Separation and detection method of echinocandin B mother nucleus and impurities thereof

Abstract

The invention discloses a separation and detection method of echinocandin B mother nucleus and impurities thereof. The separation method comprises the following steps: separating echinocandin B mother nucleus and / or impurities by adopting a high performance liquid chromatograph; wherein the high performance liquid chromatography conditions are as follows: a chromatographic column is an alkyl bonded silica gel chromatographic column or a phenyl bonded silica gel chromatographic column; carrying out isocratic elution by using 0.01-1% formic acid aqueous solution as a mobile phase A and acetonitrile or methanol as a mobile phase B; the volume ratio of the mobile phase A to the mobile phase B is (95: 5)-(99: 1). According to the separation method, ECBN and impurities or various impurities can be well separated, and the conditions of peak shape and separation degree are good.

Description

Technical Field

[0001] The invention belongs to the fields of pharmaceutical chemistry and pharmaceutical analytical chemistry, and specifically relates to a method for separating and detecting an echinocandin B mother nucleus and its impurities. Background Art

[0002] The morbidity and mortality caused by deep fungal infections (Candida, Cryptococcus neoformans, Aspergillus) are increasing year by year. At the same time, the extensive use of antifungal drugs has also led to the emergence of drug-resistant fungi. The development of safe and efficient antifungal drugs has become inevitable. Echinocandins target the fungal cell wall and destroy the integrity of the fungal cell wall to achieve the purpose of sterilization. Anidulafungin is a semi-synthetic antifungal drug of the third generation of echinocandins. Compared with caspofungin and micafungin, it has a larger distribution volume and a wider antibacterial spectrum. Due to its unique metabolic process, it does not require dosage adjustment for patients with liver and kidney dysfunction, making it have good clinical application prospects (Liu Xiaohuan, Zhang Zhaohui. Journal of Drug Analysis, 2015, 35(2): 193-202).

[0003] Anidulafungin is obtained by removing the fatty acyl chain of echinocandin B from microorganisms to obtain the echinocandin B nucleus (ECBN), and then adding new side chains through chemical synthesis. Since the echinocandin B produced by fermentation contains other impurities with similar structures, these impurities will be brought into anidulafungin through ECBN. Therefore, strict quality control of ECBN is required to ensure the safety and effectiveness of anidulafungin and to improve the purity of ECBN and anidulafungin. ECBN is a cyclic hexapeptide, and its amino acid composition is 4,5-dihydroxy-L-ornithine (1), L-threonine (2), 4-hydroxy-L-proline (3), (S)-4-hydroxy-4-(4-hydroxyphenyl)-L-threonine (4), L-threonine (5), 3-hydroxy-4-methyl-L-proline (6). The structural formula is as follows:

[0004]

[0005] At present, purification preparation combined with nuclear magnetic resonance technology is mainly used to perform structural analysis on the impurities of anidulafungin or ECBN, and mass spectrometry is only used to determine the molecular weight (Li Min, Zhang Wei, Ren Fengzhi, Leng Feng, Xie Xinyu, Guo Dong. Chinese Journal of Pharmaceutical Industry, 2020, 51(2): 259-263; Zhao LN, Wang QL, Bie Y., Lu XXJ Pharm Biomed Anal. 2017, 141: 192-199.). The purification preparation combined with nuclear magnetic resonance technology is used to perform structural analysis on the impurities of ECBN. The analysis method is cumbersome and consumes a lot of energy. Therefore, there is an urgent need to find a quick and convenient separation and detection method. Summary of the invention

[0006] In order to overcome the defects of the prior art in the separation or detection methods of ECBN and its impurities, the present invention provides a separation and detection method of echinocandin B nucleus (ECBN) and its impurities. The separation method can well separate ECBN from impurities, or separate impurities from each other, and has good peak shape and resolution.

[0007] The present invention also provides a method for identifying impurities related to the echinocandin B nucleus based on high-resolution mass spectrometry (MS, MS / MS), which can quickly identify the specific types of impurities related to the echinocandin B nucleus and quickly and conveniently identify the structure of the impurities, avoiding time-consuming and laborious sample preparation processes.

[0008] The present invention mainly solves the above technical problems through the following technical solutions.

[0009] The first aspect of the present invention provides a method for separating the mother nucleus and / or impurities of echinocandin B, which comprises the following steps: using a high performance liquid chromatograph to separate or detect a sample to be separated;

[0010] The conditions of high performance liquid chromatography are as follows: the chromatographic column is an alkyl bonded silica gel chromatographic column or a phenyl bonded silica gel chromatographic column; 0.01-1% formic acid aqueous solution is used as mobile phase A, and acetonitrile or methanol is used as mobile phase B for isocratic elution; the volume ratio of mobile phase A to mobile phase B is 95:5-99:1;

[0011] The sample to be separated is ECBN crude product or echinocandins drugs.

[0012] In the separation method, the mobile phase A is preferably a 0.1% formic acid aqueous solution.

[0013] In the separation method, the mobile phase B is preferably acetonitrile.

[0014] In the separation method, the alkyl bonded silica gel chromatographic column is preferably a C4, C8, C18 or C30 alkyl bonded silica gel chromatographic column, more preferably a Diamonsil Plus C18 column.

[0015] In the separation method, the filler particle size of the chromatographic column is preferably 5 μm; the length of the chromatographic column is preferably 250 mm; and the inner diameter of the chromatographic column is preferably 4.6 mm.

[0016] In the separation method, the flow rate of the mobile phase is preferably 0.5-2 mL / min, more preferably 1 mL / min.

[0017] In the separation method, the column temperature of the chromatographic column is preferably 25-40°C, more preferably 30°C.

[0018] In the separation method, the injection volume is preferably 1-100 μL, more preferably 5 μL.

[0019] In the separation method, the detection wavelength of the high performance liquid chromatograph is preferably 190-280 nm, more preferably 210 nm.

[0020] In the separation method, the echinocandin drug is anidulafungin, caspofungin or micafungin. The ECBN crude product is preferably prepared by the following preparation method: using echinocandin B as a substrate, using the genetically engineered bacteria disclosed in CN115537367A to carry out fermentation culture in a fermentation medium, so that the echinocandin B is converted into the echinocandin B mother nucleus, thereby obtaining the ECBN crude product. Preferably, the ECBN crude product (for example, with a concentration of 1 mg / mL) is ultrafiltered to remove the enzyme and filtered, and the supernatant is directly separated.

[0021] In the separation method, the impurity is selected from one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N. ECBN represents the echinocandin B parent nucleus. (Formula 1) or its stereoisomers; the ECBN-6I is (Formula 2) or its stereoisomers; the ECBN-6II is (Formula 3) or its stereoisomers; the impurity M is

[0022]

[0023] The second aspect of the present invention provides a method for detecting the mother nucleus of echinocandin B and its impurities, which comprises the following steps: using a high performance liquid chromatograph to detect and separate the sample to be tested.

[0024] The HPLC conditions are as follows: the chromatographic column is an alkyl or phenyl bonded silica gel chromatographic column; 0.01-1% formic acid aqueous solution is used as mobile phase A, and acetonitrile or methanol is used as mobile phase B for isocratic elution; the volume ratio of mobile phase A to mobile phase B is 95:5-99:1.

[0025] In the detection method, the mobile phase A is preferably a 0.1% formic acid aqueous solution.

[0026] In the detection method, the mobile phase B is preferably acetonitrile.

[0027] In the detection method, the alkyl bonded silica gel chromatographic column is preferably a C4, C8, C18 or C30 alkyl bonded silica gel chromatographic column, more preferably a Diamonsil Plus C18 column.

[0028] In the detection method, the particle size of the chromatographic column filler is preferably 5 μm; the length of the chromatographic column is preferably 250 mm; and the inner diameter of the chromatographic column is preferably 4.6 mm.

[0029] In the detection method, the flow rate of the mobile phase is preferably 0.5-2 mL / min, more preferably 1 mL / min.

[0030] In the detection method, the column temperature of the chromatographic column is preferably 25-40°C, more preferably 30°C.

[0031] In the detection method, the injection volume is preferably 1-100 μL, more preferably 5 μL.

[0032] In the detection method, the detection wavelength is preferably 190-280 nm, more preferably 210 nm.

[0033] In the detection method, the sample to be tested is a crude ECBN product or an echinocandin drug. The echinocandin drug may be anidulafungin, caspofungin or micafungin. The crude ECBN product is preferably prepared by the following preparation method: using echinocandin B as a substrate, the genetically engineered bacteria disclosed in CN115537367A are used for fermentation culture in a fermentation medium to convert the echinocandin B into the echinocandin B nucleus, thereby obtaining the crude ECBN product. Preferably, the crude ECBN product (e.g., a concentration of 1 mg / mL) is ultrafiltered to remove the enzyme and filtered, and the supernatant is directly detected.

[0034] The detection method may further include mass spectrometry detection, which includes the following steps: the high performance liquid chromatograph is used in combination with a mass spectrometer, and the mass-to-charge ratio (m / z) of each component separated by the high performance liquid chromatograph is detected by the mass spectrometer.

[0035] In the detection method, preferably, at least a portion of the components separated by the high performance liquid chromatography enters the mass spectrometer, and more preferably, the portion is 1 / 3.

[0036] In the detection method, the mass spectrometer is preferably a high-resolution mass spectrometer. The mass detector of the high-resolution mass spectrometer is preferably an electrostatic field orbital trap, time-of-flight or triple quadrupole mass spectrometer. The high-resolution mass spectrometer is preferably a quadrupole-time-of-flight mass spectrometer (Q-TOF).

[0037] In the detection method, the ionization mode of the mass spectrometer is preferably electrospray ionization or atmospheric pressure ionization; more preferably, when the ionization mode is electrospray ionization, the nebulizer gas temperature is 200-240°C, preferably 220°C; the nebulizer gas pressure is preferably 18-22psi, more preferably 20psi.

[0038] In the detection method, the drying gas flow rate of the mass spectrometer is preferably 15-23 L / min, more preferably 19 L / min.

[0039] In the detection method, the sheath gas flow rate of the mass spectrometer is preferably 9-13 L / min, more preferably 11 L / min.

[0040] In the detection method, the capillary voltage of the mass spectrometer is preferably 2.8-4.2 kV, more preferably 3.5 kV.

[0041] In the detection method, the nozzle voltage of the mass spectrometer is preferably 1.2-1.8 kV, more preferably 1.5 kV.

[0042] In the detection method, the scanning mode of the mass spectrometer is preferably a positive ion mode or a negative ion mode, and more preferably an ESI positive ion scanning mode.

[0043] In the detection method, the MS (primary mass spectrometer) ion scanning range is preferably 100-1700, more preferably 300-1700.

[0044] In the detection method, the MS / MS (secondary mass spectrometry) ion scanning range of the mass spectrometer is preferably 50-1700.

[0045] In the detection method, the MS / MS collision energy of the mass spectrometer is preferably 20-50 eV, more preferably 30-40 eV.

[0046] In the detection method, the impurity is preferably one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N. The impurity M and impurity N are described in the literature (Li Min, Zhang Wei, Ren Fengzhi, Leng Feng, Xie Xinyu, Guo Dong. Chinese Journal of Pharmaceutical Industry, 2020, 51(2): 259-263), and the structural formula is as follows:

[0047]

[0048] In the detection method, the ECBN-2Ser is a compound in which the threonine at position 2 of ECBN is replaced by serine or its stereoisomer, and the configuration of the other amino acids can be the same as ECBN, or can be its enantiomer or diastereomer, preferably or a stereoisomer thereof.

[0049] In the detection method, the ECBN-6I is a compound in which the 6-position of ECBN is substituted by 3-hydroxy-4-hydroxymethylproline or its stereoisomer, and the ECBN-6II is a compound in which the 6-position of ECBN is substituted by 3,5-dihydroxy-4-methylproline or its stereoisomer. The configuration of other amino acids in the ECBN-6I and ECBN-6II may be the same as that of ECBN, or may be an enantiomer or diastereomer thereof. The ECBN-6I is preferably or its stereoisomers. The ECBN-6II is preferably or a stereoisomer thereof.

[0050] In the detection method, the m / z of the mass spectrum quasi-molecular ion peak of ECBN-2Ser is between 814 and 815; preferably, the m / z of the mass spectrum quasi-molecular ion peak of ECBN-2Ser is 814.3476.

[0051] In the detection method, the m / z of the mass spectrometry quasi-molecular ion peak of ECBN-6I or ECBN-6II is between 784 and 785; preferably, the m / z of the mass spectrometry quasi-molecular ion peak of ECBN-6I or ECBN-6II is 784.3360.

[0052] Preferably, in the detection method, a total ion current diagram is obtained according to the detection results of the high performance liquid chromatograph and the mass spectrometer, and the relative retention time of ECBN-2Ser in the total ion current diagram of the sample to be tested is 6.1 min; the relative retention time of ECBN-6I or ECBN-6II in the total ion current diagram of the sample to be tested is 4.2 min;

[0053] The detection conditions of the high performance liquid chromatography are as follows: the chromatographic column model is Diamonsil Plus C18; the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, the volume ratio of mobile phase A to mobile phase B is 95:5, and isocratic elution is adopted; the injection volume is 5 μL; the column temperature is 30° C.; the detection wavelength is 210 nm; the flow rate of the mobile phase is 1 mL / min;

[0054] The detection conditions of the mass spectrometer are as follows: Q / TOF-MS adopts ESI positive ion scanning mode, the nebulizer gas temperature is 220°C, the drying gas flow rate is 19 L / min, the nebulizer pressure is 20 psi, the sheath gas flow rate is 11 L / min, the capillary voltage is 3.5 kV, and the nozzle voltage is 1.5 kV; the mass spectrometer is tuned before use, and a reference ion with an m / z of 922.00980 is selected for real-time correction during the scanning process, and the primary mass spectrometer scanning range is m / z 300-1700; the tandem mass spectrometer collision energy is 30 eV; the secondary mass spectrometer scanning range covers parent ions with m / z of 300-1700 and daughter ions with m / z of 50-1700.

[0055] The third aspect of the present invention provides an echinocandin B nucleus-related impurity, wherein the echinocandin B nucleus-related impurity is ECBN-2Ser, ECBN-6I or ECBN-6II. ECBN-2Ser is a compound in which the 2-threonine of ECBN is replaced by serine or its stereoisomer, and the configuration of other amino acids can be the same as ECBN, or can be its enantiomer or diastereomer;

[0056] ECBN-6I is a compound in which the 6-position of ECBN is substituted by 3-hydroxy-4-hydroxymethylproline or a stereoisomer thereof, and ECBN-6II is a compound in which the 6-position of ECBN is substituted by 3,5-dihydroxy-4-methylproline or a stereoisomer thereof. The configuration of other amino acids in ECBN-6I and ECBN-6II may be the same as that of ECBN, or may be an enantiomer or a diastereomer thereof.

[0057] In some embodiments, the echinocandin B nucleus (ECBN)-related impurities are selected from any of the following structures or stereoisomers thereof:

[0058]

[0059] In the present invention, the stereoisomer refers to an enantiomer or a diastereomer.

[0060] In some embodiments, the relative retention time of the ECBN-2Ser in the total ion current graph is 6.1 min; the relative retention time of the ECBN-6I or ECBN-6II in the total ion current graph is 4.2 min;

[0061] The total ion flow diagram was obtained by high performance liquid chromatography-mass spectrometry. The high performance liquid chromatography detection conditions are as follows: the chromatographic column model is Diamonsil Plus C18; the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, the volume ratio of mobile phase A to mobile phase B is 95:5, and isocratic elution is adopted; the injection volume is 5 μL; the column temperature is 30°C; the detection wavelength is 210nm; the flow rate of the mobile phase is 1mL / min;

[0062] The detection conditions of the mass spectrometer are as follows: Q / TOF-MS adopts ESI positive ion scanning mode, the nebulizer gas temperature is 220°C, the drying gas flow rate is 19 L / min, the nebulizer pressure is 20 psi, the sheath gas flow rate is 11 L / min, the capillary voltage is 3.5 kV, and the nozzle voltage is 1.5 kV; the mass spectrometer is tuned before use, and a reference ion with an m / z of 922.00980 is selected for real-time correction during the scanning process, and the primary mass spectrometer scanning range is m / z 300-1700; the tandem mass spectrometer collision energy is 30 eV; the secondary mass spectrometer scanning range covers parent ions with m / z of 300-1700 and daughter ions with m / z of 50-1700.

[0063] The fourth aspect of the present invention provides a mixed system of echinocandin B core and impurities, which includes echinocandin B core (ECBN) and impurities, wherein the impurities are selected from one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N.

[0064] Wherein, the mixed system of the echinocandin B nucleus and impurities can be a crude ECBN product or an echinocandin drug. The crude ECBN product is prepared by the following preparation method: using echinocandin B as a substrate, using the genetically engineered bacteria disclosed in CN115537367A to carry out fermentation culture in a fermentation medium, so that the echinocandin B is converted into the echinocandin B nucleus, thereby obtaining the crude ECBN product. The echinocandin drug can be anidulafungin, caspofungin or micafungin.

[0065] The structure of each impurity was confirmed by identification data obtained by combining high performance liquid chromatography and mass spectrometry, and the same applies to the following.

[0066] The detection conditions of the high performance liquid chromatography are as follows: the chromatographic column model is Diamonsil Plus C18; the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, the volume ratio of mobile phase A to mobile phase B is 95:5, and isocratic elution is adopted; the injection volume is 5 μL; the column temperature is 30° C.; the detection wavelength is 210 nm; the flow rate of the mobile phase is 1 mL / min;

[0067] The detection conditions of the mass spectrometer are as follows: Q / TOF-MS adopts ESI positive ion scanning mode, the nebulizer gas temperature is 220°C, the drying gas flow rate is 19 L / min, the nebulizer pressure is 20 psi, the sheath gas flow rate is 11 L / min, the capillary voltage is 3.5 kV, and the nozzle voltage is 1.5 kV; the mass spectrometer is tuned before use, and a reference ion with an m / z of 922.00980 is selected for real-time correction during the scanning process, and the primary mass spectrometer scanning range is m / z 300-1700; the tandem mass spectrometer collision energy is 30 eV; the secondary mass spectrometer scanning range covers parent ions with m / z of 300-1700 and daughter ions with m / z of 50-1700.

[0068] A high performance liquid chromatograph and a mass spectrometer were used in combination and a total ion current diagram was obtained according to the above detection parameters.

[0069] The ECBN-2Ser is a substance formed by replacing the threonine residue at the second position of ECBN with serine or its stereoisomer. or its stereoisomers. The identification data of ECBN-2Ser are: the relative retention time of ECBN-2Ser in the total ion current is 6.1 min, and the m / z of the mass spectrum quasi-molecular ion peak is 814.3476.

[0070] The ECBN-6I is a substance formed by replacing the 3-hydroxy-4-methyl-L-proline residue at the 6th position of ECBN with 3-hydroxy-4-hydroxymethylproline or its stereoisomer. or its stereoisomers. The ECBN-6Ⅱ is a substance formed by replacing the 3-hydroxy-4-methyl-L-proline residue at the 6th position of ECBN with 3,5-dihydroxy-4-methylproline or its stereoisomers. The ECBN-6II is or its stereoisomers. The relative retention time of ECBN-6I and ECBN-6II in the total ion current graph is 4.2 min, and the m / z of the mass spectrum quasi-molecular ion peak is 814.3476.

[0071] The structural formula of the impurity M is shown in Formula 4 above.

[0072] The structural formula of the impurity N is shown in Formula 5 above.

[0073] The fifth aspect of the present invention provides a method for identifying impurities related to the nucleus of echinocandin B, the identification method comprising the following contents:

[0074] (1) performing MS and MS / MS analysis on ECBN, and obtaining the mass spectrometry fragmentation pathway of the ECBN according to the characteristic ion information in the MS / MS spectrum; the ECBN represents the mother nucleus of echinocandin B;

[0075] (2) Using the mass spectrometry fragmentation pathway of ECBN as a control, the echinocandin B core-related impurities were subjected to MS and MS / MS analysis, and the characteristic ions of the echinocandin B core-related impurities were compared with the characteristic ions of ECBN, respectively. The types of the echinocandin B core-related impurities were identified by combining the molecular weight changes and the changed characteristic ions in the MS spectrum.

[0076] Preferably, the method for identifying impurities related to the nucleus of echinocandin B satisfies one or more of the following:

[0077] (1) The impurities of the echinocandin B core are one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N;

[0078] (2) The mass spectrometry fragmentation pathway of ECBN is as follows: (i) ECBN loses four water molecules successively to obtain fragment ions with m / z of 780.3408, 762.3313, 744.3213, and 726.3104, respectively; (ii) ECBN loses one water molecule, AA1, and AA2 to obtain fragment ions formed by AA3, AA4, AA5, and AA6, with m / z of 550.2524, and further loses one water molecule to obtain an ion with m / z 532.2415, and further loses one water molecule to obtain an ion with m / z 514.2307, and further, AA5 and AA6 form a characteristic ion with m / z 246.1468, and further fragmentation obtains a characteristic ion of AA6 with m / z 246.1468. 145.0981; (iii) ECBN loses AA1, AA2 and AA6 to obtain fragment ions formed by AA3, AA4 and AA5, with m / z of 424.1729. After losing three molecules of water in succession, ions with m / z of 406.1622, 388.1506 and 370.1408 are obtained respectively; (iv) ECBN loses one molecule of water and ammonia on AA6 and AA1 to obtain a fragment ion with m / z of 636.2522. After losing three more molecules of water, the ion fragments with m / z of 618.2420, 600.2317 and 582.2203 are obtained respectively. After the ion m / z 600.2317 is fragmented, the characteristic ion formed by AA1 and AA2 with m / z 326.1362 is obtained. This ion loses one more molecule of water to obtain an ion with m / z 308.1257;

[0079] The structural formula of the ECBN is shown below:

[0080]

[0081] (3) comparing the quasi-molecular ion peak of the echinocandin B core-related impurity with the two characteristic ions (i) and (ii) of ECBN, if it is determined that the molecular weight of the echinocandin B core-related impurity is 16 greater than the molecular weight of ECBN, then further determining through characteristic ions m / z 262.1415 and 161.0932 that the echinocandin B core-related impurity has an additional hydroxyl group relative to the 6-amino acid of ECBN, and further determining that the type of the echinocandin B core-related impurity is ECBN-6Ⅰ or ECBN-6Ⅰ;

[0082] (4) comparing the quasi-molecular ion peak of the echinocandin B parent core-related impurity with the two series of characteristic ions (i) and (iv) of the ECBN, if it is determined that the molecular weight of the echinocandin B parent core-related impurity is 14 less than the molecular weight of the ECBN, then further determining through characteristic ions m / z 312.2107 and 294.1098 that the echinocandin B parent core-related impurity has changed relative to the characteristic ion of 2-threonine of the ECBN, and determining that 2-threonine loses a methylene group and is converted into serine, and further determining that the type of the echinocandin B parent core-related impurity is ECBN-2Ser;

[0083] (5) comparing the quasi-molecular ion peak of the echinocandin B core-related impurity with the two characteristic ions (i) and (ii) of the ECBN, if it is determined that the molecular weight of the echinocandin B core-related impurity is 14 less than the molecular weight of the ECBN, then further determining through characteristic ions m / z 232.1305 and 131.0821 that the echinocandin B core-related impurity has one less methyl group relative to the 6-position amino acid of the ECBN, and further determining that the type of the echinocandin B core-related impurity is impurity M;

[0084] (6) Compare the quasi-molecular ion peak of the echinocandin B core-related impurity with the characteristic ion of ECBN. If it is determined that the molecular weight of the echinocandin B core-related impurity is 14 less than that of the ECBN, then further determine through characteristic ions m / z 232.1305 and 131.0821 that the echinocandin B core-related impurity has not changed relative to the 6-position amino acid of ECBN. Then, based on (ii) series characteristic ions m / z 536.2362, (iii) series characteristic ions m / z 410.1570, (iv) series characteristic ions m / z 622.2364 and their dehydrated ions, it is determined that the echinocandin B core-related impurity loses a methylene group relative to the 5-position threonine of ECBN and is converted into serine. Finally, it is determined that the type of the echinocandin B core-related impurity is impurity N.

[0085] The sixth aspect of the present invention provides a method for identifying impurities in the mother nucleus of echinocandin B, the identification method comprising the following contents:

[0086] (1) performing MS and MS / MS analysis on the ECBN, and obtaining the mass spectrometry fragmentation pathway of the ECBN according to the characteristic ion information in the MS / MS spectrum;

[0087] (2) Using the mass spectrometry fragmentation pathway of ECBN as a reference, the characteristic ions of the impurities were compared with the characteristic ions of ECBN, and the structure and structural formula of the impurities were obtained by combining the molecular weight change and the changed characteristic ions in the MS spectrum.

[0088] In the identification method, the impurities of the echinocandin B core are preferably one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N, wherein the structural formulas of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N are as described above.

[0089] In the identification method, the mass spectrometry fragmentation pathway of ECBN is preferably as follows: (i) ECBN loses 4 molecules of water successively to obtain fragment ions with m / z of 780.3408, 762.3313, 744.3213, and 726.3104, respectively; (ii) ECBN loses 1 molecule of water, AA1, and AA2 to obtain fragment ions formed by AA3, AA4, AA5, and AA6, with m / z of 550.2524, and further loses 1 molecule of water to obtain ions with m / z 532.2415, and further loses 1 molecule of water to obtain ions with m / z 514.2307, and AA5 and AA6 form characteristic ions with m / z 246.1468, and further fragmentation obtains characteristic ions of AA6 with m / z 246.1468. 145.0981; (iii) ECBN loses AA1, AA2 and AA6 to obtain fragment ions formed by AA3, AA4 and AA5 (), whose m / z is 424.1729, and loses three molecules of water successively to obtain ions with m / z 406.1622, 388.1506 and 370.1408 respectively; (iv) ECBN loses 1 molecule of water and ammonia on AA6 and AA1 to obtain a fragment ion with m / z 636.2522, and the m / z of the ion fragments after losing three more molecules of water are 618.2420, 600.2317 and 582.2203 respectively, and the ion m / z 600.2317 is fragmented to obtain the characteristic ion m / z 326.1362 formed by AA1 and AA2, and the ion loses one more molecule of water to obtain an ion with m / z 308.1257;

[0090]

[0091] In the identification method, the mass spectrometry fragmentation pathway of ECBN is preferably as follows Figure 4 shown.

[0092] In the identification method, when the impurity is ECBN-6Ⅰ or ECBN-6ⅠI, the structural identification method of ECBN-6Ⅰ or ECBN-6ⅠI is preferably: the quasi-molecular ion peak of ECBN-6Ⅰ or ECBN-6ⅠI is 16 larger than the molecular weight of ECBN; by comparison with the characteristic ions of ECBN, mainly the two series of ions (i) and (ii) are 16 larger than the characteristic ions of ECBN, and further by the characteristic ions m / z 262.1415 and 161.0932, it is judged that the 6-position amino acid (AA6) has an additional hydroxyl group; the ECBN-6Ⅰ or ECBN-6ⅠI is a compound in which the 6-position of ECBN is substituted by 3-hydroxy-4-hydroxymethylproline or its stereoisomer, or a compound in which the 6-position of ECBN is substituted by 3,5-dihydroxy-4-methylproline or its stereoisomer.

[0093] In the identification method, when the impurity is ECBN-6I-1, the cleavage pathway of ECBN-6I-1 is preferably as follows: Figure 7 shown.

[0094] In the identification method, when the impurity is ECBN-2Ser, the structural identification method of ECBN-2Ser is as follows: the quasi-molecular ion peak of ECBN-2Ser is 14 smaller than the molecular weight of ECBN; by comparison with the characteristic ions of ECBN, mainly the two series of ions (i) and (iv) are 14 smaller than the characteristic ions of ECBN; further, by the characteristic ions m / z 312.2107 and 294.1098, it is judged that the 2-threonine (AA2) loses a methylene group and is converted into serine, and the ECBN-2Ser is a compound in which the 2-threonine of ECBN is replaced by serine or its stereoisomer.

[0095] In the identification method, when the impurity ECBN-2Ser is ECBN-2Ser-1, the cleavage pathways (i) and (iv) of ECBN-2Ser-1 are preferably as follows: Fig. 9 shown.

[0096] In the identification method, when the impurity is impurity M, the structural identification method of the impurity M is as follows: the quasi-molecular ion peak of the impurity M is 14 smaller than the molecular weight of ECBN; by comparing with the characteristic ions of ECBN, mainly the two series of ions (i) and (ii) are 14 smaller than the characteristic ions of ECBN, and further judging from the characteristic ions m / z 232.1305 and 131.0821, the characteristic ion of the 6th amino acid (AA6) lacks a methyl group, and the structural formula of the impurity M is as described above.

[0097] In the identification method, when the impurity is impurity N, the structural identification method of the impurity N is as follows: the quasi-molecular ion peak of the impurity N is 14 smaller than the molecular weight of ECBN; by comparing with the characteristic ions m / z 232.1307 and 145.0979 of the impurity M, it is judged that AA6 has not changed, and based on (ii) series characteristic ions m / z 536.2362, (iii) series characteristic ions m / z 410.1570, (iv) series characteristic ions m / z 622.2364 and their dehydrated ions, it is judged that the 5-position threonine (AA5) loses a methylene group and is converted into serine, and the structural formula of the impurity M is as described above.

[0098] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0099] The reagents and raw materials used in the present invention are commercially available.

[0100] The positive and progressive effects of the present invention are:

[0101] (1) The present invention discloses a method for separating the echinocandin B nucleus and / or impurities based on high performance liquid chromatography-high resolution mass spectrometry, which can separate ECBN and impurities, or impurities from each other, with good peak shape and separation.

[0102] (2) The present invention provides a method for rapid detection and identification of the structure of the echinocandin B core and / or impurities, which can quickly and conveniently detect and identify the structure of the impurities and avoid time-consuming and labor-intensive preparation processes.

[0103] (3) The present invention obtains the structural formula of the echinocandin B parent core-related impurities, such as ECBN-2Ser-1, ECBN-6Ⅰ-1 or ECBN-6Ⅱ-1.

[0104] (4) The present invention is applicable to the identification of other impurities in ECBN or its derivatives such as anidulafungin. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 This is the total ion current diagram of the ECBN crude product in Example 1.

[0106] Figure 2 This is the mass spectrum (MS) of ECBN.

[0107] Figure 3 This is the secondary mass spectrum (MS / MS) of ECBN.

[0108] Figure 4 This is the mass spectrometry fragmentation rule of ECBN.

[0109] Figure 5 This is the MS graph of peak 1 impurity.

[0110] Figure 6 This is the MS / MS spectrum of peak 1 impurity.

[0111] Figure 7 This is the mass spectrometry fragmentation pattern of ECBN-6Ⅰ-1.

[0112] Figure 8 This is the MS chart of peak 4 impurity.

[0113] Fig. 9 This is the MS / MS spectrum of peak 4 impurity.

[0114] Fig.10 This is the MS / MS spectrum of ECBN-2Ser-1.

[0115] Fig.11 This is the total ion current diagram of the ECBN crude product in Comparative Example 1.

[0116] Fig.12 This is the total ion current diagram of the ECBN crude product in Comparative Example 2.

[0117] Fig.13 This is the MS / MS spectrum of peak 2 impurity.

[0118] Fig.14 This is the MS / MS spectrum of peak 3 impurity. DETAILED DESCRIPTION

[0119] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0120] The ECBN crude product involved in the present invention is a specific implementation case of the ECBN crude product provided by Yikelai Biotechnology (Shanghai) Co., Ltd., but it can also be applied to other ECBN and anidulafungin or echinocandins products.

[0121] 1. High performance liquid chromatography-high resolution mass spectrometry method

[0122] Example 1

[0123] Instrument: Agilent 1290 High Performance Liquid Chromatograph-6550Q / TOF-MS (Agilent Technologies, USA).

[0124] In this example, a crude solution of the echinocandin B nucleus (batch number: YKL) was used, and the fermentation broth (concentration of about 1 mg / ml) was prepared according to Example 5 in CN11553767A. After ultrafiltration to remove the enzyme and filtration, the supernatant was directly subjected to HPLC-MS detection, i.e., high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (HPLC-Q / TOF-MS / MS) detection.

[0125] The high performance liquid chromatography method selected a Diamonsil Plus C18 (5 μm, 4.6×250 mm) chromatographic column, the mobile phase A used a 0.1% formic acid aqueous solution, the mobile phase B used acetonitrile, the volume ratio of mobile phase A to mobile phase B was 95:5, the flow rate was set to 1 mL / min, the column temperature was 30°C, the injection volume was 5 μL, and the detection wavelength was 210 nm. The flow was split before mass spectrometry detection, and the split ratio was 2:1 (i.e., one-third of the eluent with the same relative retention time flowing out of the high performance liquid chromatograph was entered into the mass spectrometry detection).

[0126] Q / TOF-MS uses +ESI scanning mode, and other parameter settings are: nebulizer gas temperature (Gas Temp) is set to 220℃, drying gas (Drying Gas) flow rate is 19L / min, nebulizer pressure (Nebulizer) is 20psi, sheath gas flow (Sheath Gas Flow) is 11L / min, capillary voltage (Vcap) is 3.5kV, and nozzle voltage (Vnozzle) is 1.5kV. The mass spectrometer needs to be tuned before use. During the scanning process, the reference ion m / z 922.00980 is selected for real-time correction, and the primary mass spectrometer scan range is: m / z 300-1700. The tandem mass spectrometer collision energy is set to 30eV, and the Auto MS / MS scan range is: parent ion m / z 300-1700, and daughter ion m / z 50-1700.

[0127] 2. Structural analysis based on high-resolution mass spectrometry

[0128] The total ion current of crude ECBN is: Figure 1 , peak 1 is 4.2min; peak 2 is 4.8min; peak 3 is 5.1min; peak 4 is 6.1min; peak 5 is 7.0min, peak 5 is ECBN.

[0129] ECBN's MS diagram is as follows Figure 2 As shown, the m / z of the quasi-molecular ion peak is 798.3566, and the MS / MS graph is shown in Figure 3 shown.

[0130] According to the characteristic ion information in the MS / MS graph, the mass spectrometry fragmentation route of ECBN was obtained. Figure 4(1) ECBN loses 4 water molecules successively to obtain ions with m / z of 780.3408, 762.3313, 744.3213, and 726.3104 respectively; (2) ECBN loses one water molecule and (AA1+AA2) to obtain the fragment ion (AA3+AA4+AA5+AA6) with m / z of 550.2524. It loses one water molecule to obtain an ion with m / z 532.2415. It loses another water molecule to obtain an ion with m / z 514.2307 and the characteristic ion of (AA5+AA6) with m / z 246.1468. It continues to fragment to obtain the characteristic ion of AA6 with m / z 145.0981; (3) ECBN loses (AA1+AA2+AA6) to obtain a fragment ion (AA3+AA4+AA5) with an m / z of 424.1729. It loses three water molecules successively to obtain ions with m / z 406.1622, 388.1506 and 370.1408. (4) ECBN loses 1 molecule of water and ammonia on AA6 and AA1 to obtain a fragment ion with an m / z of 636.2522. After losing three more molecules of water, the m / z of the ion fragments are 618.2420, 600.2317 and 582.2203 respectively. The ion m / z 600.2317 will also break down to form the characteristic ion of (AA1+AA2) with m / z 326.1362. This ion loses one more molecule of water to obtain an ion with m / z 308.1257.

[0131]

[0132] According to the above cracking rules, the structures of other impurities in the ECBN crude product can be determined.

[0133] The MS image of peak 1 impurity is as follows Figure 5 As shown in Figure 2, the m / z of the quasi-molecular ion peak is 814.3476, which is 16 larger than the molecular weight of ECBN. Figure 6 As shown, by comparing with the characteristic ions of ECBN, it is mainly that the two series of ions (1) and (2) are 16 larger than the characteristic ions of ECBN. Further, through the characteristic ions m / z 262.1415 and 161.0932, it is judged that the characteristic ions of the amino acid at position 6 have changed, and it is further judged that a hydroxyl group has been added. There are two structural impurities, one of which is ECBN (ECBN-6Ⅰ) substituted with 3-hydroxy-4-hydroxymethylproline (position 6) or ECBN (ECBN-6Ⅱ) substituted with 3,5-dihydroxy-4-methylproline (position 6). Taking ECBN-6Ⅰ-1 as an example, Figure 7 Display the structural attributes of characteristic ion fragments.

[0134]

[0135] The MS image of peak 4 impurity is as follows Figure 8 As shown in the figure, the m / z of the quasi-molecular ion peak is 784.3360, which is 14 smaller than the molecular weight of ECBN and the same as the molecular weight of impurities M and N, indicating that one methylene group is missing. Fig. 9 As shown in the figure, by comparing with the characteristic ions of ECBN, it is mainly that the two series of ions (1) and (4) are 14 smaller than the characteristic ions of ECBN. Further, by judging from the characteristic ions m / z 312.2107 and 294.1098, it is judged that the characteristic ions of threonine at position 2 have changed, and it is judged that a methylene group is lost and converted to serine, that is, ECBN-2Ser. Taking ECBN-2Ser-1 as an example, Fig.10 Display the structural attributes of characteristic ion fragments.

[0136]

[0137] The m / z of the quasi-molecular ion peak of the impurity in peak 2 is 784.3358 ([M+H] + ), which is 14 smaller than the molecular weight of ECBN. Its MS / MS graph is shown in Fig.13 As shown in the figure, by comparing with the characteristic ions of ECBN, it is mainly that the two series of ions (1) and (2) are 14 smaller than the characteristic ions of ECBN. Further, through the characteristic ions m / z 232.1305 and 131.0821, it is judged that the characteristic ion of the amino acid at position 6 lacks a methyl group, that is, the amino acid is 3-hydroxyproline, and it is judged to be impurity M.

[0138] The m / z of the quasi-molecular ion peak of the impurity in peak 3 is 784.3361 ([M+H] + ), which is also 14 smaller than the molecular weight of ECBN. Its MS / MS graph is shown in Fig.14 As shown, by comparing with the characteristic ions m / z 232.1307 and 145.0979 of impurity M, it is judged that AA6 has not changed, and based on the characteristic ions (2) m / z 536.2362, (3) m / z 410.1570, (4) m / z 622.2364 and their dehydrated ions, it is judged that AA5 has changed from Thr to Ser, and thus it is judged to be impurity N.

[0139] Comparative Example 1

[0140] The volume ratio of the mobile phase was replaced with mobile phase A: mobile phase B = 90:10 (v / v), and the other chromatographic conditions were the same as in Example 1. The total ion current of the crude ECBN product was: Fig.11 , no significant separation was observed between the impurities and ECBN.

[0141] Comparative Example 2

[0142] The volume ratio of the mobile phase was replaced with mobile phase A: mobile phase B = 93:7 (v / v), and the other chromatographic conditions were the same as in Example 1. The total ion current of the crude ECBN product was: Fig.12 , impurity 4 and ECBN were not significantly separated.

[0143] In summary, the present invention discloses a separation or detection method for the echinocandin B nucleus (ECBN) and / or impurities based on high performance liquid chromatography-high resolution mass spectrometry, wherein the separation method can separate ECBN and impurities well. The present invention also provides a method for identifying the two impurities ECBN-2Ser, ECBN-6Ⅰ, and ECBN-6Ⅱ. ECBN-2Ser is ECBN substituted with serine (position 2). ECBN-6Ⅰ is ECBN substituted with 3-hydroxy 4-hydroxymethyl proline (position 6). ECBN-6Ⅱ is ECBN substituted with 3,5-dihydroxy 4-methyl proline (position 6). The present invention provides a method for identifying impurities in ECBN or its derivatives such as anidulafungin, which is suitable for quality control of antifungal anidulafungin drugs.

[0144] It is necessary to point out here that the above embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above contents of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for separating the mother nucleus and / or impurities of echinocandin B, characterized in that: The method comprises the following steps: using a high performance liquid chromatograph to separate a sample to be separated; The conditions of the high performance liquid chromatograph are as follows: the chromatographic column is an alkyl bonded silica gel chromatographic column or a phenyl bonded silica gel chromatographic column; 0.01-1% formic acid aqueous solution is used as mobile phase A, and acetonitrile or methanol is used as mobile phase B for isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 95:5-99:1; The sample to be separated is ECBN crude product or echinocandins drugs.

2. The method for separating the echinocandin B nucleus and / or impurities according to claim 1, characterized in that: The method for separating the echinocandin B nucleus and / or impurities satisfies one or more of the following conditions: (1) The mobile phase A is a 0.1% formic acid aqueous solution; (2) The mobile phase B is acetonitrile; (3) The alkyl bonded silica gel chromatographic column is a C4, C8, C18 or C30 alkyl bonded silica gel chromatographic column, preferably a Diamonsil Plus C18 column; (4) The filler particle size of the chromatographic column is 5 μm; (5) The length of the chromatographic column is 250 mm; (6) The inner diameter of the chromatographic column is 4.6 mm; (7) In the separation method, the flow rate of the mobile phase is 0.5-2 mL / min, preferably 1 mL / min; (8) The column temperature of the chromatographic column is 25-40°C, preferably 30°C; (9) In the separation method, the injection volume is 1-100 μL, preferably 5 μL; (10) The detection wavelength of the high performance liquid chromatograph is 190-280 nm, preferably 210 nm; (11) The echinocandin drug is anidulafungin, caspofungin or micafungin; the ECBN crude product is preferably prepared by the following preparation method: using echinocandin B as a substrate, using the genetically engineered bacteria disclosed in CN115537367A to carry out fermentation culture in a fermentation medium, so that the echinocandin B is converted into the echinocandin B mother nucleus, thereby obtaining the ECBN crude product; (12) the impurity is selected from one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N, wherein ECBN represents the echinocandin B mother nucleus; Wherein, the ECBN-2Ser is or its stereoisomers; said ECBN-6I is or its stereoisomers; the ECBN-6II is or its stereoisomers; the impurity M is The impurity N is 3. A method for detecting the mother nucleus of echinocandin B and its impurities, characterized in that: The method comprises the following steps: using a high performance liquid chromatograph to detect and separate a sample to be tested; the sample to be tested is a crude ECBN product or an echinocandins drug; Among them, the conditions of the high performance liquid chromatography are: the chromatographic column is an alkyl bonded silica gel chromatographic column or a phenyl bonded silica gel chromatographic column; 0.01-1% formic acid aqueous solution is used as mobile phase A, and acetonitrile or methanol is used as mobile phase B for isocratic elution; the volume ratio of the mobile phase A to the mobile phase B is 95:5-99:

1.

4. The method for detecting the echinocandin B parent nucleus and its impurities as claimed in claim 3, characterized in that: The method for detecting the echinocandin B parent nucleus and its impurities satisfies one or more of the following conditions: (1) In the detection method, the detection wavelength is 190-280 nm, preferably 210 nm; (2) The echinocandin drug is anidulafungin, caspofungin or micafungin; the ECBN crude product is preferably prepared by the following preparation method: using echinocandin B as a substrate, using the genetically engineered bacteria disclosed in CN115537367A to carry out fermentation culture in a fermentation medium, so that the echinocandin B is converted into the echinocandin B mother nucleus, thereby obtaining the ECBN crude product; (3) The detection method further includes mass spectrometry detection, which includes the following steps: the high performance liquid chromatograph is combined with a mass spectrometer, and the mass-to-charge ratio of each component separated by the high performance liquid chromatograph is detected by the mass spectrometer.

5. The method for detecting the echinocandin B parent nucleus and its impurities according to claim 4, characterized in that: The method for detecting the echinocandin B parent nucleus and its impurities satisfies one or more of the following conditions: (1) At least a portion of the components separated by the high performance liquid chromatography enters the mass spectrometer, preferably, the portion is 1 / 3; (2) The mass spectrometer is a high-resolution mass spectrometer; the mass detector of the high-resolution mass spectrometer is preferably an electrostatic field orbital trap, time-of-flight or triple quadrupole mass spectrometer; the high-resolution mass spectrometer is preferably a quadrupole-time-of-flight mass spectrometer; (3) The ionization mode of the mass spectrometer is electrospray ionization or atmospheric pressure ionization; preferably, when the ionization mode is electrospray ionization, the nebulizer gas temperature is 200-240°C, preferably 220°C; the nebulizer gas pressure is preferably 18-22 psi, more preferably 20 psi; (4) The drying gas flow rate of the mass spectrometer is 15-23 L / min, preferably 19 L / min; (5) The sheath gas flow rate of the mass spectrometer is 9-13 L / min, preferably 11 L / min; (6) The capillary voltage of the mass spectrometer is 2.8-4.2 kV, preferably 3.5 kV; (7) The nozzle voltage of the mass spectrometer is 1.2-1.8 kV, preferably 1.5 kV; (8) The scanning mode of the mass spectrometer is positive ion mode or negative ion mode, preferably ESI positive ion scanning mode; (9) The MS ion scanning range of the mass spectrometer is 100-1700, preferably 300-1700; (10) The MS / MS ion scanning range of the mass spectrometer is 50-1700; (11) The MS / MS collision energy of the mass spectrometer is 20-50 eV, preferably 30-40 eV; (12) The impurity is one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N; wherein the ECBN-2Ser is or its stereoisomers; said ECBN-6I is or its stereoisomers; the ECBN-6II is or its stereoisomers; the impurity M is The impurity N is 6. The method for detecting the echinocandin B parent nucleus and its impurities according to claim 5, characterized in that: The method for detecting the echinocandin B parent nucleus and its impurities satisfies one or more of the following conditions: (1) The m / z of the mass spectrum quasi-molecular ion peak of ECBN-2Ser is between 814 and 815; preferably, the m / z of the mass spectrum quasi-molecular ion peak of ECBN-2Ser is 814.3476; (2) The m / z of the mass spectrometry quasi-molecular ion peak of ECBN-6I or ECBN-6II is between 784 and 785; preferably, the m / z of the mass spectrometry quasi-molecular ion peak of ECBN-6I or ECBN-6II is 784.3360; (3) A total ion current diagram is obtained according to the detection results of the high performance liquid chromatograph and the mass spectrometer, wherein the relative retention time of ECBN-2Ser in the total ion current diagram of the sample to be tested is 6.1 min; the relative retention time of ECBN-6I or ECBN-6II in the total ion current diagram of the sample to be tested is 4.2 min; The total ion flow diagram was obtained by high performance liquid chromatography-mass spectrometry, and the high performance liquid chromatography detection conditions were as follows: the chromatographic column model was Diamonsil Plus C18; the mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was acetonitrile, the volume ratio of mobile phase A to mobile phase B was 95:5, and isocratic elution was adopted; the injection volume was 5 μL; the column temperature was 30°C; the detection wavelength was 210 nm; and the flow rate was 1 mL / min; The detection conditions of the mass spectrometer are as follows: Q / TOF-MS adopts ESI positive ion scanning mode, the nebulizer gas temperature is 220°C, the drying gas flow rate is 19 L / min, the nebulizer pressure is 20 psi, the sheath gas flow rate is 11 L / min, the capillary voltage is 3.5 kV, and the nozzle voltage is 1.5 kV; the mass spectrometer is tuned before use, and a reference ion with an m / z of 922.00980 is selected for real-time correction during the scanning process, and the primary mass spectrometer scanning range is m / z 300-1700; the tandem mass spectrometer collision energy is 30 eV; the secondary mass spectrometer scanning range covers parent ions with m / z of 300-1700 and daughter ions with m / z of 50-1700.

7. An impurity related to the parent nucleus of echinocandin B, characterized in that: The echinocandin B parent nucleus-related impurities are ECBN-2Ser, ECBN-6I or ECBN-6II, Wherein, the structural formula of ECBN-2Ser is or its stereoisomers; The structural formula of ECBN-6I is or its stereoisomers; The structural formula of ECBN-6II is or its stereoisomers; Preferably, a total ion current graph is obtained according to the detection results of a high performance liquid chromatograph and a mass spectrometer, and the relative retention time of ECBN-2Ser in the total ion current graph is 6.1 min; and / or, the relative retention time of ECBN-6I or ECBN-6II in the total ion current graph is 4.2 min; The detection conditions of the high performance liquid chromatography are as follows: the chromatographic column model is DiamonsilPlus C18; the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, the volume ratio of mobile phase A to mobile phase B is 95:5, and isocratic elution is adopted; the injection volume is 5 μL; the column temperature is 30°C; the detection wavelength is 210nm; the flow rate of the mobile phase is 1mL / min; The detection conditions of the mass spectrometer are as follows: Q / TOF-MS adopts ESI positive ion scanning mode, the nebulizer gas temperature is 220°C, the drying gas flow rate is 19 L / min, the nebulizer pressure is 20 psi, the sheath gas flow rate is 11 L / min, the capillary voltage is 3.5 kV, and the nozzle voltage is 1.5 kV; the mass spectrometer is tuned before use, and a reference ion with an m / z of 922.00980 is selected for real-time correction during the scanning process, and the primary mass spectrometer scanning range is m / z 300-1700; the tandem mass spectrometer collision energy is 30 eV; the secondary mass spectrometer scanning range covers parent ions with m / z of 300-1700 and daughter ions with m / z of 50-1700.

8. A mixed system of echinocandin B parent nucleus and impurities, characterized in that: The mixed system comprises an echinocandin B mother core (ECBN) and impurities, wherein the impurities are selected from one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N; The ECBN-2Ser is or its stereoisomers; The ECBN-6I is or its stereoisomers; The ECBN-6Ⅱ is or a stereoisomer thereof.

9. A method for identifying impurities related to the parent nucleus of echinocandin B, characterized in that: The identification method includes the following contents: (1) performing MS and MS / MS analysis on ECBN, and obtaining the mass spectrometry fragmentation pathway of the ECBN according to the characteristic ion information in the MS / MS spectrum; the ECBN represents the mother nucleus of echinocandin B; (2) Using the mass spectrometry fragmentation pathway of ECBN as a control, the echinocandin B core-related impurities were subjected to MS and MS / MS analysis, and the characteristic ions of the echinocandin B core-related impurities were compared with the characteristic ions of ECBN, respectively. The types of the echinocandin B core-related impurities were identified by combining the molecular weight changes and the changed characteristic ions in the MS spectrum.

10. The method for identifying impurities related to the nucleus of echinocandin B according to claim 9, characterized in that: The impurities of the echinocandin B parent core are one or more of ECBN-2Ser, ECBN-6I, ECBN-6II, impurity M and impurity N; And / or, the mass spectrometry fragmentation pathway of ECBN is as follows: (i) ECBN loses 4 water molecules successively to obtain ions with m / z of 780.3408, 762.3313, 744.3213, and 726.3104, respectively; (ii) ECBN loses 1 water molecule, AA1, and AA2 to obtain fragment ions formed by AA3, AA4, AA5, and AA6, with m / z of 550.2524, loses 1 water molecule to obtain ion m / z 532.2415, continues to lose 1 water molecule to obtain ion m / z 514.2307, and forms characteristic ions m / z 246.1468 with AA5 and AA6, and continues to fragment to obtain characteristic ion m / z 246.1468 of AA6. 145.0981; (iii) ECBN loses AA1, AA2 and AA6 to obtain fragment ions formed by AA3, AA4 and AA5, with an m / z of 424.1729. After losing three molecules of water, ions with m / z of 406.1622, 388.1506 and 370.1408 are obtained; (iv) ECBN loses 1 molecule of water and ammonia on AA6 and AA1 to obtain a fragment ion with an m / z of 636.2522. After losing three more molecules of water, the m / z of the ion fragments are 618.2420, 600.2317 and 582.2203, respectively. After the ion m / z 600.2317 is fragmented, the characteristic ion formed by AA1 and AA2 is obtained, with an m / z of 326.1362. This ion loses one more molecule of water to obtain an ion with m / z of 308.1257. The structural formula of the ECBN is shown below:

11. The method for identifying impurities related to the nucleus of echinocandin B according to claim 9 or 10, characterized in that: The method for identifying impurities related to the nucleus of echinocandin B satisfies one or more of the following: (1) comparing the quasi-molecular ion peak of the echinocandin B core-related impurity with the two characteristic ions (i) and (ii) of ECBN, if it is determined that the molecular weight of the echinocandin B core-related impurity is 16 greater than the molecular weight of ECBN, then further determining through characteristic ions m / z 262.1415 and 161.0932 that the echinocandin B core-related impurity has an additional hydroxyl group relative to the characteristic ion of the amino acid at position 6 of ECBN, and further determining that the type of the echinocandin B core-related impurity is ECBN-6Ⅰ or ECBN-6Ⅰ; (2) comparing the quasi-molecular ion peak of the echinocandin B parent core-related impurity with the two series of characteristic ions (i) and (iv) of the ECBN, if it is determined that the molecular weight of the echinocandin B parent core-related impurity is 14 less than the molecular weight of the ECBN, then further determining through characteristic ions m / z 312.2107 and 294.1098 that the echinocandin B parent core-related impurity has changed relative to the characteristic ion of 2-threonine of the ECBN, then determining that threonine loses a methylene group and is converted into serine, and further determining that the type of the echinocandin B parent core-related impurity is ECBN-2Ser; (3) comparing the quasi-molecular ion peak of the echinocandin B parent core-related impurity with the two characteristic ions (i) and (ii) of the ECBN, if it is determined that the molecular weight of the echinocandin B parent core-related impurity is 14 less than the molecular weight of the ECBN, then further determining through characteristic ions m / z 232.1305 and 131.0821 that the echinocandin B parent core-related impurity has one less methyl group relative to the characteristic ion of the amino acid at position 6 of the ECBN, and further determining that the type of the echinocandin B parent core-related impurity is impurity M; (4) Compare the quasi-molecular ion peak of the echinocandin B core-related impurity with the characteristic ion of the ECBN. If it is determined that the molecular weight of the echinocandin B core-related impurity is 14 less than that of the ECBN, then further determine through characteristic ions m / z 232.1305 and 131.0821 that the echinocandin B core-related impurity has not changed relative to the characteristic ion of the 6-position amino acid of ECBN. Then, according to (ii) series characteristic ions m / z 536.2362, (iii) series characteristic ions m / z 410.1570, (iv) series characteristic ions m / z 622.2364 and their dehydrated ions, it is determined that the echinocandin B core-related impurity loses a methylene group relative to the 5-position threonine of ECBN and is converted into serine. Finally, it is determined that the type of the echinocandin B core-related impurity is impurity N.

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