CN114577918A - Ultra-high performance liquid chromatography detection method for active ingredient content and fingerprint spectrum of compound sophora flavescens injection - Google Patents
Ultra-high performance liquid chromatography detection method for active ingredient content and fingerprint spectrum of compound sophora flavescens injection Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to an ultra-high performance liquid chromatography detection method for the content of active ingredients and a fingerprint of a compound sophora flavescens injectionTM C18The specification is 1.7 mu m, 2.1mm multiplied by 100 mm; the active ingredients are matrine, oxymatrine, methyl azoxymethanol primeveroside, sophocarpine, oxysophocarpine and sophoridine. The invention can provide an improved detection method of the compound radix sophorae flavescentis injection, can simultaneously determine 6 components in the compound radix sophorae flavescentis injection, and can construct a chromatographic fingerprint by using the method, thereby providing a quick and efficient technique for quality control in the compound radix sophorae flavescentis injectionThe operation method reduces the inspection workload.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an improved UPLC detection method for a compound radix sophorae flavescentis injection.
Background
The compound kuh-seng injection is a traditional Chinese medicine injection prepared by refining 2 traditional Chinese medicines of kuh-seng and rhizoma smilacis glabrae through a modern scientific method, is loaded in the national medicine standard, has the efficacies of clearing heat and promoting diuresis, cooling blood and removing toxicity, and eliminating stagnation and relieving pain, and is used for cancer swelling pain and bleeding. Modern researches show that the traditional Chinese medicine composition has various pharmacological effects of resisting tumor, resisting inflammation, relieving pain, improving the immunity of the organism and the like, and is widely used for the auxiliary treatment of severe diseases such as non-small cell lung cancer, primary liver cancer, digestive tract cancer, malignant pleural effusion and the like in clinic.
The alkaloid is the main medicinal component of the compound radix sophorae flavescentis injection. The existing compound radix Sophorae Flavescentis injection has national drug Standard (WS)3-B-2752-97-2014) only contains HPLC content determination method and fingerprint detection of matrine, oxymatrine and methyl azoxymethanol primeveroside, and lacks various scientific quality evaluation methods. The ultra-high performance liquid chromatography has the characteristics of high separation efficiency, high analysis speed, high detection sensitivity and the like, and is widely applied to detection of traditional Chinese medicines and Chinese patent medicines. By consulting the relevant literature, the research on UPLC of sophora flavescens is less, but not on the aspect of rhizoma smilacis glabrae.
Zhang Huangqin and other methods for analyzing and evaluating alkaloid and flavonoid components in Sophora flavescens flowers produced in Shanxi adopt UPLC-TQ-MS combined technology to analyze and measure 7 alkaloids and 7 flavonoid components in Sophora flavescens flowers produced in Shanxi. The method adopts acetonitrile-ammonium acetate as a mobile phase, is relatively similar to the fingerprint pattern condition in the standard, and has poor chromatographic peak shape of the injection under the condition.
In the study on alkaloid chemical component analysis and resource value of sophora flavescens seeds produced in Shanxi province, etc., the UPLC-QTOF-MS and UPLC-TQ-MS combined technology is adopted to analyze the composition and content of alkaloid resource chemical components in the mature and dry sophora flavescens seeds, and the method also adopts acetonitrile-ammonium acetate as a mobile phase, so that the method is not suitable for compound sophora flavescens injection.
Therefore, in order to more scientifically and comprehensively evaluate the internal quality of the compound radix sophorae flavescentis injection product, a method capable of simultaneously completing content determination of multiple components of the compound radix sophorae flavescentis injection and fingerprint detection is needed, a fast and efficient technical method is provided for quality control of the compound radix sophorae flavescentis injection, and the inspection workload is reduced.
Disclosure of Invention
Aiming at the technical current situation, the invention provides an improved detection method for the content of active ingredients and the fingerprint of the compound radix sophorae flavescentis injection by using UPLC. The invention comprises the following concrete steps:
the method for detecting the content of the active ingredients and the fingerprint of the compound sophora flavescens injection adopts ultra-high performance liquid chromatography for detection, and the ultra-high performance liquid chromatography condition comprises that a chromatographic column is ACQUITY UPLC CSHTM C18(ii) a The active ingredients are matrine, oxymatrine, methyl azoxymethanol primeveroside, sophocarpine, oxysophocarpine and sophoridine.
In the method of the present invention, as one embodiment, the mobile phase in the ultra high performance liquid chromatography condition is 0.15% to 0.25% potassium dihydrogen phosphate solution-methanol gradient elution, preferably 0.2% potassium dihydrogen phosphate solution-methanol gradient elution.
In the method of the present invention, as one embodiment, the elution gradient in the hplc conditions is as follows:
in the method of the present invention, as one of the embodiments, the ultra high performance liquid chromatography conditions include a column temperature of 28 ℃ to 32 ℃, preferably 30 ℃.
In the method of the present invention, as one embodiment, the ultra high performance liquid chromatography conditions include a flow rate of 0.3 ml/min.
In the method of the present invention, as one of the embodiments, the ultra high performance liquid chromatography conditions include a detection wavelength of 209nm to 213nm, preferably 211 nm;
in the method of the present invention, as one of the embodiments, the condition of the ultra high performance liquid chromatography includes a sample amount of 0.8. mu.l to 1.2. mu.l, preferably 1. mu.l.
In the method of the present invention, as one embodiment, the ultra high performance liquid chromatography further comprises:
(1) preparing a blank solution: adjusting pH of the potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, mixing 0.2% potassium dihydrogen phosphate solution-methanol at a ratio of 85: 15, and filtering.
In the method of the present invention, as one embodiment, the ultra high performance liquid chromatography further comprises:
(2) preparing a reference substance solution:
precisely weighing appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking;
precisely weighing a proper amount of sophocarpine reference substance, sophoridine reference substance and methyl azoxymethanol primeveroside reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09mg of sophocarpine, 0.08mg of sophoridine and 0.08mg of methyl azoxymethanol primeveroside per 1ml, and shaking up to obtain the final product;
precisely measuring 2ml of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to a scale with a blank solution, and shaking up to obtain the final product; alternatively, 2 parts are prepared in the same manner.
As an embodiment of the method of the present invention, the content of the reference substance in the reference substance solution may be in the following range:
the preferable range of matrine is 0.28-0.40mg, and the optimal range is 0.33 mg; the preferable range of oxymatrine is 0.72-1.06mg, and the optimal range is 0.85 mg; the preferable range of the oxysophocarpine is 0.21-0.31mg, and the optimal range is 0.25 mg; the preferable ranges of sophocarpine, sophoridine and methyl azoxymethanol primeveroside are 0.07-0.11mg, and the optimal ranges are 0.09mg, 0.08mg and 0.08mg respectively.
In the method of the present invention, as one embodiment, the ultra high performance liquid chromatography further comprises:
(3) preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding the blank solution to scale, shaking, filtering, and collecting the filtrate as the sample solution.
In the method of the present invention, as one embodiment, the method for detecting the content of the active ingredient in the compound sophora flavescens injection of the present invention comprises:
the conditions of the ultra-high performance liquid chromatography are as follows:
(1) preparing a blank solution: regulating pH value of the potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, mixing the potassium dihydrogen phosphate solution with 0.2% concentration and methanol in the ratio of 85 to 15, and filtering.
(2) Preparing a reference substance solution:
precisely weighing appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking;
precisely weighing a proper amount of sophocarpine reference substance, sophoridine reference substance and methyl azoxymethanol primeveroside reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09mg of sophocarpine, 0.08mg of sophoridine and 0.08mg of methyl azoxymethanol primeveroside per 1ml, and shaking up to obtain the final product;
precisely measuring 2ml of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to a scale with a blank solution, and shaking up to obtain the final product; alternatively, 2 parts are prepared in the same way;
(3) preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding the blank solution to scale, shaking, filtering, and collecting the filtrate as the sample solution.
(4) Injecting the blank solution, the reference substance and the sample into a liquid chromatograph in the sample injection sequence, recording the chromatogram, and calculating the content of each active ingredient by an external standard method.
In the method, as one embodiment, the method for detecting the fingerprint of the compound sophora flavescens injection is characterized by comprising the step of constructing the fingerprint of the compound sophora flavescens injection, wherein the fingerprint contains matrine, oxymatrine, methyl azoxymethanol primeveroside, sophocarpine, oxysophocarpine, sophoridine, guava acid, sophoramine and trifoliate pterocarpan glycoside.
In the method of the present invention, as one of embodiments, the method comprises: the conditions of the ultra-high performance liquid chromatography are as follows:
(1) preparing a blank solution: adjusting pH of the potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, mixing 0.2% potassium dihydrogen phosphate solution-methanol at a ratio of 85: 15, and filtering.
(2) Preparing a reference substance solution:
precisely weighing appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking;
precisely weighing a proper amount of sophocarpine reference substance, sophoridine reference substance and methyl azoxymethanol primeveroside reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09mg of sophocarpine, 0.08mg of sophoridine and 0.08mg of methyl azoxymethanol primeveroside per 1ml, and shaking up to obtain the final product;
precisely measuring 2ml of the mixed reference substance solutions I and II, placing in a 10ml measuring flask, diluting with blank solution to scale, shaking, and optionally preparing 2 parts by the same method;
(3) preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding the blank solution to scale, shaking, filtering, and collecting the filtrate as the sample solution.
(4) Injecting sample according to blank solution, reference substance, and sample to construct fingerprint containing matrine, oxymatrine, methyl azoxymethanol primeveroside, sophocarpine, oxysophocarpine, sophoridine, guava acid, sophoramine, and trifoliosid
(5) Injecting the blank solution, the reference substance and the sample into a liquid chromatograph in the sample injection sequence, recording the chromatogram, and calculating the content of each active ingredient by an external standard method.
In the method of the present invention, as one of embodiments, the method comprises:
taking oxymatrine as reference, the relative retention time of the common characteristic peaks is respectively as follows: the relative retention time of the peaks of sophoramine is 0.389 plus or minus 0.02; the peak relative retention time of methyl azoxy primrose glycoside is 0.469 +/-0.04; the relative retention time of matrine peak is 0.637 + -0.04; the peak relative retention time of sophocarpine is 0.767 + -0.03; the relative retention time of the peaks of sophoridine is 0.819 +/-0.02; the peak relative retention time of the oxysophocarpine is 0.925 + -0.02; the relative retention time of the oxymatrine peak is 1.0; the peak relative retention time of guava acid is 1.055 +/-0.03; the peak relative retention time of trifoliosid was 2.097 ± 0.05.
In the method of the present invention, as one embodiment, the method further comprises that RSD of the peak areas of oxymatrine, matrine, and oxysophocarpine should not be more than 2.0%, retention time RSD should not be more than 2.0%, RSD of the peak areas of sophocarpine, sophoridine, and primeveroside should not be more than 3.0%, retention time RSD should not more than 3.0%, the number of theoretical plates of the content detection index should not be less than 3000, and the tailing factor should not be more than 2.0.
In the present invention, as an embodiment, with reference to peak No. 7, the relative peak areas of the common characteristic peaks are: the relative peak area of the No. 1 peak is 0.032; the relative peak area of the No. 2 peak is 0.073; the relative peak area of the No. 3 peak is 0.466; the relative peak area of the No. 4 peak is 0.183; the relative peak area of the No. 5 peak is 0.107; the relative peak area of the No. 6 peak is 0.422; the relative peak area of the No. 7 peak is 1.0; the relative peak area of the No. 8 peak is 0.222; the relative peak area of the No. 9 peak is 0.056; wherein the peak 1 is sophoramine, the peak 2 is methyl azoxy primrose glycoside, the peak 3 is matrine, the peak 4 is sophocarpine, the peak 5 is sophoridine, the peak 6 is oxysophocarpine, the peak 7 is oxymatrine, the peak 8 is guava acid, and the peak 9 is trifolio pterocarpan glycoside.
Compared with the existing detection method of the compound radix sophorae flavescentis injection, the invention adopts the ultra-high performance liquid chromatography, can simultaneously determine 6 components in the compound radix sophorae flavescentis injection, and constructs the chromatographic fingerprint by using the method, thereby providing a quick and efficient technical method for quality control in the compound radix sophorae flavescentis injection and reducing the inspection workload.
Drawings
FIG. 1 is a graph showing the results of blank and negative samples in example 1.
FIG. 2 is a linear graph of 6 index components of example 1.
FIG. 3-1 is the standard control fingerprint of example 2.
FIG. 3-2 shows the sample fingerprint in example 2.
Figure 4 is a repetitive fingerprint of example 2.
Figure 5 is the precision fingerprint of example 2.
Figure 6 is the stability fingerprint of example 2.
FIG. 7-1 is a fingerprint of a different column of example 2.
Figure 7-2 is a fingerprint of a different instrument of example 2.
Fig. 7-3 are overlay spectra of 10 batches of samples in column 2 of instrument 1 of example 2.
Fig. 7-4 are control fingerprints for column 2 of instrument 1 of example 2.
Fig. 7-5 are overlay spectra of 10 batches of samples in column 3 of instrument 1 of example 2.
Figures 7-6 are control fingerprints for column 3 of instrument 1 of example 2.
Fig. 7-7 are overlay spectra of 10 batches of samples in column 1 of instrument 2 of example 2.
Fig. 7-8 are control fingerprints for column 1 of instrument 2 of example 2.
Fig. 7-9 are overlay spectra of 10 batches of samples in column 1 of instrument 3 of example 2.
Figures 7-10 are control fingerprints for column 1 of instrument 3 of example 2.
FIGS. 7-11 are overlay fingerprints of control fingerprints under different conditions of example 2.
FIG. 8 is the key point fingerprint of the production process of example 2.
FIG. 9 is a graph showing the results of example 3-1, the curves from bottom to top being: a test sample 1 and a test sample 2.
FIG. 10-1 is a graph showing the results of chromatographic condition 1 in example 3-2, the curve from bottom to top being: a sample, sophocarpine, oxysophocarpine, matrine and oxymatrine;
FIG. 10-2 is a graph showing the result of chromatographic condition 2 in example 3-2;
FIG. 10-3 is a graph showing the results of chromatographic condition 3 in example 3-2.
FIG. 10-4 is a graph showing the results of chromatographic condition 4 in example 3-2;
FIGS. 10 to 5 are graphs showing the results of chromatographic condition 5 in example 3-2, respectively;
FIGS. 10-6 are the overlay of each control sample and the test sample in example 3-2 after injection under the above conditions, wherein the chromatographic peak locations (bottom-to-top curves) are: sample, matrine, oxymatrine, sophocarpine, oxysophocarpine, sophoridine, methyl azoxymethanol primrose glycoside, guava acid, trifolibanum pterocarpus glycoside, sophoramine, N-methyl laburnine.
FIG. 11-1 is a graph showing the results of method 1 in example 3-3.
FIG. 11-2 is a graph showing the results of method 2 in example 3-3;
FIG. 11-3 is a graph showing the results of method 3 in example 3-3.
FIGS. 11-4 are graphs showing the results of method 4 of example 3-3.
FIGS. 11 to 5 are graphs showing the results of method 5 in example 3 to 3.
FIGS. 11 to 6 are graphs showing the results of the method 6 in example 3 to 3;
FIG. 12-1 is a graph showing the result of examining condition 1 in example 3-4;
FIG. 12-2 is a graph showing the results of examination of condition 2 in example 3-4;
FIG. 12-3 is a graph showing the results of examining condition 3 in example 3-4;
FIGS. 12 to 4 are graphs showing the results of examination of condition 4 in example 3 to 4;
FIGS. 12 to 5 are graphs showing the results of examination of condition 5 in examples 3 to 4;
FIGS. 12 to 6 are graphs showing the results of examination of condition 6 in examples 3 to 4;
FIGS. 12 to 7 are graphs showing the results of examination of condition 7 in examples 3 to 4.
Detailed Description
The following examples and test examples are intended to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.
Instrument
Reference substance
Test article of example 1
| Name (R) | Batch number | Source |
| Compound lightyellow |
20181138 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181034 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181139 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181203 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181204 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181209 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181212 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181213 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181214 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
| Compound lightyellow |
20181215 | SHANXI ZHENDONG PHARMACEUTICAL Co.,Ltd. |
Test article of example 2
Reagent
| Name (R) | Batch number | Source | Rank of |
| Potassium dihydrogen phosphate | 018823 | MREAD | HPLC |
| Phosphoric acid | 0160318 | BEIJING CHEMICAL PLANT | Analytical grade |
| Methanol | 10985407 902 | Merck two-in-one company, Ltd | HPLC |
Example 1 detection method of active ingredient of Compound Sophora flavescens injection
1. Chromatographic conditions, sample preparation, system applicability requirements, calculation formula, limit requirements
2. Verifying specific content
2.1 System applicability
(1) Experimental procedure
Preparing a blank solution: 0.2% potassium dihydrogen phosphate solution-methanol 85: 15 mixed solution, and filtering to obtain the final product.
Preparing a reference substance solution: precisely weighing appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking;
precisely weighing appropriate amount of sophocarpine control, sophoridine control and methyl azoxymethanol primeveroside control, adding blank solution to obtain mixed control solution II containing sophocarpine 0.09mg, sophoridine 0.08mg and methyl azoxymethanol primeveroside 0.08mg per 1ml, and shaking.
Precisely measuring 2ml of the mixed reference substance solution I and II, placing the mixed reference substance solution I and II into a 10ml measuring flask, diluting the mixed reference substance solution with a blank solution to a scale, and shaking up.
Preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection (lot number: 20181138), placing in a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting the filtrate as sample solution.
Requirement of sample introduction procedure
Sample introduction sequence
| Sequence of | Sample (I) | Number of |
| 1 | |
1 |
| 2 | |
5 needles (continuous needle insertion) |
| 3 | |
1 needle |
(2) Results reporting
And continuously injecting the control solution for 5 times to obtain peak area and RSD value of retention time.
TABLE 2.1-1 results of peak area and retention time of control solutions
TABLE 2.1-2 System suitability results
(3) Conclusion
From the results, the reference substance solution is continuously injected for 5 times, the RSDs of the peak areas of oxymatrine, matrine and oxysophocarpine are all less than 2.0%, the RSDs of the retention time are all less than 2.0%, the RSDs of the peak areas of sophocarpine, sophoridine and methyl azoxymethanol primeveroside are all less than 3.0%, and the RSDs of the retention time are all less than 3.0%; the theoretical plate number of the six index components is more than 3000, and the tailing factors are all less than 2.0, so that the requirements are met.
2.2 specificity
(1) Experimental procedure
Preparing a blank solution: 0.2% potassium dihydrogen phosphate solution-methanol 85: 15 mixed solution, and filtering to obtain the final product.
Preparing a negative sample solution: precisely measuring 1ml of each of single radix Sophorae Flavescentis injection (wild and planted) and single rhizoma Smilacis Glabrae injection, placing into 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting the filtrate as negative sample solution.
Preparing a 0.25% Tween solution: weighing 0.25g of Tween 80, adding water to dissolve to 100ml, shaking up, filtering, and taking the subsequent filtrate as 0.25% Tween solution.
Preparing a reference substance solution: refer to reference substance solution preparation method under item 2.1.
Preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing into a 50ml measuring flask, adding the blank solution to scale, and shaking up for use.
Preparing a filter membrane interference sample: taking a test solution (batch number is 20181138), and centrifuging one part; one portion was filtered and different volumes (1ml, 3ml, 5ml, 7ml, 9ml) were discarded.
Requirement of sample introduction procedure
Sample introduction sequence
(2) See figure 1 and table below for results reports.
TABLE 2.2-1 Filter interference test results (percentage area of different volumes discarded relative to centrifuged sample)
(3) Conclusion
From the results, the blank solution, the blank mobile phase and the 0.25% tween 80 solution have no interference on the sample, the single kuh-seng negative sample solution (wild kuh-seng and planted kuh-seng) has no interference on methyl azoxy methanol primrose glycoside, and the single rhizoma smilacis glabrae negative sample solution has no interference on alkaloid.
After different volumes are discarded, the percentage relative content of the index component area of the sample solution to be tested and the index component area of the centrifugal sample solution is between 95.0 and 102.0 percent, and the adsorption can be ignored.
2.3 Linearity and Range
(1) Experimental procedure
Preparing a blank solution: 0.2% potassium dihydrogen phosphate solution-methanol 85: 15 mixed solution, and filtering to obtain the final product.
Preparing a linear stock solution: precisely weighing appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking; precisely weighing sophocarpine reference substance, sophoridine reference substance, and methyl azoxymethanol primeveroside reference substance, adding blank solution to obtain mixed reference substance solution II containing 0.09mg of sophocarpine, 0.08mg of sophoridine, and 0.08mg of methyl azoxymethanol primeveroside per 1ml, and shaking.
Control solution 1: accurately weighing 0.5ml of each of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to the scale with the blank solution, and shaking up the mixed reference substance solutions.
Control solution 2: precisely measuring 1ml of each of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to the scale with the blank solution, and shaking up.
Control solution 3: precisely measuring 2ml of each of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to the scale with the blank solution, and shaking up.
Control solution 4: precisely measuring 3ml of each of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to the scale with the blank solution, and shaking up.
Control solution 5: precisely measuring 4ml of each of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to the scale with the blank solution, and shaking up.
Requirement of sample introduction procedure
Sample introduction sequence
(2) Results reporting
The regression equation, the correlation coefficient and the linear graph result of each index component are shown in fig. 2.
(3) Conclusion
The linear correlation coefficient is more than or equal to 0.999, and meets the standard.
2.4 accuracy
(1) Experimental procedure
Preparing a blank solution: 0.2% potassium dihydrogen phosphate solution-methanol 85: 15 mixed solution, and filtering to obtain the final product.
Preparing a reference substance solution: refer to reference solution preparation method under item 2.1, prepare two parts by the same method.
Preparation of 50% recovery solution: precisely measuring 0.5ml of the compound lightyellow sophora root injection, placing in a 50ml measuring flask, respectively adding 2.5ml of each of the mixed reference substance solutions I and II, adding the blank solution to the scale, shaking up, filtering to obtain a 50% recovery rate solution (3 parts prepared by the same method).
Preparation of 100% recovery solution: precisely measuring 0.5ml of the compound sophora flavescens injection, placing the compound sophora flavescens injection into a 50ml measuring flask, respectively adding 5ml of the mixed reference substance solution I and 5ml of the mixed reference substance solution II, adding the blank solution to the scale, shaking up, filtering, and taking the mixture as a solution with 100% recovery rate (3 parts are prepared by the same method).
Preparation of 150% recovery solution: precisely measuring 0.5ml of the compound sophora flavescens injection, placing the compound sophora flavescens injection into a 50ml measuring flask, respectively adding 7.5ml of the mixed reference substance solutions I and II, adding blank solutions to the scale, shaking up, filtering, and taking the mixture as a 150% recovery rate solution (3 parts prepared by the same method).
Sample introduction sequence
(2) Results reporting
TABLE 2.4-1 methyl azoxymethanol primeveroside content recovery test results
TABLE 2.4-2 matrine content recovery test results
TABLE 2.4-3 Sophocarpine content recovery test results
TABLE 2.4-4 Sophoridine content recovery test results
TABLE 2.4-5 Sophora japonica alkaloid content recovery test results
TABLE 2.4-6 Sophocarpidine oxide content recovery test results
(3) Conclusion
The recovery rate ranges of the matrine, oxymatrine, oxysophocarpine, sophocarpine, sophoridine and methyl azoxymethanol primeveroside in the test sample are all between 95% and 105%, and the 9 parts of recovery rate RSD are all less than 3%, which meets the requirement.
2.5 repeatability
(1) Experimental procedure
Preparing a blank solution: 0.2% potassium dihydrogen phosphate solution-methanol 85: 15 mixed solution, and filtering to obtain the final product.
Preparing a reference solution: refer to reference solution preparation method under item 2.1, prepare two parts by the same method.
Preparing a test solution: precisely measuring 1ml of compound radix Sophorae Flavescentis injection (lot 2081138), placing in a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting the filtrate as sample solution. 6 portions were worked up in parallel.
Requirement of sample introduction procedure
Sample introduction sequence
| Sequence of | Sample (I) | Number of |
| 1 | |
1 |
| 2 | |
5 needles (continuous test) |
| 3 | |
2 needles |
| 4 | Test article-1 |
1 |
| 5 | Test article-2 |
1 |
| 6 | Test article-3 |
1 |
| 7 | Test article-4 |
1 |
| 8 | Test article-5 |
1 |
| 9 | Test article-6 |
1 |
| 10 | |
1 needle |
(2) Results reporting
TABLE 2.5 repeatability test results
(3) Conclusion
From the results, the RSD of 6 samples containing matrine, oxymatrine and oxysophocarpine is less than 3.0%, and RSD of sophocarpine, sophoridine and methyl azoxymethanol primeveroside is less than 4.0%, indicating good repeatability of the samples.
2.6 precision
(1) Experimental procedure
Preparing a blank solution: 0.2% potassium dihydrogen phosphate solution-methanol 85: 15 mixed solution, and filtering to obtain the final product.
Preparing a reference substance solution: refer to reference solution preparation method under item 2.1, prepare two parts by the same method.
Preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection (lot number: 20181138), placing in a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting the filtrate as sample solution.
Requirement of sample introduction procedure
Sample introduction sequence
(2) Results reporting
TABLE 2.6 results of precision measurements
(3) Conclusion
As can be seen from the results, the results of the content results RSD of matrine, oxymatrine and oxysophocarpine in the test sample are all less than 3.0% and the content results RSD of sophocarpine, sophoridine and methyl azoxymethanol primeveroside are all less than 4.0% after 6 needles of continuous sample injection, which indicates that the precision of the test sample is good.
2.7 solution stability
(1) Experimental procedure
Preparing a blank solvent: 0.2% potassium dihydrogen phosphate solution-methanol 85: 15 mixed solution, and filtering to obtain the final product.
Preparing a reference substance solution: refer to reference solution preparation method under item 2.1, prepare two parts by the same method.
Preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection (lot number: 20181138), placing in a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting the filtrate as sample solution.
Requirement of sample introduction procedure
Sample introduction sequence
| Sequence of | Sample (I) | Number of |
| 1 | |
1 |
| 2 | |
5 needles (continuous test) |
| 3 | |
2 needles |
| 4 | Test article-0 |
1 |
| 5 | Test article-1 |
1 |
| 6 | Sample-2 |
1 |
| 7 | Sample-4 |
1 |
| 8 | Sample-8 |
1 |
| 9 | Sample-16 |
1 |
| 10 | Test article-24 |
1 |
| 11 | |
1 needle |
(2) Results reporting
TABLE 2.7 stability test results
(3) Conclusion
From the results, the control solution and the test solution are placed for 24 hours, the RSD of the content result of the matrine, oxymatrine and oxysophocarpine in the test is less than 3.0 percent, and the RSD of the content result of the sophocarpine, sophoridine and methyl azoxymethanol primeveroside in the test is less than 4.0 percent, which shows that the test solution is stable within 24 hours.
Calculating the percentage of the index component area at each time point to the index component area at 0 hr, comparing the result of each time point of the reference and test solutions with the initial result, and determining the relative content of the reference and test solutions
98.0-102.0%, and the solution stability is good.
2.8 durability
(1) Experimental procedure
The varied chromatographic conditions are shown in the following table.
Preparing a blank solution: 0.2% potassium dihydrogen phosphate solution-methanol 85: 15 mixed solution, and filtering to obtain the final product.
Preparing a reference substance solution: refer to reference substance solution preparation method under item 2.1.
Preparing a test solution: precisely measuring 1ml of each batch of the compound radix Sophorae Flavescentis injection, placing into a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting the filtrate as sample solution.
And (5) the requirement of a sample introduction procedure.
(different investigation conditions are all injected according to the sample injection sequence)
Sample introduction sequence
| Sequence of | Sample (I) | Number of |
| 1 | |
1 |
| 2 | |
5 |
| 3 | |
2 needles |
| 4 | Test article-1 | 1 |
| 5 | Test article-2 | 1 |
| 6 | Sample-3 to sample-9 | 1 needle for each |
| 7 | Test article-10 | 1 |
| 8 | |
1 needle |
Note: if the solution is unstable and the chromatographic condition is changed, the reference substance and the test solution need to be prepared again.
(2) Results reporting
TABLE 2.9 content durability test results
(3) Conclusion
The content of matrine, sophocarpine, oxysophocarpine and oxymatrine in the test solution is basically consistent under different conditions, the RSD value of each batch of samples is less than 3%, the RSD value of methyl azoxymethanol primrose glycoside is less than 5%, and the durability is good. The content of each batch of sophoridine samples is low under the condition of the chromatographic column 1 of the instrument 2, so that the RSD value is less than 10 percent, and the durability is in an acceptable range.
Example 2 UPLC fingerprint verification and examination of Compound radix Sophorae Flavescentis injection
1. Chromatographic conditions, elution conditions, sample preparation, etc
2 authenticating content
2.1 System applicability
(1) Experimental procedure
Preparing a blank solution: methanol-0.2% potassium dihydrogen phosphate 15: 85, and filtering.
Preparing a reference substance solution: precisely weighing appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking; precisely weighing a proper amount of sophocarpine reference substance, sophoridine reference substance and methyl azoxymethanol primeveroside reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09mg of sophocarpine, 0.08mg of sophoridine and 0.08mg of methyl azoxymethanol primeveroside per 1ml, and shaking up to obtain the final product; accurately weighing appropriate amount of trilobatin reference substance, sophoramine reference substance, and N-methylcardnine reference substance, adding blank solution to obtain mixed reference substance solution III containing 0.08mg of trilobatin, 0.08mg of sophoramine, and 0.09mg of N-methylcardnine per 1ml, and shaking. Precisely measuring the mixed reference substance solutions I, II and III by 2ml respectively, placing in a 10ml measuring flask, diluting with blank solution to scale, and shaking up to obtain the final product.
Test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding the blank solution to scale, shaking, filtering, and collecting the filtrate as the sample solution.
The order of introduction and the requirements are given in the following table.
Sample introduction sequence
| Sequence of | Sample (I) | Number of |
| 1 | |
1 |
| 2 | |
5 needles (continuous needle insertion) |
| 3 | |
1 needle |
(2) Results reporting
And (3) continuously feeding the reference substance solution for 5 times to obtain peak area and RSD value of retention time.
TABLE 2.1-1 results of peak area and retention time of control solutions
TABLE 2.1-2 System suitability results
(3) Conclusion
From the results, the reference substance solution is continuously injected for 5 times, the RSDs of the peak areas of oxymatrine, matrine and oxysophocarpine are all less than 2.0%, the RSDs of the retention time are all less than 2.0%, the RSDs of the peak areas of sophocarpine, sophoridine and methyl azoxymethanol primeveroside are all less than 3.0%, and the RSDs of the retention time are all less than 3.0%; the theoretical plate number of the six index components is more than 3000, the tailing factor is less than 2.0, and the requirements are met.
2.2 fingerprint Pattern establishment
(1) Experimental procedure
Blank solution: mixing methanol with 0.2% potassium dihydrogen phosphate at a ratio of 15: 85, and filtering.
Preparing a reference substance solution: refer to the reference substance solution preparation method under item 2.1.
Each batch of test solution: precisely measuring 1ml of each batch of the compound radix Sophorae Flavescentis injection, placing into a 50ml measuring flask, adding the blank solution to scale, shaking, filtering, and collecting the filtrate as sample solution.
The order of introduction and the requirements are given in the following table.
Sample introduction sequence
(2) Results reporting
Based on the chromatographic fingerprint of 10 batches of compound sophora flavescens injection, data processing is carried out by using 2012 edition of traditional Chinese medicine chromatographic fingerprint similarity evaluation system recommended by pharmacopoeia committee, a chromatographic peak of a sample 1(S1) is used as a reference spectrum, a median method is adopted, a time window is 0.1, and after multi-point correction, full-peak matching is carried out to generate a standard control fingerprint and a common mode. See fig. 3-1-3-2 and the following table.
TABLE 2.2-1 results of similarity of fingerprint spectra of each batch to control fingerprint
TABLE 2.2-2 results of non-common peaks for each batch
| Test article | Non-consensus peak area | Total peak area | The non-shared peak area accounts for% |
| 20181138 | 74.01 | 2242.64 | 3.30 |
| 20181139 | 66.45 | 2476.85 | 2.68 |
| 20181203 | 46.58 | 2329.11 | 2.00 |
| 20181204 | 56.14 | 2279.62 | 2.46 |
| 20181209 | 57.61 | 2291.59 | 2.51 |
| 20181212 | 53.14 | 2381.77 | 2.23 |
| 20181213 | 52.58 | 2473.87 | 2.13 |
| 20181214 | 38.36 | 2328.47 | 1.65 |
| 20181215 | 47.41 | 2395.86 | 1.98 |
| 20181034 | 39.67 | 2413.80 | 1.64 |
(3) Conclusion
By comparison with the control, it can be seen that the 1 st peak is sophoramine, the 2 nd peak is methyl azoxy primrose glycoside, the 3 rd peak is matrine, the 4 th peak is sophocarpine, the 5 th peak is sophoridine, the 6 th peak is oxysophocarpine, the 7 th peak is oxymatrine, the 8 th peak is guava acid, and the 9 th peak is trifolium pterocarpus santalin.
From the results, 10 samples have a similarity of more than 0.9 to the control fingerprint, and the ratio of the non-shared peak area is less than 5%.
2.3 reproducibility
(1) Experimental procedure
Preparing a blank solution: methanol-0.2% potassium dihydrogen phosphate 15: 85, and filtering.
Preparing a reference substance solution: refer to reference substance solution preparation method under item 2.1.
Preparing a test solution: precisely measuring 1ml of 6 parts of compound radix Sophorae Flavescentis injection of the same batch number, placing in a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting the filtrate as sample solution.
The order of injection and the requirements are given in the following table.
Sample introduction sequence
| Sequence of events | Sample (I) | Number of |
| 1 | |
1 |
| 2 | |
5 needles (continuous test) |
| 3 | Test article-1 |
1 needle |
| 4 | Test article-2 |
1 |
| 5 | Test article-3 |
1 |
| 6 | Test article-4 |
1 |
| 7 | Test article-5 |
1 |
| 8 | Test article-6 |
1 |
| 9 | |
1 needle |
(2) Results report see FIG. 4
TABLE 2.3-1 repeatability common Peak Pattern similarity results
TABLE 2.3-2 results of relative retention time of repeated consensus peaks
TABLE 2.3-3 repeatability common peak area results
(3) Conclusion
From the results, the similarity of 6 samples is greater than 0.99, and the relative retention time of each common peak and the RSD value of the peak area are less than 3%, so that the repeatability is good.
2.6 precision
(1) Experimental procedure
Preparing a blank solution: methanol-0.2% potassium dihydrogen phosphate 15: 85, and filtering.
Preparing a reference substance solution: refer to reference substance solution preparation method under item 2.1.
Preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding the blank solution to scale, shaking, filtering, and collecting the filtrate as the sample solution.
The order of injection and the requirements are given in the following table.
Sample introduction sequence
| Sequence of | Sample (I) | Number of |
| 1 | |
1 |
| 2 | Control solution | 5-pin (continuous test) |
| 3 | |
6 needles |
| 4 | |
1 needle |
(2) Results report see FIG. 5 and Table below
TABLE 2.4-1 precision consensus peak pattern similarity results
Tables 2.4-2 results of precision consensus peak relative retention time
Tables 2.4-3 precision common Peak area results
(3) Conclusion
From the results, the similarity of the sample after continuous sample introduction for 6 needles is more than 0.99, and the relative retention time of each common peak and the RSD value of the peak area are both less than 3 percent, which meets the requirements.
2.7 solution stability
(1) Experimental procedure
Preparing a blank solution: methanol-0.2% potassium dihydrogen phosphate 15: 85, and filtering.
Preparing a reference solution: refer to reference substance solution preparation method under item 2.1.
Preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding the blank solution to scale, shaking, filtering, and collecting the filtrate as the sample solution.
The sample introduction sequence and requirements are as follows.
Sample introduction sequence
| Sequence of | Sample(s) | Number of |
| 1 | |
1 |
| 2 | |
5 needles (continuous test) |
| 3 | Test article-0 |
1 needle |
| 4 | Test article-1 |
1 |
| 5 | Sample-2 |
1 |
| 6 | Sample-4 |
1 |
| 7 | Sample-8 |
1 |
| 8 | Sample-16 |
1 |
| 9 | Test article-24 |
1 |
| 10 | |
1 needle |
(2) Results report see FIG. 6 and Table below
TABLE 2.5-1 results of similarity of stability consensus peak patterns
TABLE 2.5-2 results of relative retention time of stability consensus peaks
TABLE 2.5-3 results of stability consensus peak area
(3) Conclusion
From the results, the similarity of the test sample is more than 0.99 within 24 hours, and the relative retention time of each common peak and the RSD value of the peak area are both less than 3%, so that the test sample is stable within 24 hours.
2.8 durability
(1) Experimental procedure
Preparing a blank solution: methanol-0.2% potassium dihydrogen phosphate 15: 85, and filtering.
Preparing a reference solution: refer to reference substance solution preparation method under item 2.1.
Preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing into a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting filtrate as sample solution.
Respectively taking 1 mul of the solution, injecting the solution into a high performance liquid chromatograph under different conditions, and obtaining the sample injection sequence and requirements in the following table (the sample injection sequence is used for injecting samples under different investigation conditions).
Sample introduction sequence
(3) Results reporting
TABLE 2.8-1 parameters for chromatographic Condition variation
(2) Results reporting
The durability of the test sample on the chromatographic column and the instrument was examined by using the test sample of lot 20181138, and the results are shown in FIG. 7-1 and the following table.
TABLE 2.8-2 results of similarity of common peak patterns of different chromatographic columns
Tables 2.8-3 results for common peak relative retention times for different chromatography columns
TABLE 2.8-4 results of peak area shared by different chromatographic columns
The durability results for the different instruments are shown in FIG. 7-2 and the following Table
TABLE 2.8-5 common Peak mode similarity results for different instruments
TABLE 2.8-6 results of relative retention time of common peaks for different instruments
TABLE 2.8-7 results of peak-to-peak area common to different instruments
Secondly, on the basis of the chromatographic fingerprint of 10 batches of the compound sophora flavescens injection, establishing fingerprints of different chromatographic columns and instruments respectively, generating a control fingerprint, and inspecting the similarity of the generated control fingerprints. See fig. 7-3 through 7-11 and the tables below.
TABLE 2.8-8 results of similarity (Instrument 1 column 2)
TABLE 2.8-9 results of similarity (Instrument 1 column 3)
TABLE 2.8-10 results of similarity (Instrument 2 column 1)
TABLE 2.8-11 similarity results (Instrument 3 column 1)
TABLE 2.8-12 similarity results (different control fingerprints)
(3) Conclusion
From the results, in different chromatographic column investigation results, the similarity of all common peaks is greater than 0.99, the relative retention time RSD is less than 3%, and the peak area is less than 5%; in the results of different instrument examinations, the similarity of each common peak is greater than 0.99, and the RSD of the relative retention time and the peak area is less than 10%, which indicates that the instrument has influence on the method but is within an acceptable range.
Under different investigation conditions, the similarity of 10 samples is greater than 0.99, and the similarity between the generated control fingerprints is greater than 0.99, so that the method has better durability in view of the similarity result.
2.9 Key points investigation of production Process
(1) Experimental procedure
Preparing a blank solution: methanol-0.2% potassium dihydrogen phosphate 15: 85, and filtering.
Preparing a reference substance solution: refer to reference substance solution preparation method under item 2.1.
Preparing a test solution: calculating the sampling amount according to the volume amount of each key point, wherein the key point 1 is taken from 1ml to 25ml measuring flask, the key points 2 and 6 are taken from 5ml to 50ml measuring flask, the key point 4 is taken from 2ml to 25ml measuring flask, the key point 5 is taken from 1ml to 100ml measuring flask, the rest key points are taken from 1ml to 50ml measuring flask, all samples are added with blank solution to scale, shaking up and filtering are carried out, and the subsequent filtrate is taken as the sample solution.
The sample introduction sequence and requirements are as follows.
Sample introduction sequence
(2) Results report see FIG. 8
TABLE 2.9-1 results of similarity of common peak patterns of production processes
(3) Conclusion
From the results, the similarity of the key points of the production process is between 0.451 and 0.754, and the similarity of the key points of the production process (the water-sinking liquid medicine-the samples after sterilization) is higher than 0.9.
Examples 3-1 to 3-4UPLC Condition investigation
Example 3-1 (examination of sample preparation)
The experimental steps are as follows:
3-1.1 blank solution preparation: adjusting pH of the potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, mixing 0.34% potassium dihydrogen phosphate solution-methanol at a ratio of 85: 15, and filtering.
3-1.2 preparation of control solutions: precisely measuring appropriate amount of matrine, oxymatrine, oxysophocarpine, sophocarpine, sophoridine, and methyl azoxymethanol primeveroside control, and adding blank solution to obtain control solutions of 0.2mg/ml, 0.4mg/ml, 0.2mg/ml, 0.05mg/ml, and 0.05 mg/ml.
3-1.3 preparation of test solution: taking a compound radix sophorae flavescentis injection sample, precisely measuring 0.5ml, putting the compound radix sophorae flavescentis injection sample into a 25ml measuring flask, adding a blank solution to be diluted to a scale, shaking up, and filtering to obtain a test solution 1. Taking a compound lightyellow sophora root injection sample, precisely measuring 1ml, putting into a 50ml measuring flask, adding a blank solution to dilute to a scale, shaking up, and filtering to obtain a test solution 2.
3-1.4 chromatographic conditions
A chromatographic column: a chromatographic column: ACQUITY UPLC CSHTM C181.7 μm, 2.1mm × 100 mm; detection wavelength: 211nm, column temperature: 30 ℃, flow rate: 0.3 ml/min; mobile phase A: methanol, mobile phase B: 0.2% monopotassium phosphate (pH adjusted to 3.0 with phosphoric acid) was eluted in a gradient.
3-1.5 sample injection of the sample 1 and sample injection of the sample 2 are respectively carried out by 0.5 mul and 1 mul according to the chromatographic conditions.
3-1.6 Experimental results: see fig. 9.
In conclusion, the test sample was prepared by the method for preparing test sample solution 2 (1ml of sample diluted to 50ml with blank solution).
Example 3-2 (examination of mobile phase)
The experimental steps are as follows:
3-2.1 blank solution preparation: see example 3-1 blank solution formulation.
3-2.2 preparation of reference solution: precisely measuring appropriate amount of sophoramine, guava acid, N-methyl laburnine and red sandalwood glycoside, adding blank solution to obtain reference solution of 0.05mg/ml, 0.1mg/ml, 0.05mg/ml and 0.1mg/ml, and preparing the same reference solution as in example 3-1.
3-2.3 preparation of test solution: taking a compound radix sophorae flavescentis injection sample, precisely measuring 1ml, placing the sample in a 50ml measuring flask, adding a blank solution to dilute to a scale, shaking up, and filtering to obtain a test solution.
3-2.4 chromatographic Condition Observation:
chromatographic conditions 1:
a chromatographic column: ACQUITY UPLC CSHTM C18 1.7μm，2.1mm×100mm
Detection wavelength: 211nm, column temperature: 30 ℃, flow rate: 0.4ml/min, sample size: 1 μ l
Mobile phase A: 0.01M aqueous ammonium formate solution B: acetonitrile: 0.01M aqueous ammonium formate solution (9: 1) elution conditions:
the experimental results are as follows: see fig. 10-1.
Chromatographic conditions 2:
and (3) chromatographic column: ACQUITY UPLC CSHTM C18 1.7μm，2.1mm×100mm
Detection wavelength: 211nm, column temperature: 30 ℃, flow rate: 0.3ml/min, sample size: 1 μ l
Mobile phase A: acetonitrile B: 0.34% Potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)
The experimental results are as follows: see fig. 10-2.
Chromatographic conditions 3:
the mobile phase was adjusted as follows, with the remainder being identical to chromatographic conditions 2.
Mobile phase A: methanol B: 10mmol ammonium bicarbonate (pH9.0)
The experimental results are as follows: see FIGS. 10-3
Chromatographic conditions 4:
the mobile phase was adjusted as follows, and the rest was the same as in condition 2.
Mobile phase A: methanol B: 0.34% Potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)
The experimental results are as follows: see FIGS. 10-4
Chromatographic conditions 5:
the mobile phase was adjusted as follows, and the rest was the same as in condition 2.
Mobile phase A: methanol B: 0.2% Potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)
The experimental results are as follows: referring to FIGS. 10-5, the overlay of each reference sample with the test sample after sample injection under the above conditions is shown in FIGS. 10-6.
In conclusion, the condition of methanol-0.2 percent potassium dihydrogen phosphate as a mobile phase is the best.
Examples 3-3 (mobile phase gradient survey):
the experimental steps are as follows:
3-3.1 blank solution preparation: see example 3-1 blank solution formulation.
3-3.2 preparation of test solution: see example 3-2 for test solution preparation.
3-3.3 gradient Condition Observation:
a chromatographic column: ACQUITY UPLC CSHTM C18 1.7μm，2.1mm×100mm
Flow rate: 0.3ml/min, column temperature: 30 ℃, detection wavelength: 211nm, sample size: 1 μ l
Mobile phase A: methanol B: 0.2% Potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid)
The gradient elution procedure was:
the method comprises the following steps:
the experimental results are as follows: see FIG. 11-1
The method 2 comprises the following steps:
the experimental results are as follows: see fig. 11-2
The method 3 comprises the following steps:
the experimental results are as follows: see FIGS. 11-3
The method 4 comprises the following steps:
the experimental results are as follows: see FIGS. 11-4
The method 5 comprises the following steps:
the experimental results are as follows: see FIGS. 11-5
The method 6 comprises the following steps:
the experimental results are as follows: see FIGS. 11-6
In summary, the elution process conditions of method 5 are optimal.
Examples 3 to 4 (column, column temperature and flow rate investigation)
The experimental steps are as follows:
3-4.1 preparation of blank solution: see example 3-1 blank solution formulation.
3-4.2 preparation of test solution: see example 3-1 test article solution 1 formulation.
3-4.3 Condition Observation:
examination condition 1:
a chromatographic column: agilent Poroshell 120EC-C18 4μm，4.6mm×100mm
Flow rate: column temperature 0.3 ml/min: detection wavelength at 30 ℃: 211nm sample size: 1 μ l
Mobile phase A: methanol B: 0.34% Potassium dihydrogen phosphate (phosphoric acid adjusted to pH 3.0)
The experimental results are as follows: see FIG. 12-1
Examination condition 2:
a chromatographic column: ACQUITY UPLC CSHTM C181.7 μm, 2.1mm × 100 mm; detection wavelength: 211nm, sample size: 1 μ l, flow rate: 0.3ml/min, column temperature: 35 deg.C
See example 3-3 for elution procedure, gradient conditions as in example 3-3 for examination of method 5.
The experimental results are as follows: see fig. 12-2
Examination condition 3:
sample injection amount: 0.5ul, the rest being identical to examination 2, the gradient elution program was adjusted as follows:
the experimental results are as follows: see FIGS. 12-3
Examination condition 4:
sample introduction amount: 0.5. mu.l, flow rate: 0.3ml/min, 0.28ml/min, and the rest of the same examination conditions 2, the elution program was adjusted as follows:
the experimental results are as follows: see FIGS. 12-4
Examination condition 5:
sample introduction amount: 0.5. mu.l, flow rate 0.28ml/min, and the rest of conditions 2, elution program was adjusted as follows:
the experimental results are as follows: see FIGS. 12-5
Examination of condition 6:
column temperature: 30 ℃, sample introduction: 0.5. mu.l, flow rate 0.28ml/min, and the rest of the same considerations as for condition 2, the elution procedure was adjusted as follows:
the experimental results are as follows: see fig. 12-6
Examination condition 7:
column temperature: 30 ℃, sample introduction: 0.5. mu.l, flow rate 0.25ml/min, and the rest of conditions 2, elution program was adjusted as follows:
the experimental results are as follows: see FIGS. 12-7
In summary, the preferred column is ACQUITY UPLC CSHTM C181.7 μm, 2.1 mm. times.100 mm, the optimum flow rate is 0.3ml/min, and the optimum column temperature is 30 ℃.
Claims (14)
1. The ultra-high performance liquid chromatography detection method for the active ingredient content and the fingerprint spectrum of the compound sophora flavescens injection is characterized in thatThe ultra-high performance liquid chromatography condition comprises that the chromatographic column is ACQUITY UPLC CSHTMC18(ii) a The active ingredients are matrine, oxymatrine, methyl azoxymethanol primeveroside, sophocarpine, oxysophocarpine and sophoridine.
2. The method according to claim 1, wherein the mobile phase in the ultra performance liquid chromatography conditions is a 0.15% to 0.25% potassium dihydrogen phosphate solution-methanol gradient elution, preferably a 0.2% potassium dihydrogen phosphate solution-methanol gradient elution.
3. The method of claim 2, wherein the elution gradient in the ultra performance liquid chromatography conditions is as follows:
4. the method according to claim 1, wherein the ultra performance liquid chromatography conditions comprise a column temperature of 28 ℃ to 32 ℃, preferably 30 ℃.
5. The method of claim 1, wherein the ultra high performance liquid chromatography conditions comprise a flow rate of 0.3 ml/min.
6. The method according to claim 1, wherein the ultra performance liquid chromatography conditions comprise a detection wavelength of 209nm to 213nm, preferably 211 nm.
7. The method according to claim 1, wherein the ultra performance liquid chromatography conditions comprise a sample volume of 0.8 μ l to 1.2 μ l, preferably 1 μ l.
8. The method of claim 1, wherein the ultra high performance liquid chromatography further comprises:
(1) preparing a blank solution: regulating pH value of the potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, mixing the potassium dihydrogen phosphate solution with 0.2% concentration and methanol in the ratio of 85 to 15, and filtering.
9. The method of claim 1, wherein the ultra high performance liquid chromatography further comprises:
(2) preparing a reference substance solution:
precisely weighing matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking;
precisely weighing a proper amount of sophocarpine reference substance, sophoridine reference substance and methyl azoxymethanol primeveroside reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09mg of sophocarpine, 0.08mg of sophoridine and 0.08mg of methyl azoxymethanol primeveroside per 1ml, and shaking up to obtain the final product;
precisely measuring 2ml of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to a scale with a blank solution, and shaking up to obtain the compound.
10. The method of claim 1, wherein the ultra high performance liquid chromatography further comprises:
(3) preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding the blank solution to scale, shaking, filtering, and collecting the filtrate as the sample solution.
11. The method for detecting the content of active ingredients in the compound sophora flavescens injection as claimed in any one of claims 1 to 10, wherein the method comprises the following steps:
the conditions of the ultra-high performance liquid chromatography are as follows:
(1) preparing a blank solution: regulating the pH value of the potassium dihydrogen phosphate solution to 3.0 by using phosphoric acid, filtering a mixed solution of 0.2 percent potassium dihydrogen phosphate solution and methanol, wherein the ratio of the mixed solution to the methanol is 85: 15;
(2) preparing a reference substance solution:
precisely weighing appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking;
precisely weighing a proper amount of sophocarpine reference substance, sophoridine reference substance and methyl azoxymethanol primeveroside reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09mg of sophocarpine, 0.08mg of sophoridine and 0.08mg of methyl azoxymethanol primeveroside per 1ml, and shaking up to obtain the final product;
precisely measuring 2ml of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to a scale with a blank solution, and shaking up to obtain the final product;
(3) preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting filtrate as sample solution;
(4) injecting the blank solution, the reference substance and the sample into a liquid chromatograph in the sample injection sequence, recording the chromatogram, and calculating the content of each active ingredient by an external standard method.
12. The method for detecting the fingerprint of the compound sophora flavescens injection as claimed in any one of claims 1 to 10, wherein the method comprises constructing the fingerprint of the compound sophora flavescens injection containing matrine, oxymatrine, methyl azoxymethanol primeveroside, sophocarpine, oxysophocarpine, sophoridine, psicosanoic acid, sophoramine and trifoliosid.
13. The method of claim 12, wherein the method comprises: the conditions of the ultra-high performance liquid chromatography are as follows:
(1) preparing a blank solution: regulating the pH value of the potassium dihydrogen phosphate solution to 3.0 by using phosphoric acid, filtering a mixed solution of 0.2 percent potassium dihydrogen phosphate solution and methanol, wherein the ratio of the mixed solution to the methanol is 85: 15;
(2) preparing a reference substance solution:
precisely weighing appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to obtain mixed reference substance solution I containing matrine 0.33mg, oxymatrine 0.85mg, and oxysophocarpine 0.25mg per 1ml, and shaking;
precisely weighing a proper amount of sophocarpine reference substance, sophoridine reference substance and methyl azoxymethanol primeveroside reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09mg of sophocarpine, 0.08mg of sophoridine and 0.08mg of methyl azoxymethanol primeveroside per 1ml, and shaking up to obtain the final product;
precisely measuring 2ml of the mixed reference substance solutions I and II, placing the mixed reference substance solutions in a 10ml measuring flask, diluting the mixed reference substance solutions to a scale with a blank solution, and shaking up to obtain the final product;
(3) preparing a test solution: precisely measuring 1ml of the compound radix Sophorae Flavescentis injection, placing in a 50ml measuring flask, adding blank solution to scale, shaking, filtering, and collecting the filtrate as sample solution;
(4) injecting sample according to blank solution, reference substance, and sample to construct fingerprint containing matrine, oxymatrine, methyl azoxymethanol primeveroside, sophocarpine, oxysophocarpine, sophoridine, guava acid, sophoramine, and trifoliate red sandalwood glycoside;
(5) injecting the blank solution, the reference substance and the sample into a liquid chromatograph in the sample injection sequence, recording the chromatogram, and calculating the content of each active ingredient by an external standard method.
14. The method of claim 12, wherein the method comprises:
taking oxymatrine as a reference, the relative retention time of the common characteristic peaks is respectively as follows: the relative retention time of the peaks of sophoramine is 0.389 plus or minus 0.02; the peak relative retention time of the methyl azoxy primrose glycoside is 0.469 +/-0.04; the relative retention time of matrine peak is 0.637 + -0.04; the peak relative retention time of sophocarpine is 0.767 + -0.03; the relative retention time of the peaks of sophoridine is 0.819 +/-0.02; the peak relative retention time of the oxysophocarpine is 0.925 + -0.02; the peak relative retention time of oxymatrine is 1.0; the peak relative retention time of guava acid is 1.055 +/-0.03; the peak relative retention time for trifolialin was 2.097 ± 0.05.
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| CN117007711A (en) * | 2023-08-03 | 2023-11-07 | 山东省食品药品检验研究院 | Method for detecting characteristic spectrum of matrine and related preparations thereof combined with one standard for multiple tests |
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| CN117007711B (en) * | 2023-08-03 | 2024-01-23 | 山东省食品药品检验研究院 | Method for detecting characteristic spectrum of matrine and related preparations thereof combined with one standard for multiple tests |
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