A detection method for the genotoxic impurity 3,4-dihydroxybenzaldehyde in berberine hydrochloride

Abstract

The present application discloses a method for detecting the genotoxic impurity 3,4-dihydroxybenzaldehyde in berberine hydrochloride, specifically using liquid chromatography-mass spectrometry: Dissolve and dilute the test sample with water, add an extraction agent, vortex, take the supernatant for concentration and centrifugation, evaporate to dryness, add dansyl chloride solution and neutralization solution, carry out a derivatization reaction, centrifuge, and take the supernatant as the test sample solution; Dilute 3,4-dihydroxybenzaldehyde with water, add an extraction agent, vortex, take the supernatant for concentration and centrifugation, evaporate to dryness, add dansyl chloride solution and neutralization solution, carry out a derivatization reaction, centrifuge, and take the supernatant as the reference substance solution; Inject the test sample solution and the reference substance solution for testing respectively. The detection method of the present application has good linear correlation, accuracy, and precision.

Description

Technical Field

[0001] This application relates to the field of chemical detection, and particularly to a method for detecting the genotoxic impurity 3,4-dihydroxybenzaldehyde in berberine hydrochloride. Background Art

[0002] As an alkaloid drug widely used in clinical practice, berberine hydrochloride has attracted much attention due to its significant antibacterial, anti-inflammatory, hypoglycemic and other pharmacological activities. However, during its production process, it is inevitable to introduce or generate a small amount of genotoxic impurities, such as 3,4-dihydroxybenzaldehyde. As a potential genotoxic impurity, its presence not only directly affects the safety assessment of drugs, but also may pose a potential threat to the health of patients taking drugs for a long time.

[0003] Traditional impurity detection methods, such as thin-layer chromatography (TLC) and gas chromatography (GC), have difficulty meeting the current precise detection requirements for low-concentration genotoxic impurities in terms of sensitivity, resolution and specificity. Especially for small molecule impurities such as 3,4-dihydroxybenzaldehyde with similar structures and strong polarity, the separation and quantitative analysis from the main drug berberine hydrochloride face great challenges. Therefore, it is particularly important to develop an efficient, accurate and sensitive detection method.

[0004] In recent years, liquid chromatography-mass spectrometry (LC-MS) technology has become an important tool in the field of trace impurity detection due to its high sensitivity, high resolution and excellent selectivity. By combining the high-efficiency separation ability of liquid chromatography with the high-specificity recognition ability of mass spectrometry, this technology can effectively solve the problem of trace analysis in complex matrices, especially showing significant advantages in the field of drug impurity analysis. However, there is currently no report on the application of LC-MS technology to the analysis and detection of 3,4-dihydroxybenzaldehyde in berberine hydrochloride. Summary of the Invention

[0005] This application provides a method for detecting the genotoxic impurity 3,4-dihydroxybenzaldehyde in berberine hydrochloride, which uses liquid chromatography-mass spectrometry to achieve efficient and precise detection of the impurity.

[0006] A method for detecting the genotoxic impurity 3,4-dihydroxybenzaldehyde in berberine hydrochloride provided by this application specifically adopts liquid chromatography-mass spectrometry:

[0007] Take the test sample, dissolve and dilute it with water, add an extractant, vortex, take the supernatant for concentration and centrifugation, evaporate to dryness, add dansyl chloride solution and neutralization solution, carry out a derivatization reaction, centrifuge, and take the supernatant as the test sample solution;

[0008] Dilute 3,4-dihydroxybenzaldehyde with water, add an extractant, vortex, take the supernatant for concentration and centrifugation, evaporate to dryness, add dansyl chloride solution and neutralization solution, carry out derivatization reaction, centrifuge, and take the supernatant as the reference substance solution;

[0009] Inject the test solution and the reference substance solution respectively for testing.

[0010] In this application, liquid chromatography detection is carried out according to General Rule 0512 of the Chinese Pharmacopoeia, and mass spectrometry detection is carried out according to General Rule 0431 of the Chinese Pharmacopoeia.

[0011] Preferably, the neutralization solution is a saturated sodium carbonate acetonitrile solution.

[0012] Preferably, the extractant is methyl tert-butyl ether.

[0013] Preferably, the concentration of the dansyl chloride solution is 10-30 μg / mL.

[0014] Preferably, the temperature of the derivatization reaction is 50-60 °C and the time is 25-45 min.

[0015] Preferably, the chromatographic conditions are as follows: using octadecylsilane chemically bonded silica gel as the chromatographic column packing material, using water as mobile phase A and acetonitrile as mobile phase B for gradient elution; the column temperature of the chromatographic column is 35 °C, the flow rate is 0.35 mL per minute, and the injection volume is 2 μL.

[0016] Preferably, the elution gradient changes are as follows:

[0017] 0-1 min: 80% A → 80% A, 20% B → 20% B;

[0018] 1-2 min: 80% A → 5% A, 20% B → 95% B;

[0019] 2-4 min: 5% A → 5% A, 95% B → 95% B;

[0020] 4-4.1 min: 5% A → 80% A, 95% B → 20% B;

[0021] 4.1-5 min: 80% A → 80% A, 20% B → 20% B.

[0022] Preferably, the mass spectrometry conditions are as follows: using an electrospray ionization source, using m / z 605.092 → 170.200 as the quantitative ion pair, using m / z 605.092 → 171.200 and 605.092 → 156.100 as the qualitative ion pairs, the collision energies are 32 eV, 65 eV and 93 eV respectively, the declustering voltage is 126 V, the scanning time is 150 msec, and the mass spectrometry acquisition time is 2.5 min to 3.6 min.

[0023] Preferably, the mass spectrometry conditions are as follows: positive ion detection mode is adopted, the spray voltage is 5000V, the ion source temperature is 500°C, the curtain gas is 28 psi, the nebulizing gas is 50 psi, and the auxiliary gas is 55 psi.

[0024] In summary, the present application has the following beneficial effects:

[0025] The responsiveness and stability of 3,4-dihydroxybenzaldehyde in detection are relatively low, and it is not easy to detect. In the present application, derivatization treatment is carried out on it with dansyl chloride solution, which can significantly improve the detection sensitivity and stability of this target compound. This detection method has excellent specificity, solution stability, accuracy, precision and durability. The chromatographic conditions of the detection method of the present invention can well separate the chromatographic peak of berberine hydrochloride from the chromatographic peak of the impurity 3,4-dihydroxybenzaldehyde, and have good linear correlation. The average recovery rate is 83.33% - 101.29%, and the average relative standard deviation RSD < 8.71% (n = 6). Description of the Drawings

[0026] Figure 1 It is the liquid chromatography - mass spectrometry diagram of the test solution in the example.

[0027] Figure 2 It is the liquid chromatography - mass spectrometry diagram of the reference solution in the example.

[0028] Figure 3 It is the linear regression diagram of 3,4-dihydroxybenzaldehyde in the linear correlation test.

[0029] Figure 4 It is the quantitative limit chromatogram of 3,4-dihydroxybenzaldehyde in the quantitative limit test.

[0030] Figure 5 It is the detection limit chromatogram of 3,4-dihydroxybenzaldehyde in the detection limit test. Detailed Embodiments Examples

[0031] A method for detecting the genotoxic impurity 3,4-dihydroxybenzaldehyde in berberine hydrochloride, comprising the following steps:

[0032] Take about 31 mg of the test sample, weigh it accurately, place it in a 10-mL volumetric flask, dissolve and dilute it to the mark with ultrapure water, shake well. Accurately pipette 1 mL and place it in a 5-mL centrifuge tube. Add 2 mL of methyl tert-butyl ether, vortex and shake well. Take 1 mL of the supernatant and place it in a 1.5-mL centrifuge tube. Concentrate and centrifuge at 40 °C for 25 min to evaporate to dryness. Add 0.2 mL of dansyl chloride solution (20 μg / mL), 0.2 mL of saturated sodium carbonate acetonitrile solution, and 0.6 mL of acetonitrile. Place it at 55 °C and derivatize for 30 min. Cool to room temperature, centrifuge, and take the supernatant as the test sample solution.

[0033] Take an appropriate amount of 3,4-dihydroxybenzaldehyde, dilute it with ultrapure water to prepare a solution containing 5 ng of 3,4-dihydroxybenzaldehyde per 1 mL. Accurately pipette 1 mL and place it in a 5-mL centrifuge tube. Add 2 mL of methyl tert-butyl ether, vortex and shake well. Take 1 mL of the supernatant and place it in a 1.5-mL centrifuge tube. Concentrate and centrifuge at 40 °C for 25 min to evaporate to dryness. Add 0.2 mL of dansyl chloride solution (20 μg / mL), 0.2 mL of saturated sodium carbonate acetonitrile solution, and 0.6 mL of acetonitrile. Place it at 55 °C and derivatize for 30 min. Cool to room temperature, centrifuge, and take the supernatant as the reference solution.

[0034] Chromatographic analysis was performed according to the high performance liquid chromatography method (General Principles 0512 of the Chinese Pharmacopoeia 2015 Edition). Inject the test sample solution and the reference solution respectively, record the chromatogram (as shown in Figure 1 、 Figure 2 ), and calculate the content of 3,4-dihydroxybenzaldehyde by the external standard method based on the peak area. Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler (ACQUITY UPLC BEH C18, 2.1 mm × 50 mm, 1.7 μm); Use ultrapure water as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution according to Table 1; The column temperature is 35 °C; The flow rate is 0.35 mL per minute; The injection volume is 2 μL.

[0035] See Figure 1 、 Figure 2 It can be seen that 3,4-dihydroxybenzaldehyde was not detected in the test sample of berberine hydrochloride in this application.

[0036] Table 1 Changes in elution gradient

[0037]

[0038] Mass spectrometry was performed according to the method of mass spectrometry (General Principles 0431 of Chinese Pharmacopoeia 2015 Edition). An electrospray ionization source (Turbospray) was used, with positive ion detection mode. The spray voltage was 5000 V, the ion source temperature was 500 °C, the curtain gas was 28 psi, the nebulizing gas was 50 psi, and the auxiliary gas was 55 psi. In the multiple reaction monitoring scanning mode, the ion pair m / z 605.092→170.200 was collected as the quantitative ion pair, and m / z 605.092→171.200 and 605.092→156.100 were collected as the qualitative ion pairs. The collision energies were 32 eV, 65 eV, and 93 eV respectively, the declustering voltage was 126 V, the scanning time was 150 msec, and the mass spectrometry acquisition time was 2.5 min - 3.6 min.

[0039] Verification test

[0040] Preparation before verification - Solution preparation:

[0041] Blank solution: Ultra-pure water.

[0042] 3,4-Dihydroxybenzaldehyde stock solution: Weigh about 5 mg of 3,4-dihydroxybenzaldehyde reference substance, accurately weigh it, place it in a 20 mL volumetric flask, dissolve it with ultra-pure water and dilute to the mark, and shake well.

[0043] Dansyl chloride stock solution: Weigh about 20 mg of dansyl chloride, accurately weigh it, place it in a 20 mL volumetric flask, dissolve it with acetonitrile and dilute to the mark, and shake well.

[0044] Dansyl chloride solution: Accurately measure 0.2 mL of dansyl chloride stock solution, place it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and shake well to obtain a dansyl chloride solution (about 20 μg / mL).

[0045] Reference substance stock solution: Accurately measure 1 mL of 3,4-dihydroxybenzaldehyde stock solution, place it in a 50 mL volumetric flask, dilute it to the mark with ultra-pure water, and shake well; then accurately measure 0.5 mL and place it in a 50 mL volumetric flask, dilute it to the mark with ultra-pure water, and shake well to obtain a reference substance stock solution (about 50 ng / mL).

[0046] Reference substance solution: Accurately measure 2 mL of reference substance stock solution, place it in a 20 mL volumetric flask, dilute it to the mark with ultra-pure water, and shake well to obtain a reference substance solution (about 5 ng / mL).

[0047] Test solution: Weigh about 31 mg of this product, accurately weigh it, place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with ultra-pure water, and shake well to obtain a test solution (about 3.1 mg / mL).

[0048] 100% Spiked Test Solution: Weigh approximately 31 mg of this product accurately, place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with the reference solution, shake well, and use it as the 100% spiked test solution.

[0049] Derivatization:

[0050] Take the blank solution, reference solution, test solution, and 100% spiked test solution, and perform derivatization according to the following operations respectively to obtain the blank derivative solution, reference derivative solution, test derivative solution, and spiked test derivative solution.

[0051] Derivatization: Accurately pipette 1 mL of each of the above solutions into a 5 mL centrifuge tube, add 2 mL of methyl tert-butyl ether, vortex and shake well. Take 1 mL of the supernatant and place it in a 1.5 mL centrifuge tube. Concentrate and centrifuge at 40 °C for 25 min to evaporate to dryness. Add 0.2 mL of dansyl chloride solution (20 μg / mL), 0.2 mL of saturated sodium carbonate acetonitrile solution, and 0.6 mL of acetonitrile. Place it at 55 °C for 30 min for derivatization. Cool to room temperature, centrifuge, and take the supernatant for standby.

[0052] Experiment 1: Specificity

[0053] (1) System Suitability

[0054] Inject the reference solution continuously for 6 times, and examine the RSD of the peak area.

[0055] Requirement: The RSD of the peak area of the 3,4-dihydroxybenzaldehyde derivative in the reference solution injected continuously for 6 times should be ≤ 20.0%.

[0056] Result: The RSD of the peak area of the 3,4-dihydroxybenzaldehyde derivative in the reference solution injected continuously for 6 times is 4.18%, and the results are shown in Table 1.

[0057] Table 1. Results of System Suitability Test

[0058]

[0059] (2) Interference Test

[0060] Take the blank solution, reference solution, test solution, and 100% spiked test solution, and determine them according to law, and record the chromatogram.

[0061] Requirement: The blank solution should have no interference; the impurity peaks in the test solution should have no interference with the detection.

[0062] Result: The blank solution has no interference; the impurity peaks in the test solution have no interference with the detection.

[0063] Conclusion: This method has good specificity.

[0064] Test 2: Linear Correlation

[0065] Precisely pipette 1 mL of the reference stock solution into a 5 mL centrifuge tube, add 2 mL of methyl tert-butyl ether, vortex, and mix well. Take 1 mL of the supernatant and transfer it into a 1.5 mL centrifuge tube. Concentrate and centrifuge at 40 °C for 25 min to evaporate to dryness. Add 0.2 mL of dansyl chloride solution (20 μg / mL), 0.2 mL of saturated sodium carbonate acetonitrile solution, and 0.6 mL of acetonitrile. Place it at 55 °C for 30 min for derivatization. Cool to room temperature, centrifuge, and take the supernatant as the reference stock derivatized solution.

[0066] Precisely pipette the reference stock derivatized solution and serially dilute it to prepare solutions with concentrations of 200% limit, 150% limit, 100% limit, 75% limit, 50% limit, 25% limit, and LOQ concentration.

[0067] 200% linear solution: Precisely pipette 200 μL of the reference stock derivatized solution into a centrifuge tube, add 800 μL of acetonitrile, vortex and mix well.

[0068] 150% linear solution: Precisely pipette 150 μL of the reference stock derivatized solution into a centrifuge tube, add 850 μL of acetonitrile, vortex and mix well.

[0069] 100% linear solution: Precisely pipette 100 μL of the reference stock derivatized solution into a centrifuge tube, add 900 μL of acetonitrile, vortex and mix well.

[0070] 75% linear solution: Precisely pipette 75 μL of the reference stock derivatized solution into a centrifuge tube, add 925 μL of acetonitrile, vortex and mix well.

[0071] 50% linear solution: Precisely pipette 50 μL of the reference stock derivatized solution into a centrifuge tube, add 950 μL of acetonitrile, vortex and mix well.

[0072] 25% linear solution: Precisely pipette 25 μL of the reference stock derivatized solution into a centrifuge tube, add 975 μL of acetonitrile-water, vortex and mix well.

[0073] LOQ concentration solution: Precisely pipette 10 μL of the reference stock derivatized solution into a centrifuge tube, add 990 μL of acetonitrile, vortex and mix well.

[0074] Inject the above linear solutions respectively and record the chromatograms. Perform linear regression of the peak area (y) against the solution concentration (x) to obtain the regression equation and the correlation coefficient r.

[0075] Requirements: The correlation coefficient r should be ≥ 0.990; the absolute value of the intercept should not exceed 25.0% of the response value at the 100% limit, and the RSD of the response value per unit concentration should ≤ 20.0%.

[0076] Results: The correlation coefficient r was 0.9990; the absolute value of the intercept was 4.90% of the 100% limit response value, and the RSD of the response value per unit concentration was 5.97%. The results are shown in Table 2 and Figure 3 .

[0077] Table 2. Linear Results

[0078]

[0079] Conclusion: This method showed good linearity in the range of 3,4-dihydroxybenzaldehyde concentration from 0.25 ng / ml to 5.04 ng / ml.

[0080] Experiment 3: Quantitation Limit and Detection Limit

[0081] Take the linear solution and dilute it step by step. When S / N≥3, the concentration is the detection limit; when S / N≥10, the concentration is the quantitation limit. Prepare six aliquots of the quantitation limit solution in parallel. Precisely measure 2 μL each of the detection limit and quantitation limit solutions and inject them into the liquid chromatography-mass spectrometry instrument, and record the chromatogram.

[0082] Requirements: The S / N of the chromatographic peak at the detection limit concentration should not be less than 3, the S / N of the chromatographic peak at the quantitation limit concentration should not be less than 10, and the RSD of the peak areas of the six aliquots of the quantitation limit solution should be ≤20.0%.

[0083] Results: The S / N of the peak at the quantitation limit concentration was 115.2, and the RSD of the six peak areas was 5.86%. The S / N of the peak at the detection limit concentration was 42.1. The results are shown in Table 3, Table 4 and Figure 4 、 Figure 5 .

[0084] Table 3. Quantitation Limit and Detection Limit Results

[0085]

[0086] Table 4. Repeatability Results of Quantitation Limit

[0087]

[0088] Conclusion: The percentage content corresponding to the quantitation limit of 3,4-dihydroxybenzaldehyde in this method was 0.000016%, and the percentage content corresponding to the detection limit was 0.000008%, which could meet the requirements of accurate detection.

[0089] Experiment 4: Accuracy

[0090] Take this product and design it at three concentrations of 50%, 100%, and 150% of the limit concentration, with three replicates for each concentration, a total of n = 9. Prepare 1 blank derivative solution and 1 reference substance derivative solution, and 3 test substance derivative solutions according to the aforementioned solution preparation method. Prepare the spiked test substance derivative solution according to the following method.

[0091] 50% Intermediate Spiked Solution: Accurately measure 2.5 mL of the reference stock solution, transfer it to a 50 mL volumetric flask, dilute it to the mark with ultrapure water, and shake well.

[0092] 100% Intermediate Spiked Solution: Accurately measure 10 mL of the reference stock solution, transfer it to a 100 mL volumetric flask, dilute it to the mark with ultrapure water, and shake well.

[0093] 150% Intermediate Spiked Solution: Accurately measure 7.5 mL of the reference stock solution, transfer it to a 50 mL volumetric flask, dilute it to the mark with ultrapure water, and shake well.

[0094] 50% Spiked Test Solution: Take about 31 mg of this product, accurately weigh it, place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with 50% intermediate spiked solution, and shake well; prepare three portions in parallel.

[0095] 100% Spiked Test Solution: Take about 31 mg of this product, accurately weigh it, place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with 100% intermediate spiked solution, and shake well; prepare three portions in parallel.

[0096] 150% Spiked Test Solution: Take about 31 mg of this product, accurately weigh it, place it in a 10 mL volumetric flask, dissolve and dilute it to the mark with 150% intermediate spiked solution, and shake well; prepare three portions in parallel.

[0097] Derivatization: Accurately transfer 1 mL of each of the above spiked test solutions into 5 mL centrifuge tubes, add 2 mL of methyl tert-butyl ether, vortex and shake well. Take 1 mL of the supernatant and transfer it to a 1.5 mL centrifuge tube. Concentrate and centrifuge at 40 °C for 25 min to evaporate to dryness. Add 0.2 mL of dansyl chloride solution (20 μg / mL), 0.2 mL of saturated sodium carbonate acetonitrile solution, and 0.6 mL of acetonitrile. Place it at 55 °C for derivatization for 30 min. Cool to room temperature, centrifuge, and take the supernatant for standby.

[0098] Accurately measure the above reference derivatized solution, test sample derivatized solution, and each limit spiked test sample derivatized solution and determine them according to law. Calculate the recovery rate of 3,4-dihydroxybenzaldehyde by the external standard method based on the peak area.

[0099] Requirement: The recovery rates, average recovery rate, and total recovery rate of each concentration sample solution should be between 70.0% and 130.0%; the RSD of the recovery rate of each concentration sample solution and the total recovery rate RSD ≤ 20.0%.

[0100] Result: The recovery rates, average recovery rate, and total recovery rate of each concentration sample solution are between 83.33% and 101.29%, and the RSD of the recovery rate of each concentration sample solution and the total RSD are both ≤ 8.71%. The results are shown in Table 5.

[0101] Table 5. Accuracy Results

[0102]

[0103] Conclusion: This method has good accuracy.

[0104] Test 5: Repeatability precision

[0105] According to the description of the "accuracy test", prepare 1 portion of the reference substance derivative solution, 3 portions of the test substance derivative solution, and 6 portions of the 100% limit spiked test substance derivative solution in parallel, and calculate the individual recovery rate, average recovery rate, and RSD of 3,4-dihydroxybenzaldehyde in the 6 portions of the 100% limit spiked test substance derivative solution by the external standard method.

[0106] Requirement: In the 6 portions of the 100% limit spiked test substance solution, the individual recovery rate and the average recovery rate should both be between 70.0% and 130.0%, and the recovery rate RSD ≤ 20.0%.

[0107] Result: In the 6 portions of the 100% limit spiked test substance solution, the individual recovery rate and the average recovery rate are both between 71.70% and 99.60%, and the recovery rate RSD is 11.76%. The results are shown in Table 6.

[0108] Table 6. Repeatability results

[0109]

[0110] Conclusion: This method has good repeatability.

[0111] Test 6: Precision

[0112] On different days and by different analysts, according to the description of the "accuracy test", prepare 1 portion of the reference substance derivative solution, 3 portions of the test substance derivative solution, and 6 portions of the 100% limit spiked test substance derivative solution in parallel, and calculate the individual recovery rate, average recovery rate, and RSD of 3,4-dihydroxybenzaldehyde in the 6 portions of the 100% limit spiked test substance derivative solution by the external standard method. And statistically analyze the total average recovery rate and total RSD of the repeatability and intermediate precision results.

[0113] Requirement: The recovery rate of the 6 portions of the 100% limit spiked test substance solution should be between 70.0% and 130.0%; the recovery rate RSD of the 6 portions of the 100% limit spiked test substance solution ≤ 20.0%. The total average recovery rate of the 12 portions of the 100% limit spiked test substance solution should be between 70.0% and 130.0%; the recovery rate total RSD of the 12 portions of the 100% limit spiked test substance solution ≤ 20.0%.

[0114] Results: The recoveries of 6 spiked test solution samples at 100% limit should be between 84.21% and 112.91%; the RSD of the recoveries of 6 spiked test solution samples at 100% limit was 10.86%. The total average recovery of 12 spiked test solution samples at 100% limit should be 90.99%; the total RSD of the recoveries of 12 spiked test solution samples at 100% limit was 12.82%. The results are shown in Table 7 and Table 8.

[0115] Table 7, Intermediate precision results

[0116]

[0117] Table 8, Precision results

[0118]

[0119] Conclusion: The precision of this method is good.

[0120] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art may make modifications to this embodiment that do not contribute creatively, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for detecting the genotoxic impurity 3,4-dihydroxybenzaldehyde in berberine hydrochloride, characterized in that, Use liquid chromatography-mass spectrometry (LC-MS): Dissolve and dilute the test sample with water, add the extraction agent, vortex, take the supernatant for concentration and centrifugation, evaporate to dryness, add dansyl chloride solution and neutralization solution, carry out the derivatization reaction, centrifuge, and take the supernatant as the test sample solution; Dilute 3,4-dihydroxybenzaldehyde with water, add the extraction agent, vortex, take the supernatant for concentration and centrifugation, evaporate to dryness, add dansyl chloride solution and neutralization solution, carry out the derivatization reaction, centrifuge, and take the supernatant as the reference substance solution; the extraction agent is methyl tert-butyl ether, and the neutralization solution is saturated sodium carbonate acetonitrile solution; Inject the test sample solution and the reference substance solution for detection respectively.

2. The detection method according to claim 1, wherein The concentration of the dansyl chloride solution is 10 - 30 μg / mL.

3. The detection method according to claim 1, characterized in that, The temperature of the derivatization reaction is 50 - 60 °C, and the time is 25 - 45 min.

4. The detection method according to claim 1, wherein The chromatographic conditions are as follows: Use octadecylsilane chemically bonded silica gel as the chromatographic column packing material, use water as mobile phase A, and use acetonitrile as mobile phase B for gradient elution.

5. The detection method according to claim 1, characterized in that The column temperature of the chromatographic column is 35 °C, the flow rate is 0.3 - 0.4 mL per minute, and the injection volume is 1 - 2 μL.

6. The detection method according to claim 4, wherein The elution gradient changes are as follows: 0 - 1 min: 80%A → 80%A, 20%B → 20%B; 1 - 2 min: 80%A → 5%A, 20%B → 95%B; 2 - 4 min: 5%A → 5%A, 95%B → 95%B; 4 - 4.1 min: 5%A → 80%A, 95%B → 20%B; 4.1 - 5 min: 80%A → 80%A, 20%B → 20%B.

7. The detection method according to claim 1, wherein, The mass spectrometry conditions are as follows: Use an electrospray ionization source, use m / z 605.092 → 170.200 as the quantitative ion pair, use m / z 605.092 → 171.200 and 605.092 → 156.100 as the qualitative ion pairs, the collision energies are 32 eV, 65 eV, and 93 eV respectively, the declustering voltage is 126 V, the scan time is 150 msec, and the mass spectrometry acquisition time is 2.5 min - 3.6 min.

8. The detection method according to claim 7, wherein The mass spectrometry conditions are as follows: Use the positive ion detection mode, the spray voltage is 5000 V, the ion source temperature is 500 °C, the curtain gas is 28 psi, the nebulizing gas is 50 psi, and the auxiliary gas is 55 psi.

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