CN110308213B - Method for constructing fingerprint spectrum of kidney-nourishing and fetus-growing pill and application of fingerprint spectrum in quality detection - Google Patents
Method for constructing fingerprint spectrum of kidney-nourishing and fetus-growing pill and application of fingerprint spectrum in quality detection Download PDFInfo
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Abstract
The invention provides a method for constructing a fingerprint of a kidney-nourishing and fetus-growing pill and application thereof in quality detection. The construction method comprises the following steps: selecting any one of chlorogenic acid, loganin, swertiamarin, loganin and teasel root saponin VI as a reference substance, and dissolving with an organic solvent to prepare a reference substance solution; selecting a plurality of batches of kidney-nourishing and fetus-growing pills, and dissolving the pills by using organic solvents respectively to prepare a test solution; testing the reference substance solution and the test substance solutions of the multiple batches of kidney nourishing and fetus culturing pills by adopting a high performance liquid chromatography to obtain a reference substance chromatographic peak and a test substance chromatographic peak of the multiple batches of kidney nourishing and fetus culturing pills; and (4) referring to the chromatographic peak of the reference substance, and performing similarity analysis on the chromatographic peaks of the plurality of test substances to obtain the finger print of the kidney nourishing and fetus culturing pill. The fingerprint is used as a reference fingerprint for quality detection of the kidney nourishing and fetus culturing pills, can better and comprehensively evaluate the overall quality of the kidney nourishing and fetus culturing pills, and has good repeatability, good stability and high accuracy. Can be used for inspecting the production stability and the batch consistency.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine testing, in particular to a method for constructing a finger print of a kidney nourishing and fetus culturing pill and application thereof in quality detection.
Background
The kidney-nourishing fetus-cultivating pill is a Chinese patent medicine developed by a pharmaceutical industry limited company in Guangzhou Baiyunshan according to years of clinical experience of professor Royuen Cai which is a famous gynecologic specialist in 20 th century in the first 60 th years, and consists of 15 traditional Chinese medicines of prepared rehmannia root, medlar, dodder, amomum fruit, ginseng, loranthus parasiticus, donkey-hide gelatin (fried), fleece-flower root, Chinese mugwort leaf, morinda root, largehead atractylodes rhizome, pilose asiabell root, deglued antler powder, himalayan teasel root, eucommia bark and the like, and has the effects of tonifying kidney and spleen, benefiting qi and cultivating vitality, nourishing blood and preventing miscarriage and strengthening body. Is mainly used for preventing and treating habitual abortion and threatened abortion clinically and has exact curative effect. In recent years, based on the principles of "treating both abnormal diseases and" kidney governing reproduction "in traditional Chinese medicine, the kidney-nourishing and fetus-growing pill is also commonly used for treating irregular menstruation and infertility. The quality standard of the kidney-nourishing and fetus-cultivating pill is recorded in the sixteenth volume (WS3-B-3113-98) of the drug Standard-Chinese medicine prescription preparation of Ministry of health of the people's republic of China, the original standard only carries out thin-layer identification on radix codonopsitis and radix polygoni multiflori, no content determination item exists, the specificity is poor, and the quality of the pill is difficult to comprehensively evaluate. Based on the excellent clinical curative effect, the research on the kidney nourishing and fetus culturing pills at present is mostly more focused on clinical and pharmacological research, and the quality research, such as content measurement, fingerprint information and the like, is not reported.
Therefore, the development of a quality detection method of kidney-nourishing and fetus-growing pills is urgently needed.
Disclosure of Invention
Based on the above, one of the purposes of the present application is to provide a method for constructing a fingerprint of a kidney-nourishing and fetus-growing pill.
The technical purpose is realized by the following technical scheme:
a method for constructing a finger print of a kidney-nourishing and fetus-growing pill comprises the following steps:
preparation of a reference solution: selecting any one of chlorogenic acid, loganin, swertiamarin, loganin and teasel root saponin VI as a reference substance, and dissolving with an organic solvent to prepare a reference substance solution;
preparing a test solution: selecting a plurality of batches of kidney-nourishing and fetus-growing pills, and dissolving the pills by using organic solvents respectively to prepare a test solution;
constructing a fingerprint: respectively testing the reference solution and the test solution by adopting a high performance liquid chromatography to obtain a reference chromatogram and chromatograms of a plurality of batches of kidney nourishing and fetus culturing pills; carrying out similarity analysis and correction on common peaks in the chromatograms of the kidney-nourishing and fetus-growing pills of a plurality of batches to obtain a reference fingerprint; and marking the chemical components of the chromatographic peak of the reference fingerprint by referring to the retention time of the chromatographic peak of the reference substance in the chromatogram of the reference substance to obtain the fingerprint of the kidney nourishing and fetus culturing pill for quality detection.
In some of these embodiments, the control is swertiamarin.
In some embodiments, the conditions employed for high performance liquid chromatography include:
the chromatographic column is selected from Kromasil C 18 、Venusil HILIC、Bolitimate C 18 Any one of (a); the mobile phase is acetonitrile (A) -0.1-0.5% phosphoric acid water solution (B); the elution gradient was: 0-12 min, 2% A; 12-30 min, 2% -7% A; 30-40 min, 7% -9% A; 9-15% of A for 40-60 min; 60-80 min, 15% -35% A; 80-85 min, 35% -70% A; the detection wavelength is 250-260 nm.
In some of these embodiments, the conditions employed for high performance liquid chromatography include: the flow rate is 0.8-1.2 mL/min -1 The column temperature is 28-32 ℃, and the sample injection amount is 8-12 mu L.
In some of these embodiments, the chromatography column is kromasil c 18 The mobile phase is acetonitrile (A) -0.3 percent phosphoric acid aqueous solution (B), and the detection wavelength is 254 nm; the flow rate is 1.0mL min -1 The column temperature was 30 ℃ and the amount of sample was 10. mu.L.
In some of these embodiments, the chromatography column is kromasil c 18 (5μm，4.6×250mm)。
In some embodiments, the organic solvent is selected from methanol or a methanol aqueous solution with a volume content of 20-80%; the preparation of the test solution adopts a reflux method or an ultrasonic method. Preferably, an aqueous methanol solution having a volume content of 80% is used. Preferably, sonication is used, preferably for a sonication time of 40 min.
The invention also aims to provide a quality detection method of the kidney nourishing and fetus culturing pill based on the fingerprint spectrum of the kidney nourishing and fetus culturing pill, which comprises the following steps:
preparation of a reference solution: selecting at least one of chlorogenic acid, loganin, swertiamarin, loganin and teasel root saponin VI as reference substances, dissolving with organic solvent, and preparing reference substance solution;
preparing a solution of a to-be-detected product: weighing the kidney-nourishing and fetus-growing pill to be detected, and dissolving the kidney-nourishing and fetus-growing pill by using an organic solvent to prepare a test solution;
and (3) quality detection: testing the reference substance solution and the solution to be tested by adopting a high performance liquid chromatography to obtain a reference substance chromatogram and a chromatogram of the to be tested; analyzing the common peak of the chromatogram of the sample to be tested and the fingerprint of the kidney-nourishing and fetus-growing pill, and the chromatogram of the sample to be tested and the chromatogram of the reference substance.
In some of these embodiments, the conditions employed for high performance liquid chromatography include:
the chromatographic column is selected from Kromasil C 18 、Venusil HILIC、Bolitimate C 18 Any one of (a); the mobile phase is acetonitrile (A) -0.1-0.5% phosphoric acid water solution (B); the elution gradient was: 0-50 min, 6% -11% A; 50-52 min, 11% -17% A; 52-80 min, 17% -28% A; 80-95 min, 28-40% A; the detection wavelength is 230-240 nm, 210-215 nm.
In some embodiments, the conditions employed for high performance liquid chromatography include: the flow rate is 0.8-1.2 mL/min -1 The column temperature is 28-32 ℃, and the sample injection amount is 8-12 mu L.
In some of these embodiments, the chromatography column is kromasil c 18 (ii) a The mobile phase is acetonitrile (A) -0.1% phosphoric acid water solution (B); the detection wavelength is 235nm and 212 nm; the flow rate is 1.0mL min -1 The column temperature was 30 ℃ and the sample size was 10. mu.L. The chromatographic column is preferably Kromasil C 18 (5μm，4.6×250mm)。
In some embodiments, the organic solvent is selected from methanol or a methanol aqueous solution with a volume content of 20-80%; the preparation of the test solution adopts a reflux method or an ultrasonic method. Preferably, an aqueous solution of methanol having a content of 80% by volume is used. Preferably, ultrasonic method is adopted, and the ultrasonic time is preferably 30 min.
Compared with the prior art, the invention has the following beneficial effects:
the inventor discovers that on the basis of long-term test and experience accumulation, a better fingerprint can be obtained by selecting any one of chlorogenic acid, loganin, swertiamarin, loganin and teasel root saponin VI as a reference substance, particularly selecting swertiamarin as the reference substance under the condition of proper high performance liquid chromatography, and chromatographic peaks in the fingerprint are stable and comprise chromatographic peaks of the loganin, the chlorogenic acid, the loganin, the swertiamarin and the teasel root saponin VI; the chemical components can well reflect the teasel roots which are used as ministerial drugs in the prescription of the kidney nourishing and fetus-growing pill; the established fingerprint spectrum can better and comprehensively reflect the whole quality of the kidney nourishing and fetus culturing pill, and has good repeatability, good stability and high accuracy. And moreover, the method can be used for inspecting the production stability and the batch consistency.
According to the quality detection method of the kidney nourishing and embryo culturing pill, loganin, swertiamarin and dipsacus asperoides VI are selected as reference substances, and a proper quality detection high performance liquid chromatography condition is adopted in a matching manner, particularly the quality detection high performance liquid chromatography condition is different from the high performance liquid chromatography condition used for fingerprint construction, so that the types of chemical components of the kidney nourishing and embryo culturing pill sample can be well detected, the contents of the five chemical components in the kidney nourishing and embryo culturing pill sample can be accurately measured, qualitative and quantitative detection can be well carried out on the kidney nourishing and embryo culturing pill sample to be detected, and the method is good in repeatability, good in stability and high in accuracy.
Drawings
FIG. 1 is a comparison fingerprint of kidney-nourishing and fetus-growing pill constructed in example 1;
FIG. 2 is the finger print of 11 test samples in example 1;
FIG. 3 is a chromatogram obtained by detecting kidney-nourishing and fetus-growing pill samples at different detection wavelengths;
A. a test article; B. each control; a. loganin acid; b. chlorogenic acid; c. swertisin; d. loganin; e. dipsacus asperoides saponin VI.
Detailed Description
The method for constructing fingerprint of the kidney-nourishing and fetus-growing pill and the application thereof in quality detection are further described in detail in the following by combining with specific embodiments.
1. Instrument for measuring the position of a moving object
A BT 125D model 1/10 ten thousand electronic balances and a BSA224S-CW model 1/1 ten thousand electronic balances [ sartorius scientific instruments (beijing) ltd ], a LC20A model high performance liquid chromatograph (shimadzu, japan, including an LC-20AT type Solution transmission unit, an SIL-20A type autosampler, an SPD-M20A type diode array detector, and an LC Solution chromatography workstation), a DET-50 model high speed universal pulverizer (dade traditional Chinese medicine machines ltd, greenling), an XMTD-6000 model water bath (beijing longfeng instruments ltd), a KQ-250DE model digitally controlled ultrasonic cleaner (kunshan ultrasonic instruments ltd).
2. Material
The kidney nourishing and fetus culturing pill sample is provided by a pharmaceutical industry Co., Ltd in Baiyunshan, Guangzhou (batch numbers: S00031, S00033, S00039, S00038, S00040, S00041, SF0001, V00013M, W000125, T01002, V00116);
teasel saponin VI and loganin (Duyunreisi Biotechnology Co., Ltd., batch numbers of C-014-; loganin, swertiamarin and chlorogenic acid reference substances (the batches of which are 111640-201005, 111742-200501 and 110753-200212 of China food and drug testing research institute respectively, the purities of which are respectively more than or equal to 99 percent, more than or equal to 98 percent and more than or equal to 98 percent) are specified for content determination.
Acetonitrile is chromatographically pure; other reagents are analytically pure; the water is ultrapure water.
Example 1 establishing fingerprint of Kidney-nourishing and fetus-growing pill with swertisin as reference
1.1 preparation of solutions of test and reference substances
Preparation of control solutions: precisely weighing appropriate amount of swertiamarin reference substance, adding methanol to dissolve and fixing volume to obtain the product with mass concentration of 0.1 g.L -1 The control solution of (4).
Preparation of a test solution: precisely weighing about 1.5g of kidney nourishing and fetus culturing pill powder (80 meshes, the same below), precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, tightly plugging, weighing, ultrasonically treating (with power of 250W and frequency of 40kHz) for 30min, cooling, weighing, complementing weight loss with methanol, shaking up, filtering with a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the test solution.
1.2 chromatographic conditions:
using a chromatographic column Kromasil C 18 (5 mu m, 4.6 multiplied by 250mm), the mobile phase is acetonitrile (A) -0.3 percent phosphoric acid aqueous solution (B), the gradient elution is carried out (0-12 min, 2 percent A, 12-30 min, 2-7 percent A, 30-40 min, 7-9 percent A, 40-60 min, 9-15 percent A, 60-80 min, 15-35 percent A, 80-85 min, 35-70 percent A), the flow rate is 1.0 mL/min -1 The column temperature was 30 ℃, the sample size was 10 μ L, and the detection wavelength was 254 nm.
1.3 finger print methodology investigation
(1) Precision experiment
Sample solution (lot S00039) was continuously injected 6 times under the chromatographic conditions of item 1.2, and the relative retention time and relative peak area of the common peak were analyzed.
As a result, the relative retention time and the relative peak area RSD of each common peak are respectively less than 0.2% and 3.5%, which indicates that the precision of the instrument is good.
(2) Stability test
The same sample solution (lot S00039) was injected at 0, 2, 4, 6, 12, and 24h after preparation, and the relative retention time and relative peak area of the common peak were analyzed by measuring under chromatographic conditions of 1.2.
The results show that the relative retention time of all the common peaks and the RSD value of the relative peak area are respectively less than 1.0 percent and 5.0 percent, which indicates that the test solution has good stability within 24 hours.
(3) Repeatability test
Weighing about 1.5g and 6 parts of powder of the same batch of kidney-nourishing and fetus-growing pills (batch number S00039), precisely weighing, preparing a test solution according to the method under item 1.1, measuring according to chromatographic conditions under item 1.2, and respectively analyzing the relative retention time and the relative peak area of the common peak.
The results show that the relative retention time of each common peak and the RSD value of the relative peak area are respectively less than 2.0 percent and 5.0 percent, which indicates that the repeatability is good.
1.4 fingerprint establishment and similarity evaluation
Taking 11 batches of kidney-nourishing and fetus-rearing pills (batch numbers: S00031, S00033, S00038, S00039, S00040, S00041, SF0001, T01002, V00013M, V00116, W000125, and subsequent numbers are S1-S11 in sequence), preparing sample solutions according to the method of item 1.1, and measuring according to the chromatographic conditions of step 1.2;
analyzing with a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.1 version), performing multi-point correction on chromatogram peaks, automatically matching, and generating a reference fingerprint shown in figure 1.
According to the figure 1, 15 common peaks are calibrated, and four index components are identified, namely peak 8 loganin, peak 10 chlorogenic acid, peak 11 swertiamarin and peak 12 loganin. Wherein, the peak 11 swertiamarin is taken as a reference peak, the similarity of each batch of kidney nourishing and embryo culturing pills sample and the reference fingerprint is 0.957-0.999, which shows that each batch of kidney nourishing and embryo culturing pills have good consistency, and the specific data result is shown in table 1 and fig. 2.
TABLE 1 evaluation of similarity of Kidney-nourishing and fetus-growing pills
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | | Control | |
| S1 | ||||||||||||
| 1 | 0.995 | 0.975 | 0.983 | 0.977 | 0.980 | 0.976 | 0.979 | 0.957 | 0.980 | 0.983 | 0.988 | |
| S2 | 0.995 | 1 | 0.975 | 0.983 | 0.978 | 0.980 | 0.977 | 0.990 | 0.975 | 0.990 | 0.994 | 0.994 |
| S3 | 0.975 | 0.975 | 1 | 0.999 | 0.995 | 0.998 | 0.980 | 0.976 | 0.971 | 0.980 | 0.966 | 0.988 |
| S4 | 0.983 | 0.983 | 0.999 | 1 | 0.997 | 0.999 | 0.983 | 0.981 | 0.975 | 0.990 | 0.974 | 0.993 |
| S5 | 0.977 | 0.978 | 0.995 | 0.997 | 1 | 0.997 | 0.975 | 0.974 | 0.972 | 0.980 | 0.967 | 0.989 |
| S6 | 0.980 | 0.980 | 0.998 | 0.999 | 0.997 | 1 | 0.985 | 0.979 | 0.973 | 0.980 | 0.970 | 0.991 |
| S7 | 0.976 | 0.977 | 0.98 | 0.983 | 0.975 | 0.985 | 1 | 0.986 | 0.970 | 0.980 | 0.976 | 0.987 |
| S8 | 0.979 | 0.99 | 0.976 | 0.981 | 0.974 | 0.979 | 0.986 | 1 | 0.992 | 0.990 | 0.996 | 0.996 |
| S9 | 0.957 | 0.975 | 0.971 | 0.975 | 0.972 | 0.973 | 0.970 | 0.992 | 1 | 0.990 | 0.984 | 0.989 |
| S10 | 0.977 | 0.989 | 0.981 | 0.986 | 0.983 | 0.984 | 0.976 | 0.994 | 0.994 | 1 | 0.993 | 0.997 |
| S11 | 0.983 | 0.994 | 0.966 | 0.974 | 0.967 | 0.97 | 0.976 | 0.996 | 0.984 | 0.990 | 1 | 0.993 |
| Control | 0.988 | 0.994 | 0.988 | 0.993 | 0.989 | 0.991 | 0.987 | 0.996 | 0.989 | 1 | 0.993 | 1 |
Example 2, quality detection of kidney-nourishing and fetus-growing pills by using the fingerprint constructed in example 1
2.1 preparation of solutions of test and reference substances
Preparation of a reference solution: precisely weighing appropriate amount of chlorogenic acid, loganin, swertisin, loganin and Dipsacaceae saponin VI reference substance, adding methanol to dissolve and fix volume to obtain the final product with mass concentration of 0.0204, 0.0121, 0.0332, 0.0532, 0.158 mg.mL -1 Mixing the reference solution for use. The kind of the reference substance of this example was selected with reference to the fingerprint of the kidney-nourishing and fetus-growing pill for quality test constructed in example 1, and mainly selected from chemical components in which the chemical components have been labeled according to the retention time of the chromatographic peak of the reference substance.
Preparation of a test solution: precisely weighing about 1.0g of kidney nourishing and fetus culturing pill powder, precisely weighing, placing in a 50mL conical flask with a plug, precisely adding 20mL of 80% methanol, sealing the plug, weighing the mass, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30min, cooling, weighing, complementing the weight with 80% methanol, shaking up, filtering with a 0.22 mu m microporous filter membrane, and taking out a subsequent filtrate to obtain the kidney nourishing and fetus culturing pill.
2.2 chromatographic conditions
Using a chromatographic column Kromasil C 18 (5 mu m, 4.6 multiplied by 250mm), the mobile phase is acetonitrile (A) -0.1 percent phosphoric acid water solution (B), the gradient elution is carried out (0-50 min, 6-11 percent A, 50-52 min, 11-17 percent A, 52-80 min, 17-28 percent A, 80-95 min, 28-40 percent A), the flow rate is 1.0 mL/min -1 The column temperature was 30 ℃, the sample size was 10 μ L, and the detection wavelength was 235nm, 212 nm.
Under the chromatographic conditions, the retention time of chromatographic peaks of chlorogenic acid, loganin, sweroside, loganin and dipsacoside VI in the sample is consistent with that of a reference substance, chromatographic peaks of 5 components can be well separated, and purity inspection meets requirements. Other components in the sample do not interfere with the determination of the component to be measured. See fig. 3.
2.3 assay methodology investigation
(1) Investigation of Linear relationships
Precisely sucking 2.1 items of mixed reference solution 1, 2, 5, 10, 15, 20 μ L respectively, injecting into high performance liquid chromatograph, and measuring according to 2.2 items of chromatographic conditions. Taking the sample amount as a horizontal coordinate and the peak area as a vertical coordinate, drawing a standard curve, and calculating a regression equation and a correlation coefficient, which are shown in table 2.
The result shows that 5 components have a better linear relation with the peak area within a certain mass concentration.
TABLE 2 examination of the Linear relationship between 5 index components in the kidney-nourishing and fetus-growing pill
| TargetComposition (I) | Regression equation | r | Linear range/ng |
| Strychnos acid | Y＝1440.3X+11805 | 0.9992 | 53.2～1064 |
| Swertisin | Y＝1256.5X+319.09 | 0.9999 | 33.2～664 |
| Loganin A. Loganin C.P.Loganin A. Loganin C.P.E. Loganin | Y＝1507.9X+34.223 | 0.9999 | 12.1～242 |
| Chlorogenic acid | Y＝1301.6X+844.21 | 0.9999 | 20.4～408 |
| Dipsacus asperoides saponin VI | Y＝240.49X+1324.2 | 0.9999 | 129～2580 |
(2) Precision test
The same mixed reference solution was sampled for 6 times continuously under 2.2 chromatographic conditions.
The results show that the RSD of the peak areas of chlorogenic acid, loganin, swertiamarin, loganin and teasel root saponin VI are respectively 0.85%, 0.93%, 2.03%, 0.66% and 0.68%. Indicating that the precision of the instrument is good.
(3) Stability test
The same sample solution (batch number S00038) of the kidney nourishing and embryo culturing pill is taken, and sample injection is carried out for 0, 2, 4, 6, 12 and 24 hours after sample preparation, and the sample is measured according to the chromatographic condition under 2.2.
The results show that the peak areas RSD of five components of chlorogenic acid, loganin, swertisin, loganin and teasel root saponin VI in the sample solution are respectively 3.85%, 0.51%, 0.79%, 3.01% and 1.32%. The result shows that the stability of the test solution is good within 24 hours.
(4) Repeatability test
Weighing about 1.0g of powder of the same batch of kidney nourishing and fetus culturing pills (batch number S00038), 6 parts in total, precisely weighing, preparing a test solution according to the method under item 3.2.2, and determining according to the chromatographic condition under item 3.1.
The results show that the contents of RSD in chlorogenic acid, loganin, swertisin, loganin and teasel root saponin VI are respectively 0.94%, 1.02%, 0.78%, 1.48% and 1.63%. The experimental repeatability is good.
(5) Sample application recovery test
Weighing 9 parts of kidney nourishing and fetus culturing pill powder with known content, each part is about 0.5g, precisely weighing, placing into a conical flask with a plug, respectively adding a certain amount of reference solution, preparing test solution according to the method of item 3.2.1, and measuring according to the chromatographic condition of item 3.1.
The result shows that the average value of the sample recovery rate of the five components of the loganin, the swertiamarin, the chlorogenic acid, the loganin and the teasel root saponin VI is 96.61-103.55 percent, and the RSD is less than 3.0 percent. Indicating that the accuracy of the method is good. The results are shown in Table 3.
TABLE 3 determination of recovery rate of 5 ingredients in Kidney-nourishing and fetus-growing pill (n ═ 9)
2.4 sample determination
Taking the kidney-nourishing and fetus-growing pill powder of each batch, preparing a test solution according to the method under item 2.1, wherein each sample is 3 parts in parallel, and determining according to the chromatographic condition under item 2.2.
The results are shown in Table 4. The mass fractions of loganin, chlorogenic acid, swertiamarin, loganin and teasel root saponin VI in the 11 batches of pills for nourishing kidney and raising fetus are not less than 0.61 per thousand, 0.27 per thousand, 0.46 per thousand, 0.19 per thousand and 0.81 per thousand.
TABLE 4 weight fractions (n 3)% o of 5 ingredients in the Kidney-nourishing and fetus-growing pill sample
| Batch number | Strychnos acid | Chlorogenic acid | Swertisin | Loganin A. Loganin C.P.Loganin A. Loganin C.P.E. Loganin | Dipsacus asperoides saponin VI |
| S00031 | 0.61 | 0.27 | 0.46 | 0.20 | 1.17 |
| S00033 | 0.65 | 0.31 | 0.47 | 0.19 | 1.31 |
| S00039 | 0.86 | 0.43 | 0.59 | 0.22 | 1.99 |
| S00038 | 0.89 | 0.46 | 0.59 | 0.23 | 2.02 |
| S00040 | 0.90 | 0.48 | 0.59 | 0.23 | 2.10 |
| S00041 | 0.90 | 0.47 | 0.59 | 0.22 | 1.99 |
| SF0001 | 0.93 | 0.39 | 0.65 | 0.24 | 1.70 |
| V00013M | 1.45 | 0.51 | 0.88 | 0.29 | 1.60 |
| W00125 | 0.81 | 0.32 | 0.68 | 0.24 | 0.81 |
| T01002 | 1.08 | 0.44 | 0.76 | 0.28 | 0.97 |
| V00116 | 1.01 | 0.46 | 0.84 | 0.33 | 1.34 |
When the quality detection method provided by the embodiment is adopted to test a sample to be detected, if the chromatographic peak of the sample to be detected is consistent with the fingerprint of the kidney nourishing and fetus culturing pill for quality detection, the type of the chemical components contained in the sample to be detected is in accordance with the requirement; if the chromatographic peak areas of chlorogenic acid, loganin, swertiamarin, loganin and teasel root saponin VI in the chromatographic peaks of the sample to be detected are consistent with those of the reference substance, the content of chemical components in the sample to be detected is in accordance with the requirement.
Example 3 construction of fingerprint with loganin as reference substance and quality control of Kidney nourishing and fetus growth pill
3.1 constructing a fingerprint: the steps for constructing the fingerprint map in the embodiment are the same as the embodiment 1, and the change is that loganin is used as a reference substance for constructing the fingerprint map in the embodiment.
3.2, carrying out quality detection: the fingerprint constructed in the 3.1 of the embodiment is taken as a reference map to carry out quality detection on the kidney nourishing and embryo culturing pills, and the quality detection steps are the same as the embodiment 2.
The fingerprint map construction result of this example is the same as that of example 1, and refer to fig. 1 and 2. In this example, the quality test of the kidney-nourishing and fetus-growing pill with lot number S00031 was conducted, and the results are shown in Table 4. In the process of implementing the technical scheme, the inventor also carries out the following optimization on the technical scheme:
(1) the optimization of the preparation method of the test sample comprises 1) pre-testing the optimization of the preparation method of the test sample solution for measuring the fingerprint spectrum and the content of the kidney nourishing and fetus cultivating pill, and respectively examining the extraction effects of 2 extraction modes of a reflux method and an ultrasonic method on the kidney nourishing and fetus cultivating pill. The results show that the extraction effects of the 2 extraction methods have no significant difference, and the ultrasonic extraction method is simple and easy to implement, so that the ultrasonic extraction is selected. 2) The experiment also examines the extraction solvent, compares the extraction efficiency of 5 solvents including methanol, 80% methanol, 60% methanol, 40% methanol and 20% methanol, and the result shows that the number of chromatographic peaks is large when the methanol is used as the extraction solvent in the kidney nourishing and fetus culturing pill sample, so that the methanol is used as the extraction solvent in the kidney nourishing and fetus culturing pill fingerprint sample. And when 80% methanol is used as an extraction solvent, the extraction efficiency of five components in the kidney nourishing and embryo culturing pill is higher, so that 80% methanol is selected as the extraction solvent for measuring the content of the kidney nourishing and embryo culturing pill. In addition, the ultrasonic time (20, 30 and 40min) is considered, and the result shows that the ultrasonic time is 40min when the finger print of the kidney nourishing and fetus culturing pill is determined. The five target components of the test solution for measuring the content of the kidney nourishing and embryo culturing pills are basically and completely extracted after 30min of ultrasonic extraction.
(2) The selection of chromatographic conditions comprises 1) the kidney nourishing and fetus culturing pill is a compound preparation of 15 medicines, the chemical properties of all the components are greatly different, and the experiment adopts a gradient elution mode to carry out separation and elution. The laboratory has optimized the chromatography columns of different packing, looking at 3 columns (Kromasil C) 18 ，Venusil HILIC，Bolitimate C 18 ) The results of the separation efficiency of each component to be measured showed Kromasil C 18 The chromatographic column has long service life, stable base line, good peak shape of each component to be measured and high resolution, so the column is selected as the separation column. 2) In the experiment, an acetonitrile-phosphoric acid system is used as a mobile phase, and the influence of 0.1%, 0.2%, 0.3% and 0.5% phosphoric acid on the peak shape of a chromatographic peak is respectively examined. The chromatogram of the finger print of the kidney nourishing and fetus culturing pill is better in the elution of all common peak shapes under the system of acetonitrile-0.3 percent phosphoric acid water solution; the acetonitrile-0.1% phosphoric acid water solution can make five components in the kidney nourishing and embryo cultivating pill achieve baseline separation, so the acetonitrile-0.1% phosphoric acid water solution system is selected as the mobile phase for measuring the content of the kidney nourishing and embryo cultivating pill.
(3) Selection of detection wavelength: and carrying out ultraviolet absorption full-wavelength scanning on the kidney nourishing and fetus culturing pill sample at 190-400 nm by adopting a diode array detector. The chromatogram of the finger print of the kidney nourishing and embryo culturing pill has stable base line under the wavelength of 254nm, more chromatographic peaks and larger information content, so that 254nm is selected as the detection wavelength of the finger print of the kidney nourishing and embryo culturing pill. The ultraviolet maximum absorption wavelength a of loganin and loganin acid is 235nm, the ultraviolet maximum absorption wavelength of swertiamarin is 241nm, the ultraviolet maximum absorption wavelength of chlorogenic acid is 327nm, and the ultraviolet maximum absorption wavelength of dipsacoside VI is 212 nm. In order to give consideration to the maximum absorption of 5 components of the kidney nourishing and embryo culturing pill and have a good baseline, the final detection wavelength determines that the dipsacoside VI is 212nm, and the other 4 components are detected at 235nm, so that the detection wavelengths for determining the content of the kidney nourishing and embryo culturing pill are 235nm and 212 nm.
The fingerprint spectrum method established for the first time in the experiment can be used for inspecting the production stability and batch consistency and identifying and evaluating the quality of the kidney-nourishing and fetus-growing pills; the content determination method for simultaneously determining the five index components established in the research has the advantages of good repeatability, good stability and high accuracy, and can accurately determine the content of the index components in the kidney-nourishing and fetus-growing pill sample. In conclusion, the invention establishes the quality control standard of the kidney nourishing and embryo culturing pill by combining the fingerprint qualitative determination with the multi-component synchronous determination method, and performs the quality control of the kidney nourishing and embryo culturing pill from the analysis of the whole to the local chemical components, thereby providing a basis for perfecting the quality standard and the clinical medication safety of the kidney nourishing and embryo culturing pill and laying a foundation for the further research of the spectrum effectiveness.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A method for constructing a fingerprint of a kidney-nourishing and fetus-growing pill is characterized by comprising the following steps:
preparation of a reference solution: selecting any one of chlorogenic acid, loganin, swertiamarin, loganin and teasel root saponin VI as a reference substance, and dissolving with an organic solvent to prepare a reference substance solution;
preparing a test solution: selecting a plurality of batches of kidney-nourishing and fetus-growing pills, and dissolving the pills by using organic solvents respectively to prepare a test solution;
constructing a fingerprint: respectively testing the reference solution and the test solution by adopting a high performance liquid chromatography to obtain a reference chromatogram and chromatograms of a plurality of batches of kidney nourishing and fetus culturing pills; carrying out similarity analysis and correction on common peaks in the chromatograms of the kidney-nourishing and fetus-growing pills of a plurality of batches to obtain a reference fingerprint spectrum; according to the retention time of the reference substance chromatographic peak in the reference substance chromatogram, marking the chemical components of the chromatographic peak of the reference fingerprint spectrum to obtain the fingerprint spectrum of the kidney nourishing and fetus culturing pill for quality detection;
the conditions adopted by the high performance liquid chromatography comprise:
the chromatographic column is Kromasil C 18 ；
The mobile phase is acetonitrile A-0.3 percent phosphoric acid water solution B;
the elution gradient was:
0～12min，2％A；
12～30min，2％～7％A；
30～40min，7％～9％A；
40～60min，9％～15％A；
60～80min，15％～35％A；
80～85min，35％～70％A；
the detection wavelength is 250-260 nm;
the flow rate is 0.8-1.2 mL/min -1 The column temperature is 28-32 ℃, and the sample injection amount is 8-12 mu L;
the organic solvent is selected from methanol or a methanol aqueous solution with the volume content of 20-80%.
2. The method for constructing fingerprint of kidney-nourishing and fetus-nourishing pill as claimed in claim 1, wherein said reference substance is swertiamarin.
3. The method for constructing the fingerprint of the kidney-nourishing and fetus-nourishing pill according to claim 1 or 2, wherein the detection wavelength is 254 nm; the flow rate is 1.0mL min -1 The column temperature was 30 ℃ and the amount of sample was 10. mu.L.
4. The method for constructing fingerprint of Zishen Yutai Wan according to claim 1 or 2, wherein the chromatographic column is Kromasil C 18 The mobile phase is acetonitrile A-0.3 percent phosphoric acid water solution B.
5. The method for constructing fingerprint of kidney-nourishing and fetus-nourishing pill as claimed in claim 1 or 2, wherein the sample solution is prepared by refluxing or ultrasonic method.
6. A quality detection method of the kidney nourishing and fetus-growing pill based on the fingerprint of the kidney nourishing and fetus-growing pill constructed by the construction method of any one of claims 1 to 5 is characterized by comprising the following steps:
preparation of a reference solution: selecting at least one of chlorogenic acid, loganin, swertiamarin, loganin and teasel root saponin VI as reference substances, dissolving with organic solvent, and preparing reference substance solution;
preparing a solution of a to-be-detected product: weighing the kidney-nourishing and fetus-growing pill to be detected, and dissolving the kidney-nourishing and fetus-growing pill by using an organic solvent to prepare a test solution;
and (3) quality detection: testing the reference solution and the solution to be tested by adopting a high performance liquid chromatography to obtain a reference chromatogram and a chromatogram of the to be tested; analyzing the common peak of the chromatogram of the to-be-detected product and the fingerprint of the kidney-nourishing and fetus-growing pill, and the chromatogram of the to-be-detected product and the chromatogram of the reference product;
the organic solvent is selected from methanol or a methanol water solution with the volume content of 20-80%;
the conditions adopted by the high performance liquid chromatography comprise:
the chromatographic column is selected from Kromasil C 18 、Venusil HILIC、Bolitimate C 18 Any one of (a);
the mobile phase is acetonitrile A-0.1-0.5% phosphoric acid water solution B;
the elution gradient was:
0～50min，6％～11％A；
50～52min，11％～17％A；
52～80min，17％～28％A；
80～95min，28％～40％A；
the detection wavelength is 230-240 nm and 210-215 nm;
the flow rate is 0.8-1.2 mL/min -1 The column temperature is 28-32 ℃, and the sample injection amount is 8-12 mu L.
7. The method for detecting the quality of the kidney-nourishing and fetus-nourishing pill as claimed in claim 6, wherein the chromatographic column is Kromasil C 18 (ii) a The mobile phase is acetonitrile A-0.1 percent phosphoric acid water solution B; the detection wavelength is 235nm and 212 nm; the flow rate is 1.0mL min -1 The column temperature was 30 ℃ and the amount of sample was 10. mu.L.
8. The method for detecting the quality of the kidney-nourishing and fetus-nourishing pill as claimed in claim 6 or 7, wherein the preparation of the test solution adopts a reflux method or an ultrasonic method.
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