Sample pretreatment method for determination of bile acid subtype content and application thereof

Abstract

This invention belongs to the field of bile acid detection technology, and discloses a sample pretreatment method for determining the content of bile acid subtypes and its application. The pretreatment method includes the following steps: S1, acidifying the sample to be tested with an acidifying agent to a pH of 2-4 to obtain an acidified sample; S2, eluting the acidified sample onto a mixed-mode anion exchange column to obtain an eluent; S3, evaporating the eluent and reconstituted with a rehydration solution to obtain the test sample. The test sample obtained by this pretreatment method can effectively eliminate or compensate for matrix interference when used for targeted analysis of bile acid subtypes by LC-MS / MS, thus effectively improving detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of bile acid detection technology, specifically relating to a sample pretreatment method for determining the content of bile acid subtypes and its application. Background Technology

[0002] Intrahepatic cholestasis of pregnancy (ICP) is a common liver complication in the second and third trimesters of pregnancy. It is also a core phenotype of bile metabolism disorders widely present in various chronic liver diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Its pathological essence lies in the obstruction of bile acid synthesis, secretion, and flow, leading to the accumulation of cytotoxic bile acid components in the liver, causing hepatocellular damage, inflammation, and even fibrosis.

[0003] Currently, the "gold standard" for clinical diagnosis is liver biopsy. However, due to its invasiveness, procedural risks, and special contraindications during pregnancy, patient acceptance in clinical practice is extremely low (generally less than 30%). This makes it difficult to obtain clear histological evidence in suspected cases, relying only on serological indicators for inferential diagnosis, which affects early diagnosis, severity grading, and timing of treatment.

[0004] Serum total bile acids (TBA), as the most commonly used surrogate biomarker, have limited clinical utility. Studies have shown that up to 40% of patients with hematologically or symptomatically confirmed ICP still have TBA levels within the normal reference range. This false-negative phenomenon indicates that TBA, as a aggregate indicator, is insufficient to reflect the dramatic changes in the composition ratios of different subtypes (hydrophilic / hydrophobic, primary / secondary, free / bound) within the bile acid pool. It is precisely this qualitative change, rather than a simple quantitative increase, that is directly related to the degree of liver damage.

[0005] Recent studies have clearly revealed that the toxicity of the bile acid pool is not uniform. Hydrophobic bile acid subtypes, due to their strong detergency and cell membrane-damaging abilities, have been identified as key toxic mediators leading to hepatocyte apoptosis, necrosis, and worsening cholestasis. Their serum concentrations show a significant positive correlation with liver enzyme levels, the severity of pruritus, and adverse pregnancy outcomes (such as fetal distress and preterm birth).

[0006] However, accurate monitoring of this key toxic component is limited by the sensitivity ceiling of traditional analytical techniques. High-performance liquid chromatography (HPLC), relied upon by mainstream clinical testing, typically has a detection limit of 1 ng / mL and is easily affected by complex serum matrices. This makes it difficult to accurately quantify toxic bile acid subtypes in the early stages of disease or in cases with low concentrations of certain subtypes. Summary of the Invention

[0007] The purpose of this invention is to provide a sample pretreatment method for determining the content of bile acid subtypes and its application, which can accurately determine the content of bile acid subtypes in a sample.

[0008] The following technical solutions are used to achieve the above objectives.

[0009] The first aspect of this invention provides a sample pretreatment method for determining the content of bile acid subtypes, comprising the following steps:

[0010] Includes the following steps:

[0011] S1. Acidify the sample to be tested with an acidifying agent to a pH value of 2-4 to obtain the acidified sample;

[0012] S2. Elute the acidified sample in a mixed-mode anion exchange column to obtain the eluent.

[0013] S3. Evaporate the eluent and then reconstitute it with a reconstitution solution to obtain the test sample.

[0014] In some embodiments, in step S1, the acidifying agent is at least one of formic acid, acetic acid, and phosphoric acid; preferably, the acidifying agent is formic acid, and the pH value is 2 to 2.5, more preferably 2 to 2.2.

[0015] In some embodiments, the molar concentration of the acidifying agent is 1.8M to 1.5M.

[0016] In some implementations, step S2 specifically includes the following steps:

[0017] The acidified sample was loaded onto a mixed-mode anion exchange column.

[0018] Wash successively with 3%~5% v / v ammonia solution and 10%~30% v / v methanol solution;

[0019] The eluent is then obtained by eluting with a methanol solution containing 1%~5% v / v formic acid.

[0020] In some embodiments, the formic acid concentration in the methanol solution is 1% to 3% v / v, preferably 1.8% to 2.2% v / v;

[0021] And / or, the volume ratio of the sample to be tested to ammonia is 100 μL: 2 mL to 3 mL;

[0022] And / or, the volume ratio of the sample to be tested to the methanol aqueous solution is 100 μL: 2 mL to 3 mL;

[0023] And / or, the volume ratio of the sample to be tested to a methanol solution containing 1%~5% v / v formic acid is 100 μL: 2mL~3mL.

[0024] In some embodiments, the hybrid-mode anion exchange column is an Oasis MAX column.

[0025] In some embodiments, the bile acid subtype is selected from at least one of ursodeoxycholic acid, cholic acid, glycoursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurochlic acid, lithochlic acid, and taurochlic acid, preferably a hydrophobic bile acid, and more preferably deoxycholic acid or lithochlic acid.

[0026] In some embodiments, the reconstitution solution is a 40-60% v / v aqueous methanol solution or an aqueous acetonitrile solution;

[0027] Preferably, the reconstituted solution further contains 0.08%~0.5% (v / v) formic acid;

[0028] And / or, the complex solution contains 1 µg / mL to 2 µg / mL of a stable isotope label;

[0029] And / or, the sample to be tested is serum or plasma.

[0030] The second aspect of the present invention provides the application of the sample pretreatment method for determining the content of bile acid subtypes as described above in the preparation of test samples for the detection of bile acid subtypes; or the application of the sample pretreatment method for determining the content of bile acid subtypes as described above in the detection of bile acid subtypes.

[0031] A third aspect of the present invention provides a method for detecting bile acid subtypes, comprising the following steps:

[0032] The test sample for detection was obtained according to the sample pretreatment method described above;

[0033] The test sample was separated by high performance liquid chromatography.

[0034] The sample separated by high performance liquid chromatography was then analyzed by mass spectrometry.

[0035] In some embodiments, the chromatographic conditions for the chromatographic separation include:

[0036] Mobile phase A: 8mM~12mM ammonium acetate aqueous solution, containing 0.05%~0.2% v / v formic acid;

[0037] Mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 75~85:15~25, wherein the mixed solution contains 0.05%~0.2% (v / v) formic acid;

[0038] Gradient elution was used at a flow rate of 0.1 mL / min to 0.3 mL / min;

[0039] And / or, the mass spectrometry detection is performed using a triple quadrupole mass spectrometer;

[0040] The conditions for the mass spectrometry detection include:

[0041] Ionization mode: ESI-;

[0042] Atomizing gas flow rate: 2.0L / min~4.0L / min;

[0043] Interface temperature: 280℃~320℃;

[0044] Heating gas flow rate: 8.0 L / min ~ 12.0 L / min;

[0045] DL temperature: 160℃~180℃;

[0046] Drying airflow rate: 8.0 L / min ~ 12.0 L / min;

[0047] Heating module temperature: 380℃~420℃;

[0048] Collision gas: Argon;

[0049] Scanning mode: Selective Ion Detection (SIM).

[0050] In this invention, in the method for determining the content of bile acid subtypes, we found that by using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) platform, combined with a series of sample pretreatment operations, including acidification at a specific pH value and appropriate solid-phase extraction columns and elution operations, we can effectively eliminate or compensate for the influence of matrix interference on the detection results when targeting bile acid subtypes in LC-MS / MS analysis of samples. By combining optimized chromatographic conditions and mass spectrometry parameters, we can achieve trace detection of bile acid subtypes, especially hydrophobic bile acid subtypes, effectively improving detection sensitivity. Detailed Implementation

[0051] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0052] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0053] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0054] The present invention will be described below with reference to specific embodiments.

[0055] Example 1: Sample preparation, pretreatment, detection, and analysis

[0056] I. Preparation of Control Samples

[0057] 1. Preparation of standard solutions of 13 bile acids

[0058] Accurately weigh eight bile acid reference standards: ursodeoxycholic acid (UDCA), cholic acid (CA), lithocholic acid (LCA), glycoursodeoxycholic acid (GUDCA), glycocholic acid (GCA), tauroursodeoxycholic acid (TUDCA), taurocholic acid (TCA), and taurochlic acid (TLCA). Place them in a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the stock solutions of the eight reference standards.

[0059] Take appropriate amounts of each individual reference standard stock solution and add them to a 50 ml volumetric flask. Dilute to volume with methanol to obtain a mixed reference standard stock solution. Dilute stepwise at ratios of 1:1, 2, 4, 10, 20, 40, 100, and 200 to obtain a series of mixed reference standard solutions with eight concentrations. Store at 4°C for later use.

[0060] Preparation of internal standard solution: Accurately weigh 6.5 mg of erythromycin reference standard and place it in a 50 ml volumetric flask. Dissolve and dilute to the mark with acetonitrile, and shake well to obtain a 130 μg / ml internal standard stock solution. Take 0.5 ml of this stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with acetonitrile, and shake well to obtain a 1.3 μg / ml erythromycin acetonitrile internal standard solution. Store at 4°C for later use.

[0061] II. Pretreatment of test samples for the determination of bile acid subtype content

[0062] The sample preprocessing was performed using the following method:

[0063] S1. Acidify 100 μL of sample with 1M formic acid to pH 2 to obtain the acidified sample;

[0064] S2. Load all the acidified sample into an Oasis MAX column that has been pre-activated and equilibrated with methanol and water.

[0065] Wash successively with 2.5 mL of 5% ammonia solution and 2.5 mL of methanol / water (20 / 80, v / v) to remove impurities;

[0066] S3: Elute the target analyte with 2.5 mL of methanol solution containing 2% formic acid;

[0067] S4. Collect the eluent in a clean test tube and dry it under a gentle nitrogen stream (<40°C). Avoid high temperatures (<40°C).

[0068] The dried substance was reconstituted with 100 μL of a 50% methanol aqueous solution containing 0.1% formic acid to obtain the test solution, and a stable isotope-labeled internal standard (SIL-IS) with a final concentration of 1.3 µg / mL was added to the test solution.

[0069] d4-DCA, d4-LCA, d4-GDCA, d4-GLCA, d4-TDCA;

[0070] The sample test solution was obtained.

[0071] III. Sample Testing

[0072] The sample detection solution prepared above was detected using HPLC-MS / MS tandem mass spectrometry.

[0073] The detection method includes the following steps:

[0074] The internal standard solution prepared above was mixed with multiple standard solutions and then separated and detected by liquid chromatography and mass spectrometry. Based on the detection results and the concentration of bile acids in the internal standard solution and the standard solutions, the quantitative correction equations for each bile acid were obtained.

[0075] Provide a solution containing derivatization reagents: Add 50 μL of pyridine (C) solution to the standard solution prepared above and the sample detection solution containing a stable isotope-labeled internal standard to reconstitute the sample. Then add 50 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (D) containing 1% trimethylchlorosilane (E), shake and centrifuge, and then perform a derivatization reaction in a metal bath at 65 °C for 25 min to obtain the derivative. Remove the sample and let it stand on ice for 65 s. Then centrifuge at 13000 rpm for 9 min at 4 °C and collect the supernatant into a gas chromatography vial.

[0076] The standard solution containing the derivatizing reagent prepared above is mixed with the sample test solution at a volume ratio of 1.5:1 and reacted to obtain the test sample; the test sample is then separated and detected by liquid chromatography and mass spectrometry, and the detection results of each bile acid are obtained by combining the detection results and the quantitative correction equations of each bile acid.

[0077] The specific liquid chromatography conditions are as follows:

[0078] Chromatograph: Ultra-high performance liquid chromatography system.

[0079] Chromatographic column: Reversed-phase C18 column, specifications: column length 50-100 mm, inner diameter 2.1 mm, packing particle size 1.7-1.8 μm. Specific selection:

[0080] Agilent ZORBAX Eclipse Plus C18 (2.1 × 100 mm, 1.8 μm)

[0081] Injection volume: 5 μL;

[0082] Flow rate: 0.2 mL / min;

[0083] Column temperature: 40℃;

[0084] Column oven temperature: 40 °C (optimized range: 35-45 °C).

[0085] Mobile phase:

[0086] Mobile phase A: 10 mM ammonium acetate aqueous solution containing 0.1% (v / v) formic acid.

[0087] Mobile phase B: Methanol / acetonitrile mixed solution (80 / 20 v / v) containing 0.1% (v / v) formic acid.

[0088] Elution procedure: Gradient elution is used, and the specific procedure is shown in Table 1 below. The total run time is 14 minutes, including column equilibration time.

[0089] Table 1

[0090]

[0091] 2. The specific mass spectrometry conditions are as follows:

[0092] Mass spectrometer: Triple quadrupole mass spectrometer (Thermo Fisher Scientific TSQ Quantis plus)

[0093] Ionization mode: ESI-;

[0094] Atomizing gas flow rate: 3.0 L / min;

[0095] Interface temperature: 300℃;

[0096] Heating gas flow rate: 10.0 L / min;

[0097] DL temperature: 170℃;

[0098] Drying airflow rate: 10.0 L / min;

[0099] Heating module temperature: 400℃;

[0100] Collision gas: Argon;

[0101] Scanning mode: Selective ion detection SIM

[0102] In the mass spectrometry detection, the ion source temperature is 500–550℃, the nebulizing gas pressure is 35–45 psi, the auxiliary gas pressure is 45–55 psi, the curtain gas pressure is 30–40 psi, and the spray voltage is 4500–5000 V or -4500–-5000 V.

[0103] Table 2 shows the mass spectrometry parameters and retention times for detecting various bile acids.

[0104] Table 2

[0105]

[0106] Quantitative correction equation:

[0107] The samples were injected and analyzed, and the chromatograms were recorded. The peak areas of each bile acid standard, the internal standard peak area, and the ratio of the peak area of ​​each bile acid to the internal standard peak area are shown in Table 3-10 below. Plotting the spiking concentration (X) on the x-axis and the ratio of the peak area of ​​each bile acid after blank subtraction to the internal standard peak area (Y) on the y-axis, the regression equation, linear range, and correlation coefficient were obtained, as shown in Table 11 below.

[0108] Table 3 Ursodeoxycholic acid (UDCA)

[0109]

[0110] Table 4. Bile Acids (CA)

[0111]

[0112] Table 5 Glycineursodeoxycholic acid (GUDCA)

[0113]

[0114] Table 6 Glycinecholic acid (GCA)

[0115]

[0116] Table 7 Taururose Deoxycholic Acid (TUDCA)

[0117]

[0118] Table 8 Taurocholic Acid (TCA)

[0119]

[0120] Table 9 Taurolocholic Acid (TLCA)

[0121]

[0122] Table 10 Lithocholic Acid (LCA)

[0123]

[0124] Table 11

[0125]

[0126] As shown in Table 11, within the concentration range of 1 ng / mL to 200 ng / mL, ursodeoxycholic acid, cholic acid, glycoursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurocholelic acid, lithocholic acid, and taurocholelic acid all exhibited good linear relationships. The correlation coefficients (r) of each bile acid were all between 0.9988 and 0.9998, indicating that there was a highly consistent linear correlation between the instrument response value (peak area ratio) and the theoretical concentration throughout the entire concentration range.

[0127] Example 2: Optimization of Sample Pretreatment Process

[0128] Experimental Materials and Methods

[0129] 1. Reagents and Materials

[0130] Chemicals: Formic acid, acetic acid, phosphoric acid (all LC-MS grade); methanol, acetonitrile, water (all LC-MS grade).

[0131] Standards and internal standards: a mixed standard of target hydrophobic bile acids; and a stable isotope-labeled internal standard of bile acids (d4-glycine).

[0132] Biological matrix: human serum albumin solution (40 mg / mL, dissolved in PBS, matrix analog); mixed healthy human serum (endogenous bile acids removed) purchased from commercial sources.

[0133] Solid phase extraction: Oasis MAX (Mixed-mode Anion Exchange) 96-well plate or single column.

[0134] Instruments and equipment: precision pH meter, vortex mixer, centrifuge, nitrogen blower, high performance liquid chromatograph in series with triple quadrupole mass spectrometer.

[0135] 2. Experimental Procedure

[0136] a. Take 100 μL of mixed healthy human serum sample, pre-process it, and then place it in a centrifuge tube.

[0137] Add the stable isotope internal standard working solution.

[0138] Add the target bile acid standard to prepare a series of concentrations.

[0139] b. Acidification and SPE process (sample pretreatment)

[0140] Acidification: Add a specific concentration of acidifying reagent (e.g., 1M formic acid / acetic acid / phosphoric acid) dropwise to the above sample, while precisely monitoring with a pH meter, until the target pH value (1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0) is reached. Record the volume of acid used to obtain the acidified sample.

[0141] Sample loading: Load all the acidified sample into an Oasis MAX column that has been pre-activated and equilibrated with methanol and water.

[0142] Cleaning: Cleanse sequentially with 2.5 mL of 5% ammonia solution and 2.5 mL of methanol / water (20 / 80, v / v) to remove impurities.

[0143] Elution: Elute the target analyte with 2.5 mL of methanol solution containing 2% formic acid.

[0144] Concentration and redissolution: Dry the eluent under a gentle nitrogen stream (<40°C).

[0145] The test solution was obtained by redissolving the sample in 100 μL of the initial mobile phase (50% methanol aqueous solution containing 0.1% formic acid).

[0146] c. LC-MS / MS analysis

[0147] Separation and detection were performed using liquid chromatography and mass spectrometry following the steps in Example 1.

[0148] 3. Data Analysis and Judgment Criteria

[0149] a. Sensitivity assessment

[0150] Spike recovery rate:

[0151] Calculation formula: Recovery rate (%) = (Measured concentration - Background concentration) / Spiked concentration × 100%

[0152] Judgment criteria:

[0153] Excellent (A): 85% - 115%;

[0154] Good (B): 70% - 85% or 115% - 130%;

[0155] Acceptable (C): 50% - 70%;

[0156] Unacceptable (D): <50% or >130%.

[0157] Signal-to-noise ratio and lower limit of quantitation:

[0158] LOQ: The recovery rate is required to be between 80% and 120% at this concentration, and the intra-day and inter-day precision RSD is < 20%.

[0159] Judgment: Under the same spiking concentration, the lower the LOQ value and the higher the signal-to-noise ratio, the better the sensitivity.

[0160] b. Specificity assessment

[0161] Calculation method: In a blank matrix chromatogram, within the retention time window of the target analyte, measure the average height of the baseline noise and the height of any interfering peaks.

[0162] Judgment criteria:

[0163] Excellent (A): No discernible interference peaks (peak height < 3 times baseline noise).

[0164] Good (B): Interference peaks exist, but their area is less than 20% of the LOQ concentration target analyte peak area.

[0165] Unacceptable (C): Target peak area of ​​LOQ concentration ≥ 20%, or seriously affecting the integral.

[0166] c. Overall score

[0167] The sensitivity and specificity ratings for each experimental condition were weighted and combined (sensitivity weight 60%, specificity weight 40%) to select the acidification condition with the best overall performance.

[0168] The results are shown in Table 12.

[0169] Table 12 Spiked recoveries of key bile acids and matrix effects under different acidification conditions

[0170]

[0171] As shown in Table 12, the pH value of acidification treatment has a significant impact on the sensitivity and specificity of the detection results during the pretreatment process. Only when the pH value of the sample is controlled to be 2-4 with acidification reagent, and then combined with specific solid phase extraction columns and elution operations, can the effect of high sensitivity and specificity of trace detection of deoxycholic acid and lithocholic acid be achieved.

[0172] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0173] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A sample pretreatment method for determining the content of bile acid subtypes, characterized in that, Includes the following steps: S1. Acidify the sample to be tested with an acidifying agent to a pH value of 2-4 to obtain the acidified sample; S2. Elute the acidified sample in a mixed-mode anion exchange column to obtain the eluent. S3. Evaporate the eluent and then reconstitute it with a reconstitution solution to obtain the test sample.

2. The pretreatment method as described in claim 1, characterized in that, In step S1, the acidifying agent is at least one of formic acid, acetic acid, and phosphoric acid; preferably, the acidifying agent is formic acid, and the pH value is 2 to 2.5, more preferably 2 to 2.

2.

3. The pretreatment method as described in claim 1, characterized in that, Step S2 specifically includes the following steps: The acidified sample was loaded onto a mixed-mode anion exchange column. Wash successively with 3%~5% v / v ammonia solution and 10%~30% v / v methanol solution; The eluent is then obtained by eluting with a methanol solution containing 1%~5% v / v formic acid.

4. The pretreatment method as described in claim 3, characterized in that, The formic acid concentration in the methanol solution is 1%~3% v / v, preferably 1.8%~2.2% v / v; And / or, the volume ratio of the sample to be tested to ammonia is 100 μL: 2 mL to 3 mL; And / or, the volume ratio of the sample to be tested to the methanol aqueous solution is 100 μL: 2 mL to 3 mL; And / or, the volume ratio of the sample to be tested to a methanol solution containing 1%~5% v / v formic acid is 100 μL: 2mL~3 mL.

5. The pretreatment method as described in claim 3, characterized in that, The hybrid-mode anion exchange column is an OasisMAX column.

6. The pretreatment method according to any one of claims 1-5, characterized in that, The bile acid subtypes are deoxycholic acid or lithocholic acid.

7. The pretreatment method according to any one of claims 1-4, characterized in that, The complex solution is a 40-60% v / v methanol aqueous solution or an acetonitrile aqueous solution; Preferably, the reconstituted solution further contains 0.08%~0.5% (v / v) formic acid; And / or, the complex solution contains 1 µg / mL to 2 µg / mL of a stable isotope label; And / or, the sample to be tested is serum or plasma.

8. The application of the sample pretreatment method for determining the content of bile acid subtypes according to any one of claims 1-7 in the preparation of test samples for the detection of bile acid subtypes; or the application of the sample pretreatment method for determining the content of bile acid subtypes according to any one of claims 1-7 in the detection of bile acid subtypes.

9. A method for detecting bile acid subtypes, characterized in that, Includes the following steps: The sample pretreatment method according to any one of claims 1-7 yields a test sample for detection; The test sample was separated by high performance liquid chromatography. The sample separated by high performance liquid chromatography was then analyzed by mass spectrometry.

10. The detection method as described in claim 9, characterized in that, The chromatographic conditions for the chromatographic separation include: Mobile phase A: 8mM~12mM ammonium acetate aqueous solution, containing 0.05%~0.2% v / v formic acid; Mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 75~85:15~25, wherein the mixed solution contains 0.05%~0.2% (v / v) formic acid; Gradient elution was used at a flow rate of 0.1 mL / min to 0.3 mL / min; And / or, the mass spectrometry detection is performed using a triple quadrupole mass spectrometer; The conditions for the mass spectrometry detection include: Ionization mode: ESI-; Atomizing gas flow rate: 2.0L / min~4.0L / min; Interface temperature: 280℃~320℃; Heating gas flow rate: 8.0 L / min ~ 12.0 L / min; DL temperature: 160℃~180℃; Drying airflow rate: 8.0 L / min ~ 12.0 L / min; Heating module temperature: 380℃~420℃; Collision gas: Argon; Scanning mode: Selective Ion Detection (SIM).

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