CN115420832A - GC-MS fingerprint spectrum and multi-index content determination method of litsea pungens oil - Google Patents
GC-MS fingerprint spectrum and multi-index content determination method of litsea pungens oil Download PDFInfo
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- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000001228 spectrum Methods 0.000 title claims abstract description 32
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- 239000001289 litsea cubeba fruit oil Substances 0.000 claims abstract description 66
- 238000005303 weighing Methods 0.000 claims abstract description 49
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- 239000007788 liquid Substances 0.000 claims abstract description 28
- 238000002791 soaking Methods 0.000 claims abstract description 27
- 239000002245 particle Substances 0.000 claims abstract description 15
- 239000000341 volatile oil Substances 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 238000007789 sealing Methods 0.000 claims abstract description 10
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- 239000000463 material Substances 0.000 claims abstract description 7
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- 238000012360 testing method Methods 0.000 claims description 26
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- WEEGYLXZBRQIMU-WAAGHKOSSA-N Eucalyptol Chemical compound C1C[C@H]2CC[C@]1(C)OC2(C)C WEEGYLXZBRQIMU-WAAGHKOSSA-N 0.000 claims description 7
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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Abstract
The invention discloses a GC-MS fingerprint spectrum and multi-index content determination method of litsea cubeba oil, belonging to the field of litsea cubeba oil quality, a GC-MS fingerprint spectrum and multi-index content determination method of litsea cubeba oil, and a GC-MS fingerprint spectrum determination method of litsea cubeba oil, comprising the following determination methods: s1: accurately weighing 20g of litsea pungens, placing in a flask, and respectively inspecting the influence of four factors of the crushed particle size of the medicinal materials, the material-liquid ratio, the soaking time and the extraction time on the oil yield; s2: adding appropriate amount of anhydrous sodium sulfate into the collected volatile oil, sealing overnight, centrifuging at 8000r/min for 10min the next day, collecting upper oil layer, weighing, paralleling for 3 times, and calculating oil yield; s3: the solution preparation and the analysis result can be realized by adopting the GC-MS technology to research the chemical components in the litsea cubeba oil, preliminarily defining the material basis and laying the foundation for the subsequent researches such as quality control and the like.
Description
Technical Field
The invention relates to the field of the quality of litsea cubeba oil, in particular to a GC-MS fingerprint spectrum and a multi-index content determination method of the litsea cubeba oil.
Background
The litsea pungens is dry mature fruit of cinnamomum migao in Lauraceae, also called Maotan mother, tou Murraya koenigii and Chinese Alangium, mainly produced in Guizhou, guangxi, yunnan and other places in China, and the litsea pungens oil is volatile oil obtained from the litsea pungens dry fruit by a steam distillation method, is a common medicine for minority nationalities in Guizhou, and is mainly used for treating cardiovascular diseases such as coronary heart disease, angina pectoris and the like and gastrointestinal diseases clinically.
In the aspect of quality control of the Chinese medicinal volatile oil, the GC-MS or GC technology is mainly adopted to carry out qualitative and quantitative research on the chemical components of the volatile oil. In the traditional Chinese medicine quality evaluation system, the integrity of the traditional Chinese medicine is mostly embodied by adopting a fingerprint spectrum technology. The fingerprint spectrum is combined with chemical pattern recognition to carry out deeper mining and discussion on the fingerprint spectrum data, and the method is widely used for identification and quality evaluation of traditional Chinese medicines.
At present, domestic and foreign literature reports about the litsea cubeba oil mainly focus on the aspects of extraction and purification, chemical components and pharmacological activity, and no research on pharmacokinetics and tissue distribution is found, so that in order to enable related preparations of the litsea cubeba oil to be better applied to clinic, an analysis method combining fingerprint spectrum with multi-index content measurement needs to be established to provide scientific basis for quality control and quality evaluation of the litsea cubeba oil.
Disclosure of Invention
1. Technical problem to be solved
Aiming at the problems in the prior art, the invention aims to provide a GC-MS fingerprint spectrum and a multi-index content determination method of the litsea cubeba oil, which can be realized by researching chemical components in the litsea cubeba oil by adopting a GC-MS technology, preliminarily defining the material basis of the litsea cubeba oil and laying a foundation for subsequent researches on quality control and the like.
2. Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
The GC-MS fingerprint spectrum determination method of the litsea cubeba oil comprises the following determination methods:
s1: accurately weighing 20g of litsea pungens, placing in a flask, and respectively inspecting influence of four factors of the crushed particle size of the medicinal materials, the material-liquid ratio, the soaking time and the extraction time on the oil yield;
s2: adding appropriate amount of anhydrous sodium sulfate into the volatile oil, sealing overnight, centrifuging at 8000r/min for 10min the next day, collecting upper oil layer, weighing, paralleling for 3 times, and taking the average value to calculate oil yield;
s3: establishing a GC-MS fingerprint;
s4: solutions were prepared and the results analyzed.
Further, the influence on the oil yield of the litsea pungens comprises the following factors:
influence of the crushed particle size on the oil yield: crushing litsea pungens by using a crusher, respectively inspecting the crushed litsea pungens powder by using a sieve of 10 meshes, 30 meshes, 60 meshes, 80 meshes and 100 meshes, accurately weighing 20g, soaking for 0h and extracting for 5h, comparing oil yield and determining the optimal crushed particle size, wherein the material-liquid ratio is 1;
influence of feed liquid ratio on oil yield: crushing litsea pungens by using a crusher, sieving crushed litsea pungens powder by a 60-mesh sieve, accurately weighing 20g, respectively observing a material-liquid ratio of 1;
influence of soaking time on oil yield: crushing litsea pungens by using a crusher, sieving crushed litsea pungens powder by a 60-mesh sieve, accurately weighing 20g, respectively inspecting soaking for 0h, 0.5h, 1h, 1.5h and 2h, extracting for 5h, comparing oil yield and determining optimal soaking time, wherein the material-liquid ratio is 1;
the effect of extraction time on oil yield: crushing litsea pungens by using a crusher, sieving crushed litsea pungens powder by a 60-mesh sieve, accurately weighing 20g, respectively inspecting extraction time of 2h, 3h, 4h, 5h and 6h, comparing oil yield and determining optimal extraction time, wherein the material-liquid ratio is 1.
Further, performing an extraction test of the litsea pungens oil on the result of the single-factor test, accurately weighing 20g of crushed litsea pungens, calculating the oil yield, and paralleling for 3 times;
the calculation method comprises the following steps:
further, accurately weighing the extracted litsea cubeba oil in a volumetric flask of 50mg to 50mL, adding ethyl acetate to dilute the oil to a scale, shaking up, accurately weighing 1mL to 50mL volumetric flask, adding ethyl acetate to dilute the oil to a scale, and shaking up to obtain a sample solution.
Further, the establishment of the GC-MS fingerprint comprises the following steps:
a1: GC-MS conditions:
chromatographic conditions are as follows: the split ratio is 50; no shunt sampling; the sample size is 1 mu L;
mass spectrum conditions: the ion source temperature is 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ion (EI); detecting voltage: 0.2KV; delaying the solvent for 3min; mass number scan range: 30-400 amu; a SCAN full SCAN mode;
a2: the extraction method comprises the following steps:
respectively taking 22 batches of dried litsea pungens, crushing, sieving with a 60-mesh sieve, accurately weighing 20g of powder, respectively and rapidly placing into 250mL round-bottomed flasks, adding 160mL of ultrapure water, sealing and soaking for 0.5h, connecting an extraction device for extraction for 5h, standing for 1h after extraction, collecting volatile oil, adding a proper amount of anhydrous sodium sulfate, sealing and standing overnight, centrifuging for 10min at 8000r/min the next day, collecting an upper oil layer, and keeping away from light at-20 ℃;
a3: solution preparation:
respectively and precisely weighing 0.1g of the litsea cubeba oil, placing the litsea cubeba oil in a 20mL volumetric flask to obtain stock solution, precisely weighing 1.5mL of the stock solution in the 20mL volumetric flask, and adding ethyl acetate to a constant volume to obtain a test solution;
a4: the experiment leads to the conclusion and establishes the fingerprint spectrum.
The multi-index content determination method of the litsea cubeba oil comprises the following steps:
s1: GC-MS conditions
Chromatographic conditions are as follows: a chromatographic column: an SH-Stabilwax capillary column; the temperature of a sample inlet is 220 ℃; the flow splitting ratio is 50; the sample size is 1 mu L;
mass spectrum conditions: the ion source temperature is 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ions; detecting voltage: 0.2KV; delaying the solvent for 3min; mass number scan range: 30-400 amu; SIM mode;
s2: preparing a reference substance solution;
s3: preparing an internal standard solution;
s4: preparing a test solution;
s5: and (5) inspecting, analyzing and testing.
Further, the preparation method of the reference solution in the S2 comprises the following steps: accurately weighing appropriate amount of levo-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene p-cymene, terpinolene, 2-nonanone, 4-thuja alcohol, bornyl acetate, beta-caryophyllene, 4-terpenol, (-) -myrtenal, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol, cuminaldehyde, myrtenol, 2- (4-methylphenyl) propan-2-ol, caryophyllene oxide and guaiol as reference substances, placing in a 10mL volumetric flask, adding ethyl acetate to dissolve and dilute to scale, preparing into a stock solution of levo-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene, p-cymene, terpinolene, 2-nonanone, 4-thuja alcohol, bornyl acetate, beta-caryophyllene, 4-terpene alcohol, (-) -myrtenal aldehyde, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol, cuminaldehyde, myrtenol, 2- (4-methylphenyl) propan-2-ol, caryophyllene oxide and guaiol, and storing in a refrigerator at (-20 ℃) for later use.
Further, the preparation method of the internal standard solution in S3 comprises the following steps: the cyclohexanone and naphthalene reference substances are precisely weighed and placed in a 10mL volumetric flask, ethyl acetate is added to dissolve and dilute the reference substances to the scale, and internal standard stock solutions of cyclohexanone (3.300 mg. ML-1) and naphthalene (2.710 mg. ML-1) are prepared. Respectively and precisely measuring an appropriate amount of internal standard stock solution into 100mL volumetric flasks, diluting the internal standard stock solution to the scale with ethyl acetate to prepare internal standard solution of cyclohexanone (150 mu g/mL-1) and naphthalene (3 mu g/mL-1), and storing the internal standard solution in a refrigerator at (-20 ℃) for later use.
Further, the preparation method of the test solution in S4 comprises the following steps: precisely weighing 0.1g of the litsea cubeba oil, placing the litsea cubeba oil in a 20mL volumetric flask, adding ethyl acetate to dissolve and dilute the litsea cubeba oil to a scale, precisely weighing 1mL of the solution, placing the solution in a 10mL volumetric flask, respectively and precisely adding 1mL of cyclohexanone and naphthalene internal standard solution, adding ethyl acetate to dilute the solution to the scale, shaking the solution uniformly to obtain a sample solution, and paralleling the solution for 2 times.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
(1) According to the technical scheme, firstly, a GC-MS technology is adopted to research chemical components in the litsea cubeba oil, the material basis is determined preliminarily, the foundation is laid for the follow-up research of quality control and the like, secondly, a GC-MS SIM technology is adopted to combine with chemical pattern recognition, the multi-index component content of the litsea cubeba oil samples in different producing areas is analyzed, the foundation is laid for comprehensively and effectively controlling the overall quality of the litsea cubeba oil, the quality difference marking components of the litsea cubeba oil are screened out, and a basis and a research thought are provided for improving and perfecting the quality standard of the litsea cubeba oil.
(2) According to the technical scheme, through the investigation of an extraction method of the litsea cubeba oil by a steam distillation method, the optimal extraction conditions are determined as the crushing particle size of 60 meshes, the material-liquid ratio is 1 (g: mL), the soaking time is 0.5h, and the extraction time is 5h. The verification test result shows that the extraction method is simple to operate, low in cost, stable and feasible.
(3) The technical scheme establishes GC-MS fingerprint spectrums of 22 batches of litsea cubeba oil by taking the litsea cubeba oil comparison fingerprint spectrums as comparison. The method is verified by methodology, has good precision, stability and repeatability, and can be used for fingerprint spectrum research of the litsea pungens seed oil.
(4) The GC-MS SIM method is combined with chemical pattern recognition, the quality of the litsea cubeba oil of different batches is systematically, comprehensively and integrally researched, the method is scientific, accurate, reliable and easy to implement, and references can be provided for the quality control and identification of the litsea cubeba oil.
Drawings
FIG. 1 is a GC-MS total ion flow diagram of Litsea macrocarpa oil of the present invention;
FIG. 2 shows the relative percentages of various types of compounds in the litsea cubeba oil according to the present invention;
FIG. 3 is a chromatogram of the precision test of the litsea pungens oil and a comparison fingerprint;
FIG. 4 is a chromatogram of the repeatability test of litsea cubeba oil and a comparison fingerprint of the invention;
FIG. 5 is a chromatogram and a reference fingerprint of the stability test of Litsea pungens oil of the present invention;
FIG. 6 is a comparison fingerprint of Litsea pungens oil of the present invention;
FIG. 7 is an overlay of 22 batches of litsea cubeba oil fingerprints according to the present invention;
FIG. 8 is a graph showing the effect of particle size reduction on the oil yield of Litsea pungens according to the present invention;
FIG. 9 shows the effect of feed liquid ratio on the oil yield of Litsea pungens according to the present invention;
FIG. 10 is a graph showing the effect of soaking time on the oil yield of Litsea pungens according to the present invention;
FIG. 11 shows the effect of extraction time on the oil yield of Litsea pungens according to the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention; it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.
Example 1:
referring to fig. 1-11, the GC-MS fingerprint of litsea cubeba oil is determined by the following methods:
s1: accurately weighing 20g of litsea pungens, placing in a flask, and respectively inspecting influence of four factors of the crushed particle size of the medicinal materials, the material-liquid ratio, the soaking time and the extraction time on the oil yield;
s2: adding appropriate amount of anhydrous sodium sulfate into the collected volatile oil, sealing overnight, centrifuging at 8000r/min for 10min the next day, collecting upper oil layer, weighing, paralleling for 3 times, and calculating oil yield;
s3: establishing a GC-MS fingerprint;
s4: solutions were prepared and the results analyzed.
The influence on the oil yield of the litsea pungens comprises the following factors:
influence of the crushed particle size on the oil yield: crushing litsea pungens by using a crusher, respectively inspecting the crushed litsea pungens powder by using a sieve of 10 meshes, 30 meshes, 60 meshes, 80 meshes and 100 meshes, accurately weighing 20g, soaking for 0h and extracting for 5h, comparing oil yield and determining the optimal crushed particle size, wherein the material-liquid ratio is 1;
as can be seen from fig. 8, under the extraction conditions of the feed-liquid ratio of 1 to 12, the soaking time of 0 hour, and the extraction time of 5 hours, when the pulverized particle size is 10 to 100 mesh, the oil yield of litsea cubeba increases with the increase of the pulverized particle size, and when the pulverized particle size is 60 mesh or more, the oil yield of volatile oil decreases. Therefore, the pulverized particle size of 60 mesh is preferably selected.
Influence of feed liquid ratio on oil yield: crushing litsea cubeba by using a crusher, sieving crushed litsea cubeba powder by a 60-mesh sieve, accurately weighing 20g, respectively observing the material-liquid ratio of 1;
as can be seen from fig. 9, under the extraction conditions of a crushed particle size of 60 mesh, a soaking time of 0h, and an extraction time of 5h, the litsea cubeba oil yield increased first and then decreased with the increase of the feed-liquid ratio, and when the feed-liquid ratio was 1.
Influence of soaking time on oil yield: crushing litsea cubeba by using a crusher, sieving crushed litsea cubeba powder by a 60-mesh sieve, accurately weighing 20g, respectively inspecting soaking for 0h, 0.5h, 1h, 1.5h and 2h, extracting for 5h, and comparing oil yield to determine the optimal soaking time, wherein the material-liquid ratio is 1;
as can be seen from fig. 10, under the conditions of a crushed particle size of 60 mesh, a material-to-liquid ratio of 1 to 12, and an extraction time of 5 hours, the oil yield of litsea cubeba reaches a maximum value when the soaking time is 0.5 hours as the soaking time is prolonged. Therefore, 0.5h of soaking is selected.
Effect of extraction time on oil yield: crushing the litsea cubeba by using a crusher, sieving the crushed litsea cubeba powder by a 60-mesh sieve, accurately weighing 20g of the powder with a material-liquid ratio of 1.
As can be seen from fig. 11, under the extraction conditions of a pulverized particle size of 60 mesh, a material-to-liquid ratio of 1 (g: mL), and an immersion time of 0h, the oil yield of litsea cubeba gradually increased and then decreased with the increase of the extraction time, and the oil yield reached the highest at 5h. The extraction time is too long, and a small amount of volatile oil is lost. Therefore, according to the principle of saving time, the extraction time is selected to be 5h.
Performing an extraction test of the litsea pungens oil on the result of the single-factor test, accurately weighing 20g of crushed litsea pungens, calculating the oil yield, and paralleling for 3 times;
the calculation method comprises the following steps:
accurately weighing the litsea pungens oil extracted from the solution into a volumetric flask of 50mg to 50mL, adding ethyl acetate to dilute to a scale, shaking up, accurately weighing into a volumetric flask of 1mL to 50mL, adding ethyl acetate to dilute to a scale, and shaking up to obtain the test solution.
And (3) verification test:
according to the test results, 3 parts of crushed litsea pungens are taken to perform the volatile oil extraction verification test. The extraction conditions comprise that the crushed particles are 60 meshes, 20g of litsea pungens powder is accurately weighed, the material-liquid ratio is 1 (g: mL), the soaking time is 0.5h, the extraction time is 5h, volatile oil is collected, a proper amount of anhydrous sodium sulfate is added, the mixture is sealed overnight, the mixture is centrifuged for 10min at 8000r/min on the next day, an upper oil layer is collected, and the oil yield is weighed and calculated. The extraction rates of 3 parts of the litsea cubeba oil are respectively 11.23%, 11.29% and 11.37%, the average value is 11.30%, and the RSD is 0.62%, which indicates that the method is stable and reliable for extracting the litsea cubeba oil.
And (3) identifying chemical components:
taking 1 μ L of the volatile oil sample of Litsea pungens obtained by the above factor processing method, detecting with GC-MS combined instrument, comparing the analyzed result with NIST 2020 standard mass spectrum library, selecting the peak with matching degree higher than 80, and determining the total ion flow diagram of each chemical component with the combination of related literature and manual chromatogram analysis as shown in figure 1, and the relative percentage content of each compound as shown in figure 2.
Through the investigation of the extraction method of the litsea cubeba oil by the steam distillation method, the optimal extraction conditions are determined as the crushing particle size of 60 meshes, the material-liquid ratio of 1. The verification test result shows that the extraction method is simple to operate, low in cost, stable and feasible.
The establishment of the GC-MS fingerprint comprises the following steps:
a1: GC-MS conditions:
chromatographic conditions are as follows: the split ratio is 50; no shunt sampling; the sample size is 1 mu L; the temperature program is detailed in the following table:
column oven temperature program
Mass spectrum conditions: the ion source temperature is 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ion (EI); detecting voltage: 0.2KV; delaying the solvent for 3min; mass number scan range: 30-400 amu; a SCAN full SCAN mode;
a2: the extraction method comprises the following steps:
respectively taking 22 batches of dried litsea pungens, crushing, sieving with a 60-mesh sieve, accurately weighing 20g of powder, respectively and rapidly placing into 250mL round-bottomed flasks, adding 160mL of ultrapure water, sealing and soaking for 0.5h, connecting an extraction device for extraction for 5h, standing for 1h after extraction, collecting volatile oil, adding a proper amount of anhydrous sodium sulfate, sealing and standing overnight, centrifuging for 10min at 8000r/min the next day, collecting an upper oil layer, and keeping away from light at-20 ℃;
a3: solution preparation:
respectively and precisely weighing 0.1g of the litsea cubeba oil, placing the litsea cubeba oil in a 20mL volumetric flask to obtain stock solution, precisely weighing 1.5mL of the stock solution in the 20mL volumetric flask, and adding ethyl acetate to a constant volume to obtain a test solution;
a4: the experiment is concluded and a fingerprint is established.
Test example:
and (3) precision test:
taking the same sample solution, continuously feeding sample according to GC-MS conditions for 6 times to obtain fingerprint (shown in figure 3), and calculating similarity by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.130723 edition). And comparing the 6-time fingerprint spectrum with the 1 st-time fingerprint spectrum by taking the 1 st-time fingerprint spectrum as a reference. The result shows that the similarity of the fingerprints of 6 times is greater than 0.996, which shows that the precision is good, and the detailed table is as follows:
calculation result of similarity of fingerprint precision test
And (3) repeatability test:
taking 6 parts of the same batch of test solution, detecting according to the GC-MS condition of each factor to obtain a fingerprint (shown in figure 4), and performing similarity calculation by adopting a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012.130723 version). And comparing the fingerprint of the 6 samples with the fingerprint of the 1 st sample by taking the fingerprint of the 1 st sample as a reference. The results show that the similarity of the fingerprint spectra of 6 samples is greater than 0.996, which shows good repeatability, and the details are shown in the following table:
similarity calculation result of fingerprint repeatability test
And (3) stability test:
sampling the same batch of sample solution for 0, 2, 4, 8, 12 and 24h respectively, sampling and determining according to GC-MS conditions under the item of '1.2.1', obtaining a fingerprint (shown in figure 5), and performing similarity calculation by adopting a 'traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012.130723 version)'. And comparing the fingerprint spectrum of 6 times with the fingerprint spectrum of 0h by taking the fingerprint spectrum of the 0h sample injection as a reference. The result shows that the similarity of the fingerprint spectra of 6 times is more than 0.996, which shows that the stability is good, and the detailed table is shown in the following table:
calculation result of stability similarity of fingerprint
Establishing a fingerprint spectrum:
by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.130723 edition), taking a sample spectrum as a reference spectrum, adopting an average number method, wherein the time window width is 0.1, automatically matching a full spectrum peak to generate a reference fingerprint, wherein the reference fingerprint (shown in figure 6) and 22 batches of the litsea cubeba oil superposition fingerprints (shown in figure 7).
The GC-MS fingerprint of 22 batches of litsea cubeba oil is established by taking the litsea cubeba oil comparison fingerprint as a comparison. Proved by methodology, the method has good precision, stability and repeatability, and can be used for the fingerprint spectrum research of the litsea pungens seed oil. The 22 samples are analyzed by a similarity evaluation method, the quality conditions of the litsea cubeba oil in Guizhou, yunnan and Guangxi are preliminarily known, and the fingerprint spectrum method can provide reference for the quality standard of the litsea cubeba oil.
The multi-index content determination method of the litsea cubeba oil comprises the following steps:
s1: GC-MS conditions
Chromatographic conditions are as follows: a chromatographic column: an SH-Stabilwax capillary column; the temperature of a sample inlet is 220 ℃; the flow splitting ratio is 50; the sample volume was 1 μ L (see above);
mass spectrum conditions: the ion source temperature is 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ions; detecting voltage: 0.2KV; delaying the solvent for 3min; mass number scan range: 30-400 amu; SIM mode, the following table for each component detected ion information:
multi-index component detection ion information of litsea pungens oil
S2: preparing a reference substance solution;
accurately weighing appropriate amounts of levo-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene p-cymene, terpinolene, 2-nonanone, 4-thujanol, bornyl acetate, beta-caryophyllene, 4-terpene alcohol, (-) -myrcene aldehyde, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol, cuminaldehyde, myrtenol, 2- (4-methylphenyl) propan-2-ol, caryophyllene oxide and guaiol, placing in a 10mL volumetric flask, adding ethyl acetate to dissolve and dilute to scale, preparing a stock solution of levo-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene, p-cymene, terpinolene, 2-nonanone, 4-thujanol, bornyl acetate, beta-caryophyllene, 4-terpene alcohol, (-) -myrtenonal, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol, cuminaldehyde, myrtenol, 2- (4-methylphenyl) propan-2-ol, caryophyllene oxide and guaiol, and storing the stock solution in a refrigerator at (-20 ℃) for later use;
s3: preparing an internal standard solution;
the cyclohexanone and naphthalene reference substances are precisely weighed and placed in a 10mL volumetric flask, ethyl acetate is added to dissolve and dilute the reference substances to the scale, and internal standard stock solutions of cyclohexanone (3.300 mg. ML-1) and naphthalene (2.710 mg. ML-1) are prepared. Respectively and precisely measuring an appropriate amount of internal standard stock solution into 100mL volumetric flasks, diluting the internal standard stock solution to a scale with ethyl acetate to prepare an internal standard solution of cyclohexanone (150 mu g. ML-1) and naphthalene (3 mu g. ML-1), and storing the internal standard solution in a refrigerator at (-20 ℃) for later use;
s4: preparing a test solution;
precisely weighing 0.1g of the litsea cubeba oil, placing the litsea cubeba oil in a 20mL volumetric flask, adding ethyl acetate to dissolve and dilute the litsea cubeba oil to a scale, precisely weighing 1mL of the solution, placing the solution in a 10mL volumetric flask, respectively and precisely adding 1mL of cyclohexanone and naphthalene internal standard solution, adding ethyl acetate to dilute the solution to the scale, shaking the solution uniformly to obtain a sample solution, and paralleling the solution for 2 times.
S5: and (5) inspecting, analyzing and testing.
Test example:
precisely absorbing appropriate amount of reference substance solution extracted under various factors to obtain a series of mixed reference substance solutions, and measuring by GC-MS injection. And (3) drawing a standard working curve by taking the concentration of each reference substance as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y), and calculating a regression equation as shown in the following table. R2 is more than 0.9997, which shows that 21 components have good linear relation in corresponding concentration range.
Taking the mixed reference substance solution, carrying out sample injection analysis according to GC-MS conditions under various factor conditions, carrying out continuous sample injection for 6 times within 1 day, recording peak areas, and calculating the precision within the day, wherein the results are detailed in the following table 1; precisely sucking the same mixed reference solution, continuously injecting sample for 3 days, measuring for 3 times per day, and recording peak area to obtain day precision, wherein the result is shown in Table 2 below. The daily precision and the daytime precision of the 21 components are both less than 3 percent, which indicates that the precision of the instrument is good.
TABLE 1 in-day precision tests of 21 ingredients in litsea galanga oil
TABLE 2 daytime precision tests of 21 ingredients in litsea cubeba oil
And (3) stability test:
precisely sucking to prepare a test solution, and respectively injecting samples for 0, 2, 4, 6, 8, 12 and 24 hours under the GC-MS condition to determine the peak areas of 21 components, wherein the results are detailed in the following table. The peak area RSD of the 21 components is between 1.67 and 2.63 percent, which indicates that the test solution has good stability within 24 hours.
Stability test of 21 ingredients in Litsea grandiflorum oil
And (3) repeatability test:
taking 0.1g of litsea cubeba oil, precisely weighing, preparing 6 parts of test solution in parallel, carrying out sample injection measurement according to GC-MS conditions, recording peak area, calculating content, and obtaining the result shown in the table below. The RSD of the 21 components is less than 3 percent, which shows that the method has good repeatability.
Repeatability test of 21 components of litsea pungens oil
The GC-MS SIM method is combined with chemical pattern recognition, the quality of the litsea cubeba oil of different batches is researched systematically, comprehensively and integrally, the method is scientific, accurate, reliable and feasible, and references can be provided for the quality control and identification of the litsea cubeba oil. 6 components of eucalyptol, p-cymene, sabinene, levo-beta-pinene, alpha-terpineol and caryophyllene oxide which are possibly main quality mark components of differences among big fruit ginger samples in Guizhou, yunnan and Guangxi are screened by the method.
The foregoing is only a preferred embodiment of the present invention; the scope of the invention is not limited thereto. Any person skilled in the art should be able to cover the technical scope of the present invention by equivalent or modified solutions and modifications within the technical scope of the present invention.
Claims (9)
1. The GC-MS fingerprint spectrum determination method of the litsea megacarpum oil is characterized in that: the method comprises the following determination methods:
s1: accurately weighing 20g of litsea pungens, placing in a flask, and respectively inspecting the influence of four factors of the crushed particle size of the medicinal materials, the material-liquid ratio, the soaking time and the extraction time on the oil yield;
s2: adding appropriate amount of anhydrous sodium sulfate into the collected volatile oil, sealing overnight, centrifuging at 8000r/min for 10min the next day, collecting upper oil layer, weighing, paralleling for 3 times, and calculating oil yield;
s3: establishing a GC-MS fingerprint;
s4: solutions were prepared and the results analyzed.
2. The method for determining the GC-MS fingerprint of the litsea megacarpum oil according to claim 1, wherein the method comprises the following steps: the influence on the oil yield of the litsea pungens comprises the following factors:
influence of the crushed particle size on the oil yield: crushing litsea pungens by using a crusher, respectively screening crushed litsea pungens powder by 10, 30, 60, 80 and 100 meshes, accurately weighing 20g, soaking for 0h, extracting for 5h, comparing oil yield, and determining the optimal crushing particle size, wherein the material-liquid ratio is 1;
influence of feed liquid ratio on oil yield: crushing litsea pungens by using a crusher, sieving crushed litsea pungens powder by a 60-mesh sieve, accurately weighing 20g, respectively observing a material-liquid ratio of 1;
influence of soaking time on oil yield: crushing litsea cubeba by using a crusher, sieving crushed litsea cubeba powder by a 60-mesh sieve, accurately weighing 20g, respectively inspecting soaking for 0h, 0.5h, 1h, 1.5h and 2h, extracting for 5h, and comparing oil yield to determine the optimal soaking time, wherein the material-liquid ratio is 1;
effect of extraction time on oil yield: crushing the litsea cubeba by using a crusher, sieving the crushed litsea cubeba powder by a 60-mesh sieve, accurately weighing 20g of the powder with a material-liquid ratio of 1.
3. The method for determining the GC-MS fingerprint of the litsea cubeba oil according to claim 2, wherein the GC-MS fingerprint comprises the following steps: performing an extraction test of the litsea pungens oil on the result of the single-factor test, accurately weighing 20g of crushed litsea pungens, calculating the oil yield, and performing parallel 3 times;
the calculation method comprises the following steps:
4. the method for determining the GC-MS fingerprint of the litsea cubeba oil according to claim 3, wherein the GC-MS fingerprint of the litsea cubeba oil is characterized in that: accurately weighing the extracted litsea pungens oil in a volumetric flask of 50mg to 50mL, adding ethyl acetate to dilute the oil to a scale, shaking up, accurately weighing a volumetric flask of 1mL to 50mL, adding ethyl acetate to dilute the oil to a scale, and shaking up to obtain a test solution.
5. The method for determining the GC-MS fingerprint of the litsea cubeba oil according to claim 1, wherein the GC-MS fingerprint comprises the following steps: the establishment of the GC-MS fingerprint comprises the following steps:
a1: GC-MS conditions:
chromatographic conditions are as follows: the split ratio is 50; no shunt sampling; the sample volume is 1 mu L;
mass spectrum conditions: the ion source temperature is 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ion (EI); detecting voltage: 0.2KV; delaying the solvent for 3min; mass number scan range: 30-400 amu; a SCAN full SCAN mode;
a2: the extraction method comprises the following steps:
respectively taking 22 batches of dried litsea pungens, crushing, sieving with a 60-mesh sieve, accurately weighing 20g of powder, respectively and rapidly filling into 250mL round-bottom flasks, adding 160mL of ultrapure water, sealing and soaking for 0.5h, connecting an extraction device for extraction for 5h, standing for 1h after extraction, collecting volatile oil, adding a proper amount of anhydrous sodium sulfate, sealing overnight, centrifuging at 8000r/min for 10min the next day, collecting an upper oil layer, and keeping away from light and at-20 ℃;
a3: solution preparation:
respectively and precisely weighing 0.1g of the litsea cubeba oil, placing the litsea cubeba oil in a 20mL volumetric flask to obtain stock solution, precisely weighing 1.5mL of the stock solution in the 20mL volumetric flask, and adding ethyl acetate to a constant volume to obtain a test solution;
a4: the experiment leads to the conclusion and establishes the fingerprint spectrum.
6. The multi-index content determination method of the litsea megacarpi oil is characterized by comprising the following steps: the method for measuring the content of the multiple indexes comprises the following steps:
s1: GC-MS conditions
Chromatographic conditions are as follows: and (3) chromatographic column: an SH-Stabilwax capillary column; the temperature of a sample inlet is 220 ℃; the flow splitting ratio is 50; the sample volume is 1 mu L;
mass spectrum conditions: the ion source temperature is 200 ℃; the interface temperature is 230 ℃; the ionization mode is electron bombardment ions; detecting voltage: 0.2KV; delaying the solvent for 3min; mass number scan range: 30-400 amu; a SIM mode;
s2: preparing a reference substance solution;
s3: preparing an internal standard solution;
s4: preparing a test solution;
s5: and (5) inspecting, analyzing and testing.
7. The method for measuring the multi-index content of the litsea cubeba oil according to claim 6, characterized in that: the preparation method of the reference substance solution in S2 comprises the following steps: accurately weighing appropriate amount of levo-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene p-cymene, terpinolene, 2-nonanone, 4-thuja alcohol, bornyl acetate, beta-caryophyllene, 4-terpenol, (-) -myrtenal, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol, cuminaldehyde, myrtenol, 2- (4-methylphenyl) propan-2-ol, caryophyllene oxide and guaiol as reference substances, placing in a 10mL volumetric flask, adding ethyl acetate to dissolve and dilute to scale, preparing into a stock solution of levo-beta-pinene, sabinene, myrcene, (+) -limonene, eucalyptol, gamma-terpinene, p-cymene, terpinolene, 2-nonanone, 4-thuja alcohol, bornyl acetate, beta-caryophyllene, 4-terpene alcohol, (-) -myrtenal aldehyde, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol, cuminaldehyde, myrtenol, 2- (4-methylphenyl) propan-2-ol, caryophyllene oxide and guaiol, and storing in a refrigerator at (-20 ℃) for later use.
8. The method for measuring the multi-index content of the litsea cubeba oil according to claim 6, characterized in that: the preparation method of the S3 internal standard solution comprises the following steps: precisely weighing cyclohexanone and naphthalene reference substances, placing the reference substances in a 10mL volumetric flask, adding ethyl acetate to dissolve and dilute the reference substances to the scale mark to prepare the internal standard stock solution of cyclohexanone (3.300 mg. ML-1) and naphthalene (2.710 mg. ML-1). Respectively and precisely measuring an appropriate amount of internal standard stock solution into 100mL volumetric flasks, diluting the internal standard stock solution to a scale with ethyl acetate to prepare internal standard solutions of cyclohexanone (150 mu g & mL-1) and naphthalene (3 mu g & mL-1), and storing the internal standard solutions in a refrigerator at (-20 ℃) for later use.
9. The method for measuring the multi-index content of the litsea cubeba oil according to claim 6, characterized in that: the preparation method of the test solution in S4 comprises the following steps: precisely weighing 0.1g of litsea cubeba oil, placing the litsea cubeba oil in a 20mL volumetric flask, adding ethyl acetate to dissolve and dilute the litsea cubeba oil to a scale, precisely weighing 1mL of solution, placing the solution in a 10mL volumetric flask, respectively and precisely adding 1mL of cyclohexanone and naphthalene internal standard solution, adding ethyl acetate to dilute the solution to the scale, shaking up to obtain a sample solution, and paralleling for 2 times.
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