CN110031556B - Method for rapidly identifying lipstatin metabolites in streptomyces toxytricini fermentation products - Google Patents

Method for rapidly identifying lipstatin metabolites in streptomyces toxytricini fermentation products Download PDF

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CN110031556B
CN110031556B CN201811429426.3A CN201811429426A CN110031556B CN 110031556 B CN110031556 B CN 110031556B CN 201811429426 A CN201811429426 A CN 201811429426A CN 110031556 B CN110031556 B CN 110031556B
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lipstatin
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streptomyces toxytricini
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杨煌建
张祝兰
连云阳
严凌斌
陈洲琴
程贤
王德森
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Fujian Institute of Microbiology
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Abstract

The invention provides a method for quickly identifying lipstatin metabolites in a streptomyces toxytricini fermentation product, and belongs to the technical field of analysis of microbial fermentation products. The invention aims to provide a practical, rapid and accurate method for rapidly identifying lipstatin metabolites in a streptomyces toxytricini fermentation product. By analyzing the profile of the fragment ions of the Lipstatin compounds in the streptomyces toxytricini fermentation liquor and comparing the characteristic fragment ions of the Lipstatin compounds with the characteristic fragment ions, the mass spectrum cracking rule of the same compounds is comprehensively analyzed, so that the quick structure identification of unknown Lipstatin compounds in the fermentation liquor is realized. The invention has the advantages of simple operation, high sensitivity, strong anti-interference performance and good popularization and application value.

Description

Method for rapidly identifying lipstatin metabolites in streptomyces toxytricini fermentation products
Technical Field
The invention provides a method for quickly identifying a streptomyces toxytricini fermentation product lipstatin metabolite, belonging to the technical field of microbial fermentation product analysis.
Background
Obesity has become a common disease worldwide and is a risk factor for causing various diseases (such as non-insulin-dependent diabetes mellitus, heart disease, cerebrovascular disease, respiratory system diseases and certain cancers), so research and development of safe and effective weight-reducing medicines have become a research field of world attention.
At present, most of the developed and applied weight-reducing medicines act on the central nervous system and are basically forbidden due to strong side effects. The pancreatic lipase inhibitor is the only weight-reducing drug which does not act through a central nervous system so far, reduces the decomposition and absorption of fat by selectively inhibiting pancreatic lipase in a gastrointestinal tract, has relatively high safety, and becomes a main direction of the current weight-reducing drug research.
Through the continuous efforts of countless researchers, a plurality of pancreatic lipase inhibitors with great application value are successively discovered from the secondary metabolites of microorganisms. The most successful of these is the discovery of lipstatin (lipstatin). lipstatin is obtained by screening Streptomyces toxytricini fermentation liquor, and lipstatin is used as an intermediate raw material to synthesize a tetrahydrogen derivative orlistat (orlistat, trade name cinchona, xenical) by Roche lnc. Therefore, studies of lipstatin have attracted extensive interest to researchers.
The prior patents on lipstatin mainly focus on the fermentation production, culture medium improvement process optimization and separation preparation methods of lipstatin, and are described in patents US2005089978a1, US8501444, EP0803576, WO2007134836a1, WO2004003212, CN103320481B, CN103820510 a 103820510B, CN 103820510B, etc., while there is no report on the analysis and characterization of other metabolites of lipstatin producing Streptomyces toxytricini. Research shows that in addition to the main metabolic component of lipstatin, the fermentation broth of Streptomyces toxytricini produced by lipstatin also contains other metabolites with similar polarity, and the metabolites seriously affect the later separation preparation and quality control of lipstatin. The efficient chemical structure identification and analysis method is of great importance to the research in the field of biological pharmacy, the traditional analysis mode that natural products are separated and purified firstly and then the structure of the natural products is identified by using the spectrum technology is complex, and the problems that the known components are separated repeatedly and the pure products of new components are difficult to obtain exist. However, when the High Performance Liquid Chromatography (HPLC) method is used alone for the analysis of components, it takes a long time to separate the components at a high degree, and there is a problem that no ultraviolet-absorbing component can be detected. Therefore, a practical, fast and accurate analysis and identification method needs to be established for fast analysis of unknown chemical components in the Streptomyces toxytricini fermentation liquor.
Disclosure of Invention
The invention aims to provide a practical, rapid and accurate method for rapidly identifying lipstatin metabolites in a streptomyces toxytricini fermentation product.
The invention is realized by the following steps:
a method for rapidly identifying lipstatin metabolites in a streptomyces toxytricini fermentation product comprises the following steps: (1) extracting chemical components from the streptomyces toxytricini fermentation liquor; (2) measuring the extracting solution by using a high performance liquid chromatography-electrospray-quadrupole-time of flight mass spectrometer (HPLC-ESI-Q-TOF-MS), and obtaining the retention time of each chemical component in the extracting solution according to a high performance liquid chromatogram and a total ion flow diagram of the extracting solution; (3) performing mass spectrometry on a main known compound lipstatin and an unknown compound in the extracting solution by using HPLC-ESI-Q-TOF-MS to obtain the mass spectrometry ion signal quantity of different compounds; wherein the primary Mass Spectrometry (MS) measures the exact relative molecular mass of the compound, infers the possible chemical formula composition, and is based on the secondary Mass Spectrometry (MS)2) Deducing the chemical structure of the characteristic fragment ions; (4) the mass spectrum cracking rule of each compound is obtained by comparing fragment ion signals of known compound lipstatin and unknown compound in the extracting solution, so that the compound structure identification of unknown components is realized.
Further, the extraction process of the chemical components in the streptomyces toxytricini fermentation broth comprises the following steps: firstly, fermenting and culturing microbial Streptomyces toxytricini, centrifuging or filtering plates after fermentation is finished, and collecting hypha; soaking the mycelium in 95% ethanol, stirring, and collecting ethanol extractive solution; the ethanol extract contains lipstatin metabolites. Diluting the ethanol extract, filtering with 0.22um filter core, and determining by HPLC-ESI-Q-TOF-MS.
Further, the HPLC-ESI-Q-TOF-MS method refers to that high performance liquid chromatography is carried out on a quadrupole mass spectrometer in series, and multiple reaction modes are adopted for monitoring. High performance liquid chromatography conditions: the chromatographic column is Kromasil C18(4.6mm × 250mm, 5 μm), the mobile phase is water-acetonitrile (15:85, V/V), the flow rate is 1.0ml/min, the column temperature is 30 ℃, and the sample injection amount is 10 μ l; four-stage rod mass spectrometer operating conditions: the ion source is an electrospray ion source (positive ion mode), the offset voltage of an end plate is-500V, the electrophoresis voltage of a capillary tube is 3500V, the carrier gas is helium, the atomizing and drying gas is high-purity nitrogen (99.99%), the outlet voltage of the capillary tube is 135V, the voltage of a conical hole is 65V, the desolventizing temperature is 350 ℃, the flow rate of the drying gas is 10L/min, the pressure of an atomizer is 40psi, the collision energy is 8-45V, and the scanning range is m/z 100-1700. Chemical component molecular formulas were analyzed using ChemBioDraw Ultra 13.0 and Bruker data analysis software version 4.0.
Further, the high performance liquid chromatogram and total ion flow diagram results of the extracting solution show that the extracting solution contains 8 obvious chemical components, and the Retention Time (RT) is 7.23min, 9.40min, 9.79min, 10.29min, 10.83min, 12.49min, 14.59min and 17.39min respectively.
Further, mass spectrometry of a main known compound lipstatin (RT 12.49min) in the extracting solution is carried out, wherein in a positive ion mode, the lipstatin (RT 12.49min) is a compound 6, and a molecular ion peak is [ M + H ]]+ is m/z492.3719, formula C29H49NO5；MS2Characteristic ion fragment peaks m/z 333.28,287.28,189.17,160.10,149.13,142.09,119.09 and 114.09 are shown. The molecular ion peak of the compound 1(RT 7.23min) is [ M + H]+ is m/z 508.3453, formula C29H49NO6；MS2Characteristic ion fragment peaks m/z 349.28,331.26,303.27,285.25,189.16,165.13,160.12,147.12 and 114.09 are shown. The molecular ion peak of the compound 2(RT 9.40min) is [ M + H]+ is m/z490.3537, formula C29H47NO5；MS2Characteristic ion fragment peaks m/z 331.27,285.26,187.15,160.12,149.12,142.11,133.10 and 114.09 are shown. The molecular ion peak of the compound 3(RT 9.79min) is [ M + H]+ is m/z 510.3262, formula C29H51NO6；MS2Characteristic ion fragment peaks m/z 333.28,287.28,189.16,160.11,149.08,119.09 and 114.09 are shown. The molecular ion peak of the compound 4(RT 10.29min) is [ M + H]+ is m/z490.3537, formula C29H47NO5；MS2Characteristic ion fragment peaks m/z 331.27,285.26,189.17,160.12,147.12,142.11,133.10 and 114.09 are shown. The molecular ion peak of the compound 5(RT 10.83min) is [ M + H]+ is m/z478.3533, formula C28H47NO5；MS2Characteristic ion fragment peaks m/z 333.28,287.27,189.16,149.13,146.08,128.07 and 100.08 are shown. The molecular ion peak of the compound 7(RT 14.59min) is [ M + H]+ is m/z 506.3850, formula C30H51NO5；MS2Characteristic ion fragment peaks m/z 347.28,301.29,189.16,160.10,149.13,142.09 and 114.09 are shown. The molecular ion peak of the compound 8(RT 17.39min) is [ M + H]+ is m/z494.3851, formula C29H51NO5；MS2Characteristic ion fragment peaks m/z 335.30,289.29,191.18,160.10,149.13,142.09 and 114.09 are shown.
Further, the ESI-Q-TOF-MS2 cleavage of the known compound lipstatin in the extract occurs at the compound ester bond position, forming specific ion fragment peaks m/z 333.28 (fragment a) and m/z 160.10 (fragment b), wherein fragment a is further cleaved to generate ion fragment peak m/z 287.28,189.16,149.08; fragment b then cleaved ion fragment peaks m/z 142.11 and 114.09.
Further, the structure of the unknown compound in the extracting solution is identified as follows: hydroxylipstatin (Compound 1), Dehydrolipstatin (Compound 2,4), Seco-lipstatin (Compound 3), Descemetpstatin (Compound 5), Methyllipitatin (Compound 7) and Dihydroxyllipstatin (Compound 8).
The invention has the following advantages:
1. compared with the traditional method, the invention does not need to prepare and separate a standard compound, can quickly and accurately complete the identification of chemical components in the Streptomyces toxytricini (Streptomyces toxytricini) fermentation liquor, including the structure of unknown compounds, and does not need to combine other analysis means.
2. The method can rapidly and accurately analyze the mass spectrum cracking rule of the Lipstatin, identify the structures of 7 unknown Lipstatin compounds in the fermentation liquor for the first time, enrich the pancreatic lipase inhibitor compound database, and provide accurate information for the Lipstatin separation preparation and quality control research.
3. The method has the advantages of simple operation, high sensitivity and strong anti-interference performance, and can be popularized and used for the differential analysis of microbial metabolites or other natural compounds.
Drawings
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 is an HPLC chromatogram of a Streptomyces toxytricini fermentation extract of the present invention;
FIG. 2 is a LC-MS total ion flow diagram of a Streptomyces toxytricini fermentation extract of the present invention;
FIG. 3 is a secondary mass spectrum of Lipstatin in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 4 is a second-order mass spectrum of Compound 1 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 5 is a secondary mass spectrum of Compound 2 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 6 is a second-order mass spectrum of Compound 3 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 7 is a second-order mass spectrum of Compound 4 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 8 is a second-order mass spectrum of Compound 5 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 9 is a second-order mass spectrum of Compound 7 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 10 is a second-order mass spectrum of Compound 8 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 11 is a spectrum of mass spectrum cleavage pathway of lipstatin (compound 6) in Streptomyces toxytricini fermentation extract of the present invention;
FIG. 12 is a mass spectrum of the cleavage pathway of Compound 1 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 13 is a mass spectrum of the cleavage pathway of Compound 2 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 14 is a mass spectrum of the cleavage pathway of Compound 3 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 15 is a mass spectrum of the cleavage pathway of Compound 4 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 16 is a mass spectrum of the cleavage pathway of Compound 5 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 17 is a mass spectrum of the cleavage pathway of Compound 7 in the Streptomyces toxytricini fermentation extract of the present invention;
FIG. 18 is a spectrum of mass spectrum cleavage pathway of compound 8 in Streptomyces toxytricini fermentation extract of the present invention.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Examples
Extraction of chemical components in Streptomyces toxytricini fermentation liquor
Fermenting and culturing microbial Streptomyces toxytricini (CN 108753861A, a culture medium and a method for fermenting Streptomyces toxytricini to produce lipstatin with high yield, published as 2018.11.6), centrifuging or filtering plates after fermentation is finished, and collecting hyphae; soaking the mycelium in 95% ethanol, stirring, and collecting ethanol solution; the ethanol solution contains the fermentation product of Streptomyces toxytricini (Streptomyces toxytricini), i.e. the metabolite of lipstatin. Diluting the ethanol solution, filtering with 0.22um filter core, and determining by HPLC-ESI-Q-TOF-MS.
Secondly, measuring the extracting solution by HPLC-ESI-Q-TOF-MS
The instrument comprises the following steps: agilent 1290 series high performance liquid chromatograph, 6520 series electrospray-time of flight mass spectrometry (ESI-Q-TOF-MS/MS), with ESI electrospray ion source, triple quadrupole trap mass spectrometer, and data analysis software.
Chromatographic conditions are as follows: the chromatographic column is Kromasil C18(4.6mm × 250mm, 5 μm), the mobile phase is water-acetonitrile (15:85, V/V), the flow rate is 1.0ml/min, the column temperature is 30 ℃, and the sample injection amount is 10 μ l;
mass spectrum conditions: the ion source is an electrospray ion source (positive ion mode), the offset voltage of an end plate is-500V, the electrophoresis voltage of a capillary tube is 3500V, the carrier gas is helium, the atomizing and drying gas is high-purity nitrogen, the outlet voltage of the capillary tube is 135V, the voltage of a conical hole is 65V, the desolventizing temperature is 350 ℃, the flow rate of the drying gas is 10L/min, the pressure of an atomizer is 40psi, the collision energy is 8-45V, and the scanning range is m/z 100-1700. Chemical component molecular formulas were analyzed using ChemBioDraw Ultra 13.0 and Bruker data analysis software version 4.0.
By HPLC-MS/MS analysis, ultraviolet chromatogram extracted from the extract at 210nm is basically consistent with selective ion chromatogram detected by mass spectrum (figures 1 and 2). And the positive ion mode detection is adopted, so that the total ion current chromatogram has a lower signal-to-noise ratio. HPLC and total ion flow diagram show that 8 main absorption peaks are detected at 210nm, the peak-out time is respectively 7.23, 9.40, 9.79, 10.29, 10.83, 12.49, 14.59 and 17.39min, and each absorption peak has symmetrical peak shape, proper retention time and extremely small tailing, so that each component in the extracting solution is well separated, the qualitative accuracy is ensured, and the requirement of analysis precision can be met.
Analysis of chemical components in extracting solution by Q-TOF-MS
8 components separated in FIGS. 1 and 2 were analyzed and identified by Q-TOF-MS, and Table 1 shows the exact relative molecular masses of the components and the corresponding possible structural formulae.
TABLE 1 HPLC-ESI-Q-TOF-MS/MS analysis of the components in the extract
Figure BDA0001882364530000061
Figure BDA0001882364530000071
As shown in fig. 3-10Second order Mass Spectrum (MS)2) The results showed that in positive ion mode, the molecular ion peak of Compound 1(RT 7.23min) was [ M + H]+ is m/z 508.3453, formula C29H49NO6；MS2Characteristic ion fragment peaks m/z 349.28,331.26,303.27,285.25,189.16,165.13,160.12,147.12 and 114.09 are shown. The molecular ion peak of the compound 2(RT 9.40min) is [ M + H]+ is m/z490.3537, formula C29H47NO5；MS2Characteristic ion fragment peaks m/z 331.27,285.26,187.15,160.12,149.12,142.11,133.10 and 114.09 are shown. The molecular ion peak of the compound 3(RT 9.79min) is [ M + H]+ is m/z 510.3262, formula C29H51NO6；MS2Characteristic ion fragment peaks m/z 333.28,287.28,189.16,160.11,149.08,119.09 and 114.09 are shown. The molecular ion peak of the compound 4(RT 10.29min) is [ M + H]+ is m/z490.3537, formula C29H47NO5；MS2Characteristic ion fragment peaks m/z 331.27,285.26,189.17,160.12,147.12,142.11,133.10 and 114.09 are shown. The molecular ion peak of the compound 5(RT 10.83min) is [ M + H]+ is m/z478.3533, formula C28H47NO5；MS2Characteristic ion fragment peaks m/z 333.28,287.27,189.16,149.13,146.08,128.07 and 100.08 are shown. The molecular ion peak of the compound 6(RT 12.49min) is [ M + H ]]+ is m/z492.3719, formula C29H49NO5Known compound lipstatin; MS (Mass Spectrometry)2Characteristic ion fragment peaks m/z 333.28,287.28,189.17,160.10,149.13,142.09,119.09 and 114.09 are shown. The molecular ion peak of the compound 7(RT 14.59min) is [ M + H]+ is m/z 506.3850, formula C30H51NO5；MS2Characteristic ion fragment peaks m/z 347.28,301.29,189.16,160.10,149.13,142.09 and 114.09 are shown. The molecular ion peak of the compound 8(RT 17.39min) is [ M + H]+ is m/z494.3851, formula C29H51NO5；MS2Characteristic ion fragment peaks m/z 335.30,289.29,191.18,160.10,149.13,142.09 and 114.09 are shown.
Fourthly, mass spectrum cracking rule analysis and structure identification of chemical components in extracting solution
By comparing the profile graph of the known compound lipstatin (compound 6) and the fragment ions of the unknown compound and the characteristic fragment ions in the extracting solution, the mass spectrum cracking rule of each compound is comprehensively analyzed, so that the compound structure identification of the unknown component in a complex system is realized.
ESI-Q-TOF-MS of known compound lipstatin2The cleavage of (a) occurs at the position of the ester bond of the compound, forming specific ion fragment peaks m/z 333.28 (fragment a) and m/z 160.10 (fragment b), fragment a further cleaves to generate ion fragment peak m/z 287.28,189.16,149.08; fragment b then cleaved ion fragment peaks m/z 142.11 and 114.09. The mass spectrometric cleavage pathway attributed to lipstatin (compound 6) is shown in FIG. 11, and has the following chemical structure:
Figure BDA0001882364530000081
resolution of Compound 1 Compound excimer ion [ M + H ]]+m/z 508, which is 16Da higher than that of lipstatin, is judged to be a hydroxyl group introduced into the lipstatin structure. MS of the Compound2The characteristic fragment of the part b is consistent with that of lipstatin, the ion peak signal m/z of the part a is 349, which is 16Da more than that of the part a of the lipstatin, and the structure of hydroxyl at the part a is presumed; (m/z 331,313,295,285) are all 2Da less than fragments of part a of lipstatin, and the dehydration peak is distinct, indicating that hydroxyl groups are easy to remove, possibly introduced in the form of branches, and part a, m/z189 fragments and the corresponding fragment signal (m/z 175,161,147) are present, indicating that the ethylenic moiety is unchanged; meanwhile, the characteristic fragment (m/z 165,151,135,121,107) of the compound infers that the hydroxyl is connected with the 1e part at C6H13On the long chain. The mass spectrum cleavage path of the attributive compound 1 is shown in the attached figure 12, and the chemical structure is as follows:
Figure BDA0001882364530000091
resolution of Compounds 2 and 4 two Compound excimer ions [ M + H ]]+m/z490, which is 2Da lower than lipstatin, is judged to be a loss of 2H in the structure of lipstatin to form an olefinic bond. MS of two Compounds2The characteristic fragment of part b is consistent with that of lipstatin, the signal (m/z 331,313,295,285) of part a is less than that of part a of lipstatin by 2Da, and the structure of the olefinic bond in part a is presumed; the presence of the m/z189 fragment and corresponding fragment signal (m/z 175,161,147) for the a moiety in Compound 4 suggests that the olefinic linkage 4e moiety is at C6H13On the long chain. While in Compound 2, the presence of a moiety m/z187 of the fragment suggests that the portion of the olefinic linkage 2d is at C5H11On the long chain. The mass spectrum cleavage paths of the attributive compounds 2 and 4 are respectively shown in the attached figures 13 and 15, and the chemical structures are as follows:
Figure BDA0001882364530000092
Figure BDA0001882364530000101
resolution of Compound 3 Compound excimer ion [ M + H ]]+m/z 510, the molecular weight of which is 18Da more than that of lipstatin, and the fact that H is introduced into the structure of lipstatin2And O. MS of the Compound2The characteristic fragment of the part a is consistent with the lipstatin, which shows that the combined water molecules are easy to remove, and the compound 3 is a lactone ring-opening hydrolysis product of the part a of the lipstatin by combining the structure of the lipstatin. The mass spectrum cleavage path of the attributive compound 3 is shown in the attached figure 14, and the chemical structure is as follows:
Figure BDA0001882364530000102
resolution of Compound 5 Compound excimer ion [ M + H ]]+m/z478, 14Da less than lipstatin, and determined that lipstatin has less CH in structure2. MS of the Compound2The characteristic fragment (m/z333,315,297,287,189) of part a of the sequence is identical to that of lipstatin, and the characteristic fragment (m/z 160,142,114) of part b disappears, and instead the existence of the characteristic fragment (m/z146, 128,100) is 14Da less than that of part b of lipstatin, and the part b is presumed to have less CH structure2. The mass spectrum cleavage path of the attributive compound 5 is shown in the attached figure 16, and the chemical structure is as follows:
Figure BDA0001882364530000111
resolution of Compound 7 Compound excimer ion [ M + H ]]+m/z 506 with a molecular weight 14Da higher than that of lipstatin, and the fact that CH is introduced into the structure of lipstatin2. MS of the Compound2The characteristic fragment of part b is consistent with lipstatin, and the ion peak signal m/z 347 of part a is presumed to be CH2At part a, the structure; the existence of m/z189 fragment indicates that part a1 remains, and the existence of m/z 163 fragment indicates that part a2 introduces CH2. The mass spectrum cleavage route of the attributive compound 7 is shown in the attached figure 17, and the chemical structure is as follows:
Figure BDA0001882364530000112
resolution of Compound 8 Compound excimer ion [ M + H ]]+m/z494, the molecular weight is 2Da more than that of lipstatin, and 2H is introduced into the lipstatin structure. MS of the Compound2The characteristic fragment of part b is consistent with lipstatin, the characteristic fragment of part a (m/z 335,317,299,289) is 2Da more than that of part a of lipstatin, and the structural change of part a is presumed; the m/z189 fragment disappeared, and instead the presence of the characteristic fragment m/z191, indicated that the 8d moiety was augmented with 1 saturated bond. The mass spectrum cleavage route of the assigned compound 8 is shown in figure 18, and the chemical structure is as follows:
Figure BDA0001882364530000121
although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Claims (2)

1. A method for identifying lipstatin metabolites in a streptomyces toxytricini fermentation product is characterized by comprising the following specific operations: (1) extracting chemical components from the streptomyces toxytricini fermentation liquor; (2) measuring the extracting solution by using a high performance liquid chromatography-electrospray-quadrupole-time of flight mass spectrometer HPLC-ESI-Q-TOF-MS, and obtaining the retention time of each chemical component in the extracting solution according to the high performance liquid chromatogram and the total ion flow diagram of the extracting solution; (3) performing mass spectrometry on known compounds lipstatin and unknown compounds in the extracting solution by using HPLC-ESI-Q-TOF-MS to obtain mass spectrometry ion signal quantities of different compounds, and preliminarily deducing chemical structures; (4) the mass spectrum cracking rule of each compound is obtained by comparing fragment ion signals of known compound lipstatin and unknown compound in the extracting solution, so that the compound structure identification of unknown components is realized;
the HPLC-ESI-Q-TOF-MS in the step (2) is carried out by connecting a high performance liquid chromatograph on a quadrupole mass spectrometer in series and monitoring by adopting a multi-reaction mode;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is Kromasil C18 with specification of 4.6mm × 250mm and 5 μm, the mobile phase is water-acetonitrile 15:85, V/V, flow rate is 1.0ml/min, column temperature is 30 ℃, and sample injection amount is 10 μ l;
the four-stage rod mass spectrometer has the following operating conditions: the ion source is an electrospray ion source, a positive ion mode is adopted, the offset voltage of an end plate is-500V, the electrophoresis voltage of a capillary tube is 3500V, the carrier gas is helium, the atomizing and drying gas is high-purity nitrogen, the outlet voltage of the capillary tube is 135V, the voltage of a conical hole is 65V, the desolventizing temperature is 350 ℃, the flow rate of the drying gas is 10L/min, the pressure of an atomizer is 40psi, the collision energy is 8-45V, and the scanning range is m/z 100-1700;
wherein, the result of the step (2) shows that the extracting solution contains 8 obvious chemical components according to the high performance liquid chromatogram and the total ion flow diagram of the extracting solution, and the retention time RT is respectively 7.23, 9.40, 9.79, 10.29, 10.83, 12.49, 14.59 and 17.39 min;
the mass spectrum ion signal amount of different compounds in the step (3), wherein the compound 1, RT 7.23min, the molecular ion peak is [ M + H ]]+ is m/z 508.3453, formula C29H49NO6；MS2Shows characteristic ion fragment peaks m/z 349.28,331.26,303.27,285.25,189.16,165.13,160.12,147.12 and 114.09; compound 2, RT 9.40min, molecular ion peak is [ M + H]+ is m/z490.3537, formula C29H47NO5；MS2Shows characteristic ion fragment peaks m/z 331.27,285.26,187.15,160.12,149.12,142.11,133.10, and 114.09; compound 3, RT 9.79min, molecular ion peak is [ M + H]+ is m/z 510.3262, formula C29H51NO6；MS2Characteristic ion fragment peaks m/z 333.28,287.28,189.16,160.11,149.08,119.09 and 114.09; compound 4, RT 10.29min, molecular ion peak is [ M + H]+ is m/z490.3537, formula C29H47NO5；MS2Shows characteristic ion fragment peaks m/z 331.27,285.26,189.17,160.12,147.12,142.11,133.10, and 114.09; compound 5, RT 10.83min, molecular ion peak is [ M + H]+ is m/z478.3533, formula C28H47NO5；MS2Characteristic ion fragment peaks m/z 333.28,287.27,189.16,149.13,146.08,128.07 and 100.08; compound 6 is lipstatin, RT12.49, molecular ion peak is [ M + H ]]+ is m/z492.3719, formula C29H49NO5；MS2Shows characteristic ion fragment peaks m/z 333.28,287.28,189.17,160.10,149.13,142.09,119.09, and 114.09; compound 7, RT 14.59min, molecular ion peak is [ M + H]+ is m/z 506.3850, formula C30H51NO5；MS2Shows characteristic ion fragment peaks m/z 347.28,301.29,189.16,160.10,149.13,142.09, and 114.09; compound 8, RT 17.39min, molecular ion peak is [ M + H ]]+ is m/z494.3851, formula C29H51NO5；MS2Characteristic ion fragment peaks m/z 335.30,289.29,191.18,160.10,149.13,142.09 and 114.09 are shown.
2. The method for identifying a lipstatin metabolite in a Streptomyces toxytricini fermentation product according to claim 1, wherein: wherein the extraction process of the chemical components in the streptomyces toxytricini fermentation broth in the step (1) is as follows: firstly, fermenting and culturing Streptomyces toxytricini, filtering after fermentation, and collecting hyphae; soaking the mycelium in 95% ethanol, stirring, and collecting ethanol extractive solution; the extract contains lipstatin metabolites.
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