CN109187825B - Method for measuring content of radix trichosanthis or medicine prepared by taking radix trichosanthis as raw material - Google Patents

Abstract

The invention provides a method for measuring the content of trichosanthes root or a medicine prepared by taking the trichosanthes root as a raw material, which adopts HPLC (high performance liquid chromatography) spectrogram spectrum detection and comprises the following operation steps: a. preparation of a test solution: adding diluted ethanol into a sample to be detected, performing ultrasonic treatment for 20min-40min, cooling, weighing, adding diluted ethanol to make up the lost weight, shaking, filtering, and collecting the filtrate; b. preparation of reference solutions: taking a proper amount of tryptophan reference substances, precisely weighing, and adding diluted ethanol solution to obtain the tryptophan reference substances; c. respectively and precisely absorbing the reference substance solution and the test solution, injecting the reference substance solution and the test solution into a liquid chromatograph, and eluting by using acetonitrile and water as mobile phases, wherein the chromatographic conditions are as follows: phenyl-hexyl silane bonded silica gel is used as a filling agent; acetonitrile-water (4-10:90-96) is used as a mobile phase; the flow rate is: 0.8-1.2 ml/min; the column temperature is 25-35 ℃; the detection wavelength is 224 nm; the number of theoretical plates should not be less than 3000 in terms of tryptophan peaks. The method for detecting the content of tryptophan in the radix trichosanthis has the advantages of strong operability, simple sample treatment process, simple mobile phase, high repeatability and short detection time.

Description

Method for measuring content of radix trichosanthis or medicine prepared by taking radix trichosanthis as raw material

Technical Field

The invention relates to a method for measuring the content of trichosanthes root or a medicine prepared by taking the trichosanthes root as a raw material.

Background

The trichosanthes root is a long-history and widely used traditional Chinese medicine and has the effects of clearing heat, promoting fluid, expelling pus and reducing swelling. Trichosanthin has effects of inducing labor and resisting tumor. Trichosanthis radix collected in 2005 edition "pharmacopoeia of the people's republic of China (one part) is the dried root of Trichosanthes kirilowii Maxim. of the Cucurbitaceae family, or Trichosanthes Uniflora Hao of the bilateral family, and is mainly produced in Henan, Shandong, Jiangsu, Guizhou, Anhui, etc. At present, the trichosanthes roots used all over the country have many original plants, the curative effects of the trichosanthes roots from different plant sources are not completely the same, and certain counterfeit products have certain adverse reactions.

At present, many methods for detecting trichosanthes root are reported, such as lujiangjiang, extraction and content determination of trichosanthes root polysaccharide, Tianjin pharmacy, 2001, 4 months, 13 nd 2 nd phase, extracting polysaccharide from trichosanthes root and determining the content of the polysaccharide. The method comprises extracting Trichosanthis radix polysaccharide with water extraction and ethanol precipitation method, and measuring polysaccharide content with phenol-sulfuric acid colorimetric method. The experiment proves that the content of trichosanthes polysaccharide is not very high, and the extraction method needs to be further improved. Pengkouxia, HPLC measures the cucurbitacin B content in trichosanthes root, and in 11 th stage of 11 th month in 11 th month of 2009 in China, modern journal of applied pharmacy, because cucurbitacin B has high toxicity, the toxic reaction is closely related to the dosage. The document discloses a method for detecting cucurbitacin B in trichosanthes root, and reports that amino acid is used as a detection index in trichosanthes root, such as: and assaying amino acids of the trichosanthes kirilowii Maxim and similar products thereof by thin-layer chromatography, wherein in the traditional medicine, the same mauve spots are shown on the corresponding result positions of the chromatogram of a citrulline reference substance and an alanine reference substance, and the root of the trichosanthes kirilowii Maxim has no corresponding spots on the chromatogram of the alanine reference substance. The content of citrulline in trichosanthes root medicinal materials of different producing areas, such as yangyuqin, and the like, is measured by measuring the content of citrulline in 15 trichosanthes root medicinal materials of different producing areas in the No. 20 and No. 6 of Shizhen national medicine 2009, and has a certain difference in content, but the content of citrulline in Guizhou trichosanthes root is higher, and the higher the growth cycle is, the higher the content of citrulline is.

The trichosanthes root contains more amino acid components, the existing detection method comprises citrulline and gamma-aminobutyric acid (Liuwen, the content determination of citrulline and gamma-aminobutyric acid in the trichosanthes root), and the detection is carried out by adopting a pre-column derived reverse phase high-efficiency chromatographic peak, so that the sample treatment process is complicated. In addition, tryptophan is also an important precursor substance for auxin biosynthesis in plants and commonly exists in higher plants, and meanwhile, tryptophan is widely applied to feeds (measuring the content of tryptophan in corn protein powder and piglet creep feed by an HPLC method) by adopting 0.0085mol/L sodium acetate: methanol (95: 5).

Disclosure of Invention

The invention provides a method for measuring the content of trichosanthes root or a medicine prepared by taking trichosanthes root as a raw material.

The invention provides a method for measuring the content of trichosanthes root or a medicine prepared by taking the trichosanthes root as a raw material, which adopts HPLC (high performance liquid chromatography) spectrogram spectrum detection and comprises the following operation steps:

a. preparation of a test solution:

adding diluted ethanol into a sample to be detected, performing ultrasonic treatment for 20min-40min, cooling, weighing, adding diluted ethanol to make up the lost weight, shaking, filtering, and collecting the filtrate;

b. preparation of reference solutions:

taking a proper amount of tryptophan reference substances, precisely weighing, and adding diluted ethanol solution to obtain the tryptophan reference substances;

c. respectively and precisely absorbing the reference substance solution and the test solution, injecting the reference substance solution and the test solution into a liquid chromatograph, and eluting by using acetonitrile and water as mobile phases, wherein the chromatographic conditions are as follows:

phenyl-hexyl silane bonded silica gel is used as a filling agent; acetonitrile-water (4-10:90-96) is used as a mobile phase; the flow rate is: 0.8-1.2 ml/min; the column temperature is 25-35 ℃; the detection wavelength is 224 nm; the number of theoretical plates should not be less than 3000 in terms of tryptophan peaks.

Preferably, the medicine comprises trichosanthes root medicinal material, trichosanthes root standard decoction and trichosanthes root formula granules.

Further preferably, the ultrasonic conditions in the step a are as follows: power 600W, frequency 40 kHz; the ultrasonic treatment time is 20 min.

Wherein, the length of the phenyl-hexyl silane bonded silica gel filler column in the step c is 250mm, the inner diameter is 4.6mm, and the granularity is 5 μm.

The method for measuring the content of the radix trichosanthis comprises the following steps:

a. preparing test solution by weighing about 0.5g of the powder (sieved by a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, ultrasonically treating for 20 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking up, and filtering;

b. chromatographic conditions and system applicability test with phenyl-hexylsilane bonded silica gel as filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm); acetonitrile-water (4:96) is used as a mobile phase; the detection wavelength was 224 nm. The number of theoretical plates is not less than 3000 calculated according to tryptophan peaks;

c. preparing reference substance solution by precisely weighing appropriate amount of tryptophan reference substance, and adding diluted ethanol to obtain solution containing 10 μ g per 1 ml;

d. the measuring method comprises precisely sucking 10 μ l of sample solution, injecting into liquid chromatograph, and measuring.

The method for measuring the content of the standard decoction of the radix trichosanthis comprises the following steps:

a. preparing test solution by taking about 0.1g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking up, and filtering;

b. preparing reference substance solution by precisely weighing appropriate amount of tryptophan reference substance, and adding diluted ethanol to obtain solution containing 10 μ g per 1 ml;

c. chromatographic conditions and system applicability test with phenyl-hexylsilane bonded silica gel as filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm); acetonitrile-water (5:95) is used as a mobile phase; the detection wavelength was 224 nm. The number of theoretical plates is not less than 3000 calculated according to tryptophan peaks;

d. the measuring method comprises precisely sucking 10 μ l of sample solution, injecting into liquid chromatograph, and measuring.

The invention firstly determines the tryptophan component in the trichosanthes root medicinal material, the trichosanthes root standard decoction and the trichosanthes root formula granules; separating tryptophan in radix Trichosanthis medicinal material, radix Trichosanthis standard decoction, and radix Trichosanthis granule by high performance liquid chromatography, and measuring; establishing a method for detecting tryptophan in trichosanthes root medicinal materials, decoction and formula granules.

The chromatographic column with phenyl-hexyl silane bonded silica gel as the filler is selected, the conventional chromatographic column with octadecyl silane bonded silica gel as the filler is changed, the separation condition of tryptophan is taken as an index, and the chromatographic column with phenyl-hexyl silane bonded silica gel as the filler has better separation effect, better tryptophan peak shape and simpler proportion of mobile phase components compared with the chromatographic column with octadecyl silane bonded silica gel as the filler.

The method for detecting the content of tryptophan in the radix trichosanthis has the advantages of strong operability, simple sample treatment process, simple mobile phase, high repeatability and short detection time.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 ultraviolet absorption spectrum of tryptophan

Fig. 2 mobile phase 1: 10% acetonitrile-90% water

Fig. 3 mobile phase 2: 8% acetonitrile-92% water

Fig. 4 mobile phase 3: 4% acetonitrile-96% water

FIG. 5 examination results of different column temperatures

FIG. 6 examination results of different flow rates

FIG. 7 examination result of extraction solvent

FIG. 8 examination result of extraction mode

FIG. 9 extraction of temporal findings

FIG. 10 sample size investigation

FIG. 11 investigation of specificity of radix Trichosanthis

FIG. 12 Tryptophan standard graph

FIG. 13 investigation of different instruments

FIG. 14 results of durability examination of column

Fig. 15 mobile phase 1: 10% acetonitrile-90% water

Fig. 16 mobile phase 1: 8% acetonitrile-92% water

Fig. 17 mobile phase 1: 5% acetonitrile-95% water

FIG. 18 Tryptophan ultraviolet absorption Spectroscopy

FIG. 19 results of column temperature examination

FIG. 20 flow Rate investigation results

FIG. 21 examination result of extraction solvent

FIG. 22 examination result of extraction mode

FIG. 23 extraction of time finding results

FIG. 24 solvent addition investigation

FIG. 25 sample size investigation

FIG. 26 investigation of specificity of standard decoction of radix Trichosanthis

FIG. 27 Tryptophan standard graph

FIG. 28 examination of different instruments

FIG. 29 results of examining durability of column

Detailed Description

Example 1 method for measuring the content of radix Trichosanthis

Chromatographic conditions and system applicability test with phenyl-hexylsilane bonded silica gel as filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm); acetonitrile-water (4:96) is used as a mobile phase; the detection wavelength was 224 nm. The number of theoretical plates should not be less than 3000 calculated as tryptophan peaks.

Preparation of control solution A suitable amount of tryptophan control is precisely weighed, and diluted ethanol is added to obtain a solution containing 10 μ g of tryptophan per 1 ml.

Preparing test solution by weighing about 0.5g of the powder (sieved by a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, ultrasonically treating for 20 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking up, and filtering.

The measuring method comprises precisely sucking 10 μ l of sample solution, injecting into liquid chromatograph, and measuring.

Example 2 radix Trichosanthis content determination methodology of the present invention

1 apparatus

Agilent 1200 type high performance liquid chromatograph, Shimadzu 20AD type high performance liquid chromatograph, Shimadzu 20AT type high performance liquid chromatograph;

an electronic balance: ME204E/02, MS205DU, XP26 (Mettler-Tollido instruments, Inc.);

an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);

an ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);

a chromatographic column: agilent phenyl-Hexyl 4.6X 250mm 5 μm, DiKMA phenyl-Hexyl 4.6X 250mm 5 μm, Phlom phenyl-Hexyl 4.6X 250mm 5 μm

2 reagent and medicinal material

Tryptophan control (China institute for testing biological drug products, batch No. 140686-.

Acetonitrile (Sigma, chromatographically pure); the water is ultrapure water, and other reagents are analytically pure.

3 drafting test conditions

3.1 chromatographic conditions and System suitability test

Phenyl-hexylsilane bonded silica gel was used as a filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm).

3.2 preparation of control solutions

Taking a proper amount of tryptophan reference substance, precisely weighing, and adding diluted ethanol to obtain a solution containing 10 μ g of tryptophan per 1 ml.

3.3 preparation of test solutions

Taking about 0.5g of the product powder (batch No. 010442) -1801001, sieving with a No. three sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, ultrasonically treating for 20 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking up, filtering, and taking the subsequent filtrate.

3.4 assay methods

Precisely sucking 10 μ l of the sample solution, injecting into liquid chromatograph, and measuring.

4 chromatographic Condition selection and System suitability Studies

4.1 detection wavelength determination

Based on the test conditions, a diode array detector is used for scanning tryptophan in all bands, and the determination wavelength of tryptophan content in radix Trichosanthis is 224nm, as shown in FIG. 1.

4.2 Mobile phase selection

On the basis of the test conditions set forth above, the elution effects of mobile phase 1 (10% acetonitrile: 90% water), mobile phase 2 (8% acetonitrile: 92% water), and mobile phase 3 (4% acetonitrile: 96% water) were examined and shown in FIGS. 2 to 4, respectively.

As can be seen from the figure, when acetonitrile: the tryptophan peak profile is better for water (4:96), so acetonitrile: water (4:96) is used as the mobile phase for measuring the content of the radix trichosanthis.

4.3 column temperature investigation

Based on the test conditions set forth above, the column temperatures were examined at 25 ℃, 30 ℃ and 35 ℃. Tryptophan separation degree, theoretical plate number, tailing factor, and the like were used as evaluation indexes. See fig. 5, table 1.

TABLE 1 analysis results of different column temperatures

The results show that when the column temperature is 25 ℃, 30 ℃ and 35 ℃, the chromatogram peak shape and the separation effect meet the requirements.

4.4 flow Rate investigation

On the basis of the experimental conditions drawn up above, the flow rates of 0.8ml/min, 1.0ml/min, and 1.2ml/min were examined, and the degree of separation of tryptophan chromatographic peaks, the number of theoretical plates, and the tailing factor were used as evaluation indices. See fig. 6, table 2.

TABLE 2 analysis results of different flow rates

The results show that when the flow rates are 0.8ml/min, 1.0ml/min and 1.2ml/min, the chromatogram peak shape and the separation effect meet the requirements.

In conclusion, the chromatographic conditions and the systematic adaptability test for measuring the content of the radix trichosanthis medicinal material are determined as follows: phenyl-hexylsilane bonded silica gel as a filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm); acetonitrile-water (4:96) is used as a mobile phase; the detection wavelength was 224 nm. The number of theoretical plates should not be less than 3000 in terms of tryptophan peaks.

5 investigation of preparation of test solution

5.1 examination of extraction solvent

Taking about 0.5g of the product powder (batch No. 010442) -1801001, sieving with a No. three sieve), precisely weighing, placing in a conical flask with a plug, respectively extracting with methanol, 50% methanol, ethanol and diluted ethanol, respectively and precisely adding 25ml, sealing the plug, weighing, ultrasonically treating (power 600W, frequency 40kHz) for 20 minutes, cooling, weighing again, complementing the lost weight with an extraction solvent, shaking up, filtering, and taking the subsequent filtrate. Precisely sucking 10 μ l of each sample solution, and injecting into liquid chromatograph (see FIG. 7). The tryptophan content at different solvent extractions was calculated and the results are shown in Table 3.

TABLE 3 examination of extraction solvent

The result shows that the extraction efficiency of the dilute ethanol is the highest, and the peak shape is the best, so the dilute ethanol is selected as the extraction solvent for detecting the tryptophan in the radix trichosanthis medicinal material.

5.2 examination of extraction methods

Taking about 0.5g of the product powder (batch No. 010442) -1801001, sieving with a No. three sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, sealing the plug, weighing, examining the extraction method of the sample by reflux and ultrasound respectively, extracting for 20 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking uniformly, filtering, and taking the subsequent filtrate. Precisely sucking 10 μ l of each sample solution, and injecting into liquid chromatograph (see FIG. 8). And (4) respectively calculating the tryptophan content in different extraction modes. The results are shown in Table 4.

TABLE 4 examination of extraction methods

The result shows that the tryptophan content obtained by ultrasonic extraction has no obvious difference with the reflux extraction, so the ultrasonic extraction with simple and convenient operation is selected.

5.3 extraction time study

Taking about 0.5g of the product powder (batch number: 010442) -1801001, sieving with a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, sealing the plug, weighing, performing ultrasonic treatment (power 600W and frequency 40kHz), extracting the sample for 20 minutes, 30 minutes and 40 minutes respectively, cooling, weighing again, complementing the lost weight with dilute ethanol, shaking up, filtering, and taking the subsequent filtrate. Precisely sucking 10 μ l of each sample solution, and injecting into liquid chromatograph (see FIG. 9). The tryptophan content at different extraction times was analyzed and calculated, and the results are shown in Table 5.

TABLE 5 extraction of time finding results

The results show that sufficient extraction can be achieved when the extraction time is 20 minutes. Therefore, the extraction time of the test sample is determined to be 20 minutes.

5.4 sample size investigation

Weighing about 0.5g of the product powder (lot number: 010442) -1801001, sieving with a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, sealing the plug, weighing, ultrasonically treating for 20min, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking, and filtering. Precisely sucking the sample solutions of 5 μ l, 10 μ l and 15 μ l respectively, and injecting into liquid chromatograph (see figure 10). The tryptophan separation degree, the number of theoretical plates, the tailing factor, etc. were used as evaluation indexes, and are shown in Table 6.

TABLE 6 sample size survey Performance index

The result shows that when the addition amount of the solvent is 10 mu l, the chromatogram has better peak shape and moderate peak area. Therefore, the amount of the solvent added was determined to be 10. mu.l.

In conclusion, the preparation method of the radix trichosanthis medicinal material content determination test sample is determined as follows: weighing about 0.5g of the powder (sieved by a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, sealing the plug, weighing, ultrasonically treating for 20 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking uniformly, and filtering to obtain the product.

The method for determining the trichosanthes root standard decoction content test sample comprises the following steps: precisely sucking 10 μ l of the sample solution, and injecting into a liquid chromatograph.

6 radix trichosanthis medicinal material content determination method

Chromatographic conditions and system applicability test with phenyl-hexylsilane bonded silica gel as filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm); acetonitrile-water (4:96) is used as a mobile phase; the detection wavelength was 224 nm. The number of theoretical plates should not be less than 3000 calculated as tryptophan peaks.

Preparation of control solution A suitable amount of tryptophan control is precisely weighed, and diluted ethanol is added to obtain a solution containing 10 μ g of tryptophan per 1 ml.

Preparing test solution by weighing about 0.5g of the powder (sieved by a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, ultrasonically treating for 20 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking up, and filtering.

The measuring method comprises precisely sucking 10 μ l of sample solution, injecting into liquid chromatograph, and measuring.

7 methodology examination

7.1 specificity experiments

Preparing a reference substance solvent, a test solution and a negative reference solution according to a proposed method for detection. The results are shown in FIG. 11.

The result shows that the negative solution chromatogram has no interference to the measurement of the peak to be measured, and the specificity of the method is good.

7.2 precision investigation

The control solution was sampled 6 times continuously, the peak area of tryptophan was recorded, and the RSD value was calculated, the results are shown in table 7.

TABLE 7 results of precision examination

The result shows that the peak area RSD value of the tryptophan in the precision investigation is 0.29 percent, and the instrument sample injection precision is good.

7.3 Linear relationship

The tryptophan control solutions (10.20. mu.g/ml, purity 99.9%) were each precisely aspirated at 1. mu.l, 2. mu.l, 5. mu.l, 10. mu.l, 15. mu.l, and 20. mu.l, and injected into a liquid chromatograph to obtain peak areas, and response curves were plotted with the sample volumes (X,. mu.g) as abscissa and the peak areas (Y) as ordinate, and the results are shown in Table 8 and FIG. 12.

TABLE 8 analysis of tryptophan standard curves

The results show that: when the tryptophan sample amount ranges from 0.0102 to 0.2040 mu g, the linear relation is that y is 63.4321x-0.9110, and R is²0.9996. The sample injection amount is 0.0102-0.2040 mu g, and the linear relation is good.

7.4 repeatability

About 0.5g of this product powder (lot: 010442) -1801001, sieved through a third sieve) was precisely weighed to 6 parts, and the same operator prepared a test solution according to a predetermined method, and the tryptophan content of 6 parts of the test solution was calculated, the results are shown in Table 9.

TABLE 9 results of repeated experiments

The result shows that the RSD value of the tryptophan content is 0 percent, which shows that the method has good repeatability.

7.5 intermediate precision

7.5.1 different instrumental investigations

Based on the experimental conditions, two parts of the product powder (lot number: 010442) -1801001 and sieved by a third sieve) are precisely weighed to prepare test solution, and the test solution is respectively examined on Agilent phenyl-Hexyl 4.6 multiplied by 250mm 5 mu m chromatographic columns on Agilent 1260, Agilent 1200 and Shimadzu 20AT high performance liquid chromatographs. The tryptophan content was calculated and the results obtained are shown in FIG. 13, Table 10.

TABLE 10 results of different instrumental experiments

The result shows that the RSD value of the content determination result of the tryptophan is 9.54 percent by the research of Agilent 1260, Agilent 1200 and Shimadzu 20AT high performance liquid chromatographs, which indicates that the method has good intermediate precision.

7.5.2 investigation of different persons and times

Based on the above-mentioned experimental conditions, different persons (A, B) precisely measure the powder of this product (lot No. 010442) -1801001 and through No. three sieves) at different times (T1 and T2) respectively to prepare the test sample for determination. The tryptophan content results were calculated and are shown in Table 11.

TABLE 11 investigation results of different persons and times

The result shows that different persons detect at different times, the content determination result RSD value of the tryptophan is 0%, and the method has good intermediate precision.

7.6 sample recovery

About 0.25g of a known content of test sample powder (lot No. 010442) -1801001 passing through a No. three sieve and having a tryptophan content of 0.047%) was taken, 6 parts in total were precisely weighed, a certain amount of a tryptophan control (0.1045. mu.g/ml, purity: 99.9%) was precisely added, and the test sample solution was prepared and measured by a proposed method, and the recovery rate was calculated, the results are shown in Table 12. The calculation formula is as follows.

TABLE 12 sample recovery test results

The results show that the recovery averages 105.07%, the RSD values of the results are 0.68%, and the method is accurate.

7.7 durability examination

7.7.1 chromatographic column durability test

On the basis of the experimental conditions set forth above, the columns were examined for Agilent phenyl-Hexyl 4.6X 250mm 5 μm, DiKMA phenyl-Hexyl 4.6X 250mm 5 μm, and Philomen phenyl-Hexyl 4.6X 250mm 5 μm, respectively. See fig. 14, table 13.

TABLE 13 results of column durability examination

The result shows that the analytical chromatographic indexes of different chromatographic columns are good, the RSD value of 6 measurement results is 11.84%, and the durability of the chromatographic column is good.

7.7.2 stability test

About 0.5g of this product powder (lot: 010442) -1801001, sieved through a third sieve) was precisely weighed to prepare a test solution, and the tryptophan peak areas were measured at 0h, 3h, 6h, 12h, 15h and 24h, respectively, and the results are shown in Table 15.

TABLE 15 Tryptophan stability test results

The result shows that under the experimental condition, the RSD value of the tryptophan content is 0.30%, and the stability of the test solution is good within 24 hours.

7.8 sample assay verification

The trichosanthes root medicinal material is used as a test sample, a test sample solution is prepared and measured according to a proposed method, the peak area is recorded, the content of tryptophan is calculated, and the result is shown in table 16.

TABLE 1623 lots of radix Trichosanthis results

Test example 3 measurement of the content of the Standard decoction of Trichosanthis radix of the present invention

1 Instrument and reagent

1.1 instruments

High performance liquid chromatograph: agilent 1200 type HPLC, Agilent 1260 type HPLC, Shimadzu 20AD type HPLC;

an electronic balance: ME204E/02, MS205DU, XP26 (Mettler-Tollido instruments, Inc.);

an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);

an ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);

a chromatographic column: agilent phenyl-Hexyl 4.6X 250mm 5 μm, DiKMA phenyl-Hexyl 4.6X 250mm 5 μm, Phlom phenyl-Hexyl 4.6X 250mm 5 μm;

1.2 reagent

Tryptophan reference (China institute for testing biological drug products, lot number: 140686-

Acetonitrile (Sigma, chromatographically pure); the water is ultrapure water, and other reagents are analytically pure.

2 preparing test conditions for standard decoction of radix Trichosanthis

2.1 chromatographic conditions and System suitability test

Phenyl-hexylsilane bonded silica gel as a filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm); acetonitrile-water (5:95) is used as a mobile phase, the detection wavelength is 224nm, and the number of theoretical plates is not less than 3000 according to the tryptophan peak.

2.2 preparation of control solutions

Taking a proper amount of tryptophan reference substance, precisely weighing, and adding diluted ethanol to obtain a solution containing 10 μ g of tryptophan per 1 ml.

2.3 preparation of test solutions

Weighing about 0.1g of the product powder (batch number: THFBT180201), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, ultrasonically treating for 20 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking, filtering, and collecting the subsequent filtrate.

2.4 assay

Precisely sucking 10 μ l of the sample solution, injecting into liquid chromatograph, and measuring.

3 chromatographic Condition selection and System suitability inspection

3.1 Mobile phase selection

On the basis of the experimental conditions set forth above, the classification effects of mobile phase 1 (10% acetonitrile: 90% water), mobile phase 2 (8% acetonitrile: 92% water), and mobile phase 3 (5% acetonitrile: 95% water) were examined and shown in FIGS. 15 to 17, respectively.

As can be seen from the figure, when acetonitrile: the tryptophan peak profile is better for water (5:95), so acetonitrile: water (5:95) is used as the mobile phase for measuring the content of the standard decoction of the radix trichosanthis.

3.2 detection wavelength determination

On the basis of the experimental conditions outlined above, tryptophan was scanned over the full band using a diode array detector, see FIG. 18.

The result shows that the detection wavelength is 224nm through the analysis of the spectrogram.

3.3 column temperature investigation

Based on the experimental conditions set forth above, the column temperatures were examined at 25 ℃, 30 ℃ and 35 ℃. Tryptophan separation degree, theoretical plate number, tailing factor, and the like were used as evaluation indexes. See fig. 19, table 17.

TABLE 17 analysis results of various column temperatures

The results show that when the column temperature is 25 ℃, 30 ℃ and 35 ℃, the chromatogram peak shape and the separation effect meet the requirements.

3.4 flow Rate investigation

On the basis of the experimental conditions drawn up above, the flow rates of 0.8ml/min, 1.0ml/min, and 1.2ml/min were examined, and the degree of separation of tryptophan chromatographic peaks, the number of theoretical plates, and the tailing factor were used as evaluation indices. See fig. 20, table 18.

TABLE 18 analysis results of different flow rates

The results show that when the flow rates are 0.8ml/min, 1.0ml/min and 1.2ml/min, the chromatogram peak shape and the separation effect meet the requirements.

In conclusion, chromatographic conditions and system adaptability tests for measuring the content of the standard decoction of the radix trichosanthis are determined as follows: phenyl-hexylsilane bonded silica gel as a filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm); acetonitrile-water (5:95) is used as a mobile phase; the detection wavelength was 224 nm. The number of theoretical plates should not be less than 3000 calculated as tryptophan peaks.

4 investigation of preparation of test solution

4.1 examination of extraction solvent

Weighing about 0.1g of the product powder (batch number: THFBT180201), precisely weighing, placing in a conical flask with a plug, respectively extracting with methanol, ethanol, diluted ethanol and water, precisely adding 25ml of the mixture, sealing the plug, weighing, ultrasonically treating (power 600W, frequency 40kHz) for 20 minutes, cooling, weighing again, supplementing the weight loss with extraction solvent, shaking uniformly, filtering, and taking the subsequent filtrate. The sample solutions were each drawn up 10. mu.l precisely and injected into a liquid chromatograph, as shown in FIG. 21. The tryptophan content at the different solvent extractions was calculated and the results are shown in Table 19.

TABLE 19 examination results of extraction solvent

The result shows that the dilute ethanol has the highest extraction efficiency and the best peak shape, so the dilute ethanol is selected as the extraction solvent for detecting the tryptophan in the standard decoction of the radix trichosanthis.

4.2 examination of extraction methods

Weighing about 0.1g of the product powder (batch number: THFBT180201), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, sealing the plug, weighing, examining the extraction method of the sample by reflux and ultrasound respectively, extracting for 20 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking, filtering, and taking the subsequent filtrate. Precisely sucking 10 μ l of each sample solution, and injecting into liquid chromatograph (see FIG. 22). And (4) respectively calculating the tryptophan content in different extraction modes. The results are shown in Table 19.

TABLE 19 examination results of extraction modes

The result shows that the content of tryptophan obtained by ultrasonic extraction is higher than that of the tryptophan obtained by reflux extraction, but the content of tryptophan is not obviously different, so that the ultrasonic extraction with simple operation is selected.

4.3 extraction time study

Weighing about 0.1g of the product powder (batch number: THFBT180201), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, sealing the plug, weighing, ultrasonically treating (power 600W, frequency 40kHz), extracting the sample for 20 minutes, 30 minutes and 40 minutes respectively, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking uniformly, filtering, and taking the subsequent filtrate. Precisely sucking 10 μ l of each sample solution, and injecting into liquid chromatograph (see FIG. 23). The tryptophan content at different extraction times was analyzed and calculated, and the results are shown in Table 20.

TABLE 20 extraction of temporal findings

The results showed that sufficient extraction was achieved when the extraction time was 30 minutes. Therefore, the extraction time of the test sample is determined to be 30 minutes.

4.4 solvent addition investigation

Weighing about 0.1g of the product powder (batch number: THFBT180201), precisely weighing, placing into a conical flask with a plug, precisely adding 10ml, 25ml and 50ml of dilute ethanol, respectively, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking, and filtering to obtain the final product. Precisely sucking 10 μ l of the sample solution, and injecting into liquid chromatograph (see figure 24). The evaluation indexes include the tryptophan peak area, the degree of separation, the number of theoretical plates, and the tailing factor, and are shown in Table 21.

TABLE 21 examination of Performance index by solvent addition

The result shows that when the addition amount of the solvent is 25ml, the chromatogram has better peak shape and moderate peak area. The amount of the solvent added was determined to be 25 ml.

4.5 sample size investigation

Weighing about 0.1g of the product powder (batch number: THFBT180201), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking up, and filtering to obtain the final product. Precisely sucking the sample solutions of 5 μ l, 10 μ l and 15 μ l respectively, and injecting into liquid chromatograph (see figure 25). The tryptophan separation degree, the number of theoretical plates, the tailing factor, etc. were used as evaluation indexes, and are shown in Table 22.

TABLE 22 sample size survey Performance indicators

The result shows that when the sample amount is 10 mu l, the chromatogram has better peak shape and moderate peak area. Therefore, the amount of the sample was determined to be 10. mu.l.

In conclusion, the preparation method of the test sample for measuring the content of the standard decoction of the radix trichosanthis is determined as follows: weighing about 0.1g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, sealing, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking, and filtering to obtain the final product.

The method for determining the trichosanthes root standard decoction content test sample comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, and injecting into a liquid chromatograph.

5 radix trichosanthis standard decoction content determination method

Chromatographic conditions and system applicability test with phenyl-hexylsilane bonded silica gel as filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm); acetonitrile-water (5:95) is used as a mobile phase; the detection wavelength was 224 nm. The number of theoretical plates should not be less than 3000 calculated as tryptophan peaks.

Preparation of control solution A suitable amount of tryptophan control is precisely weighed, and diluted ethanol is added to obtain a solution containing 10 μ g of tryptophan per 1 ml.

Preparing test solution by taking about 0.1g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking up, and filtering.

The measuring method comprises precisely sucking 10 μ l of sample solution, injecting into liquid chromatograph, and measuring.

6 methodology examination

6.1 specificity experiments

Preparing a test solution and a negative control solution according to a proposed method, and detecting. The results are shown in FIG. 26.

The result shows that the negative solution chromatogram has no interference to the measurement of the peak to be measured, and the specificity of the method is good.

6.2 precision investigation

The control solution was sampled 6 times continuously, the peak area of tryptophan was recorded, and the RSD value was calculated, and the results are shown in table 23.

TABLE 23 results of precision examination

The result shows that the peak area RSD value of the tryptophan in the precision investigation is 0.71 percent, and the instrument sample injection precision is good.

6.3 Linear relationship

The tryptophan control solutions (10.20. mu.g/ml, purity 99.9%) were each precisely aspirated at 1. mu.l, 2. mu.l, 5. mu.l, 10. mu.l, 20. mu.l, and 25. mu.l, and injected into a liquid chromatograph to obtain peak areas, and response curves were plotted with the sample volumes (X,. mu.g) as abscissa and the peak areas (Y) as ordinate, and the results are shown in Table 24 and FIG. 27.

TABLE 24 analysis of tryptophan standard curves

The results show that: when the tryptophan sample amount ranges from 0.0102 to 0.2549 mu g, the linear relation is that y is 5835.8163x +8.4481, and R²0.9997. The sample injection amount is 0.0102-0.2549 mu g, and the linear relation is good.

6.4 repeatability

Taking 0.1g of the product powder (batch number: THFBT180201), precisely weighing 6 parts, preparing a test solution by the same operator according to a determined method, and calculating the tryptophan content of 6 parts of the test solution, wherein the results are shown in Table 25.

TABLE 25 results of repeated experiments

The result shows that the RSD value of the tryptophan content is 1.13 percent, which shows that the method has good repeatability.

6.5 intermediate precision

6.5.1 investigation of different instruments

On the basis of the experimental conditions planned above, 0.1g of the product powder (batch number: THFBT180201) is precisely taken and precisely weighed into two parts, and the two parts are respectively examined on Agilent 1260, Agilent 1200 and Shimadzu 20AT high performance liquid chromatographs (chromatographic columns are Agilent phenyl-Hexyl 4.6 multiplied by 250mm 5 mu m). The tryptophan content was calculated and the results obtained are shown in FIG. 28, Table 26

TABLE 26 Experimental results of different instruments

The result shows that the RSD value of the content determination result of the tryptophan is 5.37 percent when the high performance liquid chromatographs of Agilent 1260, Agilent 1200 and Shimadzu 20AT are examined, which indicates that the method has good intermediate precision.

6.5.2 investigation of different persons and times

Based on the above-mentioned experimental conditions, 0.1g of powder (lot: THFBT180201) was taken by different persons (A, B) at different times (T1, T2), and two portions were precisely weighed to prepare test samples for measurement. The tryptophan content results were calculated and are shown in Table 27.

Table 27 results of different personnel and time surveys

The result shows that different persons detect at different times, the content determination result RSD value of the tryptophan is 1.77%, and the method has good intermediate precision.

6.6 sample recovery

About 0.05g of a test sample (batch number: THFBT180201, tryptophan content: 0.149%) with a known content, 6 parts in total, precisely weighed, precisely added with a certain amount of tryptophan reference sample (0.1045 μ g/ml, purity: 99.9%) respectively, prepared and measured according to a proposed method, and the recovery rate was calculated, and the results are shown in Table 28. The calculation formula is as follows:

TABLE 28 sample recovery test results

The result shows that the average recovery rate is 95.48%, the RSD value of the result is 0.83%, and the method has good accuracy.

6.7 durability examination

6.7.1 inspection of column durability

On the basis of the experimental conditions set forth above, respectively subjecting a chromatographic column Agilent Plus phenyl-Hexyl to chromatography with the thickness of 4.6 multiplied by 250mm and the thickness of 5 mu m; DiKMA phenyl-Hexyl 4.6X 250mm 5 μm, Philomen phenyl-Hexyl 4.6X 250mm 5 μm. See fig. 29, table 29.

TABLE 29 results of column durability examination

The result shows that the analytical chromatographic indexes of different chromatographic columns are good, the RSD value of 6 measurement results is 3.05%, and the durability of the chromatographic column is good.

6.7.2 stability test

The same sample solution (batch number: THFBT180201) was used to determine the tryptophan peak areas at 0h, 2h, 6h, 12h, 18h, and 24h, respectively, and the results are shown in Table 30.

TABLE 30 Tryptophan stability test results

The result shows that under the experimental condition, the RSD value of the tryptophan content is 1.37%, and the stability of the test solution is good within 24 hours.

6.8 content verification

The freeze-dried powder of the trichosanthes root standard decoction is used as a test sample, a test sample solution is prepared and measured according to a proposed method, the peak area is recorded, the content of tryptophan is calculated, and the result is shown in a table 31.

TABLE 3123 Tryptophan content of standard Trichosanthis radix decoction

7 detecting radix trichosanthis formula granules

The method is used for detecting the trichosanthes root formula granules, and the result is shown in a table.

TABLE 32 examination of the content of three batches of radix Trichosanthis granules

And (4) conclusion: the method can be used for detecting the radix Trichosanthis granule.

Claims (6)

1. A method for measuring the content of trichosanthes root or a medicine prepared by taking the trichosanthes root as a raw material is characterized by comprising the following steps: it adopts HPLC atlas detection, and its operation steps are as follows:

a. preparation of a test solution:

adding diluted ethanol into a sample to be detected, performing ultrasonic treatment for 20min-40min, cooling, weighing, adding diluted ethanol to make up the lost weight, shaking, filtering, and collecting the filtrate;

b. preparation of control solutions:

taking a proper amount of tryptophan reference substances, precisely weighing, and adding diluted ethanol solution to obtain the tryptophan reference substances;

c. respectively and precisely sucking the reference solution and the test solution, injecting the reference solution and the test solution into a liquid chromatograph, and eluting by taking acetonitrile and water as mobile phases, wherein the chromatographic conditions are as follows:

phenyl-hexyl silane bonded silica gel is used as a filling agent; acetonitrile-water with the volume ratio of 4-10:90-96 is taken as a mobile phase; the flow rate is: 0.8-1.2 ml/min; the column temperature is 25-35 ℃; the detection wavelength is 224 nm; the number of theoretical plates is not less than 3000 according to tryptophan peaks;

the radix trichosanthis is a radix trichosanthis medicinal material, and the medicine comprises radix trichosanthis standard decoction and radix trichosanthis formula granules.

2. The method for measuring according to claim 1, wherein: the ultrasonic conditions in the step a are as follows: power 600W, frequency 40 kHz; the ultrasonic treatment time is 20 min.

3. The method for measuring according to claim 1, wherein: and c, the length of the phenyl-hexyl silane bonded silica gel filler column in the step is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m.

4. The assay according to any one of claims 1 to 3, wherein: the method for measuring the content of the radix trichosanthis medicinal material comprises the following steps:

a. preparing a test solution by taking radix trichosanthis powder, sieving the powder by a third sieve, precisely weighing, placing the powder in a conical flask with a plug, precisely adding 25ml of dilute ethanol, weighing, carrying out ultrasonic treatment for 20 minutes, cooling, weighing again, supplementing the lost weight with the dilute ethanol, shaking up, and filtering;

b. the chromatographic conditions and the system applicability test use phenyl-hexyl silane bonded silica gel as a filler, the column length of a chromatographic column is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; acetonitrile-water in a volume ratio of 4:96 is taken as a mobile phase; the detection wavelength is 224nm, and the number of theoretical plates is not less than 3000 calculated according to tryptophan peaks;

c. preparing reference substance solution by precisely weighing appropriate amount of tryptophan reference substance, and adding diluted ethanol to obtain solution containing 10 μ g per 1 ml;

d. the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

5. The assay according to any one of claims 1 to 3, wherein: the method for measuring the content of the standard decoction of the radix trichosanthis comprises the following steps:

a. preparing test solution by accurately weighing Trichosanthis radix standard decoction powder, placing in conical flask with plug, adding diluted ethanol 25ml, weighing, ultrasonic treating for 30 min, cooling, weighing again, supplementing lost weight with diluted ethanol, shaking, and filtering;

b. preparing reference substance solution by precisely weighing appropriate amount of tryptophan reference substance, and adding diluted ethanol to obtain solution containing 10 μ g per 1 ml;

c. the chromatographic conditions and the system applicability test use phenyl-hexyl silane bonded silica gel as a filler, the column length of a chromatographic column is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; acetonitrile-water in a volume ratio of 5:95 is taken as a mobile phase; the detection wavelength is 224nm, and the number of theoretical plates is not less than 3000 calculated according to tryptophan peaks;

d. the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

6. The assay according to any one of claims 1 to 3, wherein: the method for measuring the content of the trichosanthes root formula particles comprises the following steps:

a. preparing a test solution by precisely weighing radix Trichosanthis granule, placing in a conical flask with a plug, precisely adding diluted ethanol 25ml, weighing, ultrasonically treating for 30 min, cooling, weighing again, supplementing the lost weight with diluted ethanol, shaking, and filtering;

b. preparing reference substance solution by precisely weighing appropriate amount of tryptophan reference substance, and adding diluted ethanol to obtain solution containing 10 μ g per 1 ml;

c. the chromatographic conditions and the system applicability test use phenyl-hexyl silane bonded silica gel as a filler, the column length of a chromatographic column is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m; acetonitrile-water in a volume ratio of 5:95 is taken as a mobile phase; the detection wavelength is 224nm, and the number of theoretical plates is not less than 3000 calculated according to tryptophan peaks;

d. the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

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