CN102435689A - Determination method of UPLC-MS (ultrahigh performance liquid chromatography-mass spectrometry) fingerprint of Radix Scutellariae medicinal material - Google Patents
Determination method of UPLC-MS (ultrahigh performance liquid chromatography-mass spectrometry) fingerprint of Radix Scutellariae medicinal material Download PDFInfo
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Abstract
The invention provides a determination method of the UPLC-MS fingerprint of a Radix Scutellariae medicinal material. The finger print which is obtained through determining by the UPLC-MS has twenty-three common peaks, and each peak is calibrated with the ion mass and the molecular formula. The determination method comprises the following steps: 1, preparing a sample solution; 2, setting chromatographic conditions and mass spectrometry conditions; 3, recording a 18min chromatogram, obtaining the extracted ion chromatogram with twenty-three common peaks according to the mass/charge ratio of the molecular ion of each peak, and carrying out fingerprint evaluation. The determination method of the invention, which adopts the UPLC to separate and the MS to detect, has the following advantages: the separation time is substantially shortened, the separation efficiency and the detection sensitivity are improved, and the accurate positioning of the common peaks can be realized. The method which has the characteristics of high sensitivity and strong specialization can be used for the identification and the quality evaluation of the Radix Scutellariae medicinal material.
Description
Technical field
The present invention relates to the assay method of traditional Chinese medicine fingerprint, specifically the structure and the standard finger-print thereof of radix scutellariae medicinal materials Ultra Performance Liquid Chromatography-mass spectrometry finger-print.Belong to the Pharmaceutical Analysis technical field.
Background technology
Traditional Chinese medicine fingerprint is certain type or the chromatogram of several constituents or the collection of illustrative plates of spectral information that can reflect Chinese crude drug or Chinese patent drug that adopts modern analytical technique to set up; The overall chemical characteristic that can reflect Chinese medicine more all sidedly; Embody its inherent total quality; Can remedy the drawback that only becomes to assign to control traditional Chinese medicine quality, become the effective means of internationally recognized control Chinese medicine or natural drug quality with minority.Yet the uncertainty of the diversity of chemical composition of Chinese materia medica and complicacy and effective substance has increased the difficulty of traditional Chinese medicine fingerprint foundation and the validity and the reappearance of finger-print.
At present, what traditional Chinese medicine fingerprint research was many is the Chemistry for Chinese Traditional Medicine finger-print, is meant the finger-print in order to sign chemical composition of Chinese materia medica characteristic that adopts chromatogram, wave spectrum and other analytical approachs to set up.High performance liquid chromatography (HPLC) has that separation efficiency height, analysis speed are fast, the characteristics of favorable reproducibility, and can be equipped with the advantage that different detector is analyzed all kinds sample, has become the generally acknowledged predominant methods of setting up traditional Chinese medicine fingerprint.The Ultra Performance Liquid Chromatography technology (UPLC) of rising in recent years is by means of theory and the principle of HPLC; Adopt the chromatographic column filler of 1.7 μ m particle diameters; Shortened to about 10 minute by original 1 hour the analysis time of a finger-print at least; And chromatographic peak capacity and stability also significantly improve, and are particularly suitable for the research of Chinese medicine and natural products, for traditional Chinese medicine fingerprint research provides new separation platform.
Ultraviolet (UV) detecting device is present and finger-print Application Research detection method is the most widely carried out in the liquid chromatography coupling.Yet the ultraviolet absorptivity of chemical constitution has additive property; When the complicated chemical composition of Chinese medicine does not obtain good separation; The response that is detected be the multicomponent absorbance adding with, measure the result and produce deviation thereby make, can not truly reflect the content of chemical composition of Chinese materia medica.And uv detection method only is fit to have the chemical composition of Chinese materia medica of uv absorption, for no uv absorption or absorb more weak composition, is difficult to effectively detect.Mass spectrum (MS) detecting device can be used for the detection of all cpds in the Chinese medicine, all can produce response preferably, is a kind of highly sensitive common detector, the more important thing is molecular weight and molecular structure information (tandem mass spectrum) that compound can be provided.Adopt mass detector,, can make it comprise the information of all target compounds through confirming the mass scanning scope of ion.Use the total ion current detecting pattern, full detail that can the synthesization compound; And then, through examining or check the mass spectrum information of total chromatographic peak, can confirm the composition situation of chromatographic peak, guarantee that chromatographic peak is simplification compound composition, has scientific preferably.Because the complicacy of chemical composition of Chinese materia medica; Guarantee that the chromatographic peak in the traditional Chinese medicine fingerprint is that single compound peaks is difficult to; This also almost is impossible, and adopts mass detector, can from TIC (TIC), extract the ion of specific m/z; Set up and extract chromatography of ions figure (EIC), thereby guaranteed that chromatographic peak is simplification compound composition.In addition; Through total peak is carried out mass spectrometry, set up the extraction chromatography of ions figure at total peak, and then pass through the one-to-one relationship of chromatographic peak and compound m/z; Realization is to the accurate location and the measurement at each total peak, thereby can remedy the shortcoming of the poor reproducibility between other method laboratory.
UPLC-MS has combined the chromatogram separating capacity and the sensitiveest, the most intelligent detectability of most excellent, in the research of Chemistry for Chinese Traditional Medicine finger-print, has development prospect and space, and advantage is outstanding, and characteristics are obvious, will become the development trend and the direction of traditional Chinese medicine fingerprint.
The root of large-flowered skullcap is a conventional Chinese medicine simply, is the dry root of labiate root of large-flowered skullcap Scutellaria baicalensis Georgi, has heat-clearing and damp-drying drug, purging intense heat and detonicating, hemostasis, antiabortive effect.The root of large-flowered skullcap mainly contains flavones ingredients such as scutelloside, wogonoside, baicalein, wogonin; " Chinese pharmacopoeia " root of large-flowered skullcap " and " Baical Skullcap root P.E " item have only carried out assay and control to its contained scutelloside down, are difficult to effectively control the total quality of radix scutellariae medicinal materials and extract thereof." on the basis of Chinese pharmacopoeia; (number of patent application: 200510029014.7), other has the method control root of large-flowered skullcap quality (number of patent application: 200510031453.1) that adopts HPLC-UV finger-print and multicomponent assay to have patent further to measure the content of baicalein, wogonin and wogonoside.But still do not utilize UPLC-MS to carry out the report of root of large-flowered skullcap quality control.The present invention is on the basis of root of large-flowered skullcap serum drug chemical research; Selection can be absorbed into the composition of blood as the determining fingerprint pattern index; And the mensuration of 10 place of production root of large-flowered skullcap finger-prints of warp, confirm total fingerprint peaks and variation range thereof, as the method for root of large-flowered skullcap quality control.
Summary of the invention
The objective of the invention is to overcome the shortcoming and defect of prior art, the method that provides a kind of UPLC-MS of utilization to carry out the radix scutellariae medicinal materials quality control, and the resulting radix scutellariae medicinal materials standard finger-print of method thus.For realizing this purpose, the present invention illustrates the blood component of going into of the root of large-flowered skullcap through root of large-flowered skullcap serum drug chemical research, goes into blood component as detecting index with the root of large-flowered skullcap, and utilizes the UPLC-MS technology to make up the radix scutellariae medicinal materials chemical fingerprint, is used for the quality control of the root of large-flowered skullcap.
The assay method of radix scutellariae medicinal materials UPLC-MS finger-print of the present invention comprises the steps:
(1) preparation of need testing solution: it is an amount of to take by weighing the radix scutellariae medicinal materials powder, accurately claims surely, places conical flask, with 70% methyl alcohol 20mL ultrasonic Extraction 10～45 minutes, puts to room temperature, filters, and filtrating places the 50mL volumetric flask; Residue is used methyl alcohol 20mL ultrasonic Extraction 10～45 minutes again, puts to room temperature, filters, and residue washs with small amount of methanol, and filtrating is incorporated in the 50mL volumetric flask, and constant volume shakes up, and centrifugal 15 minutes of 13000rpm draws supernatant, as need testing solution;
(2) condition determination: chromatography column is ACQUITY
series of columns; Moving phase is 0.1% formic acid acetonitrile-0.1% formic acid water, adopts the gradient elution mode; Flow velocity: 0.3～0.5mL/min; Column temperature: 40-45 ℃; Sample size is 1～5 μ L.Scanning of the mass spectrum mode: positive ion full scan detecting pattern; Quality of scanning scope: 100-1000mDa;
(3) assay method: draw need testing solution, inject the UPLC-MS combined instrument, measure, write down 18 minutes chromatogram.Setting the karyoplasmic ratio (m/z) at each total peak, set up the extraction chromatography of ions figure at each peak, is 1 with the scutelloside peak area, calculates the relative ratio of each peak area, carries out similarity evaluation.
Wherein, described test sample sampling amount is 0.10-0.50g, 30-45 minute ultrasonic Extraction time.
Wherein, described chromatographic column is ACQUITY
The HSST3 post, flow velocity 0.4mL/min, 40 ℃ of column temperatures, sample size 2 μ L; Or described chromatographic column is ACQUITY
C
18Post, flow velocity 0.3mL/min, 45 ℃ of column temperatures, sample size 5 μ L.
Wherein, the foundation of said extraction chromatography of ions figure: the karyoplasmic ratio according to each peak molion extracts acquisition from TIC, the karyoplasmic ratio at 23 total peaks (m/z) is respectively: peak 1, and m/z 305; Peak 2, m/z 549; Peak 3, m/z 463; Peak 4, m/z 549; Peak 5, m/z 549; Peak 6, m/z 477; Peak 7, m/z 347; Peak 8, m/z 447; Peak 9, m/z 447; Peak 10, m/z 477; Peak 11, m/z 431; Peak 12, m/z 461; Peak 13, m/z 477; Peak 14, m/z 447; Peak 15, m/z 461; Peak 16, m/z491; Peak 17, m/z 271; Peak 18, m/z 271; Peak 19, m/z 285; Peak 20, m/z 315; Peak 21, m/z 375; Peak 22, m/z 285; Peak 23, m/z 315; Wherein peak 8 is a scutelloside.
Wherein, the molecular formula at said total peak characterizes: use flight time mass spectrum and measured the accurate mass at each peak in the standard finger-print, calculate the molecular formula at each peak and distinguish as follows: peak 1, C
15H
12O
7Peak 2, C
26H
28O
13Peak 3, C
21H
18O
12Peak 4, C
26H
28O
13Peak 5, C
26H
28O
13Peak 6, C
22H
20O
12Peak 7, C
17H
14O
8Peak 8, C
21H
18O
11Peak 9, C
21H
18O
11Peak 10, C
22H
20O
12Peak 11, C
21H
18O
10Peak 12, C
22H
20O
11Peak 13, C
22H
20O
12Peak 14, C
21H
18O
11Peak 15, C
22H
20O
11Peak 16, C
23H
22O
12Peak 17, C
15H
10O
5Peak 18, C
15H
10O
5Peak 19, C
16H
12O
5Peak 20, C
17H
14O
6Peak 21, C
19H
18O
8Peak 22, C
16H
12O
5Peak 23, C
17H
14O
6
The present invention also provides the method for quality control of radix scutellariae medicinal materials; Be to derive from the meeting of 10 above places of production " radix scutellariae medicinal materials of Chinese pharmacopoeia regulation is by above-mentioned assay method record finger-print; Adopt average method production standard finger-print; Testing sample finger-print and standard finger-print are compared, and similarity is greater than 0.9, thinks the up-to-standard of said product to be measured.
The present invention utilizes finger-print to characterize the chemical constitution of radix scutellariae medicinal materials on the whole, and is the composition that is absorbed into blood through 23 total peaks in this finger-print of serum drug chemical method proof, is the potential effective constituent of root of large-flowered skullcap performance drug effect.The nuance between the different medicinal materials be can find out through relatively more total peak, the discriminating and the quality control of the radix scutellariae medicinal materials true and false, the place of production and quality are suitable for.
Compared with prior art, the present invention has the following advantages:
(1) the present invention adopts UPLC-MS as analysis means at home first, sets up root of large-flowered skullcap chemical fingerprint method of quality control, compares with the HPLC-UV analytical approach of existing report, has not only shortened analysis time greatly, has improved separation efficiency and detection sensitivity.And the information that can give molecular weight for each peak; Extract chromatography of ions figure through setting up; Can accurately calculate the peak area at each peak; And when between the laboratory, reappearing if retention time departs from, can accurately locate through the molecular weight information at each peak, thereby guarantee the reproducibility of fingerprint atlas detection method.
(2) Chinese medicine serum drug chemical research shows, behind Chinese medicine (external application and act on except the Chinese medicine of the enteron aisle) oral administration, its effective constituent only is absorbed into competence exertion pharmacological action behind the blood.The effect of Chinese medicine is multicomponent, the synergistic result of many target spots, and it is gone into blood component and all might contribute to some extent its drug effect, therefore to go into blood component as the index components that finger-print detects, more can guarantee the validity of Chinese medicine.The present invention proves that through the research of root of large-flowered skullcap serum drug chemistry 23 total peaks in this finger-print are the composition that is absorbed into blood, and with this index components as the radix scutellariae medicinal materials determining fingerprint pattern, the chemical fingerprint of foundation has more validity.
Description of drawings
Fig. 1 is that the UPLC-MS of 10 place of production radix scutellariae medicinal materials extracts chromatography of ions figure, among the figure mark 23 total peaks.
Fig. 2 is that radix scutellariae medicinal materials and rat pastille serum UPLC-MS extract chromatography of ions figure, wherein (A) rat pastille serum; (B) radix scutellariae medicinal materials; Each peak mark retention time and karyoplasmic ratio.
Fig. 3 is that the UPLC-MS that radix scutellariae medicinal materials finger-print precision is investigated extracts chromatography of ions figure.
Fig. 4 is that the UPLC-MS of radix scutellariae medicinal materials finger-print study on the stability extracts chromatography of ions figure.
Fig. 5 is that the UPLC-MS that radix scutellariae medicinal materials finger-print repeatability is investigated extracts chromatography of ions figure.
Fig. 6 is the extraction chromatography of ions figure at radix scutellariae medicinal materials UPLC-MS chromatogram and each total peak.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Following examples only are used to the present invention is described and should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.Should be understood that in addition those skilled in the art can do various changes and modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.For purpose simple and removing, hereinafter has omitted the description of known technology rightly simultaneously, in order to avoid those unnecessary details influences are to the description of present technique scheme.
The assay method of embodiment 1 radix scutellariae medicinal materials UPLC-MS finger-print.
1. instrument and reagent
Liquid chromatograph is an Acquity UPLC system of U.S. Waters company, comprises binary geopressure gradient pump, automatic sampler, column oven, PDAD.Mass spectrometer is a LCT PremierXE system of U.S. Waters company, comprises electric spray ion source, Lock-spray on-line correction system, syringe needle pump.Workstation is Masslynx V4.1.
Acetonitrile is a chromatographically pure, and water is ultrapure water, and it is pure that other reagent is analysis.
The scutelloside reference substance is available from Nat'l Pharmaceutical & Biological Products Control Institute.Medicinal material is available from tree medicinal material market, three in Harbin, and the place of production is respectively Heilungkiang, Jilin, Liaoning, Hebei, Shanxi, Shaanxi, the Inner Mongol, Anhui, Gansu, totally 10 lot sample article, and wherein the place of production, Heilungkiang is 2 batches." the Chinese pharmacopoeia check was all up to specification by version in 2010.
2. the preparation of need testing solution
Take by weighing radix scutellariae medicinal materials powder 0.10g, accurate claim surely, place conical flask, with 70% methyl alcohol 20mL ultrasonic Extraction 30 minutes, put to room temperature, filter, filtrating places the 50mL volumetric flask; Residue is used methyl alcohol 20mL ultrasonic Extraction 30 minutes again, puts to room temperature, filters, and residue washs with small amount of methanol, and filtrating is incorporated in the 50mL volumetric flask, and constant volume shakes up, and centrifugal 15 minutes of 13000rpm draws supernatant, as need testing solution.
3. finger-print testing conditions
Chromatographic condition: chromatographic column is ACQUITY
HSS T
3Post (2.1 * 100mm, 1.8 μ m); Moving phase is 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B), gradient elution (0min, 16%A; 3min, 20%A; 11min, 24%A; 13min, 38%A; 18min, 40%A; 20min, 100%A); Flow velocity: 0.4mL/min; Column temperature: 40 ℃; Sample size is 2 μ L.
Mass spectrum condition: electron spray ionisation source, positive ion scan pattern, capillary voltage 1800V; Taper hole voltage 60V, 350 ℃ of desolventizing temperature degree, 110 ℃ of source temperatures; Desolventizing gas flow rate 700L/h, taper hole gas flow rate 20L/h, leucine-brain coffee is eliminated the solution on-line correction; The quality of scanning scope is 100-1000mDa, and scan mode is a full scan.
4. the foundation of finger-print and evaluation
(1) total peak confirms
Collect the radix scutellariae medicinal materials in 10 places of production,, under above-mentioned testing conditions, analyze by " preparation of a need testing solution " method operation down.See that from testing result it is that each batch test sample is total that 23 peaks are arranged, and therefore with the total fingerprint peaks of these 23 peaks as radix scutellariae medicinal materials, from TIC, obtains the extraction chromatography of ions figure at these 23 peaks, sees Fig. 1.Wherein the peak area of scutelloside is maximum, elects as with reference to the peak, and the relative peak area at the total peak of different places of production radix scutellariae medicinal materials finger-print is seen table 1.
Flight time mass spectrum is a high resolution mass spectrum, can measure the accurate mass of ion, and then according to the molecular formula of measured deviation less than each total peak of principle calculating of 5PPM and isotope degree of fitting minimum, sees table 2.
(2) root of large-flowered skullcap is gone into confirming of blood component
Get the Wistar rat (220 ± 20g), fasting 12h, orally give root of large-flowered skullcap decocting liquid, 1h is with 20% urethane solution intraperitoneal injection of anesthesia after the administration; THPV blood sampling, the centrifugal 10min of 6000rpm gets serum 1mL, makes through using the OASIS HLB solid-phase extraction column behind the activation of 2mL methyl alcohol, the 2mL water balance in advance; With the flushing of 2mL water, discard, use the 2mL methanol-eluted fractions again, collect meoh eluate; Nitrogen flows down and dries up in 40 ℃ of water-baths, redissolves with 150 μ L methyl alcohol, and the centrifugal 15min of 13000rpm draws supernatant 5 μ L; Detect by the chromatographic condition of drafting, obtain the extraction chromatography of ions figure of 23 peak karyoplasmic ratios, see Fig. 2.
Get root of large-flowered skullcap need testing solution, detect, obtain the extraction chromatography of ions figure of 23 peak karyoplasmic ratios, see Fig. 2 by the chromatographic condition of drafting.The UPLC-MS chromatogram that compares radix scutellariae medicinal materials and pastille serum thereof, 23 total peaks confirming in the above-mentioned finger-print of result all exist in pastille serum, show that these 23 total peaks all can be absorbed into blood.
(3) finger-print similarity evaluation:
The radix scutellariae medicinal materials determining fingerprint pattern data in 10 places of production are generated the finger-print common pattern with the method for average; And be the standard finger-print of radix scutellariae medicinal materials with this common pattern; Application correlation coefficient process and included angle cosine method are carried out similarity evaluation to the finger-print of each place of production radix scutellariae medicinal materials; Each medicinal material similarity is seen table 1 all greater than 0.9 as a result.Explain that the radix scutellariae medicinal materials finger-print quality control method of setting up can apply to therapeutic dose control in the actual production, satisfies the demand of medicinal material discriminating and quality control.
5. the methodological study of finger-print
In order to investigate the reliability of analytical approach, precision, stability, the reappearance of fingerprint atlas detection method are investigated, the result proves that precision, stability and reappearance are good, can satisfy the requirement of finger-print.
(1) precision test: get with a need testing solution, detect by the testing conditions of drafting, heavy
Sample introduction is 5 times again, writes down the peak area of each chromatographic peak.With the scutelloside peak area is reference, calculates the consistance of each chromatographic peak relative peak area, and the RSD value of relative peak area all is lower than 3% as a result, calculates with similarity, and the finger-print and the common pattern of gained carried out similarity evaluation, and similarity is all greater than 0.99.See Fig. 3.
(2) stability test: get with a need testing solution, respectively 0,2,4,8,12,24h
Testing conditions by drafting detects, and writes down the peak area of each chromatographic peak.With the scutelloside peak area is reference, calculates the consistance of each chromatographic peak relative peak area, and the RSD value of relative peak area all is lower than 3% as a result, calculates with similarity, and the finger-print and the common pattern of gained carried out similarity evaluation, and similarity is all greater than 0.99.The result shows that the radix scutellariae medicinal materials need testing solution is stable in 24 hours.See Fig. 4.
(3) replica test: get 5 parts of the test samples of same lot number, press need testing solution preparation method preparation
Become need testing solution, detect, write down the peak area of each chromatographic peak by the testing conditions of drafting.With the scutelloside peak area is reference, calculates the consistance of each chromatographic peak relative peak area, and the RSD value of relative peak area all is lower than 3% as a result; Calculate with similarity; The finger-print and the common pattern of gained are carried out similarity evaluation, and similarity is seen Fig. 5 all greater than 0.99.
The mensuration of embodiment 2 radix scutellariae medicinal materials UPLC-MS finger-prints.
1. instrument and reagent
Liquid chromatograph is an Acquity UPLC system of U.S. Waters company, comprises binary geopressure gradient pump, automatic sampler, column oven, PDAD.Mass spectrometer is a LCT PremierXE system of U.S. Waters company, comprises electric spray ion source, Lock-spray on-line correction system, syringe needle pump.Workstation is Masslynx V4.1.
Acetonitrile is a chromatographically pure, and water is ultrapure water, and it is pure that other reagent is analysis.
Radix scutellariae medicinal materials is available from three trees medicinal material market, Harbin, and the place of production is respectively Heilungkiang.Meet version " Chinese pharmacopoeia regulation in 2010 through check.
2. the preparation of need testing solution
Take by weighing radix scutellariae medicinal materials powder 0.50g, accurate claim surely, place conical flask, with 70% methyl alcohol 20mL ultrasonic Extraction 45 minutes, put to room temperature, filter, filtrating places the 50mL volumetric flask; Residue is used methyl alcohol 20mL ultrasonic Extraction 45 minutes again, puts to room temperature, filters, and residue washs with small amount of methanol, and filtrating is incorporated in the 50mL volumetric flask, and constant volume shakes up, and centrifugal 15 minutes of 13000rpm draws supernatant, as need testing solution.
3. finger-print testing conditions
Chromatographic condition: chromatographic column is ACQUITY
C
18Post (2.1 * 100mm, 1.7 μ m); Moving phase is 0.1% formic acid acetonitrile (A)-0.1% formic acid water (B), gradient elution (0min, 10%A; 2min, 28%A; 6min, 32%A; 8min, 38%A; 12min, 50%A; 15min, 100%A); Flow velocity: 0.3mL/min; Column temperature: 45 ℃; Sample size is 5 μ L.
Mass spectrum condition: electron spray ionisation source, positive ion scan pattern, capillary voltage 1800V; Taper hole voltage 60V, 350 ℃ of desolventizing temperature degree, 110 ℃ of source temperatures; Desolventizing gas flow rate 700L/h, taper hole gas flow rate 20L/h, leucine-brain coffee is eliminated the solution on-line correction; The quality of scanning scope is 100-1000mDa, and scan mode is a full scan.
4. determining fingerprint pattern
Get radix scutellariae medicinal materials,, under above-mentioned testing conditions, detect, obtain the UPLC-MS chromatogram of radix scutellariae medicinal materials by " preparation of a need testing solution " method operation down.According to the molecular formula or the molion m/z at 23 total peaks confirming in the instance 1, from the UPLC-MS chromatogram of gathering, extract the chromatogram at these 23 total peaks, see Fig. 6.Though the result shows need testing solution preparation method and testing conditions have been carried out suitable change, but still can accurately locate each peak, and then calculated peak area, carried out the evaluation of finger-print through the molion m/z value of each peak representation compound.
The extraction chromatography of ions figure at radix scutellariae medicinal materials UPLC-MS chromatogram and total peak.The total peak-to-peak of confirming in peak number and the instance 1 is number consistent.
The relative peak area and the similarity evaluation result of table 1.10 place of production radix scutellariae medicinal materials finger-print
| Peak number | Heilungkiang | Jilin | Liaoning | Gansu | Anhui | Shaanxi | The Inner Mongol | Hebei | Shanxi | Heilungkiang | Common pattern |
| 1 | 0.0194 | 0.0401 | 0.0572 | 0.0274 | 0.0239 | 0.0349 | 0.0331 | 0.0175 | 0.0319 | 0.0327 | 0.0318 |
| 2 | 0.0803 | 0.1013 | 0.1106 | 0.0940 | 0.1072 | 0.1052 | 0.1463 | 0.1030 | 0.1116 | 0.1108 | 0.1070 |
| 3 | 0.0126 | 0.0105 | 0.0084 | 0.0109 | 0.0177 | 0.0232 | 0.0070 | 0.0117 | 0.0135 | 0.0136 | 0.0129 |
| 4 | 0.0563 | 0.0707 | 0.0743 | 0.0715 | 0.0711 | 0.0741 | 0.0960 | 0.0836 | 0.0809 | 0.0781 | 0.0757 |
| 5 | 0.0131 | 0.0174 | 0.0184 | 0.0113 | 0.0194 | 0.0154 | 0.0261 | 0.0101 | 0.0141 | 0.0125 | 0.0158 |
| 6 | 0.0322 | 0.0032 | 0.0024 | 0.0247 | 0.0247 | 0.1101 | 0.0079 | 0.0267 | 0.0420 | 0.0420 | 0.0316 |
| 7 | 0.0220 | 0.0824 | 0.1674 | 0.0443 | 0.0486 | 0.4387 | 0.0871 | 0.0294 | 0.0431 | 0.0429 | 0.1006 |
| 8 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 |
| 9 | 0.0974 | 0.1653 | 0.0844 | 0.1260 | 0.1487 | 0.0445 | 0.0969 | 0.1288 | 0.1405 | 0.1369 | 0.1169 |
| 10 | 0.0296 | 0.0167 | 0.0173 | 0.0457 | 0.0388 | 0.0239 | 0.0354 | 0.0386 | 0.0529 | 0.0509 | 0.0350 |
| 11 | 0.0549 | 0.0313 | 0.0850 | 0.0649 | 0.0732 | 0.0582 | 0.0610 | 0.0637 | 0.0674 | 0.0677 | 0.0627 |
| 12 | 0.2570 | 0.1878 | 0.4084 | 0.2240 | 0.3597 | 0.5172 | 0.2306 | 0.1762 | 0.2576 | 0.2529 | 0.2871 |
| 13 | 0.0355 | 0.0323 | 0.0014 | 0.0381 | 0.0476 | 0.0044 | 0.0426 | 0.0282 | 0.0416 | 0.0409 | 0.0313 |
| 14 | 0.0195 | 0.0179 | 0.0099 | 0.0209 | 0.0196 | 0.0112 | 0.0193 | 0.0241 | 0.0229 | 0.0234 | 0.0189 |
| 15 | 0.5147 | 0.3560 | 0.6972 | 0.5631 | 0.5929 | 0.5997 | 0.4942 | 0.5727 | 0.3009 | 0.5589 | 0.5250 |
| 16 | 0.0242 | 0.0127 | 0.0119 | 0.0180 | 0.0254 | 0.0165 | 0.0175 | 0.0090 | 0.0233 | 0.0228 | 0.0181 |
| 17 | 0.0223 | 0.0571 | 0.0142 | 0.0121 | 0.0285 | 0.0098 | 0.1464 | 0.0481 | 0.0229 | 0.0224 | 0.0384 |
| 18 | 0.4080 | 0.6322 | 0.4145 | 0.2963 | 0.4307 | 0.3717 | 0.8497 | 0.7617 | 0.4062 | 0.3999 | 0.4971 |
| 19 | 0.2417 | 0.7767 | 0.2973 | 0.1374 | 0.2850 | 0.2628 | 0.5718 | 0.4732 | 0.2266 | 0.2265 | 0.3499 |
| 20 | 0.0153 | 0.0740 | 0.0096 | 0.0057 | 0.0202 | 0.0185 | 0.0262 | 0.0123 | 0.0106 | 0.0108 | 0.0203 |
| 21 | 0.1262 | 0.1326 | 0.2214 | 0.1640 | 0.1048 | 0.0019 | 0.1508 | 0.0875 | 0.1631 | 0.1634 | 0.1316 |
| 22 | 0.1351 | 0.4373 | 0.1822 | 0.0614 | 0.1893 | 0.2246 | 0.2579 | 0.2031 | 0.1012 | 0.0962 | 0.1888 |
| 23 | 0.0071 | 0.0175 | 0.0142 | 0.0084 | 0.0141 | 0.0021 | 0.0230 | 0.0056 | 0.0080 | 0.0082 | 0.0108 |
| Related coefficient | 0.9914 | 0.9067 | 0.9753 | 0.9607 | 0.9906 | 0.9182 | 0.9498 | 0.9703 | 0.9705 | 0.9848 | 1.0000 |
| Included angle cosine | 0.9927 | 0.9371 | 0.9833 | 0.9701 | 0.9936 | 0.9448 | 0.9664 | 0.9789 | 0.9794 | 0.9892 | 1.0000 |
Each total peak flight time mass spectrum of table 2. root of large-flowered skullcap finger-print is measured and result of calculation
Claims (7)
1. the assay method of a radix scutellariae medicinal materials UPLC-MS finger-print comprises the steps:
(1) preparation of need testing solution: it is an amount of to take by weighing the radix scutellariae medicinal materials powder, accurately claims surely, places conical flask, with 70% methyl alcohol 20mL ultrasonic Extraction 10～45 minutes, puts to room temperature, filters, and filtrating places the 50mL volumetric flask; Residue is used methyl alcohol 20mL ultrasonic Extraction 10～45 minutes again, puts to room temperature, filters, and residue washs with small amount of methanol, and filtrating is incorporated in the 50mL volumetric flask, and constant volume shakes up, and centrifugal 15 minutes of 13000rpm draws supernatant, as need testing solution;
(2) condition determination: chromatography column is ACQUITY
series of columns; Moving phase is 0.1% formic acid acetonitrile-0.1% formic acid water, adopts the gradient elution mode; Flow velocity: 0.3～0.5mL/min; Column temperature: 40-45 ℃; Sample size is 1～5 μ L.Scanning of the mass spectrum mode: positive ion full scan detecting pattern; Quality of scanning scope: 100-1000mDa;
(3) assay method: draw need testing solution, inject the UPLC-MS combined instrument, measure, write down 18 minutes chromatogram.Setting the karyoplasmic ratio (m/z) at each total peak, set up the extraction chromatography of ions figure at each peak, is 1 with the scutelloside peak area, calculates the relative ratio of each peak area, carries out similarity evaluation.
2. method according to claim 1, wherein, described test sample sampling amount is 0.10-0.50g, 30-45 minute ultrasonic Extraction time.
3. method according to claim 1, wherein, described chromatographic column is ACQUITY
HSST
3Post, flow velocity 0.4mL/min, 40 ℃ of column temperatures, sample size 2 μ L.
4. method according to claim 1, wherein, described chromatographic column is ACQUITY
C
18Post, flow velocity 0.3mL/min, 45 ℃ of column temperatures, sample size 5 μ L.
5. method according to claim 1, wherein said extraction chromatography of ions figure is that the karyoplasmic ratio according to each peak molion extracts from TIC and obtains, the karyoplasmic ratio at 23 total peaks (m/z) is respectively: peak 1, m/z 305; Peak 2, m/z 549; Peak 3, m/z 463; Peak 4, m/z 549; Peak 5, m/z 549; Peak 6, m/z 477; Peak 7, m/z 347; Peak 8, m/z 447; Peak 9, m/z 447; Peak 10, m/z 477; Peak 11, m/z 431; Peak 12, m/z 461; Peak 13, m/z 477; Peak 14, m/z 447; Peak 15, m/z 461; Peak 16, m/z 491; Peak 17, m/z 271; Peak 18, m/z 271; Peak 19, m/z285; Peak 20, m/z 315; Peak 21, m/z 375; Peak 22, m/z 285; Peak 23, m/z 315; Wherein peak 8 is a scutelloside.
6. method according to claim 1 is wherein used flight time mass spectrum and has been measured the accurate mass at each peak in the standard finger-print, calculates the molecular formula at each peak and distinguishes as follows: peak 1, C
15H
12O
7Peak 2, C
26H
28O
13Peak 3, C
21H
18O
12Peak 4, C
26H
28O
13Peak 5, C
26H
28O
13Peak 6, C
22H
20O
12Peak 7, C
17H
14O
8Peak 8, C
21H
18O
11Peak 9, C
21H
18O
11Peak 10, C
22H
20O
12Peak 11, C
21H
18O
10Peak 12, C
22H
20O
11Peak 13, C
22H
20O
12Peak 14, C
21H
18O
11Peak 15, C
22H
20O
11Peak 16, C
23H
22O
12Peak 17, C
15H
10O
5Peak 18, C
15H
10O
5Peak 19, C
16H
12O
5Peak 20, C
17H
14O
6Peak 21, C
19H
18O
8Peak 22, C
16H
12O
5Peak 23, C
17H
14O
6
7. the method for quality control of a radix scutellariae medicinal materials; Be to derive from the meeting of 10 above places of production " radix scutellariae medicinal materials of Chinese pharmacopoeia regulation is by each described assay method record finger-print of claim 1-6; Adopt average method production standard finger-print; Testing sample finger-print and standard finger-print are compared, and similarity is greater than 0.9, thinks the up-to-standard of said product to be measured.
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| CN102735787A (en) * | 2012-06-26 | 2012-10-17 | 承德颈复康药业集团有限公司 | Novel digestion promoting tablet and its relative preparation quality detection method |
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