CN101059482A - Pericarpium Trichosanthis or Pericarpium Trichosanthis injection liquid chromatography fingerprint test method - Google Patents
Pericarpium Trichosanthis or Pericarpium Trichosanthis injection liquid chromatography fingerprint test method Download PDFInfo
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Abstract
The invention discloses a test method of liquid spectrum finger diagram of melon, or melon injection, comprising that (1), preparing melon, or melon injection into a sample solution, (2), respectively absorbing citrulline water solution and the sample solution, to be filled into a liquid spectrometer, and using the solution of phthalaldehyde and the solution of chloride aminic acid fluorenes methyl ester to process derivatization, (3), testing liquid spetrum. The inventive test method first discloses a liquid spectrum finger diagram of melon or melon injection, with high accuracy, stability and repeatability, as one analysis test method which can control the product quality, used in product quality test. The inventive method first obtains the high-effect liquid spectrum contrast finger diagram of melon and melon injection, as diagram 1 and diagram 2.
Description
Technical field
The present invention relates to a kind of product quality method of testing, relate in particular to the method for testing of the liquid-phase chromatograph finger print atlas of a kind of PERICARPIUM TRICHOSANTHIS or Pericarpium Trichosanthis injection.
Background technology
PERICARPIUM TRICHOSANTHIS is the dry mature skin of cucurbitaceous plant snakegourd (Trichosanthes kirilowii Maxim) or trichosanthes rosthornii Harms (Trichosanthes rosthomii Harms), records in 2005 editions one one of Chinese Pharmacopoeia.It mainly contains amino acid, and a small amount of volatilization wet goods.
Pericarpium Trichosanthis injection is unique PERICARPIUM TRICHOSANTHIS pharmaceutical formulation that authentication code is arranged that Shanghai No.1 Bio-Chemical Pharmacetical Industry Co., Ltd initiates at home, records in the national standard product.It is to be raw material with the PERICARPIUM TRICHOSANTHIS, through water extract-alcohol precipitation, and " 732 " type Zeo-karb by Shanghai Resin Factory again, and the sterile water solution of making.
In the prior art, as yet not relevant for the bibliographical information of the efficient liquid-phase chromatograph finger print atlas method of testing of PERICARPIUM TRICHOSANTHIS or Pericarpium Trichosanthis injection.The principal ingredient of PERICARPIUM TRICHOSANTHIS is an amino acid, but because amino acid itself does not have uv absorption, can not directly measure.But, can produce the conjugation group behind one-level amino acid and o-phthalaldehyde(OPA) (OPA), secondary amino acid and 9-fluorene methyl chloro-carbonate (FMOC) derivatization, can on ultraviolet or fluorescence detector, produce response.
Summary of the invention
The objective of the invention is to disclose the method for testing of the liquid-phase chromatograph finger print atlas of a kind of PERICARPIUM TRICHOSANTHIS or Pericarpium Trichosanthis injection.
Method of testing of the present invention may further comprise the steps:
(1) PERICARPIUM TRICHOSANTHIS or Pericarpium Trichosanthis injection are made sample solution.
Among the present invention, when tested object was the trichosanthes fruit skin, the preparation process of described step (1) was: the trichosanthes fruit leather is become powder, sieve, add water and be mixed with solution, weigh, reflux is weighed after the cooling, and water is supplied and subtracted weight loss, shakes up, and is centrifugal, gets supernatant promptly.
What wherein, the used sieve of the described step of sieving was preferable is No. two sieves; Described add ratio that the water preparation steps adopts preferable be 1g trichosanthes fruit skin/20ml water; What the time of described reflux was preferable is 30 minutes; What the rotating speed that described centrifugation step adopts was preferable is 5000-10000 rev/min.
Among the present invention, when tested object was trichosanthes fruit skin injection liquid, the preparation process of described step (1) was: trichosanthes fruit skin injection liquid dilute with water is made sample solution.
What wherein, the ratio of described dilute with water step employing was preferable is trichosanthes fruit skin injection liquor ratio water 1: 5.
(2) draw citrulline aqueous solution and sample solution respectively and inject liquid chromatograph, carry out pre-column derivatization with solution that contains o-phthalaldehyde(OPA) and the solution that contains a chloro-carbonic acid fluorenes methyl esters successively.
Among the present invention, because citrulline is one of principal ingredient in the PERICARPIUM TRICHOSANTHIS, therefore its chromatographic peak area proportion in finger-print more greatly and more stable selects it as object of reference, and uses water as solvent, and measurement result shows that this method is feasible.When tested object was the trichosanthes fruit skin, that the concentration of the described citrulline aqueous solution of step (2) is preferable was 1mg/ml; When tested object was trichosanthes fruit skin injection liquid, that the concentration of the described citrulline aqueous solution of step (2) is preferable was 0.5mg/ml.
Among the present invention, in the described step (2), that the citrulline aqueous solution of absorption and the volume of sample solution are all preferable is 1 μ l.
Among the present invention, the sample introduction program that the described pre-column derivatization step of step (2) is adopted is:
1. draw borate buffer solution, contain solution, the sample solution of o-phthalaldehyde(OPA), in air, mix.
Wherein, in the described solution that contains o-phthalaldehyde(OPA), that the concentration of o-phthalaldehyde(OPA) is preferable is 0.01g/ml.This solution can be made by following method: every 80mg o-phthalaldehyde(OPA), get the 0.4mol/L borate buffer solution 7ml of pH10.2, and acetonitrile 1ml, mercaptopropionic acid 125 μ l, mixing is promptly.
What wherein, described borate buffer solution was preferable is the 0.4mol/L borate buffer solution of pH10.2.This solution can be made by following method: the 0.4mol/L boric acid aqueous solution, regulate pH value to 10.2 promptly with the 0.4g/ml sodium hydrate aqueous solution.
2. draw the solution that contains a chloro-carbonic acid fluorenes methyl esters again, in air, mix.
Wherein, in the described solution that contains a chloro-carbonic acid fluorenes methyl esters, that the concentration of a chloro-carbonic acid fluorenes methyl esters is preferable is 0.005g/ml.This solution can be made by following method: every 50mg one chloro-carbonic acid fluorenes methyl esters, make its dissolving with an amount of acetonitrile, and be diluted with water to 10ml promptly.
3. draw water again, in air, mix.
4. sample introduction.
(3) liquid chromatogram measuring.
Among the present invention, what described liquid chromatography was preferable is high performance liquid chromatography.Among the present invention, the number of theoretical plate of high performance liquid chromatography is pressed the citrulline peak and is calculated, and should be not less than 20000.
Among the present invention, the chromatographic column that described high performance liquid chromatography adopted preferable for being the chromatographic column of filling agent with octadecylsilane chemically bonded silica (ODS), as the ODS product of U.S. Hypersil company, promptly the aperture is 5 μ m, diameter is 4.6mm, and length is the chromatographic column of 20cm.
Among the present invention, what the moving phase that described high performance liquid chromatography adopted was preferable is:
Mobile phase A can be made by following method: gets sodium acetate 10.88g, adds water 4000ml and make dissolving, add triethylamine 0.8ml, and tetrahydrofuran 24ml, mixing is regulated pH value to 7.20 with 2% glacial acetic acid solution, promptly.
Mobile phase B can be made by following method: get sodium acetate 10.88g, add water 800ml and make dissolving, regulate pH value to 7.20 with 2% glacial acetic acid solution, add acetonitrile 1400ml, methyl alcohol 1800ml, mixing are promptly.
Among the present invention, preferable as shown in table 1 of the elution program that described high performance liquid chromatography adopted:
Table 1 high performance liquid chromatography elution program
| Time (minute) | Mobile phase A (wt%) | Mobile phase B (wt%) | Flow velocity (ml/min) |
| 0 | 100 | 0 | 1 |
| 8 | 92 | 8 | 1 |
| 22 | 86 | 14 | 1 |
| 31 | 73 | 27 | 1 |
| 38 | 71 | 29 | 1 |
| 54 | 21 | 79 | 1.5 |
| 58 | 85 | 15 | 1.5 |
| 60 | 100 | 0 | 1 |
Among the present invention, that the detection wavelength that described high performance liquid chromatography adopted is preferable is 338nm, and what the column temperature that is adopted was preferable is 25 ℃.
Adopt method of testing of the present invention respectively 9 batches of PERICARPIUM TRICHOSANTHIS samples to be tested.The efficient liquid-phase chromatograph finger print atlas similarity of 9 batches of PERICARPIUM TRICHOSANTHIS samples is all more than 0.94, and total peak basically identical.Therefore, generated PERICARPIUM TRICHOSANTHIS high performance liquid chromatography reference fingerprint with these 9 batches of PERICARPIUM TRICHOSANTHIS samples through " Chinese medicine stratographic analysis and data management system " computer software (Chinese medicine biological products evaluation is developed) simulation, as shown in Figure 1.
Adopt method of testing of the present invention respectively 9 batches of Pericarpium Trichosanthis injections to be tested.The efficient liquid-phase chromatograph finger print atlas similarity of 9 batches of Pericarpium Trichosanthis injection samples is all more than 0.95, and total peak basically identical.Therefore, generated Pericarpium Trichosanthis injection high performance liquid chromatography reference fingerprint with these 9 batches of Pericarpium Trichosanthis injection samples through " Chinese medicine stratographic analysis and data management system " computer software (Chinese medicine biological products evaluation is developed) simulation, as shown in Figure 2.
In the example of the present invention, the amino acid that adopts method of testing of the present invention to record in PERICARPIUM TRICHOSANTHIS and the Pericarpium Trichosanthis injection is mainly citrulline, arginine, alanine, isoleucine, serine, leucine and phenylalanine etc.
Positive progressive effect of the present invention is: the present invention is the efficient liquid-phase chromatograph finger print atlas method of testing of disclosed PERICARPIUM TRICHOSANTHIS first or Pericarpium Trichosanthis injection.This method has stronger specificity, and precision height, good stability, favorable reproducibility, can be used as a kind of analysis test method of controlling product quality preferably and is applied to the product quality test.Adopt method of testing of the present invention to obtain the high performance liquid chromatography reference fingerprint of PERICARPIUM TRICHOSANTHIS or Pericarpium Trichosanthis injection first, as shown in Figure 1 and Figure 2.
Description of drawings
Fig. 1 is a PERICARPIUM TRICHOSANTHIS high performance liquid chromatography reference fingerprint, and peak S is the chromatographic peak of object of reference citrulline among the figure.
Fig. 2 is a Pericarpium Trichosanthis injection high performance liquid chromatography reference fingerprint, and peak S is the chromatographic peak of object of reference citrulline among the figure.
Fig. 3 is a trichosanthes fruit skin efficient liquid-phase chromatograph finger print atlas, and peak S is the chromatographic peak of object of reference citrulline among the figure, and No. 7 peak, No. 1 peak～is respectively serine, citrulline, alanine, arginine, phenylalanine, isoleucine and leucine in the finger-print.
Fig. 4 is 9 batches of trichosanthes fruit skin sample efficient liquid-phase chromatograph finger print atlas.
Fig. 5 is a trichosanthes fruit skin injection liquid efficient liquid-phase chromatograph finger print atlas, and peak S is the chromatographic peak of object of reference citrulline among the figure, and No. 7 peak, No. 1 peak～is respectively serine, citrulline, alanine, arginine, phenylalanine, isoleucine and leucine in the finger-print.
Fig. 6 is 9 batches of trichosanthes fruit skin injection liquid sample efficient liquid-phase chromatograph finger print atlas.
Fig. 7 is the trichosanthes fruit skin efficient liquid-phase chromatograph finger print atlas of 6 sample introductions in the test of method of testing precision.
Fig. 8 is the trichosanthes fruit skin injection liquid efficient liquid-phase chromatograph finger print atlas of 6 sample introductions in the test of method of testing precision.
Fig. 9 is the trichosanthes fruit skin efficient liquid-phase chromatograph finger print atlas of continuous four days sample introductions in the method for testing stability test.
Figure 10 is the trichosanthes fruit skin injection liquid efficient liquid-phase chromatograph finger print atlas of continuous four days sample introductions in the method for testing stability test.
Figure 11 in the method for testing precision test with the trichosanthes fruit skin efficient liquid-phase chromatograph finger print atlas of 6 duplicate samples of lot number.
Figure 12 in the method for testing precision test with the trichosanthes fruit skin injection liquid efficient liquid-phase chromatograph finger print atlas of 6 duplicate samples of lot number.
Embodiment
Concrete steps are:
(1) the about 1g of precision weighing trichosanthes fruit skin powder crosses sieve No. two, and precision adds water 20ml, claims to decide weight, and reflux 30 minutes after the cooling, claims to decide weight again, and water is supplied and subtracted weight loss, shakes up, and 5000～10000 rev/mins centrifugal, gets supernatant, promptly;
(2) prepare following solution:
1mg/1ml citrulline aqueous solution.
0.01g/ml o-phthalaldehyde(OPA) (OPA) solution: take by weighing o-phthalaldehyde(OPA) (OPA) 80mg, add 0.4mol/L borate buffer (pH10.2) 7ml, acetonitrile 1ml, mercaptopropionic acid 125 μ l, mixing refrigerates stand-by.
0.4mol/L borate buffer (pH10.2): get the solution that boric acid is mixed with 0.4mol/L, regulate pH value to 10.2, refrigerate stand-by with 40% sodium hydroxide solution.
0.005g/ml chloro-carbonic acid fluorenes methyl esters (FMOC) solution: take by weighing chloro-carbonic acid fluorenes methyl esters (FMOC) 50mg, make its dissolving in right amount with acetonitrile, and be diluted with water to 10ml, refrigerate stand-by.
Accurate respectively citrulline aqueous solution and each 1 μ l of sample solution of drawing, inject liquid chromatograph, with the full-automatic pre-column derivatization of 0.01g/ml o-phthalaldehyde(OPA) solution (OPA) and 0.005g/ml one chloro-carbonic acid fluorenes methyl ester solution (FMOC), pre-column derivatization sample introduction program is (bottle 1～5 is respectively 0.4mol/L borate buffer (pH10.2), 0.005g/ml one chloro-carbonic acid fluorenes methyl esters (FMOC) solution, water, borate buffer solution, water):
1. draw 5 μ l (0.4mol/L borate buffer (pH10.2)) from bottle 4, draw 1 μ l (0.01g/ml o-phthalaldehyde(OPA) (OPA) solution), draw sample solution 1 μ l, mix 7 μ l, 10 times in the air from bottle 1;
2. draw 1 μ l (0.005g/ml one chloro-carbonic acid fluorenes methyl ester solution (FMOC)) from bottle 2, mix 8 μ l, 30 times in the air;
3. from bottle 5, draw 32 μ l (water), mix 20 μ l, maximal rate, 2 times in the air;
4. sample introduction.
(3) high-performance liquid chromatogram determination, chromatographic condition is as follows:
Instrument: Agilent 1100 series of high efficiency liquid chromatographs, chromatographic work station (Chemstation);
Chromatographic column: the Hypersil-ODS chromatographic column (5 μ m, 4.6mm * 20cm);
Mobile phase A: get sodium acetate 10.88g, add water 4000ml and make dissolving, add triethylamine 0.8ml, tetrahydrofuran 24ml, mixing is regulated pH value to 7.20 promptly with 2% glacial acetic acid solution;
Mobile phase B: get sodium acetate 10.88g, add water 800ml and make dissolving, regulate pH value to 7.20 with 2% glacial acetic acid solution, add acetonitrile 1400ml, methyl alcohol 1800ml, mixing are promptly;
Detect wavelength: 338nm;
Column temperature: 25 ℃;
The gradient elution program is as shown in table 1:
Table 1 high performance liquid chromatography elution program
| Time (minute) | Mobile phase A (wt%) | Mobile phase B (wt%) | Flow velocity (ml/min) |
| 0 | 100 | 0 | 1 |
| 8 | 92 | 8 | 1 |
| 22 | 86 | 14 | 1 |
| 31 | 73 | 27 | 1 |
| 38 | 71 | 29 | 1 |
| 54 | 21 | 79 | 1.5 |
| 58 | 85 | 15 | 1.5 |
| 60 | 100 | 0 | 1 |
Integral parameter: slope sensitivity (Slope Sensitivity) 2; Peak width (peak width) 0.1; The smallest peaks area is 10, and minimum peak height is 1.5.It is as shown in table 2 that integration finishes or begin (Intergration off or on) parameter:
Table 2 integration finishes or beginning (Intergration off or on) parameter
| Relative retention time with reference peak (S) | Integration Intergration |
| 0.00 | off |
| 0.25 | on |
| 0.40 | off |
| 0.90 | on |
| 1.50 | off |
| 2.00 | on |
Record trichosanthes fruit skin efficient liquid-phase chromatograph finger print atlas as shown in Figure 3.Theoretical pedal number is calculated as 21788 by citrulline amine peak 2.By the location of 22 seed amino acid reference substances, find to contain citrulline, arginine, alanine, isoleucine, serine, leucine, phenylalanine seven seed amino acids in the PERICARPIUM TRICHOSANTHIS medicinal material.Be embodied in finger-print: No. 7 peak, No. 1 peak～is respectively serine, citrulline, alanine, arginine, phenylalanine, isoleucine and leucine in the finger-print.
According to the method for embodiment 1,9 batches of trichosanthes fruit skin samples that the Shanghai first biochemical Pharma Inc. provides are tested.The record chromatogram, as shown in Figure 4.With computer simulation similarity computed in software similarity, the result is as shown in table 3.
9 batches of trichosanthes fruit skins of table 3 sample high-efficient liquid phase chromatogram similarity
| The sample lot number | 031101 | 030709 | 030801 | 030901 | 030902 | 030903 | 030904 | 031001 | 031002 |
| Similarity | 0.993 | 0.957 | 1.000 | 0.991 | 0.998 | 0.989 | 0.996 | 0.999 | 0.996 |
With 9 batches of trichosanthes fruit skin sample efficient liquid-phase chromatograph finger print atlas advance computer mould fit to trichosanthes fruit skin high performance liquid chromatography reference fingerprint, as shown in Figure 1.
According to the method for embodiment 1, wherein step (1) is: precision is measured trichosanthes fruit skin injection liquid 2ml, puts in the 10ml measuring bottle, and thin up shakes up promptly to scale; Wherein, the concentration of citrulline aqueous solution is 0.5mg/ml, all the other steps and parameter constant.
Record trichosanthes fruit skin injection liquid efficient liquid-phase chromatograph finger print atlas as shown in Figure 5.Theoretical pedal number is calculated as 21788 by citrulline amine peak 2.By the location of 22 seed amino acid reference substances, find to contain citrulline, arginine, alanine, isoleucine, serine, leucine, phenylalanine seven seed amino acids in the Pericarpium Trichosanthis injection.Be embodied in finger-print: No. 7 peak, No. 1 peak～is respectively serine, citrulline, alanine, arginine, phenylalanine, isoleucine and leucine in the finger-print.
According to the method for embodiment 3,9 batches of trichosanthes fruit skin injection liquid samples that the Shanghai first biochemical Pharma Inc. provides are tested.The record chromatogram, as shown in Figure 6.With computer simulation similarity computed in software similarity, the result is as shown in table 4.
9 batches of trichosanthes fruit skin injections of table 4 liquid sample high-efficient liquid phase chromatogram similarity
| The sample lot number | 031104 | 030905 | 030906 | 030907 | 031001 | 031002 | 031101 | 031102 | 031103 |
| Similarity | 0.993 | 0.989 | 0.994 | 0.989 | 0.999 | 0.999 | 0.997 | 0.991 | 0.994 |
With 9 batches of trichosanthes fruit skin injection liquid sample efficient liquid-phase chromatograph finger print atlas advance computer mould fit to trichosanthes fruit skin injection liquid high performance liquid chromatography reference fingerprint, as shown in Figure 2.
Adopt the PERICARPIUM TRICHOSANTHIS sample, according to the method for embodiment 1, continuous sample introduction is analyzed 6 times, and the record chromatogram as shown in Figure 7, calculates with computer simulation similarity software for calculation, and the result is as shown in table 5, and the finger-print similarity is all more than 0.948.
The trichosanthes fruit skin high-efficient liquid phase chromatogram similarity of continuous 6 sample introductions in the test of table 5 precision
| | 1 | 2 | 3 | 4 | 5 | 6 | On average | Relative average debiation |
| Similarity | 0.955 | 0.954 | 0.952 | 0.948 | 0.951 | 0.951 | 0.952 | 0.26％ |
Adopt the Pericarpium Trichosanthis injection sample, according to the method for embodiment 3, continuous sample introduction is analyzed 6 times, and the record chromatogram as shown in Figure 8, calculates with computer simulation similarity software for calculation, and the result is as shown in table 6, and the finger-print similarity is all more than 0.99.
The trichosanthes fruit skin injection liquid high-efficient liquid phase chromatogram similarity of continuous 6 sample introductions in the test of table 6 precision
| | 1 | 2 | 3 | 4 | 5 | 6 | On average | Relative average debiation |
| Similarity | 0.993 | 0.992 | 0.992 | 0.993 | 0.993 | 0.993 | 0.993 | 0.052％ |
Above data show that this method precision is good.
The same day after PERICARPIUM TRICHOSANTHIS sample solution preparation, second and third, four days, according to the method for embodiment 1, measure, the record chromatogram, as shown in Figure 9, machine simulation similarity software for calculation calculates as calculated, and the result is as shown in table 7, and the finger-print similarity is all more than 0.955
The trichosanthes fruit skin high-efficient liquid phase chromatogram similarity of continuous four days sample introductions in table 7 stability test
| Time | First day | Second day | The 3rd day | The 4th day | On average | Relative average debiation |
| Similarity | 0.955 | 0.961 | 0.963 | 0.978 | 0.964 | 1.0％ |
The same day after PERICARPIUM TRICHOSANTHIS sample solution preparation, second and third, four days, according to the method for embodiment 3, measure, the record chromatogram, as shown in figure 10, machine simulation similarity software for calculation calculates as calculated, and the result is as shown in table 8, and the finger-print similarity is all more than 0.969.
The trichosanthes fruit skin injection liquid high-efficient liquid phase chromatogram similarity of continuous four days sample introductions in table 8 stability test
| Time | First day | Second day | The 3rd day | The 4th day | On average | Relative average debiation |
| Similarity | 0.984 | 0.97 | 0.969 | 0.969 | 0.973 | 0.8％ |
Above data show that this method has good stability.
Get 6 parts of trichosanthes fruit skin samples of same lot number, press the method for embodiment 1 and measure, the record chromatogram, as shown in figure 11, with computer simulation similarity computed in software, the result is as shown in table 8, and the similarity of finger-print is all more than 0.953.
In the table 9 reappearance test with 6 parts of trichosanthes fruit skin sample high-efficient liquid phase chromatogram similarities of lot number
| Sample |
| 1 | 2 | 3 | 4 | 5 | 6 | On average | Relative average debiation | |
| Similarity | 0.957 | 0.955 | 0.956 | 0.955 | 0.955 | 0.953 | 0.955 | 0.14％ |
Get 6 parts of trichosanthes fruit skin injection liquid samples of same lot number, press the method for embodiment 1 and measure, the record chromatogram, as shown in figure 12, with computer simulation similarity computed in software, the result is as shown in table 10, and the similarity of finger-print is all more than 0.977.
In the table 10 reappearance test with 6 parts of trichosanthes fruit skin injection liquid sample high-efficient liquid phase chromatogram similarities of lot number
| Sample |
| 1 | 2 | 3 | 4 | 5 | 6 | On average | Relative average debiation | |
| Similarity | 0.988 | 0.977 | 0.979 | 0.984 | 0.981 | 0.980 | 0.982 | 0.40％ |
Above data show that this method reappearance is good.
Among the present invention, (lot number: 0875-200104) provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, trichosanthes fruit skin and trichosanthes fruit skin injection liquid are provided by the Shanghai first biochemical Pharma Inc. citrulline.Used methyl alcohol and acetonitrile are chromatographically pure, and it is pure that all the other reagent are analysis.Agents useful for same of the present invention is all commercially available to be got.
Claims (39)
1. the method for testing of the liquid-phase chromatograph finger print atlas of PERICARPIUM TRICHOSANTHIS or Pericarpium Trichosanthis injection the steps include:
(1) PERICARPIUM TRICHOSANTHIS or Pericarpium Trichosanthis injection are made sample solution;
(2) draw citrulline aqueous solution and sample solution respectively and inject liquid chromatograph, carry out pre-column derivatization with solution that contains o-phthalaldehyde(OPA) and the solution that contains a chloro-carbonic acid fluorenes methyl esters successively;
(3) liquid chromatogram measuring.
2. method of testing according to claim 1 is characterized in that, when tested object is the trichosanthes fruit skin, the preparation process of described step (1) is: the trichosanthes fruit leather is become powder, sieve, add water and be mixed with solution, weigh, reflux, weigh after the cooling, water is supplied and is subtracted weight loss, shakes up, centrifugal, get supernatant promptly.
3. method of testing according to claim 2 is characterized in that: the used sieve of the described step of sieving is No. two sieves.
4. method of testing according to claim 2 is characterized in that: the described ratio that adds the employing of water preparation steps is 1g trichosanthes fruit skin/20ml water.
5. method of testing according to claim 2 is characterized in that: the time of described reflux is 30 minutes.
6. method of testing according to claim 2 is characterized in that: the rotating speed that described centrifugation step adopts is 5000-10000 rev/min.
7. method of testing according to claim 1 is characterized in that, when tested object was trichosanthes fruit skin injection liquid, the preparation process of described step (1) was: trichosanthes fruit skin injection liquid dilute with water is made sample solution.
8. method of testing according to claim 7 is characterized in that: the ratio that described dilute with water step adopts is trichosanthes fruit skin injection liquor ratio water 1: 5.
9. method of testing according to claim 1 is characterized in that: when tested object was the trichosanthes fruit skin, the concentration of the described citrulline aqueous solution of step (2) was 1mg/ml.
10. method of testing according to claim 1 is characterized in that: when tested object was trichosanthes fruit skin injection liquid, the concentration of the described citrulline aqueous solution of step (2) was 0.5mg/ml.
11. method of testing according to claim 1 is characterized in that: in the described step (2), the citrulline aqueous solution of absorption and the volume of sample solution all are 1 μ l.
12. method of testing according to claim 1 is characterized in that, the sample introduction program that the described pre-column derivatization step of step (2) is adopted is:
1. draw borate buffer solution, contain solution, the sample solution of o-phthalaldehyde(OPA), in air, mix;
2. draw the solution that contains a chloro-carbonic acid fluorenes methyl esters again, in air, mix;
3. draw water again, in air, mix;
4. sample introduction.
13. according to claim 1 or 12 described method of testings, it is characterized in that: in the described solution that contains o-phthalaldehyde(OPA), the concentration of o-phthalaldehyde(OPA) is 0.01g/ml.
14. method of testing according to claim 13 is characterized in that, the described solution that contains o-phthalaldehyde(OPA) can be made by following method: every 80mg o-phthalaldehyde(OPA), get the 0.4mol/L borate buffer solution 7ml of pH10.2, acetonitrile 1ml, mercaptopropionic acid 125 μ l, mixing is promptly.
15. method of testing according to claim 12 is characterized in that: described borate buffer solution is the 0.4mol/L borate buffer solution of pH10.2.
16. according to claim 14 or 15 described method of testings, it is characterized in that, the 0.4mol/L borate buffer solution of described pH10.2 can be made by following method: the 0.4mol/L boric acid aqueous solution, regulate pH value to 10.2 promptly with the 0.4g/ml sodium hydrate aqueous solution.
17. according to claim 1 or 12 described method of testings, it is characterized in that: in the described solution that contains a chloro-carbonic acid fluorenes methyl esters, the concentration of a chloro-carbonic acid fluorenes methyl esters is 0.005g/ml.
18. method of testing according to claim 17 is characterized in that, the described solution that contains a chloro-carbonic acid fluorenes methyl esters can be made by following method: every 50mg one chloro-carbonic acid fluorenes methyl esters, make its dissolving with acetonitrile, and be diluted with water to 10ml promptly.
19. method of testing according to claim 12 is characterized in that: the step 1. volume drawn of described borate buffer solution is 5 μ l.
20. method of testing according to claim 12 is characterized in that: the volume that the 1. described solution that contains o-phthalaldehyde(OPA) of step is drawn is 1 μ l.
21. method of testing according to claim 12 is characterized in that: the step 1. volume drawn of described sample solution is 1 μ l.
22. method of testing according to claim 12 is characterized in that: the number of times that mixes in the 1. described air of step is 10 times.
23. method of testing according to claim 12 is characterized in that: the volume that mixes in the 1. described air of step is 7 μ l.
24. method of testing according to claim 12 is characterized in that: the volume that the 2. described solution that contains a chloro-carbonic acid fluorenes methyl esters of step is drawn is 1 μ l.
25. method of testing according to claim 12 is characterized in that: the number of times that mixes in the 2. described air of step is 30 times.
26. method of testing according to claim 12 is characterized in that: the volume that mixes in the 2. described air of step is 8 μ l.
27. method of testing according to claim 12 is characterized in that: the step 3. volume drawn of described water is 32 μ l.
28. method of testing according to claim 12 is characterized in that: the number of times that mixes in the 3. described air of step is 2 times.
29. method of testing according to claim 12 is characterized in that: the speed of mixing in the 3. described air of step is the instrument maximal rate.
30. method of testing according to claim 12 is characterized in that: the volume that mixes in the 3. described air of step is 20 μ l.
31. method of testing according to claim 1 is characterized in that: the described liquid chromatography of step (3) is a high performance liquid chromatography.
32. method of testing according to claim 31 is characterized in that: the chromatographic column that described high performance liquid chromatography adopted is for being the chromatographic column of filling agent with the octadecylsilane chemically bonded silica.
33. method of testing according to claim 32, it is characterized in that: described is that the chromatographic column of filling agent is the ODS product of U.S. Hypersil company with the octadecylsilane chemically bonded silica, be that the aperture is 5 μ m, diameter is 4.6mm, and length is the chromatographic column of 20cm.
34. method of testing according to claim 31 is characterized in that: the moving phase that described high performance liquid chromatography adopted is:
Mobile phase A can be made by following method: gets sodium acetate 10.88g, adds water 4000ml and make dissolving, add triethylamine 0.8ml, and tetrahydrofuran 24ml, mixing is regulated pH value to 7.20 with 2% glacial acetic acid solution, promptly;
Mobile phase B can be made by following method: get sodium acetate 10.88g, add water 800ml and make dissolving, regulate pH value to 7.20 with 2% glacial acetic acid solution, add acetonitrile 1400ml, methyl alcohol 1800ml, mixing are promptly.
35. method of testing according to claim 31 is characterized in that: the elution program that described high performance liquid chromatography adopted is as shown in table 1:
Table 1 high performance liquid chromatography elution program
Time (minute) Mobile phase A (wt%) Mobile phase B (wt%) Flow velocity (ml/min)
0 100 0 1
8 92 8 1
22 86 14 1
31 73 27 1
38 71 29 1
54 21 79 1.5
58 85 15 1.5
60 100 0 1
36. method of testing according to claim 31 is characterized in that: the detection wavelength that described high performance liquid chromatography adopted is 338nm.
37. method of testing according to claim 31 is characterized in that: the column temperature that described high performance liquid chromatography adopted is 25 ℃.
The trichosanthes fruit skin high performance liquid chromatography reference fingerprint that " Chinese medicine stratographic analysis and data management system " computer software simulation that 38. method of testing according to claim 1 is measured and developed through Chinese medicine biological products evaluation obtains, as shown in Figure 1.
The trichosanthes fruit skin injection liquid high performance liquid chromatography reference fingerprint that " Chinese medicine stratographic analysis and data management system " computer software simulation that 39. method of testing according to claim 1 is measured and developed through Chinese medicine biological products evaluation obtains, institute is shown in Figure 2.
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| CN103063762A (en) * | 2012-12-21 | 2013-04-24 | 青岛谱尼测试有限公司 | Method for measuring citrulline contents in watermelon by utilizing high performance liquid chromatography |
| CN105541944A (en) * | 2016-03-02 | 2016-05-04 | 上海上药第一生化药业有限公司 | Preparation method of chemical components in trichosanthes kirilowii Maxim injection and application of chemical components |
| CN105646628A (en) * | 2016-03-02 | 2016-06-08 | 上海上药第生化药业有限公司 | Method for preparing chemical constituent in pericarpium trichosanthis injection and application of chemical constituents |
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| CN103063762A (en) * | 2012-12-21 | 2013-04-24 | 青岛谱尼测试有限公司 | Method for measuring citrulline contents in watermelon by utilizing high performance liquid chromatography |
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| CN105646628A (en) * | 2016-03-02 | 2016-06-08 | 上海上药第生化药业有限公司 | Method for preparing chemical constituent in pericarpium trichosanthis injection and application of chemical constituents |
| CN105669806A (en) * | 2016-03-02 | 2016-06-15 | 上海上药第一生化药业有限公司 | Preparation method and application of chemical component in trichosanthes kirilowii Maxim peel |
| CN105541944B (en) * | 2016-03-02 | 2017-12-26 | 上海上药第一生化药业有限公司 | The preparation method and its usage of chemical composition in Pericarpium Trichosanthis injection |
| CN109187825A (en) * | 2018-06-01 | 2019-01-11 | 四川新绿色药业科技发展有限公司 | A kind of radices trichosanthis or the content assaying method containing the drug that radices trichosanthis is raw material preparation |
| CN109187825B (en) * | 2018-06-01 | 2021-09-28 | 四川新绿色药业科技发展有限公司 | Method for measuring content of radix trichosanthis or medicine prepared by taking radix trichosanthis as raw material |
| CN108802245A (en) * | 2018-06-06 | 2018-11-13 | 四川新绿色药业科技发展有限公司 | A kind of root of Chinese trichosanthes or the detection method containing the drug that root of Chinese trichosanthes is raw material preparation |
| CN108802245B (en) * | 2018-06-06 | 2021-06-22 | 四川新绿色药业科技发展有限公司 | Method for detecting trichosanthes root or medicine prepared by taking trichosanthes root as raw material |
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Address after: 200240 Shanghai city Minhang District Jianchuan Road No. 1317 Patentee after: Add medicine to the first biochemical pharmaceutcal corporation, Ltd in Shanghai Address before: 200240 Shanghai city Minhang District Jianchuan Road No. 1317 Patentee before: Shanghai No.1 Biochemical & Pharmaceutical Co., Ltd. |