CN108445131A - The detection method of main component in a kind of edible areca-nut - Google Patents
The detection method of main component in a kind of edible areca-nut Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The present invention relates to a kind of detection method of main component in edible areca-nut, the main component includes one or more in alkaloid, phenolic compound；The method detaches sample solution using liquid chromatography, and the sample solution is prepared with the following method：Take edible areca-nut sample, crushed after being dried is added methanol, and 20~40min of ultrasonic extraction after 10~15h of soaking at room temperature takes supernatant after centrifugation.Method provided by the invention carries out qualitative, quantitative research to the main chemical compositions of edible areca-nut, and the system to establish perfect quality control and safety evaluation improves reference.
Description
Technical field
The present invention relates to plant component analysis fields, and in particular to the detection method of main component in a kind of edible areca-nut.
Background technology
Betel nut (Areca catechu L.) is Palmae Areca aiphyllium, originates in Malaysia, is now divided extensively
It includes the countries such as China, India, Indonesia, Malaysia, New Guinea to be distributed in south east asia.Its seed and shell can
Chinese medicine and the treatment for being used for various disease.
Betel nut is the fourth-largest chewing preference in the whole world after cigarette, wine and coffee, is had more than in global range at present
600000000 people's chewing betel nuts, chewing population are mainly distributed on South Asia and south east asia.Regional, the Bin in India and Pakistan etc.
Bulky seed would generally together be involved in Xin Xian Lao leaves with white lime, tobacco and other essence condiment and be used for chewing.In India
The street corner in each city, which is seen everywhere on booth, to be sold Yong being wrapped in betel nut seed and honey, cardamom, fennel, cumin etc. in Lao leaves
The product for the small triangle that various fragrance are folded into.Research shows that chewing betel nut seed can lead to the generation of carcinoma of mouth, therefore Bin
Bulky seed was also classified as level-one carcinogenic substance in 2003 by the International Agency for Research on Cancer of WHO subordinate.
China's edible areca-nut is had been more than two thousand years of history, it is not only daily preference, but also is wedding and day
It often communicates important carrier, therefore forms unique betel nut culture.But it is different from chewing seed, in Hainan and Taiwan,
Common eating method is to choose tender small betel nut to be cut into several valves, and a Lao leaf is mixed per valve, is rolled into after applying last layer white lime
Chewing after a branch of.And in Hunan, immature betel nut shell is made by baking, steeping, boiling, steaming, sending out the techniques such as system and stewing system
For the commercial product of chewing.Currently, 90% or more the produced betel nut in Hainan is to supply Hunan betel nut enterprise in a manner of dry fruit or fresh fruit
Industry, which is processed into edible areca-nut, chews block, and Hunan edible areca-nut yield alreadys exceed 200,000 tons, Hainan and Hunan two places betel nut annual value of production
More than 40,000,000,000.
Four kinds of arecaline, guvacoline, arecaidine, guvacine characteristic areca alkaloids are considered as Bin
Lead to the main component of oral cavity carcinogenesis in bulky seed.In Hunan edible areca-nut and seed products Leaf-feeding insects, harvest time with
And processing method is different, contained by alkaloid component content it is necessarily different, different influences is also will produce to human body,
Detrimental effect such as carcinogenicity can not lump together.So far, not studies have reported that the biology in the edible areca-nut of Hunan
Alkali is measured.In addition, plant is research shows that contain more phenols component in betel nut shell and seed.But currently without any
Document report carries out quantitative study to the phenols component in betel nut seed or shell Related product.Therefore, it is quite necessary to establish
The content of alkaloid and phenols component in edible areca-nut is carried out while being measured to the method for one system, is the comprehensive quality of edible areca-nut
Control and safety evaluatio provide reference.
Invention content
It is an object of the invention to overcome the deficiencies of existing technologies, a kind of detection side of main component in edible areca-nut is provided
Method.
Edible areca-nut of the present invention is sold by Hunan market and chews edible areca-nut, and the two kinds of productions of Chinese olive and cigarette fruit are specifically included
Product；Wherein, Chinese olive and cigarette fruit chewing position are the shell of betel nut, and cigarette fruit has fire-cureing by oak in process, and
Chinese olive does not have then.
The targeted main component of the present invention includes one or more in alkaloid, phenolic compound.The alkaloid
It specifically includes：It is one or more in arecaline, guvacoline, arecaidine, guvacine, food can also be included in
With the few methyl nicotinate of content in betel nut.The phenolic compound includes：Catechin, epicatechin, vanillic acid, former catechu
It is one or more in acid, P-hydroxybenzoic acid, glycyrrhizic acid, Isorhamnetin, cyanidenon.
The method of the invention detaches sample solution using liquid chromatography, and the sample solution uses such as lower section
Method is prepared：Taking edible areca-nut sample, crushed after being dried is added methanol, ultrasonic extraction 20 after 10~15h of soaking at room temperature~
40min takes supernatant after centrifugation.The present invention is extracted using methanol as solvent and by largely putting into practice discovery through appropriate
It is best to the extraction effect of target component after immersion.
When the main component is one or more in arecaline, guvacoline, arecaidine, guvacine
When, the liquid chromatography uses cation exchange column, with acetonitrile and concentration 0.1~0.3%, the phosphoric acid that pH value is 2.5~3.5
The mixed solvent of aqueous solution composition is mobile phase, is eluted；The in the mixed solvent, the percent by volume of acetonitrile is 75~
85%.
In practical applications, the method includes steps in detail below：
(1) reference substance for taking the main component is configured to multiple reference substance solutions according to concentration gradient；
(2) the multiple reference substance solution is detected respectively using the liquid chromatography, obtains multiple chromatograms；
With a concentration of abscissa of each reference substance solution, using the chromatographic peak area in its corresponding chromatogram as ordinate, mark is drawn
Directrix curve；
(3) sample solution is prepared, the sample solution is detected according to step (2) identical method, is obtained
Chromatogram finds out chromatographic peak area；The corresponding peak area of the sample solution is substituted into standard curve obtained by step (2), is asked
Obtain the content of the main component.
When the main component be catechin, epicatechin, vanillic acid, protocatechuic acid, P-hydroxybenzoic acid, glycyrrhizic acid,
When one or more compounds in Isorhamnetin, cyanidenon and methyl nicotinate, the liquid chromatography uses C18 silica gel
Column, the mixed solvent formed using methanol and 0.05~0.15% aqueous formic acid are eluted as mobile phase.Preferably, it uses
Following gradient elution program elution：The percent by volume of 0~5min, methanol are 20%；5~6min, the percent by volume of methanol
It is 20~60%；The percent by volume of 6~9min, methanol are 60~90%；The percent by volume of 9~20min, methanol is
90%.
After the present invention is detached using the liquid chromatography, further it is detected using mass spectrography；The mass spectrum
The condition of method includes：Ion source is ESI；Nebulizer pressure, 50~70psi；Dryer temperature is 280~320 DEG C；Spray nozzle voltage
For 3900~4100V；Detection pattern is more reactive ion detection patterns.
In practical applications, the method includes steps in detail below：
(1) reference substance for taking the main component is configured to multiple reference substance solutions according to concentration gradient；
(2) the multiple reference substance solution is detected respectively, obtains multiple spectrograms；With the dense of each reference substance solution
Degree is abscissa, using the peak area in its corresponding spectrogram as ordinate, draws standard curve；
(3) sample solution is prepared, the sample solution is detected according to step (2) identical method, is obtained
Spectrogram finds out peak area；The corresponding peak area of the sample solution is substituted into standard curve obtained by step (2), is acquired described
The content of main component.
Method provided by the invention carries out qualitative, quantitative research to the main chemical compositions of edible areca-nut, perfect to establish
The system of quality control and safety evaluation improves reference.
Description of the drawings
Fig. 1 is that 1 four alkaloid standard curves of gained of embodiment are as shown in Figure 1；Wherein, Figure 1A is guvacoline, figure
1B is arecaline, and Fig. 1 C are guvacine, and Fig. 1 D are arecaidine；
Fig. 2 is Hunan edible areca-nut alkaloid characteristic finger-print.
Fig. 3 is the extraction chromatography of ions figure under target component MRM patterns when LC-MS/MS is detected；Wherein Fig. 3 A are that mixed mark is molten
Liquid, Fig. 3 B are sample solution；
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Embodiment 1
1, the instrument used in the present embodiment and reagent include：
Agilent 1290LC high performance liquid chromatographs (Agilent companies of the U.S. are equipped with G1311C type quaternary pumps,
G1329B type injectors, G1316A types temperature control box and G4214 types multiwavelength detector), the strong sun of WTpolysulfonix-SCX from
Sub- exchange column (Sai Fen Science and Technology Ltd.s, 4.6 × 250mm, 5 μm), (city of Kunshan is super for KQ2200DB types numerical control ultrasonic cleaner
Sound Instrument Ltd.), PH-220 type pen types PH meters (Hangzhou Qi Wei Instrument Ltd.), H1650 table model high speed centrifuges (lake
Nan Xiangyi Laboratory Instruments development corporation, Ltd.), AE1650 types electronic balance (Mettler Toledo Inc.).
Arecoline hydrobromide (Baoji time bio tech ltd, lot number：20161119), hydrochloric acid removes first betel nut
Alkali (Canadian TRC companies, lot number：4-ISU-161-2), hydrobromic acid guvacoline (Canadian TRC companies, lot number：3-MP-
45-3), arecaidine (Alfa Aesar companies of Britain, lot number：10114762).Chromatography methanol, acetonitrile are purchased from German Merk
Company.Phosphoric acid (chromatographically pure) is purchased from Tianjin recovery fine chemistry industry research institute.Ammonium hydroxide (analysis is pure) is purchased from the examination of Chinese medicines group chemistry
Agent Co., Ltd.13 batches of Hunan Chinese olive betel nuts and 27 batches of cigarette fruit betel nuts are collected in the different manufacturer in Hunan respectively；10 are not
With the feedstock capture of the batch roasting family different from Hainan Province Tunchang, Lingshui and three ground of Qionghai；
2, main component in edible areca-nut is detected as follows, the main component is：Arecaline goes first
Arecaline, arecaidine and guvacine；The method is specially：
(1) precision weighs hydrobromic acid guvacoline 6.3mg, Arecoline hydrobromide 33.6mg, hydrochloric acid and removes first betel nut respectively
Secondary alkali 19.0mg and hydrochloric acid arecaidine 16.0mg in 10ml volumetric flasks, with after chromatography methanol constant volume to graduation mark each control
The storing solution of product, 4 DEG C of refrigerations are spare；Reference substance storing solution is taken to configure the reference substance solution of 5 various concentrations from high to low；
(2) separation detection is carried out with the following method：Chromatographic column WTpolysulfonix-SCX columns (4.6 × 250mm, 5 μ
m)；Mobile phase A CN:0.2% phosphate aqueous solution (adjusting PH=3.0 with ammonium hydroxide)=81:19(v/v)；Flow velocity 1ml/min；Column temperature
30℃；10 μ l of sample size；Obtain multiple chromatograms；With a concentration of abscissa of each reference substance solution, with its corresponding chromatography
Chromatographic peak area in figure is ordinate, draws standard curve；
(3) accurately weighed sample powder 0.5g, is placed in 25ml conical flask with cover, and 10ml chromatography methanol, room is added in precision
Ultrasonic extraction 30min after temperature is impregnated 12 hours, wipes dry conical flask and lets cool to room temperature, supply the quality of less loss, taken after shaking up
Clear liquid, high speed centrifugation 10min (13000r/min) obtain sample solution；According to the identical method of step (2) to the sample solution
It is detected, obtains chromatogram, find out chromatographic peak area；The corresponding peak area of the sample solution is substituted into obtained by step (2)
In standard curve, the content of the main component is acquired.
Four alkaloid standard curves are as shown in Figure 1 obtained by the present embodiment；Wherein, Figure 1A is guvacoline, Tu1BWei
Arecaline, Fig. 1 C are guvacine, and Fig. 1 D are arecaidine；Related coefficient (the r of four alkaloid standard curves2) respectively
It is 0.9993,0.9994,0.9993 and 0.9992, illustrates that each reference substance linear relationship in sample introduction concentration range is good.Detection
Limit and quantitative limit are respectively defined as 3 times and 10 times of signal-to-noise ratio S/N, and specific detection limit and quantitative limit are as shown in table 1.
The detection limit and quantitative limit of 1 reference substance of table
| Reference substance | Related coefficient (r2) | The range of linearity (μ g/ml) | Detection limit (μ g/ml) | Quantitative limit (μ g/ml) |
| Guvacoline | 0.9993 | 0.481-120.1 | 0.0192 | 0.0726 |
| Arecaline | 0.9994 | 2.650-662.5 | 0.0133 | 0.0530 |
| Guvacine | 0.9993 | 0.886-221.5 | 0.0354 | 0.1329 |
| Arecaidine | 0.9992 | 1.017-254.3 | 0.0565 | 0.2034 |
3, precision, repeatability and stability test
A betel nut sample (S6, lot number 20170608), continuous sample introduction 6 are prepared by above-mentioned test solution preparation method
It is secondary, measure four alkaloids content to investigate the precision of content measuring method, as a result measure guvacoline, arecaline,
Guvacine and the RSD of arecaidine content are respectively 0.15%, 0.83%, 1.51% and 1.74%, the reservation at each peak
Time RSD is respectively 0.34%, 0.35%, 0.33% and 0.29% (being shown in Table 2).In order to investigate the repeatability of measurement method, press
Above-mentioned test solution preparation method is parallel to prepare 6 parts of betel nut sample (S6, lot number 20170608), and it is bent that peak area is substituted into standard
Its alkaloid is measured in line respectively and with the repeatability of RSD evaluation methods, measures guvacoline, arecaline, remove first Bin
Bulky secondary alkali and the RSD of arecaidine content are respectively 0.60%, 0.16%, 0.12% and 1.61%, the retention time RSD at each peak
Respectively 0.03%, 0.04%, 0.03% and 0.03% (being shown in Table 3).Preparing a betel nut sample in the same way, (S6 is criticized
Number 20170608), respectively at 0,2,4,6,8,12,24 h, sample introduction is analyzed carries out assay, investigates the stability of method, respectively waits for
The RSD for detecting component content is respectively 2.42%, 0.23%, 0.97% and 0.28%, and the RSD of respective peaks retention time is respectively
0.46%, 0.48%, 0.34% and 0.34% (being shown in Table 4).These are the result shows that the precision of instrument, repeatability and stability are good
Good, which is suitable for the assay of four kinds of areca alkaloids.
2 Precision test result (content of table:Mg/ml, retention time:min)
3 repetitive test result (content of table:Mg/ml, retention time:min)
4 stability test result (content of table:Mg/ml, retention time:min)
4, it is loaded recovery test
6 parts of the betel nut sample (P7 lot numbers 20170601) of known content is taken, every part of 0.5g is accurately weighed, is separately added into suitable
Reference substance known to content is measured, sample is prepared by 1 the method for embodiment, then the sample size after liquid phase measuring sample-adding, is returned
Yield=(measured value-actual value)/actual value * 100%.The sample recovery rate of different reference substances 100.89-107.96% it
Between.Show that the content measuring method is reliable.Sample-adding recovery test data are shown in Table 5.
Table 5 is loaded recovery test result
Using above-mentioned foundation assay method to 13 batches of Chinese olive betel nuts of collection, 27 batches of cigarette fruit betel nuts and 10
The raw material shell of batch carries out assay, and every batch of sample repeats sample introduction 3 times, 10 μ l of each sample introduction.According to the knot of assay
Fruit finds, in four kinds of areca alkaloids in Hunan edible areca-nut cigarette fruit and the raw material of Chinese olive and Hunan edible areca-nut all with
The content highest of arecaline, guvacoline content are minimum.Any areca alkaloids in edible areca-nut Chinese olive and cigarette fruit contain
Amount will be less than the content (P in its raw material<0.01).The content of total areca alkaloids is respectively in edible areca-nut Chinese olive and cigarette fruit
3.007 and 3.027mg/g, far below the 5.258mg/g in former shell.This explanation can be substantially reduced its Bin in edible process
The content of bulky alkaloid.In addition to this, two kinds of Hunan eat the content of four kinds of areca alkaloids between Chinese olive and cigarette fruit betel nut simultaneously
No difference of science of statistics illustrates finally not making significant difference to areca alkaloids content whether sootiness.
Embodiment 2
The integrated signal of the chromatogram of 40 batches of Hunan edible areca-nuts is imported into auspicious " the Chinese medicine chromatographic fingerprint figure for waiting developments of grandson state
Compose similarity 2004A evaluation systems " software.After being sheared to 0-5min non-alkaloids part, it is by median method setting S1
With reference to spectrum, time window 0.2 generates Hunan edible areca-nut alkaloid characteristic finger-print and sees Fig. 2, each batch of edible areca-nut with it is right
It is all higher than 0.982 according to the similarity between collection of illustrative plates, illustrates that similarity is good.Cigarette fruit betel nut and the life of Chinese olive betel nut are also demonstrated simultaneously
The alkaloids content result that there was no significant difference.
Embodiment 3
1, the instrument used in the present embodiment and reagent include：
(Agilent Science and Technology Ltd.s of the U.S. are equipped with G4024A to Agilent 1290Infinity high performance liquid chromatographs
Type quaternary pump, G4226A type injectors, G1316C types temperature control box and G4212 types multiwavelength detector), tandem mass spectrum is
The triple level four bars mass spectrographs of 6460 types of Agilent (Agilent Science and Technology Ltd.s of the U.S., ion source are electron spray),
Agilent Elipseplus C18 chromatographic columns (1.8 μm, 2.1 × 50mm, Agilent Science and Technology Ltd.s of the U.S.)；Data are adopted
Collection and processing use Aglilent Masshunter Workstation Quantitative Analysis softwares (Version
B.07.01, Agilent Science and Technology Ltd.s), KQ2200DB types numerical control ultrasonic cleaner (the limited public affairs of city of Kunshan's ultrasonic instrument
Department), H1650 table model high speed centrifuges (Hunan Xiang Yi Laboratory Instruments development corporation, Ltd.), AE1650 type electronic balances (Mei Te
Le-support benefit company).
Catechin (lot number：20161221), epicatechin (lot number：20161026), vanillic acid (lot number：20170322)、
Cyanidenon (lot number：16122801), protocatechuic acid (lot number：16041301), Isorhamnetin (lot number：20160528), niacin
Methyl esters (C10014322) is purchased from Baoji time bio tech ltd, glycyrrhizin (lot number：drk-1566-910-86-9)
Purchased from Chengdu moral it is sharp can bio tech ltd, P-hydroxybenzoic acid be laboratory self-control.All reference substances are detected through HPLC
Purity >=98%.Chromatography methanol, acetonitrile are purchased from German Merk companies.Phosphoric acid (chromatographically pure) is purchased from Tianjin recovery fine chemistry industry
Research institute.Ammonium hydroxide (analysis is pure) is purchased from Sinopharm Chemical Reagent Co., Ltd..It is different that 40 batches of edible areca-nuts are collected in Hunan
Manufacturer；The feedstock capture of the 10 different batches roasting family different from Hainan Province Tunchang, Lingshui and three ground of Qionghai；4 batches of Chinese medicines
The shell of areca nut and 10 batches of Chinese medicine betel nuts are bought respectively in the different pharmacy in the whole nation.
2, main component in edible areca-nut is detected as follows, the main component is：Catechin, table
Theine, vanillic acid, methyl nicotinate, protocatechuic acid, P-hydroxybenzoic acid, glycyrrhizic acid, Isorhamnetin and cyanidenon；The method
Specially：
(1) respectively accurately weighed catechin 14.0mg, epicatechin 9.1mg, vanillic acid 7.5mg, methyl nicotinate 11.9mg,
Protocatechuic acid 7.7mg, P-hydroxybenzoic acid 15.4mg, glycyrrhizic acid 6.6mg, Isorhamnetin 6.3mg and cyanidenon 5.6mg in
In 10ml volumetric flasks, with after chromatography methanol dilution to scale each ingredient reference substance storing solution, it is spare in 4 DEG C of refrigerations；Take control
Product storing solution configures the reference substance solution of 5 various concentrations from high to low；
(2) liquid phase chromatogram-mass spectrometry combination method is used to carry out separation detection, specially：
Chromatographic column Agilent Elipse plus C18 (1.8 μm, 2.1 × 50mm, the U.S. Agilent limited public affairs of science and technology
Department)；Column temperature：30℃；Mobile phase：The aqueous formic acid that A phases are 0.1%, B phases are 100% methanol；Elution program is：0~
5min, 20%B；5~6min, 20~60%B；6~9min, 60~90%B；9~20min 90%B；Run time is afterwards
7min；Flow velocity is 0.3ml/min；Sample size is 1 μ l；
Mass Spectrometry Conditions：Ion source is ESI；Nebulizer pressure, 60psi；Dryer temperature (Gas Temp), 300 DEG C；It is dry
Throughput (Gas Flow), 11L/min；Spray nozzle voltage (Capillary), 4000V；Detection mode detects for more reactive ions
(Multiple Reaction Monitoring, MRM) pattern；
The present embodiment obtain under MRM patterns shift to an earlier date chromatography of ions figure see Fig. 3；Wherein Fig. 3 A are mixed mark solution；Fig. 3 B are sample
Product solution；With a concentration of abscissa of each reference substance solution, using the chromatographic peak area in its corresponding chromatogram as ordinate,
Standard curve is drawn, it is specific as shown in table 6；
6 regression curve of table
| Standard items | Regression curve | Coefficient R2 | The range of linearity (μ g/ml) |
| Protocatechuic acid | Y=474.44x+211.99 | 0.9989 | 0.462-9.240 |
| Catechin | Y=1103.9x+772.05 | 0.9982 | 0.280-70.000 |
| Methyl nicotinate | Y=30745x+1398.3 | 0.9982 | 0.038-3.808 |
| P-hydroxybenzoic acid | Y=427.4x+3691.2 | 0.9967 | 7.392-73.920 |
| Vanillic acid | Y=481.28x+140.26 | 0.9991 | 0.300-30.000 |
| Epicatechin | Y=1317.7x+22.767 | 0.9995 | 0.022-5.460 |
| Glycyrrhizic acid | Y=3635.5x+51.094 | 0.9998 | 0.026-0.660 |
| Cyanidenon | Y=2661.5x+32.021 | 0.9998 | 0.004-0.560 |
| Isorhamnetin | Y=7460.4x+30.196 | 0.9996 | 0.005-0.126 |
(3) accurately weighed sample powder 0.5g, is placed in 25ml conical flask with cover, and 10ml chromatography methanol, room is added in precision
Ultrasonic extraction 30min after temperature is impregnated 12 hours, wipes dry conical flask and lets cool to room temperature, supply the quality of less loss, taken after shaking up
Clear liquid, high speed centrifugation 10min (13000r/min) obtain sample solution；According to the identical method of step (2) to the sample solution
It is detected, obtains chromatogram, find out chromatographic peak area；The corresponding peak area of the sample solution is substituted into obtained by step (2)
In standard curve, the content of the main component is acquired.
Reference substance storing solution is taken to be diluted to the reference substance solution of various concentration from high to low, it is then each to be checked in MRM patterns
Sample introduction is analyzed under the optimal Mass Spectrometry Conditions of survey ingredient, and sample size is 1 μ l, with reference substance sample introduction concentration (μ g/ml) for abscissa, color
Spectral peak area is that ordinate draws standard curve and obtains regression equation；It is respectively signal-to-noise ratio S/ to define detection limit and quantitative limit simultaneously
3 times of N and 10 times, take the detection limit and quantitative limit of each ingredient of the continuous dilution metering of reference substance solution, each ingredient to be detected online
Property within the scope of linearly dependent coefficient between 0.9967~0.9996, illustrate that each ingredient to be detected is linear good in the range of linearity
It is good.
2, precision, repeatability and stability test
Sample (S6) is taken to carry out being prepared into test solution, 6 progress of continuous sample introduction by the preparation method of test solution
Analysis measures the content of each ingredient to be measured to be investigated to the precision of assay method, the results showed that 9 ingredients to be detected
The RSD of precision is respectively 1.35%, 2.40%, 2.13%, 0.82%, 3.28%, 2.16%, 1.75%, 4.06%,
3.85%.The result illustrates that the content assaying method precision is good.
In order to investigate the repeatability of method, S6 samples are taken to prepare 6 parts by test solution preparation method is parallel, successively to 6
Sample introduction is analyzed for the progress of part sample solution, measures the chromatographic peak area of each ingredient to be measured, and then learns each component repeatability to be measured
RSD is respectively 1.63%, 4.14%, 3.819%, 1.71%, 4.19%, 3.48%, 1.33%, 3.84%, 4.28%.Explanation
This method repeatability is good.
Prepare a sample solution (S6) by method prepared by test solution, respectively at 0h, 2h, 4h, 6h, 8h, 12h,
Sample introduction measures for 24 hours, the RSD of each Component peak area to be measured is respectively 1.62%, 3.36%, 0.59%, 0.82%, 2.98%,
4.73%, 1.66%, 2.65% and 1.93%, illustrate that sample solution is stablized interior for 24 hours.
3, it is loaded recovery test
6 parts of betel nut sample (S13) known to content is taken, accurate addition is comparable with each component content to be measured in sample respectively
Reference substance solution prepares test solution by method prepared by test solution, and then sample introduction is analyzed, and sample size is 1 μ l, is calculated
Sample recovery rate, the mean sample recovery rate of each ingredient to be measured between 86.40%~107.06%, RSD be 0.79%~
4.93%.
Utilize the different Hunan edible areca-nut sample and 10 of the method 40 batches to collection of the assay of above-mentioned foundation
It criticizes different edible areca-nut raw materials and carries out assay, every batch of sample repeats sample introduction 3 times, 10 μ l of each sample introduction.In recent years, domestic
Four areca alkaloids are all focused mainly on to the chemical constitution study of betel nut Related product outside, in conjunction with HPLC and LC-MS/MS couples
The testing result of 13 kinds of chemical compositions is it is found that other than four kinds of areca alkaloids in betel nut sample, catechin and para hydroxybenzene first
Acid is also the main component in betel nut and its raw material, and in the feed, the content of catechin is even than guvacoline, arecaidine
Content with guvacine will be high, and average content 1.24mg/g accounts for the 17.82% of detected ingredient total content,
In Chinese olive and cigarette fruit, catechin content difference is larger.The content of P-hydroxybenzoic acid is less than in edible areca-nut Chinese olive and cigarette fruit
Arecaline, guvacine and arecaidine but the content for being higher than guvacoline, average content are respectively 0.36mg/
G and 0.31mg/g, percentage are respectively 9.63% and 9.00%.
Although above having used general explanation, specific implementation mode and experiment, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (9)
1. the detection method of main component in a kind of edible areca-nut, which is characterized in that the main component includes alkaloid, phenols
It is one or more in compound；
The method detaches sample solution using liquid chromatography, the sample solution prepare with the following method and
At：Take edible areca-nut sample, crushed after being dried is added methanol, 20~40min of ultrasonic extraction after 10~15h of soaking at room temperature, centrifugation
After take supernatant.
2. according to the method described in claim 1, it is characterized in that, the main component be alkaloid, specifically include：Betel nut
It is one or more in alkali, guvacoline, arecaidine, guvacine.
3. according to the method described in claim 2, it is characterized in that, the liquid chromatography uses cation exchange column, with second
The mixed solvent for the phosphate aqueous solution composition that nitrile is 2.5~3.5 with concentration 0.1~0.3%, pH value is mobile phase, is eluted；
The percent by volume of the in the mixed solvent, acetonitrile is 75~85%.
4. according to the method in claim 2 or 3, which is characterized in that include the following steps：
(1) reference substance for taking the main component is configured to multiple reference substance solutions according to concentration gradient；
(2) the multiple reference substance solution is detected respectively using the liquid chromatography, obtains multiple chromatograms；With each
It is bent to draw standard using the chromatographic peak area in its corresponding chromatogram as ordinate for a concentration of abscissa of a reference substance solution
Line；
(3) sample solution is prepared, the sample solution is detected according to step (2) identical method, obtains chromatography
Figure, finds out chromatographic peak area；The corresponding peak area of the sample solution is substituted into standard curve obtained by step (2), institute is acquired
State the content of main component.
5. according to the method described in claim 4, it is characterized in that, further including：The chromatogram of multiple sample solutions is obtained, accordingly
Draw the finger-print of the main component.
6. according to the method described in claim 1, it is characterized in that, the main component is Alkaloid and/or phenol generalization
Close object；The Alkaloid is methyl nicotinate, and the phenolic compound includes：Catechin, epicatechin, vanillic acid, former youngster
It is one or more in boheic acid, P-hydroxybenzoic acid, glycyrrhizic acid, Isorhamnetin, cyanidenon.
7. according to the method described in claim 6, it is characterized in that, the liquid chromatography use C18 silicagel columns, with methanol with
The mixed solvent of 0.05~0.15% aqueous formic acid composition is mobile phase, is eluted；
It is preferred that being eluted using following gradient elution program：The percent by volume of 0~5min, methanol are 20%；5~6min, methanol
Percent by volume be 20~60%；The percent by volume of 6~9min, methanol are 60~90%；9~20min, the volume of methanol
Percentage is 90%.
8. the method described according to claim 6 or 7, which is characterized in that after being detached using the liquid chromatography, into one
Step is detected using mass spectrography；
The condition of the mass spectrography includes：Ion source is ESI；Nebulizer pressure, 50~70psi；Dryer temperature be 280~
320℃；Spray nozzle voltage is 3900~4100V；Detection pattern is more reactive ion detection patterns.
9. wanting the method described in 6~8 any one according to right, which is characterized in that include the following steps：
(1) reference substance for taking the main component is configured to multiple reference substance solutions according to concentration gradient；
(2) the multiple reference substance solution is detected respectively, obtains multiple spectrograms；With a concentration of of each reference substance solution
Abscissa draws standard curve using the peak area in its corresponding spectrogram as ordinate；
(3) sample solution is prepared, the sample solution is detected according to step (2) identical method, obtains spectrogram,
Find out peak area；The corresponding peak area of the sample solution is substituted into standard curve obtained by step (2), acquire it is described mainly at
The content divided.
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