A method for structural analysis of an echinocandin b or impurities in a parent nucleus thereof

Abstract

The application discloses a structural analysis method of echinocandins B or impurities in a mother nucleus thereof. The structural analysis method comprises the following steps: S1, performing primary mass spectrum scanning on echinocandins B or the mother nucleus thereof to obtain parent ions [M+H] + and parent ions [M+Na] + ; S2, performing secondary mass spectrum scanning on the parent ions [M+H] + and parent ions [M+Na] + of echinocandins B or the mother nucleus thereof to obtain ion sets I and II; S3, sequentially performing the primary mass spectrum scanning and the secondary mass spectrum scanning on the impurities to obtain ion sets III and IV; and S4, comparing the ion sets III and IV with the ion sets I and II respectively to analyze chemical structural formulas of the impurities. The structural analysis method explores a new analysis technology, and especially can quickly and directly judge the change of specific amino acids of the impurities in echinocandins B or the mother nucleus thereof, and further accurately analyzes chemical structural formulas of more impurities.

Description

Technical Field

[0001] This invention relates to a method for structural analysis of echinocandin B or impurities in its parent nucleus. Background Technology

[0002] Invasive fungal infections caused by Candida or Aspergillus species pose a serious life-threatening risk to immunocompromised patients. Echinocandins strongly inhibit the activity of 1,3-β-D-glucan synthase, essential for fungal cell wall biosynthesis, thus exhibiting antifungal activity against these pathogens. Anidulafungin, a semi-synthetic drug derived from echinocandin B (ECB), is currently used clinically to treat fungal infections. Echinocandins are synthesized by non-ribosomal peptide synthases (NRPS) and are a group of structurally similar and closely related lipopeptides produced almost exclusively by filamentous fungi such as Aspergillus delacroxii or Aspergillus rugulosus. These compounds consist of a cyclic hexapeptide backbone and an N-terminal fatty acyl (FA) chain. Six amino acids in the cyclic hexapeptide are hydroxylated, four of which are non-protein amino acids. Incomplete hydroxylation of amino acids and variations in the FA chain during biosynthesis contribute to the diversity of echinocandin components. The main component of echinocandin is echinocandin B (ECB), whose structural formula is shown below:

[0003]

[0004] ECB is composed of linoleyl (C) 18 H 31 It is composed of O and the following six amino acids:

[0005] AA1: 4R,5R-dihydroxy-L-ornithine (dhOrn);

[0006] AA2 and AA5: L-threonine (Thr);

[0007] AA3: 4R-hydroxy-L-proline (4Hyp);

[0008] AA4: 3S,4S-dihydroxy-L-homotyrosine (dhhTyr);

[0009] AA6: 3S-hydroxy-4S-methyl-L-proline (4Me3Hyp).

[0010] The hexapeptide ring is formed by the condensation of the δ-NH2 group of AA1 and the carboxyl group of AA6. Other homologous components, such as ECC and ECD, have also been reported. In ECC, AA4 is replaced by 3S-hydroxy-L-homotyrosine (hhTyr), and in ECD, AA1 and AA4 are Orn and hhTyr, respectively. These echinocandin homologues may be transferred as impurities into the anidulafungin active pharmaceutical ingredient. Typically, the structure of echinocandins is determined after component purification using methods such as UV-IR spectroscopy, NMR, mass spectrometry, amino acid analysis, and fatty acid analysis.

[0011] Echinocandin contains a variety of impurities, and the usual method for chemical structure identification is nuclear magnetic resonance (NMR), which requires purification to obtain a certain amount of the compound before it meets the detection requirements. Separating all the impurities in echinocandin and preparing a quantity that meets the detection requirements undoubtedly increases the difficulty and cost of identification.

[0012] Researchers in this field have been exploring methods for structural identification of echinocandin analogues:

[0013] The article "Echinocandins: structural diversity, biosynthesis, and development of antimycotics" published by Hüttel et al. summarizes the structures of echinocandins and their impurities. Table S3 records that ECBpro was identified solely by mass spectrometry as proline (Pro) at position AA3, dihydroxyhigh-tyrosine (dhhTyr) at position AA4, and 4-methylproline (4MePro) at position AA6. However, the mass spectrometry identification process is not detailed, and the correctness of the chemical structure is questionable. The authors of the article also explicitly stated that the structure was confirmed solely by mass spectrometry, indicating that they themselves were uncertain about the correctness of the chemical structure confirmed by mass spectrometry.

[0014] In their article "Isolation, identification and characterization of potential impurities of anidulafungin", Zhao et al. disclosed that they identified four impurities, including open-chain hydrolysis products, serine (AA5) derivatives, and hydroxyproline (AA6) derivatives, using ESI-MS and one-dimensional and two-dimensional NMR.

[0015] CN119269658A discloses a structural analysis platform based on HPLC-Q-TOF-MS / MS for the analysis of echinocandin parent nucleus (ECBN). It utilizes [M+H]... +Four fragmentation pathways of ECBN were identified by secondary mass spectrometry scanning of the parent ion and the resulting fragment ion set. Four impurities were then separated by high-performance liquid chromatography (HPLC) and further analyzed using the [M+H] fractions of the corresponding impurities. + The daughter ion sets obtained by secondary mass spectrometry scanning of the parent ion were compared with those of ECBN, while also referencing the fragmentation pathways of ECBN. The daughter ion sets of these four impurities all shared the same fragmentation pathways as ECBN, with two identical fragmentation pathways. The difference in the difference between the daughter ion sets and the molecular weight difference of the parent ion indicated a change in the amino acid. Furthermore, these four impurities showed only one group change compared to ECBN, and their fragmentation pathways were largely the same as ECBN. However, for unknown impurities with multiple amino acid variations, their fragmentation pathways may differ from those of known compounds, increasing the difficulty of resolving impurities in ECBN or ECB and potentially leading to unresolved cases. Therefore, in the process of using mass spectrometry to resolve the chemical structures of echinocandin analogs, further research is needed to develop different analytical methods to identify more unknown impurities. Summary of the Invention

[0016] To overcome the shortcomings of existing technologies that may fail to resolve the chemical structures of echinocandin analogues using mass spectrometry, this invention provides a method for structural analysis of echinocandin B or impurities in its core. This method explores a new analytical technique that can quickly and directly determine changes in specific amino acids in echinocandin B or its core impurities, thereby accurately resolving the chemical structures of more impurities.

[0017] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0018] This invention provides a method for structural analysis of impurities in echinocandin B or its parent nucleus, the method comprising the following steps:

[0019] S1: Primary mass spectrometry scan of echinocandycin B or its parent nucleus yielded the parent ion [M+H]. + and the parent ion [M+Na] + ;

[0020] S2: The precursor ion [M+H] for the echinocandin B or its parent nucleus. + and the parent ion [M+Na] + Secondary mass spectrometry scans were performed to obtain daughter ion sets I and II;

[0021] S3: Perform the first-stage mass spectrometry scan and the second-stage mass spectrometry scan sequentially on the impurity to obtain daughter ion sets III and IV; wherein, daughter ion set III is the parent ion [M+H] of the impurity. +The daughter ion set obtained by performing a secondary mass spectrometry scan, wherein the daughter ion set IV is the parent ion [M+Na] of the impurity. + The set of daughter ions obtained by performing a secondary mass spectrometry scan;

[0022] S4: Compare the sub-ion sets III and IV with the sub-ion sets I and II respectively to resolve the chemical structural formula of the impurity.

[0023] The researchers of this invention, when analyzing the structural formulas of certain impurities, used the ECB precursor ion [M+H] as a reference. + When performing secondary mass spectrometry scans to obtain mass spectrometry data for structural analysis, some impurities with significantly different molecular weights from echinocandin B, i.e., impurities with significant changes in amino acid sites, exhibit fragmentation pathways that differ considerably from ECB, making it difficult to determine their structure using the ECB parent ion [M+H]. + The chemical structure of the impurity was determined through its cleavage pathway. However, for impurities with a large number of hydroxyl groups, it was impossible to determine which amino acid the hydroxyl group was located on. Particularly concerning was the fourth amino acid, which not only had many hydroxyl groups but also rarely lost alone. Therefore, the structure of the fourth amino acid could not be determined by the characteristic ion at the small molecule end or the molecular weight of the lost amino acid. Researchers unexpectedly discovered that the parent ion [M+Na] of echinocandin B and several of its impurities... + When performing secondary mass spectrometry scans, they all share a common characteristic: the removal of the fourth amino acid from the parent ion. The high relative abundance of the structural daughter ions and corresponding characteristic daughter ions allows for rapid determination of the structural formula of the fourth amino acid in impurities. Experimental verification has shown that this method is also applicable to the structural analysis of impurities in ECBN, enabling rapid identification of changes in the fourth amino acid position in ECBN impurities.

[0024] In this invention, those skilled in the art will know that the parent ion [M+H]+ and the parent ion [M+Na]+... + The "M" in the mass spectrometry typically represents the molecular weight of the compound detected by the primary mass spectrometry. In this invention, the primary mass spectrometry detects echinocandin B or its parent nucleus, as well as the impurities mentioned above.

[0025] In this invention, the first-stage mass spectrometry scan and the second-stage mass spectrometry scan can be in full-scan mode.

[0026] As those skilled in the art will know, the primary mass spectrometry and the secondary mass spectrometry are typically performed using a quadrupole time-of-flight mass spectrometer.

[0027] Preferably, the parameter settings of the quadrupole time-of-flight mass spectrometer satisfy one or more of the following conditions:

[0028] (1) The atomizer temperature is 300-350℃, for example, 320℃;

[0029] (2) The atomizing gas flow rate is 6-10 L / min, for example 8 L / min;

[0030] (3) The spray pressure is 30-40 psi, for example 35 psi;

[0031] (4) The sheath gas temperature is 300-400℃, for example 350℃;

[0032] (5) The sheath flow rate is 8-15 L / min, for example 11 L / min;

[0033] (6) The capillary pressure is 3-5kV, for example 3.5kV;

[0034] (7) The nozzle pressure is 0.5-2kV, for example, 1kV;

[0035] (8) The breaking voltage is 150-200V, for example 175V.

[0036] In this invention, the mass-to-charge ratio set during the primary mass spectrometry scan can be any conventional value in the art, as long as it accurately measures the molecular weight of the echinocandy B or its parent nucleus and impurities. In a preferred embodiment of this invention, the mass-to-charge ratio set during the primary mass spectrometry scan is 500-1500.

[0037] In this invention, the mass-to-charge ratio of the mass spectrometry range set during the secondary mass spectrometry scan is preferably 50-1500.

[0038] In this invention, the parent ion [M+H] is used. + When performing the secondary mass spectrometry scan, the collision energy is preferably 30-50 eV, more preferably 35 eV, 40 eV or 45 eV.

[0039] In this invention, the parent ion [M+Na] is used. + When performing the secondary mass spectrometry scan, the collision energy is preferably 60-90 eV, such as 70 eV, 75 eV, 80 eV, or 85 eV. Those skilled in the art know that if the collision energy is too high, a set of daughter ions with smaller molecular weights will be obtained; if the collision energy is too low, the number of daughter ions obtained will be small, and they will mainly be daughter ions with higher molecular weights. Through multiple experiments, the developers of this invention have verified that within the 60-90 eV range, a parent ion [M+Na] containing echinocandin B or its parent nucleus or its impurities with a relative abundance of 5-30% or higher can be observed. + Characteristic ions with a difference of 152 Da or 136 Da and their characteristic ions after neutral loss of acetaldehyde.

[0040] In a specific embodiment of the present invention, the sub-ion set II includes sub-ion P, and the sub-ion P has a [M+Na] ratio greater than that of the echinocandin B. + The molecular weight of the parent ion is less than 152 Da, indicating that the fourth amino acid of the echinocandin B is dihydroxyhigh tyrosine; the daughter ion set II also includes daughter ions with a molecular weight 44 Da less than that of daughter ion P, indicating that daughter ion P has undergone neutral loss of acetaldehyde.

[0041] In a specific embodiment of the present invention, the daughter ion set IV includes daughter ion P', wherein daughter ion P' is more [M+Na] than the impurity. + The molecular weight of the parent ion is less than 152 Da or 136 Da; when the molecular weight is less than 152 Da, the fourth amino acid of the impurity is dihydroxyhigh tyrosine, and when the molecular weight is less than 136 Da, the fourth amino acid of the impurity is monohydroxyhigh tyrosine; the daughter ion set IV also includes daughter ions with a molecular weight 44 Da smaller than that of the daughter ion P'.

[0042] In this invention, the absolute value of the difference between the molecular weight of the impurity and the molecular weight of the echinocanthoma B or its parent nucleus is preferably 0, 2, 14, 16 or a multiple thereof or a combination thereof, such as 28, 30, 32 or 48.

[0043] In this invention, those skilled in the art will understand that the impurity generally refers to a compound with a structure similar to that of the echinocandin B or its parent nucleus. The differences between the impurity and the echinocandin B or its parent nucleus include differences in the number of hydroxyl groups, the number of methyl or methylene groups, or the number of double bonds.

[0044] In specific embodiments of the present invention, the impurities include one or more of the following compounds: ECB-1, ECB-2, ECB-3, ECB-4, ECB-5, ECB-5', ECB-7, ECB-8, ECB-9, ECB-10, ECB-11, ECB-12, and ECB-13. In this invention, those skilled in the art will understand that the structural formulas resolved from mass spectrometry detection results are only chemical structural formulas and do not include stereoscopic structural formulas. The stereoscopic structural formulas of the impurities are for reference only and are not intended to limit the scope of the invention. Specifically, the molecular weight of ECB-1 is 1031 Da, and the chemical structural formula of ECB-1 is as follows:

[0045] The molecular weight of ECB-2 is 1057 Da, and the chemical structural formula of ECB-2 is as follows:

[0046] The ECB-3 has a molecular weight of 1045 Da and its chemical structural formula is as follows:

[0047] The ECB-4 has a molecular weight of 1033 Da and its chemical structural formula is as follows:

[0048] The ECB-5 has a molecular weight of 1075 Da and its chemical structural formula is as follows:

[0049] The ECB-5' has a molecular weight of 1075 Da and its chemical structural formula is as follows:

[0050] The ECB-7 has a molecular weight of 1045 Da and its chemical structural formula is as follows:

[0051] The ECB-8 has a molecular weight of 1043 Da and its chemical structural formula is as follows:

[0052] The ECB-9 has a molecular weight of 1043 Da and its chemical structural formula is as follows:

[0053] The ECB-10 has a molecular weight of 1043 Da and its chemical structural formula is as follows:

[0054] The ECB-12 has a molecular weight of 1027 Da and its chemical structural formula is as follows:

[0055] The ECB-13 has a molecular weight of 1011 Da and its chemical structural formula is as follows:

[0056] In this invention, the impurities may be those generated during the preparation of the echinocandin B or its parent nucleus. The preparation method can be conventional in the art and generally does not require special limitation, including but not limited to microbial fermentation.

[0057] In this invention, the impurities can be separated from the echinocandin B or its parent nucleus by high performance liquid chromatography.

[0058] The high-performance liquid chromatography column can be an alkyl-bonded silica column, such as a C18 alkyl-bonded silica column.

[0059] The mobile phase of the high-performance liquid chromatography preferably comprises 0.01-0.2% formic acid aqueous solution and acetonitrile containing 0.01-0.2% formic acid, for example, 0.1% formic acid aqueous solution and acetonitrile containing 0.1% formic acid. The 0.01-0.2% formic acid aqueous solution is used as mobile phase A, and the acetonitrile containing 0.01-0.2% formic acid is used as mobile phase B.

[0060] The high-performance liquid chromatography (HPLC) preferably employs gradient elution. The preferred gradient elution program is: 0-20 min 40% mobile phase B; 20-35 min 40-50% mobile phase B; 35-45 min 50-90% mobile phase B; 45-50 min 90% mobile phase B; 50-51 min 90-40% mobile phase B; 51-58 min 40% mobile phase B.

[0061] The detection wavelength of the high-performance liquid chromatography can be 190-280 nm, preferably 210 nm.

[0062] Another aspect of the present invention provides a related impurity in echinocandycin B, characterized in that the related impurity in echinocandycin B includes one or more of the following compounds:

[0063]

[0064]

[0065] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0066] The positive and progressive effects of this invention are as follows: the structural analysis method of this invention explores a new technology that can quickly and directly determine the changes of specific amino acids, especially the changes of the 4th amino acid (i.e., AA4), in impurities in echinocandy B or its parent nucleus, and can accurately resolve the chemical structural formulas of more impurities. Attached Figure Description

[0067] Figure 1 This is the total ion chromatogram of the ECB sample solution.

[0068] Figure 2 MS plot of ECB and its counterpart with the parent ion [M+H] + MS / MS chromatograms during secondary mass spectrometry scanning. Among them, Figure 2 A is the MS plot of ECB. Figure 2 B represents the parent ion [M+H]. + MS / MS chromatogram during secondary mass spectrometry scanning.

[0069] Figure 3 The precursor ion for ECB is [M+H].+ ECB fragmentation pathways were analyzed from MS / MS data.

[0070] Figure 4 For the parent ion [M+Na] + MS / MS chromatogram of ECB during secondary mass spectrometry scanning.

[0071] Figure 5 The parent ion for ECB is [M+Na]. + ECB fragmentation pathways were analyzed from MS / MS data.

[0072] Figure 6 For the parent ion [M+Na] + MS / MS chromatogram of ECB-6 during secondary mass spectrometry scanning.

[0073] Figure 7 For the parent ion [M+H] + MS / MS chromatogram of ECB-7 during secondary mass spectrometry scanning.

[0074] Figure 8 For the parent ion [M+Na] + MS / MS chromatogram of ECB-7 during secondary mass spectrometry scanning.

[0075] Figure 9 For [M+H] + MS / MS chromatogram of dehydrated ions of ECB-9 at m / z 996.5632 in the mode.

[0076] Figure 10 The ECB-9 fragmentation pathway was determined based on MS / MS data of the dehydration ion m / z 996.5632 of ECB-9.

[0077] Figure 11 The precursor ion for ECB-10 is [M+H]. + The ECB-10 fragmentation pathway was determined by resolving MS / MS data.

[0078] Figure 12 For the parent ion [M+H] + MS / MS chromatogram of ECB-13 during secondary mass spectrometry scanning.

[0079] Figure 13 The precursor ion for ECB-13 is [M+H]. + A fragmentation pathway of ECB-13 was identified by parsing MS / MS data.

[0080] Figure 14 The precursor ion for ECB-13 is [M+H]. + Another fragmentation pathway of ECB-13 was identified by parsing MS / MS data.

[0081] Figure 15 For the parent ion [M+Na] + MS / MS chromatogram of ECB-13 during secondary mass spectrometry scanning.

[0082] Figure 16 The parent ion of ECB-13 is [M+Na]. + ECB fragmentation pathways were analyzed from MS / MS data. Detailed Implementation

[0083] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0084] The following are definitions of some terms used in this application.

[0085] In this article, the term "mass spectrometry data" includes data information generated by a mass spectrometer, typically containing m / z values, also known as molecular weight, on the x-axis and intensity values ​​on the y-axis.

[0086] In this article, the term "mass spectrometry" refers to a technique for ionizing chemical substances and sorting them according to their mass-to-charge ratio. Primary mass spectrometry (MS) analysis involves ionizing a chemical substance to produce charged ions, and measuring the mass-to-charge ratio of these charged ions. Secondary mass spectrometry (MS / MS) analysis typically involves ionizing a chemical substance to produce charged ions, selecting a precursor ion and causing it to dissociate through collision-induced dissociation to produce daughter ions, and measuring the mass-to-charge ratio of these daughter ions.

[0087] In this article, the term "parent ion" refers to a charged molecular moiety that can dissociate to form fragments (one or more of which may be charged) and one or more neutral species.

[0088] In this article, the term "daughter ion" refers to ions with different mass-to-charge ratios produced by the chemical bond breaking after the parent ion undergoes collision-induced dissociation.

[0089] In this article, the term "m / z" (mass-to-charge ratio) is the ratio of mass to charge, which is the ratio of the largest molecular mass to the smallest charge (i.e., 1 charge) obtained in the spectrum. It can also be expressed as the molecular weight (Da, i.e., relative molecular weight) of the parent ion or daughter ion.

[0090] The chemical reagents and instruments used in the examples in this article are as follows:

[0091] ECB sample (batch number: ECB-2401) was provided by Yikelai Biotechnology (Group) Co., Ltd.

[0092] Acetonitrile and formic acid of HPLC-MS grade were purchased from Sigma-Aldrich (St. Louis, Mo, USA);

[0093] Distilled water is further purified by the Milli-Q water purification system (Millipore, Bedford, MA, USA).

[0094] Preparation of ECB sample solution: Dissolve the ECB sample in 10% acetonitrile solution to a concentration of 2 mg / mL.

[0095] HPLC-Q / TOF-MS / MS analyses were performed on an Agilent 1290 high-performance liquid chromatograph and a 6545 quadrupole-time-of-flight mass spectrometer (Agilent Technologies, California, USA), equipped with a binary pump, autosampler, column oven, and dual-jet electrospray ionization source. A Masshunter workstation was used to control the instruments and process the mass spectrometry data.

[0096] Example 1: Chromatographic separation and mass spectrometry detection of ECB and its impurities

[0097] (1) Chromatographic separation

[0098] ECB and its components were separated using a Diamonsil Plus C18 column (5 μm, 4.6 × 250 mm) with 0.1% formic acid in water as mobile phase A and acetonitrile containing 0.1% formic acid as mobile phase B. Gradient elution was performed under the following conditions:

[0099] 0–20min 40% B; 20–35min 40–50% B; 35–45min 50–90% B; 45–50min 90% B; 50–51min 90–40% B; 51–58min 40% B.

[0100] The flow rate was 0.5 mL / min, the injection volume of ECB sample solution was 2 μL, the column temperature was maintained at 40℃, and the detection wavelength was set to 210 nm.

[0101] After the above chromatographic separation, the substances with peaks 1-12 were obtained. After comparison with the ECB standard, peak 8 was identified as echinocandycin B, and the rest were impurities to be tested.

[0102] (2) Mass spectrometry detection

[0103] In positive ion mode, Agilent 6545Q / TOF-MS was used to perform mass spectrometry detection on the chromatographically separated echinocandin B and its impurities.

[0104] The mass spectrometry detection conditions were as follows: the nebulizer temperature of the DualJet ESI source was set to 320℃, and the sheath gas temperature was set to 350℃. The nebulizer gas flow rate was 8 L / min, and the sheath gas flow rate was 11 L / min. The capillary pressure was 3.5 kV, the nozzle pressure was 1.0 kV, and the fragmentation voltage was 175 V. In full scan mode, the mass range of MS (primary mass spectrometry) was set to a mass-to-charge ratio (m / z) of 500-1500, and the mass range of MS / MS (secondary mass spectrometry) was set to 50-1500 m / z, with acquisition rates of 4 mass spectra per second and 2 mass spectra per second, respectively. [M+H] + and [M+Na] + Echinochrome B and its impurities were detected by MS / MS using these as precursor ions, yielding different daughter ion sets. When the target ion for MS / MS was [M+H]... + The collision energy was set to 40 eV, and the target object detected by the MS / MS was [M+Na]. + The collision energy was set to 70 eV. During the analysis, m / z 922.0098 (C 18 H 19 O6N3P3F 24 + () is used as an internal calibration reference ion.

[0105] (3) Chemical structural formula analysis of ECB impurities

[0106] S1, the molecular weight of ECB and its impurities was determined by the above MS detection, and the parent ion [M+H] was obtained. + and the parent ion [M+Na] + ;

[0107] S2, the precursor ion for ECB [M+H] + and the parent ion [M+Na] + The second-order mass spectrometry scan in the above mass spectrometry detection was performed to obtain daughter ion set I and daughter ion set II, respectively, and two different fragmentation pathways were summarized based on daughter ion set I and daughter ion set II, respectively.

[0108] S3 is the precursor ion for ECB impurities [M+Na]. + and [M+H] + The second-order mass spectrometry scan in the above-mentioned mass spectrometry detection was performed to obtain daughter ion sets III and IV, respectively. The chemical structural formula of the impurity of echinocandycin B was determined by comparing the daughter ion sets III and IV of the impurity with the daughter ion set of ECB.

[0109] Example 2: Fracturing pathway of ECB

[0110] (1) The parent ion of ECB [M+H] + Fragmentation pathways resolved after secondary mass spectrometry scanning

[0111] All six amino acids in ECB contain hydroxyl groups, making them prone to dehydration during collisions detected by MS / MS. Due to intrasource cleavage, [M+H-H2O] is generated during MS ionization. + The dehydration ions. dhOrn and dhhTyr, containing adjacent hydroxyl groups, are considered more likely to dehydrate to form enols, which then isomerize to ketone structures. 4Hyp, 4Me3Hyp, and Thr dehydrate to form vinyl groups. Therefore, in [M-nH2O+H]... + In (n=1, 2, 3, 4) ion clusters, a molecular weight difference of 18 Da can generally be detected.

[0112] like Figure 1 The image shows the total ion current chromatogram of the ECB sample solution. Peak 8 represents ECB, and peaks 1-7 and 9-12 represent impurities in ECB. Figure 2 The image shows the mass spectrometry detection data for ECB. Figure 2 In A, m / z 1060.5815 represents the parent ion [M+H]. + The m / z value of 1082.5636 indicates the parent ion [M+Na]. + The m / z value of 1098.5354 indicates the parent ion [M+K]. + The m / z value of 1042.5710 indicates the parent ion [M+H-H2O]. + . Figure 2 The m / z ratios and relative abundances of specific daughter ions in B are shown in Table 1 below.

[0113] Table 1

[0114]

[0115]

[0116] according to Figure 2 The set of daughter ions of B leads to the derivation of three fragmentation pathways of ECB, such as... Figure 3 As shown ( Figure 3 In this context, nH2O represents 1-3 water molecules.

[0117] In the first cleavage pathway, the cyclic hexapeptide structure remains intact. ECB loses one water molecule to generate a daughter ion with m / z 1042.5732, and then undergoes continuous dehydration to form daughter ions with m / z 1024.5625, 1006.5517, and 988.5404. Based on the difference between daughter ion m / z 1024.5625 and daughter ion m / z 762.3316, it is determined that FA and one water molecule split from daughter ion m / z 1042.5732 to form ECBN ions, and then lose multiple water molecules to produce daughter ions with m / z 744.3228 and 726.3113.

[0118] The second cleavage pathway involves FA fragment ions and their subsequent product ions. After ECB fragmentation and removal of the (AA1+AA2) residues, FA is transferred to AA3, yielding (AA3+AA4+AA5+AA6+FA) residue daughter ions with m / z values ​​of 830.4932, 812.4816, and 794.4715, respectively, and their dehydrated ions. Notably, AA6 contains an amide residue instead of a carboxyl group. The (AA3+AA4+AA5+AA6+FA) residue loses FA and undergoes dehydration to yield (AA3+AA4+AA5+AA6) residue daughter ions with m / z values ​​of 568.2635, 550.2523, 532.2416, and 514.2302, or loses AA6 and undergoes dehydration to yield (AA3+AA4+AA5+FA) residue daughter ions with m / z values ​​of 686.4017, 668.3915, and 650.3821. The daughter ions m / z 568.2635 and m / z 686.4017 can both be cleaved to yield daughter ions of (AA3+AA4+AA5) residues and their dehydrated ions m / z 424.1722, 406.1617, 388.1515, and 370.1400. The daughter ions m / z 686.4017 and 424.1722, along with the consecutive dehydrated ions, are also referred to as (AA2+AA3+AA4+FA) and (AA2+AA3+AA4) residues, because AA2 and AA5 are both threonine (Thr). The daughter ion clusters of (AA2+AA3+AA4) or (AA3+AA4+AA5) residues are the most abundant in MS / MS spectra, providing a breakthrough for elucidating the structures of other components. Fragment ions of the (AA3+AA4) residue with m / z values ​​of 323.1244, 305.1140, and 287.1024 were also detected, while m / z 246.1451 belonged to the (AA5+AA6) residue. Low molecular weight fragment ions with m / z values ​​of 131.0814 and 145.0975 were identified as fragment ions of 4Hyp(AA3) and 4Me3Hyp(AA6). Characteristic ions of the (AA1+AA2+AA3+AA4+AA5+FA) residue were detected with molecular weights of m / z 898.4822, 880.4739, and 862.4611, with AA1 ending in an aldehyde group. It is inferred that the amide bond between AA1 and AA6 breaks, a nitrogen molecule is lost from AA1, forming an enol, which then transforms into an aldehyde group. The fragment ion at m / z 898.4822 lost (AA1+AA2+AA3+FA) residues and water to generate the characteristic ion at m / z 275.1022 (AA4+AA5).

[0119] The third fragmentation pathway involves product ions from the ECB cyclic moiety. The (AA1+AA2+AA3+AA4+AA5) fragment ion m / z 618.2413 and subsequent dehydration ions m / z 600.2310 and 582.2210 are generated by the loss of (AA6+FA+H2O+NH3) residues from the ECB. Other fragment ions have been detected and shown in [the diagram / information]. Figure 3 For example, the (AA2+AA3+AA4+AA5) residues have m / z values ​​of 525.2197, 507.2107, and 489.1987; (AA1+AA2+AA3) have m / z values ​​of 326.1355 and 308.1247; (AA2+AA3) have m / z values ​​of 215.1024 and 197.0925; and (AA1+AA2) have m / z values ​​of 213.0876 and 195.0772. The (AA1+AA2) and (AA2+AA3) ions can be used to determine the amino acid at the 2-position.

[0120] The characteristic ions in the three cleavage pathways of ECB can be used to identify changes in amino acids or FAs, laying the foundation for rapid analysis of the structure of other components.

[0121] (2) The parent ion of ECB [M+Na] + Fragmentation pathways resolved by secondary mass spectrometry

[0122] Based on the parent ion [M+H] + The dissociation mode and based on the parent ion [M+Na] + The dissociation modes are completely different. The parent ion of ECB is [M+Na]. + The MS / MS spectra obtained from the second-order mass spectrometry scan are as follows: Figure 4 As shown, the specific daughter ion m / z and their relative abundances are shown in Table 2 below. Parent ion [M+Na] + The ECB fragmentation pathway under the dissociation mode is as follows: Figure 5 As shown.

[0123] Specifically, the parent ion [M+Na] + Two consecutive ion cluster peaks with a difference of 44 Da were observed during the fragmentation process, suggesting that threonine underwent neutral loss of acetaldehyde, accompanied by H-transfer of acetaldehyde. The parent ion [M+Na]+ with m / z 1082.5608 of ECB successively lost two acetaldehydes, generating ions with m / z 1038.2853 and 994.5081. A cyclic ion with m / z 803.3037 was obtained by losing (FA+NH3), followed by the loss of acetaldehyde, AA1, and AA2, forming daughter ions with m / z 759.2783, 715.2507, 691.2913, and 590.2408, etc. Another fragmentation pathway involves [M+Na]. +The ECB component lost 2-hydroxy-2-(4-hydroxyphenyl)acetaldehyde from AA4 via H transfer, yielding the ion m / z 930.5157 and its successive acetaldehyde-removed daughter ions m / z 886.4864 (relative abundance 34.9%) and 842.4593 (relative abundance 26.21%). These daughter ion peaks can directly determine the amino acid structure on AA4 and are easily identifiable due to their high relative abundance. The characteristic daughter ion m / z 930.5157 also lost two acetaldehydes to generate m / z 886.4864 and 842.4593, subsequently losing (FA+NH3), acetaldehyde, AA1, AA2, (AA3+AA4), and AA5.

[0124] Table 2

[0125]

[0126]

[0127] Example 3: Chemical structural analysis of ECB impurities

[0128] The identified ECBs and their impurities are designated as ECB-No. in peak order. Information on ECBs and their impurities, including nomenclature, mass-to-charge ratio (m / z), amino acids, and fatty acids (FAs), is shown in Table 3.

[0129] Table 3

[0130]

[0131]

[0132] Analysis of Peak 1 (named ECB-1)

[0133] Peak 1, m / z 1032.5485 ([M+H]) + ), m / z 1054.5302([M+Na] + The molecular weight of FA is 28 Da smaller than that of ECB. Therefore, the molecular formula of FA is deduced to be C. 16 H 27 The O group is missing two -CH2 groups compared to the linoleoyl group in ECB. This component is believed to be aculeacin A or an isomer thereof.

[0134] Analysis of Peak 2 (named ECB-2)

[0135] The parent ion of peak component 2 is [M+H]. + The m / z value is 1058.5657, [M+Na] +The m / z of FA is 1080.5479, and its molecular weight is 2 Da smaller than that of ECB. The product ions containing FA are all 2 Da smaller than the corresponding ions of ECB, for example, m / z 904.4985, 828.4718, 684.3837 and their dehydrated ions. Therefore, it can be inferred that FA has three vinyl groups, and its molecular formula is C. 18 H 29 O.

[0136] Analysis of peak 3 (named ECB-3)

[0137] The molecular weight m / z of ECB-3 is 1046.5644 ([M+H]). + ) and 1068.5459([M+Na] + It is 14 Da smaller than ECB. This change still occurs in the FA portion, which has one less -CH2 group compared to ECB. The molecular formula of FA in this component is C 17 H 29 O.

[0138] Elucidation of peak 4 (containing two components, named ECB-4 and ECB-5)

[0139] One of the components was named ECB-4, with a molecular weight m / z of 1034.5650 ([M+H]). + ) and 1056.5473([M+Na] + This is 26 Da smaller than ECB. Cyclic ions such as (AA3+AA4+AA5+AA6) at m / z 568.2620, (AA3+AA4+AA5) or (AA2+AA3+AA4) at m / z 424.1724, and (AA1+AA2) at m / z 213.0879 are almost identical to those in ECB, indicating that the six amino acids in the composition remain unchanged. Based on the characteristic ion of m / z 844.4824 resulting from the loss of 1-(4-hydroxyphenyl)ethane-1,2-diol, and the characteristic ions of m / z 854.4545 and 836.4459 resulting from the loss of (AA6+NH3+H2O), the structural change of 26 Da smaller than ECB occurs at the FA group, suggesting that this component contains a double bond at the FA group, which is a hexadecanoyl group (C). 16 H 29 O).

[0140] The other component was named ECB-5, with an m / z of 1076.5755 [M+H]. + And m / z 1098.5571[M+Na] +The diagnostic ions (m / z 566.2450 and 548.2363) of (AA3+AA4+AA5+AA6) are 16 Da higher than those of ECB, suggesting an additional OH group. Furthermore, the daughter ion (Medhpro(AA6)) with m / z 145.09754 was not detected. A high-intensity daughter ion (m / z 161.0925) is present, 16 Da higher than AA6 in ECB, suggesting that the amino acid at position 6 is methylproline (4Medhpro) containing two hydroxyl groups. Mass spectrometry cannot definitively determine the position of the new hydroxyl group in AA6, but this can be confirmed using purified NMR.

[0141] Analysis of peak 5 (named ECB-6)

[0142] ECB-6 has the same parent ion as ECB, namely [M+H] at m / z 1060.5800. + [M+Na] with m / z 1082.5636 + And the parent ion [M+H] + During secondary mass spectrometry scanning, the daughter ion set generated using the dehydrated ion of ECB (m / z 1042.5743) as a precursor was identical to that of ECB, and also contained other characteristic ion sets identical to those of ECB. However, [M+H]... + The abundance of the ion peak is approximately [M+Na]. + The ion peak is 1%. This isomer of ECB is thought to undergo a ring-opening reaction at the hemiacetal amine between dhOrn and 4Me3Hyp under alkaline hydrolysis, forming a five-membered ring product. ECB-6 is based on the parent ion [M+Na]. + MS / MS detection results obtained during secondary mass spectrometry scanning, such as Figure 6 As shown, its fragmentation pathway is similar to that of ECB. Figure 5 Same, parent ion [M+Na] + The 2-hydroxy-2-(4-hydroxyphenyl)acetaldehyde that loses AA4 yields a daughter ion with m / z 930.5169 (relative abundance 23.18). Simultaneously, the daughter ion with m / z 930.5169 continuously loses acetaldehyde, yielding daughter ions with m / z 886.4911 (relative abundance 34.95%) and 842.4644 (relative abundance 40.88%).

[0143] Analysis of peak 6 (named ECB-7)

[0144] The parent ion of ECB-7 is [M+H]. + The m / z value is 1046.5653, [M+Na] + Its m / z is 1068.5477, and its molecular weight is 14 Da less than that of ECB, presumably due to the absence of one methylene group. The parent ion is [M+H].+ MS / MS test results as follows Figure 7 As shown in the results, the m / z values ​​of the (AA3+AA4+AA5) residue daughter ions (410.1553, 392.1456, 374.1355) and (AA5+AA6) daughter ion (232.1303) are also 14 Da smaller than those of the ECB ion. Meanwhile, similar to ECB, there are still high-intensity daughter ions with m / z values ​​of 145.0973 for AA6, 326.1353 and 308.1256 for (AA1+AA2+AA3), and 213.0872 and 195.0765 for (AA1+AA2), indicating that AA1, AA2, AA3, and AA6 have not changed. These data suggest that AA5 has been replaced by serine. Furthermore, the ECB-7 parent ion [M+Na]... + The ion m / z 916.4991 (relative abundance 34.92%), which differs from 1068.5477 by 152 Da, and the ion m / z 872.4771 (relative abundance 30.44%) after the removal of acetaldehyde, also exist, indicating that AA4 and ECB are the same. Figure 8 As shown.

[0145] The parent ion [M+H] obtained from peak 7 + (m / z 1046.5653), parent ion [M+Na] + The impurity (m / z 1068.5477) and its daughter ions (m / z 135.0809, 119.0859, 107.0858) are not associated with ECB and therefore are not analyzed.

[0146] Elucidation of peak 9 (containing 3 components, named ECB-8, ECB-9, and ECB-10)

[0147] The precursor ion of ECB-8 is [M+H]. + The m / z value is 1044.5846, [M+Na] +The m / z of this component is 1066.5658, suggesting that it has one less hydroxyl group than ECB. The characteristic ions (AA5+AA6) with m / z of 246.1448 and (AA1+AA2+AA3) with m / z of 326.1342 and 308.1241 are the same as those in ECB. However, the residues containing AA4 are all 16 Da less than those in ECB, for example, the (AA3+AA4+AA5+AA6) residues have m / z of 534.2575 and 516.2414; the (AA3+AA4+AA5) or (AA2+AA3+AA4) residues have m / z of 408.1743, 390.1646, and 372.1539; and the (AA3+AA4) residues have m / z of 307.1297 and 289.1174. Therefore, it can be inferred that AA4 is a monohydroxy high tyrosine (hhTyr), and ECB-8 should be ECC or its isomer.

[0148] The parent ion of ECB-9 is [M+H]. + The relative abundance at m / z 1014.5717 is approximately [M+Na]. + m / z 1036.5564, representing 1% of the relative abundance. In [M+H] + In this mode, the dehydration ion with m / z 996.5632 was selected as the precursor ion for secondary mass spectrometry scanning. Its MS / MS and fragmentation pathway were as follows: Figure 9 and Figure 10 The results show that the daughter ions (AA5+AA6) with m / z 246.1450, AA6 with m / z 145.0966, and AA3 with m / z 131.0810 are the same as those in ECB, confirming that the amino acids at positions 3, 5, and 6 are identical to those in ECB. Furthermore, the residues (AA3+AA4) with m / z 307.1276 and 289.1179 are 16 fewer than their corresponding counterparts in ECB. Since AA3 remains unchanged, AA4 is inferred to be hhTyr. Additionally, the residues (AA1+AA2+AA3) with m / z 296.1237 and 278.1126, and (AA1+AA2) with m / z 183.0764 are 30 fewer than their corresponding counterparts in ECB, leading to the conclusion that AA1 is hOrn and AA2 is Ser. Other characteristic ions, such as (AA1+AA2+AA3+AA4+AA5) with m / z of 590.2450, 572.2349, and 554.2224, and (AA3+AA4+AA5+AA6) with m / z of 552.2677, were further used to confirm the structure of this component named ECB-9.

[0149] In addition, Figure 9Ion pairs with a molecular difference of 14 Da compared to ECB-9 were found. For example, (AA3+AA4+AA5+AA6) has an m / z of 538.2487, while ECB-9 has an m / z of 552.2677; (AA5+AA6) has an m / z of 232.1287, while ECB-9 has an m / z of 246.1450; (AA1+AA2) has m / z of 197.0919 and 179.0797, while ECB-9 has an m / z of 183.0764. The presence of these ion pairs may suggest certain changes in the structural features or biosynthetic pathways of ECB-9 and its related compounds. It is inferred that an isomer of ECB-9, named ECB-10, exists at peak 9, with AA5 being converted to Ser. The cleavage pathway of ECB-10 is as follows... Figure 11 As shown, both ECB-9 and ECB-10 can produce characteristic ions with m / z values ​​of 394.1591 and 408.1748, respectively. In ECB-9, the ion with m / z 408.1748 belongs to (AA3+AA4+AA5), while the ion with m / z 394.1591 belongs to (AA2+AA3+AA4). In ECB-10, the assignments of these two ions are reversed. It is difficult to determine whether the hydroxyl group is located at the γ or δ position of hOrn or at the β or γ position of hhTyr. In this study, the γ position of hOrn and the β position of hhTyr are used as examples.

[0150] Analysis of peak 10 (named ECB-11)

[0151] The component at peak 10 has the same [M+H] composition as ECB-8. + (m / z = 1044.5875), [M+Na] + (m / z is 1066.5688) and diagnostic ions, therefore it was identified as an isomer of ECB-8 and defined as ECB-11.

[0152] Analysis of peak 11 (named ECB-12)

[0153] ECB-12 [M+H] + For m / z 1028.5903, [M+Na] + With an m / z of 1050.5735, its molecular weight is 32 Da smaller than ECB, presumably due to having two fewer hydroxyl groups. [M+H] +In this mode, the product ions obtained from the cleavage of the dehydrated ion m / z 1010.5842 show the same ions as ECB-10, such as (AA3+AA4) residues at m / z 307.1289 and 289.1185, (AA1+AA2+AA3) residues at m / z 310.1409 and 292.1303, and (AA1+AA2) residues at m / z 197.0928 and 179.0822. Therefore, ECB-12 has hOrn at the AA1 position and hhTyr at the AA4 position.

[0154] Analysis of peak 12 (named ECB-13)

[0155] The precursor ion of ECB-13 is [M+H]. + (m / z = 1012.5970) and [M+Na] + The m / z value of 1034.5786 indicates that the molecular weight of ECB-13 is 48 Da less than that of ECB, presumably due to the absence of three hydroxyl groups.

[0156] The precursor ion of ECB-13 is [M+H]. + Obtain as Figure 12 The MS / MS spectra shown and the corresponding mass spectrometry data shown in Table 4 indicate that the fragmentation pathways of both chemical structures match the daughter ion sets obtained from the secondary mass spectrometry scan. The two chemical structures and their fragmentation pathways are as follows: Figure 13 and 14 As shown.

[0157] Table 4

[0158]

[0159]

[0160] like Figure 12 and Figure 13 As shown, based on the relatively abundant fragment ion m / z 115.0869, AA3 was identified as proline, and AA4 was identified as dihydroxyhigh tyrosine (dhhTyr). This also allows for the analysis of related fragment ions of (AA2+AA3+AA4+AA5), (AA3+AA4+AA5), and (AA3+AA4). Therefore, if the chemical structure of ECB-13 is as follows... Figure 13 As shown, following this pyrolysis pathway is also reasonable.

[0161] like Figure 14As shown, the intensities of the daughter ion clusters m / z 605.4289 and its dehydrated ions, and 504.3812 and its dehydrated ions are significantly higher. The ions of m / z 605.4289 and 504.3812 differ by 101 Da, which corresponds to the molecular weight of the Thr residues. Meanwhile, the ion of m / z 706.4763 is 101 Da more abundant than that of m / z 605.4289. These results indicate that the daughter ion of m / z 706.4763 contains two Thrs residues. Furthermore, it is speculated that the ion of m / z 377.3170 is (AA1+FA), while the AA1 of m / z 115.0869 is ornithine (Orn). Therefore, the daughter ion at m / z 706.4763 is (AA1+AA2+AA5+AA6+FA) residues, which then lose Thr of AA2 or AA5, yielding m / z 605.4289 and m / z 504.3812. Figure 8 First, AA2 is lost, then AA5. The residues (AA2+AA3+AA4+AA5), (AA3+AA4+AA5), and (AA3+AA4) and their dehydrated ions help to deduce that AA4 is a monohydroxy homotyrosine (hhTyr). ECB-13 should be ECD or its isomer. Unlike other ECB components, fragment ions linking FA and AA1 were found in the MS / MS of ECB-13, and FA did not transfer to other amino acids (AAs). Therefore, the mechanism by which FA transfers to other amino acids requires further investigation.

[0162] As can be seen from the above, if only the parent ion [M+H] is considered... + Obtain as Figure 12 The MS / MS spectrum analysis showed two possible chemical structures, making it impossible to determine the chemical structure of ECB-13.

[0163] Next, based on [M+Na] + The fragmentation pathway determined the chemical structure of ECB-13. The parent ion of ECB-13, [M+Na], was also determined. + Perform a secondary mass spectrometry scan to obtain MS / MS spectra (e.g.) Figure 15 The mass spectrometry data of ECB-13 are shown in Table 5 below. The fragmentation pathway of ECB-13 is as follows: Figure 16 The parent ion is [M+Na]. + The resulting daughter ions, with m / z values ​​of 898.5282, 854.5018, and 810.4759, and relative abundances of 8.13%, 40.81%, and 100%, were easily identifiable. They correspond to ions that have lost 2-(4-hydroxyphenyl)acetaldehyde and acetaldehyde, confirming that AA4 of ECB-13 is hydroxyhigh tyrosine (hhTyr) and AA3 is 4-hydroxyproline (4Hyp).

[0164] Table 5

[0165]

[0166]

[0167] This invention establishes a system based on the parent ion [M+H]. + and [M+Na] + The chemical structural formulas of impurities in echinocandin B were analyzed using secondary mass spectrometry data. The mass spectrometry detection conditions of this invention were used to detect the parent ion [M+Na] of echinocandin B and its impurities. + At that time, there was a relatively high abundance of acetaldehyde and a neutral loss of 2-hydroxy-2-(4-hydroxyphenyl)acetaldehyde on AA4, which were easily and quickly identified. Although in the ECB parent ion [M+H] + Similar losses of 1-(4-hydroxyphenyl)ethane-1,2-diol (AA4) are also observed, but their relative abundance is low. The default integration threshold of the mass spectrometer needs to be increased to slightly below this daughter ion abundance to detect the characteristic daughter ion peak. This significantly increases the number of daughter ion sets, undoubtedly increasing the difficulty of chemical structure analysis. Furthermore, this characteristic is not obvious in the structure analysis of ECB-13, and there is no such characteristic daughter ion in ECB-7, making it unsuitable as an effective and easily identifiable feature for analyzing impurities in other unknown chemical structures. Additionally, in [M+H]... + During the fragmentation process, the FA linked to AA1 is transferred to other amino acid residues, while [M+Na]... + When used as a precursor, FA remains attached to AA1. These two dissociation modes are important for accurately determining the chemical structure of impurities and can mutually verify each other during structure identification. This invention identified 13 impurities of echinocandin B, including ECC, ECD (or their isomers), and several novel impurities: ECB-2, ECB-4, ECB-5, ECB-7, ECB-9, EA10, and ECB-12. These novel compounds demonstrate the diversity of ECB impurities, which may possess different antibacterial activities. In summary, this study provides an effective analytical method for identifying the chemical structures of impurities in echinocandin B or its parent nucleus, which can be used for production process monitoring, final product quality control, and potential new drug development.

[0168] It should be noted that the above embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for structural analysis of echinocandin B or its parent nucleus impurities, characterized in that, It includes the following steps: S1: Primary mass spectrometry scan of echinocandycin B or its parent nucleus yielded the parent ion [M+H]. + and the parent ion [M+Na] + ; S2: The precursor ion [M+H] for the echinocandin B or its parent nucleus. + and the parent ion [M+Na] + Secondary mass spectrometry scans were performed to obtain daughter ion sets I and II; S3: Perform the first-stage mass spectrometry scan and the second-stage mass spectrometry scan sequentially on the impurity to obtain daughter ion sets III and IV; wherein, daughter ion set III is the parent ion [M+H] of the impurity. + The daughter ion set obtained by performing a secondary mass spectrometry scan, wherein the daughter ion set IV is the parent ion [M+Na] of the impurity. + The set of daughter ions obtained by performing a secondary mass spectrometry scan; S4: Compare the sub-ion sets III and IV with the sub-ion sets I and II respectively to resolve the chemical structural formula of the impurity.

2. The structural analysis method as described in claim 1, characterized in that, The mass spectrometry range during the first-stage mass spectrometry scan is set to a mass-to-charge ratio of 500-1500. And / or, the mass spectrometry range during the secondary mass spectrometry scan is set to a mass-to-charge ratio of 50-1500; And / or, the primary mass spectrometry and the secondary mass spectrometry are performed using a quadrupole time-of-flight mass spectrometer.

3. The structural analysis method as described in claim 2, characterized in that, The parameter settings of the quadrupole time-of-flight mass spectrometer must satisfy one or more of the following conditions: (1) The atomizer temperature is 300-350℃, for example, 320℃; (2) The atomizing gas flow rate is 6-10 L / min, for example 8 L / min; (3) The spray pressure is 30-40 psi, for example 35 psi; (4) The sheath gas temperature is 300-400℃, for example 350℃; (5) The sheath flow rate is 8-15 L / min, for example 11 L / min; (6) The capillary pressure is 3-5kV, for example 3.5kV; (7) The nozzle pressure is 0.5-2kV, for example, 1kV; (8) The breaking voltage is 150-200V, for example 175V.

4. The structural analysis method as described in claim 1, characterized in that, The parent ion [M+H] + When performing a secondary mass spectrometry scan, the collision energy is 30-50 eV, for example, 35 eV, 40 eV or 45 eV; And / or, the parent ion [M+Na] + When performing a secondary mass spectrometry scan, the collision energy is 60-90 eV, such as 70 eV, 75 eV, 80 eV or 85 eV.

5. The structural analysis method as described in claim 1, characterized in that, The daughter ion set II includes daughter ion P, which is more abundant than the parent ion [M+Na] of the echinocandin B. + The molecular weight is less than 152 Da, indicating that the fourth amino acid of the echinosin B is dihydroxyhigh tyrosine; The daughter ion set IV includes daughter ion P', which is more than the parent ion [M+Na] of the impurity. + The molecular weight is less than 152 Da or 136 Da; when it is less than 152 Da, the fourth amino acid of the impurity is dihydroxyhigh tyrosine, and when it is less than 136 Da, the fourth amino acid of the impurity is monohydroxyhigh tyrosine.

6. The structural analysis method as described in claim 5, characterized in that, The sub-ion set II also includes sub-ions with a molecular weight 44 Da smaller than that of sub-ion P; The sub-ion set IV also includes sub-ions with a molecular weight 44 Da smaller than that of the sub-ion P'.

7. The structural analysis method according to any one of claims 1-6, characterized in that, The absolute value of the difference between the molecular weight of the impurity and the echinocanthoma B is 0, 2, 14, 16 or a multiple thereof or a sum of these values, for example 28, 30, 32 or 48. And / or, the impurities differ from the echinocandin B or its parent nucleus in the following chemical structures: different number of hydroxyl groups, different number of methyl or methylene groups, or different number of double bonds.

8. The structural analysis method according to any one of claims 1-6, characterized in that, The impurities in the echinocandin B include one or more of the following compounds:

9. The structural analysis method according to any one of claims 1-6, characterized in that, The impurities were separated from the echinocandin B or its parent nucleus by high performance liquid chromatography; The conditions for the high-performance liquid chromatography preferably satisfy one or more of the following conditions: (1) The high performance liquid chromatography column is an alkyl-bonded silica column, such as a C18 alkyl-bonded silica column; (2) The high performance liquid chromatography includes gradient elution using a mobile phase consisting of 0.01%-0.2% formic acid aqueous solution as mobile phase A and acetonitrile containing 0.01%-0.2% formic acid as mobile phase B. For example, mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is for example acetonitrile containing 0.1% formic acid. The preferred gradient elution program is 0–20 min 40% mobile phase B; 20–35 min 40–50% mobile phase B; 35–45 min 50–90% mobile phase B; 45–50 min 90% mobile phase B; 50–51 min 90–40% mobile phase B; 51–58 min 40% mobile phase B. (3) The detection wavelength of the high performance liquid chromatography is 190-280nm, preferably 210nm.

10. A related impurity in echinocandin B, characterized in that, The impurities in the echinocandin B include one or more of the following compounds:

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