CN104034821A - Methods for detecting and authenticating tartary buckwheat or tartary buckwheat product - Google Patents
Methods for detecting and authenticating tartary buckwheat or tartary buckwheat product Download PDFInfo
- Publication number
- CN104034821A CN104034821A CN201410298710.7A CN201410298710A CN104034821A CN 104034821 A CN104034821 A CN 104034821A CN 201410298710 A CN201410298710 A CN 201410298710A CN 104034821 A CN104034821 A CN 104034821A
- Authority
- CN
- China
- Prior art keywords
- peaks
- buckwheat
- sample
- detection method
- peak
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides methods for detecting and authenticating tartary buckwheat or a tartary buckwheat product. The tartary buckwheat or the tartary buckwheat product is detected through an ultra-performance liquid chromatography. The UPLC (ultra-performance liquid chromatography) method is easy to operate, high in sensitivity, high in stability and high in repetitiveness; all chromatographic peaks in a tartary buckwheat sample can be effectively separated; the method can be used for detecting the tartary buckwheat or the relevant product; furthermore, the samples mixed with barley and wheat which do not contain flavonoid can be authenticated.
Description
Technical field
The present invention relates to detection and the discrimination method of bitter buckwheat or its product, be specifically related to the Ultra Performance Liquid Chromatography detection method of bitter buckwheat and delicatessen food thereof or intermediate product.
Background technology
Bitter buckwheat Fagopyrum tataricum (L.) Gaer is polygonaceae Polygonaceae Fagopyrum Fagopyrum medicinal and edible plant.As food, bitter-buckwheat nutritive is abundant, is rich in protein, fat, vitamin, trace element and several amino acids etc.; As medicine, bitter buckwheat has hypoglycemic, antifatigue, anti-ageing and prevent and treat the effect of hypertension, coronary heart disease.Because edibility, nutritive value and the medical value of bitter buckwheat are higher, be therefore deeply subject to consumer's favor, the huge market demand.
But a lot of consumers lack the understanding to bitter buckwheat, part businessman mixes the spurious with the genuine whereby, adulterates to seek exorbitant profit with fake and poor products.Nowadays adulterated means numerous and complicated, adulterated problem often occurs, hinder the development progress of bitter buckwheat industry, threaten people's health, if quality supervision and control technology does not improve, do not have again relevant laws and regulations to carry out Constraints Management, will certainly cause great injury to the people's healthy and interests, so research and develop, accuracy of detection is high, easy to operate, detection method is imperative efficiently.
Ultra Performance Liquid Chromatography (Ultra Performance Liquid Chromatography UPLC) (having another name called ultrahigh pressure liquid phase chromatogram, hypervelocity liquid chromatography) is a brand-new classification in separation science, UPLC is by means of theory and the principle of HPLC (high efficiency liquid phase color method), contain the brand new technicals such as granule filler, very low system bulk and fast detecting means, increased flux, sensitivity and the chromatographic peak capacity analyzed.Li Xin etc., utilize UPLC to measure general flavone compound in duck wheat, and the condition of gradient elution of its use is:
A: methyl alcohol, B:0.1% adds aqueous acid
The method can record rutin in sample, Quercetin and Kaempferol content (Li Xin measures flavone compound in duck wheat etc. Ultra Performance Liquid Chromatography-Quadrupole Mass Spectrometry. food industry science and technology, 4 phases in 2011).But complicated component in bitter buckwheat, only to the assay of three kinds of compositions wherein, can not comprehensively reflect the quality of bitter buckwheat or its product.
Summary of the invention
The object of the present invention is to provide the detection method of a kind of bitter buckwheat or its product; Another object of the present invention is to provide its discrimination method.
Particularly, the invention provides the detection method of bitter buckwheat or its product, it detects by Ultra Performance Liquid Chromatography method, comprises following operation steps:
(1) get sample to be checked, taking methyl alcohol or methanol aqueous solution as solvent extraction, collect extract, prepare need testing solution;
(2) need testing solution is injected to Ultra Performance Liquid Chromatography instrument (or hypervelocity liquid chromatograph, ultrahigh pressure liquid phase chromatograph), detect; Wherein, chromatographic condition is as follows:
Chromatographic column: octadecylsilane chemically bonded silica is filler;
Detect wavelength: 275～285nm, be preferably 280nm;
Mobile phase: methyl alcohol-0.1～0.3% phosphate aqueous solution system, be preferably 0.1% phosphate aqueous solution system, its gradient program is as follows:
In upper table, the form of presentation of " X～Y ", refer to the arbitrary time point (comprising endpoint value) comprising in X to Y time range, in concrete operations, get a concrete time point in its scope, for example 2～3min, can choose the concrete time point such as 2min, 2.5min, 3min wherein.
Further, eluent gradient program is as follows:
Further, in the described methanol aqueous solution of step (1), methanol concentration is 60～80%v/v, is 70% ± 2%v/v.
Further, the particle diameter of described chromatographic column filler is 1.7～3.5 μ m, is 1.7～1.8 μ m, more preferably 1.8 μ m.
Further, described column size is: 2.1 × 100mm.
In a specific embodiment of the present invention, the chromatographic column model using is: ACQUITY UPLC HSS T3,1.8 μ m, 2.1 × 100mm.
Further, described flow rate of mobile phase is 0.2ml/min.
Further, chromatographic column column temperature is 30 DEG C.
Wherein, in described Ultra Performance Liquid Chromatography method, taking one or both the composition in rutin, Quercetin as reference substance, by external standard method or fingerprint pattern technology, treat sample product and detect.
Wherein, in described Ultra Performance Liquid Chromatography method, adopt fingerprint chromatogram technology to detect testing sample, its concrete operations are: need testing solution gained finger-print and reference fingerprint are compared, calculated similarity.
In described reference fingerprint, there are 7 common characteristic peaks, taking rutin as reference, the relative retention time at each common characteristic peak is respectively: No. 1 peak: 0.3346 ± 0.47, No. 2 peaks: 0.3894 ± 0.37, No. 3 peaks: 1.0000, No. 4 peaks: 1.3544 ± 0.22, No. 5 peaks: 1.6148 ± 0.18, No. 6 peaks: 2.7698 ± 0.44, No. 7 peaks: 2.8596 ± 0.44.
Further, in described reference fingerprint, taking rutin as reference, the relative peak area at each common characteristic peak is respectively peak No. 1: 0.02404 ± 1.16, No. 2 peaks: 0.02699 ± 0.12, No. 3 peaks: 1.0000, No. 4 peaks: 0.06836 ± 0.29, No. 5 peaks: 0.2210 ± 0.57, No. 6 peaks: 0.1062 ± 1.44, No. 7 peaks: 0.2038 ± 0.56.
Further, described reference fingerprint as shown in Figure 3.
The present invention also provides the discrimination method of bitter buckwheat or its product, and it has following operation steps:
Get sample to be checked, adopt above-mentioned fingerprint chromatogram technology to detect, need testing solution gained finger-print and reference fingerprint are compared, calculate similarity; Similarity is more than 0.9, and sample to be checked is bitter buckwheat or not adulterated bitter buckwheat product.
In a concrete embodiment of the present invention, the similarity of multiple batches of sample all reaches more than 0.95.These samples are pure buckwheat powder.
Described in the present invention, not adulterated bitter buckwheat product refers to the bitter buckwheat product that does not mix non-bitter buckwheat sample, and wherein, non-bitter buckwheat sample is the sample that does not contain flavones ingredient.
In a concrete embodiment of the present invention, described not adulterated bitter buckwheat product refers to and does not mix barley or/and the bitter buckwheat product of wheat, as mixes barley or/and the bitter buckwheat product of the massfraction of wheat more than 2%.
Bitter buckwheat product described in the present invention refers to buckwheat powder.Certainly, except buckwheat powder, other commercially available prod taking bitter buckwheat as primary raw material also can be detected by the inventive method, as bitter buckwheat tea, Tartary buckwheat dried noodles, bitter buckwheat biscuit, the saqima of bitter buckwheat class etc.
Easy and simple to handle, the highly sensitive and good stability, reproducible of UPLC method of the present invention, each chromatographic peak in bitter buckwheat sample effectively can be separated, can be used for the detection to bitter buckwheat or its Related product, and can realize the discriminating that does not contain the sample such as barley, wheat of flavonoids to mixing.
Obviously,, according to foregoing of the present invention, according to ordinary skill knowledge and the means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make amendment, replacement or the change of other various ways.
By the form of specific embodiment, foregoing of the present invention is described in further detail again below.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following embodiment.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Brief description of the drawings
The UPLC figure of Fig. 1 mixing reference substance, wherein, 1. rutin 2. Quercetin 3. Kaempferols
The finger-print of 22 batches of pure buckwheat powders of Fig. 2 and the common pattern of generation wherein, 3. rutin 5. Quercetins
Fig. 3 reference fingerprint
7 pure buckwheat powders not of the same race of Fig. 4 and finger-print and the reference fingerprint of mixing pearling cone meal
7 pure buckwheat powders not of the same race of Fig. 5 and finger-print and the reference fingerprint of mixing wheat flour
The pure buckwheat powder of Fig. 6 and the cluster analysis dendrogram of mixing pearling cone meal
The pure buckwheat powder of Fig. 7 and the cluster analysis dendrogram of mixing wheat flour
The finger-print comparative analysis of the UPLC of the pure buckwheat powder of Fig. 8 and commodity buckwheat powder
The cluster analysis dendrogram of the pure buckwheat powder of Fig. 9 and commodity buckwheat powder
Figure 10 gradient 1 gained chromatogram
Figure 11 gradient 2 gained chromatograms
Figure 12 gradient 3 gained chromatograms
The pure buckwheat powder 275nm of Figure 13 chromatogram
The pure buckwheat powder 280nm of Figure 14 chromatogram
The pure buckwheat powder 285nm of Figure 15 chromatogram
Figure 16 barley sample 275nm chromatogram that adulterates
Figure 17 barley sample 280nm chromatogram that adulterates
Figure 18 barley sample 285nm chromatogram that adulterates
Figure 19 wheat samples 275nm chromatogram that adulterates
Figure 20 wheat samples 280nm chromatogram that adulterates
Figure 21 wheat samples 285nm chromatogram that adulterates
Figure 22 commodity buckwheat powder 275nm chromatogram
Figure 23 commodity buckwheat powder 280nm chromatogram
Figure 24 commodity buckwheat powder 285nm chromatogram
Embodiment
The instrument using in the specific embodiment of the invention is as follows:
Waters Acquity UPLC H-Class System type Ultra Performance Liquid Chromatography instrument, comprise Acquity UPLC QSM, Acquity UPLC Sample Manager FTN, Acquity UPLC PDA Detector and Empower3 workstation (Waters company of the U.S.); Milli-Q (Millipore company of the U.S.); METTLER AE240 electronic analytical balance (plum Teller-Tuo benefit (Shanghai) Instrument Ltd.); W210B thermostat water bath (Shen Shun bio tech ltd, Shanghai)
The detection method (external standard method) of the bitter buckwheat of embodiment 1 the present invention or its Related product
(1) preparation of reference substance solution
Precision takes reference substance rutin 10mg and Quercetin 1mg in 10mL volumetric flask respectively, by methanol constant volume, is made into concentration and is respectively 1mg/mL, the reference substance stock solution of 0.1mg/mL.
(2) preparation of need testing solution
Precision takes bitter buckwheat or commercially available bitter buckwheat series products (crossing No. 5 sieves of pharmacopeia) 0.2g, is placed in tool plug conical flask, adds 70% methyl alcohol, room temperature ultrasonic (250W, 40KHZ) 30min, and collecting ultrasonic extract is need testing solution.Sample introduction is front with the organic membrane filtration of 0.22 μ m.
(3) detect
By in need testing solution and reference substance solution difference injecting chromatograph, detect according to following chromatographic condition:
Chromatographic column: ACQUITY UPLC HSS T3 (1.8 μ m, 2.1 × 100mm); Mobile phase composition: methyl alcohol-0.1% phosphoric acid solution linear gradient elution; Flow velocity: 0.2mL/min; Detect wavelength: 280nm; Sample size: 0.8 μ L; Column temperature: 30 DEG C; Elution program sees the following form:
By external standard method, detect data Criterion curve with reference substance, calculate the content of each index components in testing sample.
The detection method (finger-print counter point) of the bitter buckwheat of embodiment 2 the present invention or its Related product
(1) preparation of need testing solution
Precision takes bitter buckwheat or commercially available bitter buckwheat series products (crossing No. 5 sieves of pharmacopeia) 0.2g, is placed in tool plug conical flask, adds 70% methyl alcohol, room temperature ultrasonic (250W, 40KHZ) 30min, and collecting ultrasonic extract is need testing solution.Sample introduction is front with the organic membrane filtration of 0.22 μ m.
(2) detect
By in need testing solution difference injecting chromatograph, detect according to following chromatographic condition, then the reference fingerprint shown in gained test sample finger-print and Fig. 3 is compared, calculate similarity, can realize the detection to bitter buckwheat to be measured or bitter buckwheat series products.If similarity is more than 0.9, testing sample is bitter buckwheat or not adulterated bitter buckwheat series products, as does not mix the buckwheat powder of barley, wheat.
Chromatographic condition
Chromatographic column: ACQUITY UPLC HSS T3 (1.8 μ m, 2.1 × 100mm); Mobile phase composition: methyl alcohol-0.1% phosphoric acid solution linear gradient elution; Flow velocity: 0.2mL/min; Detect wavelength: 280nm; Sample size: 0.8 μ L; Column temperature: 30 DEG C; Elution program sees the following form:
Embodiment 3 UPLC chromatographic technique of the present invention method for building up is investigated
Adopt UPLC chromatographic technique to set up buckwheat powder and mix the UPLC finger-print of buckwheat powder, calculate the finger-print of institute's test sample product and the similarity of reference substance finger-print, whether the buckwheat powder sample room of specifying separate sources has identical ultra high efficiency liquid phase fingerprint characteristic.Application system cluster analysis (HCA) method, the buckwheat powder that simulation is mixed to pearling cone meal and wheat flour is mixed sample and is carried out fingerprint analysis, with this to pure buckwheat powder with mix buckwheat powder sample and differentiate.
1 instrument, reagent and material
1.1 instrument
Waters Acquity UPLC H-Class System type Ultra Performance Liquid Chromatography instrument, comprise Acquity UPLC QSM, Acquity UPLC Sample Manager FTN, Acquity UPLC PDA Detector and Empower3 workstation (Waters company of the U.S.); Milli-Q (Millipore company of the U.S.); METTLER AE240 electronic analytical balance (plum Teller-Tuo benefit (Shanghai) Instrument Ltd.); W210B thermostat water bath (Shen Shun bio tech ltd, Shanghai)
1.2 materials and reagent
The buckwheat powder sample (the bitter buckwheat of Chengdu University experimental plot) of 22 batches of separate sources; Pearling cone meal (Nanyang Xing Nongzhong industry company limited), the wheat flour (the western grain and oil local products in Wuwei are sold Ltd) mixed; Commodity buckwheat powder (Z1 Chengdu food factory, Z2 organic food company limited, Z3 Tianjin Food Co., Ltd, Z4 Goats in Liangshan Prefecture food Ltd, Z5 Shanxi Ltd); Control substance of Rutin (lot number: MUST-12040302, Man Site bio tech ltd, Chengdu, Sichuan Province); Kaempferol reference substance (lot number: MUST-11041101, Man Site bio tech ltd, Chengdu, Sichuan Province); Quercetin reference substance (lot number: 100081-200907, National Institute for Food and Drugs Control); Chromatogram methyl alcohol (Fisher Scientific company); Methyl alcohol (analyzing pure); Phosphoric acid (Ke Miou reagent, chromatographically pure), distilled water (Watson).
2 test methods
The preparation of 2.1 reference substance solution
Respectively precision take reference substance rutin 10mg, Quercetin 1mg and Kaempferol 1mg, in 10mL volumetric flask, by methanol constant volume, be made into concentration and be respectively 1mg/mL, the reference substance stock solution of 0.1mg/mL and 0.1mg/mL.
The preparation of 2.2 need testing solutions
Precision takes bitter buckwheat sample (crossing No. 5 sieves of pharmacopeia) 0.2g respectively, be placed in tool plug conical flask, add 70% methyl alcohol 20mL, ultrasonic (250W at ambient temperature, 40KHZ) 30min, leave standstill after 5min, cross leaching filtrate, it is need testing solution that centrifugal filtrate 15min is got to supernatant.Sample introduction is front with the organic membrane filtration of 0.22 μ m.
2.3 chromatographic condition
Chromatographic column: ACQUITY UPLC HSS T3 (1.8 μ m, 2.1 × 100mm); Mobile phase composition: methyl alcohol-0.1% phosphoric acid solution linear gradient elution; Flow velocity: 0.2mL/min; Detect wavelength: 280nm; Sample size: 0.8 μ L; Column temperature: 30 DEG C; Elution program sees the following form:
Table 1 gradient elution program
3 methodological studies
3.1 replica test
Precision takes 5 parts of No. 1, the western buckwheats of buckwheat powder sample, prepare need testing solution and measure according to " 2.2 " item method, taking the relative retention time of rutin as reference, the relative standard deviation (RSD) who calculates each total peak relative retention time and relative peak area is all less than 4%, illustrates that the repeatability of the method is good.3.2 precision test
Accurate No. 1 need testing solution of the western buckwheat of buckwheat powder sample of drawing " 2.2 " item, continuous sample introduction 5 times, taking the relative retention time of rutin as reference, the relative standard deviation (RSD) who calculates each total peak relative retention time and relative peak area is all less than 2.7%, illustrates that this instrument precision is good.
3.3 stability test
Accurate No. 1 need testing solution of the western buckwheat of buckwheat powder sample of drawing " 2.2 " respectively at 0,4,8,16,32h measures, taking the relative retention time of rutin as reference standard, the relative retention time at each total peak is all less than 3% with the relative standard deviation (RSD) of relative reservation peak area, illustrates that this test liquid is stable in 32h.
The structure of 4 buckwheat powder UPLC finger-prints
The buckwheat powder of 22 batches of separate sources is prepared to need testing solution according to 2.2, carry out ultra high efficiency liquid phase analysis, collection Ultra Performance Liquid Chromatography figure according to 2.3.Utilize chromatographic fingerprints of Chinese materia medica similarity evaluation software (2004A version), adopt Auto-matching mode to generate the finger-print (S1-S22) of 22 batches of buckwheat powder samples, generate the reference fingerprint (R) of 22 batches of buckwheat powder samples with median method, obtain 7 total peaks of the finger-print of 22 batches of buckwheat powders.Reference substance chromatogram and sample finger-print are respectively referring to Fig. 1,2.
The similarity analysis of 5 buckwheat powder UPLC finger-prints
Fingerprint similarity is an important parameter of traditional Chinese medicine quality control, and traditional Chinese medicine fingerprint similarity is usually used in the true and false of Chinese medicine and differentiates research and quality control.This test utilizes similarity evaluation (2004A version) to evaluate the finger-print between institute's test sample product, generate reference fingerprint with the finger-print data of 22 batches of pure buckwheat powder samples, and the similarity of the finger-print to each batch of sample is evaluated with this.Its result (table 2) shows: the similarity of sample finger-print and reference fingerprint is all greater than 0.954, and the pure buckwheat powder sample of separate sources has identical UPLC fingerprint characteristic.
The fingerprint similarity of the buckwheat powder that table 2 is 22 batches
6 pure buckwheat powders with mix the finger-print comparative analysis after barley, wheat flour
6.1 mix the finger-print comparative analysis after pearling cone meal
As shown in Figure 4, S1-S7 is pure buckwheat powder, and S8-S13 is the buckwheat powder of mixing barley, at about 14min place, mixes the peak that buckwheat powder has all occurred that pure buckwheat powder does not have, and whether mixes the discriminating foundation of pearling cone meal using this peak as buckwheat powder.
6.2 mix the finger-print comparative analysis after wheat flour
As shown in Figure 5, S1-S7 is pure buckwheat powder, and S8-S13 is the buckwheat powder of mixing wheat, at about 14min place, mixes the peak that buckwheat powder has all occurred that pure buckwheat powder does not have, and whether mixes the discriminating foundation of wheat flour using this peak as buckwheat powder.
7 hierarchial-cluster analysis
Cluster analysis is the scientific and effective method of one that with the thought of " things of a kind come together, people of a mind fall into the same group ", institute's research object is divided into the group of same alike result.When cluster analysis, taking the similarity of sample as basis, according to test objective and requirement, can select different statistics and clustering method; First find out according to a batch data or index the statistic that can measure similarity degree between these data or index; Then using statistic as the foundation of dividing classification, first sample large some similarity degrees is polymerized to a class, and sample less other similarity degrees is polymerized to another kind of, until all samples all polymerization is complete, last according to the relation of the similarity size between all kinds of, make complete cluster analysis pedigree chart.Just can draw the relevant information of sample class according to the pedigree chart providing.
This experimental simulation is mixed pearling cone meal, wheat flour, and select normalization transform as clustering method, lance distance as measuring distance method, the standardization of the variable class method of average, carries out cluster analysis.Selected total fingerprint peaks is more stable, and peak area is larger, and separate with adjacent peaks better, the peak that retention time is more placed in the middle, setting rutin is with reference to peak.Then the matrix, forming with the relative peak area (with reference to peak) at common characteristic peak and non-total peak carries out cluster analysis.
7.1 mix the discriminating of mixing buckwheat powder of pearling cone meal
Because not containing the Flavonoid substances such as rutin in pearling cone meal, therefore intend mixing pearling cone meal in pure buckwheat powder, the flavones ingredient content in buckwheat powder can reduce.Therefore this test simulation is prepared 6 buckwheat powder samples of mixing 2%, 5%, 10%, 15%, 20%, 25% pearling cone meal.Extract the flavone compound in these samples according to 2.2 method, and carry out the fingerprint analysis of ultra high efficiency liquid phase.Adopt DPS software, with normalization conversion-lance distance-variable method of average, data are carried out to standardization statistical study according to the matrix of the relative peak area composition of chromatographic peak, the cluster analysis dendrogram of gained is shown in Fig. 6:
7 pure buckwheat powders not of the same race of table 2 and the relative total peak area of mixing pearling cone meal
Note: A1-A6 is followed successively by the buckwheat powder that mixes 2%, 5%, 10%, 15%, 20%, 25% pearling cone meal
Known from Fig. 6: No. 2, western buckwheat, bitter No. 5, wild salty No. 3, Guizhou Province, No. 1, river buckwheat, Yunnan drought hardship, river buckwheat can be divided into a class No. 2, and this class is all pure buckwheat powder; A1, A2, A3, A4, A5, A6 can be divided into a large class, and this class is all the buckwheat powder of mixing pearling cone meal.Thus, buckwheat powder and the pure buckwheat powder of in the time that the amount of mixing reaches 2%, mixing pearling cone meal are distinguished significantly.
7.2 mix the discriminating of mixing buckwheat powder of wheat flour
Because not containing the Flavonoid substances such as rutin in wheat flour, therefore intend mixing wheat flour in pure buckwheat powder, the flavones ingredient content in buckwheat powder can reduce.Therefore this test simulation is prepared 6 buckwheat powder samples of mixing 2%, 5%, 10%, 15%, 20%, 25% wheat flour.Extract the flavone compound in these samples according to 2.2 method, and carry out the fingerprint analysis of ultra high efficiency liquid phase according to 4.Adopt DPS software, with normalization conversion-lance distance-variable method of average, data are carried out to standardization statistical study according to the matrix of the relative peak area composition of chromatographic peak, the cluster analysis dendrogram of gained is shown in Fig. 7:
7 pure buckwheat powders not of the same race of table 3 and the relative total peak area of mixing wheat flour
Note: B1-B6 is followed successively by the buckwheat powder of mixing as 2%, 5%, 10%, 15%, 20%, 25% wheat flour
Known from Fig. 7: No. 2, western buckwheat, No. 2, western buckwheat, bitter No. 5, wild salty No. 3, Guizhou Province, No. 1, river buckwheat, Yunnan drought hardship, river buckwheat can be divided into a class No. 2, and this class is all pure buckwheat powder; B1, B2, B3, B4, B5, B6 can be divided into a large class, and this class is all the buckwheat powder of mixing pearling cone meal.Thus, buckwheat powder and the pure buckwheat powder of in the time that the amount of mixing reaches 2%, mixing wheat flour distinguish significantly.
The quality assessment research of 8 pure buckwheat powders and commodity buckwheat powder
The preparation of 8.1 testing samples and detection are analyzed
Get appropriate pure buckwheat powder and commodity buckwheat powder and prepare liquid to be measured according to the method for 2.2, carry out gradient elution according to the chromatographic conditions of 2.3, gather the UPLC collection of illustrative plates (seeing Fig. 8) of pure buckwheat powder and commodity buckwheat powder, carry out cluster analysis according to the methods of 7 to detecting data.
In Fig. 8, finger-print, taking S1 sample as reference, adopts the mode of multiple spot contrast, Auto-matching to generate reference fingerprint.As shown in Figure 8, S1-S6 is pure buckwheat powder, S7-S11 is commodity buckwheat powder, in about 12.8min place (relative retention time: 2.8596), commodity buckwheat powder and pure buckwheat powder to go out peak situation obviously different, pure buckwheat powder has obvious peak to occur, and commodity buckwheat powder does not have peak to occur, using this peak as a standard of bitter buckwheat quality assessment.
The relative total peak area of 6 pure buckwheat powders not of the same race of table 4 and commodity buckwheat powder
Note: Z1-A5 is the commodity buckwheat powder of different manufacturers
Adopt DPS2000 Software of Fuzzy Clustering Analysis, with translation data conversion regime not, absolute value clustering distance, method of average clustering method carries out cluster analysis.Known from cluster analysis Fig. 9: No. 2, western buckwheat, bitter No. 5, wild salty No. 3, Guizhou Province, No. 1, river buckwheat, Yunnan drought hardship, river buckwheat can be divided into a class No. 2, and Z1, Z2, Z3, Z4, Z5 are divided into a class, and this two large class can distinguish significantly.
9 results and discussion
(1) the present invention has set up the UPLC finger-print of buckwheat powder, and measurement result shows that the finger-print of 22 batch samples and the similarity of reference substance finger-print are all greater than 0.954, illustrates that the buckwheat powder sample room of different cultivars has identical UPLC fingerprint characteristic.Application system cluster analysis (HCA) method, the buckwheat powder that simulation is mixed to pearling cone meal and wheat flour is mixed sample and is carried out fingerprint analysis result and show: the buckwheat powder that mixes pearling cone meal or wheat flour can be differentiated.The finger-print of mixing from barley, wheat and the finger-print of pure buckwheat powder can visually see, and both have obvious difference, and also draw same conclusion by cluster analysis.By finding the comparative analysis of pure buckwheat powder and commodity buckwheat powder, in finger-print, can clearly observe 12.8min (relative retention time: 2.8596) locate commodity buckwheat powder and pure buckwheat powder to go out peak situation obviously different, pure buckwheat powder has obvious peak to occur, and commodity buckwheat powder does not have peak to occur, this peak can be used as a standard of bitter buckwheat quality assessment; Find that by cluster analysis No. 2, western buckwheat, bitter No. 5, wild salty No. 3, Guizhou Province, No. 1, river buckwheat, Yunnan drought hardship, No. 26 pure buckwheat powders of river buckwheat can be divided into a class again, 5 commodity buckwheat powders such as Z1, Z2, Z3, Z4, Z5 are divided into a class, this two large class can distinguish significantly, illustrates that pure buckwheat powder and commodity buckwheat powder may there are differences on the content of component content or flavones ingredient.
(2) impact of different eluent gradients on testing result:
A, gradient 1:
The pure buckwheat powder chromatogram of this gradient gained is shown in Figure 10.
B, gradient 2
The pure buckwheat powder chromatogram of this gradient gained is shown in Figure 11.
C, gradient 3
The pure buckwheat powder chromatogram of this gradient gained is shown in Figure 12.
Figure 10～12 are more known with reference fingerprint of the present invention: gradient 1,2 gained chromatograms differ greatly with contrasting fingerprint image, and the peak negligible amounts separating, degree of separation is not good, should not serve as the mobile phase condition of finger-print; And gradient 3 gained chromatograms and reference fingerprint similarity are high, and it is consistent with reference fingerprint to go out peak number amount, and each chromatographic peak degree of separation is good.Therefore, the comprehensive above-mentioned conclusion of the present invention, obtaining being suitable for bitter buckwheat, to carry out the mobile phase condition of UPLC detection as follows:
In upper table, the form of presentation of " X～Y ", refers to the arbitrary time point (comprising endpoint value) comprising in X to Y time range.
(3) impact of different detection wavelength on testing result:
The present invention, taking pure bitter buckwheat, doping barley, doping wheat and commercially available commodity buckwheat powder as detecting sample, investigates detecting wavelength, and result is referring to Figure 13～24.As seen from the figure, chromatogram and the 280nm chromatogram degree of approximation of various detection samples 275, under 285nm wavelength is higher, and therefore, it can be 275～285nm that the present invention selects the scope that detects wavelength.
Comprehensive the above results is known, easy and simple to handle, the highly sensitive and good stability, reproducible of UPLC method of the present invention, each chromatographic peak in bitter buckwheat sample effectively can be separated, can be used for the detection to bitter buckwheat or its Related product, and can realize the discriminating that does not contain the sample such as barley, wheat of flavonoids to mixing.
Claims (10)
1. the detection method of bitter buckwheat or its product, is characterized in that: it detects by Ultra Performance Liquid Chromatography method, comprises following operation steps:
(1) get sample to be checked, taking methyl alcohol or methanol aqueous solution as solvent extraction, collect extract, prepare need testing solution;
(2) need testing solution is injected to Ultra Performance Liquid Chromatography instrument, detect; Wherein, chromatographic condition is as follows:
Chromatographic column: octadecylsilane chemically bonded silica is filler
Detect wavelength: 275～285nm, be preferably 280nm
Mobile phase: methyl alcohol-0.1～0.3% phosphate aqueous solution system, be preferably 0.1% phosphate aqueous solution system, its gradient program is as follows:
2. detection method according to claim 1, is characterized in that: described eluent gradient program is as follows
3. detection method according to claim 1, is characterized in that: in the described methanol aqueous solution of step (1), methanol concentration is 60～80%v/v, is 70% ± 2%v/v.
4. detection method according to claim 1, is characterized in that: the particle diameter of described chromatographic column filler is 1.7～3.5 μ m, is 1.7～1.8 μ m, more preferably 1.8 μ m.
5. according to the detection method described in claim 1 or 4, it is characterized in that: described column size is: 2.1 × 100mm; The chromatographic column model using is: ACQUITY UPLC HSS T3,1.8 μ m, 2.1 × 100mm.
6. detection method according to claim 1, is characterized in that: described flow rate of mobile phase is 0.2ml/min; Chromatographic column column temperature is 30 DEG C.
7. according to the detection method described in claim 1～6 any one, it is characterized in that: in described Ultra Performance Liquid Chromatography method, taking one or both the composition in rutin, Quercetin as reference substance, by external standard method or fingerprint pattern technology, treat sample product and detect.
8. according to the detection method described in claim 1～7 any one, it is characterized in that: in described Ultra Performance Liquid Chromatography method, adopt fingerprint chromatogram technology to detect testing sample, its concrete operations are: need testing solution gained finger-print and reference fingerprint are compared, calculated similarity.
In described reference fingerprint, there are 7 common characteristic peaks, taking rutin as reference, the relative retention time at each common characteristic peak is respectively: No. 1 peak: 0.3346 ± 0.47, No. 2 peaks: 0.3894 ± 0.37, No. 3 peaks: 1.0000, No. 4 peaks: 1.3544 ± 0.22, No. 5 peaks: 1.6148 ± 0.18, No. 6 peaks: 2.7698 ± 0.44, No. 7 peaks: 2.8596 ± 0.44.
9. detection method according to claim 8, it is characterized in that: in described reference fingerprint, taking rutin as reference, the relative peak area at each common characteristic peak is respectively peak No. 1: 0.02404 ± 1.16, No. 2 peaks: 0.02699 ± 0.12, No. 3 peaks: 1.0000, No. 4 peaks: 0.06836 ± 0.29, No. 5 peaks: 0.2210 ± 0.57, No. 6 peaks: 0.1062 ± 1.44, No. 7 peaks: 0.2038 ± 0.56; Preferably, described reference fingerprint as shown in Figure 3.
10. the discrimination method of bitter buckwheat or its product, is characterized in that: it has following operation steps:
Get sample to be checked, adopt method described in claim 8 or 9 to detect, need testing solution gained finger-print and reference fingerprint are compared to calculating similarity; Similarity is more than 0.9, and sample to be checked is bitter buckwheat or not adulterated bitter buckwheat product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410298710.7A CN104034821B (en) | 2014-06-26 | 2014-06-26 | The detection of bitter buckwheat or its product and discrimination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410298710.7A CN104034821B (en) | 2014-06-26 | 2014-06-26 | The detection of bitter buckwheat or its product and discrimination method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104034821A true CN104034821A (en) | 2014-09-10 |
| CN104034821B CN104034821B (en) | 2016-04-06 |
Family
ID=51465670
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410298710.7A Expired - Fee Related CN104034821B (en) | 2014-06-26 | 2014-06-26 | The detection of bitter buckwheat or its product and discrimination method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104034821B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106841428A (en) * | 2017-01-04 | 2017-06-13 | 华中农业大学 | A kind of discrimination method of organic liquid milk |
| CN108562671A (en) * | 2018-05-31 | 2018-09-21 | 广东出入境检验检疫局检验检疫技术中心 | A method of coffee is detected based on efficient liquid-phase chromatograph finger print atlas |
| CN108982684A (en) * | 2018-06-26 | 2018-12-11 | 西南民族大学 | A method of detection and identification stone lattice dish |
| CN110221007A (en) * | 2019-06-04 | 2019-09-10 | 广西壮族自治区水牛研究所 | Adulterated detection method in a kind of identification buffalo's milk |
| CN111679020A (en) * | 2020-07-01 | 2020-09-18 | 中国农业科学院作物科学研究所 | HPLC (high Performance liquid chromatography) detection method for content of main flavonoid compounds in buckwheat |
| CN113358788A (en) * | 2021-06-09 | 2021-09-07 | 劲牌有限公司 | Method for identifying authenticity of tartary buckwheat wine based on fingerprint spectrum |
| CN114371228A (en) * | 2021-11-17 | 2022-04-19 | 西南民族大学 | Method for distinguishing pseudodongfeng grass or dongfeng grass as Zhuang medicinal material |
-
2014
- 2014-06-26 CN CN201410298710.7A patent/CN104034821B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106841428A (en) * | 2017-01-04 | 2017-06-13 | 华中农业大学 | A kind of discrimination method of organic liquid milk |
| CN108562671A (en) * | 2018-05-31 | 2018-09-21 | 广东出入境检验检疫局检验检疫技术中心 | A method of coffee is detected based on efficient liquid-phase chromatograph finger print atlas |
| CN108982684A (en) * | 2018-06-26 | 2018-12-11 | 西南民族大学 | A method of detection and identification stone lattice dish |
| CN110221007A (en) * | 2019-06-04 | 2019-09-10 | 广西壮族自治区水牛研究所 | Adulterated detection method in a kind of identification buffalo's milk |
| CN111679020A (en) * | 2020-07-01 | 2020-09-18 | 中国农业科学院作物科学研究所 | HPLC (high Performance liquid chromatography) detection method for content of main flavonoid compounds in buckwheat |
| CN113358788A (en) * | 2021-06-09 | 2021-09-07 | 劲牌有限公司 | Method for identifying authenticity of tartary buckwheat wine based on fingerprint spectrum |
| CN114371228A (en) * | 2021-11-17 | 2022-04-19 | 西南民族大学 | Method for distinguishing pseudodongfeng grass or dongfeng grass as Zhuang medicinal material |
| CN114371228B (en) * | 2021-11-17 | 2023-05-23 | 西南民族大学 | Method for distinguishing strong medicinal materials false east wind grass or east wind grass |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104034821B (en) | 2016-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104034821B (en) | The detection of bitter buckwheat or its product and discrimination method | |
| CN106990214B (en) | A method of evaluation Chinese medicine quality | |
| CN106483213B (en) | The method for building up and finger-print of Taohe Chengqi decoction composition finger-print | |
| CN105675739B (en) | A kind of construction method for the HPLC characteristic spectrums for treating anemopyretic cold Chinese medicine | |
| CN107389813A (en) | Rascal, dried orange peel, the dried immature fruit of citron orange and the method for Fructus Aurantii are differentiated based on chemical classification and UPLC Tof MS | |
| CN102141555B (en) | Method for establishing high-pressure liquid-phase fingerprinting of evodia and extractives thereof | |
| CN109406645A (en) | A kind of Antisathmatic oral liquid for child epheday intermedia, the detection method for frying semen armeniacae amarae, Radix Glycyrrhizae, radix scutellariae | |
| CN106370763B (en) | UPLC method for detecting kudzu root, kudzu root extract and kudzu root preparation component | |
| CN103115973B (en) | Method for judging consistency of traditional Chinese medicine batches | |
| CN111272904B (en) | Construction method and application of characteristic spectrum of medicine terminalia chebula and fructus chebulae tomentosa | |
| CN101419200B (en) | HPLC fingerprint identification method for origin ginseng protection | |
| CN103472148B (en) | Fingerprint spectrum detection method of Chinese medicine composition preparation | |
| CN102608248B (en) | Relinqing granules and polygonum capitatum thin-layer fingerprint chromatogram determination method | |
| CN101721475A (en) | Quality control method for Yaotongning capsules | |
| CN104090040A (en) | HPLC method for rapidly detecting main active ingredients of lithocarpus polysachyus rehd | |
| CN111948330B (en) | Detection method of finger-print of Renzhu stomach-invigorating granules | |
| CN103063766A (en) | Construction method of Chinese herbal medicine Naoshuantong preparation high performance liquid chromatography (HPLC) finger-print and application thereof | |
| CN106093229B (en) | A kind of method and its application differentiating the fleece-flower root based on UPLC finger-print pattern-recognitions | |
| CN103344717A (en) | Method for establishing rhizoma bletillae high performance liquid chromatography (HPLC) fingerprint spectrum and standard fingerprint spectrum thereof | |
| CN103926350A (en) | Inspection method of rehabilitation liquid formulation fingerprint and standard fingerprint | |
| CN106323903A (en) | Establishment and detection methods of infrared-spectroscopy fingerprint map of Pu'er tea extract | |
| CN105588888A (en) | Traditional Chinese medicine fingerprint spectrum quality control and measuring method | |
| CN102890125A (en) | Building method and mass detection method for fingerprint of total alkaloid components of cortex mori radicis medicinal material or cortex mori radicis extract | |
| CN115326974A (en) | Quality double-label visual quality control technology of fructus aurantii medicinal material | |
| CN107917894A (en) | A kind of method of tocolysis ball near infrared detection mass content |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160406 Termination date: 20190626 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |