performing ultra-high performance liquid chromatography-mass spectrometry analysis to quantitatively analyze the content of oxidized lipids and precursor compounds. The application is suitable for simultaneously analyzing the content of oxidized lipids and polyunsaturated fatty acids in breast milk, milk powder and other dairy products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological separation and analysis, and particularly relates to a method for simultaneously analyzing common free oxidized lipids and precursor compounds thereof in breast milk or milk powder samples. BACKGROUND

[0002] Oxidized lipids are highly bioactive compounds produced by autoxidation of polyunsaturated fatty acids (PUFAs) (arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) and alpha-linolenic acid (ALA)) or enzymatic pathways (cyclooxygenase (COX), lipoxygenase (LOX) or cytochrome P450 (CYP)). PUFAs and their metabolites (oxidized lipids) are key regulators of intracellular signaling affecting inflammatory responses, and have become one of the research hotspots due to their multiple important functions in the human body.

[0003] Polyunsaturated fatty acids are the most important fatty acids in breast milk, which play a crucial role in the visual, immune, cognitive and motor development of newborns. The concentration of oxidized lipids in breast milk can represent a marker of the inflammatory state of the mammary gland. The most studied oxidized lipids are eicosanoids derived from AA metabolism through enzymatic pathways, which are mainly responsible for pro-inflammatory processes. Other common oxidized lipids include octadecanoids extracted from LA and ALA, eicosanoids extracted from dihomo-gamma-linolenic acid (DGLA) and EPA, and eicosanoids extracted from adrenic acid (AdA) and DHA. Breast milk oxidized lipids have been shown to regulate the development and immunity of infants and play a multiple regulatory role in infant development. Infant formula, as the best substitute for breast milk, has always taken breast milk as the golden criterion for production. In order to improve the design of infant formula simulating breast milk, it is particularly important to develop an oxidized lipid and precursor compound analysis method with simple operation, high sensitivity, good selectivity and high detection throughput.

[0004] The most common methods currently used include immunoassay, gas chromatography mass spectrometry (GC-MS) or liquid chromatography mass spectrometry (LC-MS) and the like. Immunoassay is usually limited to one or a few metabolites at a time, requires a long analysis time, and can lead to cross-reactions. The GC-MS method has high sensitivity and selectivity, but requires a large sample volume and a chemical derivatization step. The ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS) method has become the preferred method for oxidized lipids due to its high sensitivity and selectivity and ability to achieve high-throughput detection. The most similar liquid phase conditions to the present scheme are as follows: 0.1% acetic acid-water and acetonitrile: isopropanol (90:10) are used as mobile phases A and B, respectively, the analysis column is an Acquity UPLC BEHC18 column, and PUFAs (ESI+) and oxidized lipids (ESI-) are determined by two gradients: for PUFAs, the gradient is run for 19 minutes, starting with 70% mobile phase A, at a flow rate of 0.3 mL / min. For oxidized lipids, the gradient is run for 25 minutes, starting with 90% eluent A, at a flow rate of 0.3 mL / min.

[0005] The main extraction methods include protein precipitation (PPT), liquid-liquid extraction (LLE) and solid phase extraction (SPE) and derivatization. SPE, due to the presence of various types of adsorbents and solvents, is currently the most commonly used extraction method for analyzing oxidized lipids. The most similar step in the pretreatment step to the present scheme is as follows: 1 mL of milk is added to methanol for protein precipitation and centrifuged, the supernatant is mixed with 10 mL of 0.1% formic acid-water, the sample is loaded onto an Oasis PRiME HLB (30 mg) column activated with 1 mL of methanol and 1 mL of 0.1% formic acid-methanol / water solution (5:95, v / v) beforehand, then washed with 1 mL of 0.1% formic acid-methanol / water solution (5:95, v / v) and dried under high vacuum, and the target is eluted twice with 600 μL of acetonitrile:methanol (9:1, v / v). The eluate is evaporated under a stream of nitrogen, and the sample is reconstituted in 100 μL of methanol-water solution (1:1, v / v) and transferred to a glass vial for LC-MS / MS analysis.

[0006] However, the above analysis methods can only be optimized for oxidized lipids or PUFAs single group of compounds, and cannot achieve simultaneous determination of oxidized lipids and PUFAs. For the determination of fatty acids, a derivatization step is usually required, so separate determination not only takes a long time, but also causes large instrument wear, and greatly increases the amount of solvent used.

[0007] The pretreatment method for breast milk samples is mainly improved based on biological samples such as serum, but the high content of lipids in breast milk and the interference molecules can cause the SPE column to be blocked, thereby affecting the extraction efficiency. Gouveia-Figueira et al. found that compared with other types of biological samples (such as tissue, cell extract and plasma), the use of the above method for detecting oxidized lipids in plasma resulted in a significant decrease (close to 50%) in the extraction efficiency of breast milk. Therefore, we optimized a non-derivatization method for simultaneous and efficient SPE extraction of oxidized lipids and polyunsaturated fatty acids.

[0008] In summary, the present application provides a method for simultaneously analyzing a variety of oxidized lipids and their precursor compounds in breast milk or milk powder samples based on the SPE-UPLC-MS / MS method without derivatization, high selectivity and high sensitivity, which provides important technical support for the evaluation of oxidized lipids and their precursor compounds in breast milk and other biological samples and related biological research. SUMMARY

[0009] In view of the problems existing in the prior art, the purpose of the present application is to design an LC-MS / MS method for simultaneously analyzing common free oxidized lipids and their precursor compounds in breast milk and milk powder samples. Compared with the existing method, the sample pretreatment method of the present application does not require derivatization, and the SPE method is used to reduce the matrix effect, and the UPLC-MS / MS conditions can realize the complete separation of 26 kinds of oxidized lipids and their precursor compounds and provide high sensitivity and high selectivity, thereby providing a new reference method for the analysis of oxidized lipids and polyunsaturated fatty acid content in breast milk and formula milk powder.

[0010] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0011] A method for simultaneously analyzing free oxidized lipids and their precursor compounds, comprising the following steps:

[0012] (1) taking a sample, adding methanol, vortexing and then standing;

[0013] (2) centrifuging the sample treated in step (1), collecting the supernatant, adding formic acid-water solution and mixing;

[0014] (3) taking Oasis PRiME HLB, pre-activating the solid phase extraction column with 1 mL of methanol, and equilibrating with 1 mL of 0.1% formic acid-water solution; purifying the sample treated in the above step (2) using the pre-activated and equilibrated Oasis PRiME HLB solid phase extraction column, and then using formic acid-water solution and formic acid-containing methanol-water solution to rinse the column, finally eluting with acetonitrile / methanol, and taking the eluate and standing overnight before centrifuging and taking the supernatant;

[0015] (4) The supernatant obtained in step (3) is analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain multiple reaction monitoring data, and the possible presence of oxidized lipids and precursor compounds in the sample is screened by using the retention time locking and characteristic ion pair locking methods, and the content thereof is quantified by external standard method.

[0016] The method, in step (1), the sample is a dairy solution; the volume ratio of the sample to methanol is 0.5:1 or 0.5:1.5; the vortexing time is 10-30 min, and the standing condition is temperature-20℃, time 30-60 min.

[0017] The method, the sample is breast milk or milk powder solution.

[0018] The method, the concentration of the milk powder is 0.5g / mL.

[0019] The method, in step (2), the centrifugal treatment condition is temperature 4℃, speed 10000-13000rpm, time 15-30min; the concentration of the formic acid-water solution is 0.1%, and the volume ratio of the formic acid-water solution to the sample is 10:0.5.

[0020] The method, in step (3), the specification of the Oasis PRiME HLB is 1cc, 30mg; the concentration of the formic acid-water solution is 0.1%, the concentration of the formic acid-containing methanol-water solution is 10%, and the concentration of the formic acid is 0.1%; the volume ratio of acetonitrile / methanol is 9:1; the volume ratio of the formic acid-water solution, the formic acid-containing methanol-water solution and acetonitrile / methanol is 1:1:0.3.

[0021] The method, in step (4), the model of the ultra-high performance liquid chromatography-tandem mass spectrometer is UPLC-MS 8060 of Shimadzu; the analysis condition of the ultra-high performance liquid chromatography-tandem mass spectrometer is that the analysis column used is ACQUITY UPLC Premier BEH C18(2.1×100mm, 1.7μm), and the mobile phase for oxidized lipid analysis is that phase A is 0.1% formic acid-water, and phase B is isopropanol / acetonitrile solution(v / v, 1:9).

[0022] The method, in step (4), the gradient elution procedure of the ultra performance liquid chromatography-tandem mass spectrometry analysis is as follows: the initial proportion of A phase is 60%, and the proportion of B phase is 40%, and the proportion is maintained to 7.0 min; the proportion of B is increased to 55% within 3 min; the proportion of B is increased to 85% within 1 min; the proportion of B is increased to 100% within 1 min, and then maintained for 3 min; the proportion of B is maintained to 40% at 15.01 min, and maintained to 18.0 min, the flow rate of the mobile phase is always 0.2 mL / min during the whole elution process; the column oven temperature is 40 DEG C; the injection volume is 1 mu L; and the injection needle cleaning liquid is methanol / water (v / v, 1:1).

[0023] The method, in step (4), the mass spectrometry in the ultra performance liquid chromatography-tandem mass spectrometry analysis is operated in an electrospray negative or positive ion mode (ESI - / + ), the interface temperature is 300 DEG C, the heating block temperature is 400 DEG C, the desolvation temperature is 250 DEG C, the atomization gas flow is 3.0 L / min, the heating gas flow is 10.0 L / min, and the drying gas flow is 10.0 L / min.

[0024] The method, in step (4), the data acquisition mode is a multiple reaction monitoring mode (MRM).

[0025] Compared with the prior art, the method has the following advantages:

[0026] 1. The sample is pretreated by using an Oasis PRiME HLB (1cc, 30mg) solid phase extraction column, and the optimized solid phase extraction step can reduce the sample matrix effect.

[0027] 2. The ACQUITY UPLC Premier BEH C18 chromatographic column is used, the chromatographic column has high sensitivity, the optimized liquid phase condition can realize complete separation of 26 kinds of oxidized lipids and polyunsaturated fatty acids (containing 5 groups of isomers) within 18 minutes, and formic acid is used as an additive, so that the response and reproducibility of the polyunsaturated fatty acids are greatly enhanced without affecting the determination of the oxidized lipids.

[0028] 3. The technical scheme in the application can be applied to simultaneous analysis of the contents of oxidized lipids and polyunsaturated fatty acids in breast milk, milk powder and other dairy products. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1Total ion chromatogram of 26 free oxidized lipids and their precursor compounds based on premier BEH C18 column separation;

[0030] Figure 2 Extraction ion chromatogram of 26 free oxidized lipids and their precursor compounds based on premier BEH C18 column separation;

[0031] Figure 3 Standard curve of 26 free oxidized lipids and their precursor compounds;

[0032] Figure 4 Matrix effect of 26 free oxidized lipids and their precursor compounds in breast milk and milk powder samples;

[0033] Figure 5 Response and stability of 26 free oxidized lipids and their precursor compounds under different additives;

[0034] Figure 6 Relative recovery of 26 free oxidized lipids and their precursor compounds under different elution volumes (200, 300, 400 μL) (normalized to 200 μL);

[0035] Figure 7 Separation effect and response value of 26 free oxidized lipids and their precursor compounds based on premier BEH C18 and BEH C18 columns. DETAILED DESCRIPTION

[0036] The present application will be further described below in conjunction with the accompanying drawings and examples.

[0037] In this example, breast milk and infant formula milk powder samples were selected, and oxidized lipids and polyunsaturated fatty acids in the samples were extracted and analyzed. The following examples are used to illustrate the present application, but not to limit the scope of use of the present application.

[0038] Example 1:

[0039] 1) Take 0.5 mL of breast milk and milk powder solution (0.5 g of milk powder sample dissolved in 1.0 mL of warm water) respectively, add 1.0 mL of methanol, vortex for 10 min, and place at -20℃ for 30 min.

[0040] 2) The sample obtained in step 1) was placed in a refrigerated centrifuge, centrifuged at 13000 rpm for 30 min at 4°C, and the supernatant was collected in a new centrifuge tube. 10 mL of 0.1% formic acid-water solution was added, and the mixture was mixed to prepare for the solid phase extraction step.

[0041] 3) The sample was pretreated using an Oasis PRiME HLB (1 cc, 30 mg) solid phase extraction column. The column was activated with 1 mL of methanol and equilibrated with 1 mL of 0.1% formic acid-water solution. The sample obtained in step 2) was purified using the pre-equilibrated Oasis PRiME HLB (1 cc, 30 mg) solid phase extraction column. After loading the sample, the column was rinsed with 1 mL of 0.1% formic acid-water solution and 1 mL of 10% methanol-water solution (containing 0.1% formic acid), respectively. Finally, 300 μL (2*150 μL) of acetonitrile / methanol (9:1, v / v) was used for elution. The eluate was placed at -20°C overnight, and the supernatant was collected by centrifugation before being tested.

[0042] 4) The sample obtained in step 3) was analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to obtain multiple reaction monitoring (MRM) data. The sample was screened for possible oxidized lipids using retention time locking and characteristic ion pair locking, and the content was quantified by external standard method.

[0043] The UPLC-MS / MS in step 4) above was Shimadzu UPLC-MS 8060, and the analysis column used was ACQUITY UPLC Premier BEH C18 (2.1*100 mm, 1.7 μm). The mobile phase for oxidized lipid analysis was 0.1% formic acid-water for phase A and isopropanol / acetonitrile solution (v / v, 1:9) for phase B. The gradient elution program was as follows: the initial proportion was 60% A and 40% B, which was maintained until 7.0 min; it was increased to 55% B within 3 min; it was increased to 85% B within 1 min; it was increased to 100% B within 1 min and maintained for 3 min; it was 40% B from 15.01 min to 18.0 min. The flow rate of the mobile phase was always 0.2 mL / min throughout the elution process; the column oven temperature was 40°C; the injection volume was 1 μL; and the injection needle cleaning solution was methanol / water (v / v, 1:1). The mass spectrometer was operated in negative / positive ion mode (ESI - / + ), the interface temperature was 300°C, the heating block temperature was 400°C, the desolvation temperature was 250°C, the nebulization gas flow was 3.0 L / min, the heating gas flow was 10.0 L / min, and the dry gas flow was 10.0 L / min. The data acquisition mode was multiple reaction monitoring mode (MRM). The monitoring ion pairs and corresponding collision energy parameters in the detection method for the content analysis of oxidized lipids and their precursor compounds in the sample are shown in Table 1 below. For example, the monitoring ion pairs for the detection of 13-HODE and 9-HODE were m / z 255.2 / 237.2 and m / z 255.2 / 237.2, respectively, and the corresponding collision energy parameters were 35 eV and 35 eV, respectively.Figure 1 Figure 1 shows the total ion chromatogram of 26 free oxylipins and their precursors based on a premier BEH C18 column. (A) is the total ion chromatogram of 26 free oxylipins and their precursors standards based on a premier BEH C18 column; (B) is the total ion chromatogram of 26 free oxylipins and their precursors in a breast milk sample based on a premier BEH C18 column. As shown in Figure 1, the 26 free oxylipins and their precursors were completely separated. Figure 2 Figure 2 shows the extracted ion chromatogram of 26 free oxylipins and their precursors based on a premier BEH C18 column. (A) is the extracted ion chromatogram of 26 free oxylipins and their precursors standards based on a premier BEH C18 column. Figure 1 (A) and Figure 2 Figure 3 shows that 26 target analytes including 5 groups of isomers (PGA2-PGJ2-PGH2, PGE1-PGD1, PGE2-PGD2, 12(13)-DiHOME-9(10)-DiHOME, 12(13)-EpOME-9(10)-EpOME) were completely separated. Figure 1 Figure 4 shows that 5 PUFAs and 7 oxylipins were detected in breast milk. (A) shows that 5 PUFAs were detected in breast milk; (B) shows that 7 oxylipins were detected in breast milk.

[0044] Table 1. Oxylipins and their precursors monitoring ion pairs and collision energy parameters

[0045]

[0046]

[0047] Prostaglandins (PGs), Thromboxane (TXs), Hydroxy-eicosatetraenoic acids (HETEs), Hydroxy-eicosapentaenoic acids (HEPEs), Hydroxy-docosahexaenoic acid (HDHA), Dihydroxy-octadecenoic acid (DiHOME), Epoxy-octadecenoic acid (EpOME), Leukotrienes (LTs), Epoxy-eicosatrienoic acids (EET), Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA), Arachidonic acid, Linoleic acid, Linolenic acid

[0048] 5) Calculate the content of oxidized lipids in the sample according to the standard curve, and the detection limit of the standard solution at S / N = 3 and the quantification limit at S / N = 10: the standard curve of 26 kinds of free oxidized lipids and their precursor compounds is shown in Figure 3

[0049] Table 2. The content range of oxidized lipids and their precursor compounds in breast milk and milk powder samples and the detection limit and quantification limit of the analysis method

[0050]

[0051]

[0052] The results in Table 2 show that the detection limits of oxidized lipids and PUFAs are 0.01-2.10 ng / mL and 0.02-0.59 ng / mL, respectively. Five kinds of polyunsaturated fatty acids and seven kinds of oxidized lipids are detected in breast milk and milk powder: the content of free PUFAs and oxidized lipids in breast milk (average) is 6.5-55.7 times that in the same volume of milk powder, especially the content of PUFAs (EPA, DHA, AA, LA and ALA) in breast milk is much higher than that in milk powder. The oxidized lipids detected in breast milk and milk powder are mostly LA metabolites, followed by EPA and AA metabolites, which is consistent with their high precursor content.

[0053] As​Figure 4 The matrix effects of 26 free oxidized lipids and their precursors in breast milk and milk powder samples were investigated. The results showed that the matrix effects of breast milk ranged from 72% to 125% with RSDs from 1.2% to 15%. For milk powder solutions, the matrix effects ranged from 77% to 112% with RSDs from 1.7% to 10.1% except for 11,12-EET which was 49%.

[0054] Comparative Example 1:

[0055] The method for simultaneous analysis of common free oxidized lipids and their precursors in breast milk samples was the same as the procedure of Example 1, except that the additive formic acid was changed to acetic acid. Compared with Example 1, the responses and stabilities of 26 free oxidized lipids and their precursors under different additives were shown in Table 1. Figure 5 The results showed that the addition of 0.1% acetic acid could increase the response of most prostaglandins by 30% to 46%, PGF2 α -EA by 323%, DiHOME by 46% to 51%, LTE4 and TXB2 by 116% and 43%, respectively. However, the addition of 0.1% formic acid greatly increased the response of 5 PUFAs (by 861% to 4219%), the response of EpOME increased by 14% to 64%, and the response of 11,12-EET increased by 263%. In addition, for most target analytes, the reproducibility of formic acid as an additive was better than that of acetic acid. Finally, considering the quantitative requirements of simultaneous analysis and the detection results of actual samples, 0.1% formic acid was selected as the additive of mobile phase A.

[0056] Comparative Example 2:

[0057] The method for simultaneous analysis of common free oxidized lipids and their precursors in breast milk samples was the same as the procedure of Example 1, except that the elution solvent 300 μL was changed to 200 μL and 400 μL. Compared with Example 1, the relative recoveries of 26 free oxidized lipids and their precursors were shown in Table 2. Figure 6 The results showed that the recovery was the worst with 0.2 mL eluent, while there was no significant difference between 300 and 400 μL eluent. Therefore, 0.3 mL was selected as the elution volume under the premise of saving elution solvent and time.

[0058] Comparative Example 3:

[0059] The method for simultaneous analysis of common free oxidized lipids and their precursors in breast milk samples was the same as the procedure of Example 1, except that Premier BEH C18 was replaced by BEH C18 column. Compared with Example 1, the separation effect and response of 26 free oxidized lipids and their precursors were shown in Table 3. Figure 7Compared with the traditional BEH C18 column, the Premier BEH C18 column with MaxPeak high-performance surface technology improved the recovery, sensitivity and reproducibility of the analytes, especially for the five PUFAs. With the Premier BEH C18 column, the peak intensity and reproducibility were increased by 14%-78% and 1%-16%, respectively. Finally, the Premier BEH C18 column with the best separation effect and sensitivity was selected for the analysis of oxidized lipids and their precursors.

Claims

1. A method for simultaneous analysis of free oxidized lipids and their precursor compounds, characterized in that: The following steps are involved: (1) Take a sample, add methanol, vortex, and let it stand; (2) centrifuging the sample after treatment in step (1), collecting the upper layer of the sample supernatant, adding a formic acid-water solution, and mixing; (3) Pre-activate an Oasis PRiME HLB solid-phase extraction cartridge with 1 mL of methanol and equilibrate with 1 mL of 0.1% formic acid-water solution. Purify the sample treated in step (2) above using the pre-activated and equilibrated Oasis PRiME HLB solid-phase extraction cartridge. After loading the sample onto the cartridge, rinse the cartridge with formic acid-water solution and then with formic acid-containing methanol-water solution. Finally, elute with acetonitrile / methanol. Allow the eluate to stand overnight and centrifuge to obtain the supernatant. (4) analyzing the supernatant obtained in step (3) by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain multiple reaction monitoring data, screening the presence of oxidized lipids and their precursor compounds in the sample by retention time locking and characteristic ion pair locking, and quantifying their contents by external standard method; The conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry analysis in step (4) are as follows: the analytical column used is an ACQUITY UPLC Premier BEH C18 2.1×100 mm, 1.7 μm, the mobile phase for oxidized lipid analysis is: phase A is 0.1% formic acid-water, and phase B is isopropanol / acetonitrile solution v / v, 1:9; the gradient elution program for the ultra-high performance liquid chromatography-tandem mass spectrometry analysis in step (4) is: the starting ratio is 60% phase A and 40% phase B, maintained for 7.0 min; increased to 55% B within 3 min; increased to 85% B within 1 min; increased to 100% B within 1 min and maintained for 3 min; 15.01 min, 40% B maintained until 18.0 min; The free oxidized lipids and their precursor compounds include PGA2, PGJ2, PGD2, PGE2, PGH2, 12-HETE, 12-HEPE, 9(10)-DiHOME, 12(13)-DiHOME, 9(10)-EpOME, 12(13)-EpOME, 14-HDHA, PGD1, PGE1, PGF1α, PGF2α-EA, LTE4, TXB2, 6-ketoPGF1α, 11,12-EET, PGF2α, EPA, DHA, Linoleic acid, Arachidonic acid, and Linolenic acid.

2. The method according to claim 1, wherein The sample in step (1) is a dairy solution; the volume ratio of the sample to methanol is 0.5:1 or 0.5:1.5; the vortex time is 10 to 30 minutes, and the static conditions are: temperature -20°C, time 30 to 60 minutes.

3. The method according to claim 2, wherein The sample is breast milk or milk powder solution.

4. The method according to claim 3, wherein The concentration of the milk powder is 0.5 g / mL.

5. The method according to claim 1, wherein The conditions for the centrifugal treatment in step (2) are: temperature 4° C., rotation speed 10,000-13,000 rpm, time 15-30 min; the concentration of the formic acid-water solution is 0.1%, and the volume ratio of the formic acid-water solution to the sample is 10:0.

5.

6. The method according to claim 1, wherein The specification of the Oasis PRiME HLB described in step (3) is 1 cc, 30 mg; the concentration of the formic acid-water solution for rinsing the column is 0.1%, the concentration of the methanol-water solution containing formic acid for rinsing the column is 10%, and the concentration of formic acid is 0.1%; the volume ratio of the acetonitrile / methanol is 9:1; the volume ratio of the formic acid-water solution for rinsing the column, the methanol-water solution containing formic acid for rinsing the column, and acetonitrile / methanol is 1:1:0.

3.

7. The method according to claim 1, wherein The model of the ultra-high performance liquid chromatography-tandem mass spectrometry instrument in step (4) is Shimadzu's UPLC-MS 8060.

8. The method according to claim 1, wherein The flow rate of the mobile phase during the entire elution process in step (4) was always 0.2 mL / min; the column oven temperature was 40° C.; the injection volume was 1 μL; and the injection needle cleaning solution was methanol / water v / v, 1:

1.

9. The method according to claim 1, wherein The ultra-high performance liquid chromatography-tandem mass spectrometry analysis in step (4) was performed in the electrospray negative or positive ion mode (ESI - / + ) with an interface temperature of 300°C, a heating block temperature of 400°C, a desolvation temperature of 250°C, a nebulizing gas flow rate of 3.0 L / min, a heating gas flow rate of 10.0 L / min, and a drying gas flow rate of 10.0 L / min.

10. The method according to claim 1, wherein The data collection method in step (4) is a multiple reaction monitoring mode.

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