CN105301157A - Quality control method of related substances of methanesulfonic acid kukoamine B - Google Patents
Quality control method of related substances of methanesulfonic acid kukoamine B Download PDFInfo
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Abstract
The invention relates to a quality control method of related substances of methanesulfonic acid kukoamine B. The quality control method comprises the following steps of 1, preparation of a system applicable solvent: using an impurity 1, an impurity 2, an impurity 3 and methanesulfonic acid kukoamine B to prepare the system applicable solvent; 2, preparation of a test product solvent: using the raw medicine of the methanesulfonic acid kukoamine B to prepare the test product solvent; 3, preparation of a contrast solvent: using the test product solvent to prepare the contrast solvent; 4, respectively injecting the system applicable solvent, the contrast solvent and the test product solvent into a liquid chromatograph, so as to obtain chromatograph maps; 5, according to the chromatograph maps of the system applicable solvent, the contrast solvent and the test product solvent, calculating to obtain the contents of known impurities, unknown impurities and total impurities in the test product solvent. The quality control method has the characteristics that the reproducibility is good, the accuracy is high, and the like.
Description
Technical field
The invention belongs to medical art, be specifically related to a kind of methane-sulforic acid Kukoamine B related substance method of quality control.
Background technology
Kukoamine B (KukoamineB), shown under chemical constitution:
Natural products Kukoamine B (KukoamineB) is the alkaloid compound of separation and Extraction from the root bark of Chinese wolf-berry first such as ShinjiFunayama.Kukoamine B effectively can cause pyemic bacterial pathogens correlation molecule-lipopolysaccharide (endotoxin/lipopolysaccharide by antagonism, and DNA of bacteria (CpGDNA) LPS), excellent activity is shown to treatment pyemia, and effect is obviously better than positive control medicine, thus has good patent medicine prospect.
Kukoamine B easily makes its related substance increase because of the change of the conditions such as acid, alkali, oxidation in preparation and put procedure.
In research process, our the separation and purification impurity of following three kinds of Kukoamine Bs:
Impurity 1:
Impurity 2:
Impurity 3:
Kukoamine B is not mixed Quality Research at present, study report in great detail also not about Kukoamine B impurity method of quality control.In order to better study the pharmaceutical properties of Kukoamine B, improve the drug quality of Kukoamine B.The method of quality control of experimenter to the related substance of Kukoamine B is studied.Have found a kind of precision high, simple to operate, applicability is strong, may be used for the quality control of above-mentioned three kinds of impurity in Kukoamine B and related preparations.
Summary of the invention
The object of the invention is the detection method of content providing a kind of methane-sulforic acid Kukoamine B.
The liquid phase chromatography that the present invention sets up detects the related substance of methane-sulforic acid Kukoamine B, is located known impurities by system suitability solution, uses the Self-control method containing correction factor to calculate known impurity level, makes the control of related substance more strict and accurate.
Detection method of content of the present invention, comprises the following steps:
Step 1, prepared by system suitability solution: get impurity 1, impurity 2, impurity 3 and methane-sulforic acid Kukoamine B and be mixed with system suitability solution;
Step 2, prepared by need testing solution: get methane-sulforic acid Kukoamine B bulk drug and be mixed with need testing solution;
Step 3, the preparation of contrast solution: get need testing solution and be mixed with contrast solution;
Step 4, respectively by system suitability solution, contrast solution and need testing solution injection liquid chromatography, obtains chromatogram;
Step 5, according to the chromatogram of system suitability solution, contrast solution and need testing solution, through calculating the content of each known impurities, unknown impuritie and total impurities in need testing solution;
Wherein, the chromatographic condition of liquid chromatography is as follows:
Chromatographic column: enlightening horse DiamondC18 (250mm × 4.6mm, 5 μm);
Mobile phase: methyl alcohol-20mM TBAH aqueous solution;
Determined wavelength: 280nm;
Column temperature: 30 DEG C;
Flow velocity: 1ml/min
Sample size 10 μ L.
In chromatographic condition, mobile phase is methyl alcohol-20mM TBAH aqueous solution, and the two volume ratio is 10:90-20:80, adjusts the pH value range of solution for 2.5-3.5 with strong aqua.
Concrete, detection method of the present invention, step is as follows:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B, impurity 1, impurity 2, each 1-4mg of impurity 3, with being placed in 12.5-50ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 12.5-50mg, be placed in 12.5-50ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 0.5-2ml, puts in 50-200ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: accurate absorption said system applicability solution, contrast solution and need testing solution each 5-20 μ l respectively, injection liquid chromatography, obtains chromatogram;
Step 5, according to the chromatogram of system suitability solution, contrast solution and need testing solution, through calculating the content of each known impurities, unknown impuritie and total impurities in need testing solution;
Wherein, the chromatographic condition of liquid chromatography is as follows:
Chromatographic column: enlightening horse DiamondC18, model 250mm × 4.6mm, 5 μm;
Mobile phase: methyl alcohol-20mM TBAH aqueous solution;
Determined wavelength: 280nm;
Column temperature: 30 DEG C;
Flow velocity: 1ml/min;
Sample size 10 μ L;
Mobile phase is methyl alcohol-20mM TBAH aqueous solution, and the two volume ratio is 10:90-20:80, adjusts the pH value range of solution for 2.5-3.5 with strong aqua.
Preferably, detection method of the present invention, step is as follows:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 25mg, be placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: accurate absorption said system applicability solution, contrast solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, obtains chromatogram, as shown in Figure 1.
Step 5, according to the chromatogram of system suitability solution, contrast solution and need testing solution, through calculating the content of each known impurities, unknown impuritie and total impurities in need testing solution.
Wherein, the chromatographic condition of liquid chromatography is as follows:
Chromatographic column: enlightening horse DiamondC18 (250mm × 4.6mm, 5 μm);
Mobile phase: methyl alcohol-20mM TBAH aqueous solution;
Determined wavelength: 280nm;
Column temperature: 30 DEG C;
Flow velocity: 1ml/min
Sample size 10 μ L.
In chromatographic condition, mobile phase is methyl alcohol-20mM TBAH aqueous solution, and the two volume ratio is 10:90-20:80, adjusts the pH value range of solution for 2.5-3.5 with strong aqua.
Detection method of the present invention obtains after being through great many of experiments screening and checking, and screening process is as follows:
1, system suitability
Get impurity 1, impurity 2, impurity 3, methane-sulforic acid Kukoamine B reference substance be appropriate, be dissolved in water, make containing impurity 1, impurity 2, impurity 3, methane-sulforic acid Kukoamine B concentration be the sample of 80 μ g/ml as system suitability solution, sample introduction measures.
Table 1 system suitability result
| Material title | Concentration (μ g/ml) | Retention time (min) | Relative retention time | Degree of separation |
| Impurity 1 | 80 | 3.293 | 0.32 | 5.45 |
| Impurity 2 | 80 | 13.526 | 1.34 | 5.70 |
| Impurity 3 | 80 | 28.233 | 2.79 | 15.20 |
| Methane-sulforic acid Kukoamine B | 80 | 10.123 | 1.00 | 19.73 |
Table 2 system suitability result
| Sample introduction sequence | Impurity 1 | Impurity 2 | Impurity 3 | Methane-sulforic acid Kukoamine B |
| 1 | 8.74 | 63.69 | 104.26 | 68.00 |
| 2 | 8.78 | 63.69 | 99.53 | 68.97 |
| 3 | 8.43 | 63.67 | 94.35 | 69.04 |
| 4 | 8.45 | 63.71 | 91.42 | 69.57 |
| 5 | 8.36 | 62.56 | 86.76 | 70.10 |
| 6 | 8.35 | 62.95 | 84.90 | 70.37 |
| Average peak area | 8.52 | 63.38 | 93.54 | 69.34 |
| RSD(％) | 2.25 | 0.79 | 7.95 | 1.24 |
As can be seen from upper table result we: the peak-to-peak degree of separation of each chromatogram conforms with the regulations, and peak area relative standard deviation conforms with the regulations.
2, specificity test
2.1 interference and location tests
Get need testing solution, each dirt solution, blank solvent sample introduction respectively respectively, each impurity and main peak all can reach and be separated completely, and blank solvent not interference measurement.
2.2 destructive test
The method of getting in this product according to the form below carries out acid, alkali, high temperature and illumination destructive test, checks catabolite with above-mentioned chromatographic condition.
Table 3 degradation experiment table
As seen from the figure, under this law chromatographic condition, can detect each related substance after destruction and catabolite well, and impurity peaks is completely separable, concrete data are in table 4.
Table 4 degradation experiment result gathers
Therefore, adopt above-mentioned chromatographic condition feasible to the related substance and catabolite thereof that measure this product.
3, detectability quantitative limit checking
Under this experiment condition, by impurity reference substance solution dilution sample detection, calculate with signal to noise ratio (S/N ratio) S/N=3, the detection of impurity 1 is limited to 37.5ng, and the detection of impurity 2 is limited to 6.25ng, and the detection of impurity 3 is limited to 4ng, and chromatogram is shown in accompanying drawing; With signal to noise ratio (S/N ratio) S/N=10 calculate, impurity 1 be quantitatively limited to 75ng, impurity 2 is quantitatively limited to 25ng, impurity 3 be quantitatively limited to 16ng.
4, linear verification
Take impurity 1 respectively, impurity 2, impurity 3, methane-sulforic acid Kukoamine B reference substance are appropriate, by water-soluble solution and dilution makes that every 1ml is about impure 1, impurity 2, impurity 3, methane-sulforic acid Kukoamine B are the solution of 0.1mg, storing solution in contrast, precision measures contrast storing solution and is placed in measuring bottle in right amount respectively, be diluted with water to scale, prepare 6 parts of solution concentrations are followed successively by impurity 1, impurity 2, impurity 3, each 1,5,10,15,20, the 50 μ g/ml of methane-sulforic acid Kukoamine B.Get each strength solution 10 μ l, by the operation of primary colors spectral condition, measurement result is in table 5.
Table 5 linear relationship test findings table (n=6, concentration: μ g/ml)
Make regression straight line with peak area (A) to concentration (C), methane-sulforic acid Kukoamine B linear equation is: Y=3.3231X+0.0857, r=0.9999; Impurity 1 linear equation is: Y=0.4548X-0.8892, r=0.9994; Impurity 2 linear equation is: Y=3.2311X-0.6878, r=0.9999; Impurity 3 linear equation is: Y=7.3862X-0.1955, r=0.9999.Therefore, impurity 1, impurity 2, impurity 3, methane-sulforic acid Kukoamine B concentration have good linear relationship between respective concentration.Linear relation slope ratio calculates, and impurity 1 impurity peaks correction factor f value is 7.3, and impurity 2 impurity peaks correction factor f value is 1.03, and impurity 3 impurity peaks correction factor f value is 0.45.
5, precision checking
5.1, repeatability
By carrying out substantive approach for 6 times to same sample solution continuous sample introduction, there is good repeatability.Get reference substance solution and be mixed with impure 1, that impurity 2, impurity 3, methane-sulforic acid Kukoamine B concentration are 50 μ g/ml solution, by primary colors spectral condition sample introduction, repeat 6 times, record peak area, the results are shown in Table 6.
Table 6 related substance replica test result
| Sample introduction sequence | Impurity 1 | Impurity 2 | Impurity 3 |
| 1 | 8.74 | 63.69 | 104.26 |
| 2 | 8.78 | 63.69 | 99.53 |
| 3 | 8.43 | 63.67 | 94.35 |
| 4 | 8.45 | 63.71 | 91.42 |
| 5 | 8.36 | 62.56 | 86.76 |
| 6 | 8.35 | 62.95 | 84.90 |
| RSD(％) | 2.25 | 0.79 | 7.95 |
Result shows, repeatability is good.
5.2 precision
By configuring 6 sample solutions and the precision of carrying out testing substantive approach meets the requirements.The results are shown in Table 7.
Table 7 related substance Precision test result
| Sample introduction sequence | Impurity 1 | Impurity 2 | Impurity 3 | Total assorted (%) |
| 1 | Do not detect | 0.40 | 0.20 | 0.87 |
| 2 | Do not detect | 0.41 | 0.20 | 0.83 |
| 3 | Do not detect | 0.41 | 0.21 | 0.92 |
| 4 | Do not detect | 0.40 | 0.21 | 0.90 |
| 5 | Do not detect | 0.41 | 0.21 | 0.88 |
| 6 | Do not detect | 0.42 | 0.20 | 0.81 |
| RSD(％) | --- | 1.53 | 2.44 | 4.53 |
Result shows that precision is good.
5.3 Intermediate precision
For investigating random fluctuation factor to the impact of precision, another analyst sets up system on different high performance liquid chromatograph, again prepares 6 sample solutions and detects, confirm with this Intermediate precision that the method is good.
Table 8 related substance Intermediate precision test findings
| Sample introduction sequence | Impurity 1 | Impurity 2 | Impurity 3 | Total assorted (%) |
| 1 | Do not detect | 0.40 | 0.19 | 0.81 |
| 2 | Do not detect | 0.39 | 0.18 | 0.80 |
| 3 | Do not detect | 0.39 | 0.19 | 0.80 |
| 4 | Do not detect | 0.39 | 0.19 | 0.80 |
| 5 | Do not detect | 0.40 | 0.20 | 0.79 |
| 6 | Do not detect | 0.38 | 0.20 | 0.80 |
| 7 | Do not detect | 0.40 | 0.20 | 0.87 |
| 8 | Do not detect | 0.41 | 0.20 | 0.83 |
| 9 | Do not detect | 0.41 | 0.21 | 0.92 |
| 10 | Do not detect | 0.40 | 0.21 | 0.90 |
| 11 | Do not detect | 0.41 | 0.21 | 0.88 |
| 12 | Do not detect | 0.42 | 0.20 | 0.81 |
| RSD(％) | --- | 2.82 | 4.73 | 5.47 |
Result shows that Intermediate precision is good.
6, stability of solution
Get need testing solution, time sample introductions different under above-mentioned chromatographic condition measures, and the results are shown in Table 9.
Table 9 sample solution stability test result
| Time (hour) | Impurity 1 (A) | Impurity 2 (A) | Impurity 3 (A) | Methane-sulforic acid Kukoamine B (A) |
| 0 | Do not detect | 12.00 | 12.88 | 3370.29 |
| 2 | Do not detect | 11.58 | 13.50 | 3408.21 |
| 4 | Do not detect | 11.43 | 13.10 | 3378.04 |
| 6 | Do not detect | 11.31 | 13.79 | 3374.54 |
| 8 | Do not detect | 11.50 | 12.96 | 3376.10 |
| 10 | Do not detect | 11.25 | 13.64 | 3388.09 |
| RSD(％) | --- | 2.33 | 2.86 | 0.41 |
In 10 hours, the change of impurity 1, impurity 2, impurity 3 and methane-sulforic acid Kukoamine B peak area is all less, and in interpret sample, each impurity is good at 10 hours internal stabilities.
7, accuracy
Record the recovery by 50%, 100%, 150% 3 each impurity of variable concentrations adding index in need testing solution, with substantive approach, there is good accuracy.
Get every 1ml containing about impurity 1, impurity 2, impurity 3 concentration are that the reference substance solution of 50 μ g is as impurity storing solution.Prepare 9 parts of need testing solutions, add each 3 parts of 2.5ml, 5.0ml, 7.5ml impurity storing solution respectively, be diluted with water to scale, get 10 μ l injecting chromatographs, measure by above-mentioned chromatographic condition, calculate the recovery, the results are shown in Table 10.Result shows, the recovery of high, medium and low three kinds of concentration all conforms with the regulations.
Table 10 related substance determination of recovery rates result
Recovery test shows: accurately and reliably, error in allowed limits for the method.
8, durability
Get need testing solution sample introduction under different pH value mobile phase, different wave length, different in flow rate, different mobile phase ratio, different chromatographic column and different column temperature condition respectively, record degree of separation, post are imitated and calculate content.The results are shown in Table 11, in mobile phase, the change of organic phase ratio is less to Influence on test result, and the change of other condition substantially without impact, shows that system robustness is good on result.
Table 11 determination of related substances serviceability test measurement result
By above-mentioned proof procedure and the result, prove that this chromatographic condition can detect the related substance of methane-sulforic acid Kukoamine B accurately and effectively, the method is simple and feasible.
Detection method of content of the present invention, relative to prior art, has the following advantages:
This detection method of content can detect the related substance of sample effectively, and the content of each known impurities is calculated by the Self-control method of the correction up factor, reach the object more adequately controlling product related impurities, there is reproducible, precision high, improve the quality of product, for the security of product and validity provide reliable foundation.
Figure of description:
Accompanying drawing 1 methane-sulforic acid Kukoamine B and three contamination levels product high-efficient liquid phase chromatograms;
Accompanying drawing 2 embodiment 1 sample detection high-efficient liquid phase chromatogram
Accompanying drawing 3 embodiment 2 sample detection high-efficient liquid phase chromatogram
Accompanying drawing 4 embodiment 3 sample detection high-efficient liquid phase chromatogram
Accompanying drawing 5 embodiment 4 sample detection high-efficient liquid phase chromatogram
Accompanying drawing 6 embodiment 5 sample detection high-efficient liquid phase chromatogram
Embodiment
By following specific embodiment, the present invention is further illustrated, but not as prior art of the present invention.
Embodiment 1:
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution (15:85) (pH3.0 adjusted by strong aqua); Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.Take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain system suitability solution, precision measures 10 μ l and injects high performance liquid chromatograph, record chromatogram.
In the test of trying out property of system, the peak-to-peak degree of separation of each chromatogram should meet the requirements, and number of theoretical plate is not less than 2000 by methane-sulforic acid Kukoamine B, and wherein the relative retention time of impurity 1 is about 0.32; The relative retention time about 1.34 of impurity 2; The relative retention time of impurity 3 is about 2.79.Get contrast solution 10 μ l and inject high performance liquid chromatograph, regulate detection sensitivity, make major component chromatogram peak height be about 20% of full scale.Precision measures need testing solution and each 10 μ l of contrast solution, injects high performance liquid chromatograph respectively, and record chromatogram is to 3 times of major component peak retention time.
The preparation of need testing solution takes methane-sulforic acid Kukoamine B 25mg, is placed in 25ml measuring bottle, adds water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution.
Reference substance solution is prepared precision and is measured need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution.
Determination method is accurate respectively draws above-mentioned contrast solution and each 10 μ l of need testing solution, injection liquid chromatography, record chromatogram, if any impurity peaks in need testing solution chromatogram, calculate impurity content by correction up factor Self-control method, after impurity 1 takes advantage of correction factor 7.3, peak area must not be greater than contrast solution peak area (1.0%); After impurity 2 takes advantage of correction factor 1, peak area must not be greater than contrast solution peak area (1.0%); After impurity 3 takes advantage of correction factor 0.45, peak area must not be greater than contrast solution peak area (1.0%), other single contaminant peak areas must not be greater than contrast solution peak area (1.0%), and each impurity peak area sum must not be greater than 3 times (3.0%) of the area of contrast solution main peak.As shown in Figure 2.
Embodiment 2
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution (10:90) (pH3.0 adjusted by strong aqua); Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.Take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain system suitability solution, precision measures 10 μ l and injects high performance liquid chromatograph, record chromatogram.
In the test of trying out property of system, the peak-to-peak degree of separation of each chromatogram should meet the requirements, and number of theoretical plate is not less than 2000 by methane-sulforic acid Kukoamine B, and wherein the relative retention time of impurity 1 is about 0.32; The relative retention time about 1.34 of impurity 2; The relative retention time of impurity 3 is about 2.79.Get contrast solution 10 μ l and inject high performance liquid chromatograph, regulate detection sensitivity, make major component chromatogram peak height be about 20% of full scale.Precision measures need testing solution and each 10 μ l of contrast solution, injects high performance liquid chromatograph respectively, and record chromatogram is to 3 times of major component peak retention time.
The preparation of need testing solution takes methane-sulforic acid Kukoamine B 25mg, is placed in 25ml measuring bottle, adds water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution.
Reference substance solution is prepared precision and is measured need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution.
Determination method is accurate respectively draws above-mentioned contrast solution and each 10 μ l of need testing solution, injection liquid chromatography, record chromatogram, if any impurity peaks in need testing solution chromatogram, calculate impurity content by correction up factor Self-control method, after impurity 1 takes advantage of correction factor 7.3, peak area must not be greater than contrast solution peak area (1.0%); After impurity 2 takes advantage of correction factor 1, peak area must not be greater than contrast solution peak area (1.0%); After impurity 3 takes advantage of correction factor 0.45, peak area must not be greater than contrast solution peak area (1.0%), other single contaminant peak areas must not be greater than contrast solution peak area (1.0%), and each impurity peak area sum must not be greater than 3 times (3.0%) of the area of contrast solution main peak.Shown in accompanying drawing 3.
Embodiment 3
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution (20:80) (pH3.0 adjusted by strong aqua); Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.Take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, be diluted with water to scale again, shake up, obtain system suitability solution, precision measures 10 μ l and injects high performance liquid chromatograph, record chromatogram, in the test of trying out property of system, the peak-to-peak degree of separation of each chromatogram should meet the requirements, and number of theoretical plate is not less than 2000 by methane-sulforic acid Kukoamine B, and wherein the relative retention time of impurity 1 is about 0.32; The relative retention time about 1.34 of impurity 2; The relative retention time of impurity 3 is about 2.79.Get contrast solution 10 μ l and inject high performance liquid chromatograph, regulate detection sensitivity, make major component chromatogram peak height be about 20% of full scale.Precision measures need testing solution and each 10 μ l of contrast solution, injects high performance liquid chromatograph respectively, and record chromatogram is to 3 times of major component peak retention time.
The preparation of need testing solution takes methane-sulforic acid Kukoamine B 25mg, is placed in 25ml measuring bottle, adds water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution.
Reference substance solution is prepared precision and is measured need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution.
Determination method is accurate respectively draws above-mentioned contrast solution and each 10 μ l of need testing solution, injection liquid chromatography, record chromatogram, if any impurity peaks in need testing solution chromatogram, calculate impurity content by correction up factor Self-control method, after impurity 1 takes advantage of correction factor 7.3, peak area must not be greater than contrast solution peak area (1.0%); After impurity 2 takes advantage of correction factor 1, peak area must not be greater than contrast solution peak area (1.0%); After impurity 3 takes advantage of correction factor 0.45, peak area must not be greater than contrast solution peak area (1.0%), other single contaminant peak areas must not be greater than contrast solution peak area (1.0%), and each impurity peak area sum must not be greater than 3 times (3.0%) of the area of contrast solution main peak.As shown in Figure 4.
Embodiment 4
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution (15:85) (pH2.5 adjusted by strong aqua); Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.Take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, be diluted with water to scale again, shake up, obtain system suitability solution, precision measures 10 μ l and injects high performance liquid chromatograph, record chromatogram, in the test of trying out property of system, the peak-to-peak degree of separation of each chromatogram should meet the requirements, and number of theoretical plate is not less than 2000 by methane-sulforic acid Kukoamine B, and wherein the relative retention time of impurity 1 is about 0.32; The relative retention time about 1.34 of impurity 2; The relative retention time of impurity 3 is about 2.79.Get contrast solution 10 μ l and inject high performance liquid chromatograph, regulate detection sensitivity, make major component chromatogram peak height be about 20% of full scale.Precision measures need testing solution and each 10 μ l of contrast solution, injects high performance liquid chromatograph respectively, and record chromatogram is to 3 times of major component peak retention time.
The preparation of need testing solution takes methane-sulforic acid Kukoamine B 25mg, is placed in 25ml measuring bottle, adds water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution.
Reference substance solution is prepared precision and is measured need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution.
Determination method is accurate respectively draws above-mentioned contrast solution and each 10 μ l of need testing solution, injection liquid chromatography, record chromatogram, if any impurity peaks in need testing solution chromatogram, calculate impurity content by correction up factor Self-control method, after impurity 1 takes advantage of correction factor 7.3, peak area must not be greater than contrast solution peak area (1.0%); After impurity 2 takes advantage of correction factor 1, peak area must not be greater than contrast solution peak area (1.0%); After impurity 3 takes advantage of correction factor 0.45, peak area must not be greater than contrast solution peak area (1.0%), other single contaminant peak areas must not be greater than contrast solution peak area (1.0%), and each impurity peak area sum must not be greater than 3 times (3.0%) of the area of contrast solution main peak.As shown in Figure 5.
Embodiment 5
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution (15:85) (pH3.5 adjusted by strong aqua); Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.Take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, be diluted with water to scale again, shake up, obtain system suitability solution, precision measures 10 μ l and injects high performance liquid chromatograph, record chromatogram, in the test of trying out property of system, the peak-to-peak degree of separation of each chromatogram should meet the requirements, and number of theoretical plate is not less than 2000 by methane-sulforic acid Kukoamine B, and wherein the relative retention time of impurity 1 is about 0.32; The relative retention time about 1.34 of impurity 2; The relative retention time of impurity 3 is about 2.79.Get contrast solution 10 μ l and inject high performance liquid chromatograph, regulate detection sensitivity, make major component chromatogram peak height be about 20% of full scale.Precision measures need testing solution and each 10 μ l of contrast solution, injects high performance liquid chromatograph respectively, and record chromatogram is to 3 times of major component peak retention time.
The preparation of need testing solution takes methane-sulforic acid Kukoamine B 25mg, is placed in 25ml measuring bottle, adds water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution.
Reference substance solution is prepared precision and is measured need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution.
Determination method is accurate respectively draws above-mentioned contrast solution and each 10 μ l of need testing solution, injection liquid chromatography, record chromatogram, if any impurity peaks in need testing solution chromatogram, calculate impurity content by correction up factor Self-control method, after impurity 1 takes advantage of correction factor 7.3, peak area must not be greater than contrast solution peak area (1.0%); After impurity 2 takes advantage of correction factor 1, peak area must not be greater than contrast solution peak area (1.0%); After impurity 3 takes advantage of correction factor 0.45, peak area must not be greater than contrast solution peak area (1.0%), other single contaminant peak areas must not be greater than contrast solution peak area (1.0%), and each impurity peak area sum must not be greater than 3 times (3.0%) of the area of contrast solution main peak.As shown in Figure 6.
Claims (9)
1. a detection method of content for methane-sulforic acid Kukoamine B, is characterized in that, comprises the following steps:
Step 1, prepared by system suitability solution: get impurity 1, impurity 2, impurity 3 and methane-sulforic acid Kukoamine B and be mixed with system suitability solution;
Step 2, prepared by need testing solution: get methane-sulforic acid Kukoamine B bulk drug and be mixed with need testing solution;
Step 3, the preparation of contrast solution: get need testing solution and be mixed with contrast solution;
Step 4, respectively by system suitability solution, contrast solution and need testing solution injection liquid chromatography, obtains chromatogram;
Step 5, according to the chromatogram of system suitability solution, contrast solution and need testing solution, through calculating the content of each known impurities, unknown impuritie and total impurities in need testing solution;
Wherein, the chromatographic condition of liquid chromatography is as follows:
Chromatographic column: enlightening horse DiamondC18, model 250mm × 4.6mm, 5 μm;
Mobile phase: methyl alcohol-20mM TBAH aqueous solution;
Determined wavelength: 280nm;
Column temperature: 30 DEG C;
Flow velocity: 1ml/min,
Sample size 10 μ L,
Wherein, the chemical constitution of described impurity 1-3 is as follows:
Impurity 1:
Impurity 2:
Impurity 3:
2. detection method of content according to claim 1, is characterized in that, in chromatographic condition, mobile phase is methyl alcohol-20mM TBAH aqueous solution, and the two volume ratio is 10:90-20:80, adjusts the pH value range of solution for 2.5-3.5 with strong aqua.
3. detection method of content according to claim 1, is characterized in that, comprises the following steps:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B, impurity 1, impurity 2, each 1-4mg of impurity 3, with being placed in 12.5-50ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 12.5-50mg, be placed in 12.5-50ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 0.5-2ml, puts in 50-200ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: accurate absorption said system applicability solution, contrast solution and need testing solution each 5-20 μ l respectively, injection liquid chromatography, obtains chromatogram;
Step 5, according to the chromatogram of system suitability solution, contrast solution and need testing solution, through calculating the content of each known impurities, unknown impuritie and total impurities in need testing solution;
Wherein, the chromatographic condition of liquid chromatography is as follows:
Chromatographic column: enlightening horse DiamondC18, model 250mm × 4.6mm, 5 μm;
Mobile phase: methyl alcohol-20mM TBAH aqueous solution;
Determined wavelength: 280nm;
Column temperature: 30 DEG C;
Flow velocity: 1ml/min;
Sample size 10 μ L;
Mobile phase is methyl alcohol-20mM TBAH aqueous solution, and the two volume ratio is 10:90-20:80, adjusts the pH value range of solution for 2.5-3.5 with strong aqua.
4. detection method of content according to claim 1, is characterized in that, comprises the following steps:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 25mg, be placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: accurate absorption said system applicability solution, contrast solution and each 10 μ l of need testing solution respectively, injection liquid chromatography, obtains chromatogram;
Step 5, according to the chromatogram of system suitability solution, contrast solution and need testing solution, through calculating the content of each known impurities, unknown impuritie and total impurities in need testing solution;
Wherein, the chromatographic condition of liquid chromatography is as follows:
Chromatographic column: enlightening horse DiamondC18, model 250mm × 4.6mm, 5 μm;
Mobile phase: methyl alcohol-20mM TBAH aqueous solution;
Determined wavelength: 280nm;
Column temperature: 30 DEG C;
Flow velocity: 1ml/min;
Sample size 10 μ L;
Mobile phase is methyl alcohol-20mM TBAH aqueous solution, and the two volume ratio is 10:90-20:80, adjusts the pH value range of solution for 2.5-3.5 with strong aqua.
5. detection method of content according to claim 1, is characterized in that, step is as follows:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 25mg, be placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: the above-mentioned contrast solution of accurate absorption and each 10 μ l of need testing solution respectively, injection liquid chromatography, record chromatogram, calculates, to obtain final product;
Wherein, the chromatographic condition of liquid chromatography is filling agent with octadecylsilane chemically bonded silica as follows; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution=15:85, adjusts pH to 3.0 with strong aqua; Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.
6. detection method of content according to claim 1, is characterized in that, step is as follows:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 25mg, be placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: the above-mentioned contrast solution of accurate absorption and each 10 μ l of need testing solution respectively, injection liquid chromatography, record chromatogram, calculates, to obtain final product;
Wherein, the chromatographic condition of liquid chromatography is as follows: be filling agent with octadecylsilane chemically bonded silica, and mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution=10:90, adjusts pH to 3.0 with strong aqua; Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.
7. detection method of content according to claim 1, is characterized in that, step is as follows:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 25mg, be placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: the above-mentioned contrast solution of accurate absorption and each 10 μ l of need testing solution respectively, injection liquid chromatography, record chromatogram, calculates, to obtain final product;
Wherein, the chromatographic condition of liquid chromatography is as follows: be filling agent with octadecylsilane chemically bonded silica; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution=20:80, adjusts pH to 3.0 with strong aqua; Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.
8. detection method of content according to claim 1, is characterized in that, step is as follows:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 25mg, be placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: the above-mentioned contrast solution of accurate absorption and each 10 μ l of need testing solution respectively, injection liquid chromatography, record chromatogram, calculates, to obtain final product;
Wherein, the chromatographic condition of liquid chromatography is as follows: be filling agent with octadecylsilane chemically bonded silica; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution=15:85, adjusts pH to 2.5 with strong aqua; Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.
9. detection method of content according to claim 1, is characterized in that, step is as follows:
Step 1, prepared by system suitability solution: take methane-sulforic acid Kukoamine B 25mg, impurity 1, impurity 2, each 2mg of impurity 3, with being placed in 25ml measuring bottle, adding water and making dissolving in right amount, then being diluted with water to scale, shaking up, obtaining system suitability solution;
Step 2, prepared by need testing solution: take methane-sulforic acid Kukoamine B 25mg, be placed in 25ml measuring bottle, add water and make dissolving in right amount, then be diluted with water to scale, shake up, obtain need testing solution;
Step 3, the preparation of contrast solution: precision measures need testing solution 1ml, puts in 100ml measuring bottle, is diluted with water to scale, shake up, obtain contrast solution;
Step 4, injection liquid chromatography: the above-mentioned contrast solution of accurate absorption and each 10 μ l of need testing solution respectively, injection liquid chromatography, record chromatogram, calculates, to obtain final product;
Wherein, the chromatographic condition of liquid chromatography is as follows: be filling agent with octadecylsilane chemically bonded silica; Mobile phase is methyl alcohol-20mM 4-butyl ammonium hydrogen sulfate aqueous solution=15:85, adjusts pH to 3.5 with strong aqua; Column temperature is 30 DEG C; Determined wavelength 280nm; Flow velocity is 1ml/min.
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| CN112505175A (en) * | 2020-11-20 | 2021-03-16 | 西安乐析医疗科技有限公司 | Method for determining content of impurities in peritoneal dialysis solution |
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