WO2012129847A1

WO2012129847A1 - Methods for simultaneously determinating multi-component of simotang and fingerprint building method thereof

Info

Publication number: WO2012129847A1
Application number: PCT/CN2011/075808
Authority: WO
Inventors: 易跃能; 刘令安; 王长虹; 程雪梅; 杨华; 邓义德; 刘东文
Original assignee: 湖南汉森制药股份有限公司
Priority date: 2011-03-25
Filing date: 2011-06-16
Publication date: 2012-10-04
Also published as: CN102221590A; CN102221590B

Abstract

Provided are methods for simultaneously determinating multi-component of Simotang and fingerprint building method thereof. The Simotang is prepared from vladimiria root, bitter orange, spicebush root and areca seed. In the method the Simotang sample solution is detected by HPLC, with a column of octadecyl silane bonded silica gel column, acetonitrile as the mobile phase A, 0.1%(by volume)formic acid aqueous solution as the mobile phase B, using gradient elution, and with the detection wavelength of 283nm. The fingerprint of the Simotang is built by the said determination method, and then by computer simulation. The determination method is simple with high specificity, excellent stability, precision, reproducibility and average recovery, and can detect a variety of active ingredients of the Simotang, thereby, scientific and rational fingerprint can be obtained, and effectiveness, security, stability and homogeneity of the Simotang can be completed and accurately evaluated.

Description

Simultaneous determination of multi-index components of Si Mo Tang preparation and its fingerprint mapping method

Technical field

The invention relates to a method for simultaneously determining a multi-index component of a Si Mo Tang preparation, and a method for constructing a fingerprint of a Si Mo Tang preparation. Background technique

The four-bred Chinese medicine includes four kinds of herbs: woody, oyster shell, black peony and betel nut. The side has the effect of reducing qi and reducing the pain, eliminating the pain and eliminating pain. It can be used for infants and young children with milk stagnation syndrome (see bloating, abdominal pain, crying, anorexia, diarrhea or constipation), middle-aged qi stagnation, food dysfunction (certificate) See abdominal fullness, abdominal pain, constipation), and the recovery of gastrointestinal function after abdominal surgery. In addition, there are reports of successful cases for the treatment of neonatal jaundice, gastroesophageal reflux, viral enteritis, constipation-type irritable bowel syndrome and toxic intestinal paralysis.

In the prior art, according to the traditional Chinese medicine prescription of Si Mo Tang, various preparations of the Si Mo Tang preparation have been prepared. The usual preparation process is generally as follows: The four kinds of original medicinal materials are extracted by steam distillation to extract the aromatic water, and the aromatic water is enriched in the volatile oil, and then directly used or packaged for later use, and then the slag is extracted with water to obtain an aqueous extract, and the water is extracted. The liquid can be concentrated and dried to obtain a dry paste powder, and the above-mentioned aromatic water or volatile oil, as well as an aqueous extract or a dry paste powder, and an appropriate auxiliary material is selected, thereby preparing an oral liquid, a dropping pill, an emulsifier, a capsule, a soft capsule, a tablet, A variety of dosage forms such as granules.

For example, Chinese Patent No. 94110842.2 (Publication No. CN1106288) discloses a four-mill soup mouth liquid. It adopts Chinese traditional medicine betel nut 10~25%, woody incense 10~25%, oyster shell 20~30%, black peony 20~30%, take the above-mentioned medicinal herbs to extract aromatic components, and save; the slag is added with water to cook, and the filtrate is combined. , Concentrate, add ethanol to stand, filter the clear liquid, recover the ethanol under reduced pressure, add water to boil, filter to remove the residue, add the aromatic component water and appropriate amount of auxiliary materials to the filtrate, filter, dispense and sterilize.

The four-mill soup includes betel nut, woody, clam shell and black peony. The ingredients are complex and diverse. According to reports in the literature, the main components of woody agaric include acarids, alkaloids, body and amino acids, and the main components of the clam shell include flavonoid glycosides. Olefin and alkaloids, the main components of black peony include steroids and alkaloids, and the main components of betel nut include alkaloids, tannins and amino acids.

However, the existing quality standard of Si Mo Tang Oral Liquid is the Ministry of Standards WS-10040 (ZD-0040) -2002. The standard "identification" item uses TLC method to identify woody and betel nut: Take the finished product of Si Mo Tang Oral Liquid, The organic solvent is used for extraction, and is prepared into a test solution after pretreatment; the reference drug is extracted with a suitable organic solvent, and is prepared into a reference drug solution after pretreatment; the test solution and the reference drug solution are plated on a thin layer of silica gel G. The plate is developed with a suitable developing agent, and the coloring agent is developed to identify the medicine. "Content determination" item was determined by high performance liquid chromatography with the content of naringin as an indicator: octadecyl silicon germanium bonded silica as a filler; methanol-1% acetic acid (38: 62) as mobile phase; detection wavelength It is 283 nm. The finished product of Si Mo Tang Oral Liquid is eluted by D _1Q1 macroporous adsorption resin column, and the eluent is collected to prepare a test solution; the reference substance of naringin is prepared as a reference solution; the naringin is calculated by external standard method content. This standard does not quantitatively detect other active ingredients, and it has certain limitations and cannot fully reflect the quality level of traditional Chinese medicine products.

Fingerprint technology is a newly developed method for quality control of traditional Chinese medicine. However, the usual fingerprints remain in the evaluation of fingerprint identification or overall similarity, focusing on macroscopic features, and do not reflect well on microscopic features such as specific chromatographic peak chemical information and material content. Simultaneous determination of combined fingerprinting techniques with multiple indicator components is a quality control method developed in parallel. It combines spectral efficacy research methods and separation techniques to identify the main active components (or major active ingredient groups) of traditional Chinese medicines, and to determine the index components for key monitoring of fingerprints. Simultaneous determination of multiple fingerprints combined with fingerprint technology not only focuses on macroscopic features, but also monitors the content of multiple active ingredients. It can achieve microscopic and macroscopic organic combination, conforming to the overall, macroscopic and complex characteristics of traditional Chinese medicine, and can evaluate and control traditional Chinese medicine as a whole. The quality of the preparation is one of the key technologies for the modernization of traditional Chinese medicine.

For the Si Mo Tang preparation, it is urgent to establish a detection method, construct a multi-indicator component and simultaneously measure the joint fingerprint pattern technique to more comprehensively and accurately monitor the quality of the Si Mo Tang preparation.

Summary of the invention The technical problem to be solved by the present invention is to overcome the defects of the quality detection method of the existing four-mill soup preparation, and to comprehensively reflect the defects of the quality of the Si Mo Tang preparation, and to provide a multi-index component of the Si Mo Tang preparation. Simultaneous determination method, and construction method of fingerprint of Si Mo Tang preparation.

The invention determines the main index components of the fingerprint to be monitored by the spectral effect method and the separation and purification technology, namely, deisopoldine, isophylloside, naringin, hesperidin, neohesperidin and sorbus Potassium acid. After a glimpse of the experiment, a test method with strong specificity, simple operation, and good stability, precision, reproducibility and sample recovery rate was established, thus establishing a scientific and rational four-mill soup preparation for the first time. Multiple indicator component fingerprints.

The method for simultaneously determining the multi-index component of the Si Mo Tang preparation of the present invention comprises the following steps: the test solution of the Si Mo Tang preparation is subjected to high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography are performed; The column is an 18-inch silicon germanium bonded silica column, the mobile phase A is acetonitrile, and the mobile phase B is 0.1% by volume aqueous formic acid. The elution gradient is shown in Table 1. The detection wavelength is

In the present invention, the traditional Chinese medicine prescription of the Si Mo Tang is an existing Chinese medicine prescription, which comprises four kinds of herbs of woody, oyster shell, black peony and betel nut, and the ratio of parts by weight of each medicinal material is generally 100: 50-150: 50- 150: 50~150. The preparation of the four-mill soup can be the preparation of the existing four-mill soup according to various dosage forms prepared by the traditional Chinese medicine of Si Mo Tang, such as oral liquid (such as Si Mo Tang oral liquid disclosed in Chinese Patent No. CN1106288), dropping pills, emulsification. Agents, capsules (including soft capsules), tablets or granules, and the like. The above-mentioned four-mill soup preparation can be as follows The method comprises the following steps: preparing the volatile component extracted by the steam distillation method from the original medicinal material and the aqueous extract obtained by extracting the slag by water extraction and alcohol precipitation, and preparing each dosage form according to the existing method.

In the present invention, the test solution of the above-mentioned Si Mo Tang preparation can be prepared by a conventional pretreatment method of a chromatographic test solution. Specifically, for the Si Mo Tang Oral Liquid, it can be directly filtered through a microporous membrane, and the filtrate can be diluted without dilution or diluted to 100 times the original volume (the dilution solvent can be water or a mobile phase A and B with a volume ratio of 1:1). The mixed solution), that is, the test solution. The pore diameter of the microporous membrane can be selected according to conventional conditions, and is generally 0.22 to 0.8 μm, preferably 0.45 μm. Various types of microporous membranes are suitable for use in the present invention, such as polyethersulfone microporous membranes. For the four-mill soup preparations of other dosage forms (such as other liquid preparations or solid preparations), the solution in which the Si Mo Tang preparation is dissolved may be sonicated, cooled, and then filtered through a microporous membrane (same as described above), and the filtrate is supplied. Sample solution. Wherein, the solvent in the solution in which the Si Mo Tang preparation is dissolved may be methanol, ethanol, 40% by volume or more of methanol aqueous solution, 40% by volume of aqueous ethanol solution, chloroform, ethyl acetate, diethyl ether, petroleum Ether, acetone or cyclohexane, the first four are preferred. The amount of the solvent used is such that the concentration of the Si Mo Tang preparation is suitable for chromatographic detection. Specifically, according to the concentration of the above-mentioned four-milling oral liquid for the test solution, the same concentration can be calculated according to the dosage of the other four-milling preparations. The ultrasonic treatment conditions are preferably 160 to 250 W, a frequency of 40 kHz, and an ultrasonic wave of 20 to 60 minutes.

In the present invention, the injection amount of the high-performance liquid chromatography detection is selected according to the conventional knowledge in the field of chromatographic detection, and is generally 2 to 50 μM, preferably 5 to 2 (^L is preferred.

In the present invention, the column can be an existing 18-inch silicon germanium bonded silica column of various types and specifications, such as 4.6mmX 250mm, 4.6mm X 150mm, etc. 4.6mmX 250mm. The present invention is particularly preferably a Venusil XBP-C ₁₈ column (4.6 mm X 250 mm) or an Agilent ZORBAX SB-C ₁₈ column (4.6 mm X 250 mm), most preferably the former.

In the present invention, the elution gradient is as shown in Table 1. In order to obtain the best separation effect, when different octadecyl silicon germanium bonded silica gel columns are used, the optimum selection can be made within the range shown in Table 1. When a particularly preferred Venusil XBP-C ₁₈ column (4.6 mm X 250 mm) or an Agilent ZORBAX SB-C ₁₈ column (4.6 mm X 250 mm) of the present invention is used, the elution gradient as shown in Table 2 is most preferred. Table 2

In the present invention, the column temperature has little effect on the separation effect, and a temperature range which can be used by a conventional column can be used, and is generally 15 to 50 ° C, preferably 20 to 40 ° C.

In the present invention, the flow rate has little effect on the separation effect, and can be selected according to a conventional method in the art, and is generally 0.5 to 1.5 mL / min, preferably 0.8 to 1.2 mL / min.

The number of theoretical plates of the high performance liquid chromatography of the present invention is not less than 3,000 based on naringin.

The invention further relates to a method for constructing a fingerprint of a Si Mo Tang preparation, which comprises the following steps: using the above method to detect the Si Mo Tang preparation, and then using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system software (National Pharmacopoeia Commission 2004A 1.0 version) Simulate the fingerprint of the four mill soup preparations.

Wherein, the number of samples of the tested Si Mo Tang preparation is more than the number of samples required to establish the minimum fingerprint, generally 10 to 30, and the invention is illustrated by taking 26 batches of samples as an example.

Among them, the traditional Chinese medicine chromatographic fingerprint similarity evaluation system software simulation generates the fingerprint of the Si Mo Tang preparation as usual.

The above preferred conditions can be arbitrarily combined on the basis of the common knowledge in the art, that is, the preferred examples of the present invention.

The reagents and starting materials used in the present invention are commercially available.

The positive effect of the invention is: The invention provides a method for simultaneously determining the multi-index component of the Si Mo Tang preparation and the method for constructing the fingerprint of the Si Mo Tang preparation on the basis of clarifying the main active ingredients of the Si Mo Tang. The measuring method of the invention has strong specificity, simple operation, excellent stability, precision, reproducibility and sample recovery rate, and can comprehensively detect various main active ingredients in the Si Mo Tang preparation, thereby constructing science Reasonable four-milled soup fingerprint, complete and accurate evaluation of four mills The effectiveness, safety, stability and quality uniformity of the soup preparation. DRAWINGS

Fig. 1 is a chromatogram obtained by using four different chromatographic columns to determine the solution of the tetrarishamine oral solution for the test solution. 1A is employed Venusil XBP-C ₁₈ column (4.6mmX250mm), using FIG. IB is a Agilent ZORBAX SB-C ₁₈ column (4.6mm X 250mm).

2 is a chromatogram of a sample solution and a standard of the Si Mo Tang Oral Liquid in Example 3. Figure 2A shows the sample solution of the Si Mo Tang Oral Liquid, and Figure 2B shows the standard. Among them, 2 is norbibeldine, 3 is new North American erioside, 4 is isophylloside, 5 is naringin, 6 is hesperidin, 8 is neohesperidin, 10 is potassium sorbate .

Figure 3 is a chromatogram of HPLC analysis of different concentrations of acetonitrile -0.1% aqueous formic acid (the mobile phase conditions of the present invention) in Example 5-1, wherein A, B, C and D are Table 3-1, respectively.

Chromatograms of the elution gradients shown in 3-2, 3-3, and 3-4.

Figure 4 is a chromatogram of HPLC analysis of different gradients of acetonitrile-ammonium acetate buffer (compared to mobile phase conditions) in Example 5-2, wherein A, B, C and D are Tables 4-1 and 4-, respectively. 2,

Chromatograms of the elution gradients shown in 4- 3 and 4-4; 1 is the chromatographic peak of potassium sorbate.

Figure 5 is a quantitative limit detection chromatogram of the third standard (norgosole, metapoverin, naringin, hesperidin, neohesperidin and potassium sorbate) in Example 8. In Fig. 5A, 4 is isoquertoside, 5 is naringin, 6 is hesperidin, 8 is neohesperidin, 10 is potassium sorbate, and in Fig. 5B, 2 is norbibeldine.

Figure 6 is a chromatogram of the finished product of 26 batches of Si Mo Tang Oral Liquid in Example 9 (Fig. 6A), and a fingerprint of Si Mo Tang Oral Liquid (Fig. 6B). detailed description

The invention is further illustrated by the following examples, which are not intended to limit the invention. Determination of the detection index component in the reference embodiment

Using blood medicinal chemistry, pharmacokinetics and other means and methods, the blood components (flavonoids, lactones, alkaloids) in the Si Mo Tang preparations were identified, and the monomer components with higher content of blood components were identified. Separation and purification are carried out, and the index component of the fingerprint map to be monitored is determined by the carbon enthalpy propulsion rate index.

Monomer component activity test: Each monomer component was mixed with physiological saline (pH-assisted solution if necessary), and administered to a solution of 0.30 ml/10 g body weight in a volume of 10 ml. The dosage is shown in Table 3. , respectively, to determine the carbon sputum propulsion rate of each group (Liu Lingan, Cai Ying, Yan Xiaoyuan, et al, the effect of Si Mo Tang on gastrointestinal motility in mice with different functional status] [J]. TCM Herald, 2009, 15(12): 64-66 ), as shown in Table 3: Table 3

* P < 0.5 compared with the blank group, ** P < 0.1 compared with the blank group.

According to the above data, arecoline is the main monomer in the four-mill soup to promote small bowel movements. Peel, naringin, hesperidin, neohesperidin and norbiphordine also play a role.

In addition, potassium sorbate is a preservative for Si Mo Tang Oral Liquid. Compared with other preservatives, it has better antibacterial and antiseptic effects and lower toxicity. Although potassium sorbate is not an active ingredient of Si Mo Tang preparation, its content can be controlled to meet the needs of the quality stability of the preparation during the effective period, and to comprehensively control the quality of the Si Mo Tang preparation. Therefore, potassium sorbate as a characteristic indicator component is of great significance for the quality control of Si Mo Tang preparation.

Example 1 Determination of Detection Wavelength

1. Materials and methods:

Test solution of Si Mo Tang preparation: Si Mo Tang Oral Liquid (Hunan Hansen Pharmaceutical Co., Ltd., batch number 090436) 5 pieces, mixed evenly, using microporous membrane (0.45μηι, polyethersulfone, SCAA-101 water) The phase needle filter) is filtered, which is the test solution.

High performance liquid chromatography detection: Precision extraction of 5 L of the test solution, injection into high performance liquid chromatography for determination. Chromatographic conditions: column was Venusil XBP-C ₁₈ column (4.6mm X 250mm), a column temperature of 30 ° C, a flow rate of lml min- ^1, acetonitrile mobile phase A, mobile phase B 0.1% by volume aqueous formic acid The elution gradient is shown in Table 2. The detection wavelength is 190~400 nm, and the peaks in 45 min and their retention time and peak area are recorded.

Table 2

2, the experimental results

On-line detection of the test solution in the ultraviolet region of 190~400 nm by using a diode array detector revealed that the obtained peaks were mainly flavonoids in the clam shell and the raw materials in the black peony. Alkaloids. In the black medicinal herb, the maximum absorption is at 280 nm. The ultraviolet absorption behavior of flavonoids in the oyster shell is very similar. The absorption is the strongest at 283 nm, and the information provided is the most abundant. The ratio is suitable, so a comprehensive selection of 283 nm is used as the detection wavelength.

Example 2 Optimization of the column

1, materials and methods

The test solution of the Si Mo Tang preparation was the same as in Example 1.

High performance liquid chromatography detection: The column was a Venusil XBP-C ₁₈ column (4.6 mm X 250 mm) or an Agilent ZORBAX SB-C ₁₈ column (4.6 mm X 250 mm) with a detection wavelength of 283 nm, and other conditions were the same as in Example 1.

2, the experimental results

The chromatogram obtained using a Venusil XBP-C ₁₈ column (4.6 mm X 250 mm) is shown in Figure 1A, and the chromatogram obtained using an Agilent ZORBAX SB-C ₁₈ column (4.6 mm X 250 mm) is shown in Figure 1B. In contrast, the Venusil XBP-C ₁₈ column (4.6mm X 250mm) column is used for chromatographic separation, and the eluted peak is more stable (see Figure 1), so the Venusil XBP-C ₁₈ column (4.6mm X 250mm) is preferred. column.

Example 3 Identification of characteristic peaks and reference peaks

1, materials and methods

The test solution of the Si Mo Tang preparation was the same as in Example 1.

Preparation of standard products: Accurately weigh naringin, hesperidin, neohesperidin, isophylloside, norbiphordine, potassium sorbate, accurately weighed, add methanol to make each 1 ml LMG containing naringin, hesperidin 25μ _§, neohesperidin 0.45 mg, isonaringin 80μ _§, methyl isobutyl wave set to 45μ _§, potassium sorbate 0.2mg solution, that was the standard.

High-performance liquid chromatography detection method: respectively, accurately extract the standard product and the test solution 5μ1, respectively, and inject them into high performance liquid chromatography for measurement. The column was a Venusil XBP-C ₁₈ column (4.6 mm X 250 mm) with a detection wavelength of 283 nm, and other conditions were the same as in Example 1. 2, the experimental results

The chromatograms of the test solution and the standard of the Si Mo Tang Oral Liquid are shown in Fig. 2A and Fig. 2B, respectively. Among them, 2 is norbibelol (retention time: 13.1min), 3 is new North American ergoside (retention time: 19.9 min), 4 is isoquerment (retention time: 21.7 min), 5 is pomelo Decoction (retention time: 22.6 min), 6 is hesperidin (retention time: 23.2 min), 8 is neohesperidin (retention time: 24.0 min), and 10 is potassium sorbate (retention time: 25.1 min).

Example 4 Detection time

1, materials and methods

The test solution of the Si Mo Tang preparation was the same as in Example 1.

High performance liquid chromatography detection method: The detection time was 70 min, and other conditions were the same as in Example 3.

2, the experimental results

The peak of 70 min was recorded, and there was no peak after 40 min, suggesting that the peaks of the four mills were completely within 40 min. Therefore, it is appropriate to select a detection time of 40 to 50 minutes.

Example 5 Elution conditions

1. Different gradients of mobile phase conditions of the acetonitrile-0.1% formic acid aqueous solution of the invention

The column is a Venusil XBP-C ₁₈ column (4.6mmX 250mm) column, the mobile phase A is acetonitrile, and the mobile phase B is 0.1% by volume aqueous formic acid. The elution gradient is shown in Table 3-1, 3-2, 3-3. As shown in Figures 3-4, the column temperature is 30 ° C, the flow rate is 1.0 ml min ^-1 , the detection wavelength is 283 nm, and the chromatogram is shown in Figures 3A, B, C and D.

Table 3-1

Table 3-2 Time (minutes) mobile phase (v/v%)

0 to 5 10% mobile phase A and 90% mobile phase B

5 to 23 linear gradient changes to: 25% mobile phase A and 75% mobile phase B

23 to 35 linear gradient changes to: 92% mobile phase A and 8% mobile phase B

35 to 45 92% mobile phase A and 8% mobile phase B

Table 3-3

Time (minutes) mobile phase (v/v%)

0 to 5 15% mobile phase A and 85% mobile phase B

5 to 20 linear gradient changes to: 40% mobile phase A and 60% mobile phase B

20 to 32 linear gradient changes to: 100% mobile phase A and 0% mobile phase B

32 to 40 100% mobile phase A and 0% mobile phase B

Table 3-4

It can be seen from the above experiments that under the elution condition of the present invention, when the proportion of the mobile phase changes within a certain range, the influence on the resolution is small, and the relative peak order of each component is relatively uniform, and the condition has certain durability. Sex, so it is more convenient and practical to determine the acetonitrile and 0.1% formic acid system.

2. Comparison of mobile phase conditions

The column was a Venusil XBP-C ₁₈ column (4.6 mm X 250 mm) with a column temperature of 30 ° C, a flow rate of ¹ mL-min" ¹ , and a detection wavelength of 283 nm.

Comparison of mobile phase and elution gradient: Prepare with acetonitrile (mobile phase A) - ammonium acetate buffer (mobile phase C) as mobile phase, ammonium acetate buffer: ammonium acetate 1.5g + 2ml glacial acetic acid + 1000ml water. Elution was performed using the elution gradients shown in Tables 4-1, 4-2, 4-3, and 4-4. The chromatograms are shown in Figure 4A and Figure 4B, respectively. Figures 4C and 4D are shown.

Table 4-1

As shown in Fig. 4, B, C and D, the above-mentioned acetonitrile-ammonium acetate buffer was used as the mobile phase, and the retention time of the chromatographic peak changed greatly. Taking potassium sorbate as an example, when the gradient elution conditions change, the retention time of the chromatographic peak changes greatly, and it may partially overlap with the chromatographic peaks of naringin, hesperidin or neohesperidin in the clam shell or Completely overlapping. Therefore, the method established using this mobile phase lacks certain durability. Moreover, the instrument and column maintenance requirements are higher after elution with buffer salts.

Example 6 Effect of Flow Rate and Column Temperature on Separation Effect

The column was a Venusil XBP-C ₁₈ column (4.6 mm X 250 mm) column, mobile phase A was acetonitrile, and mobile phase B was a 0.1% by volume aqueous solution of formic acid. HPLC analysis was carried out according to the elution gradient of Example 1. Flow rate and column temperature, as well as retention time, symmetry factor, resolution and theoretical plate number As shown in Table 5;

table 5

It can be seen from Table 5 that the flow rate and column temperature have little effect on the separation effect of the method of the present invention, and the above conditions Both can achieve better separation results.

Example 7 Pretreatment method of various four-mill soup preparations

(1) Pretreatment of Si Mo Tang Tablets

Take 20 tablets of four-milled soup, grind it in a mortar, weigh 2g into a lOOmL volumetric flask, add 90mL of methanol to sonicate for 30min, add methanol to the mark after cooling, shake well, use 0.22μηι polyethersulfone microporous filter The membrane was filtered, and the filtrate was diluted 50 times to obtain a test solution.

(2) Pretreatment of Si Mo Tang Capsules

Take 20 capsules of Si Mo Tang, grind it in a mortar, weigh 2g into a lOOmL volumetric flask, add 90mL of ethanol to sonicate for 30min, then add ethanol to the scale after cooling, shake well, use 0.45μηι polytetrafluoroethylene microporous filter The membrane was filtered, and the filtrate was diluted 60 times to obtain a test solution.

(3) Pretreatment of Si Mo Tang Pills

Take 20 tablets of Si Mo Tang Dropping Pills, grind it in a mortar, weigh 2g into a 100mL volumetric flask, add 90mL volume to 60% methanol aqueous solution for 30min, dissolve, add 60% methanol solution to the mark after cooling, shake well It was filtered through a 0.80 μηι cellulose acetate microporous membrane, and the filtrate was diluted 80 times to obtain a test solution.

(4) Pretreatment of Si Mo Tang Soft Capsules

Take 50 capsules of four-milled soft capsules, take out the contents, weigh 2g of content into a lOOmL volumetric flask, add 90mL of methanol for 30min to dissolve, add methanol to the mark after cooling, shake well, use 0.45μηι polytetrafluoroethylene microporous The membrane is filtered, and the filtrate is diluted 70 times to obtain a test solution.

(5) Pretreatment of Si Mo Tang Granules

Take 20 packets of Si Mo Tang granules, grind and grind, weigh 2g into a lOOmL volumetric flask, add 90mL 70% ethanol aqueous solution to sonicate for 30min, then add 70% ethanol solution to the mark after cooling, shake well, 0.45μηι The fluoroethylene microporous membrane was filtered, and the filtrate was diluted 80 times to obtain a test solution.

Example 8 Methodological study on the detection method of the four-mill soup preparation of the present invention

The high performance liquid chromatography conditions of the following experiment were the same as in Example 3.

1, system adaptability test Materials and methods were the same as in Example 3. The number of theoretical plates in the assay is not less than 3,000 based on naringin.

2, standard curve and linear range

Take naringin, hesperidin, neohesperidin, isophylloside, norbiphordine, potassium sorbate, accurately weighed, set separately in different measuring flasks, add methanol to make each 1ml separately A solution containing 1.0164 mg of naringin, 0.2412 mg of hesperidin, 0.9644 mg of new hesperidin, 0.4024 mg of isoquertoside, 0.4324 mg of norbipoldine, and 2.5236 mg of potassium sorbate. Shake well. liquid. Each solution was diluted according to the concentrations in Tables 6-1, 6-2, 6-3, 6-4, 6-5, 6-6, and detected by HPLC. The injection volume was 5μ1. The data is shown in Table 6-1~6- 2. Draw a standard curve, see Table 7.

Table 6-1 Noisoprene standard curve data

Table 6-2 Isophylline standard curve data

Table 6-6 K-sorbate standard curve data C g/mL 1261.8 504.72 252.36 100.944 50.472 10.0944 5.0472 2.5326 0.6309

A 20854.9 8781.7 4448.3 1802.8 954 183.4 92 45.5 11.68 Standard curve and linear range of each indicator component

3. Limit of quantitation (LOQ) and limit of detection (LOD)

The standard samples (norgosine, isophylloside, naringin, hesperidin, neohesperidin and potassium sorbate) are shown in Table 8. The signal-to-noise ratio S/N= 10 or 3, which are the limit of quantitation and detection limit of each of the above indicators. The quantitative limit chromatograms of the various indicator components are shown in Figures 5A and 5B.

Table 8 Quantitative limits (LOQ) and detection limits (LOD) for each indicator

4, precision inspection

Three standard solutions of different concentrations were taken. The concentrations of norbipotrel were 43.24 g/mL, 86.48 g/mL, and 172.96 g/mL, respectively. The concentrations of isoquertoside were 40.24 g/mL and 80.48, respectively. g/mL, 402.4μ _§ /ηΛ, the concentration of naringin was 101.64 g/mL, 203.3 (Vg/mL, 1016.4 g/mL, the concentration of hesperidin was 24.12 g/mL, 48.24 g/mL, respectively. At 241.2 g/mL, the concentrations of neohesperidin were 96.44 g/mL, 192.88 g/mL, and 964.4 g/mL, respectively. The potassium sorbate concentrations were 50.472 g/mL, 252.36 g/mL, and 504.72 g/mL, respectively. The sample volume was 5 L, and the concentration of each group was repeated 6 times. HPLC detection, determination of the average peak area, calculation of RSD. Three consecutive days of testing, counting Calculate RSD, the results are shown in Tables 9 and 10.

Table 9 Intraday precision measurement results (n=6)

It can be seen from Table 9 that on the same day, the RSD of each concentration component of each indicator component is less than 3%, indicating that the precision of the measurement method is good within days.

Table 10 Daytime precision measurement results (n=6) Peak area average peak area average peak area average indicator component RSD %

(Day 1) (Day 2) (Day 3)

447.63 458.3 449.2 0.76 norbibeldine 923.60 939.1 901.6 1. 33

1858.85 1889.8 1861.7 1. 17

300.82 295.2 313.2 1.85 Isophylline 588.97 599.1 602.3 0.97

2976.03 3052.2 2969 2.57

812.92 795.3 844.1 1.88 Naringin 1649.98 1677.2 1686.4 0.94

8157.45 7996 7675.1 2.17

222.68 217.7 230.6 1.84 Hesperidin 437.28 444.4 446.7 0.93

2217.87 2091.8 2196.7 2.15

879.68 858.5 910.1 1.77 New hesperidin 1769.98 1799.8 1810.4 0.96

9032.72 8979.8 9183.6 0.69

962.02 961.9 989 1.36 potassium sorbate 4583.25 4489.1 4448.3 1.44

9150.50 9419.6 9483.6 1.72

This indicates that the measurement method has good daytime precision.

5, sample stability

Take the Si Mo Tang Oral Liquid (Hunan Hansen Pharmaceutical Co., Ltd., batch number 090436), prepare the test solution according to the method of Example 1, and perform HPLC detection. The results are shown in Table 11.

Table 11 Stability measurement results Time (h) 0 4 6 8 15 18 24 RSD % norbiphorol 538.1 505.2 537.8 539.8 512.9 547.8 537.9 2.97 Isophylline 776.8 745.7 776.7 779.5 752.9 784.7 764.7 1.91 Naringin 8003.7 7983.9 8023.2 8034.9 8057.1 8110.6 8029.1 0.51 Hesperidin 263 250.3 262.5 263.8 251.3 265.5 255.9 2.43 Neohesperidin 4236.3 4230.4 4250.9 4258.9 4266.4 4294.1 4258.4 0.49 Potassium Sorbate 20607.6 20653.5 20679.1 20743.3 20745.4 20472 20641.67 0.46 As can be seen from Table 11, the sample is stable within 24 h, RSD is 0.46%~ 2.97%.

6, repeatability experiment

Take Si Mo Tang Oral Liquid (Hunan Hansen Pharmaceutical Co., Ltd., batch number 090436), and prepare 6 samples for the test solution according to the method of Example 1, and perform HPLC detection. The results are shown in Table 12.

Table 12 Repeatability results

As can be seen from Table 12, the RSD of each indicator component is 0.24% to 2.36%, indicating that the detection method has good repeatability.

7, sample recovery experiment

Precisely weighed the standard naringin, hesperidin, neohesperidin, isophylloside, norbiphordine, potassium sorbate, and added methanol to make 1.0164mg of naringin per orange. 0.2412 mg of dermatan, 90,864 mg of hesperidin, 0.4024 mg of isoquertoside, 0.432.4 mg of norbiphordine, sorbic acid Potassium 2.5236mg solution, shake well, that is, the respective stock solution, spare.

Precisely measure the appropriate amount of Si Mo Tang Oral Liquid (Hunan Hansen Pharmaceutical Co., Ltd., batch number 090436), filter the pretreatment according to the method of Example 1, and place it in a 25 mL volumetric flask. For each indicator component, add the standard sample containing 100% of the index component in the sample solution, add methanol to the mark 10ml, shake it, and perform HPLC measurement. Calculate the recovery rate according to the following formula. See Table 13.

Addition recovery rate %= (measured amount - the amount of the indicator component contained in the test solution) / actual addition amount χ ΐοο% Table 13-1 Sample test results of deisomethodazole

Table 13-3 Results of different naringenin sample recovery experiments Sample volume average content addition amount measurement amount recovery rate average recovery rate RSD

(mL) (mg/mL) (mg) (mg) (%) (%) (%)

1.006 2.0668 103.86

1.006 2.0487 102.06

1.006 1.9880 96.02

10 0.1022 101.52 2.78

1.006 2.0460 101.79

1.006 2.0482 102.01

1.006 2.0620 103.38

Table 13-4 Results of hesperidin recovery test

Table 13-5 Results of new hesperidin sample recovery test

Table 13-6 Experimental results of potassium sorbate sample recovery Sample amount average content addition amount measured amount recovery rate average recovery RSD

(mL) (mg/mL) (mg) (mg) (%) rate (%) (%)

3.0283 6.0537 99.78

3.0283 6.0309 97.03

3.0283 6.0791 100.62

2.5 1.2128 99.27 1.41

3.0283 6.0334 99.11

3.0283 6.0428 98.42

3.0283 6.0800 100.65

As can be seen from Tables 13-1 to 13-6, the detection method of the present invention has a good sample recovery rate for each index component.

Example 9 Construction of Fingerprint of Si Mo Tang Preparation

The sample was prepared in 26 batches of Si Mo Tang Oral Liquid, and the test solution was prepared according to the method of Example 3, and subjected to HPLC detection. The chromatogram is shown in Fig. 6A. Fig. 6B is a fingerprint of the four-milling oral liquid.

Selection of characteristic fingerprint peaks: According to the determination results of 26 batches of Si Mo Tang oral liquid, with the naringin standard as the reference peak, 9 characteristic fingerprint peaks were identified from 20 characteristic fingerprint peaks, namely, the de-isolated wave Erding (No. 3), New North American Glycyrrhizin (No. 6), Isoflavone (No. 7), Naringin (No. 8), Hesperidin (No. 9), Neohesperidin (No. 11) Potassium sorbate (No. 13), Chuan Chenshen (No. 18) and Red Tangerine (No. 19).

The establishment of fingerprint mapping standards: using the Chinese Pharmacopoeia Committee recommended traditional Chinese medicine chromatographic fingerprint similarity evaluation system software operating specifications (2004A 1.0 version), using the mean method, the experimental data of 26 batches of Si Mo Tang oral liquid products, through Select the peak, set the matching mode, automatically match the peaks, and then set the standard template for the peak difference evaluation and overall similarity evaluation. According to the results of 26 batches of samples, the fingerprint of the TCM Tangkou solution was generated by the chromatographic fingerprint similarity evaluation system software of Chinese medicine (Fig. 6B). The similarity results of the chromatograms of each sample compared with the fingerprint are shown in Table 14. The retention time of each characteristic peak in the fingerprint is based on the standard value of the naringin standard, and the relative retention times of the other peaks are shown in Table 15.

Table 14 Similarity evaluation of 26 batches compared to the common mode Sample number batch vendor similarity

SI 070109 Hunan Hansen Pharmaceutical Co., Ltd. 1.000

S2 070419 Hunan Hansen Pharmaceutical Co., Ltd. 0.999

S3 070706 Hunan Hansen Pharmaceutical Co., Ltd. 0.998

S4 071017 Hunan Hansen Pharmaceutical Co., Ltd. 0.999

S5 080121 Hunan Hansen Pharmaceutical Co., Ltd. 1.000

S6 080421 Hunan Hansen Pharmaceutical Co., Ltd. 0.997

S7 080712 Hunan Hansen Pharmaceutical Co., Ltd. 0.998

S8 081036 Hunan Hansen Pharmaceutical Co., Ltd. 0.988

S9 090225 Hunan Hansen Pharmaceutical Co., Ltd. 0.992

S10 090628 Hunan Hansen Pharmaceutical Co., Ltd. 0.991

Sl l 091026 Hunan Hansen Pharmaceutical Co., Ltd. 0.997

S12 091243 Hunan Hansen Pharmaceutical Co., Ltd. 0.995

S13 090424 Hunan Hansen Pharmaceutical Co., Ltd. 0.998

S14 090425-1 Hunan Hansen Pharmaceutical Co., Ltd. 0.996

S15 090426-1 Hunan Hansen Pharmaceutical Co., Ltd. 0.988

S16 090435-1 Hunan Hansen Pharmaceutical Co., Ltd. 0.996

S17 090436-1 Hunan Hansen Pharmaceutical Co., Ltd. 0.998

S18 060311-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.969

S19 060601-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.997

S20 060905-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.996

S21 061203-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.993

S22 070308-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.999

S23 070624-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.994

S24 070918-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.997

S25 071201-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.995

S26 080306-2 Hunan Zhongda Yuma Pharmaceutical Co., Ltd. 0.999 Based on the above test results, combined with the technical requirements of traditional Chinese medicine fingerprints, the preparation of the four-mill soup preparations meeting the following standards is a preparation that meets the quality standards: The chromatographic peaks in the figure should be consistent with the characteristic peaks of the fingerprint of Si Mo Tang Oral Liquid, and the similarity is not less than 0.90. Table 15 Relative retention schedule of each characteristic peak in the multi-index fingerprint of Si Mo Tang (minutes)

Claims

Rights request

A method for simultaneously determining a multi-indicator component of a four-mill soup preparation, characterized in that it comprises the following steps: the test solution of the Si Mo Tang preparation is subjected to high performance liquid chromatography, and the high-efficiency liquid is used; The chromatographic conditions of the chromatographic method are as follows: the column is an 18-inch silicon germanium bonded silica gel column, the mobile phase A is acetonitrile, and the mobile phase B is 0.1% by volume aqueous formic acid. The elution gradient is shown in Table 1

The detection wavelength was 283 nm.

2. The method according to claim 1, wherein: the preparation of the four-mill soup is mainly prepared from the following parts by weight of the original medicine: 100 parts of woody, 50 to 150 parts of clam shell, 50 to 150 A portion of black medicine and 50 to 150 parts of areca.

3. The method according to claim 2, wherein: the Si Mo Tang preparation is a Si Mo Tang preparation prepared by the following method: a volatile component extracted by steam distillation of the original medicine, and The aqueous extract of the dregs extracted by water extraction and alcohol precipitation is prepared into an oral liquid, a dropping pill, an emulsifier, a capsule, a tablet or a granule according to a conventional method.

4. The method according to claim 1, wherein: the test solution of the tetrabred soup preparation is prepared as follows:

When the said Si Mo Tang preparation is Si Mo Tang Oral Liquid, the Si Mo Tang Oral Liquid is directly filtered through a microporous membrane, and the filtrate is diluted or diluted to 100 times or less of the original volume, which is a test sample. Solution When the preparation of the Si Mo Tang preparation is other than the Si Mo Tang oral liquid, the solution in which the Si Mo Tang preparation is dissolved is sonicated, cooled, and then filtered through a microporous membrane, and the filtrate is used as a test sample. Solution.

The method according to claim 1, wherein the high-performance liquid chromatography detects an injection amount of 2 to 50 μM, preferably 5 to 20 μM.

6. The method according to claim 1, wherein: the column temperature detected by the high performance liquid chromatography is 15 to 50 ° C, preferably 20 to 40 ° C; and/or, The flow rate detected by high performance liquid chromatography is 0.5 to 1.5 mL/min, preferably 0.8 to 1.2 mL/min.

7. The method according to claim 1 or 6, wherein the octadecyl silicon germanium bonded silica gel column is a Venusil XBP-C ₁₈ column or an Agilent ZORBAX SB-C ₁₈ column.

8. The method according to claim 7, wherein: the octadecylsilicone-bonded silica gel column has a diameter of 4.6 mm X 250 mm or 4.6 mm X 150 mm.

The method according to claim 7 or 8, wherein: the elution gradient is as shown in Table 2;

Table 2

A method for constructing a fingerprint of a four-milled soup preparation, characterized in that it comprises the following steps: using the method according to any one of claims 1 to 9 to detect a preparation of a Si Mo Tang, and then using a chromatographic fingerprint similar to a traditional Chinese medicine; The evaluation system software simulates the fingerprint of the four-mill soup preparation.