CN113030302B - Method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in vinasse - Google Patents
Method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in vinasse Download PDFInfo
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Abstract
The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in vinasse. Aiming at the problems that the method for effectively separating and identifying the cyclic peptide and the cyclic peptide ethyl ester in the distilled grains cannot provide basis for researching cyclic peptide components in the distilled spirit, the invention provides a method for separating and identifying the cyclic peptide and the cyclic peptide ethyl ester in the distilled grains, which utilizes pretreatment means such as liquid-liquid extraction, silica gel column chromatography and the like to extract and separate the cyclic peptide and the cyclic peptide ethyl ester in the distilled grains, and adopts a high-resolution high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry technology to identify the cyclic peptide and the cyclic peptide ethyl ester based on the similarity of mass-to-charge ratio, relative isotope abundance, isotope proportion and the like and secondary mass spectrometry characteristic ions. The method qualitatively analyzes the novel compounds of cyclic peptide and cyclic peptide ethyl ester in the vinasse, and provides technical support for the development and application of more compounds.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in vinasse.
Background
The cyclic peptides are peptides formed by condensation of peptide bonds among amino acid molecules, and amino groups and carboxyl groups at two ends of the cyclic peptides can be subjected to peptide bond condensation again under certain conditions to form stable cyclic peptides. The cyclic peptide of the small molecule has small molecular weight and simple structure, can directly enter cells in the original shape through the permeation of cell membranes, participates in the growth, development, physiological functions and the like of the cells, and has wide biological activity. Cyclic peptide ethyl esters are generally produced by the reaction of an alcohol with a carboxyl group exposed outside the ring, which is easily reacted with an acidic amino acid among amino acids constituting the cyclic peptide, and the dehydration and esterification of the hydroxyl group.
The distillers' grains are the fermentation product of grain material with certain amount of yeast powder and through long-term solid fermentation. The solid brewed white spirit is distilled product of distilled grains, and in the distillation process, the components which are difficult to volatilize and are not volatilized in the distilled grains are also carried into the white spirit along with the steam through the distilled grain layer. Therefore, the active cyclopeptide component in the distilled spirit can provide a certain basis for the active cyclopeptide component in the distilled spirit, and the cyclopeptide component in the distilled spirit can be further researched by researching the active cyclopeptide component in the distilled spirit, so that a certain theoretical basis is provided for solving the action mechanism of the distilled spirit on the human body.
However, because the content of the cyclic peptide component in the distillers ' grains is very low, the starch, sugar, proteins, acids and alcohols in the distillers ' grains are high, and it is very difficult to separate the cyclic peptide and the cyclic peptide ethyl ester from the distillers ' grains; the newly discovered compounds are not standard to be contrasted, and the identification of the unknown cyclopeptides and cyclopeptides ethyl ester compounds is more difficult.
At present, related reports of separating and identifying cyclic peptides and cyclic peptide ethyl esters in vinasse are not seen, and development of a method capable of separating and identifying cyclic peptides and cyclic peptide ethyl esters in vinasse is needed.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the prior art does not see the problem that a method capable of effectively separating and identifying the cyclopeptides and the cyclopeptides ethyl esters in the distilled spirit cannot provide basis for researching the cyclopeptides in the distilled spirit.
The technical scheme for solving the technical problems is as follows: a method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in distiller's grains is provided. The method comprises the following steps:
a. extraction of cyclic peptides and cyclic peptide ethyl esters in vinasse: soaking distiller's grains in methanol or ethanol, extracting, filtering, concentrating the filtrate to obtain concentrated solution, removing impurities from the concentrated solution by petroleum ether or diethyl ether, extracting with ethyl acetate, and concentrating the extract to obtain crude extract;
b. separation of cyclic peptides and cyclic peptide ethyl esters: separating the crude extract by normal phase chromatographic column, collecting and concentrating in segments to obtain target sample;
c. identification of cyclic peptides and cyclic peptide ethyl esters: and identifying the target sample by adopting a liquid chromatography-mass spectrometry technology.
In the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in the vinasse, the ratio of the volume of methanol or ethanol to the weight of the vinasse in the step a is 1:1.5-3.
In the method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in the vinasse, the volume ratio of petroleum ether or diethyl ether to the concentrated solution in the step a is 1-2:1.
In the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in vinasse, the volume ratio of ethyl acetate to concentrated solution in the step a is 1-2:1.
Preferably, in the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in distillers' grains, the step a is repeated for 2-3 times during extraction.
Wherein, in the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in vinasse, the normal phase chromatography in the step b meets at least one of the following:
the stationary phase adopts normal phase silica gel;
normal phase silica gel is 100-300 meshes;
ethyl acetate and n-butanol are used as eluting reagents;
gradient washing is sequentially carried out according to the volume ratio of ethyl acetate to n-butanol of 100:0, 80:1, 50:1 and 20:1.
Wherein, in the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in the vinasse, the concentration temperature of the step b is 50-80 ℃.
In the method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in the vinasse, the high performance liquid chromatography in the step c adopts reverse phase chromatography, and the organic phase is methanol or acetonitrile.
Further, in the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in distillers' grains, the high performance liquid chromatography adopts gradient elution, and the mobile phase is: 0-15 min: 10-100% v/v methanol; 15.1 to 22min:100% v/v methanol; 22.1 to 24 minutes: 10% v/v methanol; the elution was stopped.
Further, in the method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in distillers' grains, the chromatographic column of the high performance liquid chromatography is a C18 column; preferably, the chromatographic column is a ZORBAX Eclipse Plus C column; preferably, the ZORBAX Eclipse Plus C column is 100mm long, 2.1mm in inner diameter and 1.8 μm in particle size.
Further, in the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in vinasse, the temperature of the high performance liquid chromatography column is 20-30 ℃, the flow rate is 0.1-0.4 mL/min, and the sample injection amount is 1-10 uL; preferably, the column temperature is 30 ℃, the flow rate is 0.2mL/min, and the sample injection amount is 2uL.
Further, in the method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in distillers grains, the aqueous phase contains 0.1g/L formic acid or acetic acid.
In the method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in vinasse, the mass spectrum in the step c is quadrupole time-of-flight mass spectrum, and positive mode detection is adopted.
Further, in the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in distillers grains, the detection conditions of the mass spectrum are as follows: adopting an electrospray ion source, wherein the temperature of drying gas is 300-350 ℃, the flow rate of drying gas is 8-12L/min, the atomization gas pressure is 30-40 psig, the capillary voltage is 3500-4000 v, and the capillary outlet voltage is 60-110 v; preferably, the drying gas temperature is 325 ℃, the drying gas flow is 10L/min, the atomizing gas pressure is 40psig, the capillary voltage is 4000v, and the capillary outlet voltage is 90v.
The distillers' grains are five grains of the five-grain strong aromatic Chinese liquor, and are obtained by adding strong aromatic Chinese liquor Daqu powder into five grains of rice, corn, glutinous rice, sorghum and wheat and carrying out solid state fermentation.
Wherein the cyclic peptide is cyclic tripeptide; preferably a ring (Leu-Glu-Leu) or a ring (Glu-Leu-GABA).
Wherein the cyclic peptide ethyl ester is cyclic (Leu-Glu) -ethyl ester, cyclic (Phe-Glu) -ethyl ester, cyclic (Pro-Glu) -ethyl ester, cyclic (Glu-Tyr) -ethyl ester, cyclic (Glu-Ala) -ethyl ester, cyclic (Leu-Glu-Val) -ethyl ester or cyclic (Leu-Glu-Leu) -ethyl ester.
The beneficial effects of the invention are as follows:
the invention provides a method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in vinasse, which utilizes pretreatment means such as liquid-liquid extraction, silica gel column chromatography and the like to extract and separate the cyclic peptide and cyclic peptide ethyl ester in the vinasse, and uses a high-resolution high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry combination technology to identify the cyclic peptide and cyclic peptide ethyl ester based on similarity of mass-to-charge ratio, relative isotope abundance, isotope proportion and the like and secondary mass spectrometry characteristic ions. The method can rapidly and accurately identify the cyclic peptide and the cyclic peptide ethyl ester in the distilled spirit, is simple and convenient to operate, and provides a theoretical basis for identifying the cyclic peptide component in the distilled spirit.
Drawings
FIG. 1 is a secondary mass spectrum of cyclo (Leu-Glu) -ethyl ester in example 2;
FIG. 2 is a secondary mass spectrum of cyclo (Phe-Glu) -ethyl ester in example 2;
FIG. 3 is a secondary mass spectrum of cyclo (Pro-Glu) -ethyl ester in example 2;
FIG. 4 is a secondary mass spectrum of cyclo (Glu-Tyr) -ethyl ester in example 2;
FIG. 5 is a secondary mass spectrum of cyclo (Glu-Ala) -ethyl ester in example 2;
FIG. 6 is a secondary mass spectrum of cyclo (Leu-Glu-Val) -ethyl ester in example 2;
FIG. 7 is a secondary mass spectrum of cyclo (Leu-Glu-Leu) -ethyl ester in example 2;
FIG. 8 is a secondary mass spectrum of the ring (Leu-Glu-Leu) in example 2;
FIG. 9 is a graph of the secondary mass spectrum of cyclo (Glu-Leu-GABA) in example 2;
FIG. 10 is a diagram of the structure of cyclo (Leu-Glu) -ethyl ester in example 2;
FIG. 11 is a diagram showing the structure of cyclo (Phe-Glu) -ethyl ester in example 2;
FIG. 12 is a block diagram of cyclo (Pro-Glu) -ethyl ester in example 2;
FIG. 13 is a diagram of the structure of cyclo (Glu-Tyr) -ethyl ester in example 2;
FIG. 14 is a diagram of the structure of cyclo (Glu-Ala) -ethyl ester in example 2;
FIG. 15 is a diagram showing the structure of cyclic (Leu-Glu-Val) -ethyl ester in example 2;
FIG. 16 is a diagram showing the structure of cyclic (Leu-Glu-Leu) -ethyl ester in example 2;
FIG. 17 is a diagram showing the structure of a ring (Leu-Glu-Leu) in example 2;
FIG. 18 is a diagram showing the structure of cyclic (Glu-Leu-GABA) structure in example 2.
Detailed Description
The invention provides a method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in vinasse, wherein the vinasse refers to vinasse of five-grain strong-flavor white spirit, and the vinasse is prepared by adding five grains of rice, corn, glutinous rice, sorghum and wheat into strong-flavor white spirit Daqu powder and carrying out solid state fermentation.
The invention firstly separates and identifies the cyclic tripeptide compound from the vinasse to obtain the cyclic tripeptide compound: ring (Leu-Glu-Leu) and ring (Glu-Leu-GABA) are also first identified to obtain cyclic dipeptide ethyl esters and cyclic tripeptide ethyl compounds in distillers' grains: cyclic (Leu-Glu) -ethyl ester, cyclic (Phe-Glu) -ethyl ester, cyclic (Pro-Glu) -ethyl ester, cyclic (Glu-Tyr) -ethyl ester, cyclic (Glu-Ala) -ethyl ester, cyclic (Leu-Glu-Val) -ethyl ester, cyclic (Leu-Glu-Leu) -ethyl ester.
The content of the cyclic peptide and the cyclic peptide ethyl ester compound in the distilled grain is very low, and the invention provides technical basis for further researching the existence of the substances in distilled liquor components by separating and identifying the substances in the distilled grain.
The invention uses high-resolution high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to analyze and identify the target, which is very difficult. The technology has no existing compound spectrum library, and needs to splice compound structures according to mass-to-charge ratio, isotope, molecular ions, secondary fragment characteristic ions and other mass spectrum information of the compound to perform analysis and identification of unknown substances so as to qualify the compound.
In this context, the present invention developed a method for the separation and identification of cyclic tripeptides and cyclic tripeptidyl ethyl esters in distillers grains. The method is simple and applicable, and has low cost.
Specifically, the method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in vinasse comprises the following steps:
a. extraction of cyclic peptides and cyclic peptide ethyl esters in vinasse: soaking distiller's grains in methanol or ethanol, extracting, filtering, concentrating the filtrate to obtain concentrated solution, removing impurities from the concentrated solution by petroleum ether or diethyl ether, extracting with ethyl acetate, and concentrating the extract to obtain crude extract;
b. separation of cyclic peptides and cyclic peptide ethyl esters: separating the crude extract by normal phase chromatographic column, collecting and concentrating in segments to obtain target sample;
c. identification of cyclic peptides and cyclic peptide ethyl esters: and identifying the target sample by adopting a liquid chromatography-mass spectrometry technology.
In the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in the vinasse, the ratio of the volume of methanol or ethanol to the weight of the vinasse in the step a is 1:1.5-3.
In the method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in the vinasse, the volume ratio of petroleum ether or diethyl ether to the concentrated solution in the step a is 1-2:1.
In the method for separating and identifying cyclic peptide and cyclic peptide ethyl ester in vinasse, the volume ratio of ethyl acetate to concentrated solution in the step a is 1-2:1.
Preferably, the extraction is repeated 2 to 3 times during the extraction.
Wherein the normal phase chromatography of step b satisfies at least one of the following:
the stationary phase adopts normal phase silica gel;
normal phase silica gel is 100-300 meshes;
ethyl acetate and n-butanol are used as eluting reagents;
gradient washing is sequentially carried out according to the volume ratio of ethyl acetate to n-butanol of 100:0, 80:1, 50:1 and 20:1.
Wherein the concentration temperature in the step b is 50-80 ℃.
Wherein, the high performance liquid chromatography in the step c adopts reverse phase chromatography, and the organic phase is methanol or acetonitrile.
Further, the high performance liquid chromatography adopts gradient elution, and the mobile phase is: 0-15 min: 10-100% v/v methanol; 15.1 to 22min:100% v/v methanol; 22.1 to 24 minutes: 10% v/v methanol; the elution was stopped.
Further, the chromatographic column of the high performance liquid chromatography is a C18 column; preferably, the chromatographic column is a ZORBAX Eclipse Plus C column; preferably, the ZORBAX Eclipse Plus C column is 100mm long, 2.1mm in inner diameter and 1.8 μm in particle size.
Further, the temperature of the high performance liquid chromatography column is 20-30 ℃, the flow rate is 0.1-0.4 mL/min, and the sample injection amount is 1-10 uL; preferably, the column temperature is 30 ℃, the flow rate is 0.2mL/min, and the sample injection amount is 2uL.
Further, the aqueous phase contains 0.1g/L formic acid or acetic acid.
The mass spectrum in the step c is quadrupole flight time mass spectrum, and positive mode detection is adopted.
Further, the detection conditions of the mass spectrum are as follows: adopting an electrospray ion source, wherein the temperature of drying gas is 300-350 ℃, the flow rate of drying gas is 8-12L/min, the atomization gas pressure is 30-40 psig, the capillary voltage is 3500-4000 v, and the capillary outlet voltage is 60-110 v; preferably, the drying gas temperature is 325 ℃, the drying gas flow is 10L/min, the atomizing gas pressure is 40psig, the capillary voltage is 4000v, and the capillary outlet voltage is 90v.
Firstly, alcohol solvents (methanol or ethanol) are selected to soak the vinasse, and the cyclic peptide and cyclic peptide ethyl ester (hereinafter referred to as target objects) in the vinasse can be effectively extracted by ultrasonic extraction; the cyclic peptide and the cyclic peptide ethyl ester belong to medium polar compounds, and the cyclic peptide ethyl ester compounds in the distillers' grains can be effectively dissolved into an alcohol system volume system by extracting the cyclic peptide and the cyclic peptide ethyl ester compounds by utilizing the alcohol system volume with medium and strong polarity.
Then properly concentrating, increasing the concentration of a target object in the extract, and performing liquid-liquid extraction on the extract concentrate by using weak-polarity petroleum ether, so that weak-polarity components such as higher fatty acid, higher fatty acid ester and the like in the extract concentrate are effectively removed; a large amount of weak polar higher fatty acid and higher fatty acid ester compounds exist in the distilled grains, and according to a similar compatibility principle, weak polar petroleum ether or diethyl ether is utilized for liquid-liquid extraction, so that weak polar components such as higher fatty acid, higher fatty acid ester and the like in the extraction concentrate can be effectively removed, and medium polar cyclic peptide and cyclic peptide ethyl ester compounds can be effectively reserved.
Then, the ethyl acetate with medium polarity is used for liquid-liquid extraction, the target object belongs to medium polarity, and according to the similar compatibility principle, the ethyl acetate can effectively extract the target object;
and finally, eluting by utilizing silica gel normal phase column chromatography, wherein ethyl acetate and normal butanol are proportioned according to a proper proportion, and eluting target substances sequentially, wherein the impurity components with strong polarity are reserved on the silica gel column, so that the effective separation between the target substances and the impurities with strong polarity is realized. After the target is concentrated and the eluent is removed, methanol is dissolved, and identification is carried out by using a high-resolution high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry technology based on similarity of mass-to-charge ratio, isotope relative abundance, isotope proportion and the like and secondary mass spectrometry characteristic ions, so that the result is accurate. The method can qualitatively analyze the novel compounds of the cyclic peptide and the cyclic peptide ethyl ester in the vinasse, and provides technical support for the development and application of more compounds.
The following examples are provided to further illustrate embodiments of the present invention and are not intended to limit the scope of the invention to the examples.
The following examples use Agilent 1290LC-6520QTOF liquid chromatograph-mass spectrometer, the rest of the raw materials and equipment are all known products, and are obtained by purchasing commercial products.
Example 1 extraction separation of cyclic peptide and cyclic peptide ethyl ester in vinasse
Weighing 1500g of distiller's grains, ultrasonically extracting with 3L of methanol for 30min, filtering, repeating twice, mixing filtrates, and rotary evaporating at 60deg.C for concentrating to obtain 600mL of extractive concentrate.
Extracting the concentrated solution with petroleum ether 0.6L for two times, extracting with ethyl acetate 1L for three times, collecting ethyl acetate extract, and concentrating by rotary evaporation at 60deg.C to obtain cyclic peptide and cyclic peptide ethyl ester crude extract.
Uniformly stirring the crude extract and 100-200 meshes of equal amount normal phase silica gel, and carrying out normal phase column chromatography on a 200-300 meshes of normal phase silica gel column. Using ethyl acetate and n-butanol as eluting reagents, and sequentially performing gradient elution according to the following proportion: 100:0, 80:1, 50:1, 20:1, ethyl acetate: n-butanol, v/v; collecting the eluent, concentrating at 60 ℃ to obtain the separated target substance.
Example 2 identification of objects
Dissolving the target substance separated in the embodiment 1 by methanol, filtering by a membrane, and feeding a sample to a high performance liquid chromatography mass spectrometer.
High performance liquid chromatography conditions: the chromatographic column is ZORBAX Eclipse Plus C column, the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 μm; the column temperature is 25 ℃, the flow rate is 0.2mL/min, and the sample injection amount is 2uL; the mobile phase is methanol and water (containing 0.1g/L formic acid), and gradient elution is carried out: 0-15 min: 10-100% v/v methanol; the method comprises the steps of carrying out a first treatment on the surface of the 15.1 to 22min:100% v/v methanol; 22.1 to 24 minutes: 10% v/v methanol; the elution was stopped.
The detection conditions of the mass spectrum are as follows: the electrospray ion source, positive mode, drying gas temperature of 325 ℃, drying gas flow of 10L/min, atomizing gas pressure of 40psig, capillary voltage of 4000v, capillary outlet voltage of 90v are used.
And identifying target objects based on similarity of mass-to-charge ratio, relative isotope abundance, isotope proportion and the like and secondary mass spectrometry characteristic ions, wherein the information of each target object is shown in the table 1 below.
TABLE 1 isolation of identified target information
From the above table, 9 target substances, namely two cyclic peptide rings (Leu-Glu-Leu) and two cyclic peptides (Glu-Leu-GABA) were obtained by separating and identifying from distiller's grains, and 7 cyclic dipeptide ethyl esters and cyclic tripeptide ethyl compounds were obtained: cyclic (Leu-Glu) -ethyl ester, cyclic (Phe-Glu) -ethyl ester, cyclic (Pro-Glu) -ethyl ester, cyclic (Glu-Tyr) -ethyl ester, cyclic (Glu-Ala) -ethyl ester, cyclic (Leu-Glu-Val) -ethyl ester, cyclic (Leu-Glu-Leu) -ethyl ester.
The secondary mass spectrogram of each target substance is shown in figures 1-9, and the structural formula is shown in figures 10-18.
According to the embodiment, the method can effectively separate and identify various cyclic peptides and cyclic peptide ethyl esters in the distilled spirit, and can provide basis for identifying relevant components in the distilled spirit. The identification method is simple to operate, suitable for popularization and high in application value.
Claims (12)
1. A method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in distillers grains, comprising the steps of:
a. extraction of cyclic peptides and cyclic peptide ethyl esters in vinasse: soaking distiller's grains in methanol or ethanol, extracting, filtering, concentrating the filtrate to obtain concentrated solution, removing impurities from the concentrated solution by petroleum ether or diethyl ether, extracting with ethyl acetate, and concentrating the extract to obtain crude extract;
b. separation of cyclic peptides and cyclic peptide ethyl esters: separating the crude extract by normal phase chromatographic column, collecting and concentrating in segments to obtain target sample;
c. identification of cyclic peptides and cyclic peptide ethyl esters: identifying a target sample by adopting a liquid chromatography-mass spectrometry technology;
the ratio of the volume of methanol or ethanol to the weight of the vinasse during the extraction in the step a is 1:1.5-3;
the volume ratio of petroleum ether or diethyl ether to the concentrated solution in the step a is 1-2:1;
the volume ratio of the ethyl acetate to the concentrated solution in the step a is 1-2:1;
the step a is repeated for 2 to 3 times during the extraction;
the normal phase chromatography described in step b satisfies the following:
the stationary phase adopts normal phase silica gel;
normal phase silica gel is 100-300 meshes;
ethyl acetate and n-butanol are used as eluting reagents;
sequentially carrying out gradient elution according to the volume ratio of ethyl acetate to n-butanol of 100:0, 80:1, 50:1 and 20:1;
9 target substances are obtained from the distillers' grains through separation and identification: cyclic Leu-Glu-Leu, cyclic Glu-Leu-GABA, cyclic Leu-Glu-ethyl ester, cyclic Phe-Glu-ethyl ester, cyclic Pro-Glu-ethyl ester, cyclic Glu-Tyr-ethyl ester, cyclic Glu-Ala-ethyl ester, cyclic Leu-Glu-Val-ethyl ester, cyclic Leu-Glu-Leu-ethyl ester.
2. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 1, wherein: the concentration temperature in the step b is 50-80 ℃.
3. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 1, wherein: the liquid chromatography in the step c adopts reverse phase chromatography, and the organic phase is methanol or acetonitrile.
4. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 1, wherein: the liquid chromatography adopts gradient elution, and the mobile phase is as follows: 0-15 min: 10-100% v/v methanol; 15.1 to 22min:100% v/v methanol; 22.1 to 24 minutes: 10% v/v methanol; the elution was stopped.
5. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 1, wherein: the chromatographic column of the liquid chromatograph is a C18 column.
6. The method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in stillage according to claim 5, wherein: the chromatographic column is a ZORBAX Eclipse Plus C18 column.
7. The method for separating and identifying cyclic peptides and cyclic peptide ethyl esters in stillage according to claim 6, wherein: the ZORBAX Eclipse Plus C column is 100mm long, 2.1mm in inner diameter and 1.8 μm in particle size.
8. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 1, wherein: the temperature of the liquid chromatographic column is 20-30 ℃, the flow rate is 0.1-0.4 mL/min, and the sample injection amount is 1-10 uL.
9. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 8, wherein: the column temperature was 30deg.C, the flow rate was 0.2mL/min, and the sample injection amount was 2uL.
10. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 1, wherein: and c, the mass spectrum is quadrupole flight time mass spectrum, and positive mode detection is adopted.
11. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 1, wherein: the detection conditions of the mass spectrum are as follows: and an electrospray ion source is adopted, the temperature of the drying gas is 300-350 ℃, the flow rate of the drying gas is 8-12L/min, the atomization pressure is 30-40 psig, the capillary voltage is 3500-4000 v, and the capillary outlet voltage is 60-110 v.
12. The method for the separation and identification of cyclic peptides and cyclic peptide ethyl esters in vinasse according to claim 11, wherein: the drying gas temperature was 325 ℃, the drying gas flow rate was 10L/min, the atomizing gas pressure was 40psig, the capillary voltage was 4000v, and the capillary outlet voltage was 90v.
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