CN106324143B - The structure and detection method of product volatile ingredients fingerprint among WANGLAOJI LIANGCHA - Google Patents
The structure and detection method of product volatile ingredients fingerprint among WANGLAOJI LIANGCHA Download PDFInfo
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Abstract
The present invention relates to a kind of construction methods and detection method of WANGLAOJI LIANGCHA volatile ingredients fingerprint, belong to herbal tea plant beverage Quality Control Technology field.The construction method includes the following steps：(1) preparation of inner mark solution：Precision weighs palmitic acid, and with ethyl acetate dissolving and constant volume is to get inner mark solution；(2) preparation of test solution：Precision measures WANGLAOJI LIANGCHA, is extracted with ethyl acetate, takes ethyl acetate supernatant to get test solution；(3) accurate respectively to draw above-mentioned inner mark solution and test solution, gas chromatograph for determination is injected after mixing, obtains the WANGLAOJI LIANGCHA volatile ingredients fingerprint with common characteristic peaks.The construction method and detection method can comprehensively characterize each ingredient being added in WANGLAOJI LIANGCHA, and have the characteristics that stability is good, can the quality of Efficient Characterization WANGLAOJI LIANGCHA and the quality of monitoring product.
Description
Technical field
The present invention relates to herbal tea plant beverage Quality Control Technology fields, more particularly to product among a kind of WANGLAOJI LIANGCHA
The construction method and detection method of volatile ingredients fingerprint.
Background technology
South of the Five Ridges herbal tea culture possesses long history product and accumulates and folk custom intension.The ancient times south of the Five Ridges is hundred ground more, more miasma, dye
Person is not dead without having.The south of the Five Ridges people have accumulated the preciousness for improving health care, preventing and curing diseases in the continuous fight with extreme natural environment
Experience.They according to local weather, water and soil characteristic, with some with it is clearing heat and detoxicating, promote the production of body fluid to quench thirst, fire dehumidifying and other effects of dispelling
Based on Lingnan features Chinese herbal medicine, long-term practice is passed through under tcm health preserving theoretical direction, creates various " herbal teas ",
For preventing and curing diseases, unique scenery of a Lingnan Culture is formd.
WANGLAOJI LIANGCHA is the earliest guangdong herbal tea of history, firstly appears in 1828 (clean the street light 8 years), has more than 180 so far
Year history, is acknowledged as herbal tea first ancestor, there is the title of " medicinal herb tea king ".Nearest decades, with modern production equipment and first inlet pipe
Great change has occurred in the introducing of reason theory, herbal tea industry, keeps herbal tea paving in the past and drinks the situation of herbal tea and is broken, and herbal tea is processed to
Drink the more easily plurality of specifications such as granule, tea bag, Tetra Pak, canned.
WANGLAOJI LIANGCHA using water, white granulated sugar, celestial grass, egg flower, chrysanthemum, Prunella vulgaris, Radix Glycyrrhizae, honeysuckle, strophanthus divaricatus as raw material,
It is extracted, concentration, prepare, sterilizing, packaging etc. main process be made, cool taste is tasty and refreshing, it is cold and hot drink all preferably, by
Consumer's likes.Comprehensively to reflect and monitoring the inherent quality of the kind, promotes the development of the kind, the kind should be carried out
Finger-print research.
Invention content
The present invention provides the construction method and detection method of a kind of WANGLAOJI LIANGCHA centre product volatile ingredients fingerprint,
It is detected and monitors to the quality to product among WANGLAOJI LIANGCHA, realize the purpose of general warranty WANGLAOJI LIANGCHA quality.
A kind of construction method of WANGLAOJI LIANGCHA centre product volatile ingredients fingerprint, includes the following steps：
(1) preparation of inner mark solution：
Precision weighs palmitic acid, and with ethyl acetate dissolving and constant volume is to get inner mark solution；
(2) preparation of test solution：
Precision measures product among WANGLAOJI LIANGCHA, is extracted with ethyl acetate, takes ethyl acetate supernatant molten to get test sample
Liquid；
(3) accurate respectively to draw above-mentioned inner mark solution and test solution, gas chromatograph for determination is injected after mixing, is obtained
Product volatile ingredients fingerprint among WANGLAOJI LIANGCHA with common characteristic peaks.
The construction method of product volatile ingredients fingerprint, accumulates for a long time by the present inventor among above-mentioned WANGLAOJI LIANGCHA
Experience and many experiments after, it is Extraction solvent to have chosen ethyl acetate, palmitic acid as the internal reference object in finger-print, with
Other common characteristic peaks match, and can preferably characterize each ingredient being added in product among WANGLAOJI LIANGCHA, and with steady
Qualitative good feature.
In one of the embodiments, in step (3), the determination condition of gas chromatograph is as follows：
Chromatographic column：Polyglycol polymer is the capillary chromatographic column of stationary phase；
Temperature program：Initial temperature is 50 DEG C -70 DEG C, and 220 DEG C -240 DEG C are warming up in a manner of gradient increased temperature
Detector：Fid detector.
It is the capillary chromatographic column of stationary phase as stationary phase using polyglycol polymer, then the side for passing through gradient increased temperature
Formula can characterize the volatile ingredient in WANGLAOJI LIANGCHA in gas chromatogram, and it is cool that foundation can preferably characterize king Lao Ji
The finger-print of product volatile ingredient quality among tea.
In one of the embodiments, in step (3), the determination condition of gas chromatograph further includes：
Carrier gas：Nitrogen；
Column flow：2.8ml/min-3.2ml/min；
Hydrogen flowing quantity：35ml/min-45ml/min；
Air mass flow：350ml/min-450ml/min；
Injector temperature：210℃-240℃；
Detector temperature：240℃-260℃.
Above-mentioned testing conditions are selected, the volatile ingredient in the product of WANGLAOJI LIANGCHA centre can have preferable on finger-print
Separating effect.
The mode of the gradient increased temperature is in one of the embodiments,：Initial temperature is 60 DEG C, 5min is kept, with 5 DEG C/min
195 DEG C are warming up to, then 230 DEG C are warming up to 2 DEG C/min, keeps 10min.
Above-mentioned gradient increased temperature condition is selected, can make the volatile ingredient among WANGLAOJI LIANGCHA in product in gas-chromatography
Separation, and there is preferable peak type, separating degree and separating effect, to preferably with product among finger-print characterization WANGLAOJI LIANGCHA
In each volatile ingredient in the chemical composition of Chinese materia medica group contained.
In one of the embodiments, in step (3), the common characteristic peaks are 13, opposite with palmitic acid to protect
It is respectively 0.25-0.27,0.32-0.34,0.35-0.36,0.39-0.41,0.49-0.50,0.51-0.52,0.53- to stay the time
0.54,0.74-0.75,0.76,0.77-0.78,0.81-0.83,0.91-0.93,1.27-1.28.Select above-mentioned retention time
Chromatographic peak as common characteristic peaks, it is not only representative, but also have the advantages that favorable reproducibility.
In one of the embodiments, in the common characteristic peaks, using the chromatographic peak of palmitic acid as internal reference peak, the palm fibre is set
The relative retention time of the chromatographic peak of palmitic acid acid is 1.0, and the relative peak area for the characteristic peak that above-mentioned relative retention time is 0.76 is
1.0, structure obtains product volatile ingredients fingerprint among the WANGLAOJI LIANGCHA being made of above-mentioned common characteristic peaks.
After the characteristics of fully having investigated each common characteristic peaks, select the chromatographic peak of internal standard palmitic acid as internal reference peak, and
Relative peak area with No. 9 peak that relative retention time is 0.76 is 1, is had when fingerprint similarity is evaluated preferable
Evaluation effect.
In one of the embodiments, in step (2), it is according to volume ratio with ethyl acetate by product among WANGLAOJI LIANGCHA
1: 1.5-2.5 volume ratio carries out ultrasonic extraction, then takes ethyl acetate supernatant up to test solution.
The invention also discloses a kind of detection methods of product volatile ingredients fingerprint among WANGLAOJI LIANGCHA, including with
Lower step：
(1) preparation of inner mark solution：
Precision weighs palmitic acid, and with ethyl acetate dissolving and constant volume is to get inner mark solution；
(2) preparation of test solution：
Precision measures product among WANGLAOJI LIANGCHA, is extracted with ethyl acetate, takes ethyl acetate supernatant molten to get test sample
Liquid；
(3) accurate respectively to draw above-mentioned inner mark solution and test solution, gas chromatograph is injected after mixing to be detected.
The detection method of product volatile ingredients fingerprint among above-mentioned WANGLAOJI LIANGCHA, using palmitic acid as finger-print
In object of reference, and have the characteristics that stability is good.
The test solution chromatogram and above-mentioned construction method that step (3) detection is obtained in one of the embodiments,
Obtained finger-print compares, and calculates similarity, evaluates the quality of product among WANGLAOJI LIANGCHA.
In one of the embodiments, in step (3), the determination condition of gas chromatograph is as follows：
Chromatographic column：Polyglycol polymer is the capillary chromatographic column of stationary phase；
Temperature program：Initial temperature is 60 DEG C, keeps 5min, is warming up to 195 DEG C with 5 DEG C/min, then be warming up to 2 DEG C/min
230 DEG C, keep 10min.
Carrier gas：Nitrogen；
Column flow：2.8ml/min-3.2ml/min；
Hydrogen flowing quantity：35ml/min-45ml/min；
Air mass flow：350ml/min-450ml/min；
Injector temperature：210℃-240℃；
Detector：Fid detector；
Detector temperature：240℃-260℃.
Using above-mentioned testing conditions, the volatile ingredient among WANGLAOJI LIANGCHA in product can be made to divide in gas-chromatography
From, and there is preferable peak type, separating degree and separating effect, to more preferably be able to contain in finger-print characterization WANGLAOJI LIANGCHA
Volatile ingredient in chemical composition of Chinese materia medica group.
Compared with prior art, the invention has the advantages that：
The construction method of product volatile ingredients fingerprint, has chosen palmitic acid among a kind of WANGLAOJI LIANGCHA of the present invention
As internal standard, i.e. internal reference object, there is preferable evaluation effect when evaluating fingerprint similarity, with the good spy of stability
Point the quality of product and can monitor the quality of product among Efficient Characterization WANGLAOJI LIANGCHA, also, also be that spectrum effect is learned
Research lay a good foundation.
In particular, control of the present invention to WANGLAOJI LIANGCHA volatile ingredient, can preferably monitor product special flavour substance
The case where, provide technical support for the mouthfeel guarantee of WANGLAOJI LIANGCHA.
Also, the construction method further define test solution preparation and each step in entire building process it is optimal
Parameter, the quality of energy Efficient Characterization WANGLAOJI LIANGCHA centre product, is conducive to the quality for monitoring product.
A kind of detection method of WANGLAOJI LIANGCHA volatile ingredients fingerprint of the present invention, it is old can comprehensively to characterize king
Volatile ingredient in lucky herbal tea, and have the characteristics that stability is good, can among Efficient Characterization WANGLAOJI LIANGCHA product matter
Amount, and be conducive to monitor the quality of product.The detection method is easy with method, stablize, precision is high, favorable reproducibility excellent
Point；It can quickly and accurately differentiate that the true and false of product is good and bad.
Description of the drawings
Fig. 1 is the gas chromatogram of n-hexane part in embodiment 1；
Fig. 2 is the gas chromatogram at 1 petrochina ether position of embodiment；
Fig. 3 is the gas chromatogram at ether position in embodiment 1；
Fig. 4 is the gas chromatogram of ethyl acetate extract in embodiment 1；
Fig. 5 is second of gas chromatogram extracted of 5ml ethyl acetate in embodiment 1；
Fig. 6 is the gas chromatogram of OV-1701 pillars in embodiment 1；
Fig. 7 is the gas chromatogram that split ratio is 20: 1 in embodiment 1；
Fig. 8 is the gas chromatogram that split ratio is 10: 1 in embodiment 1；
Fig. 9 is the gas chromatogram that split ratio is 5: 1 in embodiment 1；
Figure 10 is internal standard palmitic acid and sample gas chromatogram in embodiment 1；
Figure 11 be embodiment 1 in 10 batches of samples gas chromatogram with compare spectrogram；
Figure 12 is the gas chromatogram of Chinese mesona herb in embodiment 1；
Figure 13 is the gas chromatogram of frangipanis in embodiment 1；
Figure 14 is the gas chromatogram of honeysuckle in embodiment 1；
Figure 15 is the gas chromatogram of Prunella vulgaris in embodiment 1；
Figure 16 is the gas chromatogram of strophanthus divaricatus in embodiment 1；
Figure 17 is the gas chromatogram of Radix Glycyrrhizae in embodiment 1；
Figure 18 is the gas chromatogram of chrysanthemum in embodiment 1；
Figure 19 is the gas chromatogram of different negative medicinal materials and sample in embodiment 1；
Figure 20 is the gas chromatogram of ethyl acetate blank solvent in embodiment 1；
Figure 21 is the gas chromatogram of sample in embodiment 1；
Figure 22 is the gas chromatogram in sample 120min in embodiment 1.
Specific implementation mode
To facilitate the understanding of the present invention, below with reference to relevant drawings to invention is more fully described.It gives below
Presently preferred embodiments of the present invention is gone out.But the present invention can realize in many different forms, however it is not limited to be retouched herein
The embodiment stated.Keep the understanding to the disclosure more thorough complete on the contrary, purpose of providing these embodiments is
Face.
Unless otherwise defined, all of technologies and scientific terms used here by the article and belong to the technical field of the present invention
The normally understood meaning of technical staff is identical.Used term is intended merely to description tool in the description of the invention herein
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
Embodiment 1
The present embodiment is the construction method of WANGLAOJI LIANGCHA volatile ingredients fingerprint.
One, instrument and equipment
Japanese Shimadzu GC-2010AF gas chromatographs, fid detector, GC solution chromatographic work stations, high pure nitrogen,
The high-purity hydrogen generators of GH-400 (Beijing Zhong Xinghui profit developments in science and technology Co., Ltd), GA-380A low-noise airs pump are (emerging in Beijing
Hui Li developments in science and technology Co., Ltd).
Two, material and reagent
Product (totally 10 batch) are provided by Wanglaoji Pharmaceutical Co., Ltd., Guangzhou City among WANGLAOJI LIANGCHA.N-hexane is (wide
Dongguang Science and Technology Co., Ltd. of China), petroleum ether (Tianjin Zhi Yuan chemical reagent Co., Ltd), ether (Guangzhou chemical reagent
Factory), ethyl acetate (Guangzhou Chemical Reagent Factory) be analysis it is pure.Reference substance palmitic acid is purchased from Shanghai Mike's beautiful jade, and purity is 99%
More than.
Three, method
(1), conditional filtering.
A, sample treatment screens.
4 kinds of n-hexane, petroleum ether, ether, ethyl acetate organic solvents are respectively adopted to product among WANGLAOJI LIANGCHA in experiment
Sample carry out liquid-liquid extraction, be detected with following chromatographic conditions：Chromatographic column be DB-WAX capillary columns (30m × 0.25mm,
0.25μm)；Carrier gas is high pure nitrogen；1 μ l of sample size；Split ratio 10: 1；250 DEG C of detector temperature；230 DEG C of injector temperature；
Column flow 3.0ml/min；Hydrogen flowing quantity 40.0ml/min；Air mass flow 400ml/min；Temperature program：60 DEG C of initial temperature is kept
5min is warming up to 195 DEG C with 5 DEG C/min, then is warming up to 230 DEG C with 2 DEG C/min, keeps 10min.Measurement obtains Fig. 1-Fig. 4 institutes
The gas chromatogram shown.
As can be seen from the results, n-hexane and petroleum ether extraction position chromatographic peak quantity is few and abundance is small, i.e., both
Solvent is poor to volatile ingredient extraction effect in sample；The chromatographic peak information at ether extraction position does not have that ethyl acetate extract is more,
Especially show low-boiling compound part, and the overall abundance of chromatographic peak is also high without ethyl acetate extract, thus it is final determining
Best extractant is ethyl acetate.
Product sample solution among the WANGLAOJI LIANGCHA of 5ml is extracted twice with 10ml, 5ml ethyl acetate solvent respectively, by two
For secondary extract liquor respectively into gas phase analysis, secondary extract liquor gas chromatogram is as shown in Figure 5.
The result shows that most chromatographic peaks disappear in secondary 5ml extract liquors, i.e., once extracted with 10ml ethyl acetate
Taking can extract completely substantially.
B, gas phase analysis method optimizes.
According to above-mentioned chromatographic condition, above-mentioned DB-WAX pillars are replaced with OV-1701 pillars, carry out analysis measurement, gas phase
Chromatogram is as shown in Figure 6.
The analysis result of above-mentioned OV-1701 pillars and DB-WAX pillars is compared, the results show that the selectivity of DB-WAX pillars
And separating effect is better than OV-1701 pillars (Fig. 4, Fig. 6).
According to above-mentioned chromatographic condition, analysis measurement, gas-chromatography are carried out with different split ratios (20: 1,10: 1,5: 1)
Figure is as shown in figs. 7 to 9.The result shows that the sample introduction moderate concentration that split ratio is 10: 1, the separating effect of chromatogram and intuitive upper table
Now preferably.
C, the selection of object of reference.
Palmitic acid chemical property is more stable, and the peak area for confirming it to other chemical compositions is investigated through experiment without dry
It disturbs, palmitic acid is finally added in sample test liquid and is used as internal reference object (internal standard) by experiment.
(2), the construction method of finger-print.
1, the preparation of inner mark solution.
Palmitic acid 2mg is taken, accurately weighed, ethyl acetate is settled to 2ml, as inner mark solution.
2, the preparation of test solution.
Product test solution among WANGLAOJI LIANGCHA：Precision draws product sample 5ml among the WANGLAOJI LIANGCHA of each batch, second
Acetoacetic ester 10ml extractions, divide and take ethyl acetate layer, be concentrated into 2ml to get test solution.
Single raw medicinal material test liquid：7 taste medicinal material (celestial grass, egg flower, chrysanthemum, Prunella vulgaris, Radix Glycyrrhizae, honeysuckle, cloth are taken respectively
Slag leaf) about 1g, it is accurately weighed, add ethyl acetate 25ml, ultrasonic extraction 15min, centrifuge, take supernatant to get.
3, finger-print is established.
(1), testing conditions.
Gas Chromatographic Determination condition is as follows：
Chromatographic column：It is stationary phase that DB-WAX capillary columns (30m × 0.25mm, 0.25 μm), which are polyglycol polymer,
Capillary chromatographic column；
Temperature program：Initial temperature is 60 DEG C, keeps 5min, is warming up to 195 DEG C with 5 DEG C/min, then be warming up to 2 DEG C/min
230 DEG C, keep 10min.
Carrier gas：Nitrogen；
Sample size：1μl；
Split ratio：10∶1；
Column flow：3ml/min；
Hydrogen flowing quantity：40ml/min；
Air mass flow：400ml/min；
Detector：Fid detector；
Injector temperature：230℃；
Detector temperature：250℃.
Product sample test liquid 1.0ml and 60 μ l of inner mark solution, mixing shake up among accurate absorption WANGLAOJI LIANGCHA respectively, essence
1 μ l of close absorption inject gas chromatograph, are measured according to above-mentioned condition, obtain the gas of 10 different batches samples shown in Figure 11
Phase chromatogram (S1-S10 indicates 10 different batches samples respectively in figure).
The 1 μ l of single raw medicinal material test liquid prepared in accurate aspiration step 2 respectively again, inject gas chromatograph, according to
Above-mentioned condition measures, and carries out each single medicinal material check experiment, is studied at the correlation of flavour of a drug and sample room each group, determines
The flavour of a drug ownership of main chromatographic peak.Respectively obtain Chinese mesona herb, frangipanis, honeysuckle, Prunella vulgaris, cloth as shown in Figure 12-Figure 18
Slag leaf, Radix Glycyrrhizae and chrysanthemum gas chromatogram.
The intermediate product sample that different medicinal materials are negative is prepared, is handled according to the processing method of above-mentioned determination, gas is injected
Chromatography is measured according to above-mentioned condition, respectively obtains negative Chinese mesona herb as shown in figure 19, negative frangipanis, negative gold and silver
Flower, negative Prunella vulgaris, negative strophanthus divaricatus, negative Radix Glycyrrhizae and negative chrysanthemum gas chromatogram.
(2), it analyzes.
A, the calibration of 10 batches of sample common characteristic peaks.
According to the method described above, select the WANGLAOJI LIANGCHA centre product of 10 different batches (by Guangzhou Wang Laoji medicine company shares
Co., Ltd provides), it is measured, obtains the gas chromatogram of 10 different batches samples (S1-S10) as shown in figure 11,
The file of gas chromatograph results is imported into " similarity evaluation " (2004A editions), it is folded to obtain sample
Add figure and control collection of illustrative plates, as shown in figure 11, R is control collection of illustrative plates in figure., 13 common characteristic peaks are tentatively demarcated, are with palmitic acid
Internal reference object (peaks S), calculates the relative retention time of each characteristic peak and the peaks S；With the peak area at No. 9 peaks for 1, calculate other each shared
The results are shown in table below for the relative peak area of characteristic peak, relative retention time and opposite reservation peak area.
The relative retention time value at each shared peak in 1.10 batches of samples of table
| Peak number | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | Mean value |
| 1 | 0.26 | 0.26 | 0.26 | 0.26 | 0.26 | 0.26 | 0.26 | 0.26 | 0.26 | 0.26 | 0.26 |
| 2 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 | 0.33 |
| 3 | 0.36 | 0.36 | 0.36 | 0.36 | 0.36 | 0.35 | 0.35 | 0.35 | 0.36 | 0.35 | 0.36 |
| 4 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 |
| 5 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 |
| 6 | 0.51 | 0.51 | 0.51 | 0.51 | 0.51 | 0.51 | 0.51 | 0.51 | 0.51 | 0.51 | 0.51 |
| 7 | 0.53 | 0.53 | 0.53 | 0.53 | 0.53 | 0.53 | 0.53 | 0.53 | 0.53 | 0.53 | 0.53 |
| 8 | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 | 0.75 |
| 9 | 0.76 | 0.76 | 0.76 | 0.76 | 0.76 | 0.76 | 0.76 | 0.76 | 0.76 | 0.76 | 0.76 |
| 10 | 0.77 | 0.77 | 0.77 | 0.77 | 0.77 | 0.77 | 0.77 | 0.77 | 0.77 | 0.77 | 0.77 |
| 11 | 0.82 | 0.82 | 0.82 | 0.82 | 0.82 | 0.82 | 0.82 | 0.82 | 0.82 | 0.82 | 0.82 |
| 12 | 0.92 | 0.92 | 0.92 | 0.92 | 0.92 | 0.92 | 0.92 | 0.92 | 0.92 | 0.92 | 0.92 |
| S | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| 13 | 1.28 | 1.28 | 1.27 | 1.27 | 1.27 | 1.27 | 1.28 | 1.28 | 1.28 | 1.28 | 1.28 |
The opposite of each shared peak retains peak area value in 2.10 batches of samples of table
As can be seen that the relative retention time basic one of 10 batches of king's Lao Ji sample characteristic fingerprint peaks in from the above
It causes, but its relative peak area value has certain difference, i.e. the content of each volatile ingredient has difference in 10 batches of samples.
B, sample fingerprint map analyzing.
Using each single medicinal material check experiment, each group is studied at the correlation of flavour of a drug and sample room, is determined main
The flavour of a drug of chromatographic peak belong to, and each medicinal material chromatogram is shown in Figure 12-Figure 18.
Analyze chromatography graph discovery：No. 1 peak has embodiment, 2, No. 3 peaks mainly to be contributed by Radix Glycyrrhizae, chrysanthemum in each medicinal material, and 13
Number peak other medicinal materials in addition to Radix Glycyrrhizae, Prunella vulgaris, which have, to be embodied, and the chromatographic peak in sample chromatogram figure between 15~40min fail at
Respective home is found in 7 taste medicinal materials of side, it may be because sample is to be obtained by production process to cause this result, and medicinal material is
Solid powder, without preparation processing, so finally obtained chromatographic peak information difference.
Judged as shown in figure 19 according to the reduction degree of chromatographic peak peak area by each medicinal material feminine gender and sample controls
Its main Chinese medicinal materials belongs to.
The results show that 1,4,7,8, No. 13 peak is embodied jointly by 7 taste medicinal materials, 2, No. 3 peaks are by other outside strophanthus divaricatus, Chinese mesona herb
Medicinal material contribute, 5, No. 6 peaks by Chinese mesona herb outside other medicinal materials contribution, No. 9 peaks mainly by strophanthus divaricatus, chrysanthemum, frangipanis contribute, 10
Number peak is mainly contributed by Chinese mesona herb, No. 11 peaks mainly by honeysuckle outside the contribution of other medicinal materials, No. 12 peaks are mainly by strophanthus divaricatus, chicken
Egg flower is contributed.
According to the above ingredients and negative experimental analysis it is found that the finger-print can embody composition WANGLAOJI LIANGCHA 7
The information of taste medicinal material observes the missing degree of chromatographic peak in each negative medicinal material it is found that chrysanthemum, frangipanis volatilize to WANGLAOJI LIANGCHA
Property ingredients fingerprint contribution it is maximum, and the contribution of Chinese mesona herb is then minimum, is seen below to the ownership of each common characteristic peaks in sample
Shown in table.
The ownership of characteristic peak in 3. gas-phase fingerprint pattern of table
| Peak number | Ownership | Peak number | Ownership |
| 1 | 7 taste medicinal materials | 8 | 7 taste medicinal materials |
| 2 | Radix Glycyrrhizae, chrysanthemum | 9 | Strophanthus divaricatus, chrysanthemum, frangipanis |
| 3 | Radix Glycyrrhizae, chrysanthemum | 10 | Chinese mesona herb |
| 4 | 7 taste medicinal materials | 11 | In addition to honeysuckle |
| 5 | In addition to Chinese mesona herb | 12 | Strophanthus divaricatus, frangipanis |
| 6 | In addition to Chinese mesona herb | 13 | In addition to Radix Glycyrrhizae, Prunella vulgaris |
| 7 | 7 taste medicinal materials |
C, 10 batches of sample fingerprint similarity evaluations.
10 batches of intermediate product sample GC collection of illustrative plates are carried out with " similarity evaluation " (2004A editions)
Evaluation, similarity calculation result see the table below shown.
4. similarity evaluation of table
| Sample lot number | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 |
| Similarity | 0.987 | 0.989 | 0.988 | 0.993 | 0.99 | 0.997 | 0.996 | 0.982 | 0.998 | 0.985 |
In from the above as can be seen that this experiment using the 10 batches of WANGLAOJI LIANGCHAs of gas chromatography pair centre product samples into
Row fingerprint map analyzing wherein the most chromatographic peaks that can be detected are detached well, and has been demarcated 13 and has been shared
Fingerprint peaks are characterized collection of illustrative plates.By with ingredients and negative control experiments, as a result 13 shared fingerprint peaks can belong into side 7
In taste medicinal material, i.e., the WANGLAOJI LIANGCHA volatile ingredients fingerprint that this experiment is established can express WANGLAOJI LIANGCHA into 7 taste medicines of side
The Global Information of material can be used for its quality evaluation.And the collection of illustrative plates similarity of 10 batches of WANGLAOJI LIANGCHA samples is said 0.98 or more
The WANGLAOJI LIANGCHA volatile ingredients fingerprint and the similarity for compareing collection of illustrative plates that bright experiment is established are higher, can embody its general character
Feature can be used for controlling its quality.
4, methodological study.
(1) blank test
1 μ l of ethyl acetate blank solution are drawn, gas chromatograph is injected, measures, sees according to the chromatographic condition of above-mentioned foundation
Influence situations of the Cha Yisuanyizhirongjifeng to sample chromatogram peak.Experiment chromatogram (Figure 20 and Figure 21) shows that ethyl acetate is molten
Agent peak is mainly the chromatographic peak before 3min, and the influence to sample chromatogram peak is little.
(2) precision test.
Same test solution (S3) is taken, is measured by said determination method, continuous sample introduction 5 times, each shared peak is opposite to be retained
The RSD values of time are respectively less than 0.05%, and the RSD values of relative peak area are respectively less than 4.12%, show that the precision of instrument is good.
(3) repetitive test.
It takes with a collection of 6 parts of sample to be tested (S3), is measured by said determination method, the RSD of each shared peak relative retention time
Value is respectively less than 0.05%, and the RSD values of relative peak area are respectively less than 4.83%, shows that the repeatability of this method is good.
(4) stability test.
Take same test solution (S3), by said determination method, respectively at 0,2,4,8,12, sample introduction is analyzed for 24 hours, as a result
The RSD values of each shared peak relative retention time are respectively less than 0.05%, and the RSD values of relative peak area are respectively less than 4.72%, show to supply
Test sample solution is good in internal stability for 24 hours.
(5) analysis time is investigated.
1 μ l of test solution are drawn, gas chromatograph is injected, 120min is run according to above-mentioned chromatographic condition, records chromatography
Scheme (Figure 22).Can be seen that after 59min from the 2h chromatograms of sample does not have chromatographic peak appearance, i.e. volatile ingredient in sample
Appearance is complete before 59min, therefore acquisition time is determined as 59min.
Embodiment 2
The detection method of product volatile ingredients fingerprint among WANGLAOJI LIANGCHA.
The present embodiment is detected WANGLAOJI LIANGCHA using product volatile ingredients fingerprint among WANGLAOJI LIANGCHA, has
Body is as follows：
One, instrument and equipment
With embodiment 1.
Two, material and reagent
It is another that product sample (being provided by Wanglaoji Pharmaceutical Co., Ltd., Guangzhou City), Qi Tacai among 10 batches of WANGLAOJI LIANGCHAs are provided
Material is with reagent with embodiment 1.
Three, method
1, the preparation of inner mark solution：With embodiment 1.
2, the preparation of test solution：With embodiment 1.
3, it detects.
(1), chromatographic condition：With embodiment 1.
Product sample test liquid 1.0ml and 60 μ l of inner mark solution, mixing shake up among accurate absorption WANGLAOJI LIANGCHA respectively, essence
1 μ l of close absorption inject gas chromatograph, are measured according to above-mentioned condition, obtain the gas chromatogram of 10 different batches samples
(2), evaluation analysis.
10 batches of WANGLAOJI LIANGCHA samples are detected respectively using above-mentioned detection method, record chromatogram and import Chinese medicine
Chromatography similarity evaluation system software (2004A editions processing), compares trace analysis with what embodiment 1 obtained, calculates similarity, phase
It see the table below like degree result of calculation.
5. similarity calculation result of table
It can be seen from the results above that each batch of sample similarity is all higher than 0.90.Show WANGLAOJI LIANGCHA volatile ingredient
The similarity of finger-print is higher, can be used as WANGLAOJI LIANGCHA quality control and evaluation index.
Embodiment 3
The present embodiment is detected WANGLAOJI LIANGCHA using WANGLAOJI LIANGCHA volatile ingredients fingerprint, with embodiment
2 is essentially identical, the difference is that：
In the preparation of WANGLAOJI LIANGCHA sample in the present embodiment, do not feed intake strophanthus divaricatus, Radix Glycyrrhizae, frangipanis, gold and silver respectively
Negative strophanthus divaricatus, negative Radix Glycyrrhizae, negative frangipanis, negative honeysuckle, feminine gender is made in flower, chrysanthemum, Prunella vulgaris or celestial grass respectively
Chrysanthemum, negative Prunella vulgaris, negative celestial grass WANGLAOJI LIANGCHA defective work, then with above-mentioned finger-print to the WANGLAOJI LIANGCHA not
Certified products are detected, and are recorded chromatogram and are imported Chinese medicine chromatography similarity evaluation system software (2004A editions processing), with implementation
The control trace analysis that example 1 obtains calculates similarity, the results are shown in table below as shown in figure 19.
The similarity of table 6 each negative sample and common pattern
It can be seen from the results above that the similarity of defective work is respectively less than 0.9.Show WANGLAOJI LIANGCHA volatile ingredient
Finger-print can be used as the control of WANGLAOJI LIANGCHA quality of intermediate amount and evaluation index.Also, energy Efficient Characterization king of the invention is old
The quality of lucky herbal tea is conducive to the quality for monitoring product；Easy with method, stable, precision height, favorable reproducibility；Can quickly,
Accurately differentiate that the true and false of product is good and bad.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (6)
1. the construction method of product volatile ingredients fingerprint among a kind of WANGLAOJI LIANGCHA, it is characterised in that：Including following step
Suddenly：
(1) preparation of inner mark solution：
Precision weighs palmitic acid, and with ethyl acetate dissolving and constant volume is to get inner mark solution；
(2) preparation of test solution：
Precision measures product among WANGLAOJI LIANGCHA, is extracted with ethyl acetate, takes ethyl acetate supernatant to get test solution；
(3) accurate respectively to draw above-mentioned inner mark solution and test solution, gas chromatograph for determination is injected after mixing, is had
Product volatile ingredients fingerprint among the WANGLAOJI LIANGCHA of common characteristic peaks；In step (3), the determination condition of gas chromatograph
It is as follows：
Chromatographic column：Polyglycol polymer is the capillary chromatographic column of stationary phase；
Detector：Fid detector；
Carrier gas：Nitrogen；
Column flow：2.8ml/min-3.2ml/min；
Hydrogen flowing quantity：35ml/min-45ml/min；
Air mass flow：350ml/min-450ml/min；
The mode of the gradient increased temperature is：Initial temperature is 60 DEG C, keeps 5min, is warming up to 195 DEG C with 5 DEG C/min, then with 2 DEG C/min
230 DEG C are warming up to, 10min is kept.
2. the construction method of product volatile ingredients fingerprint, feature among WANGLAOJI LIANGCHA according to claim 1
It is, in step (3), the determination condition of gas chromatograph further includes：
Injector temperature：210℃-240℃；
Detector temperature：240℃-260℃.
3. the construction method of product volatile ingredients fingerprint, feature among WANGLAOJI LIANGCHA according to claim 1
It is, in step (3), the common characteristic peaks are 13, and the relative retention time with palmitic acid is respectively 0.25-0.27,
0.32-0.34,0.35-0.36,0.39-0.41,0.49-0.50,0.51-0.52,0.53-0.54,0.74-0.75,0.76,
0.77-0.78,0.81-0.83,0.91-0.93,1.27-1.28.
4. the construction method of product volatile ingredients fingerprint, feature among WANGLAOJI LIANGCHA according to claim 3
It is, in the common characteristic peaks, using the chromatographic peak of palmitic acid as internal reference peak, sets the opposite reservation of the chromatographic peak of the palmitic acid
Time is 1.0, and the relative peak area for the characteristic peak that above-mentioned relative retention time is 0.76 is 1.0, and structure is obtained by above-mentioned shared
Product volatile ingredients fingerprint among the WANGLAOJI LIANGCHA that characteristic peak is constituted.
5. the construction method of product volatile ingredients fingerprint, feature among WANGLAOJI LIANGCHA according to claim 1
It is, with ethyl acetate is 1 according to volume ratio by product among WANGLAOJI LIANGCHA in step (2):1.5-2.5 volume ratio carry out
Ultrasonic extraction then takes ethyl acetate supernatant, and 4-6 times of concentration is to get test solution.
6. the detection method of product volatile ingredients fingerprint among a kind of WANGLAOJI LIANGCHA, which is characterized in that including following step
Suddenly：
(1) preparation of inner mark solution：
Precision weighs palmitic acid, and with ethyl acetate dissolving and constant volume is to get inner mark solution；
(2) preparation of test solution：
Precision measures product among WANGLAOJI LIANGCHA, is extracted with ethyl acetate, takes ethyl acetate supernatant to get test solution；
(3) accurate respectively to draw above-mentioned inner mark solution and test solution, gas chromatograph is injected after mixing to be detected；Using
The determination condition of claim 1 is detected；The test solution chromatogram that step (3) detection is obtained and claim 1
The finger-print that construction method obtains compares, and calculates similarity, evaluates the quality of product among WANGLAOJI LIANGCHA.
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Application publication date: 20170111 Assignee: GUANGZHOU WANGLAOJI FOOD Co.,Ltd. Assignor: GUANGZHOU WANGLAOJI PHARMACEUTICAL Co.,Ltd. Contract record no.: X2021440000022 Denomination of invention: Fingerprint construction and detection method of volatile components in Wanglaoji herbal tea intermediate Granted publication date: 20181002 License type: Common License Record date: 20210122 |