CN101344507A - High Performance Liquid Chromatography detection method for distinguishing arillus longan quality - Google Patents
High Performance Liquid Chromatography detection method for distinguishing arillus longan quality Download PDFInfo
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Abstract
The invention provides a high-efficiency liquid chromatogram test method used for distinguishing the quality of longan pulp, which comprises the following concrete steps: a sample to be tested (fresh longan pulp or produced dried longan) is processed by cutting and grinding, and ultrasonically extracted from added water, an extracted material is filtered by a microporous film and measured by a high-efficiency liquid chromatogram instrument, and a chromatogram map of the sample to be tested is obtained; the chromatogram map of the sample to be tested is compared with a chromatogram map of standard longan pulp, thus scientifically discerning the sample to be tested between true and false; the chromatogram map of standard longan pulp is established according to a separation method of 10 types of standard nucleotide products; the content of various nucleotide components in the standard longan pulp can be computed according to the standard curve; and consequently the measuring method of the sample to be tested can realize the measurement according to measuring methods of standard nucleotide products and standard longan pulp, and can distinguish the test sample between true and false. The high-efficiency liquid chromatogram test method can accurately and scientifically discern the tested sample between true and false, and evaluate the quality of the tested sample, and has high sensitivity, and good stability and repeatability as well as maneuverability.
Description
Technical field
The present invention relates to agricultural product and differentiate, particularly relate to a kind of high-efficiency liquid chromatography method for detecting of differentiating that arillus longan is true and false.
Background technology
By document 1 (pharmacopoeia commission of Ministry of Health of the People's Republic of China. Chinese Pharmacopoeia version (an one) [M] in 2000. Beijing. the .2000. of Chemical Industry Press), document 2 (China of passing away. arillus longan is mixed pseudo-differentiate [J]. the time precious traditional Chinese medical science traditional Chinese medicines, 1999,355.) and document 3 (Ji Chengmao 10 (5):, Wu Wenhai, Zhang Hezhen. the discriminating [J] of arillus longan and imperial litchi meat. Strait Pharmaceutical Journal, 1996,52) and document 4 (Sun Fali 8 (2):, Xu Lingchuan, the horse duckweed. the discriminating [J] of dried longan pulp and pseudo-product thereof. traditional Chinese medicines are differentiated, 1998,9 (4): 384) as can be known, longan (Dimocarpus longan Lour) be first announcement of health ministry be health food be again the special fruit of tonic, enjoy the laudatory title of southern genseng.Arillus longan is commonly called as longan, and as traditional health food and tonic, at home and abroad circulation is very big on the market, and fake and inferior product is a lot.Report is arranged: useful plant that belongs to Sapindaceae such as poisonous imperial litchi meat or litchi meat pretend to be longan to sell on the market, have only the discrimination method from the mode of appearance at present, differentiate the true and false of arillus longan.
By document 5 (Xiao Weiqiang, HuangBing Xiong, Wang Xiaorong, Deng several nucleosides materials [J] in the .HPLC method mensuration arillus longan. Food Science, 2007,28 (1): 234-237.), document 6 (Xiao Weiqiang, Dai Hongfen, HuangBing Xiong, etc. the HPLC of 9 kinds of ucleosides compositions analyzes [J] in the arillus longan. Hua Zhong Agriculture University's journal, 2007,26 (5): 722-726.), document 7 (HuangBing Xiong, Li Jianguang, Wang Xiaorong waits the HPLC quantitative measurement [J] of multiple nucleosides in the .18 kind longan. guangdong agricultural science, 2008, (3): 67-69) as can be known, the nucleosides material of high-load is arranged in the arillus longan, with adenosine (adenos ine), the content of guanosine (guanosine) and uridine (uridine) is the highest.
But, do not appear in the newspapers at home about the true and false high-efficiency liquid chromatography method for detecting of arillus longan.
Summary of the invention
Technical matters to be solved by this invention is: provide a kind of high-efficiency liquid chromatography method for detecting of differentiating the arillus longan quality, so that the foundation of discriminating is provided for scientific evaluation arillus longan quality.
The present invention solves its technical matters and adopts following technical scheme:
The high-efficiency liquid chromatography method for detecting of discriminating arillus longan quality provided by the invention, specifically: earlier to shredding milled processed for test agent, for test agent is fresh longan pulp or the dried longan made, add the water Extraction by Ultrasound then, extract is behind micro-pore-film filtration, measure with the high performance liquid chromatography standard determination method of having set up, must supply the test agent chromatogram, to compare with standard longan pulp chromatogram for the test agent chromatogram at last, with this to for the test agent true and false evaluation of making science; Standard arillus longan chromatogram is to set up according to the separation method of 10 kinds of nucleosides standard items.
The high-efficiency liquid chromatography method for detecting of discriminating arillus longan quality provided by the invention, its application in differentiating the longan pulp true and false.
The present invention compared with prior art has following main advantage:
1. have obvious characteristics: its adenosine, guanosine and uridine are ucleosides characteristic materials more stable in the sample.This method is highly sensitive, stability and favorable reproducibility, and through several different separation methods tests, above-mentioned three kinds of nucleosides materials all can be separated well, and stable content (the application only chooses wherein a kind of separation method).
2. differentiate and compare from outward appearance, tissue morphology with conventional art adopts, this method from arillus longan include, stable nucleosides material distinguishes, therefore technical have stability and a globality.
3. this method is a kind of discriminating means of comprehensive integral, has both avoided the mistake of naked eyes from judging in appearance, can exactly, scientifically judge the true and false of detected sample again.The typical curve of combined standard product can also be determined in the sample the accurate content of main nucleosides material, thus the quality of judgement sample.
4. this method also has operability, and the common liquid chromatograph that can satisfy the said determination condition all can satisfy separation condition (needs binary gradient elution and column temperature control system).
That the present invention utilizes that high-efficiency liquid chromatography method for detecting has is highly sensitive, good stability, repeatability and practical characteristics, planting longan cultivars stone gorge with the master in Guangdong is analysis of material, water-soluble substances in the stone gorge (comprising multiple nucleosides composition) is separated, plan to build the high-efficiency liquid chromatography method for detecting of upright stone gorge longan, thereby the foundation of discriminating is provided for scientific evaluation longan pulp quality in the same way.
Description of drawings
Fig. 1 mixes nucleosides standard items chromatogram.Among Fig. 1 successively be: cytimidine, uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, thymidine and adenosine (content is 2 μ g/mL) according to retention time.
Fig. 2 is multiple nucleosides and the water-soluble substances high-efficient liquid phase chromatogram that obtains after stone gorge longan sample (bright sample) separates.
Embodiment
The high-efficiency liquid chromatography method for detecting of discriminating arillus longan quality provided by the invention, specifically: earlier to shredding milled processed for test agent, for test agent is fresh longan pulp or the dried longan made, add the water Extraction by Ultrasound then, extract is behind micro-pore-film filtration, utilize the high-performance liquid chromatogram determination method of having set up to separate again, must supply the test agent chromatogram, at last with comparing with standard longan pulp chromatogram for the test agent chromatogram, with this as the true and false scientific evaluation of confession test agent; Standard arillus longan chromatogram is to set up according to the separation method of 10 kinds of nucleosides standard items.
When utilizing the high-performance liquid chromatogram determination technology, the chromatographic condition that is adopted is: the anti-phase water-based post 5 μ m of Nucleosil C18,250mm * 4mm; Moving phase is the KH of 0.005mol/L
2PO
4And methyl alcohol; The binary gradient elution, flow velocity 1mL/min; Detect wavelength 260nm, 35 ℃ of column temperatures.
The present invention differentiates the high-efficiency liquid chromatography method for detecting of arillus longan quality, can adopt the method that may further comprise the steps:
(1) separation of ucleosides constituent reference standard sample:
Accurately take by weighing each standard model 2.5mg, be accurate to 0.00001g, water dissolving and constant volume are to 5mL, the hybrid standard sample solution is diluted with water to 50 μ g/mL, be 2 times of dilutions later on, do typical curve, this typical curve can be used for the content of each nucleosides in the calculation sample, quantitatively the confession test agent is carried out the quality evaluation of longan pulp with this; Carry out the separation of 10 kinds of standard models then with chromatographic condition, obtain 10 kinds of standard model chromatograms.
(2) processing of confession test agent:
A. the fresh longan that will gather really removes the peel and examines, and deposits in-20 ℃, and is standby; Take by weighing standby longan pulp 0.9～1.1g, be accurate to 0.001g; Move into after shredding milled processed in the 25mL volumetric flask, add the water of 10～15mL, carry out ultrasonic Treatment twice, wherein, ultrasonic Treatment is 20 minutes for the first time, stops to carry out the ultrasonic Treatment second time after 10 minutes again, and the time is 10 minutes; Then, water is settled to scale, and sample introduction is preceding with 0.5 μ m micro porous filtration membrane filtration.
Perhaps,
B. the fresh longan pulp that will gather is made semi-dry type longan, and dried longan is dried to constant weight through 70 ℃, and take by weighing 0.32g and be accurate to 0.0001g, the doing of arillus longan: wet is 1: 5.8～6.5; Move in the 25mL volumetric flask after shredding milled processed again, add the water of 14～16mL, soaked 3 hours, ultrasonic Treatment was settled to scale after 30 minutes, and sample introduction is preceding with 0.5 μ m micro porous filtration membrane filtration.
Described fresh arillus longan is taken from Fruit Tree Inst., Guangdong prov. Academy of Agricultural Sciences orchard, comprises that standard longan pulp sample is through this expert appraisal Guangdong main breed stone gorge longan for test agent and standard longan pulp sample.
(3) foundation of confession test agent chromatogram:
The test agent that supplies after handling is carried out efficient liquid phase chromatographic analysis, and each sample repeats 3 times; Under selected chromatographic condition, obtain satisfied separation for test agent, obtain multiple water-soluble substances, the nucleosides more than 7 kinds is wherein arranged; And that is set up contains a plurality of peaks for the test agent chromatogram, and bigger peak has 6, wherein when adenosine, guanosine, uridine peak higher (perhaps peak area is bigger), represents that promptly the content of these three kinds of nucleosides is also higher; According to typical curve and peak value, can calculate the content of these three kinds of nucleosides then.
(4) relatively:
To compare for test agent chromatogram and the standard longan pulp chromatogram of having set up by above-mentioned same step, utilizing examination adenosine, guanosine and uridine is that ucleosides characteristic material more stable in the standard longan pulp sample is differentiated the true and false for test agent.
The high-efficiency liquid chromatography method for detecting of discriminating arillus longan quality provided by the invention, its application in differentiating the longan pulp true and false.Described longan pulp is a Guangdong main breed stone gorge longan pulp, simultaneous adaptation in Guangdong, other kind on ground such as Guangxi, Yunnan, Fujian.
The invention will be further described below in conjunction with example and accompanying drawing.
This example adopts high performance liquid chromatography (HPLC) method, the ucleosides composition HPLC quantitative analysis method of having set up standard stone gorge longan pulp.The result: 10 kinds of ucleosides ingredient standard product can be separated fully, Guangdong main breed stone gorge longan pulp water-soluble extractive chromatogram has about 30 peaks under similarity condition, wherein adenosine, guanosine and uridine content are higher, and adenosine, guanosine and uridine content can be used as the true and false detection method of scientific evaluation stone gorge longan product in above standard stone gorge longan pulp chromatogram and the confession test agent.Specific as follows:
1. materials and methods
1.1. ucleosides constituent reference standard sample, instrument and reagent
1.1.1. standard model
Standard model has 10 kinds, is respectively: cytimidine (cytosine), purchase company in Fluka; Uracil (uracil), cytidine (cytidine), hypoxanthine (hypoxanthine), inosine (inosine), thymidine (thymidine), thymine (thymine), they are purchased in source, Shanghai consor thing Science and Technology Ltd.; Uridine, guanosine (adenine) are purchased the bio tech ltd difficult to understand in the Shanghai uncle; Adenosine is purchased the sigma company in the U.S..
1.1.2. key instrument and reagent
1100 type high performance liquid chromatographs (U.S. Agilent company): band Agilent chem workstation and VWD detecting device, manual injector, 20uL quantity tube.
Separating column (German MACHEREY-NAGEL company): filler is Nucleosil C18, reversed-phase column (water-based post, 5 μ m, the cutting ferrule post of 250mm * 4mm).
Methyl alcohol (chromatographic grade, U.S. Fisher company): experimental water is that distilled water gets through the filtration of Millipore water purifior; It is pure that all the other reagent are homemade analysis.
1.2. the separation of ucleosides constituent reference standard sample:
The separation of 10 kinds of standard models is with reference to Xiao Weiqiang, and the method for HuangBing Xiong etc. is improved (seeing document 5-7).
Accurately take by weighing each standard items 2.5mg, be accurate to 0.00001g, water dissolving and constant volume are to 5mL, and the hybrid standard sample solution is diluted with water to 50 μ g/mL, is 2 times of dilutions later on, does typical curve; Carry out the separation of 10 kinds of standard models then with chromatographic condition, obtain 10 kinds of standard model chromatograms.
Chromatographic condition: the anti-phase water-based post 5 μ m of Nucleosil C18,250mm * 4mm; Moving phase is the KH of 0.005mol/L
2PO
4And methyl alcohol; The binary gradient elution, flow velocity 1mL/min; Detect wavelength 260nm, 35 ℃ of column temperatures.
1.3. standard stone gorge longan sample collecting and processing:
The stone gorge longan pulp of gathering Guangdong Province's longan main breed maturity stage is as research material.The sample collecting place is orchard, the applicant place (Fruit Tree Inst., Guangdong prov. Academy of Agricultural Sciences orchard, sample are through the Guangdong of this expert appraisal main breed stone gorge longan).Adopt following method then:
Method one: longan peeling and the nuclear that will newly adopt, take by weighing the arillus longan that (is accurate to 0.001g) about 1g, put into the 25ml volumetric flask after shredding grinding, add the water about 10ml, Extraction by Ultrasound 20 minutes, stop after 10 minutes ultrasonic Treatment 10 minutes, water is settled to scale again, before the sample introduction with the micro-pore-film filtration of 0.5 μ m.
Method two: the fresh longan pulp that will gather is made semi-dry type longan, and dried longan is dried to constant weight through 70 ℃, and take by weighing 0.32g and be accurate to 0.0001g, the doing of arillus longan: wet is 1: 5.8～6.5; Move in the 25mL volumetric flask after shredding milled processed again, add the water of 14～16mL, soaked 3 hours, ultrasonic Treatment was settled to scale after 30 minutes, and sample introduction is preceding with 0.5 μ m micro porous filtration membrane filtration.
1.4. precision, degree of accuracy and sample stability test
2. result and analysis:
2.1.10 plant ucleosides ingredient standard product separation condition
Chromatographic condition:
Mobile phase A: be the KH of 0.005mol/L
2PO
4, Mobile phase B: be methyl alcohol, flow velocity 1mL/min detects wavelength 260nm.Binary gradient elution: 100% A 5min, 3% B 5min, 10% B 2mim (0.8mL/min), 20% B 4mim (0.6mL/min), 32% B 3mim (0.7mL/min), 48% B 3mim (0.8mL/min), 100% B 5min, 100% B 5min, 100% A 5min, 35 ℃ of column temperatures, 20 μ L quantity tubes.
By selected chromatographic condition, the separating resulting of mixed nucleus glycoside ingredient standard product as shown in Figure 1.According to Fig. 1,10 kinds of standard items are under selected chromatographic condition, and standard items have obtained separating completely.
Among Fig. 1 successively be: cytimidine, uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, thymidine and adenosine (being 2 μ g/mL) according to retention time.
2.2. standard stone gorge longan sample separation result:
Sample treatment and separation condition by 1.3 and 2.1 are carried out HPLC with the arillus longan of taking a sample and are analyzed, and repeat 3 times.Standard stone gorge arillus longan sample separation the results are shown in Figure 2.
As shown in Figure 2: under selected chromatographic condition, sample can obtain satisfied separation, contains the water-soluble substances (comprising the nucleosides more than 7 kinds) about 30 kinds, compares with the standard control collection of illustrative plates, and adenosine, guanosine and uridine content are higher in the sample.
2.3. precision, degree of accuracy and sample stability measurement result
According to previous studies, the relative standard deviation of 9 kinds of hybrid standard nucleosides retention time and peak area in 48h respectively 0.133～1.156% and 1.153-3.023% between, illustrate that the hybrid standard product are stable in 48h, stable and precision is high for test instrument.The standard items recovery is between 91.00～107.29%, and recovery relative standard deviation (is seen document 4-5) between 1.411～4.587%.Sample is unstable after ultrasonic Treatment, under the ice bath preservation condition, measures in 8 hours, should not place and spend the night.
Claims (8)
1. high-efficiency liquid chromatography method for detecting of differentiating the arillus longan quality, it is characterized in that: earlier to shredding milled processed for test agent, for test agent is fresh longan pulp or the dried longan made, add the water Extraction by Ultrasound then, extract is measured with the high performance liquid chromatography standard determination method of having set up behind micro-pore-film filtration, must supply the test agent chromatogram, to compare with standard arillus longan chromatogram for the test agent chromatogram, with this to for the test agent true and false evaluation of making science; Standard arillus longan chromatogram is to set up according to the separation method of 10 kinds of nucleosides standard items.
2. high-efficiency liquid chromatography method for detecting according to claim 1 is characterized in that the chromatographic condition that is adopted is when utilizing the high-performance liquid chromatogram determination technology: the anti-phase water-based post 5 μ m of Nucleosil C18,250mm * 4mm; Moving phase is the KH of 0.005mol/L
2PO
4And methyl alcohol; The binary gradient elution, flow velocity 1mL/min; Detect wavelength 260nm, 35 ℃ of column temperatures.
3. high-efficiency liquid chromatography method for detecting according to claim 1 and 2 is characterized in that described high-efficiency liquid chromatography method for detecting, specifically is to adopt the method that may further comprise the steps:
(1) separation of ucleosides constituent reference standard sample:
Accurately take by weighing each standard model 2.5mg, be accurate to 0.00001g, water dissolving and constant volume are to 5mL, and the hybrid standard sample solution is diluted with water to 50 μ g/mL, is 2 times of dilutions later on, does typical curve; Carry out the separation of 10 kinds of standard models then with chromatographic condition, obtain 10 kinds of standard model chromatograms; 10 kinds of standard models are respectively cytimidine, uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, thymidine and adenosine;
(2) processing of confession bar product:
A. peeling of fresh longan and the nuclear that will gather are deposited in-20 ℃, and is standby; Take by weighing standby longan pulp 0.9～1.1g, be accurate to 0.001g; After shredding milled processed, move in the 25mL volumetric flask again, add the water of 10～15mL, carry out ultrasonic Treatment twice; Then, water is settled to scale, uses the micro porous filtration membrane filtration before the sample introduction,
Perhaps,
B. the fresh longan pulp that will gather is made semi-dry type longan, and dried longan is dried to constant weight through 70 ℃, and take by weighing 0.32g and be accurate to 0.0001g, the doing of arillus longan: wet is 1: 5.8～6.5; Move in the 25mL volumetric flask after shredding milled processed again, add the water of 14～16mL, soaked 3 hours, ultrasonic Treatment was settled to scale after 30 minutes, used the micro porous filtration membrane filtration before the sample introduction;
(3) foundation of confession test agent chromatogram:
The test agent that supplies after handling is carried out efficient liquid phase chromatographic analysis, and each sample repeats 3 times; Under selected chromatographic condition, obtain satisfied separation for test agent, obtain multiple water-soluble substances, the nucleosides more than 7 kinds is wherein arranged; And that is set up contains a plurality of peaks for the test agent chromatogram, and bigger peak has 6, when wherein higher the or peak area in the peak of adenosine, guanosine, uridine is big, represents that promptly the content of these three kinds of nucleosides is also higher; According to typical curve and peak value, calculate the content of these three kinds of nucleosides then;
(4) relatively:
To compare for test agent chromatogram and the standard arillus longan chromatogram of having set up by above-mentioned same step, utilizing adenosine, guanosine and uridine is that ucleosides characteristic material more stable in the standard longan pulp sample is differentiated the true and false for test agent.
4. high-efficiency liquid chromatography method for detecting according to claim 3, it is characterized in that: described twice ultrasonic Treatment of step (2), wherein, ultrasonic Treatment is 20 minutes for the first time, stop to carry out the ultrasonic Treatment second time after 10 minutes again, the time is 10 minutes.
5. high-efficiency liquid chromatography method for detecting according to claim 3 is characterized in that: the described micropore filtering film of step (2) is 0.5 μ m micropore filtering film.
6. high-efficiency liquid chromatography method for detecting according to claim 3 is characterized in that: the described fresh arillus longan of step (2) is taken from Fruit Tree Inst., Guangdong prov. Academy of Agricultural Sciences orchard, comprises for test agent and standard longan pulp sample.
7. high-efficiency liquid chromatography method for detecting according to claim 3 is characterized in that: according to the content of each nucleosides in the described typical curve calculation sample of step (1), quantitatively the confession test agent is carried out the quality evaluation of longan pulp with this.
8. the purposes according to the described high-efficiency liquid chromatography method for detecting of arbitrary claim in the claim 1 to 7 is characterized in that: the application in differentiating the longan pulp true and false.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103439442A (en) * | 2013-08-21 | 2013-12-11 | 上海交通大学医学院附属仁济医院 | Method for determining content of inosine in blood plasma |
| CN108037220A (en) * | 2018-01-26 | 2018-05-15 | 上海上药第生化药业有限公司 | A kind of separation method of nucleosides and alkali radical species and its application |
| CN108061763A (en) * | 2017-11-30 | 2018-05-22 | 广西中医药大学 | Multicomponent content assaying method in longan leaf |
| CN108333280A (en) * | 2018-01-26 | 2018-07-27 | 上海上药第生化药业有限公司 | The separation method of nucleosides material and its application in a kind of Pericarpium Trichosanthis injection |
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| CN103439442A (en) * | 2013-08-21 | 2013-12-11 | 上海交通大学医学院附属仁济医院 | Method for determining content of inosine in blood plasma |
| CN103439442B (en) * | 2013-08-21 | 2014-09-17 | 上海交通大学医学院附属仁济医院 | Method for determining content of inosine in blood plasma |
| CN108061763A (en) * | 2017-11-30 | 2018-05-22 | 广西中医药大学 | Multicomponent content assaying method in longan leaf |
| CN109856254A (en) * | 2018-01-11 | 2019-06-07 | 广西中医药大学 | The method for building up and its finger-print of longan leaf ethyl acetate extract HPLC finger-print |
| CN108037220A (en) * | 2018-01-26 | 2018-05-15 | 上海上药第生化药业有限公司 | A kind of separation method of nucleosides and alkali radical species and its application |
| CN108333280A (en) * | 2018-01-26 | 2018-07-27 | 上海上药第生化药业有限公司 | The separation method of nucleosides material and its application in a kind of Pericarpium Trichosanthis injection |
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Open date: 20090114 |