CN112798709A - Method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology and application - Google Patents
Method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology and application Download PDFInfo
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- CN112798709A CN112798709A CN202011617102.XA CN202011617102A CN112798709A CN 112798709 A CN112798709 A CN 112798709A CN 202011617102 A CN202011617102 A CN 202011617102A CN 112798709 A CN112798709 A CN 112798709A
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Abstract
The present disclosure provides a method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology and application thereof, wherein the method comprises: and detecting the sample to be detected after the crushing treatment by adopting a GC-IMS technology. Compared with the prior art, the method can not carry out pretreatment on the sample, does not need extraction concentration and other treatments, can directly carry out sample injection detection by directly crushing the sample, greatly saves the detection time and has high accuracy.
Description
Technical Field
The invention relates to the technical field of authenticity identification of donkey-hide gelatin, in particular to a method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on a GC-IMS technology and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
For a long time, the key material of donkey-hide gelatin is donkey skin, which is derived from donkey of an equine family. Donkey-hide gelatin belongs to one of a few traditional Chinese medicines with definite curative effects. The colla Corii Asini contains gelatin, protein, lysine, histidine, arginine, threonine and microelements, and has effects of resisting anoxia, cold and fatigue, increasing in vivo calcium intake, and promoting hematopoiesis. Has been considered as a health food for health preservation and clinical treatment.
However, in recent years, the donkey-hide resources are in short supply, and donkey-hide gelatin manufacturers adopt low-value skins such as cow hide gelatin, horse hide gelatin and pigskin to replace donkey hide gelatin in order to solve the problem of raw material shortage and reduce cost, so that the benefits of consumers are seriously damaged, and the market order is disturbed. The methods of infrared, near infrared, HPLC and the like are adopted to identify the donkey-hide gelatin, and the problems of inaccurate judgment result, lack of persuasion and the like often exist. The conventional methods for identifying the authenticity of the donkey-hide gelatin which are reported at present are PCR and specific peptide identification technologies.
However, the inventors found that extracting characteristic peptides, performing comparative verification of sequences and structures, and detecting methods are complex and time-consuming. In particular, both PCR and specific peptide identification techniques require a specific method for pre-treating the sample, which is very complicated and requires a corresponding standard peptide. Therefore, it is important to develop a method for identifying donkey-hide gelatin, which is simpler, short in time consumption and high in accuracy.
Disclosure of Invention
In order to solve the problems in the prior art, the purpose of the disclosure is to provide a method for distinguishing donkey-hide gelatin from other animal source gelatin based on a GC-IMS technology and application thereof.
Specifically, the technical scheme of the present disclosure is as follows:
in a first aspect of the present disclosure, the present disclosure provides a method for distinguishing donkey-hide gelatin from other animal-derived gums based on GC-IMS technology, comprising: and detecting the sample to be detected after the crushing treatment by adopting a GC-IMS technology.
In a second aspect of the disclosure, the disclosure provides an application of a method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology in identifying the authenticity of donkey-hide gelatin.
One or more technical schemes in the disclosure have the following beneficial effects:
(1) the donkey-hide gelatin and other animal source gelatin are distinguished by utilizing a gas chromatography-ion mobility spectrometry (GC-IMS) technology for the first time, and the advantages of the gas chromatography are just embedded with the weak potential of the ion mobility spectrometry through the combination of the ion mobility spectrometry and the gas chromatography, so that the advantages are exerted respectively, the advantages are made up, and the effects of adding one and being more than two are obtained.
(2) The traditional method for identifying the authenticity of the donkey-hide gelatin needs to carry out a complicated pretreatment process on a sample to be detected, and the detection process is complex and consumes long time. However, the GC-IMS is used for identifying the authenticity of the donkey-hide gelatin, so that the sensitivity is higher, the detection speed is higher, the analysis cost is lower, no chemical reagent is needed for carrying out complex pretreatment on a sample to be detected, and only the sample to be detected needs to be crushed to directly sample. The detection method is simpler, and the time and the cost are greatly saved.
(3) The detection method has the advantages of high sensitivity (the detection limit reaches ppt), high detection speed (25-35 minutes), and accurate qualitative and quantitative determination.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.
Embodiments of the present disclosure are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: three-dimensional spectra of volatile components in the samples of example 1;
FIG. 2: spectrum of volatile matter composition in sample of example 1 (top view);
FIG. 3: comparison of the difference spectrum of the volatile substance components in the sample of example 1;
FIG. 4: the Gallery Plot fingerprint spectrum of the sample of example 1;
FIG. 5: PCA analysis of all samples of example 1;
FIG. 6: qualitative analysis of Library Search of example 1.
Detailed Description
The disclosure is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, most of the existing methods for identifying donkey-hide gelatin extract theoretical characteristic peptides, and compare and verify sequences and structures, and the detection method is complex and consumes a long time. In particular, both PCR and specific peptide identification techniques require a specific method for pre-treating the sample, which is very complicated and requires a corresponding standard peptide. In order to improve the efficiency of donkey-hide gelatin identification, save time and cost and improve accuracy, the present disclosure provides a method and application for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology.
In one embodiment of the present disclosure, the present disclosure provides a method for distinguishing donkey-hide gelatin from other animal-derived gums based on GC-IMS technology, comprising: and detecting the sample to be detected after the crushing treatment by adopting a GC-IMS technology.
Wherein the sample to be tested can be DHG (donkey hide gelatin), CHG (bovine hide gelatin), HHG (horse hide gelatin) or PHG (pig hide gelatin).
The sample to be detected is firstly pre-separated by gas chromatography, the concentration of the analyte is controlled, impurities in the analyte are separated, then secondary separation in the true sense is realized by re-separation of ion mobility spectrometry, the obtained retention time and migration time can accurately carry out two-dimensional qualitative analysis on the analyte, and the analyte can be quantitatively analyzed by knowing the ion signal peak intensity in the ion mobility spectrometry. After data is processed by software, the fingerprint spectrum of trace volatile organic compounds can be rapidly obtained, sample classification is carried out by combining the main components of the analytes, the difference among the substances is clear at a glance, the qualitative and quantitative analysis on a single substance is simple and easy to implement, and a very visual analysis result is provided.
In one embodiment of the present disclosure, the present disclosure provides a method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology, which specifically includes the following steps:
(1) weighing samples to be detected DHG (donkey hide gelatin), CHG (bovine hide gelatin), HHG (horse hide gelatin) and PHG (pig hide gelatin), crushing, placing in a 20mL headspace bottle, incubating, injecting via headspace, and performing gas chromatography-ion mobility spectrometry to obtain volatile component information of various samples.
(2) Collecting the gas phase retention time and the ion migration time of the volatile components, drawing a gas phase ion migration chromatogram and carrying out qualitative analysis;
(3) collecting volatile component signal peaks, and producing fingerprint spectrums of all samples for comparison;
(4) and carrying out similarity analysis on the sample by PCA principal component analysis.
Further, in the step (1), the crushing treatment method comprises a tissue triturator, a flour mill, a wall breaking machine and the like.
Further, in the step (1), the amount of the sample to be detected is 1-3g, preferably 2 g.
Further, in step (1), the sample to be tested placed in the headspace bottle is incubated at a temperature of 50-70 ℃, preferably, at a temperature of 60 ℃.
Further, in the step (1), the incubation time is 10-30min, preferably 15 min.
Further, in the step (1), the sample amount is 400-.
Further, in the step (1), gas chromatography-ion mobility spectrometry is adopted, and the gas chromatography conditions are as follows: a chromatographic column: MXT-515 m ID of 0.53 mm; the column temperature is 50-70 ℃; carrier gas: n is a radical of2(ii) a Flow rate of carrier gas: 0-2min, 2 ml/min; 2-20min, 100ml/min
Most preferably, the column temperature of the column is 60 ℃.
Further, in the gas chromatography-ion mobility spectrometry analysis, the Ion Mobility Spectrometry (IMS) conditions are as follows:
the temperature of the migration tube is 40-50 ℃; carrier gas/drift gas: n is a radical of2(ii) a Drifting airflow rate: 140-160 mL/min.
Preferably, the temperature of the migration tube is 45 ℃.
Preferably, the drift gas flow rate is 150 mL/min.
Further, the temperature of the injection needle is 60-70 ℃, preferably, the temperature of the injection needle is 65 ℃; the sample injection amount is 300-600. mu.L, preferably 500. mu.L.
Further, in the step (1), the testing time of the gas chromatography-ion mobility spectrometry is 20-40min, and preferably, the testing time is 30 min.
Further, in the step (2), the gas phase ion mobility chromatogram and qualitative analysis can be used for qualitatively analyzing the substances through a built-in NIST database and an IMS database.
Further, in the step (3), the fingerprint of each sample is produced by selecting all volatile component signal peaks by using a Gallery Plot plug box of LAV software to form the fingerprint of each sample.
In one embodiment of the disclosure, the disclosure provides an application of a method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology in identifying the authenticity of the donkey-hide gelatin.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.
Example 1
Sample treatment: samples DHG (donkey hide gelatin), CHG (bovine hide gelatin), HHG (horse hide gelatin) and PHG (pig hide gelatin) are crushed, 2g of samples are weighed respectively and placed in a 20mL headspace bottle, incubated at 60 ℃ for 15 minutes and injected with 500 mL.
An analytical instrument:
flavor analyzer
Chromatographic conditions are as follows:
a chromatographic column: MXT-515 m ID of 0.53 mm; column temperature: 60 ℃; carrier/drift gas N2(ii) a The carrier gas flow rates were as follows:
| Time | E1 | E2 | R |
| 00:00,000 | 150mL/min | 2ml/min | rec |
| 02:00,000 | 150mL/min | 2ml/min | - |
| 20:00,000 | 150mL/min | 100ml/min | Stop |
the conditions for gas phase ion mobility chromatography were:
analysis time: 20 min; type of column: MXT-515 m ID of 0.53 mm; the column temperature is 60 ℃; drift tube temperature 45 ℃, carrier/drift gas: n is a radical of2Drift airflow rate: 140-160 mL/min; the temperature of the injection needle is 65 ℃, and the injection amount is 500 mu L.
Then, data analysis was performed:
the detection based on gas phase-ion mobility spectrometry uses the matched analysis software of instruments including LAV (laboratory Analytical viewer) and three plugins as well as GC × IMS Library Search, and can respectively analyze samples from different angles. The functions of the various software and plug-ins are as follows:
1) and (5) LAV: used for checking and analyzing a spectrogram, wherein each point in the spectrogram represents a volatile organic compound; after a standard curve is established, quantitative analysis can be carried out.
2) Reporter plug-in: spectral differences between samples were directly compared (two-dimensional top view and three-dimensional spectra).
3) Gallery Plot insert: and comparing fingerprint spectrograms, and visually and quantitatively comparing the difference of the volatile organic compounds among different samples.
4) Dynamic PCA plug-in: and the dynamic principal component analysis is used for clustering and analyzing the samples and quickly determining the type of the unknown sample.
5) GC × IMS Library Search: the NIST database and the IMS database built in the application software can perform qualitative analysis on the substances.
The grouped data of gas phase-ion mobility spectrometry are further subjected to multivariate statistical analysis by using SIMCA 14.1 software, and the multivariate statistical analysis methods used include PCA analysis, OPLS-DA analysis and PLS-DA analysis. And performing confidence test on the result of the OPLS-DA analysis for 200 times, screening potential differential metabolites under the condition that the VIP value is greater than 1 in two groups with better separation results and better confidence test results in the multivariate statistical analysis, and performing further analysis and explanation on the potential differential metabolites qualitatively serving as final differential metabolites.
The software was able to automatically generate a three-dimensional spectrum (fig. 1, retention time, migration time and peak intensity) and a two-dimensional top view (fig. 2 and 3, retention time and migration time) of the sample volatile species. The differences of volatile organic compounds in different samples can be visually seen from the three-dimensional graph, and the differences and changes of volatile substances can be more obviously reflected by combining the corresponding two-dimensional top view.
And (4) conclusion: from fig. 4, the overall comparison and analysis of all samples can be performed, and the composition difference of volatile substances between samples is very obvious and intuitive, such as octanone, nonanone, decanone and the like, in the CHG sample, which is much higher than other samples; the relative content of hexanal, heptanal, nonanal, butyraldehyde and other substances in the DHG is far higher than that of other samples; HHG and PHG have certain similarity, and the relative contents of volatile components are relatively close.
Principal Component Analysis (PCA) analysis:
as shown in FIG. 5, the characteristic differences and clustering trends among the samples can be seen, the DHG is farthest from other samples, the characteristic differences are most obvious, the HHG and the PHG are relatively close to each other, and the characteristics have certain similarity.
The GC-IMS analysis technology can clearly distinguish donkey-hide gelatin from horse skin, pigskin and cow skin gelatin.
Qualitative analysis of volatile organic compounds in sample
The qualitative analysis of substances can be performed by NIST database and IMS database built in the application software, Table 1. identified characteristic odor substances of colla Corii Asini and other animal source gums
Specific metabolites in donkey-hide gelatin
Specific metabolite in equine hide glue
Specific metabolite in pigskin glue
Specific metabolite in oxhide gelatin
| Compound | Name of Chinese | CAS# | Formula | MW |
| 2-Decanone | 2-decanones | C693549 | C10H20O | 156.3 |
| 2-nonanone-M | 2-nonanones | C821556 | C9H18O | 142.2 |
| 2-nonanone-D | 2-nonanone dimer | C821556 | C9H18O | 142.2 |
| 2-Octanone-M | 2-octanones | C111137 | C8H16O | 128.2 |
| Benzaldehyde | Benzaldehyde | C100527 | C7H6O | 106.1 |
| 2-Octanone-D | 2-octanone dimer | C111137 | C8H16O | 128.2 |
| 2-heptanone | 2-heptanone | C110430 | C7H14O | 114.2 |
| 2-Hexanone-D | 2-hexanone dimers | C591786 | C6H12O | 100.2 |
| 2-Hexanone-M | 2-hexanones | C591786 | C6H12O | 100.2 |
| Propanoic acid | Propionic acid | C79094 | C3H6O2 | 74.1 |
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology is characterized by comprising the following steps: and detecting the sample to be detected after the crushing treatment by adopting a GC-IMS technology.
2. A method for distinguishing donkey-hide gelatin from other animal-derived gelatin based on GC-IMS technology is characterized by comprising the following steps:
(1) weighing DHG, CHG, HHG and PHG to be detected, crushing, placing in a 20mL headspace bottle, incubating, introducing sample via headspace, and performing gas chromatography-ion mobility spectrometry to obtain volatile component information of various samples.
(2) Collecting the gas phase retention time and the ion migration time of the volatile components, drawing a gas phase ion migration chromatogram and carrying out qualitative analysis;
(3) collecting volatile component signal peaks, and producing fingerprint spectrums of all samples for comparison;
(4) and carrying out similarity analysis on the sample by PCA principal component analysis.
3. The method for distinguishing donkey-hide gelatin from gelatin of other animal sources based on the GC-IMS technology as claimed in claim 1, wherein in the step (1), the crushing treatment method comprises a tissue triturator, a flour mill or a wall breaking machine;
further, in the step (1), the amount of the sample to be detected is 1-3g, preferably 2 g;
further, in step (1), the sample to be tested placed in the headspace bottle is incubated at a temperature of 50-70 ℃, preferably, at a temperature of 60 ℃.
4. The method for distinguishing donkey-hide gelatin from other animal-derived gums based on the GC-IMS technology as claimed in claim 1, wherein in the step (1), the incubation time is 10-30min, preferably 15 min;
further, in the step (1), the sample injection amount is 400-;
further, in the step (1), gas chromatography-ion mobility spectrometry is adopted, and the gas chromatography conditions are as follows: a chromatographic column: MXT-515 m ID of 0.53 mm; the column temperature is 50-70 ℃; carrier gas: n is a radical of2(ii) a Flow rate of carrier gas: 0-2min, 2 ml/min; 2-20min, 100 ml/min; most preferably, the column temperature of the column is 60 ℃.
5. The GC-IMS based method of claim 1The method for preparing the glue and other animal source glue is characterized in that in gas chromatography-ion mobility spectrometry, the Ion Mobility Spectrometry (IMS) conditions are as follows: the temperature of the drift tube is 40-50 ℃; carrier gas/drift gas: n is a radical of2(ii) a Drifting airflow rate: 140-160 mL/min.
6. The method for distinguishing donkey-hide gelatin from other animal-derived gums based on GC-IMS technology as claimed in claim 1 wherein the drift tube temperature is 45 ℃; preferably, the drift gas flow rate is 150 mL/min; further, the temperature of the injection needle is 60-70 ℃, preferably, the temperature of the injection needle is 65 ℃; the sample injection amount is 300-600. mu.L, preferably 500. mu.L.
7. The method for distinguishing donkey-hide gelatin from other animal-derived gums according to claim 1, wherein the GC-IMS technique is used in step (1), wherein the GC-IMS test time is 20-40min, preferably 30 min.
8. The method for distinguishing colla Corii Asini from other animal-derived gums based on GC-IMS of claim 1 wherein in step (2) the gas phase ion mobility chromatogram and qualitative analysis is performed by built-in NIST database and IMS database to perform qualitative analysis of the substances.
9. The method for distinguishing colla Corii Asini from other animal-derived gums according to claim 1 using GC-IMS wherein in step (3) each sample fingerprint is generated by selecting all the volatile component signal peaks using the Gallery Plot box of the LAV software to form each sample fingerprint.
10. Use of a method for distinguishing between donkey-hide gelatin and other animal-derived gums according to any of claims 1 to 9 based on GC-IMS technology for the identification of the authenticity of donkey-hide gelatin.
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