Structural analysis method for impurities in pneumocandin B0 crude product

Abstract

The invention discloses a structural analysis method for impurities in a pneumocandin B0 crude product. The structural analysis method comprises the following steps: (1) separating a pneumocandins B0 crude product to obtain pneumocandins B0 and a plurality of impurities; (2) respectively carrying out primary mass spectrum detection and secondary mass spectrum detection on pneumocandin B0 and each impurity to obtain mass spectrum data; (3) determining a cracking mode according to the mass spectrum data of the pneumocandin B0; (4) analyzing a chemical structural formula by taking a splitting decomposition mode of pneumocandin B0 as a reference and combining mass spectrum data obtained by secondary mass spectrum detection of each impurity; in the step (2), the pneumocandin B0 is subjected to secondary mass spectrometry detection with different collision energy for more than two times, and the collision energy is 20-45 eV. According to the structural analysis method disclosed by the invention, a plurality of new impurities are analyzed by utilizing mass spectrum data obtained by secondary mass spectrum detection, the analysis process is simple and efficient, and the structural analysis method has important significance on quality control of pneumocandin B0.

Description

TECHNICAL FIELD

[0001] The application relates to a method for analyzing the structure of impurities in crude Pneumocandin B0. BACKGROUND

[0002] Human invasive fungal infection is usually associated with high mortality, so the selection of antifungal drugs is crucial for patients. Pneumocandins are a class of antifungal lipopeptides produced by the filamentous fungus Glarea lozoyensis (formerly Zalerion arboricola), which has a high bactericidal effect on Candida albicans. Since this class of compounds exerts a pharmacodynamic effect by inhibiting the synthesis of beta-(1, 3)-D-glucan, it is not easy to develop drug resistance. Merk company synthesized caspofungin by modifying the side chain on the basis of retaining the cyclic hexapeptide mother nucleus of Pneumocandin B0 (PB0), thereby improving its antibacterial activity and reducing hemolysis and toxicity to mammalian cells. It was approved for marketing by FDA in 2001.

[0003] The structural formula of Pneumocandin B0 and its analogues is as follows:

[0004]

[0005] According to the structural formula of Pneumocandin B0, from the N-terminal, there are 4R, 5R-dihydroxy-L-ornithine (AA1), 2S, 3R-L-threonine (AA2), 4R-hydroxy-L-proline (AA3), 3S, 4S-dihydroxy-L-homotyrosine (AA4), 3R-hydroxy-L-glutamine (AA5), and 3S-hydroxy-L-proline (AA6). The 5-amino group of AA1 and the carboxyl group of AA6 form a ring through an amide bond (from the perspective of AA1, this is a hemiaminal structure), and the fatty acyl chain (FA) connected to the 2-amino group is a 10, 12-dimethyl myristoyl group. PB0 is an intermediate of caspofungin acetate and is one of the important raw materials in current biological and pharmaceutical products.

[0006] In addition to Pneumocandin B0, Glarea lozoyensis also produces other secondary metabolites with similar structures during fermentation. For example, when the number and position of the hydroxyl groups on AA1 are different, PB2, PB5, and PB6 components are derived; when AA2 is converted into serine, PB0 serine analogues are produced; when AA4 has no hydroxyl group at the 4 position, PB1 is produced; and when AA6 is 4-methyl-3-hydroxyproline, other Pneumocandin A compounds are produced. Zhang Hongzhi et al. published an article entitled “Preparation of high performance liquid chromatography for purification of Pneumocandin impurities B1 and B5” in Chinese Journal of Antibiotics, which basically lists the currently identified impurities.

[0007] The structure of PB0 was determined by comparing NMR and mass spectrometry data with that of Numocodin A0, while its stereoconfiguration was confirmed by X-ray diffraction, amino acid derivatization, and comparison with chemically synthesized fatty acids. Currently, the structure of Numocodin components is mainly identified through separation and preparation followed by one-dimensional and two-dimensional NMR techniques. For example, Zeng Fanzhou et al. prepared and confirmed Numocodin CO, Sun Xinqiang et al. identified the serine analogue of PB0 (i.e., AA2 converted from threonine to serine), and Zhang Hongzhi et al. confirmed PB1 and PB5. Under alkaline conditions, the 2-amino group of AA1 attacks the semiamine aldehyde, thereby opening the parent nucleus to form 4,5-dihydroxyproline, yielding PB6b.

[0008] In the aforementioned prior art, the chemical formulas of the impurities in Numocontin B0 were mainly determined by nuclear magnetic resonance (NMR), with mass spectrometry used as an auxiliary means to confirm molecular weight. NMR determination of the structure of unknown compounds requires the preparation of a certain amount of high-purity impurities to be tested, and the functional groups where H or C are located are also identified one by one based on the chemical shifts of the NMR detection results. This is extremely difficult for compounds with complex structures. In addition, the preparation of a certain amount of high-purity impurities is not only time-consuming but also consumes a large amount of solvent.

[0009] The applicant's published strategy for resolving the core structure of echinocandin B based on high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (HPLC-QQMS) identified four impurities in echinocandin B using mass spectrometry data. Compared to echinocandin B, nemocontin B0 has a more complex structure and a larger molecular weight, making mass spectrometry analysis of impurities in nemocontin B0 more challenging.

[0010] Although many impurities in Numocontin B0 have been identified, numerous unknown impurities remain to be discovered. Without understanding the structural formulas of these impurities, the therapeutic efficacy of caspofungin may be affected, and serious side effects may occur. Therefore, rapidly confirming the chemical structural formulas of the various impurities in Numocontin B0 is a pressing technical problem that needs to be solved. Summary of the Invention

[0011] To overcome the shortcomings of existing methods for structural analysis of new impurities in crude numococcal B0, which are difficult, time-consuming, and require significant solvent input, this invention provides a method for structural analysis of impurities in numococcal B0. This invention first separates multiple impurities from numococcal B0, and then successfully resolves the chemical structures of new impurities in numococcal B0 based on characteristic ion data obtained through mass spectrometry.

[0012] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0013] One aspect of the present invention provides a method for structural analysis of impurities in crude Numococtin B0, which includes the following steps:

[0014] (1) The crude Numocontin B0 was separated to obtain Numocontin B0 and several impurities;

[0015] (2) The Numocontin B0 and each of the impurities were detected by first-stage mass spectrometry and second-stage mass spectrometry to obtain mass spectrometry data;

[0016] (3) Determine the fragmentation mode of the nimococtin B0 based on the mass spectrometry data of the nimococtin B0;

[0017] (4) Using the cleavage mode of the aforementioned Numocontin B0 as a reference, the chemical structural formula of each of the impurities is determined by combining the mass spectrometry data of each of the impurities in step (2).

[0018] In step (2), the nimocontin B0 is subjected to secondary mass spectrometry detection at two or more different collision energies, the collision energies being 20-45 eV.

[0019] In this invention, the purity of the crude Numococtin B0 is preferably 80%-95% or higher, for example, 90%. This purity is determined by HPLC, and the mass percentage of the analyte is calculated using area normalization. This method can only detect impurities detectable by HPLC; impurities not detectable by this method are not included. The crude Numococtin B0 can be prepared using conventional methods in the art or can be commercially available.

[0020] In this invention, the separation is preferably performed using high performance liquid chromatography.

[0021] The high-performance liquid chromatography (HPLC) method preferably employs gradient elution. Gradient elution refers to a process in which the composition of a mobile phase containing two or more solvents of different polarities changes continuously or intermittently during elution; gradient elution can be linear or non-linear. Conversely, during isocratic elution, the composition of the chromatographic mobile phase remains constant. In this invention, the gradient elution is preferably a linearly changing gradient elution.

[0022] As those skilled in the art will know, the mobile phase of the high-performance liquid chromatography (HPLC) typically includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous phase and mobile phase B is an organic phase, respectively. Mobile phase A is preferably a 0.05-0.2% (v / v) aqueous solution of formic acid, for example, a 0.1% (v / v) aqueous solution of formic acid. Mobile phase B is preferably acetonitrile. The preferred elution conditions for the HPLC are as follows:

[0023] During the 0-20 min period, the mobile phase consists of 60% by volume mobile phase A and 40% by volume mobile phase B;

[0024] During the period of 20-35 minutes (excluding 20 minutes), the mobile phase consists of 50-60% by volume mobile phase A and 40-50% by volume mobile phase B.

[0025] During the period of 35-40 minutes (excluding 35 minutes), the mobile phase consists of 10-50% by volume of mobile phase A and 50-90% by volume of mobile phase B.

[0026] The total volume of the mobile phase remains at 100% as it changes over the corresponding time interval.

[0027] The flow rate of the mobile phase is preferably 0.1-1 mL / min, and more preferably 0.5 mL / min.

[0028] The chromatographic column used in the high-performance liquid chromatography (HPLC) method is preferably a C18 column, specifically a Diamonsil Plus C18 column in this embodiment. The Diamonsil Plus C18 column has dimensions of 5 μm and 4.6 × 250 mm.

[0029] When performing the high performance liquid chromatography method, the injection volume of the crude Numocontin B0 can be as small as possible to meet the subsequent mass spectrometry detection, without special limitation. It can be less than 10 μL but not 0 μL, for example 2 μL, 3 μL or 5 μL.

[0030] In performing the high-performance liquid chromatography (HPLC) method, the column temperature can be conventional in the art, generally below 50°C, for example, 40°C. In this invention, the ultraviolet detection wavelength can be conventional in the art, generally approximately 210 nm.

[0031] In this invention, the detection conditions of the primary mass spectrometer can be conventional in the art, as long as the molecular weight of the nimococtin B0 and each of the impurities can be obtained.

[0032] In this invention, the scanning range of the primary mass spectrometer can be 150-2000 m / z.

[0033] In this invention, during step (2) of the secondary mass spectrometry detection of the nimocotin B0, the difference between the two or more different collision energies is between 5-20 eV, for example, 10 eV or 15 eV. Preferably, the collision energy of one secondary mass spectrometry detection is 25-35 eV, for example, 30 eV; and the collision energy of another secondary mass spectrometry detection is preferably 35-45 eV, for example, 40 eV.

[0034] In this invention, in step (2), each impurity is preferably detected by secondary mass spectrometry at two or more different collision energies, wherein the collision energy is preferably 20-45 eV. The difference between the two or more different collision energies is preferably between 5-20 eV, for example, 10 eV or 15 eV. The collision energy of one secondary mass spectrometry detection is preferably 25-35 eV, for example, 30 eV; the collision energy of the other secondary mass spectrometry detection is preferably 35-45 eV, for example, 40 eV.

[0035] In this invention, the detection conditions of the secondary mass spectrometer preferably also satisfy one or more of the following conditions:

[0036] (1) The atomizer temperature is 300-350℃, for example, 320℃;

[0037] (2) The atomizing gas flow rate is 6-10 L / min, for example 8 L / min;

[0038] (3) The spray pressure is 30-40 psi, for example 35 psi;

[0039] (4) The sheath gas temperature is 300-400℃, for example 350℃;

[0040] (5) Sheath flow rate 8-15 L / min, for example 11 L / min;

[0041] (6) The capillary voltage is 3-5kV, for example 3.5kV;

[0042] (7) The nozzle voltage is 0.5-2kV, for example, 1kV;

[0043] (8) The capillary outlet pressure is 150-200V, for example 175V.

[0044] In a specific embodiment of the present invention, the primary mass spectrometer and the secondary mass spectrometer are quadrupole time-of-flight tandem mass spectrometers.

[0045] This invention first optimizes the elution conditions of high-performance liquid chromatography (HPLC) to separate Numococcal B0 and several impurities from crude Numococcal B0. In a specific embodiment, 11 impurities were separated. Furthermore, the detection conditions, such as collision energy, were screened for secondary mass spectrometry (MS / MS), and a suitable MS / MS detection method was determined. Two MS / MS tests with different collision energies were performed to obtain specific ion peaks and their information. Based on these ion peaks and their information, multiple fragmentation pathways of Numococcal B0 were successfully plotted. It was discovered that during the fragmentation process, the fatty acyl chain (FA) of Numococcal B0 can transfer from AA1 dihydroxyornithine to other amino acids, and the component substituted with monohydroxyornithine can form an imine ion. This provides a clear reference for the chemical structural analysis of the 11 separated impurities. Unexpectedly, eight new impurities were discovered that had not been previously disclosed in reports related to Numococcal B0 impurity research, constituting an innovative invention with significant implications for the quality control of Numococcal B0.

[0046] In this invention, in step (3), the fragmentation method refers to the fragmentation of the parent ion of Numococtin B0 into daughter ions during the secondary mass spectrometry detection. The fragmentation method is to deduce the breaking sites of the chemical bonds in Numococtin B0 and the structural formula after the breaking by using the molecular weight of the daughter ions obtained by the secondary mass spectrometry detection. Thus, the fragmentation pathway from Numococtin B0 to fragmentation into daughter ions is drawn, which serves as a clear reference for resolving the chemical structural formula of unknown impurities.

[0047] In a specific embodiment of the present invention, based on the mass spectrometry data obtained from the secondary mass spectrometry detection of the Numococtin B0, its fragmentation modes are mainly identified as including the following six types:

[0048] Cleavage method 1: Numocontin B0 is dehydrated and loses FA, but retains the cyclic peptide structure of Numocontin B0;

[0049] Cleavage method 2: Cleavage of high tyrosine residues in Numocontin B0, but retaining the cyclic peptide structure of Numocontin B0;

[0050] Cleavage method three: The removal of AA1 and AA2 from the nemocodin B0 causes the cyclic peptide structure of the nemocodin B0 to cleave, and FA is transferred to other amino acids;

[0051] Cleavage method four: Numocontin B0 loses (AA6+FA+H2O+NH3) to obtain the cleaved cyclic peptide structure;

[0052] Cleavage method 5: Numocontin B0 removes (AA1+AA6+FA) to obtain the cleaved cyclic peptide structure;

[0053] Cleavage method six: Numocontin B0 removes (AA6+H2O+NH3) to obtain the cleaved cyclic peptide structure.

[0054] In this invention, the removal of “FA”, “AA1”-“AA6”, “H2O”, and “NH3” indicates the removal of the corresponding groups from Numococtin B0 or ​​its impurities. FA represents a fatty acyl chain group, and the molecular formula of Numococtin B0 is C0. 16 H 31 O. Wherein, “AA1”-“AA6” represent amino acids at positions 1-6. In Numocontin B0, AA1 represents 4R,5R-dihydroxy-L-ornithine residue; AA2 represents 2S,3R-L-threonine residue; AA3 represents 4R-hydroxy-L-proline residue; AA4 represents 3S,4S-dihydroxy-L-high tyrosine residue; AA5 represents 3R-hydroxy-L-glutamine residue; and AA6 represents 3S-hydroxy-L-proline.

[0055] The first fragmentation method is derived as follows: Based on the molecular weight of the daughter ion (1047.56) obtained by secondary mass spectrometry and the daughter ions (1029.55, 1011.54, and 993.53) after continuous dehydration, it can be inferred that Numocontin B0 will first undergo dehydration. Based on the molecular weight of the daughter ion (791.32) obtained by secondary mass spectrometry and the daughter ions (773.31 and 755.30) after continuous dehydration, it can be inferred that Numocontin B0 will lose one molecule of water and then the FA group.

[0056] The second fragmentation method was derived as follows: Based on the molecular weight fragment 911.51 obtained by secondary mass spectrometry detection, and the fragment ions 893.50 and 875.49 after continuous dehydration, it can be concluded that Numocontin B0 loses a group with a molecular weight of 154. This is consistent with the composition of Numocontin B0. Group matching.

[0057] The third fragmentation method is derived as follows: The molecular weight of the fragment ion obtained by secondary mass spectrometry is 835.48, along with the fragment ions 817.47 and 799.46 after continuous dehydration. The molecular weight difference between 835.48 and Numocontin B0 indicates that fragment ion 835.48 is an ion that has lost AA1 and AA2 but retains the fatty acyl chain on AA1. Therefore, it can be determined that FA has transferred to other amino acids. According to the structural formula, the amino acid accepting FA is AA3, and AA6 is a 3-hydroxyproline amidated residue. Next, the fragment ion represented by 835.48 will undergo FA loss and continuous dehydration, and AA6 loss and continuous dehydration, ultimately resulting in further fragmentation to obtain low molecular weight fragment ions such as AA3, AA6, and (AA5+AA6).

[0058] The fourth fragmentation method was derived through the following process: the secondary mass spectrometry detected a fragment ion with a molecular weight of 679.26 and fragment ions 661.25, 643.24, and 625.22 after continuous dehydration. The molecular weight difference between 679.26 and Numocontin B0 is 386, suggesting that the removed ion is (AA6+FA+H2O+NH3). Furthermore, 679.26 is exactly the molecular weight of the (AA1-AA5) ion, verifying this deduction. Simultaneously, the secondary mass spectrometry also detected low molecular weight fragment ions 326.13, 215.10, and 213.09, which match the molecular weights of the (AA1+AA2+AA3), (AA2+AA3), and (AA1+AA2) ions, respectively.

[0059] Among them, the fifth fragmentation method is derived through the following process: the secondary mass spectrometry detection obtains a daughter ion with a molecular weight of 568.22 and daughter ions 550.21 and 532.20 after continuous dehydration. Based on the molecular weight difference between daughter ion 568.22 and Numocontin B0, it can be known that the characteristic ion represented by 568.22 is the ion (AA2-AA5) after the removal of (AA1+AA6) and FA.

[0060] Among them, the sixth fragmentation method is derived through the following process: the secondary mass spectrometry detection obtained a daughter ion with a molecular weight of 917.49 and daughter ions 899.48, 881.46 and 863.45 after continuous dehydration. It can be seen that the daughter ion represented by 917.49 is the ion after the removal of (AA6+H2O+NH3).

[0061] In this invention, the number of the plurality of impurities is preferably two or more, for example, four, eight or eleven.

[0062] In this invention, based on the fragmentation mechanism of the nimocodin B0, and combined with the primary and secondary mass spectrometry detection results of the impurities, it can be confirmed that several of the impurities preferably include one or more of the following compounds:

[0063] (1) Compound of formula PB0-1 and / or isomers of compound of formula PB0-1:

[0064] Wherein, the isomers of the compound of formula PB0-1 have the same chemical structure as the compound of formula PB0-1 at -C 15 H 29 The double bonds in the groups are in different positions or configurations; in a specific embodiment of the present invention, the isomers of the compound of formula PB0-1 include two, named PB0-2 and PB0-5 respectively;

[0065] (2) Compound of formula PB0-3:

[0066]

[0067] (3) Compound of formula PB0-4 or compound of formula PB0-4':

[0068]

[0069] (4) Compound of formula PB0-6:

[0070]

[0071] (5) Compounds of formula PB0-7 or PB0-7':

[0072]

[0073] (6) Compound PB0-8:

[0074]

[0075] (7) Compound of formula PB0-9 and / or isomers of compound of formula PB0-9:

[0076]

[0077] The chemical structures of compound PB0-1 and its isomers PB0-2 and PB0-5 were confirmed by performing primary and secondary mass spectrometry on the isolated PB0-1, PB0-2, and PB0-5. Primary mass spectrometry determined the molecular weight of the impurity to be 1063.55, which is 2 less than that of Numocontin B0. In the molecular weight dataset of the daughter ions obtained by secondary mass spectrometry, the molecular weights of the daughter ions after removing FA and different small peptides are basically consistent with the molecular weight dataset of the corresponding daughter ions of Numocontin B0; however, the molecular weights of the daughter ions containing FA (915.47, 909.49, 815.45, 703.39) and their dehydrated daughter ions are all 2 less than the molecular weights of the daughter ions containing FA in Numocontin B0, indicating that the fatty acyl chain contains a double bond.

[0078] The chemical structure of compound PB0-3 was confirmed by performing primary and secondary mass spectrometry on the isolated PB0-3. Primary mass spectrometry determined the molecular weight of the impurity to be 1051.55, which is 14 less than that of Numocontin BO, indicating the absence of a methylene group. Secondary mass spectrometry analysis revealed that the molecular weight of the daughter ions was 14 less than that of PB0, compared to corresponding characteristic ions of FA. For example, the ions after the loss of (AA1+AA2) and FA transfer were 821.46, the ions after the removal of high-tyrosine residues were 897.48 and 879.48, and the daughter ions after the removal of AA6 were 903.47 and 885.46, indicating that the molecular formula at the FA end is C. 15 H 29 O.

[0079] The chemical structure of compound PB0-4 or PB0-4' was confirmed by performing primary and secondary mass spectrometry on the isolated PB0-4 or PB0-4'. Primary mass spectrometry determined the molecular weight of the impurity to be 1065.57, the same as that of Numocodin B0. Secondary mass spectrometry revealed the following fragments: ion 597.25 representing (AA3+AA4+AA5+AA6) and its dehydrated fragments; ion 467.18 representing (AA3+AA4+AA5) and its dehydrated fragments; and ion 568.22 representing (AA2+AA3+AA4+AA5) and its dehydrated fragments. These fragments were essentially consistent with the secondary mass spectrometry results for Numocodin B0, indicating that the structure of the five amino acids AA2-AA6 in PB0-4 or PB0-4' remained unchanged, meaning the AA1 structure was altered. Unlike the secondary mass spectrometry results of Numocodin B0, the secondary mass spectrometry results of formula PB0-4 or PB0-4' include 793.34 and its dehydrated ion, which has a molecular weight 2 greater than the 791.34 fragment ion after dehydration and FA removal in fragmentation mode one of Numocodin B0. This is because the AA1 structure in formula PB0-4 or PB0-4' changes, resulting in a molecular weight increase of 2, presumably due to the conversion of dihydroxyornithine to monohydroxyornithine. Furthermore, the secondary mass spectrometry results of formula PB0-4 or PB0-4' include a series of fragment ions with a molecular weight one larger than that of 911.52 and its dehydrated ion (after the removal of high tyrosine residues) in Numocodin B0, as well as 917.48 and its dehydrated ion (after the removal of the (AA6+NH3+H2O) characteristic ion), indicating that the fatty acyl chain contains one hydroxyl group.

[0080] The chemical structure of compound PB0-6 was confirmed by performing primary and secondary mass spectrometry on the isolated compound PB0-6. The primary mass spectrometry determined the molecular weight of the impurity to be 1051.56, which is 14 less than that of nimocontin B0, suggesting the absence of a methylene group. Based on the chemical structure of nimocontin B0, the site where a methylene group is lost is typically FA or threonine at position 2. The molecular weight data of the fragment ions obtained from the secondary mass spectrometry analysis, comparing the fragment ions 777.30 and 759.29 after the loss of FA with the characteristic ion 199.07 (AA1+AA2), suggests that the threonine at position 2 is converted to serine. Furthermore, the fragment ions 817.47 and 687.40 after FA transfer are essentially identical to the relevant ions of PB0, indicating no change at the FA end. Therefore, the difference between compound PB0-6 and PB0 is that the threonine at position 2 has lost a methylene group.

[0081] The chemical structure of compound PB0-7 or PB0-7' was confirmed by performing primary and secondary mass spectrometry on the isolated compound PB0-7 or PB0-7'. The primary mass spectrometry determined the molecular weight of the impurity to be 1049.58, which is 16 smaller than that of PB0. In the molecular weight data of the daughter ions obtained by the secondary mass spectrometry, the impurity's (AA3+AA4+AA5+AA6) ion (597.25), (AA3+AA4+AA5) ion (467.18), its dehydration ions, and the corresponding FA transfer ions (835.48, 817.47, 799.46, etc.) are essentially consistent with the relevant ions of PB0, indicating that the four amino acids AA3-AA6 and FA have not changed. The characteristic ions of (AA1+AA2+AA3+AA4+AA5) are 663.27, 645.25, and 627.24, and the ions of (AA1+AA2+AA3) are 310.14 and 292.13, and the ions of (AA1+AA2) are 197.09 and 179.08. Therefore, it can be confirmed that the compound PB0-7 or PB0-7' differs from PB0 in that the amino acid at position 1 is changed from 4,5-dihydroxyornithine to 4 or 5-hydroxyornithine.

[0082] The chemical structure of compound PB0-8 was confirmed as follows: Primary mass spectrometry analysis of the isolated PB0-8 determined its molecular weight to be 1063.56, which is 2 less than that of nimocontin B0. Secondary mass spectrometry analysis of this component yielded the following fragments: (AA3+AA4+AA5+AA6) fragment ion 597.25 and its dehydrated fragment ion; (AA3+AA4+AA5) fragment ion 467.18 and its dehydrated fragment ion; FA transfer ion 817.47; and (AA2+AA3+AA4+AA5) fragment ion 568.22 and its dehydrated fragment ion. These fragments are essentially consistent with those detected in nimocontin B0, indicating that the five amino acids AA2-AA6 and FA remained unchanged. This suggests that a change in the amino acid at position AA1 resulted in a molecular weight PB0-8 that is 2 less than that of nimocontin B0. Secondary mass spectrometry analysis of this component also revealed a daughter ion 211.07 with the same molecular weight as (AA1+AA2) and its dehydrated daughter ion, which is 2 less than (AA1+AA2) in Numocontin B0. Combined with the properties of the structural formula itself, it can be confirmed that 4,5-dihydroxyornithine is converted into 4-carbonyl-5-hydroxyornithine.

[0083] The chemical structures of compound PB0-9 and its isomers PB0-10 and PB0-11 were confirmed as follows: Primary mass spectrometry analysis of the isolated compound PB0-9 and its isomers revealed a molecular weight of 1049.58, which is 16 smaller than that of nimocontin B0. Secondary mass spectrometry analysis of this fraction showed the presence of (AA2+AA3+AA4+AA5) ion 568.23 and its dehydrated ion, and (AA3+AA4+AA5) ion 467.18 and its ion, indicating that the structural formulas of the four amino acids AA2-AA5 remained unchanged. The absence of (AA5+AA6) ion 275.14 and related ions of (AA3+AA4+AA5+AA6) in the secondary mass spectrometry analysis indicates that the amide bond at AA5 and AA6 breaks, leading to the ring-opening of the cyclic peptide and the formation of a linear lipopeptide. The secondary mass spectrometry results of this component showed the presence of newly emerging, highly abundant fragment ions 583.41 and 482.36, as well as a dehydrated fragment ion, which were not present in other components. Furthermore, the molecular weight difference between fragment ions 583.41 and 482.36 is 101, meaning they differ by one threonine residue. Therefore, fragment ion 583.41 is inferred to be a fragment ion of (AA1+AA2+AA6+FA), and 482.36 is also a fragment ion of (AA1+AA6+FA). Based on the structural formula of PB0, the lone pair electrons on the amino group at AA1 readily attack the carbon atom connecting the hydroxyl group to AA1, causing AA1 to transform from hydroxylated ornithine to 5-aminoproline. This ultimately affects the stability of the amide bond at AA5 and AA6, leading to its breakage, thus resolving the impurities in the compound shown in formula PB0-9.

[0084] In a specific embodiment of the present invention, when performing the secondary mass spectrometry detection, the mass spectrometry data of Numococtin B0 includes a set of daughter ions with molecular weights of 1047.56, 791.32, 911.51, 835.48, 597.25, 705.41, 467.18, 275.14, 131.08, 86.06, 679.26, 568.22, 326.13, 215.10, 213.09, 917.49, and 319.09. Preferably, the molecular weight dataset of the daughter ions of Numococtin B0 also includes the molecular weights after one, two, or three dehydration cycles of the aforementioned daughter ions.

[0085] In a specific embodiment of the present invention, during the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-1 includes a set of daughter ions with molecular weights of 915.47, 909.49, 815.46, and 703.39. Preferably, the mass spectrometry data of the compound of formula PB0-1 also includes the molecular weights of the aforementioned daughter ions after one, two, or three dehydration processes. In a specific embodiment of the present invention, the mass spectrometry data of the isomers of the compound of formula PB0-1 are the same as those of the compound of formula PB0-1.

[0086] In a specific embodiment of the present invention, when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-3 includes a set of daughter ions with molecular weights of 821.46, 897.48, 879.48, 903.47, and 885.46. Preferably, the mass spectrometry data of the compound of formula PB0-3 also includes the molecular weights of the aforementioned daughter ions after one, two, or three dehydration processes.

[0087] In a specific embodiment of the present invention, when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-4 or the compound of formula PB0-4' includes a set of daughter ions with molecular weights of 597.25, 467.18, 568.22, 793.34, 663.26, 215.10, 131.08, 113.07, 911.52, and 917.48. Preferably, the mass spectrometry data of the compound of formula PB0-4 or the compound of formula PB0-4' further includes the molecular weight after the aforementioned daughter ions have undergone one, two, or three dehydration processes.

[0088] In a specific embodiment of the present invention, when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-6 includes a set of daughter ions with molecular weights of 777.30, 199.07, 817.47, and 687.40. Preferably, the mass spectrometry data of the compound of formula PB0-6 also includes the molecular weights of the aforementioned daughter ions after one, two, or three dehydration processes.

[0089] In a specific embodiment of the present invention, when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-7 or the compound of formula PB0-7' includes a set of daughter ions with molecular weights of 597.25, 467.18, 835.48, 663.27, 310.14, and 197.09. Preferably, the mass spectrometry data of the compound of formula PB0-7 or the compound of formula PB0-7' further includes the molecular weight of the aforementioned daughter ions after one, two, or three dehydration processes.

[0090] In a specific embodiment of the present invention, when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-8 includes a set of daughter ions with molecular weights of 597.25, 467.18, 817.47, 568.22, 659.22, 324.12, and 211.07. Preferably, the mass spectrometry data of the compound of formula PB0-8 also includes the molecular weights of the aforementioned daughter ions after one, two, or three dehydration processes.

[0091] In a specific embodiment of the present invention, during the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-9 includes a set of daughter ions with molecular weights of 568.23, 467.18, 583.41, 482.36, 895.51, and 930.52. The secondary mass spectrometry data of the compound of formula PB0-9 does not include daughter ions with molecular weights of 275.1 and 597.3 and their dehydrated ions. Preferably, the mass spectrometry data of the compound of formula PB0-9 also includes the molecular weights of the aforementioned daughter ions after one, two, or three dehydrations. In a specific embodiment of the present invention, the mass spectrometry data of the isomers of the compound of formula PB0-9 are the same as those of the compound of formula PB0-9.

[0092] 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.

[0093] The positive and progressive effects of this invention are as follows: By using specific mass spectrometry detection conditions, the fragmentation pattern of nimocodin B0 is plotted. Based on the fragmentation pattern of nimocodin B0 and the primary and secondary mass spectrometry data of the separated impurities, multiple new impurities are analyzed, which is of great significance for the quality control of nimocodin B0. Attached Figure Description

[0094] Figure 1 The TIC chromatogram is shown for the crude Numocontin B0 solution in Example 1.

[0095] Figure 2 The first-order mass spectrum of Numocontin B0 isolated in Example 1.

[0096] Figure 3The secondary mass spectrum of Numocontin B0 obtained in Example 1, with a secondary detection condition of 30 eV.

[0097] Figure 4 The secondary mass spectrum of Numocontin B0 obtained in Example 1, with a secondary detection condition of 40 eV.

[0098] Figure 5 The secondary mass spectrum of Numocontin B0 obtained in Example 1, with a secondary detection condition of 50 eV.

[0099] Figure 6 The cleavage pathway of the nimocontin B0 isolated in Example 1.

[0100] Figure 7 The cleavage pathway of PB0-4 isolated in Example 1.

[0101] Figure 8 The pyrolysis pathway of PB0-8 isolated in Example 1.

[0102] Figure 9 The cleavage pathway of PB0-9 isolated in Example 1. Detailed Implementation

[0103] 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.

[0104] The following are definitions of some terms used in this application.

[0105] 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.

[0106] In this article, the term "mass spectrometry" refers to an analytical technique that ionizes a chemical substance and sorts it according to its mass-to-charge ratio. First-order mass spectrometry involves ionizing the chemical substance to produce charged ions and measuring the mass-to-charge ratio of these charged ions. Second-order mass spectrometry typically involves ionizing the chemical substance to produce charged ions, selecting a precursor ion and causing collision-induced dissociation to produce daughter ions, and measuring the mass-to-charge ratio of these daughter ions.

[0107] In this article, the term "daughter ion," also known as "characteristic ion," refers to fragment ions generated during the mass spectrometry analysis process. In secondary mass spectrometry analysis, there is a clear relationship between the parent ion generated during ionization and the daughter ions generated during dissociation, as well as the measured ion spectral peaks.

[0108] 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.

[0109] The instruments and reagents used in the following examples were: Agilent 1290 high-performance liquid chromatograph-6545Q / TOF-MS; Milli-Q water purification system (Millipore, Bedford, MA). Numococtin B0 crude product (HPLC purity 95%). Formic acid (LC-MS grade) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., acetonitrile (LC-MS grade) was purchased from Sigma-Aldrich Reagent Co., Ltd., and ultrapure water (prepared using a Milli-Q system).

[0110] Example 1

[0111] The crude nimocontin B0 was separated by chromatography and detected by mass spectrometry using an Agilent 1290 high performance liquid chromatograph-6545Q / TOF-MS tandem instrument.

[0112] For HPLC separation, 0.1% formic acid aqueous solution was selected as mobile phase A, and acetonitrile as mobile phase B, with gradient elution: 0-20 min, mobile phase B: 40%; 20-35 min, mobile phase B: 40%-50%; 35-45 min, mobile phase B: 50%-90%; 45-50 min, mobile phase B: 90%; 50-51 min, mobile phase B: 90%-40%; 51-58 min, mobile phase B: 40%; flow rate: 0.5 mL / min (split before mass spectrometry detection, split ratio 2:1). A Diamonsil Plus C18 column (5 μm, 4.6 × 250 mm) was used, with a column temperature of 40℃. The injection volume was 2 μL, and the UV detection wavelength was 210 nm. The results obtained by HPLC detection in tandem with TOF-MS are as follows: Figure 1 The TIC diagram shown.

[0113] like Figure 1 As shown, the above HPLC conditions can effectively separate Numococtin B0 and several of its impurities. Figure 1 Peak 7, as indicated in the text, is Numocontin B0. The other peaks 1-6 and 8-12 represent different impurities.

[0114] Mass spectrometry detection of Numocontin B0: Q / TOF-MS was performed in +ESI scan mode. Other parameter settings were as follows: nebulizer temperature (Gas Temp) was set to 320℃, nebulizer flow rate (Gas Flow) was 8 L / min, nebulizer pressure (Nebulizer) was 35 psi, sheath gas temperature (Sheath Gas Temp) was 350℃, sheath gas flow rate (Sheath Gas Flow) was 11 L / min, capillary voltage (Vcap) was 3.5 kV, nozzle voltage (Nozzle Voltage) was 1.0 kV, and capillary outlet voltage (Fragmentor) was 175 V. The mass spectrometer required tuning before use. During the scan, a reference ion of m / z 922.0098 was selected for real-time calibration. The primary mass spectrometry scan range was m / z 150-2000. The collision energies for secondary mass spectrometry were set to 30 eV, 40 eV, and 50 eV. The AutoMS / MS scan range was: mother ion m / z 500-1500, daughter ion m / z 50-1500.

[0115] For the impurities represented by peaks 1-6 and 8-12, the secondary mass spectrometry collision energies were set to 40 eV and 50 eV, respectively, and the other mass spectrometry detection parameters were the same as those for the mass spectrometry detection of Numocontin B0.

[0116] The mass spectrometry results for Numocontin B0 are shown below. Figure 2 As shown. The results of secondary mass spectrometry detection of Numocontin B0 are as follows. Figure 3 , Figure 4 and Figure 5 As shown, the detection results correspond to collision energies of 30 eV, 40 eV, and 50 eV, respectively. Figures 3-5 The mass spectrometry data represented in the table are rounded to two decimal places and summarized in Table 1 below.

[0117] Depend on Figures 3-5 It is evident that the secondary mass spectrometry data obtained using a collision energy of 50 eV mostly consist of low-molecular-weight fragment ions, making it impossible to deduce the fragmentation mechanism of Numococtin B0. Furthermore, the chemical structures of the impurities represented by peaks 1-6 and 8-12 could not be resolved. When determining the fragmentation mechanism of Numococtin B0 and resolving the chemical structures of impurities, the fragmented fragment ions should be selected as the base peaks for derivation analysis. The parent ion and its dehydration peaks should not be used as base peaks. For example, although the molecular weight data of fragment ions obtained from collisions at 30 eV and 40 eV both contain a fragment ion of 1029.6, this fragment ion is a parent ion at 30 eV and is not used as a base peak. Data obtained at 40 eV is required for the analysis in this invention. On the other hand, although the mass spectrometry used for analysis… Figure 1Generally, the parent ion needs to be fragmented to 1 / 3 of the base peak. The 40 eV mass spectrum meets this requirement. However, the secondary mass spectrum measured at 40 eV lacks some key daughter ion peaks, making it impossible to resolve the fragmentation pathway of nimocontin B0 and the chemical structure of the impurities. Mass spectrometry data obtained from 30 eV detection is also needed as a key auxiliary analysis to realize the present invention.

[0118] Table 1

[0119]

[0120]

[0121]

[0122]

[0123] Note: When the same substance is detected by mass spectrometry, even if the mass spectrometry detection conditions are exactly the same, the decimal places of the molecular weights of the parent ion or daughter ion obtained by detection may be different. However, if the digits before the decimal point and the two digits after the decimal point are the same, they should be regarded as the same ion.

[0124] (1) Analysis of the cleavage pathway of neomont B0

[0125] Based on the detection results of NeomoContin B0 (PB0) primary and secondary mass spectrometry, the following results were obtained through analysis: Figure 6 The shown fragmentation pathway is analyzed in the following manner:

[0126] Because PB0 contains a large number of hydroxyl groups, especially the ortho-dihydroxyl groups in AA1 and AA4, it is speculated that during the cleavage process, dehydration first occurs to form an enol structure, followed by isomerization to a keto structure. Other groups such as AA6, AA3, AA5, and AA2 can also dehydrate under high vacuum and form an enone structure with the ortho-carbonyl group, thus allowing the observation of ion clusters formed by continuous dehydration (M-18) in secondary mass spectrometry. Based on whether the cyclic peptide core structure is preserved, three cleavage pathways are summarized as follows:

[0127] 1) Retaining the parent nucleus structure, PB0, after losing one water molecule, yields an ion with m / z 1047.5604. Further dehydration yields characteristic ions with m / z 1029.5503, 1011.5389, and 993.5285. Ion m / z 1047.5604 loses a fatty acyl ion (FA) and one water molecule to obtain a parent nucleus fragment ion with m / z 791.3208. Further dehydration yields characteristic ions with m / z 773.3090 and 755.2984. PB0, through the cleavage of high-tyrosine residue ions, yields the PB0 imine ion with m / z 911.5058. Further dehydration yields m / z 893.4968 and 875.4864.

[0128] 2) During the cleavage process of PB0, amino acid residues in the cyclic peptide are removed to form ions that do not retain the parent nucleus structure. During the analysis, it was discovered that the FA ion can transfer to other amino acids in the cyclic peptide. After PB0 removes the (AA1+AA2) residue, FA may transfer to the 3-position 4-hydroxyproline to obtain fragment ions with m / z 835.4780. Further dehydration yields m / z 817.4702 and 799.4593. Notably, AA6 at this point is an amidated 3-hydroxyproline residue. Ion m / z 835.4780 can subsequently lose FA and undergo continuous dehydration to obtain fragment ions m / z 597.2506, 579.2406, 561.2295, and 543.2193, or lose AA6 residue ions and undergo continuous dehydration to obtain m / z 705.4062, 687.3959, and 669.3854. The characteristic ions m / z 467.1771, 449.1669, 431.1564, and 413.1454 of (AA3+AA4+AA5) can be obtained by the co-fragmentation of characteristic ions m / z 597.2506 and m / z 705.4062. Meanwhile, at the low molecular weight end, characteristic amidation ions (AA5+AA6) with m / z 275.1351 and 257.1245, and characteristic ions (AA3 and AA6) with m / z 131.0815 and 86.0601 were observed. These characteristic ions are very helpful in determining the changes in hydroxyproline at the 3 and 6 positions.

[0129] 3) After losing (AA6+FA+H2O+NH3), PB0 yields a characteristic ion m / z of 679.2569, and further dehydration yields characteristic ions m / z of 661.2456, 643.2352, and 625.2237. Subsequently, after losing amino acid residues at different sites and undergoing dehydration, characteristic ions m / z of (AA2+AA3+AA4+AA5) are obtained: m / z of 568.2242, 550.2138, and 532.2030; characteristic ions m / z of (AA1+AA2+AA3) are: m / z of 326.1344 and 308.1242; characteristic ions m / z of (AA2+AA3) are: m / z of 215.1024 and 197.0916; and characteristic ions m / z of (AA1+AA2) are: m / z of 213.0871 and 195.0764. The characteristic ions mentioned above, especially (AA1+AA2), are crucial for determining changes in threonine at position 2. If PB0 retains FA and loses (AA6+H2O+NH3) and undergoes continuous water loss, characteristic ions with m / z 917.4844, 899.4762, 881.4649, and 863.4542 are obtained. It should be noted that this series of ions may be formed by the hydroxyl group of 5-hydroxyglutamine attacking the amide bond to form a four-membered ring and losing one molecule of NH3. Characteristic ion m / z 917.4844 loses (FA+AA1+AA2+AA3) and H2O to obtain (AA4+AA5), with characteristic ion m / z 319.0922.

[0130] (2) Detection results of impurities in neomont B0 and structural analysis process

[0131] By analyzing the characteristic ions in the three cleavage pathways of PB0, the amino acids that have undergone changes can be identified, providing a basis for the rapid resolution of the structures of other components. In the cleavage pathways of PB0, fatty acyl chains can transfer from dihydroxyornithine to other amino acids, and components substituted with monohydroxyornithine can form imine ions. Based on the cleavage patterns of PB0, the structures of 11 components were resolved. For the first time, the following were discovered: PB components containing unsaturated fatty acyl chains (PB0-1, PB0-2, PB0-5); PB components containing hydroxylated fatty acyl chains (PB0-4 or PB0-4'); a component with carbonylated ornithine (PB0-8); and components with ring-opening of a cyclic peptide (ring-opening at amino acids positions 5 and 6) and the conversion of amino acid position 1 to 5-aminoproline (PB0-9, PB0-10, PB0-11).

[0132] Peak 1 is the H+ ion peak [M+H]. + The value is m / z 1063.5547, with the addition of the Na ion peak [M+Na]. +With an m / z of 1085.5355, the molecular weight is 2 smaller than that of PB0, suggesting that this component has 2 fewer hydrogen atoms than PB0. Secondary mass spectrometry analysis revealed that the parent nucleus after the loss of FA and the fragment ions of various small peptides were largely consistent with the relevant ions of PB0. However, fragment ions containing FA, such as the de-(AA1+H2O+NH3) ion (m / z 915.4725), the de-homotyrosine residue ion (m / z 909.4934), the FA transfer ions (m / z 815.4556 and m / z 703.3947), and their dehydrated ions, were all 2 fewer than the relevant ions of PB0, indicating the presence of an unsaturated alkenyl group at the FA terminus. This component was named PB0-1.

[0133] Peak 2 is the H+ ion peak [M+H]. + The value is m / z 1063.5553, with the addition of the Na ion peak [M+Na]. + With an m / z of 1085.5361, the molecular weight is 2 smaller than that of PB0, suggesting that this component has 2 fewer hydrogens than PB0. The fragment ion is the same as the related ion of peak 1, indicating that there is also a double bond at the FA end. Therefore, this component is named PB0-2.

[0134] Peak 3 is the H+ ion peak [M+H]. + The value is m / z 1051.5552, with the addition of the Na ion peak [M+Na]. + With an m / z of 1073.5346, the molecular weight is 14 smaller than that of PB0, suggesting that this component lacks one methylene group compared to PB0. Comparison with corresponding ions of PB0 reveals that the ions containing FA are primarily 14 smaller, such as the ions after the loss of (AA1+AA2) and FA transfer (m / z 821.4607), the ions after the removal of high-tyrosine residues (m / z 897.4829, 879.4804), and the daughter ions after the removal of AA6 (m / z 903.4718, 885.4597), indicating that the molecular formula at the FA end is C. 15 H 29 O represents pentadecanoyl, and this component is named PB0-3.

[0135] Peak 4 is the H+ ion peak [M+H]. + The value is m / z 1065.5706, with a Na ion peak [M+Na]. +The molecular weight is 1087.5525, the same as PB0; the base peak is 1047.5600, therefore it preferentially fragments in the secondary mass spectrometer, and its structure can be deduced together with the fragment ions of the H-added ion peak. The characteristic ions of this fraction, m / z 597.2509, 579.2397, 561.2295 (AA3+AA4+AA5+AA6) and m / z 467.1764, 449.1663, 431.1552 (AA3+AA4+AA5), are basically consistent with the corresponding characteristic ions of PB0, indicating that the four amino acids AA3-AA6 have not changed. In addition, the characteristic ions m / z 568.2226 and 550.2125 further indicate that the threonine at position 2 has not changed. The m / z values ​​of the dehydration ions at the parent nucleus (793.3354, 775.3241, 757.3187) are 2 greater than the corresponding characteristic ion at the parent nucleus (791.3208) of PB0, suggesting that the dihydroxyornithine at position 1 is converted to monohydroxyornithine, indicating that no neutral dehydration occurred at this position. The m / z values ​​of the dehydration characteristic ions (AA1+AA2+AA3+AA4+AA5) (663.2605, 645.2474, 627.2395) and the m / z values ​​of the ions (AA1+AA2) (215.1036, 197.0933, 179.0807, which should be distinguished from the characteristic ions of ((AA2+AA3)) are 2 greater than the corresponding ions at PB0, further supporting the assumption that position 1 is monohydroxyornithine. Mass spectrometry cannot yet confirm whether the hydroxyl group is at position 4 or 5 of AA1; the fragmentation pathway is described in [link to fragmentation pathway description]. Figure 7 As shown, the hydroxyl group at AA1 is located at position 5 for illustration. Further analysis revealed daughter ions with m / z values ​​of 131.0812 and 113.0712 at the lower molecular weight end. The daughter ion m / z 131.0812 may be a characteristic ion of hydroxyprolyl amide at positions 3 or 6, or it could be monohydroxyornithine. The m / z 113.0712 was not found at the lower molecular weight end of PB0, indicating that hydroxyprolyl amide at positions 3 or 6 is less likely to lose one molecule of water and tends to lose (NH3 + CO) to obtain the hydroxycyclopentimine ion m / z 86.0597, while monohydroxyornithine readily obtains the dehydration daughter ion m / z 113.0712. (See...) Figure 7 Based on the H-ion peak of this component, it is inferred that FA should contain one hydroxyl group. The characteristic ions m / z 911.5162, 893.4977, 875.4783 after the removal of high tyrosine residues and the characteristic ions m / z 917.4825, 899.4715, 881.4586, 863.4477 after the loss of (AA6+NH3+H2O) and continuous water loss confirm that FA has one additional hydroxyl group. The position of the hydroxyl group at the end of FA has not yet been confirmed by mass spectrometry. This component is named PB0-4 or PB0-4'.

[0136] Peak 5 is the H+ ion peak [M+H].+ The value is m / z 1063.5558, with the addition of a Na ion peak [M+Na]. + The value is m / z 1085.5367, and the fragment ion peaks are basically consistent with peaks 1 and 2. It is inferred that there is a double bond at the FA end, and it is named PB0-5.

[0137] Peak 6 is the H+ ion peak [M+H]. + The value is m / z 1051.5568, with the addition of the Na ion peak [M+Na]. + With an m / z of 1073.5372, the molecular weight is 14 smaller than that of PB0, suggesting that this component lacks one methylene group. The most likely sites for methylene loss are FA and threonine at position 2. Comparison of the fragment ions (m / z 777.3036, 759.2940) after FA loss and the characteristic ion (m / z 199.0708) of (AA1+AA2) after FA loss indicates that threonine at position 2 is converted to serine. Furthermore, the fragment ions (m / z 817.4703, 687.3963) after FA transfer are essentially identical to the relevant ions of PB0, indicating that the FA terminus remains unchanged. Sun et al. reported a similar PB0 serine analogue, whose elution position precedes that of PB0; this should be the same compound identified in this study, named PB0-6.

[0138] Peak 8 is the H+ ion peak [M+H]. + The value is m / z 1049.5764, with a Na ion peak [M+Na]. +The molecular weight is 1071.5576 with a base peak of 1031.5664, and the molecular weight is 16 smaller than that of PB0. The (AA3+AA4+AA5+AA6) ion m / z 597.2496, the (AA3+AA4+AA5) ion m / z 467.1774, and their dehydration ions and corresponding FA transfer ions m / z 835.4793, 817.4697, and 799.4600 are basically consistent with the relevant ions of PB0, indicating that the four amino acids AA3-AA6 and FA have not changed. The characteristic ion m / z of (AA1+AA2+AA3+AA4+AA5) is 663.2628, 645.2510, 627.2401, and the ion m / z of (AA1+AA2+AA3) is 310.1401, 292.1290, and the ion m / z of (AA1+AA2) is 197.0924, 179.0813. This confirms that the amino acid at position 1 is converted from 4,5-dihydroxyornithine to 4 or 5-hydroxyornithine. The relative elution position of B5 reported by Zhang et al. is similar to that of this component, both being after PB0. This component may be PB5. In this study, it is named PB0-7 or PB0-7' based on the difference between 4- or 5-hydroxyornithine. Both PB0-4 and PB0-7 are monohydroxyornithine components, and their base peak is a dehydration peak. This should be noted when determining the molecular weight, which also indicates that this type of component is prone to intrasource cleavage to generate a dehydration peak.

[0139] Peak 9 is the H+ ion peak [M+H]. + The value is m / z 1063.5550, with the addition of the Na ion peak [M+Na]. + The m / z value is 1085.5376, and the molecular weight is 2 less than that of PB0. The m / z values ​​of the (AA3+AA4+AA5+AA6) ion, (AA3+AA4+AA5) ion, and (AA3+AA4+AA5) ion (467.1765), as well as the dehydration ion and FA transfer ion (m / z 817.46975) of this component, are basically consistent with the relevant ions of PB0, indicating that the four amino acids AA3-AA6 and FA have not changed. The m / z values ​​of the (AA2+AA3+AA4+AA5) ion (568.2236 and 550.2128) are basically consistent with those of PB0, indicating that AA2-AA5 have not changed. It is inferred that the hydroxyl group in dihydroxyornithine at position 1 is converted to a carbonyl group. The characteristic ion m / z of (AA1+AA2+AA3+AA4+AA5) is 659.2270. Combined with the dehydration ions m / z of (AA1+AA2+AA3) (324.1177, 306.1083) and the ions m / z of (AA1+AA2) (211.0708, 193.0628), it can be confirmed that the amino acid at position 1 is converted from 4,5-dihydroxyornithine to 4-carbonyl-5-hydroxyornithine. The cleavage pathway is described in [link to cleavage pathway description]. Figure 8As shown, it is named PB0-8. The cleavage pattern of this component differs from that of PB0, especially in the formation process of m / z 659.2270 and its dehydration ions. It is possible that the 5-position hydroxyl group is removed during the cleavage process to form an imine, and then subsequent ions are generated.

[0140] Peak 10 is the H+ ion peak [M+H]. + The value is m / z 1049.5757, with the addition of a Na ion peak [M+Na]. + The m / z value is 1071.5566, and the molecular weight is 16 less than PB0. Compared to component peak 8, the fragment ion peaks are significantly different, and the base peak is a hydrogenation ion peak rather than a dehydration peak. The characteristic ions (m / z) of (AA2+AA3+AA4+AA5) at 568.2255, 550.2170, and 532.2029, and the ions (m / z) of (AA3+AA4+AA5) at 467.1773, 449.1653, and 431.1559, indicate that AA2-AA5 remained unchanged. The secondary mass spectrum did not show the characteristic ion (m / z 275.1351) of (AA5+AA6) or related ions of (AA3+AA4+AA5+AA6), suggesting that the amide bond at AA5 and AA6 broke, leading to the ring-opening of the cyclic peptide and the formation of a linear lipopeptide. Ions m / z 583.4065 and 482.3585, and dehydration ions m / z 565.3938 and 464.3427 are newly emerging ions with high abundance, not found in other components. The molecular weight difference between ions m / z 583.4065 and 482.3585 is 101, meaning they differ by one threonine residue. Therefore, it is inferred that ion m / z 583.4065 is a fragment ion of (AA1+AA2+AA6+FA), and m / z 482.3585 is a fragment ion of (AA1+AA6+FA). The structural formulas and fragmentation pathways are shown in [reference needed]. Figure 9 As shown in the diagram. According to the structural formula of PB0, the lone pair electrons on the amino group at AA1 readily attack the carbon atom connecting the hydroxyl group to AA1, causing AA1 to transform from hydroxylated ornithine to 5-aminoproline. This ultimately affects the stability of the amide bond at AA5 and AA6, leading to its breakage, and is named PB0-9. The analytical process can be found in [link to analytical process]. Figure 9The characteristic ions m / z 895.5134 and 877.5053 after the removal of high-tyrosine residues also verify the rationality of this component structure. It is noteworthy that the fragment ion m / z 930.5184 may be formed by the transfer of the acyl group of hydroxyglutamine at position 5 to hydroxyproline at position 6, followed by the removal of the hydroxyglutamine residue ion. The currently discovered ring-opening peptide is PB6b, which is formed by the 2-amino group of AA1 attacking the semiamine aldehyde to open the parent nucleus and form 4,5-dihydroxyproline. The precursor of PB0-9 may be a 5-hydroxyornithine component, in which the 2-amino group of 5-hydroxyornithine attacks the carbon at position 4 to remove one molecule of water to form 5-aminoproline, while AA5 and AA6 undergo ring opening to form PB0-9.

[0141] Peak 11 is the H+ ion peak [M+H]. + The value is m / z 1049.5767, with the addition of a Na ion peak [M+Na]. + The molecular weight is 1071.5577, the same as the component at peak 10, and its fragment ions such as m / z 930.5195, 895.5116, 583.4052, 568.2249, and 482.3587 are also basically consistent with the component. It is inferred that this component is its isomer and named PB0-10.

[0142] Peak 12 is the H+ ion peak [M+H]. + The value is m / z 1049.5765, with the addition of the Na ion peak [M+Na]. + With an m / z of 1071.5576, and information such as fragment ions that are the same as those in peaks 10 and 11, it should be an isomer, named PB0-11.

[0143] It should be pointed out 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 impurities in crude nimococcal B0, characterized in that, It includes the following steps: (1) The crude Numocontin B0 was separated to obtain Numocontin B0 and several impurities; (2) The Numocontin B0 and each of the impurities were detected by first-stage mass spectrometry and second-stage mass spectrometry to obtain mass spectrometry data; (3) Determine the fragmentation mode of the nimococtin B0 based on the mass spectrometry data of the nimococtin B0; (4) Using the cleavage mode of the aforementioned Numocontin B0 as a reference, the chemical structural formula of each of the impurities is determined by combining the mass spectrometry data of each of the impurities in step (2). In step (2), the nimocontin B0 is subjected to secondary mass spectrometry detection at two or more different collision energies, the collision energies being 20-45 eV.

2. The structural analysis method as described in claim 1, characterized in that, In step (1), the separation is performed using high performance liquid chromatography (HPLC); wherein the HPLC method employs gradient elution. The mobile phase of the high-performance liquid chromatography preferably includes mobile phase A and mobile phase B; mobile phase A is an aqueous formic acid solution; mobile phase B is acetonitrile; wherein mobile phase A is preferably an aqueous formic acid solution with a volume concentration of 0.05-0.2%, for example, an aqueous formic acid solution with a volume concentration of 0.1%.

3. The structural analysis method as described in claim 2, characterized in that, In step (1), the gradient elution conditions are as follows: During the 0-20 min period, the mobile phase consists of 60% by volume mobile phase A and 40% by volume mobile phase B; During the period of 20-35 minutes (excluding 20 minutes), the mobile phase consists of 50-60% by volume mobile phase A and 40-50% by volume mobile phase B. During the period of 35-40 minutes (excluding 35 minutes), the mobile phase consists of 10-50% by volume of mobile phase A and 50-90% by volume of mobile phase B.

4. The structural analysis method as described in claim 2, characterized in that, The high-performance liquid chromatography method uses a C18 column; And / or, the column temperature of the high performance liquid chromatography is below 50°C, preferably 40°C.

5. The structural analysis method as described in claim 1, characterized in that, In step (2), when performing secondary mass spectrometry detection on the Numocontin B0, the difference between two or more different collision energies is between 5-20 eV, for example 10 eV or 15 eV; The collision energy for one of the secondary mass spectrometry detections is preferably 25-35 eV, for example 30 eV; the collision energy for the other secondary mass spectrometry detection is preferably 35-45 eV, for example 40 eV.

6. The structural analysis method as described in claim 5, characterized in that, In step (2), each impurity is subjected to secondary mass spectrometry detection at two or more different collision energies, wherein the collision energy is preferably 20-45 eV; The difference between the energies of the two or more different collisions is preferably between 5 and 20 eV, for example, 10 eV or 15 eV; The collision energy for one of the secondary mass spectrometry detections is preferably 25-35 eV, for example 30 eV; the collision energy for the other secondary mass spectrometry detection is preferably 35-45 eV, for example 40 eV.

7. The structural analysis method as described in claim 1, characterized in that, When the secondary mass spectrometer detects the Numococtin B0 and / or each of the impurities, it satisfies 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 capillary outlet pressure is 150-200V, for example 175V.

8. The structural analysis method according to any one of claims 1-7, characterized in that, In step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of Numocontin B0 includes a set of daughter ions with molecular weights of 1047.56, 791.32, 911.51, 835.48, 597.25, 705.41, 467.18, 275.14, 131.08, 86.06, 679.26, 568.22, 326.13, 215.10, 213.09, 917.49, and 319.

09.

9. The structural analysis method according to any one of claims 1-7, characterized in that, In step (3), the impurities include one or more of the following compounds: (1) Compound of formula PB0-1 and / or isomers of compound of formula PB0-1: Wherein, the isomers of the compound of formula PB0-1 have the same chemical structure as the compound of formula PB0-1 at -C 15 H 29 The double bonds in the groups are in different positions or configurations; (2) Compound of formula PB0-3: (3) Compound of formula PB0-4 or compound of formula PB0-4': (4) Compound of formula PB0-6: (5) Compounds of formula PB0-7 or PB0-7': (6) Compound PB0-8: (7) Compound of formula PB0-9 and / or isomers of compound of formula PB0-9:

10. The structural analysis method as described in claim 9, characterized in that, In step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-1 includes a set of daughter ions with molecular weights of 915.47, 909.49, 815.46 and 703.39; And / or, in step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the isomers of the compound of formula PB0-1 include a set of daughter ions with molecular weights of 915.47, 909.49, 815.46 and 703.39; And / or, in step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-3 includes a set of daughter ions with molecular weights of 821.46, 897.48, 879.48, 903.47 and 885.46; And / or, in step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-4 or the compound of formula PB0-4' includes a set of daughter ions with molecular weights of 597.25, 467.18, 568.22, 793.34, 663.26, 215.10, 131.08, 113.07, 911.52 and 917.48; And / or, in step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-6 includes a set of daughter ions with molecular weights of 777.30, 199.07, 817.47 and 687.40; And / or, in step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-7 or the compound of formula PB0-7' includes a set of daughter ions with molecular weights of 597.25, 467.18, 835.48, 663.27, 310.14 and 197.09; And / or, in step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-8 includes a set of daughter ions with molecular weights of 597.25, 467.18, 817.47, 568.22, 659.22, 324.12 and 211.07; And / or, in step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the compound of formula PB0-9 includes a set of daughter ions with molecular weights of 568.23, 467.18, 583.41, 482.36, 895.51 and 930.52; And / or, in step (2), when performing the secondary mass spectrometry detection, the mass spectrometry data of the isomers of the compound of formula PB0-9 include a set of daughter ions with molecular weights of 568.23, 467.18, 583.41, 482.36, 895.51 and 930.52.

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