CN103808822A - LC-QTOF (Liquid Chromatography-Quadrupole Time Of Flight) analysis method for distinguishing resveratrol of different resources - Google Patents
LC-QTOF (Liquid Chromatography-Quadrupole Time Of Flight) analysis method for distinguishing resveratrol of different resources Download PDFInfo
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- 235000021283 resveratrol Nutrition 0.000 title claims abstract description 188
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- 238000004458 analytical method Methods 0.000 title claims description 15
- 239000007788 liquid Substances 0.000 title claims description 5
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Abstract
The invention belongs to the field of analytic chemistry, resveratrol from polygonum cuspidatum and resveratrol from grape vine are taken as research objects, a liquid chromatography-mass spectrometry technology (LC-QTOF) with high sensitivity and high resolution is adopted for providing more characteristics of compound structural information, the difference of micro components in resveratrol samples is researched, four marking compounds which can distinguish resveratrol of different sources are found, the difference of the micro components in the resveratrol from polygonum cuspidatum and resveratrol from grape vine is further confirmed, a reference method is provided for distinguishing resveratrol samples of different raw material sources, and great significance for identifying the real property of the natural healthcare food, namely, resveratrol, is achieved, and meanwhile the technical method can be also widely popularized and applied to real property detection and identification standard of imported and exported agriculture products and biologic raw materials, technical demonstration for identification on the real property of the natural product raw material is provided, and establishment of reference standards is achieved.
Description
Technical field
The invention belongs to analytical chemistry field, a kind of LC-QTOF(liquid chromatography/level Four bar-flight time mass spectrum coupling of identifying resveratrol raw material sources be provided) analytical approach.
Background technology
That resveratrol has is antitumor, angiocardiopathy preventing, immunological regulation and the biologically active widely such as antibacterial, and the special health-care efficacy that it has makes it very popular on market.Along with the biologically active of resveratrol is familiar with gradually, more and more health products manufacturing enterprise all has an optimistic view of the functional of resveratrol very much, and it is expected to constantly widen as the application surface of end product.Present stage, domestic and international market is increasing to the demand of resveratrol, and what resveratrol will be more extensive, safer is applied in the middle of every field.
Conventionally there are chemosynthesis, microorganism, biosynthesizing and extracted form natural plant in the source of resveratrol.The production resveratrol that chemosynthesis energy is a large amount of, but there is the problems such as environmental pollution in various degree in the whole bag of tricks of present stage chemosynthesis resveratrol; And the input of microorganism fermenting organism synthesizing resveratrol technology is larger, on market, form not yet on a large scale industrialization prospect.Therefore on market, 98% raw material level resveratrol mainly stems from natural plants giant knotweed to extract and obtains and synthetic acquisition, but the product that has 5%-20% different size on market respectively from giant knotweed and other plant as grapevine etc.
Polygonum cuspidatum Sieb. et Zucc starting material itself contain a small amount of resveratrol, but it contains the polygonin up to 3% left and right, can obtain the resveratrol of high-load by the method for biological glycolysis, so giant knotweed is the very important starting material of commercial production resveratrol.The product specification that in Polygonum cuspidatum Sieb. et Zucc, resveratrol is mainly developed is 50%, 98%, and product price is about 800-3000 unit/kilogram left and right, has comparatively wide application market.But because giant knotweed is the one in Chinese traditional medicine, not medicinal and edible plant, abroad as Japan, the resveratrol product that extracts exploitation from giant knotweed can only be applied to field of medicaments, in food service industry, uses and has certain law bottleneck.
And the Resveratrol content in grapevine source relatively low (content is generally tens to hundreds of ppm), the exploitation of its product relatively lags behind in China, but its security of resveratrol Related product take medicinal and edible plant-grapevine as development of raw materials can obtain accepting more widely in the international market, can in field of medicaments, use, also can be widely applied to such as in health products, field of food.Simultaneously, in grapevine, not only contain resveratrol, but also contain abundant resveratrol oligomer, as ε-viniferin, α-viniferin, Miyabenol C etc., these resveratrol oligomer can transform to monomer, thereby improve the content of resveratrol.In grapevine, resveratrol production specification is generally 5% or 15-20%, price is respectively 200 Euros/kilogram and 1000 Euros of/kilogram of left and right, far above traditional giant knotweed source resveratrol product price, although expensive, be subject to favor and the welcome of healthy food manufacturer.
The greatest differences of the resveratrol product price that different plant origins are produced causes some illegal businessmans on market to gain high profits to be applied to health products with the more expensive grapevine source resveratrol of the resveratrol personation in relatively cheap giant knotweed source, in field of food, on market, exist and add highly purified giant knotweed source resveratrol to phenomenon in the resveratrol in low-purity grapevine source, thereby cause consumer's healthy infringement and the threat being subject to a certain extent, also upset the international and domestic market order of Related product, cause trade dispute in various degree.On international market, clearly stipulate, the product with medicinal health can not add in the middle of health food, therefore, for guaranteeing that the common people are healthy, reply international trade barrier, safeguard the international fame of China's product, safeguard that national economy social stability and international trade develop in a healthy way, the real property in resveratrol products material in urgent need of strengthening source characterizes and recognition technology research.
The analyzing detecting method of resveratrol that at present should be with the most use is as follows:
(1) high performance liquid chromatography (HPLC)
The polarity of resveratrol is less, generally adopts reversed-phase high-performance liquid chromatography analysis in analyzing and testing, often adopts the conventional mobile phase analysis such as acetonitrile-phosphoric acid water, methyl alcohol-glacial acetic acid.All-purpose detector comprises ultraviolet spectrophotometer, diode array, galvanochemistry, fluorescent scanning detection etc.The people such as Wu Bo apply High Performance Liquid Chromatography with Fluorescence Detection set up a kind of quick, sensitive, be applicable to suitable in fruit, to highlight veratryl alcohol and glycosides thereof quantitative analysis method, this method is well used.
(2) LC-MS technology (LC-MS)
Because resveratrol is close with its derivant structure, and Resveratrol Metabolins is lower at plant intensive amount, single analytical technology is generally difficult to obtain result accurately, therefore LC-MS technology becomes for analyzing one of important means of this type of complex component, the people such as Yang Runtao are using Lichrospher C18 post as analyzing chromatographic column, acetonitrile-water is mobile phase, adopt electric spray ion source, inner mark method ration and multiple-reaction monitoring pattern, under selected condition, set up the LC-MS quantitative analysis method that polydatin in blood plasma and metabolic product thereof are measured simultaneously.LC-QTOF refers to liquid chromatography-level Four bar-flight time mass spectrum, LC-MS refers to liquid chromatography-single-stage mass spectrum, LC-QTOF technology be a kind of in recent years just rise more advanced, more reliably, mass-spectrometric technique more accurately, obtainable compound information is much more a lot of than LC-MS.
(3) gas chromatography mass spectrometry technology (GC-MS)
The people such as Luan Tiangang are by the preprocess method that adopts methyl-monosilaneization to combine with solid phase micro-extraction technique (SPME), application GC-MS technology is carried out qualitative analysis to 19 kinds of polar organic matters in grapevine wine and polyphenol compound, trans-resveratrol has been carried out to quantitative test simultaneously.
(4) capillary electrophoresis (CE)
The electrophoretic that has at present quantitatively detected research for resveratrol comprises capillary zone electrophoresis, non Aqueous Capillary Electrophoresis etc., can separate detection to the positive and negative isomeride of resveratrol simultaneously.Capillary Electrophoresis detects the technology of resveratrol by combining with continuous sample introduction technology and solid phase extraction techniques, has realized the robotization preprocessing process of sample.The people such as Cao Jia, take benzyl trimethyl iodate amine as interior mark, have set up in 7 minutes and have made resveratrol and polydatin reach all good methods of baseline separation and linear dynamic range and detection limit effect.Zheng Yanpeng etc. adopt three (methylol) aminomethane-boric acid (THAM-H3BO3) as supporting electrolyte, methyl alcohol is as separating medium, electricity consumption is led detection technique the resveratrol in giant knotweed has been carried out to determination and analysis, and result shows to adopt non Aqueous Capillary Electrophoresis electricity to lead to detect its interfering material separating effect when resveratrol is measured and is better than gas chromatography mass spectrometry technology.
(5) thin layer scanning chromatography (TLC)
The people such as Zhou Guohai are take chloroform: acetone: acetic acid: water (4:4:0.5:0.2) is as developping agent, and silica G, as thin layer adsorbent, has been carried out methodological study to the assay of the each position of giant knotweed resveratrol.The people such as Shu Youqin are take benzene: methyl alcohol: formic acid (10:5:1) is as developping agent, adopt first the polyamide film phase that fixes, utilize fluorescent scanning quantitative analysis to measure 4 kinds of isomeride of giant knotweed resveratrol, result shows that it measures the fix thin layer chromatography scanning of phase of remolding sensitivity silica gel and improved 100 times nearly, and resveratrol and content of isomer thereof that this method equally also can be applicable in other samples are measured.
In addition, also comprise Second Order Differential Simple Oscillographic voltammetry for the detection method of resveratrol, electrochemical method, enzyme linked immunosorbent assay, ion resonance spectroscopic methodology, electron paramagnetic resonance method etc. at present.
Up to now, relative less with the research of quality control about the identification of resveratrol raw material sources real property, the Study of recognition of the external resveratrol to different plant origins rarely has report, domestic only Liu Dai beautiful jades etc. are studied grapevine source resveratrol sample, adopt high performance liquid chromatography to set up the analysis of spectra of grapevine source resveratrol sample, and main chromatographic peak in spectrogram is identified and assay, and the giant knotweed that commute is obscured source resveratrol sample carries out archen detection, in result demonstration grapevine resveratrol sample, contain its endemic element ε-viniferin, ε-viniferin and resveratrol are proportional to be present in plant extracts, and should not detect archen in the resveratrol sample in grapevine source.
The concrete grammar to the research of grape resveratrol extract of domestic Liu Dai beautiful jade is: utilize high performance liquid chromatography to set up the analysis collection of illustrative plates of grape source resveratrol extract, its main chromatographic peak resveratrol and ε-viniferin are identified, and set up content assaying method.
The high efficiency liquid phase atlas analysis condition of grape source resveratrol extract is:
Chromatographic column: C18,250mm*4.6mm, 5um
Flow velocity: 1.0ml/min column temperature: 25 ℃ are detected wavelength: 310nm
Mobile phase: A:1% glacial acetic acid aqueous solution B:1% glacial acetic acid methanol solution
The content assaying method of resveratrol and ε-viniferin is:
High efficiency liquid phase testing conditions: mobile phase: acetonitrile-0.1% phosphate aqueous solution (30:70), detects wavelength: 303nm
Chromatographic column: C18,150mm*4.6mm, 5um flow velocity: 1.0ml/min
The content assaying method of archen is:
High efficiency liquid phase testing conditions: mobile phase: methyl alcohol-0.1% phosphate aqueous solution (85:15), detects wavelength: 254nm
Chromatographic column: C18,150mm*4.6mm, 5um flow velocity: 1.0ml/min
In Liu Dai beautiful jade article, adopt different analytical approachs to carry out analysis and assay to resveratrol, ε-grape element and archen in grape resveratrol extract, its result shows, in the resveratrol in grape source, contain its endemic element ε-grape element, and be present in plant extracts with resveratrol is proportional, and should not detect archen in the resveratrol extract of grape source.
The people's such as Liu Dailin research method is only studied for the resveratrol extract in grape source, draw in the resveratrol of grape source and contain its endemic element ε-grape element, and the content to archen in the resveratrol of grape source of the property guessed is measured, result shows in the resveratrol of grape source should not detect archen, so using ε-grape element and the foundation of two kinds of compounds of archen as difference grape source resveratrol.If highly purified giant knotweed resveratrol and grape resveratrol are mixed, adopt this rule cannot distinguish the true source of resveratrol, this method is not also carried out comparative study by other raw material sources resveratrols as giant knotweed source resveratrol,, in the recognition technology of resveratrol, there is certain defect in the resveratrol that can not clearly how to differentiate different material source.
The method is to a certain extent for the resveratrol product in identification grapevine source provides reference and reference, but do not form the integral framework of a set of identification separate sources resveratrol real property, how the method can not clearly differentiate the resveratrol in different material source, has certain defect in the recognition technology of resveratrol.Therefore, for further improving the analyzing detecting method of resveratrol and the resveratrol recognition technology in different material source, in-depth is to resveratrol real property Study of recognition, work out a set of complete resveratrol Study of recognition method, its product quality management control is significant.
Summary of the invention
The technical problem to be solved in the present invention is: for the deficiencies in the prior art, a kind of LC-QTOF analytical approach of distinguishing different resveratrol is provided, can distinguish the separate sources of resveratrol by same analytical approach, the real property identification of natural health care-resveratrol is had great importance.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is:
A LC-QTOF analytical approach for distinguishing different resveratrol, concrete steps are:
(1) preparation detects sample: get Gentrin Knotweed P.E sample and grapevine extract sample that resveratrol mass content is identical, add alcohol to dissolve, be mixed with the solution that sample quality concentration is identical, ultrasonic 25-35min at 25 ℃-30 ℃, leave standstill cooling, filtering with microporous membrane, filtrate is as detecting sample;
(2) detect: detection sample is carried out to liquid chromatography/level Four bar-flight time mass spectrum method for combined use and analyze, obtain liquid chromatogram and mass spectrogram and data; Wherein, analysis condition control is as follows:
A) liquid-phase chromatographic analysis condition
Chromatographic column: XUnion C18;
Mobile phase: acetonitrile-water;
Gradient elution: 0-30min, in mobile phase, the volumetric concentration of acetonitrile is 10%-50%;
30-40min, in mobile phase, the volumetric concentration of acetonitrile is 50%-95%;
40-42min, in mobile phase, the volumetric concentration of acetonitrile is 95%-10%;
42-50min, in mobile phase, the volumetric concentration of acetonitrile is 10%;
Flow velocity: 0.1-0.3mL/min;
Column temperature: 25 ℃-35 ℃;
Detect wavelength: 287nm;
B) mass spectrophotometry condition
Ion gun: ESI atomization gas: 50-60psig;
Scan mode: negative ion mode dry gas flow velocity: 5-7L/min;
Sheath temperature degree: 400 ℃; Capillary voltage: 3000V;
Sheath gas velocity: 10-14L/min; Cracking voltage: 125-145V;
Taper hole voltage: 50-70V; Mass scanning scope: 100-1700m/z, every 0.2s gathers 1 collection of illustrative plates;
(3) to collection of illustrative plates and data analysis:
To the giant knotweed collecting and grapevine source resveratrol mass spectrogram and data analysis, obtain total peak;
(4) characterization compound is confirmed
Utilize the compound information at total peak in giant knotweed, the grapevine source resveratrol finger-print that mass spectrophotometry obtains, further total ion current figure is carried out to the ion extraction according to accurate molecular weight, obtain EIC figure, in contrast EIC figure, go out peak difference, obtain having the characterization compound of notable difference between the two;
(5) judgement
Judge the separate sources of resveratrol according to characterization compound.
Step (3) is described to be preferably as follows collection of illustrative plates and data analysis:
Adopt Agilent Mass Hunter analysis software to the giant knotweed collecting and grapevine source resveratrol mass spectrogram and data analysis, obtain 11 total peaks, specific as follows:
In the resveratrol mass spectrogram of giant knotweed source, appearance time is that the peak of 10.26mi is polygonin, appearance time is that the peak of 16.02min is 3-methyl-5-hydroxyl-7-methoxyl-chromone, appearance time is that the peak of 17.087min is archen-8-O-β-D-Glucose glycosides, appearance time is that the peak of 17.627min is Physcion-1-O-β-D-Glucose glycosides, appearance time is that the peak of 20.2min is Physcion-1-O-β-D-Glucose glycosides, appearance time is that the peak of 22.093min is Kaempferol, and the peak that appearance time is 34.487min is archen;
In the resveratrol mass spectrogram of grapevine source; appearance time is that the peak of 18.427min is grape element A; appearance time is that the peak of 19.4min is grape element H, and the peak that appearance time is 20.953min is ε-grape element, and the peak that appearance time is 22.867min is α-grape element;
The characterization compound of step (4) is confirmed to be preferably as follows:
Utilize the compound information at total peak in giant knotweed, the grapevine source resveratrol finger-print that mass spectrophotometry obtains, further total ion current figure is carried out to the ion extraction according to accurate molecular weight, obtain EIC figure, in the resveratrol extract in giant knotweed source, be 20.200min place in retention time, 34.827min located respective peaks, be respectively Physcion-1-O-β-D-Glucose glycosides and archen, and archen peak is higher, and there is no chromatographic peak at 20.200min place in the resveratrol extract of grapevine source, also lose peak shape at 34.827min place; ε-grape element and α-grape element are carried out to EIC while extracting, grapevine source resveratrol extract is that 21.368min and 23.253min place have respective peaks in retention time, and giant knotweed source resveratrol extract does not have respective peaks in corresponding retention time, therefore, obtain having 4 characterization compounds of notable difference between the two, they are respectively Physcion-1-O-β-D-Glucose glycosides in the resveratrol extract in giant knotweed source and ε-grape element and the α-grape element in archen and grapevine source resveratrol extract;
Step (5) determination methods is preferably as follows:
By above-mentioned analysis, in EIC figure, only there is respective peaks at 20.200min place and 34.827min place, the resveratrol of originating for giant knotweed; Only there is respective peaks at 21.368min and 23.253min place, the resveratrol of originating for grapevine; All not going out peak at above four appearance times, is the resveratrol in other source; All going out peak at above four appearance times, is both potpourri.
Analysis conditional control is as follows:
A) liquid-phase chromatographic analysis condition
Chromatographic column: XUnion C18;
Mobile phase: acetonitrile-water;
Gradient elution: 0-30min, in mobile phase, the volumetric concentration of acetonitrile is 10%-50%;
30-40min, in mobile phase, the volumetric concentration of acetonitrile is 50%-95%;
40-42min, in mobile phase, the volumetric concentration of acetonitrile is 95%-10%;
42-50min, in mobile phase, the volumetric concentration of acetonitrile is 10%;
Flow velocity: 0.2mL/min;
Column temperature: 30 ℃;
Detect wavelength: 287nm;
Sample size: 5 μ L
B) mass spectrophotometry condition
Ion gun: ESI atomization gas: 55psig;
Scan mode: negative ion mode dry gas flow velocity: 6L/min;
Sheath temperature degree: 400 ℃; Capillary voltage: 3000V;
Sheath gas velocity: 12L/min; Cracking voltage: 135V;
Taper hole voltage: 65V; Mass scanning scope: 100-1700m/z, every 0.2s gathers 1 collection of illustrative plates.
The mass content of preferred described resveratrol is 5%, described sample quality concentration 10mg/100mL.
Compared with prior art, advantage of the present invention is:
1, this analytical approach can be distinguished by a kind of analytical approach the separate sources of resveratrol, particularly in the resveratrol in grapevine source, distinguish the resveratrol that whether contains giant knotweed source, prevent that the phenomenon that highly purified giant knotweed source resveratrol is added in the resveratrol in low-purity grapevine source from occurring.
2, high, reproducible, the good stability of this analytical approach accuracy.
Accompanying drawing explanation
Fig. 1 is 5% giant knotweed resveratrol TIC figure;
Fig. 2 is 5% grape resveratrol TIC figure;
Fig. 3 is 98% giant knotweed resveratrol TIC figure;
Fig. 4 is that 5% giant knotweed RES extracts TIC figure;
Fig. 5 is that 5% giant knotweed RES extracts Physcion-1-O-β-D-Glucose glycosides EIC figure;
Fig. 6 is that 5% giant knotweed RES extracts archen EIC figure;
Fig. 7 is that 5% giant knotweed RES extracts ε-grape element EIC figure;
Fig. 8 is that 5% giant knotweed RES extracts α-grape element EIC figure;
Fig. 9 is that 5% grape RES extracts TIC figure;
Figure 10 is that 5% grape RES extracts Physcion-1-O-β-D-Glucose glycosides EIC figure;
Figure 11 is that 5% grape RES extracts archen EIC figure;
Figure 12 is that 5% grape RES extracts ε-grape element EIC figure;
Figure 13 is that 5% grape RES extracts α-grape element EIC figure.
Embodiment
Below in conjunction with embodiment, the present invention is described further.
1 materials and methods
1.1 experiment material
1.1.1 specimen material
Eight batches of the same place of production (Yichang) different times Gentrin Knotweed P.Es (Resveratrol content is 5%), eight batches of the same place of production (Yongan, Hunan) different times grapevine extracts (Resveratrol content is 5%), the same place of production (Yichang) different times Gentrin Knotweed P.E (Resveratrol content is 98%) provides by Hunan Micolta Bioresource Inc..
1.1.2 instrument and equipment
Agilent1290 liquid chromatography-G6530 series four Agilent companies of the U.S.
Level bar-flight time mass spectrum combined instrument
Milipore company of the ultrapure water equipment U.S.
Electronic analytical balance Shanghai Mettler-Tolede Instrument Ltd.
KQ5200DE type numerical control Vltrasonic device Kunshan Ultrasonic Instruments Co., Ltd.
1.1.3 main agents
Methyl alcohol (Anhui Shi Lian special solvent incorporated company) is that chromatographically pure, acetonitrile (Merck company) are chromatographically pure; Institute's water is laboratory self-control ultrapure water (total organic carbon TOC≤5ppb);
Resveratrol (RES, >=99%) and polygonin (Polydatin, >=96%), Kaempferol (Kaempferol, >=96%), archen (Emodin, >=98%) standard model are all purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
1.2 experimental technique
1.2.1 mix reference substance solution preparation
It is appropriate that precision takes standard items resveratrol, polygonin, Kaempferol, archen respectively, be placed in the brown volumetric flask of 100mL, add appropriate chromatogram methyl alcohol to dissolve, keep 25 ℃ ultrasonic, until sample dissolves completely, leave standstill cooling constant volume, chromatogram methyl alcohol dilution for storing solution, being configured to concentration is the mixing reference substance solution of 10-50 μ g/mL, crosses the organic miillpore filter of 0.22 μ m, for subsequent use.
1.2.2 test liquid solution preparation
Precision takes 5%, 98% Gentrin Knotweed P.E, 5% the each 10mg of grapevine extract sample, is placed in the brown volumetric flask of 100mL, adds appropriate chromatogram methyl alcohol to dissolve, keep 25 ℃, ultrasonic 30min, leaves standstill cooling constant volume, cross the organic miillpore filter of 0.22 μ m, filtrate is for subsequent use as need testing solution.
1.2.3 chromatographiccondition
Chromatographic column: XUnion C18 (2.8 μ m, 4.6 × 150mm) (Hua Puxinchuan Science and Technology Ltd.);
Mobile phase: acetonitrile (B)-water (A); Gradient elution (0-30min, 10%-50%B; 30-40min, 50%-95%B; 40-42min, 95%-10%B, 42-50min, 10%B)
Flow velocity: 0.2mL/min;
Column temperature: 30 ℃;
Detect wavelength: 287nm;
Sample size: 5 μ L
1.2.4 mass spectrophotometry condition
Ion gun: ESI atomization gas: 55psig;
Scan mode: negative ion mode dry gas flow velocity: 6L/min;
Sheath temperature degree: 400 ℃; Capillary voltage: 3000V;
Sheath gas velocity: 12L/min; Cracking voltage: 135V;
Taper hole voltage: 65V; Mass scanning scope: 100-1700m/z, every 0.2s gathers 1 collection of illustrative plates.
2 results and analysis
2.1 methodological study
2.1.1 precision
Get Gentrin Knotweed P.E (Resveratrol content 5%) No. 1, sample (HZ1), according to 1.2.2 item below legal system available test sample solution, continuous sample introduction 6 times, records sample chromatogram figure.Result shows that the relative retention time RSD value of main chromatographic peak resveratrol is 0.52%, and the RSD value of relative peak area is 1.59%, as shown in table 3.1, meets characteristic spectrum requirement, shows that the precision of instrument is good.
Table 3.1 Precision Experiment data
2.1.2 repeated
Get No. 1 (HZ1) 6 parts of Gentrin Knotweed P.E (Resveratrol content 5%) sample, according to 1.2.2 item below legal system available test sample solution, respectively sample is detected, record sample chromatogram figure.In chromatogram, the relative retention time RSD value of main chromatographic peak resveratrol is 0.25%, and the RSD value of relative peak area is 0.82%, as shown in table 3.2, meets characteristic spectrum requirement, shows that the repeatability of method is good.
The repeated data of table 3.2
2.1.3 stability
Get Gentrin Knotweed P.E (Resveratrol content 5%) No. 1, sample (HZ1), according to 1.2.2 item below legal system available test sample solution, respectively 0,1,2,4,6,12,18,24h detects, and records sample chromatogram figure.The relative retention time RSD value that calculates main chromatographic peak resveratrol is 0.47%, and the RSD value of relative peak area is 2.66%, as shown in table 3.3, meets characteristic spectrum requirement, shows that test sample solution is stable in 24h.
The repeated data of table 3.3
2.1.4 lowest detectable limit
Under selected chromatogram and mass spectrum condition, in the time of S/N=3, the lowest detectable limit of the archen in giant knotweed and grapevine extract sample to be calculated, result shows that the lowest detection of archen is limited to 0.569ng/mL.
2.2 mass spectrometric data collections
By the test solution of the giant knotweed source resveratrol of 8 batches 5% of same place of production different times, the grapevine source resveratrol of 8 batches 5% and 8 batches of 98% giant knotweed source resveratrol extract samples by time of-flight mass spectrometer auto injection, carry out mass spectrum and high performance liquid chromatography data acquisition, the total ion current figure (TIC) of all kinds of samples, as Figure 1-3.
5% giant knotweed source resveratrol, 5% grapevine source resveratrol and the difference of 98% giant knotweed source resveratrol between total ion current figure are all larger.Because 98% giant knotweed source resveratrol has passed through meticulous processing purifying in art production process, its sample impurities amount is less, so the giant knotweed of 98% giant knotweed source resveratrol sample peak-to-average force ratio 5% and grapevine source resveratrol sample collection of illustrative plates obviously reduce; Between 5% giant knotweed resveratrol and 5% grapevine resveratrol sample spectrogram there is larger difference in peak shape, in the situation that analytical approach is definite, the distribution in time of peak on 5% giant knotweed source resveratrol sample total ion current figure is comparatively even, and peak and peak-to-peak degree of separation are also better, and 5% grapevine source resveratrol sample is on total ion current figure, the time that compound goes out peak all early, goes out peak before concentrating on 30min, and the comparatively intensive state of gathering of the distribution of peak on time shaft.
In 2.3 characteristic spectrums, chemical composition is inferred
Adopt Agilent Mass Hunter analysis software to analyze 5% giant knotweed collecting and 5% grapevine source resveratrol sample data, adopt standard control to confirm the method for retention time, and the element composition of each accurate molecular weight is retrieved, the theoretical molecular and the each compound that take into full account each compound element composition are surveyed absolute error≤10 × 10 between molecular weight
-6principle, retain element composition that retrieval obtains and with giant knotweed and grapevine chemical composition data storehouse in each compound be analyzed, obtain altogether the information of 11 chemical compositions in the each total peak of resveratrol sample finger-print, grapevine source of 5% giant knotweed source resveratrol sample finger-print and 5%, in table 3.4.
Chemical composition mass spectrometric data in table 3.4 giant knotweed, grapevine resveratrol sample
11 compound Information in Mass Spectras shown in table 3.4 are all inferred gained according to standard control confirmation or accurate molecular weight, and each compound inference analysis process is as follows:
(1) polygonin t
rthe peak of=10.260min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z389.132.The polygonin compound element set existing in giant knotweed becomes C
20h
22o
8, relative molecular mass theoretical value is 390.1315, and measured value is 390.132, and error is 1.3ppm, meanwhile, adopts the contrast of polygonin reference substance to confirm, and retention time is coincide at 10.260min and reference substance retention time, therefore infer that this compound is polygonin.
(2) resveratrol t
rthe peak of=16.02min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z227.0792.Its compound element set of resveratrol becomes C
14h
12o
3, relative molecular mass theoretical value is 228.0786, and measured value is 228.0792, and error is 2.6ppm, meanwhile, adopts the contrast of resveratrol reference substance to confirm, and retention time is consistent with reference substance retention time at 16.02min, therefore infer that this compound is resveratrol.
(3) 3-methyl-5-hydroxyl-7 methoxyl-chromone t
rthe peak of=17.087min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z205.0585.According to document
[60]report, its compound element set becomes C
11h
10o
4, relative molecular mass theoretical value is 206.0579, and measured value is 206.0585, and error is 1.9ppm, infers that accordingly this compound is 3-methyl-5-hydroxyl-7 methoxyl-chromones.
(4) archen-8-O-β-D-Glucose glycosides t
rthe peak of=17.627min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z431.1062.According to document
[60]report, its compound element set becomes C
21h
20o
10, relative molecular mass theoretical value is 432.1056, and measured value is 432.1062, and error is 1.4ppm, infers that accordingly this compound is archen-8-O-β-D-Glucose glycosides.
(5) Physcion-1-O-β-D-Glucose glycosides t
rthe peak of=20.2min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z445.1218.According to document
[60]report, its compound element set becomes C
22h
22o
10, relative molecular mass theoretical value is 446.1213, and measured value is 446.1218, and error is 1.1ppm, infers that accordingly this compound is Physcion-1-O-β-D-Glucose glycosides.
(6) Kaempferol t
rthe peak of=22.093min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z285.0483.The polygonin compound element set existing in giant knotweed becomes C
15h
10o
6, relative molecular mass theoretical value is 286.0477, and measured value is 286.0483, and error is 2.1ppm, meanwhile, adopts the contrast of Kaempferol reference substance to confirm, and retention time is identical with reference substance retention time at 22.093min, therefore infer that this compound is Kaempferol.
(7) archen t
rthe peak of=34.827min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z269.0534.Its compound element set becomes C
15h
10o
5, relative molecular mass theoretical value is 270.0528, and measured value is 270.0534, and error is 2.2ppm, meanwhile, adopts the contrast of archen reference substance to confirm, and retention time is consistent with reference substance retention time at 34.827min, therefore infer that this compound is archen.
(8) grape element t
rthe peak of=18.427min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z469.1371.According to document
[61]report, grape element A compound element set becomes C
28h
22o
7, relative molecular mass theoretical value is 470.1336, and measured value is 470.1371, and error is 7.4ppm, infers that accordingly this compound is grape element A.
(9) grape element t
rthe peak of=19.4min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z905.2682.According to document
[61]report, the grape existing in grape element H compound element set becomes C
56h
42o
12, relative molecular mass theoretical value is 906.2676, and measured value is 906.2682, and error is 0.6ppm, infers that accordingly this compound is grape element H.
(10) ε-grape element t
rthe peak of=20.953min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z454.According to document
[61]report, the ε existing in grape-grape element compound element set becomes C
28h
22o
6, relative molecular mass theoretical value is 454.1416, and measured value is 454.1422, and error is 1.3ppm, infers that accordingly this compound is ε-grape element.
(11) α-grape element t
rthe peak of=20.953min is at ESI
-under pattern, obtain the quasi-molecular ions of accurate molecular weight m/z677.1895.According to document
[61]report, the α existing in grape-grape element compound element set becomes C
42h
30o
9, relative molecular mass theoretical value is 678.189, and measured value is 678.1895, and error is 0.7ppm, infers that accordingly this compound is α-grape element.
2.4 characterization compounds are confirmed
The giant knotweed that utilizes mass spectrophotometry to obtain, the compound information at total peak in the resveratrol finger-print of grapevine source, further total ion current figure (TIC) is carried out to the ion extraction (EIC) according to accurate molecular weight, from Fig. 4-13, while extraction by the accurate molecular weight to Physcion-1-O-β-D-Glucose glycosides and archen, in the resveratrol extract in giant knotweed source, be that 34.827min place of 20.200min place has respective peaks in retention time, and archen peak height is higher, and there is no chromatographic peak at 20.200min place in the resveratrol extract of grape source, also lose peak shape at 34.827min place, ε-grape element and α-grape element are carried out to EIC while extracting, grape source resveratrol extract is that 21.368min and 23.253min place have respective peaks in retention time, and giant knotweed source resveratrol extract does not have the appearance of respective peaks in corresponding retention time.Therefore, infer 11 compound information that obtain according to mass spectrum, contrast giant knotweed sample finger-print and grape sample finger-print, search checking by UPLC-QTOF analysis software molecular formula, tentatively obtain having 4 characterization compounds of notable difference between the two, they are respectively ε-grape element and α-grape element in Physcion-1-O-β-D-Glucose glycosides, archen and the grape in giant knotweed.In 11 kinds of compounds that mass spectrum is inferred, the existence of Physcion-1-O-β-D-Glucose glycosides and archen in the resveratrol of grape source, can not be detected, and giant knotweed source resveratrol can not detect ε-grape element and 2 kinds of compounds of α-grape element.Relative content in the resveratrol sample that two kinds of compounds of wherein archen and ε-grape element are originated with grape in giant knotweed source respectively is separately higher.So, can be take archen and ε-two kinds of compounds of grape element as main, and distinguish giant knotweed and grape separate sources resveratrol in conjunction with Physcion-1-O-β-D-Glucose glycosides and α-grape element as 4 kinds of characterization compounds.
3 discuss
This experiment utilizes the high efficiency ultra high efficiency liquid phase of high-resolution time-of-flight mass spectrometry instrument to conduct in-depth research the resveratrol sample identification method in giant knotweed and grapevine source, and 11 the compound information in giant knotweed and grape source resveratrol sample that obtained are polygonin, resveratrol, 3-methyl-5-hydroxyl-7-methoxyl-chromone, archen-8-O-β-D-Glucose glycosides, Physcion-1-O-β-D-Glucose glycosides, Kaempferol, archen, grape element A, grape element H, ε-grape element and α-grape element, and tentatively found and can distinguish giant knotweed, 4 potential mark compounds of grapevine source resveratrol are Physcion-1-O-β-D-Glucose glycosides, archen, ε-grape element and α-grape element, wherein take archen and ε-grape element as main, Physcion-1-O-β-D-Glucose glycosides and α-grape element are the auxiliary resveratrol of distinguishing giant knotweed and grape separate sources, for the raw material sources of resveratrol and the discriminating of quality provide scientific basis, also for the quality control of other medicinal materials provides certain reference.Adopt UPLC-Q/TOF technology to analyze the resveratrol in grapevine and giant knotweed source, the total ion spectrogram of resveratrol sample in contrast different material source, it is carried out to daughter ion TIC extraction, can obtain raw material sources more accurately and characterize spectrogram and more accurate compound information, for accuracy and the reliability of experimental result provide technical guarantee.Simultaneously, sign by LC-MS technology to different plant origin resveratrols, and to potential marker compounds in the resveratrol sample in grapevine and two kinds of sources of giant knotweed determine, for the identification of the true raw material sources of resveratrol provides simple to operate, effective method.
Claims (5)
1. a LC-QTOF analytical approach for distinguishing different resveratrol, is characterized in that, concrete steps are:
(1) preparation detects sample: get Gentrin Knotweed P.E sample and grapevine extract sample that resveratrol mass content is identical, add alcohol to dissolve, be mixed with the solution that sample quality concentration is identical, ultrasonic 25-35min at 25 ℃-30 ℃, leave standstill cooling, filtering with microporous membrane, filtrate is as detecting sample;
(2) detect: detection sample is carried out to liquid chromatography/level Four bar-flight time mass spectrum method for combined use and analyze, obtain liquid chromatogram and mass spectrogram and data; Wherein, analysis condition control is as follows:
A) liquid-phase chromatographic analysis condition
Chromatographic column: XUnion C18;
Mobile phase: acetonitrile-water;
Gradient elution: 0-30min, in mobile phase, the volumetric concentration of acetonitrile is 10%-50%;
30-40min, in mobile phase, the volumetric concentration of acetonitrile is 50%-95%;
40-42min, in mobile phase, the volumetric concentration of acetonitrile is 95%-10%;
42-50min, in mobile phase, the volumetric concentration of acetonitrile is 10%;
Flow velocity: 0.1-0.3mL/min;
Column temperature: 25 ℃-35 ℃;
Detect wavelength: 287nm;
B) mass spectrophotometry condition
Ion gun: ESI atomization gas: 50-60psig;
Scan mode: negative ion mode dry gas flow velocity: 5-7L/min;
Sheath temperature degree: 400 ℃; Capillary voltage: 3000V;
Sheath gas velocity: 10-14L/min; Cracking voltage: 125-145V;
Taper hole voltage: 50-70V; Mass scanning scope: 100-1700m/z, every 0.2s gathers 1 collection of illustrative plates;
(3) to collection of illustrative plates and data analysis:
To the giant knotweed collecting and grapevine source resveratrol mass spectrogram and data analysis, obtain total peak;
(4) characterization compound is confirmed
Utilize the compound information at total peak in giant knotweed, the grapevine source resveratrol finger-print that mass spectrophotometry obtains, further total ion current figure is carried out to the ion extraction according to accurate molecular weight, obtain EIC figure, in contrast EIC figure, go out peak difference, obtain having the characterization compound of notable difference between the two;
(5) judgement
Judge the separate sources of resveratrol according to characterization compound.
2. a kind of LC-QTOF analytical approach of distinguishing different resveratrol according to claim 1, is characterized in that, analysis condition control is as follows:
A) liquid-phase chromatographic analysis condition
Chromatographic column: XUnion C18;
Mobile phase: acetonitrile-water;
Gradient elution: 0-30min, in mobile phase, the volumetric concentration of acetonitrile is 10%-50%;
30-40min, in mobile phase, the volumetric concentration of acetonitrile is 50%-95%;
40-42min, in mobile phase, the volumetric concentration of acetonitrile is 95%-10%;
42-50min, in mobile phase, the volumetric concentration of acetonitrile is 10%;
Flow velocity: 0.2mL/min;
Column temperature: 30 ℃;
Detect wavelength: 287nm;
Sample size: 5 μ L
B) mass spectrophotometry condition
Ion gun: ESI atomization gas: 55psig;
Scan mode: negative ion mode dry gas flow velocity: 6L/min;
Sheath temperature degree: 400 ℃; Capillary voltage: 3000V;
Sheath gas velocity: 12L/min; Cracking voltage: 135V;
Taper hole voltage: 65V; Mass scanning scope: 100-1700m/z, every 0.2s gathers 1 collection of illustrative plates.
3. according to the LC-QTOF analytical approach of a kind of distinguishing different resveratrol described in claim 1 or 2, it is characterized in that, the mass content of described resveratrol is 5%, described sample quality concentration 10mg/100mL.
4. according to the LC-QTOF analytical approach of a kind of distinguishing different resveratrol described in claim 1 or 2, it is characterized in that, step (3) is described as follows to collection of illustrative plates and data analysis:
Adopt Agilent Mass Hunter analysis software to the giant knotweed collecting and grapevine source resveratrol mass spectrogram and data analysis, obtain 11 total peaks, specific as follows:
In the resveratrol mass spectrogram of giant knotweed source, appearance time is that the peak of 10.26mi is polygonin, appearance time is that the peak of 16.02min is 3-methyl-5-hydroxyl-7-methoxyl-chromone, appearance time is that the peak of 17.087min is archen-8-O-β-D-Glucose glycosides, appearance time is that the peak of 17.627min is Physcion-1-O-β-D-Glucose glycosides, appearance time is that the peak of 20.2min is Physcion-1-O-β-D-Glucose glycosides, appearance time is that the peak of 22.093min is Kaempferol, and the peak that appearance time is 34.487min is archen;
In the resveratrol mass spectrogram of grapevine source, appearance time is that the peak of 18.427min is grape element A, appearance time is that the peak of 19.4min is grape element H, and the peak that appearance time is 20.953min is ε-grape element, and the peak that appearance time is 22.867min is α-grape element.
5. according to the LC-QTOF analytical approach of a kind of distinguishing different resveratrol described in claim 1 or 2, it is characterized in that, the described characterization compound of step (4) is respectively Physcion-1-O-β-D-Glucose glycosides in the resveratrol extract in giant knotweed source and ε-grape element and the α-grape element in archen and grapevine source resveratrol extract.
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| CN107561035A (en) * | 2017-07-06 | 2018-01-09 | 成都中医药大学 | A kind of detection method in commercially available Radix Notoginseng powder powder position source |
| CN108362805A (en) * | 2018-02-28 | 2018-08-03 | 安徽瑞思威尔科技有限公司 | A kind of method that UPLC-Q-Tof/MS measures four kinds of natural products in health liquor simultaneously |
| CN108865916A (en) * | 2017-05-08 | 2018-11-23 | 天津华泰至成医药科技发展有限公司 | For converting general bacterium of ε-grape element and application thereof for resveratrol |
| CN108982703A (en) * | 2018-08-23 | 2018-12-11 | 中国检验检疫科学研究院 | A kind of LC-MS detection method of polyphenols |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865916A (en) * | 2017-05-08 | 2018-11-23 | 天津华泰至成医药科技发展有限公司 | For converting general bacterium of ε-grape element and application thereof for resveratrol |
| CN108865916B (en) * | 2017-05-08 | 2022-01-04 | 天津华泰至成医药科技发展有限公司 | Pantoea for converting resveratrol into epsilon-viniferin and application thereof |
| CN107561035A (en) * | 2017-07-06 | 2018-01-09 | 成都中医药大学 | A kind of detection method in commercially available Radix Notoginseng powder powder position source |
| CN108362805A (en) * | 2018-02-28 | 2018-08-03 | 安徽瑞思威尔科技有限公司 | A kind of method that UPLC-Q-Tof/MS measures four kinds of natural products in health liquor simultaneously |
| CN108982703A (en) * | 2018-08-23 | 2018-12-11 | 中国检验检疫科学研究院 | A kind of LC-MS detection method of polyphenols |
| CN108982703B (en) * | 2018-08-23 | 2021-05-14 | 中国检验检疫科学研究院 | A kind of liquid-mass spectrometry detection method of polyphenols |
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