CN103698427B - Infantile seven-element tea preparation medium-polarity component fingerprint detection method and construction method - Google Patents
Infantile seven-element tea preparation medium-polarity component fingerprint detection method and construction method Download PDFInfo
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Abstract
The invention discloses an infantile seven-element tea preparation medium-polarity component fingerprint detection method and construction method. The detection method comprises the following steps: (1) preparation of a control solution; (2) preparation of a test sample solution; and (3) detection by a high performance liquid chromatograph. The method can effectively detect the quality of the infantile seven-element tea preparation, and is beneficial to monitoring the quality of the product; and the method is simple and stable, has the advantages of high precision and high reproducibility, and can quickly and accurately identify the authenticity and quality of the product.
Description
Technical field
The present invention relates to Chinese medicine preparation method of quality control, particularly relate to detection method and the construction method of Xiao ' erqixingcha preparation medium-polarity component fingerprint.
Background technology
Xiao ' erqixingcha preparation is with Chinese crude drug coix seed, rice bud, hawthorn, lophatherum gracile, yncaria stem with hooks, cicada slough, Radix Glycyrrhizae for raw material, and after water extraction is got, pharmaceutical technology is routinely made.Having appetizing disappears stagnant, effect of heat-clearing arresting convulsion, and for infantile malnutrition heat-transmission, indigestion, do not feel like eating, agitation is easily shied, uneasy sleeping at night, and stool is not smooth, scanty drak urine.Xiao ' erqixingcha preparation has recorded Xiao ' erqixingcha electuary (particle), Xiao ' erqixingcha syrup in Drug Standard of Ministry of Public Health of the Peoples Republic of China Traditional Chinese medicine historical preparation the 9th, is the Chinese medicine preparation that paediatrics is commonly used, deeply welcomes by patient.Now have the formulation list marketings such as Xiao ' erqixingcha oral liquid.
Pharmacopoeia of the People's Republic of China version one in 2010 mainly adopts thin-layered chromatography to differentiate whether contain hawthorn, Radix Glycyrrhizae, adopt the content of its liquiritin of high effective liquid chromatography for measuring.
Zeng Changqing etc. at " Xiao ' erqixingcha electuary quality standard research ", Chinese patent drug, 03 phase in 2006: report and adopt thin-layer chromatography to differentiate Xiao ' erqixingcha electuary rice bud, hawthorn, Radix Glycyrrhizae and yncaria stem with hooks, measure the method for its rhynchophyllin content with RP-HPLC.
Xiao Shuxiong etc. are " Xiao ' erqixingcha syrup quality standard improves research ", Chinese patent drug, 07 phase in 2008, report the indentification by TLC adopting thin-layered chromatography to formulate Xiao ' erqixingcha syrup yncaria stem with hooks, Radix Glycyrrhizae, by the method for high effective liquid chromatography for measuring Radix Glycyrrhizae acid mono-ammonium content.
Li Dong etc. are " HPLC measures quercetin content in Xiao ' erqixingcha electuary ", and Chinese pharmacoeconomics, 06 phase in 2012, reports the method adopting HPLC method to measure quercetin content in Xiao ' erqixingcha electuary.
Above method is all differentiate or assay individual components in Xiao ' erqixingcha preparation, needs to be further improved Xiao ' erqixingcha quality of the pharmaceutical preparations control method.
Summary of the invention
Based on this, be necessary for the problems referred to above, a kind of detection method and construction method of Xiao ' erqixingcha preparation medium-polarity component fingerprint are provided.
Concrete technical scheme is as follows:
A detection method for Xiao ' erqixingcha preparation medium-polarity component fingerprint, adopts high performance liquid chromatography to detect, comprises the following steps:
(1) preparation of reference substance solution:
Liquiritin, chlorogenic acid, apiolin reference substance are respectively got 5mg, is the dissolve with ethanol solution of 72-78% with concentration of volume percent respectively and is settled to 10ml, shaking up, obtain reference substance solution;
(2) preparation of need testing solution:
Get Xiao ' erqixingcha sample, upper macroporous resin column, first use distilled water wash-out, discard water lotion; Be eluted to colourless with the ethanolic solution that concentration of volume percent is 72-78% again, collect ethanol washing lotion; By described ethanol washing lotion reduced pressure concentration, miillpore filter filters, and obtains need testing solution; Described Xiao ' erqixingcha sample be Xiao ' erqixingcha syrup, Xiao ' erqixingcha oral liquid or with distilled water dissolve after Xiao ' erqixingcha particle;
(3) measure:
Reference substance solution, need testing solution described in sample introduction respectively, injects high performance liquid chromatograph and measures.
Wherein in some embodiments, the condition of the high performance liquid chromatograph in described step (3) is: chromatographic column with carbon octadecylsilane base bonded silica gel for filler, mobile phase: concentration of volume percent be 0.1% acetic acid aqueous solution be mobile phase A, acetonitrile is Mobile phase B, adopts gradient elution mode; Flow velocity: 1mL/min; Determined wavelength: 254nm.
Present invention also offers a kind of construction method of Xiao ' erqixingcha preparation medium-polarity component fingerprint, comprise the following steps:
(1) preparation of reference substance solution:
Liquiritin, chlorogenic acid, apiolin reference substance are respectively got 5mg, is the dissolve with ethanol solution of 72-78% with concentration of volume percent respectively and is settled to 10ml, shaking up, obtain reference substance solution;
(2) preparation of need testing solution:
Get Xiao ' erqixingcha sample, upper macroporous resin column, first use distilled water wash-out, discard water lotion; Be eluted to colourless with the ethanolic solution that concentration of volume percent is 72-78% again, collect ethanol washing lotion; By described ethanol washing lotion reduced pressure concentration, miillpore filter filters, and obtains need testing solution; Described Xiao ' erqixingcha sample be Xiao ' erqixingcha syrup, Xiao ' erqixingcha oral liquid or with distilled water dissolve after Xiao ' erqixingcha particle;
(3) reference substance solution, need testing solution described in difference sample introduction, inject high performance liquid chromatograph and measure, build finger-print, obtain the Xiao ' erqixingcha preparation medium-polarity component fingerprint be made up of 3 common characteristic peaks.
Wherein in some embodiments, described step (3) is respectively reference substance solution, need testing solution described in sample introduction, injection high performance liquid chromatograph measures, with liquiritin peak for reference peak, its relative retention time, relative peak area are 1.0, build the Xiao ' erqixingcha preparation medium-polarity component fingerprint obtaining being made up of 3 common characteristic peaks.
Wherein in some embodiments, the condition of the high performance liquid chromatograph in described step (3) is: chromatographic column with carbon octadecylsilane base bonded silica gel for filler, mobile phase: concentration of volume percent be 0.1% acetic acid aqueous solution be mobile phase A, acetonitrile is Mobile phase B, adopts gradient elution mode; Flow velocity: 1mL/min; Determined wavelength: 254nm.
The present invention compared to the advantage of prior art and beneficial effect is:
Inventor is through great many of experiments and the permanent experience accumulated, determine detection method and the construction method of Xiao ' erqixingcha preparation medium-polarity component fingerprint, and the optimized parameter determined in the preparation (as above macroporous resin column, be the ethanolic solution wash-out etc. of 72-78% with concentration of volume percent) of need testing solution in detection method and construction method and whole testing process and building process, the quality of energy Efficient Characterization Xiao ' erqixingcha preparation, is conducive to the quality of monitoring product; Have that method is easy, stable, precision is high, the advantage of favorable reproducibility; Can differentiate that the true and false of product is good and bad quickly and accurately.
Accompanying drawing explanation
Fig. 1 is the original fingerprint collection of illustrative plates of Xiao ' erqixingcha 10 batch sample, and wherein S1-S10 is respectively 10 batches of Xiao ' erqixingcha samples, and horizontal ordinate is retention time, and ordinate is peak height;
Fig. 2 is the HPLC finger-print of Xiao ' erqixingcha sample, wherein, and s1-need testing solution; S2-liquiritin reference substance solution; S3-chlorogenic acid reference substance solution; S4-apiolin reference substance solution, horizontal ordinate is retention time, and ordinate is peak height;
Fig. 3 is the finger-print after Xiao ' erqixingcha 10 batch sample coupling, and wherein S1-S10 is respectively 10 batches of Xiao ' erqixingcha samples, and R is: the reference fingerprint of Xiao ' erqixingcha middle polarity composition, and horizontal ordinate is: retention time, and ordinate is: peak height;
Fig. 4 is the reference fingerprint of Xiao ' erqixingcha middle polarity composition, and horizontal ordinate is: retention time, and ordinate is: peak height.
Embodiment
Below embodiments of the invention are described in detail.
Embodiment 1
The structure of Xiao ' erqixingcha preparation medium-polarity component fingerprint
1 instrument and equipment
Shimadzu LC-20AD high performance liquid chromatograph (Shimadzu Corporation);
Sartorius CP225D analytical balance (d=0.01mg) (German Sartorius company);
Ratavapor R-II Rotary Evaporators (BUCHI Labortechnik AG of Switzerland);
The positive empty pump (Yuhua Instrument Co., Ltd., Gongyi City) of SHZ-D (III) circulating water type;
RTHW electric jacket (Yuhua Instrument Co., Ltd., Gongyi City);
SK-1 rapid mixer (instrument plant of Jiamei of Community of Jin Tan County city);
2 materials and reagent
Xiao ' erqixingcha particulate samples (being provided by Wanglaoji Pharmaceutical Co., Ltd., Guangzhou City);
3 kinds of flavonoids reference substances: liquiritin, chlorogenic acid, apiolin (Nat'l Pharmaceutical & Biological Products Control Institute);
Ethanol (analyzing pure, Tianjin Zhi Yuan chemical reagent company limited);
Anhydrous acetic acid (analyzing pure, Tianjin Zhi Yuan chemical reagent company limited);
Acetonitrile (chromatographically pure, Merk company);
3 methods
The preparation of 3.1 reference substance solution
Liquiritin, chlorogenic acid, apiolin reference substance are respectively got 5mg, accurately weighed, be that the dissolve with ethanol solution of 75% is in 10ml volumetric flask respectively with a small amount of concentration of volume percent, and be settled to scale, shake up, obtain reference substance solution (comprising liquiritin reference substance solution, chlorogenic acid reference substance solution, apiolin reference substance solution).
The pre-service of 3.2AB-8 macroreticular resin
AB-8 macroreticular resin is first washed till clear liquid with the ethanolic solution that concentration of volume percent is 95%, then is washed till without alcohol taste with distilled water.
The preparation of 3.3 need testing solutions
Get Xiao ' erqixingcha sample particle 3g, dissolve (consumption of distilled water is for being enough to dissolve Xiao ' erqixingcha sample particle) with distilled water, upper AB-8 macroporous resin column, first uses 100ml distilled water wash-out, discards water lotion; Be eluted to colourless with the ethanolic solution that concentration of volume percent is 75% again, collect ethanol washing lotion; Above-mentioned ethanol washing lotion is evaporated to 10ml, and miillpore filter (0.45 μm) filters, and obtains Xiao ' erqixingcha need testing solution.
3.4 chromatographic condition
Chromatograph: Shimadzu high performance liquid chromatograph
Chromatographic column:
c18 post (4.6mm × 250mm, 5 μm); Chromatographic column with carbon octadecylsilane base bonded silica gel for filler
Mobile phase: concentration of volume percent is acetic acid aqueous solution (A)-acetonitrile (B) gradient elution of 0.1%
The gradient elution of table 1 mobile phase
Flow velocity: 1ml/min;
Determined wavelength: 254nm;
Determination method: sample introduction reference substance solution, need testing solution 10 μ L respectively, injects high performance liquid chromatograph, measures each chromatogram.
4.110 batch samples have the demarcation at peak
Adopt similarity evaluation software (2004A version) the original HPLC collection of illustrative plates to 10 batch samples to analyze, see Fig. 1, the preparation medium-polarity component fingerprint such as flavonoids have 3, peak.Wherein, No. 2 peaks (liquiritin) are the strongest, degree of separation is high, with No. 2 peaks for reference peak, its relative retention time, relative peak area are 1.0, calculate the preparation medium-polarity component fingerprint such as 3 flavonoidss in 10 batch samples (S1-S10) and have relative retention time (see table 2) and the relative peak area (see table 3) at peak.
Table 2 relative retention time
| P1 | P2（S） | P3 | |
| S1 | 0.5190 | 1.0000 | 1.5613 |
| S2 | 0.5120 | 1.0000 | 1.5562 |
| S3 | 0.5113 | 1.0000 | 1.5507 |
| S4 | 0.5174 | 1.0000 | 1.5432 |
| S5 | 0.5130 | 1.0000 | 1.5506 |
| S6 | 0.5126 | 1.0000 | 1.5575 |
| S7 | 0.5135 | 1.0000 | 1.5449 |
| S8 | 0.5149 | 1.0000 | 1.5604 |
| S9 | 0.5125 | 1.0000 | 1.5527 |
| S10 | 0.5125 | 1.0000 | 1.5527 |
| Average | 0.51387 | 1.0000 | 1.55302 |
| RSD（%） | 0.46 | 0 | 0.37 |
Table 3 relative peak area
| P1 | P2 | P3 | |
| S1 | 0.7491 | 1.0000 | 0.1112 |
| S2 | 0.6099 | 1.0000 | 0.1005 |
| S3 | 0.6663 | 1.0000 | 0.0946 |
| S4 | 0.5847 | 1.0000 | 0.0964 |
| S5 | 0.6553 | 1.0000 | 0.1034 |
| S6 | 0.6745 | 1.0000 | 0.0977 |
| S7 | 0.6908 | 1.0000 | 0.094 |
| S8 | 0.6773 | 1.0000 | 0.0966 |
| S9 | 0.7325 | 1.0000 | 0.1002 |
| S10 | 0.7281 | 1.0000 | 0.1002 |
| Average | 0.67685 | 1.0000 | 0.09948 |
| RSD（%） | 7.34 | 0 | 4.81 |
4.2 sample fingerprint map analyzings
By to middle polarity composition reference substance collection of illustrative plates (liquiritin, chlorogenic acid, apiolin) and need testing solution atlas analysis (see figure 2)s such as 3 kinds of flavonoidss, middle polarity compositions such as having 3 flavonoidss can be recognized in finger-print and have peak, account for 11.86% of total peak area, wherein No. 1 peak is chlorogenic acid, No. 2 peaks are liquiritin, and No. 3 peaks are apiolin.
4.310 batch sample fingerprint similarity is evaluated
Similarity evaluation software (2004A version) is adopted to evaluate 10 batch sample HPLC collection of illustrative plates, with relative area average for contrast, with S9 sample for Similarity Measure is carried out in reference.Before coupling, coupling after and generate reference fingerprint be respectively Fig. 1, Fig. 3, Fig. 4.Similarity Measure is table 4.
More each batch sample and the similarity with reference to collection of illustrative plates (S9), similarity, all between 0.97 ~ 1, illustrates that each batch sample is higher with the similarity with reference to collection of illustrative plates; More each batch sample and the similarity contrasting collection of illustrative plates (Fig. 4), similarity, all between 0.98 ~ 1, illustrates that each batch sample is higher with the similarity contrasting collection of illustrative plates.
Table 4 Similarity Measure
Embodiment 2
Xiao ' erqixingcha preparation medium-polarity component fingerprint is utilized to detect Xiao ' erqixingcha
1 instrument and equipment
With embodiment 1.
2 materials and reagent
Separately get 10 batches of Xiao ' erqixingcha particulate samples (being provided by Wanglaoji Pharmaceutical Co., Ltd., Guangzhou City), other materials and reagent are with embodiment 1.
3 methods
The preparation of 3.1 reference substance solution
With embodiment 1.
The pre-service of 3.2AB-8 macroreticular resin
With embodiment 1.
The preparation of 3.3 need testing solutions
With embodiment 1.
3.4 chromatographic condition
With embodiment 1.
Measure: sample introduction reference substance solution, need testing solution 10 μ L respectively, inject high performance liquid chromatograph, measure each chromatogram.
Above-mentioned detection method is utilized to detect 10 batches of Xiao ' erqixingcha formulation samples respectively, record chromatogram also imports Chinese medicine chromatogram similarity evaluation system software (process of 2004A version), with contrast trace analysis, calculate similarity (see table 5), each batch sample similarity is all greater than 0.9.Show that the similarity of the middle polarity compositions such as Xiao ' erqixingcha flavonoids is higher, can as Xiao ' erqixingcha quality control and evaluation index.
Table 5 Similarity Measure
The present invention adopts HPLC chromatography to establish the fingerprint map analyzing of the middle polarity compositions such as Xiao ' erqixingcha flavonoids, determines the preparation medium-polarity component fingerprint such as 10 batches of Xiao ' erqixingcha flavonoidss.Have the middle polarity composition characteristics such as 3 flavonoidss in finger-print and have peak, be respectively chlorogenic acid, liquiritin, apiolin.
The present invention adopts similarity evaluation software (2004A version) to carry out similarity analysis evaluation to the 10 batches of Xiao ' erqixingcha sample HPLC collection of illustrative plates detected by the inventive method.More each batch sample and the similarity with reference to collection of illustrative plates, similarity, all between 0.97 ~ 1, illustrates that each batch sample is higher with the similarity with reference to collection of illustrative plates; More each batch sample and the similarity contrasting collection of illustrative plates, similarity all (specifically asks for an interview table 4) between 0.98 ~ 1, illustrates that each batch sample is higher with the similarity contrasting collection of illustrative plates.Result shows that the similarity of the middle polarity compositions such as Xiao ' erqixingcha flavonoids is higher, can as Xiao ' erqixingcha quality control and evaluation index.
As from the foregoing, the quality of energy Efficient Characterization Xiao ' erqixingcha preparation of the present invention, is conducive to the quality of monitoring product; Have that method is easy, stable, precision is high, favorable reproducibility; Can differentiate that the true and false of product is good and bad quickly and accurately.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (3)
1. a detection method for Xiao ' erqixingcha preparation medium-polarity component fingerprint, is characterized in that, adopts high performance liquid chromatography to detect, comprises the following steps:
(1) preparation of reference substance solution:
Liquiritin, chlorogenic acid, apiolin reference substance are respectively got 5mg, is the dissolve with ethanol solution of 72-78% with concentration of volume percent respectively and is settled to 10ml, shaking up, obtain reference substance solution;
(2) preparation of need testing solution:
Get Xiao ' erqixingcha sample, upper macroporous resin column, first use distilled water wash-out, discard water lotion; Be eluted to colourless with the ethanolic solution that concentration of volume percent is 72-78% again, collect ethanol washing lotion; By described ethanol washing lotion reduced pressure concentration, miillpore filter filters, and obtains need testing solution; Described Xiao ' erqixingcha sample be Xiao ' erqixingcha syrup, Xiao ' erqixingcha oral liquid or with distilled water dissolve after Xiao ' erqixingcha particle;
(3) measure:
Reference substance solution, need testing solution described in sample introduction respectively, injects high performance liquid chromatograph and measures;
The condition of described high performance liquid chromatograph is: chromatographic column with carbon octadecylsilane base bonded silica gel for filler, mobile phase: concentration of volume percent be 0.1% acetic acid aqueous solution be mobile phase A, acetonitrile is Mobile phase B, adopts gradient elution mode; Flow velocity: 1mL/min; Determined wavelength: 254nm;
The mobile phase condition of described gradient elution is as follows: described mobile phase is made up of mobile phase A and Mobile phase B, wherein, the variation tendency of Mobile phase B is: from 0.01min to 2min, and B is changed to 6% by 0%, be changed to 10% to 20min subsequently, 27min is changed to 13%, 48min and is changed to 18%, 80min and is changed to 40%, 90min is changed to 50%, 105min is changed to 80%, is reduced to 4% again to 115min, maintains 4% to 118min.
2. a construction method for Xiao ' erqixingcha preparation medium-polarity component fingerprint, is characterized in that, comprises the following steps:
(1) preparation of reference substance solution:
Liquiritin, chlorogenic acid, apiolin reference substance are respectively got 5mg, is the dissolve with ethanol solution of 72-78% with concentration of volume percent respectively and is settled to 10ml, shaking up, obtain reference substance solution;
(2) preparation of need testing solution:
Get Xiao ' erqixingcha sample, upper macroporous resin column, first use distilled water wash-out, discard water lotion; Be eluted to colourless with the ethanolic solution that concentration of volume percent is 72-78% again, collect ethanol washing lotion; By described ethanol washing lotion reduced pressure concentration, miillpore filter filters, and obtains need testing solution; Described Xiao ' erqixingcha sample be Xiao ' erqixingcha syrup, Xiao ' erqixingcha oral liquid or with distilled water dissolve after Xiao ' erqixingcha particle;
(3) reference substance solution, need testing solution described in difference sample introduction, inject high performance liquid chromatograph and measure, build finger-print, obtain the Xiao ' erqixingcha preparation medium-polarity component fingerprint be made up of 3 common characteristic peaks;
The condition of described high performance liquid chromatograph is: chromatographic column with carbon octadecylsilane base bonded silica gel for filler, mobile phase: concentration of volume percent be 0.1% acetic acid aqueous solution be mobile phase A, acetonitrile is Mobile phase B, adopts gradient elution mode; Flow velocity: 1mL/min; Determined wavelength: 254nm;
The mobile phase condition of described gradient elution is as follows: described mobile phase is made up of mobile phase A and Mobile phase B, wherein, the variation tendency of Mobile phase B is: from 0.01min to 2min, and B is changed to 6% by 0%, be changed to 10% to 20min subsequently, 27min is changed to 13%, 48min and is changed to 18%, 80min and is changed to 40%, 90min is changed to 50%, 105min is changed to 80%, is reduced to 4% again to 115min, maintains 4% to 118min.
3. the construction method of Xiao ' erqixingcha preparation medium-polarity component fingerprint according to claim 2, it is characterized in that, step (3) is respectively reference substance solution, need testing solution described in sample introduction, injection high performance liquid chromatograph measures, with liquiritin peak for reference peak, its relative retention time, relative peak area are 1.0, build the Xiao ' erqixingcha preparation medium-polarity component fingerprint obtaining being made up of 3 common characteristic peaks.
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