Detection method of small molecule amine

Abstract

The invention provides a method for detecting small molecule amine through liquid chromatography. According to the method, anhydride is adopted as a derivatization reagent and is derived into a compound with a chromogenic group, analysis is conducted through UV, the derivatization reaction speed is high, redundant impurities are not generated, related substances and the content of the related substances can be accurately detected, the problems that an ESLD detector cannot detect the related substances, the GC upper peak pattern is poor, response is low, interference is large, and higher sensitivity is achieved are solved. And the analysis problem of small molecule amine similar compounds is improved.

Description

[0001] This application claims priority to an earlier application filed by the applicant with the China National Intellectual Property Administration on August 6, 2024, with patent application number 202411071390.1 and entitled "A Method for Detecting Small Molecule Amines". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention relates to a drug analysis and detection method, specifically to a method for detecting small molecule amines. Background Technology

[0003] Small molecule amines, such as cyclic secondary amines, show no response on UV detectors and are difficult to detect. They may show a response on ESLD detectors, but related substances cannot be detected. On GC, the peak shape is poor, the response is low, and there is significant interference. This invention uses acid anhydrides as derivatizing reagents to derivatize them into compounds with chromogenic groups, which are then analyzed using UV. This derivatization reaction is fast, does not produce unnecessary impurities, and can accurately detect related substances and their content.

[0004] Existing technologies use ELSD, CAD detectors, or MS detectors for detection. ELSD has low sensitivity, CAD has incomplete related substances and some impurities may not show up as peaks, and MS is not compatible with mobile phases containing non-volatile salts, and it is not recommended to use mobile phases containing trifluoroacetic acid additives for a long time. Summary of the Invention

[0005] To improve the above-mentioned technical problems, the present invention provides a method for detecting small molecule amines, comprising the following steps: taking a mixed solution of small molecule amine and acid anhydride, and detecting it by liquid chromatography.

[0006] According to an embodiment of the present invention, the small molecule amine may be a primary amine or a secondary amine, and the secondary amine may be selected from cyclic secondary amines, such as 6-azaspiro[2.5]octane and 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

[0007] According to an embodiment of the present invention, the acid anhydride is selected from phthalic anhydride, succinic anhydride, and maleic anhydride.

[0008] According to an embodiment of the present invention, an alkali may be added to the mixed solution.

[0009] According to an embodiment of the present invention, the alkali is selected from at least one of triethylamine, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, or cesium carbonate.

[0010] According to an embodiment of the present invention, an acid may be added to the mixed solution.

[0011] According to an embodiment of the present invention, the acid is selected from at least one of formic acid, acetic acid, or hydrochloric acid.

[0012] According to an embodiment of the invention, the pH of the mixed solution is 3-6, for example 4-5.

[0013] According to an embodiment of the present invention, the concentration of the small molecule amine in the mixed solution is 0.01-2 mg / mL, for example 0.05-1 mg / mL, such as 0.2 mg / mL or 0.5 mg / mL.

[0014] According to an embodiment of the present invention, the concentration of the acid anhydride in the mixed solution is 0.1-10 mg / mL, for example 0.5-5 mg / mL, such as 1 mg / mL or 2.5 mg / mL.

[0015] According to an embodiment of the present invention, the concentration of the alkali in the mixed solution is 0.5-10 mg / mL, for example 1-8 mg / mL, such as 1.68 mg / mL or 4.19 mg / mL.

[0016] According to an embodiment of the present invention, the concentration of the acid in the mixed solution is 0.0001-0.1 mL / mL, for example 0.001-0.05 mL / mL, such as 0.01 mL / mL or 0.004 mL / mL.

[0017] According to an embodiment of the present invention, the solvent in the mixed solution is selected from at least one of acetonitrile and DMF, such as a mixed solvent of acetonitrile and DMF, preferably with a volume ratio of acetonitrile to DMF of 3:1.

[0018] According to an embodiment of the present invention, the mobile phase A in the liquid chromatography is an aqueous solution of trifluoroacetic acid (volume concentration of 0.05%), and the mobile phase B is acetonitrile.

[0019] According to an embodiment of the present invention, the elution program of the liquid chromatography is gradient elution, which is divided into three gradient elutions. The volume ratio of mobile phase A to mobile phase B in the first gradient eluent is (70-98):10, for example, 90:10. The volume ratio of mobile phase A to mobile phase B in the second gradient eluent is 5:(80-98), for example, 5:95. The volume ratio of mobile phase A to mobile phase B in the third gradient eluent is (70-98):10, for example, 90:10.

[0020] According to an embodiment of the present invention, the time for the first gradient elution accounts for 10%-50% of the data acquisition time, the time for the second gradient elution accounts for 10%-35% of the data acquisition time, and the time for the third gradient elution accounts for 0-35% of the data acquisition time.

[0021] According to an embodiment of the present invention, the chromatographic column in the liquid chromatography is selected from a C18 column, such as a Wasters XBridge RP 18 column or an Agilent Zorbax SB-C18 column.

[0022] According to an embodiment of the present invention, the detector in the liquid chromatography is a UV detector.

[0023] According to an embodiment of the present invention, the acid anhydride is a solution of the acid anhydride, and its solvent is the solvent in the mixed solution.

[0024] According to an embodiment of the present invention, the alkali is a solution of alkali, and the solvent is the solvent in the mixed solution.

[0025] According to an embodiment of the present invention, the detection method includes the following steps: adding acid to a mixed solution of a small molecule amine, anhydride and base, and then taking a sample for detection by liquid chromatography.

[0026] According to an embodiment of the present invention, the detection method includes the following steps: dissolving a small molecule amine in an alkaline solution, adding an acid anhydride solution, mixing (e.g., sonicating), adding an acid, adding a solvent, mixing, and then taking a sample for detection by liquid chromatography.

[0027] Beneficial effects

[0028] This invention provides a method for detecting small molecule amines by liquid chromatography. The method uses acid anhydrides as derivatizing reagents to derivatize the amines into compounds with chromogenic groups, which are then analyzed using UV chromatography. This derivatization reaction is fast, produces no unnecessary impurities, and accurately detects related substances and their concentrations. It avoids the problems of ESLD detectors failing to detect related substances, poor peak shapes, low response, and significant interference on GC, and offers higher sensitivity, thus improving the analysis of small molecule amine-like compounds (especially cyclic secondary amines). Attached Figure Description

[0029] Figure 1 The chromatogram of 6-azaspiro[2.5]octane (QR059DS-SM-03) was obtained by the method of Example 1.

[0030] Figure 2 The chromatogram for detecting impurity QR059DS-SM-03-IM-02 using the method of Example 1 is shown.

[0031] Figure 3 The chromatogram for detecting impurity QR059DS-SM-03-IM-03 by the method of Example 1 is shown.

[0032] Figure 4 This is a graph showing the linear fit between the concentration of the test sample and the peak area in Example 2.

[0033] Figure 5 The chromatogram for the detection of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane by the method of Example 5 is shown. Detailed Implementation

[0034] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0035] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0036] Example 1

[0037] 6-Zazaspiro[2.5]octane: (QR059DS-SM-03);

[0038] Experimental methods:

[0039]

[0040]

[0041] Solution preparation

[0042] diluent and mobile phase preparation

[0043] Mobile phase A: 0.05% trifluoroacetic acid aqueous solution

[0044] Accurately measure 0.5 ml of trifluoroacetic acid into 1000 ml of purified water, mix thoroughly, and degas by sonication for 10 min.

[0045] Mobile phase B: Acetonitrile

[0046] Diluent 1: Acetonitrile: DMF = 3:1

[0047] Diluent 2: Weigh 4.19 g of triethylamine into a 100 mL volumetric flask, dilute with diluent 1 to a final volume, and mix well.

[0048] Succinic anhydride solution: Weigh 2.5g of succinic anhydride into a 100mL volumetric flask, dilute to volume with diluent 1, and mix well.

[0049] Blank solution: Transfer 5.0 ml of diluent 2 to a 50 mL volumetric flask, add 5.0 mL of succinic anhydride solution, shake well, sonicate for 10 min, add 0.5 ml of formic acid, dilute to volume with diluent 1, and mix well.

[0050] Test solution: Accurately weigh approximately 25 mg of QR059DS-SM-03 and place it in a 50 ml volumetric flask. Add 5.0 ml of diluent 2 to dissolve it, then add 5.0 mL of succinic anhydride solution, shake well, sonicate for 10 min, add 0.5 ml of formic acid, and dilute to volume with diluent 1. Mix well. (0.5 mg / ml)

[0051] Impurity localization solution 1: Take approximately 5 mg of QR059DS-SM-03-IM-02, place it in a 10 ml volumetric flask, add 1.0 ml of diluent 2 to dissolve it, add 1.0 ml of succinic anhydride solution, shake well, sonicate for 10 min, add 0.1 ml of formic acid, dilute to volume with diluent 1, and mix well. (0.5 mg / ml)

[0052] Impurity localization solution 2: Take about 5 mg of QR059DS-SM-03-IM-03, place it in a 10 ml volumetric flask, add 1.0 ml of diluent 2 to dissolve it, add 1.0 ml of succinic anhydride solution, shake well, sonicate for 10 min, add 0.1 ml of formic acid, dilute to volume with diluent 1, and mix well. (0.5 mg / ml).

[0053] After the system stabilizes, inject the sample according to the injection sequence list (Table 1), record the chromatogram, and see the chromatogram of the test sample. Figure 1 The chromatograms of QR059DS-SM-03-IM-02 and QR059DS-SM-03-IM-03 are shown below. Figure 2 and Figure 3 .

[0054] Table 1

[0055] Serial Number Injection solution Number of injections 1 blank solution ≥1 2 Test solution 1 3 Impurity positioning solution 1 1 4 Impurity positioning solution 2 1

[0056] Example 2

[0057] Following the method of Example 1, test solutions of QR059DS-SM-03 at different concentrations were prepared for detection. The peak areas of the target derivatives are shown in Table 2 below, and the relationship between concentration and peak area of ​​the derivatives is as follows. Figure 4 As shown:

[0058] Table 2

[0059] name QR059DS-SM-03 concentration (mg / ml) Derivative peak area L-50％ 0.2975 4240.24 L-100％ 0.5235 6409.85 L-120％ 0.6245 7670.71 L-150％ 0.7520 8702.67 L-200％ 0.9730 10278.1

[0060] The results showed that the concentration of the derivative was linearly related to the peak area of ​​the derivative.

[0061] Example 3

[0062] Following the method in Example 1, the test solution of QR059DS-SM-03 was allowed to stand, and samples were taken at different times for testing. The results are shown in Table 3 below:

[0063] Table 3

[0064] name Peak area of ​​derivatives Recovery rate A-100%-1-0h 6172.95 / A-100%-1-1h 6102.74 98.86％ A-100%-1-2h 6134.9 99.38％ A-100%-1-4h 6138.52 99.44％ A-100%-1-8h 6126.77 99.25％ A-100%-1-3 days 6124.26 99.21％

[0065] The results show that the derivatives obtained by the method of the present invention have high stability in the system.

[0066] Example 4

[0067] Referring to the method in Example 1, the difference is:

[0068] Reference solution: Accurately weigh approximately 20 mg of reference standard QR059DS-SM-03, place it in a 100 ml volumetric flask, add 4.0 ml of diluent 2 to dissolve it, add 4.0 ml of succinic anhydride solution, shake well, sonicate for 10 min, add 0.4 ml of formic acid, dilute to volume with diluent 1, mix well, and prepare two parallel aliquots. (0.2 mg / ml)

[0069] Test solution: Accurately weigh approximately 20 mg of test sample QR059DS-SM-03, place it in a 100 ml volumetric flask, add 4.0 ml of diluent 2 to dissolve, add 4.0 ml of succinic anhydride solution, shake well, sonicate for 10 min, add 0.4 ml of formic acid, dilute to volume with diluent 1, mix well, and prepare two parallel solutions. (0.2 mg / ml)

[0070] Once the system is stable, inject the sample according to the injection sequence list and record the chromatogram.

[0071] Serial Number Injection solution Number of injections 1 blank solution ≥1 2 Reference solution 1 5 3 Reference solution 2 2 4 Test solution 1 1 5 Test solution 2 1 6 Reference solution 1 1

[0072] Calculation formula

[0073]

[0074] In the formula: W STD —The sample weight of the reference standard, in mg;

[0075] P—Content of reference standard;

[0076] A SP —Peak area of ​​QR059DS-SM-03 in the test solution;

[0077] V SP —The dilution volume of the test solution, in mL;

[0078] A STD —The average peak area of ​​QR059DS-SM-03 in reference solution 1 for five consecutive tests;

[0079] V STD —The dilution volume of the reference standard, mL;

[0080] W Sp—The sample weight, in mg.

[0081] Example 5

[0082] 6,6-Dimethyl-3-azabicyclo[3.1.0]hexane Test methods

[0083]

[0084] Solution preparation

[0085] diluent and mobile phase preparation

[0086] Mobile phase A: 0.03% TFA aqueous solution

[0087] Accurately measure 300 μl of trifluoroacetic acid into 1000 ml of purified water, mix thoroughly, and degas by sonication for 10 min.

[0088] Mobile phase B: Acetonitrile

[0089] Diluent 1 / Blank Solvent: ACN

[0090] Needle washing solution: ACN:H2O = 50:50 (V / V)

[0091] Add 250 ml of water and 250 ml of acetonitrile to the mobile phase bottle, mix well, and degas by sonication.

[0092] Succinic anhydride solution: Weigh 2.5g of succinic anhydride into a 100mL volumetric flask, dilute to volume with diluent 1, and mix well.

[0093] Test solution: Weigh approximately 10 mg of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane accurately, place it in a 10 ml volumetric flask, and dilute to volume with succinic anhydride solution (0.1 mg / ml).

[0094] After the system stabilizes, inject the sample according to the injection sequence list, record the chromatogram, and see... Figure 5 The target peak is 2.7 minutes.

[0095] Serial Number Injection solution Number of injections 1 blank solution ≥1 2 Test solution 1

[0096] The above description provides an exemplary account of the implementation methods of the technical solution disclosed herein. It should be understood that the scope of protection of this disclosure is not limited to the above-described embodiments. Any modifications, equivalent substitutions, or improvements made by those skilled in the art within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this application.

Claims

1. A method for detecting small molecule amines, comprising the following steps: A mixed solution of small molecule amines and acid anhydrides was taken and detected by liquid chromatography; The small molecule amine can be a primary amine or a secondary amine, and the secondary amine can be selected from cyclic secondary amines, such as 6-azaspiro[2.5]octane and 6,6-dimethyl-3-azabicyclo[3.1.0]hexane.

2. The method according to claim 1, characterized in that, The acid anhydride is selected from phthalic anhydride, succinic anhydride, and maleic anhydride.

3. The method according to claim 1 or 2, characterized in that, An alkali may be added to the mixed solution; Preferably, the alkali is selected from at least one of triethylamine, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, or cesium carbonate.

4. The method according to any one of claims 1-3, characterized in that, Acid is added to the mixed solution; Preferably, the acid is selected from at least one of formic acid, acetic acid, or hydrochloric acid.

5. The method according to any one of claims 1-4, characterized in that, The pH of the mixed solution is 3-6, for example 4-5.

6. The method according to any one of claims 1-5, characterized in that, The concentration of the small molecule amine in the mixed solution is 0.01-2 mg / mL, for example 0.05-1 mg / mL.

7. The method according to any one of claims 1-6, characterized in that, The concentration of the acid anhydride in the mixed solution is 0.1-10 mg / mL, for example 0.5-5 mg / mL; Preferably, the concentration of the alkali in the mixed solution is 0.5-10 mg / mL, for example 1-8 mg / mL; Preferably, the concentration of the acid in the mixed solution is 0.0001-0.1 mL / mL, for example, 0.001-0.05 mL / mL.

8. The method according to any one of claims 1-7, characterized in that, The solvent in the mixed solution is selected from at least one of acetonitrile and DMF, such as a mixed solvent of acetonitrile and DMF, preferably with a volume ratio of acetonitrile to DMF of 3:

1.

9. The method according to any one of claims 1-8, characterized in that, In the liquid chromatography, mobile phase A is an aqueous solution of trifluoroacetic acid (volume concentration of 0.05%), and mobile phase B is acetonitrile; Preferably, the elution program of the liquid chromatography is gradient elution, which is divided into three gradient elutions. The volume ratio of mobile phase A to mobile phase B in the first gradient eluent is (70-98):10, for example, 90:

10. The volume ratio of mobile phase A to mobile phase B in the second gradient eluent is 5:(80-98), for example, 5:

95. The volume ratio of mobile phase A to mobile phase B in the third gradient eluent is (70-98):10, for example, 90:

10. Preferably, the first gradient elution time accounts for 10%-50% of the data acquisition time, the second gradient elution time accounts for 10%-35% of the data acquisition time, and the third gradient elution time accounts for 0-35% of the data acquisition time. Preferably, the chromatographic column in the liquid chromatography is selected from C18 columns, such as Wasters XBridge RP 18 columns or Agilent Zorbax SB-C18 columns; Preferably, the detector in the liquid chromatograph is a UV detector.

10. The method according to any one of claims 1-8, characterized in that, The method includes the following steps: adding acid to a mixed solution of small molecule amine, acid anhydride and base, and then taking a sample for detection by liquid chromatography; Preferably, the method includes the following steps: dissolving a small molecule amine in an alkaline solution, adding an acid anhydride solution, mixing (e.g., sonicating), adding an acid, adding a solvent, mixing, and then taking a sample for detection by liquid chromatography.

Images