CN106018587A - Quality control method of Yixuean granules - Google Patents
Quality control method of Yixuean granules Download PDFInfo
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- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a quality control method of Yixuean granules. With protocatechuic acid serving as a reference substance, high performance liquid chromatography is adopted for carrying out gradient elution, and a fingerprint spectrum of a common pattern of the Zhuang medicine Blumea riparia material is obtained. The fingerprint spectrum has 21 chromatographic peaks in total and is capable of fully reflecting the chemical components of Yixuean granules and large in information amount, and the method is high in precision, repeatability and stability and has great significance on quality control over Yixuean granules and the clinical effect of Yixuean granules.
Description
Technical field
The present invention relates to drug tests, the method for quality control of her blood peace granule a kind of.
Background technology
Her blood peace granule is a kind of Chinese patent medicine treating gynecologic blood diseases, and Main Ingredients and Appearance is Herba Blumeae Balsamiferae, Herba Leonuri, prolongs
Rhizoma Corydalis, Radix Glycyrrhizae etc., have the effect such as promoting blood circulation and hemostasis, promoting the circulation of QI to relieve pain.Clinically for postpartum lochiorrhea, manually flow
Puerperal, metrorrhagia was clean, differential diagnosis in tcm belongs to syndrome of blood stasis person.
For ensureing the quality of her blood peace granule and the safe and effective of clinical application, the research of its quality standard and foundation are day by day
Important.The quality standard of her existing blood peace granule, the thin layer including Herba Blumeae Balsamiferae, Rhizoma Corydalis, Radix Glycyrrhizae differentiates,
With the content of high effective liquid chromatography for measuring protocatechuic acid, but the method lacks the discriminating to Herba Leonuri, to Rhizoma Corydalis,
The mensuration of Radix Glycyrrhizae is also only limitted to observation measurements.
Summary of the invention
The invention provides the method for quality control of her blood a kind of peace granule, comprise to Herba Blumeae Balsamiferae, Herba Leonuri thin
Layer discrimination method, and the finger printing of her blood peace granule, provide important references for her quality control of blood peace granule.
For achieving the above object, the technical scheme is that
The method of quality control of her blood peace granule a kind of, comprises the following steps:
(1) with protocatechuic acid and protocatechualdehyde for comparison, with chloroform, ethyl acetate, formic acid mixed solution for launching
Agent differentiates the Herba Blumeae Balsamiferae composition in her blood peace granule；
(2) with stachydrine hydrochloride for comparison, her is differentiated with acetone, ethyl acetate, hydrochloric acid mixed solution for developing solvent
Herba Leonuri composition in blood peace granule；
(3) with protocatechuic acid as object of reference, her blood peace granule extracting solution is detected by high performance liquid chromatography, it is thus achieved that her blood
The finger printing of peace granule；Wherein, the extraction process of her blood peace granule extracting solution: take her blood peace granule, be dissolved in water,
Add dilute hydrochloric acid regulation pH, stir evenly, stand, filter, add water saturation n-butanol extraction 3～6 times, point take out n-butyl alcohol
Extracting solution, merges n-butanol extracting liquid, stands, and divides and takes acquisition n-butyl alcohol liquid, reclaims n-butyl alcohol, and it is molten that residue adds methanol
Solve, centrifugal, take supernatant, obtain her blood peace granule extracting solution.Regulation pH dilute hydrochloric acid be mass fraction be 9.5%～10.5%
Hydrochloric acid.
Further, the concrete operation method of described step (1) is:
(11) Herba Blumeae Balsamiferae thin layer differentiates the preparation of need testing solution: takes her blood peace granule, adds water, make Yi Xuean
Grain dissolution, adds dilute hydrochloric acid, stirs, and filters, and add diethyl ether extraction 2～3 times, it is thus achieved that ether extracted liquid, merges
Ether extracted liquid, adds water after washing 2 times, discards water liquid, divides and takes ether extracted liquid, is evaporated, and residue adds methanol and is allowed to
Dissolve, differentiate need testing solution as Herba Blumeae Balsamiferae thin layer；Wherein, dilute hydrochloric acid be mass fraction be 9.5%～10.5%
Hydrochloric acid；
(12) protocatechuic acid reference substance solution: protocatechuic acid reference substance adds methanol and makes protocatechuic acid reference substance solution；
(13) protocatechualdehyde reference substance solution: protocatechualdehyde reference substance adds methanol and makes protocatechualdehyde reference substance solution；
(14) take above-mentioned Herba Blumeae Balsamiferae thin layer and differentiate need testing solution, protocatechuic acid reference substance solution, protocatechualdehyde
Reference substance solution point is on same silica gel thin-layer plate, and the chloroform with volume ratio as 6:3:5, ethyl acetate, formic acid mix
Solution is that developing solvent launches, and takes out, dries, inspect under uviol lamp.
Further, the concrete operation method of described step (2) is:
(21) Herba Leonuri thin layer differentiates the preparation of need testing solution: taking her blood peace granule, add dehydrated alcohol, shaking carries
Taking, filter, the filtrate obtained is evaporated, and residue adds anhydrous alcohol solution, differentiates test sample as Herba Leonuri thin layer
Solution；
(22) stachydrine hydrochloride reference substance solution: stachydrine hydrochloride reference substance adds dehydrated alcohol and makes stachydrine hydrochloride pair
According to product solution；
(23) take above-mentioned Herba Leonuri thin layer and differentiate need testing solution, stachydrine hydrochloride reference substance solution, put in same silicon
On glue lamellae, the acetone with volume ratio as 6:6:1, ethyl acetate, hydrochloric acid mixed solution launch for developing solvent, take
Going out, dry, spray is to improve bismuth potassium iodide test solution, and heating makes spot development clear, and Herba Leonuri thin layer differentiates that test sample is molten
Liquid chromatography, with on the corresponding position of stachydrine hydrochloride reference substance solution chromatograph, shows the speckle of same color.
Further, the concrete operation method of described step (3) is:
(31) preparation of object of reference solution: protocatechuic acid standard substance, adds methanol constant volume, prepares object of reference solution.
(32) preparation of her blood peace granule extracting solution；
(33) chromatographic condition of high performance liquid chromatograph:
Flowing phase: methanol-0.1% glacial acetic acid；
Gradient elution program: time gradient
0min→11min→16min→29min→30min→35min→45min→55min→70min→95min；Corresponding
Gradient eluent form than by mobile phase A methanol and Mobile phase B 0.1% glacial acetic acid by volumes below, mobile phase A
Percentage by volume gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70
%, the percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55 of Mobile phase B
% → 55% → 30%；
Ultraviolet detection wavelength: 256nm；
Flow velocity: 1.0mL min-1；
Column temperature: 25 DEG C.
(34) measure: draw her blood peace granule extracting solution and inject high performance liquid chromatograph, according to the color of step (33)
Spectral condition measures, and obtains finger printing.
Preferably, the concrete operation method that her blood peace granule extracting solution described extracts is: weigh her blood peace granule 3～8g,
The 50mL that adds water dissolves, and adds dilute hydrochloric acid 0.2～0.4mL, stirs evenly, and stands 15～30min, filters, adds the positive fourth of water saturation
Alcohol extracts 5～6 times, each 15～20mL, divides and takes n-butanol extracting liquid, merges, and stands 1～1.5h, divides and takes acquisition
N-butyl alcohol liquid, reclaims n-butyl alcohol, and residue adds methanol 4～6mL, centrifugal, takes supernatant, obtains her blood peace granule and extracts
Liquid.It is furthermore preferred that the concrete operation method that her blood peace granule extracting solution described extracts is: weigh her blood peace granule 5g,
The 50mL that adds water dissolves, and adds dilute hydrochloric acid 0.3mL, stirs evenly, and stands 20min, filters, adds water saturation n-butanol extraction
5 times, each 15mL, divide and take n-butanol extracting liquid, merge, stand 1h, divide and take acquisition n-butyl alcohol liquid, recovery is just
Butanol, residue adds methanol 5mL, centrifugal, takes supernatant, obtains her blood peace granule extracting solution.
Further, described finger printing has chromatographic peak 21, and its Average residence time is respectively as follows:
No. 1 peak 3.234min, No. 2 peak 5.962min, No. 3 peak 11.023min, No. 4 peak 15.353min, No. 5 peaks
16.521min, No. 6 peak 24.498min, No. 7 peak 27.158min, No. 8 peak 30.877min, No. 9 peak 32.29min,
No. 10 peak 34.752min, No. 11 peak 36.182min, No. 12 peak 37.968min, No. 13 peak 38.708min, 14
Number peak 56.509min, No. 15 peak 58.989min, No. 16 peak 59.670min, No. 17 peak 66.895min, No. 18
Peak 68.358min, No. 19 peak 69.319min, No. 20 peak 85.217min, No. 21 peak 91.016min.Further
, her component of blood peace granule described includes Herba Blumeae Balsamiferae, Herba Leonuri, Rhizoma Corydalis and Radix Glycyrrhizae, wherein No. 1 peak～21
Number peak derives from Herba Blumeae Balsamiferae, and No. 9 peaks derive from Herba Leonuri, and 10, No. 11 peaks derive from Rhizoma Corydalis, 6,14,
15, No. 16 peaks derive from Radix Glycyrrhizae.
Preferably, in described step (33), chromatographic column be Phenomenex Gemini C18 post (4.6mm × 250mm,
5 μm), guard column is Phenomenex C18 (4 × 3.0mm).
Preferably, in described step (33), theoretical cam curve, in terms of protocatechuic acid peak, is not less than 5000.
The method of quality control of her blood peace granule above-described, optimizes the indentification by TLC side of Herba Blumeae Balsamiferae
Method, supplements the TLC Identification of Herba Leonuri, determines the fingerprint of her blood peace granule simultaneously.
In the fingerprint of her blood peace granule, with protocatechuic acid as object of reference, by high performance liquid chromatograph, adopt
It is that flowing carries out gradient elution mutually with methanol-0.1% glacial acetic acid, it is thus achieved that finger printing, total chromatograph in finger printing
21, peak, wherein 21 total peaks derive from Herba Blumeae Balsamiferae, and 1 total peak derives from Herba Leonuri, and 2 have
Peak derives from Rhizoma Corydalis, and 4 total peaks derive from Radix Glycyrrhizae, can fully react the change of four kinds of crude drug in her blood peace granule
Study point, can be used for the quality of four kinds of crude drug of the overall evaluation and differentiate the quality of four kinds of crude drug, for controlling Yi Xuean
The quality of granule provides strong theoretical foundation.The present invention uses the side that indentification by TLC and finger printing combine
Method, quickly analyzes her blood peace granule, method repeatability, good stability, the quality control to her blood peace granule
Significant with clinical efficacy.
Accompanying drawing explanation
Fig. 1 is the chromatogram using Welchrom C18 post (4.6mm × 250mm, 5 μm) to obtain in embodiment 1；
Fig. 2 is the chromatogram using Hypersil C18 post (4.6mm × 250mm, 5 μm) to obtain in embodiment 1；
Fig. 3 is the color using Phenomenex Gemini C18 post (4.6mm × 250mm, 5 μm) to obtain in embodiment 1
Spectrogram；
Fig. 4 is that in embodiment 1, employing methanol-water is the chromatogram that flowing obtains mutually；
Fig. 5 is that in embodiment 1, employing acetonitrile-water is the chromatogram that flowing obtains mutually；
Fig. 6 is that in embodiment 1, employing methanol-0.1% phosphoric acid is the chromatogram that flowing obtains mutually；
Fig. 7 is that in embodiment 1, employing methanol-0.05% glacial acetic acid is the chromatogram that flowing obtains mutually；
Fig. 8 is that in embodiment 1, employing methanol-0.1% glacial acetic acid is the chromatogram that flowing obtains mutually；
Fig. 9 is that in embodiment 1, employing acetonitrile-0.1% glacial acetic acid is the chromatogram that flowing obtains mutually；
Figure 10 is the chromatogram that the embodiment 1 a length of 230nm of medium wave obtains；
Figure 11 is the chromatogram that the embodiment 1 a length of 256nm of medium wave obtains；
Figure 12 is the chromatogram that the embodiment 1 a length of 280nm of medium wave (0～20min)-279nm (20～30min) obtains；
Figure 13 is the chromatogram that the embodiment 1 a length of 320nm of medium wave obtains；
Figure 14 is the chromatogram that the embodiment 1 a length of 360nm of medium wave obtains；
Figure 15 is that in embodiment 1, column temperature is 20 DEG C of chromatograms obtained；
Figure 16 is that in embodiment 1, column temperature is 25 DEG C of chromatograms obtained；
Figure 17 is that in embodiment 1, column temperature is 30 DEG C of chromatograms obtained；
Figure 18 is that in embodiment 1, column temperature is 35 DEG C of chromatograms obtained；
Figure 19 is that in embodiment 1, flow velocity is 0.8mL min-1The chromatogram obtained；
Figure 20 is that in embodiment 1, flow velocity is 1.0mL min-1The chromatogram obtained；
Figure 21 is that in embodiment 1, flow velocity is 1.2mL min-1The chromatogram obtained；
Figure 22 is to detect, after Extraction solvent extraction, the chromatogram obtained with water in embodiment 2；
Figure 23 is to detect, after Extraction solvent extraction, the chromatogram obtained with methanol in embodiment 2；
Figure 24 is to detect, after Extraction solvent extraction, the chromatogram obtained with ethanol in embodiment 2；
Figure 25 is to detect, after extractant extraction, the chromatogram obtained with water-saturated n-butanol in embodiment 2；
Figure 26 is to detect, after extractant extraction, the chromatogram obtained with chloroform in embodiment 2；
Figure 27 is to detect, after extractant extraction, the chromatogram obtained with ether in embodiment 2；
Figure 28 is to detect, after extractant extraction, the chromatogram obtained with ethyl acetate in embodiment 2；
Figure 29 is the finger printing of her blood peace granule in embodiment 3；
Figure 30 is the chromatogram of object of reference in embodiment 3；
Figure 31 is herba blumea riparia chromatogram in embodiment 3；
Figure 32 is motherwort medicinal wood color spectrogram in embodiment 3；
Figure 33 is corydalis tuber medicinal material chromatogram in embodiment 3；
Figure 34 is licorice medicinal materials chromatogram in embodiment 3；
Figure 35 is the chromatogram that Herba Blumeae Balsamiferae TLC Identification obtains；
Figure 36 is the chromatogram that Herba Leonuri TLC Identification obtains；
Figure 37 is the coupling figure of her blood peace granule of 10 lot numbers in embodiment 5.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, but protection scope of the present invention is not limited to following reality
Execute example:
The experiment material of following example, instrument and reagent prepare as follows:
Experiment material: protocatechuic acid standard substance (Nat'l Pharmaceutical & Biological Products Control Institute, for assay, lot number:
110809-200503)。
Instrument: Agilent Agilent1260 high performance liquid chromatograph, be contained in line vacuum degassing machine (G-1311C),
Quaternary gradient pump (G-1311C), standard automatic sampler (G-1329B), intelligent column oven (G-1316A),
(U.S.'s Agilent Technologies is limited for variable-wavelenght detector (G-1314B), Agilent1260Infinity chromatographic work station
Company)；SB3200T ultrasonic washing instrument (ultrasonic company limited must be believed in Shanghai)；Millipore Simplicity-185
Ultra-pure water instrument (Mi Libo company of the U.S.)；LG16-W high speed micro centrifuge (Beijing Medical Centrifugal Machine Factory)；BT224S
Electronic analytical balance (Beijing Sai Duolisi instrument system company limited).
Reagent: methanol, acetonitrile (chromatographically pure, Fisher Scientific company of the U.S.)；Glacial acetic acid (HPLC, sky
Ke Miou chemical reagent company limited of Jinshi City)；Water is ultra-pure water；Other reagent is analytical pure.
Embodiment 1 finger printing: the optimization of chromatographic condition
The preparation method of her blood peace granule extracting solution: weigh she blood peace granule 5g, the 50mL that adds water and dissolve, add dilute salt
Acid 0.3mL, stirs evenly, and stands 20min, filters, adds water saturation n-butanol extraction 5 times, and each 15mL divides and takes
N-butanol extracting liquid, merges, and stands 1h, divides and takes acquisition n-butyl alcohol liquid, reclaims n-butyl alcohol, and residue adds methanol 5mL,
Centrifugal, take supernatant, obtain her blood peace granule extracting solution.
The selection of 1-1 chromatographic column
Other condition determinations of high performance liquid chromatograph are identical, investigated Welchrom C18 post (4.6mm × 250mm,
5 μm), Hypersil C18 post (4.6mm × 250mm, 5 μm) and Phenomenex Gemini C18 post
(4.6mm × 250mm, 5 μm) three kinds of chromatographic columns, obtained chromatogram is divided into as shown in Figure 1, Figure 2, Figure 3 shows,
As seen from Figure 3, Phenomenex Gemini C18 post (4.6mm × 250mm, 5 μm) separating degree is best,
Peak shape is symmetrical and sharp-pointed, and baseline is more steady.
The selection of 1-2 flowing phase
Other condition determinations of high performance liquid chromatograph are identical, investigated methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid,
Methanol-0.05% glacial acetic acid, methanol-0.1% glacial acetic acid, acetonitrile-0.1% glacial acetic acid carry out gradient elution mutually for flowing, institute
The chromatogram obtained respectively as shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, through comparing it can be seen that with
Methanol-0.1% glacial acetic acid system is that the chromatogram absworption peak of flowing phase separates preferably, and baseline is more steady.
The selection of 1-3 detection wavelength
Other condition determinations of high performance liquid chromatograph are identical, investigated 230nm, 256nm, 280nm, 320nm,
Chromatogram under 360nm absorbing wavelength, chromatogram respectively as shown in Figure 10, Figure 11, Figure 12, Figure 13 and Figure 14,
Through comparing it can be seen that at 256nm chromatogram show more peak information, and signal is preferable, gained chromatograph
In figure, each peak shape is preferable, baseline is steady.
The selection of 1-4 column temperature
Other condition determinations of high performance liquid chromatograph are identical, investigated 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C different
Column temperature, chromatogram is respectively as shown in Figure 15, Figure 16, Figure 17 and Figure 18, through comparing it can be seen that each when 25 DEG C
Peak separating effect is best.
The selection of 1-5 flow velocity
Other condition determinations of high performance liquid chromatograph are identical, investigated 0.8mL min-1、1.0mL·min-1、
1.2mL·min-1Different in flow rate, chromatogram respectively as shown in Figure 19, Figure 20 and Figure 21, through comparing it can be seen that
Flow velocity is 1.0mL min-1Time each peak separating effect best.
Embodiment 2 finger printing: the optimization of sample-pretreating method
The chromatographic condition of high performance liquid chromatograph: chromatographic column uses Phenomenex Gemini C18 post
(4.6mm × 250mm, 5 μm)；Guard column uses Phenomenex C18 (4 × 3.0mm)；Flowing phase: methanol
-0.1% glacial acetic acid；Gradient elution program: time gradient
0min→11min→16min→29min→30min→35min→45min→55min→70min→95min；Corresponding
Gradient eluent form than by mobile phase A methanol and Mobile phase B 0.1% glacial acetic acid by volumes below, mobile phase A
Percentage by volume gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70
%, the percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55 of Mobile phase B
% → 55% → 30%；；Detection wavelength: 256nm；Flow velocity: 1.0mL min-1；Column temperature: 25 DEG C；Sample size:
5μL。
The selection of 2-1 Extraction solvent
Weigh her blood peace granule 5g, select water, methanol and three kinds of Extraction solvent of ethanol, use identical extracting method
Her blood peace granule is extracted, has extracted after same extraction, concentration, dissolution process, by efficient liquid phase
Chromatograph detects, obtained chromatogram respectively as shown in Figure 22, Figure 23, Figure 24, through comparing it can be seen that
During with water for Extraction solvent, showing more peak information, the active substance of sample is completely dissolved, and peak separating degree is good.
The selection of 2-2 extractant
Weigh her blood peace granule 5g, after obtaining extracting solution with water for Extraction solvent, select water-saturated n-butanol, chloroform,
Ether, ethyl acetate are that extractant extracts, and extracting process is identical, has extracted through same concentration, dissolving
After process, being detected by high performance liquid chromatograph, obtained chromatogram is respectively such as Figure 25, Figure 26, Figure 27
Shown in Figure 28, through comparing it can be seen that the sample of chloroform extraction, peak information is little, the sample of ether extraction,
Peak information is less and peak area is less, and ethyl acetate is the most similar to water-saturated n-butanol extraction gained collection of illustrative plates, but face, peak
Long-pending less, and it is easier to emulsifying during ethyl acetate extraction, therefore water-saturated n-butanol effect of extracting is optimal.
The selection of 2-3 extraction times
Weigh her blood peace granule 5g, with water as Extraction solvent, extract for extractant with water-saturated n-butanol, respectively
Investigated extraction 3 times, 4 times, 5 times, 6 times, other extracting process are identical, extracted through same concentration,
After dissolution process, being detected by high performance liquid chromatograph, experiment shows the former catechu extracting 5 times with extracting 6 times
Acid on peak area without larger difference；It is indicated above that extraction 5 times the most substantially can be her blood peace granule water soluble ingredient
Extract completely, therefore extraction is advisable with 5 times.
The selection of 2-4 extraction quantity
Weigh her blood peace granule 5g, with water as Extraction solvent, extract 5 times for extractant with water-saturated n-butanol,
Other extracting process are identical, investigate extraction quantity and are respectively the effect of extracting of 10mL, 15mL, 20mL, and extraction completes
After same concentration, dissolution process, being detected by high performance liquid chromatograph, experiment shows that extraction quantity is 15mL
With the protocatechuic acid that extraction quantity is 20mL on peak area without larger difference.It is indicated above that extraction quantity be 15mL
Substantially her blood peace granule water soluble ingredient can be extracted completely.
The quality testing of her blood peace granule of embodiment 3
The preparation of 3-1 object of reference solution: weigh protocatechuic acid standard substance 0.81mg and put in 10mL volumetric flask, add methanol
Make dissolving in right amount and be settled to scale, shaking up, obtaining the protocatechuic acid standard solution that concentration is 0.081mg mL-1
The preparation of her blood peace granule extracting solution of 3-2: weigh she blood peace granule 5g, the 50mL that adds water and dissolve, add dilute hydrochloric acid
0.3mL, stirs evenly, and stands 20min, filters, adds water saturation n-butanol extraction 5 times, and each 15mL divides and takes positive fourth
Alcohol extract, merges, and stands 1h, divides and takes acquisition n-butyl alcohol liquid, reclaims n-butyl alcohol, and residue adds methanol 5mL, from
The heart, takes supernatant, obtains her blood peace granule extracting solution.
The chromatographic condition of 3-3 high performance liquid chromatograph:
Chromatographic column: Phenomenex Gemini C18 post (4.6mm × 250mm, 5 μm)；
Guard column: Phenomenex C18 (4 × 3.0mm)；
Flowing phase: methanol-0.1% glacial acetic acid；
Gradient elution program: time gradient
0min→11min→16min→29min→30min→35min→45min→55min→70min→95min；Corresponding
Gradient eluent form than by mobile phase A methanol and Mobile phase B 0.1% glacial acetic acid by volumes below, mobile phase A
Percentage by volume gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70
%, the percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55 of Mobile phase B
% → 55% → 30%；
Ultraviolet detection wavelength: 256nm；
Flow velocity: 1.0mL min-1；
Column temperature: 25 DEG C.
The mensuration of her blood peace granule extracting solution of 3-4: draw she blood peace granule extracting solution 10 μ L and inject high performance liquid chromatography
Instrument, measures according to the chromatographic condition of step 3-3, obtains finger printing, as shown in figure 29.By " Chinese medicine chromatograph
Fingerprint similarity evaluates system A version " software, the relevant parameter of finger printing is carried out Auto-matching, demarcates
She has peak by 21 fingerprints of blood peace granule, and its Average residence time is respectively as follows: No. 1 peak 3.234min, No. 2 peaks
5.962min, No. 3 peak 11.023min, No. 4 peak 15.353min, No. 5 peak 16.521min, No. 6 peak 24.498min,
No. 7 peak 27.158min, No. 8 peak 30.877min, No. 9 peak 32.29min, No. 10 peak 34.752min, No. 11 peaks
36.182min, No. 12 peak 37.968min, No. 13 peak 38.708min, No. 14 peak 56.509min, No. 15 peaks
58.989min, No. 16 peak 59.670min, No. 17 peak 66.895min, No. 18 peak 68.358min, No. 19 peaks
69.319min, No. 20 peak 85.217min, No. 21 peak 91.016min.
The mensuration of 3-5 object of reference solution；Accurate 5 μ L object of reference solution of drawing inject high performance liquid chromatograph, according to step
The chromatographic condition of rapid 3-3 measures, and obtains the collection of illustrative plates of object of reference, as shown in figure 30, protocatechuic acid chromatographic peak and test sample
In finger printing, the retention time at No. 5 peaks is consistent, as with reference to peak.
3-6 fingerprint has the demarcation at peak
Press 3-2 respectively and make Herba Blumeae Balsamiferae, Herba Leonuri, Rhizoma Corydalis and the need testing solution of licorice medicinal materials, by 3-3
Chromatographic condition sample introduction is analyzed, and assert total peak according to each chromatographic peak retention time and ultra-violet absorption spectrum feature,
Shown in Figure 31, No. 1 peak～No. 21 peaks derive from Herba Blumeae Balsamiferae, and shown in Figure 32, No. 9 peaks derive from
Herba Leonuri, shown in Figure 33,10, No. 11 peaks derive from Rhizoma Corydalis, shown in Figure 34,6,14,15,
No. 16 peaks derive from Radix Glycyrrhizae.
The TLC Identification of 3-7 Herba Blumeae Balsamiferae:
Herba Blumeae Balsamiferae thin layer differentiates the preparation of need testing solution: her blood taking three lot numbers pacifies each 5g of granule, adds water
30mL, makes her blood peace grain dissolution, adds dilute hydrochloric acid 0.25mL, stir, filter, and add diethyl ether extraction 3 times,
Every time 15mL, merges ether extracted liquid, and add water washing 2 times, and each 15mL discards water liquid, point takes ether and carries
Taking liquid, be evaporated, residue adds methanol 1mL and is allowed to dissolve, and differentiates need testing solution as Herba Blumeae Balsamiferae thin layer；
Protocatechuic acid reference substance solution: protocatechuic acid reference substance adds methanol and makes 0.30mg mL-1 solution；
Protocatechualdehyde reference substance solution: protocatechualdehyde reference substance adds methanol and makes 0.30mg mL-1 solution；
Negative control solution: her the blood peace particulate samples of feminine gender taking scarce Herba Blumeae Balsamiferae is differentiated to supply by Herba Blumeae Balsamiferae thin layer
Test sample solution preparation method is made in the same way of negative control solution.
Test according to thin layer chromatography (Chinese Pharmacopoeia one annex VI B of version in 2010), draw above-mentioned Herba Blumeae Balsamiferae
Thin layer differentiates that need testing solution, protocatechuic acid reference substance solution, each 15 μ L points of protocatechualdehyde reference substance solution are in same
On silica gel thin-layer plate, the chloroform with volume ratio as 6:3:5, ethyl acetate, formic acid mixed solution launch for developing solvent,
It is placed in the pre-saturated expansion cylinder of developing solvent, launches, take out, dry, inspect under ultra-violet lamp (254nm),
Inspecting result as shown in figure 35, A is the negative control solution of 3-7 item, and B is protocatechuic acid reference substance solution, C
For protocatechualdehyde reference substance solution, D, E, F are that the Herba Blumeae Balsamiferae thin layer of three lot numbers differentiates need testing solution,
Visible Herba Blumeae Balsamiferae thin layer differentiate need testing solution chromatograph with protocatechuic acid reference substance, protocatechualdehyde reference substance chromatograph
On relevant position, show the speckle of same color, and good separating effect, favorable reproducibility.
The TLC Identification of 3-8 Herba Leonuri:
Herba Leonuri thin layer differentiates the preparation of need testing solution: her blood taking three lot numbers pacifies each 15g of granule, is ground to
100～300 mesh, add dehydrated alcohol 50ml, and shaking is extracted 30 minutes, filters, and filtrate is evaporated, and residue adds anhydrous
Ethanol 1ml makes dissolving, differentiates need testing solution as Herba Leonuri thin layer；
The preparation of stachydrine hydrochloride reference substance solution: every 1ml anhydrous alcohol solution stachydrine hydrochloride 0.1mg makes hydrochloric acid
Stachydrine reference substance solution；
The preparation of negative control sample solution: her blood taking scarce Herba Leonuri pacifies granule, differentiates for examination according to Herba Leonuri thin layer
The preparation method of product solution carries out preparing negative control sample solution.
Test according to thin layer chromatography (Chinese Pharmacopoeia one annex VI B of version in 2010), take above-mentioned Herba Leonuri thin layer mirror
The each 10 μ L of other need testing solution, stachydrine hydrochloride reference substance solution, put on same silica gel thin-layer plate, with volume ratio
It is that developing solvent launches for the acetone of 6:6:1, ethyl acetate, hydrochloric acid mixed solution, is placed in the pre-saturated expansion of developing solvent
In cylinder, taking out, dry, spray is to improve bismuth potassium iodide test solution, and heating makes spot development clear, as shown in figure 36,
G, H, I are that the Herba Leonuri thin layer of three batches of the present embodiment differentiates need testing solution, and J is the hydrochloric acid of the present embodiment
Stachydrine reference substance solution, K is the negative control sample solution of 3-8 item, and Herba Leonuri thin layer differentiates need testing solution color
Compose with on the corresponding position of stachydrine hydrochloride reference substance solution chromatograph, show the speckle of same color, and separating effect
Good, favorable reproducibility.
The methodological study of embodiment 4 fingerprint
The preparation of 4-1 object of reference solution: weigh protocatechuic acid standard substance 0.81mg and put in 10mL volumetric flask, add methanol
Make dissolving in right amount and be settled to scale, shaking up, obtaining the protocatechuic acid standard solution that concentration is 0.081mg mL-1
The preparation of her blood peace granule extracting solution of 4-2: weigh she blood peace granule 5g, the 50mL that adds water and dissolve, add dilute hydrochloric acid
0.3mL, stirs evenly, and stands 20min, filters, adds water saturation n-butanol extraction 5 times, and each 15mL divides and takes positive fourth
Alcohol extract, merges, and stands 1h, divides and takes acquisition n-butyl alcohol liquid, reclaims n-butyl alcohol, and residue adds methanol 5mL, from
The heart, takes supernatant, obtains her blood peace granule extracting solution.
The chromatographic condition of 4-3 high performance liquid chromatograph:
Chromatographic column: Phenomenex Gemini C18 post (4.6mm × 250mm, 5 μm)；
Guard column: Phenomenex C18 (4 × 3.0mm)；
Flowing phase: methanol-0.1% glacial acetic acid；
Gradient elution program: time gradient
0min→11min→16min→29min→30min→35min→45min→55min→70min→95min；Corresponding
Gradient eluent form than by mobile phase A methanol and Mobile phase B 0.1% glacial acetic acid by volumes below, mobile phase A
Percentage by volume gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70
%, the percentage by volume gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55 of Mobile phase B
% → 55% → 30%；
Ultraviolet detection wavelength: 256nm；
Flow velocity: 1.0mL min-1；
Column temperature: 25 DEG C.
4-4 Precision Experiment
Take her blood peace granule (lot number 120303), prepare her blood same by the preparation of her the blood peace granule extracting solution of 4-2
Peace granule extracting solution, is measured by the chromatographic condition of 4-3, continuous sample introduction 6 times, records chromatogram, investigates the essence of instrument
Density.The results are shown in Table 1, table 2, result shows that the relative retention time RSD value of each chromatographic peak is 0.02%～1.13%
Between, each chromatographic peak relative peak area RSD value, between 1.02%～2.92%, shows that the precision of instrument is good.
Her blood of table 1 peace granule finger printing Precision Experiment-relative retention time
Her blood of table 2 peace granule finger printing Precision Experiment-relative peak area
4-5 repeated experiment
Take 6 parts of her blood peace granule (lot number 120303), by the preparation of her the blood peace granule extracting solution of 4-2 prepare same she
Blood peace granule extracting solution, is measured by the chromatographic condition of 4-3, records chromatogram, investigate the repeatability of test method.Knot
Fruit is shown in Table 3, in 4,6 parts of samples of table the relative retention time RSD value of each chromatographic peak between 1.03%～1.64%, colors
Spectral peak relative peak area RSD value, between 1.04%～3.09%, shows that the repeatability of experimental technique is good.
Her blood of table 3 peace granule finger printing repeated experiment-relative retention time
Her blood of table 4 peace granule finger printing repeated experiment-relative peak area
4-6 stability experiment
Take her blood peace granule (lot number 120303), prepare her blood same by the preparation of her the blood peace granule extracting solution of 4-2
Peace granule extracting solution, is measured by the chromatographic condition of 4-3, respectively 0,2,4,8,16,24h detect, examine
Examine the stability of her blood peace granule extracting solution.The results are shown in Table 5, table 6, the relative retention time RSD value of each chromatographic peak
Between 0.07%～1.76%, the relative peak area RSD value of each chromatographic peak, between 1.45%～3.11%, shows her
Blood peace granule extracting solution 24h internal stability is good.
Her blood of table 5 peace granule finger printing stability experiment-relative retention time
Her blood of table 6 peace granule finger printing stability experiment-relative peak area
The collection of her blood peace granule finger printing of embodiment 5 10 batch
Her blood taking 10 lot numbers pacifies granule, prepares by her preparation of blood peace granule extracting solution of 4-2 in embodiment 4
Her blood same pacifies granule extracting solution, measures by the chromatographic condition of 4-3, records her blood of 10 lot numbers and pacifies granule HPLC
Chromatogram, with " technology of Chinese medicine finger printing research requires (provisional) " regulation, sets up her blood peace granule
Finger printing, her the coupling figure of blood peace granule of 10 lot numbers is shown in Figure 37.
Similarity evaluation: use the similarity evaluation software that Chinese Pharmacopoeia committee is recommended
2004A version, carries out overall merit to HPLC collection of illustrative plates, and similarity evaluation the results are shown in Table 7.
Her blood of table 7 peace granule HPLC fingerprint similarity result of calculation
Determine with reference to peak (S) by the method for 3-5 in embodiment 3, using with reference to the retention time at peak and peak area as 1,
Calculate fingerprint respectively and have a relative retention time at peak and relative peak area is shown in Table 8, table 9.
Her blood of 8 10 lot numbers of table is pacified particulate chromatography figure and is had peak relative retention time
Her blood of 9 10 lot numbers of table is pacified particulate chromatography figure and is had peak relative peak area
Experimental data shows, it is the most higher that test sample has peak relative peak area RSD% value, and 10 batches of her blood peaces
The similarity of grain HPLC finger printing, the most only more than 0.82, is not up to " the technology of Chinese medicine finger printing research
Require (provisional) " regulation in more than 0.9 requirement.It is probably raw medicinal material to receive region, weather, environment, adopt
Between the time receiving, the impact of storage etc. or product formulation technique is unstable etc. that reason causes each constituent content proportional difference.Should
Study its influence factor, standardized planting further, keep quality of medicinal material stable, strengthen product quality in production technology
Control, be just effective to ensure that the steady quality of preparation.
Claims (10)
1. the method for quality control of her a blood peace granule, it is characterised in that comprise the following steps:
(1) with protocatechuic acid and protocatechualdehyde for comparison, her blood is differentiated with chloroform, ethyl acetate, formic acid mixed solution for developing solvent
Herba Blumeae Balsamiferae composition in peace granule；
(2) with stachydrine hydrochloride for comparison, differentiate in her blood peace granule with acetone, ethyl acetate, hydrochloric acid mixed solution for developing solvent
Herba Leonuri composition；
(3) with protocatechuic acid as object of reference, her blood peace granule extracting solution is detected by high performance liquid chromatography, it is thus achieved that the finger of her blood peace granule
Stricture of vagina collection of illustrative plates；Wherein, the extraction process of her blood peace granule extracting solution: take her blood peace granule, be dissolved in water, add dilute hydrochloric acid regulation pH,
Stir evenly, stand, filter, add water saturation n-butanol extraction 3～6 times, point take out n-butanol extracting liquid, merge n-butanol extracting liquid,
Standing, divide and take acquisition n-butyl alcohol liquid, reclaim n-butyl alcohol, residue adds methanol and dissolves, centrifugal, takes supernatant, obtains her blood and pacifies granule
Extracting solution.
The method of quality control of her blood peace granule the most according to claim 1, it is characterised in that:
The concrete operation method of described step (1) is:
(11) Herba Blumeae Balsamiferae thin layer differentiates the preparation of need testing solution: takes her blood peace granule, adds water, and makes her blood peace grain dissolution,
Adding dilute hydrochloric acid, stir, filter, add diethyl ether extraction 2～3 times, it is thus achieved that ether extracted liquid, merges ether extracted liquid, adds water
After washing 2 times, discarding water liquid, divide and take ether extracted liquid, be evaporated, residue adds methanol and is allowed to dissolve, thin as Herba Blumeae Balsamiferae
Layer differentiates need testing solution；
(12) protocatechuic acid reference substance solution: protocatechuic acid reference substance adds methanol and makes protocatechuic acid reference substance solution；
(13) protocatechualdehyde reference substance solution: protocatechualdehyde reference substance adds methanol and makes protocatechualdehyde reference substance solution；
(14) take above-mentioned Herba Blumeae Balsamiferae thin layer and differentiate need testing solution, protocatechuic acid reference substance solution, protocatechualdehyde reference substance solution
Point is on same silica gel thin-layer plate, and the chloroform with volume ratio as 6:3:5, ethyl acetate, formic acid mixed solution launch for developing solvent,
Take out, dry, inspect under uviol lamp.
The method of quality control of her blood peace granule the most according to claim 1, it is characterised in that:
The concrete operation method of described step (2) is:
(21) Herba Leonuri thin layer differentiates the preparation of need testing solution: taking her blood peace granule, add dehydrated alcohol, shaking is extracted, and filters,
The filtrate obtained is evaporated, and residue adds anhydrous alcohol solution, differentiates need testing solution as Herba Leonuri thin layer；
(22) stachydrine hydrochloride reference substance solution: stachydrine hydrochloride reference substance adds dehydrated alcohol and makes stachydrine hydrochloride reference substance solution；
(23) take above-mentioned Herba Leonuri thin layer to differentiate need testing solution, stachydrine hydrochloride reference substance solution, put on same silica gel thin-layer plate,
Acetone with volume ratio as 6:6:1, ethyl acetate, hydrochloric acid mixed solution launch for developing solvent, take out, dry, and spray with improvement
Bismuth potassium iodide test solution, heating makes spot development clear, and Herba Leonuri thin layer differentiates that need testing solution chromatograph is compareing with stachydrine hydrochloride
On the corresponding position of product solution chromatograph, the speckle of display same color.
The method of quality control of her blood peace granule the most according to claim 1, it is characterised in that:
The concrete operation method of described step (3) is:
(31) preparation of object of reference solution: protocatechuic acid standard substance, adds methanol constant volume, prepares object of reference solution.
(32) preparation of her blood peace granule extracting solution；
(33) chromatographic condition of high performance liquid chromatograph:
Flowing phase: methanol-0.1% glacial acetic acid；
Gradient elution program: time gradient
0min→11min→16min→29min→30min→35min→45min→55min→70min→95min；Corresponding gradient
Eluent is formed than by mobile phase A methanol and Mobile phase B 0.1% glacial acetic acid by volumes below, the percentage by volume of mobile phase A
Gradient 5% → 16% → 17% → 30% → 34% → 34% → 35% → 45% → 45% → 70%, the volume hundred of Mobile phase B
Mark gradient 95% → 84% → 83% → 70% → 66% → 66% → 65% → 55% → 55% → 30%；
Ultraviolet detection wavelength: 256nm；
Flow velocity: 1.0mL min-1；
Column temperature: 25 DEG C.
(34) measure: draw her blood peace granule extracting solution and inject high performance liquid chromatograph, measure according to the chromatographic condition of step (33),
Obtain finger printing.
5. according to the method for quality control of her the blood peace granule described in claim 1 or 4, it is characterised in that:
The concrete operation method that her blood peace granule extracting solution described extracts is: weighing her blood peace granule 3～8g, the 50mL that adds water dissolves, adds
Dilute hydrochloric acid 0.2～0.4mL, stirs evenly, and stands 15～30min, filters, and adds water saturation n-butanol extraction 5～6 times, each 15～20mL,
Divide and take n-butanol extracting liquid, merge, stand 1～1.5h, divide and take acquisition n-butyl alcohol liquid, recovery n-butyl alcohol, residue adds methanol 4～6mL,
Centrifugal, take supernatant, obtain her blood peace granule extracting solution.
The method of quality control of her blood peace granule the most according to claim 1, it is characterised in that:
Described finger printing has chromatographic peak 21, and its Average residence time is respectively as follows:
No. 1 peak 3.234min, No. 2 peak 5.962min, No. 3 peak 11.023min, No. 4 peak 15.353min, No. 5 peak 16.521min,
No. 6 peak 24.498min, No. 7 peak 27.158min, No. 8 peak 30.877min, No. 9 peak 32.29min, No. 10 peak 34.752min,
No. 11 peak 36.182min, No. 12 peak 37.968min, No. 13 peak 38.708min, No. 14 peak 56.509min, No. 15 peaks
58.989min, No. 16 peak 59.670min, No. 17 peak 66.895min, No. 18 peak 68.358min, No. 19 peak 69.319min,
No. 20 peak 85.217min, No. 21 peak 91.016min.
The method of quality control of her blood peace granule the most according to claim 6, it is characterised in that:
The component of her blood peace granule described includes Herba Blumeae Balsamiferae, Herba Leonuri, Rhizoma Corydalis and Radix Glycyrrhizae, wherein No. 1 peak～source, No. 21 peaks
In Herba Blumeae Balsamiferae, No. 9 peaks derive from Herba Leonuri, and 10, No. 11 peaks derive from Rhizoma Corydalis, source, 6,14,15, No. 16 peaks
In Radix Glycyrrhizae.
The method of quality control of her blood peace granule the most according to claim 4, it is characterised in that:
In described step (33), chromatographic column is Phenomenex Gemini C18 post (4.6mm × 250mm, 5 μm), guard column
For Phenomenex C18 (4 × 3.0mm)..
The method of quality control of her blood peace granule the most according to claim 4, it is characterised in that:
In described step (33), theoretical cam curve, in terms of protocatechuic acid peak, is not less than 5000.
10. according to the method for quality control of her the blood peace granule described in any one of claim 1,4,5, it is characterised in that:
The concrete operation method that her blood peace granule extracting solution described extracts is: weighs she blood peace granule 5g, the 50mL that adds water and dissolves, adds dilute
Hydrochloric acid 0.3mL, stirs evenly, and stands 20min, filters, adds water saturation n-butanol extraction 5 times, and each 15mL divides and takes n-butyl alcohol
Extracting solution, merges, and stands 1h, divides and takes acquisition n-butyl alcohol liquid, reclaims n-butyl alcohol, and residue adds methanol 5mL, centrifugal, takes supernatant
Liquid, obtains her blood peace granule extracting solution.
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