CN104569195B - A kind of method of flavonoids effective constituent content in toothpaste detected containing baikal skullcap root - Google Patents
A kind of method of flavonoids effective constituent content in toothpaste detected containing baikal skullcap root Download PDFInfo
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Abstract
The invention discloses the method for flavonoids effective constituent content in a kind of toothpaste detected containing baikal skullcap root: get toothpaste, adsorbent of molecular sieve, the drying agent anhydrous sodium sulfate containing baikal skullcap root, add root of large-flowered skullcap mixed sample, add in mortar after mixing and grind, obtain the solid mixture after grinding; Solid mixture after grinding adds in solid-phase extraction column, with eluent, collects eluent, by eluent vortex, centrifugal, gets supernatant liquor and be flavonoids effective constituent extract; Get the sampling analysis of flavonoids effective constituent extract Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of flavonoids effective constituent extract; Contrast with typical curve convert, deduct mixed sample after calculate containing the root of large-flowered skullcap toothpaste in scutelloside, wogonoside, wogonin content.The present invention creatively using molecular sieve as adsorbent, under micro-system, carry out extraction detect, efficient, environmental protection ground can extract and detect the content containing the flavonoids effective constituent in root of large-flowered skullcap toothpaste.
Description
Technical field
The invention belongs to natural drug and extract detection field.It relates to a kind of Trace bio-element detection method of toothpaste, and specifically, it relates to by molecular sieve KIT-6 as adsorbent, in conjunction with micro-matrix solid phase dispersion abstraction technique, is used for extracting the new method of detection containing flavonoids effective constituent content in the toothpaste of baikal skullcap root.
Background technology
Root of large-flowered skullcap this product is the dry root of lip standing grain bucket plant Huang extraction (ScutellariabaicalensisGeorgi).There is heat-clearing and damp-drying drug, purging intense heat and detonicating, hemostasis and antiabortive effect, be usually used in the treatment that damp and hot ruffian is full, rush down the illnesss such as dysentery, jaundice, cough with lung heat, carbuncle sore tumefacting virus, fetal irritability.Alone or be used for inflammatory allergic disease and the gynecological disease etc. such as respiratory tract infection, acute bacillary dysentery, virus hepatitis with other drug matching clinically, be widely used bulk medicinal materials on tcm clinical practice.The root of large-flowered skullcap is the famous traditional Chinese medicine of China, and history tree is all on the books.Scutelloside, wogonoside and wogonin are biological flavone constituents main in the root of large-flowered skullcap.Scutelloside is one of principle active component of the root of large-flowered skullcap, is the major quality controlling index components of the root of large-flowered skullcap and preparation thereof.According to pharmaceutical research, scutelloside has antimicrobial, antiallergic action, antitumor, step-down and calmness, cholagogic, protects the liver and the effect such as spasmolysis and immunity moderation.
At present to the pre-treating method that toothpaste is analyzed, usually based on simple ultrasonic extraction.Although the method is simple, comparatively low to the extraction efficiency of target molecule, and affect comparatively large by matrix interference, make analysis result corresponding lower.And the defect that these class methods exist also is apparent: use virulent organic solvent in experimentation and large usage quantity, it is very long to extract the time used, and extraction efficiency is low, is harmful to experiment operator health, environmental pollution is serious, deviates from the proposal of " Green Chemistry ".
For solving an above-mentioned difficult problem, along with the development of science and technology, many Novel extraction method, also arise at the historic moment.Wherein just there is matrix solid phase dispersion abstraction technique.Matrix solid phase dispersion extraction (MSPD) is the novel sample preparation technology of the one be based upon on Solid-Phase Extraction basis, is a popular domain of recent domestic research, is widely used in the fields such as food, environment, medicine, chemical industry, commodity inspection.
The matrix solid phase dispersion abstraction technique of contemporary literature report, particularly for natural product field, the amount of its adsorbent, medicinal material consumption and and the large usage quantity of eluant, eluent, and be virulent organic solvent, the health of operator is harmful to, can pollutes again environment.And in the matrix solid phase dispersion abstraction technique of routine, used adsorbent is most based on silica gel, florisil silica, C18 reverse phase silica gel etc.
Up to now, using molecular sieve KIT-6 as eluent, and binding matrix solid phase dispersion abstraction technique, the active component in toothpaste matrix extracts context of detection, and there is not been reported, and the method is extracted for root of large-flowered skullcap toothpaste and detected this research field blank out especially.
Summary of the invention
The object of the invention is to seek one than conventional method toothpaste extraction more rapidly and effectively detection technique, make it be applied to root of large-flowered skullcap toothpaste and extract detection.Invention main points be creatively using molecular sieve as adsorbent, be applied in matrix solid phase dispersion extraction field.Target molecule in the adsorbent that molecular sieve extracts as matrix solid phase dispersion and matrix solid phase dispersion potpourri adsorbs and combines, thus target molecule is separated from complex matrices, obtains the eluent containing target molecule.And the present invention implements in a micro-system, medicinal material used and experimental drug reagent are all in microgram rank.
The present invention specifically provides a kind of using molecular sieve as adsorbent, and binding matrix solid phase dispersion abstraction technique, extracts the method for target molecule in micro-root of large-flowered skullcap toothpaste.Can be used for extracting the flavonoids effective constituent detected in micro-toothpaste.
The technical solution used in the present invention is:
Detect the method for flavonoids effective constituent content in the toothpaste containing baikal skullcap root, described flavonoids effective constituent is one or more in scutelloside, wogonoside, wogonin, said method comprising the steps of:
Get the toothpaste containing baikal skullcap root, adsorbent of molecular sieve, drying agent anhydrous sodium sulfate, add root of large-flowered skullcap mixed sample, described toothpaste, mass ratio 1:1 ~ 5 of adsorbent of molecular sieve: described toothpaste, the mass ratio of drying agent is 1:1 ~ 4, described root of large-flowered skullcap mixed sample is scutelloside, wogonoside and wogonin standard items add in methyl alcohol the hybrid standard product solution prepared and obtain, the described mixed root of large-flowered skullcap closes the scutelloside contained in standard specimen, the quality of wogonoside or wogonin and the mass ratio of toothpaste are independently 0.05 ~ 0.2mg/g separately, add in mortar after above-mentioned substance mixing and grind, obtain the solid mixture after grinding, get solid-phase extraction column, the bottom of solid-phase extraction column adds sieve plate, adds in solid-phase extraction column by the solid mixture after grinding, filler tamps rear top and adds sieve plate again, the solid-phase extraction column filled in is placed on solid-phase extracting instrument, gets eluant, eluent, inject in solid-phase extraction column, open the aspiration pump of solid-phase extracting instrument, wash-out is carried out to solid-phase extraction column, collects eluent, after wash-out terminates, by eluent vortex, centrifugal, get supernatant liquor and be flavonoids effective constituent extract, get the sampling analysis of flavonoids effective constituent extract Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of flavonoids effective constituent extract,
Then with the reference substance solution of the reference substance of scutelloside preparation variable concentrations, detect according to flavonoids effective constituent extract similarity condition Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of scutelloside reference substance, with the sample size of scutelloside reference substance for horizontal ordinate, with the peak area of chromatographic peak in the liquid chromatogram of scutelloside reference substance for ordinate makes scutelloside typical curve, make the typical curve of wogonoside and wogonin by same method; The peak area of each chromatographic peak and the typical curve of each composition in liquid chromatogram according to flavonoids effective constituent extract, calculate the content of scutelloside total in flavonoids effective constituent extract, wogonoside, wogonin, after deducting the content of each composition in root of large-flowered skullcap mixed sample respectively, obtain the content of each composition extracted in flavonoids effective constituent extract from toothpaste, corresponding conversion obtains the content of scutelloside, wogonoside, wogonin in the toothpaste containing the root of large-flowered skullcap.
The preferred KIT-6 of described adsorbent of molecular sieve.
Molecular sieve KIT-6 of the present invention can according to the synthesis of method below:
85g concentrated hydrochloric acid (35wt%) is dissolved in 682g water, add 30gP123 (polyethylene oxide-polypropyleneoxide-polyethylene oxide triblock copolymer) to dissolve, after add 34g normal butyl alcohol, add 447g sodium silicate solution again, composition in described sodium silicate solution is: the water of the silicon dioxide of 5.0wt%, the sodium oxide molybdena of 1.6wt% and 93.4wt%, 1 day is stirred at 298K lower magnetic force, under 298K, leave standstill 1 day after stirring terminates, filtration, Washing of Filter Cake, be drying to obtain molecular sieve KIT-6.
Above-mentioned steps is at Jo, Changbum; Kim, Kyoungsoo; Ryoo, Deng .SynthesesofhighqualityKIT-6andSBA-15mesoporoussilicasus inglow-costwaterglass, throughrapidquenchingofsilicatestruc-tureinacidicsolutio n [J] .MicroporousAndMesoporousMaterials, 2009,124 (1-3): mention in 45-51. well known to a person skilled in the art method.
The mass ratio of toothpaste of the present invention and adsorbent of molecular sieve is 1:1 ~ 5, is preferably 1:4.
The mass ratio of described toothpaste and drying agent is 1:1 ~ 4, is preferably 1:2.
Milling time of the present invention is 30 ~ 90s, is preferably 60s.
Solid-phase extraction column of the present invention has different volumes specification, and select the solid-phase extraction column of suitable volumes according to the volume of the solid mixture after toothpaste and the grinding such as adsorbent of molecular sieve, drying agent, this well known to a person skilled in the art.
Described eluant, eluent is methyl alcohol, acetonitrile, acetone or ethyl acetate, is preferably methyl alcohol.
The volumetric usage of described eluant, eluent preferably counts 0.016 ~ 0.028mL/mg with the quality of toothpaste, preferred 0.02mL/mg.
Described centrifugal preferably at the centrifugal 5min of 13000rpmin.
In the inventive method, root of large-flowered skullcap mixed sample is scutelloside, wogonoside and wogonin standard items add in methyl alcohol the hybrid standard product solution prepared and obtain, adding root of large-flowered skullcap mixed sample is to get rid of the interference of toothpaste matrix to active component better, obtain more significantly chromatographic peak, especially for the effective constituent that concentration in toothpaste is lower, actual concentrations can be detected more accurately and effectively.
This is the means that those skilled in the art commonly use.
The quality of the scutelloside, wogonoside or the wogonin that contain in described root of large-flowered skullcap mixed sample and the mass ratio of toothpaste are independently 0.05 ~ 0.2mg/g separately, scutelloside in general root of large-flowered skullcap mixed sample, wogonoside can not be identical with the concentration of wogonin, but in general preferred root of large-flowered skullcap mixed sample, scutelloside, wogonoside and wogonin are identical mass concentration, are convenient to follow-up conversion.
Further, the quality of the scutelloside, wogonoside and the wogonin that contain in described root of large-flowered skullcap mixed sample is 0.1mg/g with the mass ratio of toothpaste separately.
Further, in preferred described root of large-flowered skullcap mixed sample, the concentration of scutelloside, wogonoside and wogonin is 50 μ g/mL separately.
Further, the inventive method, for micro-toothpaste sample, preferably operates according to the following steps:
Take root of large-flowered skullcap toothpaste 25mg, KIT-6100mg, anhydrous sodium sulfate 50mg, add 50 μ L root of large-flowered skullcap mixed samples, in described root of large-flowered skullcap mixed sample, the concentration of scutelloside, wogonoside and wogonin is 50 μ g/mL separately, above-mentioned substance mixing is added in mortar and grinds 60s, obtain the solid mixture after grinding; Get the solid-phase extraction column of 1mL specification, the bottom of solid-phase extraction column adds sieve plate, solid mixture after grinding is added in solid-phase extraction column, filler tamps rear top and adds sieve plate again, the solid-phase extraction column filled in is placed on solid-phase extracting instrument, get the methyl alcohol of 0.5mL, in injection solid-phase extraction column, open the aspiration pump of solid-phase extracting instrument, wash-out is carried out to solid-phase extraction column, collect eluent, after wash-out terminates, put into centrifuge by after eluent vortex 10s, the centrifugal 5min of 13000rpm, get supernatant liquor and be flavonoids effective constituent extract; Get the sampling analysis of flavonoids effective constituent extract Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of flavonoids effective constituent extract;
Then with the reference substance solution of the reference substance of scutelloside preparation variable concentrations, detect according to flavonoids effective constituent extract similarity condition Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of scutelloside reference substance, with the sample size of scutelloside reference substance for horizontal ordinate, with the peak area of chromatographic peak in the liquid chromatogram of scutelloside reference substance for ordinate makes scutelloside typical curve, make the typical curve of wogonoside and wogonin by same method; The peak area of each chromatographic peak and the typical curve of each composition in liquid chromatogram according to flavonoids effective constituent extract, calculate the content of scutelloside total in flavonoids effective constituent extract, wogonoside, wogonin, after deducting the content of each composition in root of large-flowered skullcap mixed sample respectively, obtain the content of each composition extracted in flavonoids effective constituent extract from toothpaste, corresponding conversion obtains the content of scutelloside in the toothpaste containing the root of large-flowered skullcap, wogonoside, wogonin.
The detectability of the flavonoids effective constituent that the present invention detects in the root of large-flowered skullcap toothpaste of trace can reach scutelloside 0.0385ng, wogonoside 0.0359ng, wogonin 0.0177ng, has well extract Detection results to micro-example.
The invention has the advantages that:
1. the inventive method genus is applied in matrix solid phase dispersion extraction field first using molecular sieve KIT-6 as adsorbent, there is originality, in periodical patent at home and abroad, all do not report the case using this material as adsorbent, and during the extraction program being applied to toothpaste detects, also belong to the first.
2. this method is carried out extraction and is detected under have employed micro-system, and the consumption of the toothpaste of employing, adsorbent, eluant, eluent is extremely low, and this invention can be made to can be applicable to determination of trace field.Especially the consumption of organic solvent only 0.5mL that method of the present invention uses, this just makes this technical scheme extremely low to the murder by poisoning of operating personnel, is also reduced to almost nil to the pollution of environment.
3. molecular sieve KIT-6 of the present invention possess that a series of excellence gives prominence to physicochemical property: be first the nonhazardous to human body and environment, pollution-free, meet the concept that " Green Chemistry " is advocated completely, and the chemical stability of KIT-6 own and good thermal stability, to human non-toxic, free from environmental pollution, do not fire, make the experimental situation of operating personnel safe and reliable.
4. operation environment safety provided by the invention, operation steps is short and sweet, and conventional efficient is high, and operating personnel can carry out operation without the need to professional training.
Namely the present invention creatively provides a kind of Trace bio-element detection method of Chinese medicine, the method adopts molecular sieve as adsorbent, binding matrix solid phase dispersion abstraction technique, can efficient, environmental protection extract and detect the content containing the flavonoids effective constituent in root of large-flowered skullcap toothpaste.
Accompanying drawing explanation
Fig. 1 is the process chart of matrix solid phase dispersion extracting process of the present invention.
Fig. 2 is the matrix solid phase dispersion effect of extracting histogram investigating different quality ratio between root of large-flowered skullcap toothpaste and adsorbent.In figure, 1,2,3 represent effective constituent different in root of large-flowered skullcap toothpaste respectively, are respectively: 1: scutelloside, 2: wogonoside, 3: wogonin.In figure, horizontal ordinate is the mass ratio of root of large-flowered skullcap toothpaste and adsorbent.
Fig. 3 is the matrix solid phase dispersion effect of extracting histogram under the different milling time of investigation.In figure, 1,2,3 represent effective constituent different in root of large-flowered skullcap toothpaste respectively, are respectively: 1: scutelloside, 2: wogonoside, 3: wogonin.In figure, horizontal ordinate is milling time, unit s.
Fig. 4 is the matrix solid phase dispersion effect of extracting histogram investigating variety classes eluant, eluent.In figure, 1,2,3 represent effective constituent different in root of large-flowered skullcap toothpaste respectively, are respectively: 1: scutelloside, 2: wogonoside, 3: wogonin.In figure, A, B, C, D represent different eluant, eluents respectively, are respectively: A: methyl alcohol, B: acetonitrile, C: acetone, D: ethyl acetate.
Fig. 5 is the matrix solid phase dispersion effect of extracting histogram investigating different quality ratio between root of large-flowered skullcap toothpaste and drying agent.In figure, 1,2,3 represent effective constituent different in root of large-flowered skullcap toothpaste respectively, are respectively: 1: scutelloside, 2: wogonoside, 3: wogonin.In figure, horizontal ordinate is the mass ratio of root of large-flowered skullcap toothpaste and drying agent.
Fig. 6 is the matrix solid phase dispersion effect of extracting histogram investigating different volumes eluant, eluent.In figure, 1,2,3 represent effective constituent different in root of large-flowered skullcap toothpaste respectively, are respectively: 1: scutelloside, 2: wogonoside, 3: wogonin.In figure, horizontal ordinate represents the volume of eluant, eluent, and unit is mL.
Fig. 7 is the histogram that the different adsorbent species of investigation affects matrix solid phase dispersion effect of extracting.In figure, 1,2,3 represent effective constituent different in root of large-flowered skullcap toothpaste respectively, are respectively: 1: scutelloside, 2: wogonoside, 3: wogonin.A, B, C, D represent different adsorbents, are respectively A:400 order silica gel, B:200-300 order silica gel, the reverse silica gel of C:KIT-6, D:C18.
Fig. 8 is the liquid chromatogram of the different sample of the present invention.In figure, 1,2,3 represent effective constituent different in root of large-flowered skullcap toothpaste respectively, are respectively: 1: scutelloside, 2: wogonoside, 3: wogonin.A figure represents the liquid chromatogram that concentration is the standard items mixed liquor of 100 μ g/mL, B figure represents the inventive method to the liquid chromatogram extracting the flavonoids effective constituent extract obtained in root of large-flowered skullcap toothpaste, and C figure represents saliva sample extracts the extract obtained liquid chromatogram according to the inventive method.
Embodiment
By following instance, detection method provided by the present invention is made a more detailed description.Due to its applied range, therefore specific embodiments is also many, and the discussion below in conjunction with several example is further elaborated content of the present invention.But protection scope of the present invention is not limited thereto.
Get respectively scutelloside, wogonoside, wogonin reference substance appropriate, accurately weighed, mixing is put in brown measuring bottle, add methyl alcohol and make the solution of every lmL containing scutelloside 500 μ g, wogonoside 500 μ g, wogonin 500 μ g, obtain reference substance solution, and dilution obtains the mixed mark of reference substance of variable concentrations, in following examples.
Embodiment 1
Respectively with root of large-flowered skullcap toothpaste: adsorbent=1:1,1:2,1:3,1:4,1:5 (m/m) ratio takes root of large-flowered skullcap toothpaste 25mg and KIT-625mg, 50mg, 75mg, 100mg, 125mg, and respectively with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (concentration of scutelloside, wogonoside and wogonin is 50 μ g/mL separately) of the 50 μ L roots of large-flowered skullcap respectively, add in mortar respectively after above-mentioned substance mixing and grind 60s.Added respectively in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after filler adds, the top of filler adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
Embodiment instrument is WatersAcquityUplcH-Class.Chromatographic column: ACQUITY
bEHC18,1.7 μm, 2.1 × 50mm (Waters), determined wavelength: 280nm, column temperature: 40 DEG C, sample size: 1.0 μ L, flow velocity 0.4mL/min, mobile phase: A:0.1v% formic acid water, B: acetonitrile.Gradient elution: 0 ~ 2min, 30% ~ 40%B; 2 ~ 4min, 40% ~ 50%B; 4 ~ 6min, 50% ~ 60%B; 6 ~ 8min, 60% ~ 100%B; 8 ~ 9min, 100% ~ 100%B, 9 ~ 10min, 100% ~ 30%B.Chromatographic condition in subsequent embodiment is all identical with embodiment 1.
Experimental result is as following table 1, and the data in table 1 are peak area.
Table 1.
1 be scutelloside, 2 be wherein wogonoside, 3 for wogonin.
The Solid-Phase Extraction effect histogram of different root of large-flowered skullcap toothpaste and adsorbent mass ratio as shown in Figure 2.Result display is along with the increase of adsorbent amount, and effect of extracting has the trend of rising, reaches optimum at ratio 1:4 place, and continue the amount increasing adsorbent, effect of extracting tends towards stability, even slightly downtrending.Possible reason is, along with the increase of adsorbent amount, can adsorb the active component in hybrid solid better, but the adsorbable total amount of active component is limited, after adsorbent amount is increased to optimal value, effect of extracting trend is mild, illustrate active component substantially adsorb by adsorbent.And too much adsorbent also can cause active component desorb from adsorbent to become difficulty, makes effect of extracting have downtrending on the contrary.
Embodiment 2
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes each 4 parts of root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and respectively with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap respectively, add respectively in mortar after above-mentioned substance mixing and grind the different time (30s, 45s, 60s, 75s, 90s).Added respectively in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
Experimental result is as following table 2, and the data in table 2 are peak area.
Table 2.
1 be scutelloside, 2 be wherein wogonoside, 3 for wogonin.
Different milling times on the impact of matrix solid phase dispersion effect of extracting as shown in Figure 3.Result display is along with the increase of milling time, and effect of extracting becomes large gradually, and to optimal value 60s, along with the increase of milling time, effect of extracting has a declining tendency on the contrary.Possible reason is, fully grinding contacts active component in hybrid solid more fully by contributing to adsorbent, thus increases effect of extracting.But excessive grinding can make active component be forced to be squeezed in aperture less, fine and close in adsorbent, make wash-out become difficulty, reduce effect of extracting on the contrary.
Embodiment 3
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes each 4 points of root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and respectively with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap respectively, add in mortar respectively after above-mentioned substance mixing and grind 60s.Added respectively in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the different types of eluant, eluent of 0.5mL (methyl alcohol, acetonitrile, acetone, ethyl acetate), inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
Experimental result is as following table 3, and the data in table 3 are peak area.
Table 3.
1 be scutelloside, 2 be wherein wogonoside, 3 for wogonin; A is methyl alcohol, B is acetonitrile, C is acetone, D is ethyl acetate.
The matrix solid phase dispersion effect of extracting histogram of variety classes eluant, eluent is shown in Fig. 4.Result shows, the very large difference that attracted each other of different types of adsorbent and active component, and ethyl acetate is best to the elute effect of wogonin, but can not show a candle to methyl alcohol, Comprehensive Experiment result to both elute effect in addition, selects methyl alcohol as best eluant, eluent.
Embodiment 4
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes each 4 parts of root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and respectively with different root of large-flowered skullcap toothpaste: drying agent (m/m) ratio (1:1,1:2,1:3,1:4) takes anhydrous sodium sulfate 25mg, 50mg, 75mg, 100mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap respectively, add in mortar respectively after above-mentioned substance mixing and grind 60s.Added respectively in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
Experimental result is as following table 4, and the data in table 4 are peak area.
Table 4.
1 be scutelloside, 2 be wherein wogonoside, 3 for wogonin.
The matrix solid phase dispersion effect of extracting of root of large-flowered skullcap toothpaste and drying agent different proportion as shown in Figure 5.Result shows, and drying agent can, by water adsorption contained in toothpaste, make the moisture contained in eluent tail off, thus the effect of extracting improved, within the specific limits, the increase of drying agent can increase effect of extracting, and when exceeding certain proportion, the increase of drying agent has inhibition to effect of extracting on the contrary, possible reason is, too much drying agent, in wash-out link, hinders the contact between eluent and active component, the active component be eluted is reduced, thus reduces effect of extracting.
Embodiment 5
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes each 4 parts of root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and respectively with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap respectively, add in mortar respectively after above-mentioned substance mixing and grind 60s.Added respectively in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of different volumes (0.4mL, 0.5mL, 0.6mL, 0.7mL), inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
Experimental result is as following table 5, and the data in table 5 are peak area.
Table 5.
1 be scutelloside, 2 be wherein wogonoside, 3 for wogonin.
Fig. 6 is shown in the impact of different volumes eluant, eluent on matrix solid phase dispersion effect of extracting.Result shows, eluant, eluent volume is larger, the concentration of gained eluent is lower, chromatography peak integration area is less, but when eluant, eluent volume is too small, eluent can be made abundant not to the desorb of active component, can completely active component desorb from adsorbent not got off, affect final effect of extracting, Comprehensive Experiment result, adopt 0.5mL eluent as top condition.
Embodiment 6
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes root of large-flowered skullcap toothpaste 25mg and different types of adsorbent (400 order silica gel, 200-300 order silica gel, KIT-6, C18 reverse phase silica gel) each 4 parts of 100mg, and respectively with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap respectively, add in mortar respectively after above-mentioned substance mixing and grind 60s.Added respectively in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
Experimental result is as following table 6, and the data in table 6 are peak area.
Table 6.
1 be scutelloside, 2 be wherein wogonoside, 3 for wogonin; A is 400 order silica gel, B is 200-300 order silica gel, C be KIT-6, D is C18 reverse phase silica gel.
The matrix solid phase dispersion effect of extracting histogram of variety classes adsorbent as shown in Figure 7.Result shows, and the adsorption effect of KIT-6 is best, and possible reason is, compares other adsorbents, and the aperture junction structure of its inside of KIT-6 makes itself to possess larger specific surface area, can adsorption activity composition better.
Withinday precision
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap, above-mentioned substance mixing adds in mortar grinds 60s.Added in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
Continuous sample introduction 6 times within the same day.
Day to day precision
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap, above-mentioned substance mixing adds in mortar grinds 60s.Added in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
By this sample continuous sample introduction 3 days, every day 2 times.
In a few days, day to day precision experimental result gathers as following table 7:
Table 7.
1 be scutelloside, 2 be wherein wogonoside, 3 for wogonin.
Repeatability is investigated
With reference to following experiments step, parallelly do 5 groups, as investigation.
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap, above-mentioned substance mixing adds in mortar grinds 60s.Added in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.Repeated experiment the results are shown in Table 9.
Toothpaste assay
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (50 μ g/mL) of the 50 μ L roots of large-flowered skullcap, above-mentioned substance mixing adds in mortar grinds 60s.Added in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor, be flavonoids effective constituent extract, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.Obtain the liquid chromatogram of extract.
Saliva contents measures
With saliva sample: adsorbent=1:4 (m/m) ratio takes saliva 25mg and KIT-6100mg, and with saliva: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, add the mixed mark (scutelloside, wogonoside, wogonin concentration is separately 50 μ g/mL) of the 50 μ L roots of large-flowered skullcap, above-mentioned substance mixing adds in mortar grinds 60s.Added in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Getting supernatant liquor, is saliva extract, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.
Fig. 8 is the liquid chromatogram of different sample.In figure, 1,2,3 represent effective constituent different in root of large-flowered skullcap toothpaste respectively, 1 be scutelloside, 2 are wherein wogonoside, 3 for wogonin; A figure represents the liquid chromatogram that concentration is the standard items mixed liquor of 100 μ g/mL, B figure represents the inventive method extracts the flavonoids effective constituent extract obtained liquid chromatogram to root of large-flowered skullcap toothpaste, and C figure represents saliva sample extracts the saliva extract obtained liquid chromatogram according to the inventive method.
Specification Curve of Increasing:
With the mixing reference substance solution of the reference substance of scutelloside, wogonoside, wogonin preparation variable concentrations, detect by embodiment 1 similarity condition Ultra Performance Liquid Chromatography, obtain the chromatogram of three's reference substance, with the sample size of three's reference substance for horizontal ordinate, with the peak area of chromatographic peak in the chromatogram of three's reference substance solution for ordinate, make the typical curve of three.
According to the peak area of each chromatographic peak and the typical curve of each composition in the liquid chromatogram of flavonoids effective constituent extract and saliva extract, calculate scutelloside total in flavonoids effective constituent extract and saliva extract, wogonoside, the content of wogonin, after deducting the content (50 μ g/mL) of each composition in root of large-flowered skullcap mixed sample respectively, obtain in flavonoids effective constituent extract, extracting the content from each composition of toothpaste and each component content of extraction in saliva, corresponding conversion obtains scutelloside in the toothpaste containing the root of large-flowered skullcap, wogonoside, the content of wogonin, and scutelloside in saliva sample, wogonoside, the content of wogonin.Content detection the results are shown in Table 9.
The typical curve of 3 kinds of compositions and detectability and quantitative limit as shown in table 8 below:
Table 8.
1 be scutelloside, 2 be wherein wogonoside, 3 for wogonin.
The recovery is tested
With root of large-flowered skullcap toothpaste: adsorbent=1:4 (m/m) ratio takes root of large-flowered skullcap toothpaste 25mg and KIT-6100mg, and with root of large-flowered skullcap toothpaste: drying agent=1:2 (m/m) ratio takes anhydrous sodium sulfate 50mg, and above-mentioned substance mixing adds in mortar grinds 60s.Added in 1mL specification solid phase extraction column by solid mixture after mixing, the bottom of pillar adds sieve plate in advance, and after adding, the top of pillar adds sieve plate again.The pillar filled in is placed on solid-phase extracting instrument.Measure the methyl alcohol of 0.5mL, inject in pillar, start the aspiration pump (air pressure is-28.0bar) of solid-phase extracting instrument, wash-out is carried out to pillar.Eluent 1.5mL specification centrifuge tube is collected.After wash-out terminates, put into centrifuge by centrifuge tube after liquid vortex 10s, the centrifugal 5min of 13000rpm.Get supernatant liquor dress sample, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analysis.With the Data Comparison of " toothpaste assay " part, calculate the recovery.
Repeatability, assay, recovery experimental result gather as following table 9:
Table 9.
Wherein: 1 be scutelloside, 2 be wogonoside, 3 for wogonin.
Result shows, the repeatability of the inventive method is good, and the recovery is high, and detection accuracy is high.
Claims (10)
1. detect the method for flavonoids effective constituent content in the toothpaste containing baikal skullcap root, described flavonoids effective constituent is one or more in scutelloside, wogonoside, wogonin, said method comprising the steps of:
Get toothpaste, adsorbent of molecular sieve, the drying agent anhydrous sodium sulfate containing baikal skullcap root, add root of large-flowered skullcap mixed sample, mass ratio 1:1 ~ 5 of described toothpaste, adsorbent of molecular sieve; The mass ratio of described toothpaste, drying agent is 1:1 ~ 4, described root of large-flowered skullcap mixed sample is scutelloside, wogonoside and wogonin standard items add in methyl alcohol the hybrid standard product solution prepared and obtain, the quality of the scutelloside, wogonoside or the wogonin that contain in described root of large-flowered skullcap mixed sample and the mass ratio of toothpaste are independently 0.05 ~ 0.2mg/g separately, add in mortar after the described toothpaste containing baikal skullcap root, adsorbent of molecular sieve, drying agent anhydrous sodium sulfate and the mixing of root of large-flowered skullcap mixed sample and grind, obtain the solid mixture after grinding; Get solid-phase extraction column, the bottom of solid-phase extraction column adds sieve plate, adds in solid-phase extraction column by the solid mixture after grinding, filler tamps rear top and adds sieve plate again, the solid-phase extraction column filled in is placed on solid-phase extracting instrument, gets eluant, eluent, inject in solid-phase extraction column, open the aspiration pump of solid-phase extracting instrument, wash-out is carried out to solid-phase extraction column, collects eluent, after wash-out terminates, by eluent vortex, centrifugal, get supernatant liquor and be flavonoids effective constituent extract; Get the sampling analysis of flavonoids effective constituent extract Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of flavonoids effective constituent extract;
Then with the reference substance solution of the reference substance of scutelloside preparation variable concentrations, detect according to flavonoids effective constituent extract similarity condition Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of scutelloside reference substance, with the sample size of scutelloside reference substance for horizontal ordinate, with the peak area of chromatographic peak in the liquid chromatogram of scutelloside reference substance for ordinate makes scutelloside typical curve, make the typical curve of wogonoside and wogonin by same method; The peak area of each chromatographic peak and the typical curve of each composition in liquid chromatogram according to flavonoids effective constituent extract, calculate the content of scutelloside total in flavonoids effective constituent extract, wogonoside, wogonin, after deducting the content of each composition in root of large-flowered skullcap mixed sample respectively, obtain the content of each composition extracted in flavonoids effective constituent extract from toothpaste, corresponding conversion obtains the content of scutelloside, wogonoside, wogonin in the toothpaste containing the root of large-flowered skullcap.
2. the method for claim 1, is characterized in that described adsorbent of molecular sieve is KIT-6.
3. the method for claim 1, is characterized in that the mass ratio of described toothpaste and adsorbent of molecular sieve is 1:4.
4. the method for claim 1, is characterized in that the mass ratio of described toothpaste and drying agent is 1:2.
5. the method for claim 1, is characterized in that described eluant, eluent is methyl alcohol, acetonitrile, acetone or ethyl acetate.
6. the method for claim 1, is characterized in that described eluant, eluent is methyl alcohol.
7. the method for claim 1, is characterized in that the volumetric usage of described eluant, eluent counts 0.016 ~ 0.028mL/mg with the quality of toothpaste.
8. the method for claim 1, is characterized in that the volumetric usage of described eluant, eluent counts 0.02mL/mg with the quality of toothpaste.
9. the method for claim 1, is characterized in that the quality of scutelloside, wogonoside and the wogonin contained in described root of large-flowered skullcap mixed sample is 0.1mg/g with the mass ratio of toothpaste separately.
10. the method for claim 1, is characterized in that described method operates according to the following steps:
Take root of large-flowered skullcap toothpaste 25mg, KIT-6100mg, anhydrous sodium sulfate 50mg, add 50 μ L root of large-flowered skullcap mixed samples, in described root of large-flowered skullcap mixed sample, the concentration of scutelloside, wogonoside and wogonin is 50 μ g/mL separately, root of large-flowered skullcap toothpaste 25mg, KIT-6100mg, anhydrous sodium sulfate 50mg and the mixing of 50 μ L root of large-flowered skullcap mixed samples are added in mortar and grinds 60s, obtain the solid mixture after grinding; Get the solid-phase extraction column of 1mL specification, the bottom of solid-phase extraction column adds sieve plate, solid mixture after grinding is added in solid-phase extraction column, filler tamps rear top and adds sieve plate again, the solid-phase extraction column filled in is placed on solid-phase extracting instrument, get the methyl alcohol of 0.5mL, in injection solid-phase extraction column, open the aspiration pump of solid-phase extracting instrument, wash-out is carried out to solid-phase extraction column, collect eluent, after wash-out terminates, put into centrifuge by after eluent vortex 10s, the centrifugal 5min of 13000rpm, get supernatant liquor and be flavonoids effective constituent extract; Get the sampling analysis of flavonoids effective constituent extract Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of flavonoids effective constituent extract;
Then with the reference substance solution of the reference substance of scutelloside preparation variable concentrations, detect according to flavonoids effective constituent extract similarity condition Ultra Performance Liquid Chromatography instrument, obtain the liquid chromatogram of scutelloside reference substance, with the sample size of scutelloside reference substance for horizontal ordinate, with the peak area of chromatographic peak in the liquid chromatogram of scutelloside reference substance for ordinate makes scutelloside typical curve, make the typical curve of wogonoside and wogonin by same method; The peak area of each chromatographic peak and the typical curve of each composition in liquid chromatogram according to flavonoids effective constituent extract, calculate the content of scutelloside total in flavonoids effective constituent extract, wogonoside, wogonin, after deducting the content of each composition in root of large-flowered skullcap mixed sample respectively, obtain the content of each composition extracted in flavonoids effective constituent extract from toothpaste, corresponding conversion obtains the content of scutelloside, wogonoside, wogonin in the toothpaste containing the root of large-flowered skullcap.
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