Patent Application
20240044851
The present application belongs to the field of a pharmaceutical technology, and specifically relates to an improved method for detecting a content and fingerprint of active ingredients in Compound Kushen Injection.
A Compound Kushen Injection is a traditional Chinese medicine injection refined by modern scientific methods from two traditional Chinese medicines, Sophora flavescens and Heterosmilax yunnanensis Gagnep. It is included in the National Drug Standards and has the effects of clearing heat, promoting dampness, cooling blood, detoxifying, dispersing nodules and relieving pain. It is used for treating cancer pain and bleeding. Modern researches have shown that it has various pharmacological effects such as anti-tumor effect, anti-inflammatory effect, analgesic effect, and enhancing immunity of a body. It is widely used in clinical practice as an adjuvant therapy for severe diseases such as a non-small cell lung cancer, a primary liver cancer, a gastrointestinal cancer, and a malignant pleural effusion.
The main components of Sophora flavescens are alkaloids and flavonoids. Modern researches have shown that Sophora flavescens alkaloids have multiple pharmacological effects and are the main pharmacological components of the Compound Kushen Injection. At present, there are few research reports on Heterosmilax yunnanensis Gagnep both at home and abroad, and research on its chemical composition, quality, and pharmacology is relatively limited.
The existing national drug standard for Compound Kushen Injection (WS3-B-2752-97-2014) includes HPLC methods for the determination of contents of matrine and oxymatrine (Radix Sophora flavescens), and macrozamin (Heterosmilax yunnanensis Gagnep), respectively. The former method is relatively cumbersome in sample preparation, while the latter method has low column utilization. At the same time, in the detection conditions of fingerprints, there is a significant damage to the chromatographic column, and the overall spectrum has a poor peak shape. All the three determination conditions cause damage to the chromatographic column and are time-consuming and labor-intensive. Therefore, the detection and fingerprint methods for Compound Kushen Injection need to be modified and improved.
Li Huali et al. disclosed, in “Simultaneous Determination of the Contents of 6 Alkaloids in Sophora Flavescens Dispensing Granules by HPLC-DAD Method”, the establishment of an HPLC-DAD method for simultaneous determination of the contents of 6 active alkaloids in Sophora Flavescens Dispensing Granules, including Sophoranol n-oxide, oxymatrine, sophoridine, oxysophocarpine, matrine, and sophocarpine. It can be seen from the chromatographic conditions that it is relatively similar to the fingerprint chromatogram conditions included in the National Drug Standards. When examining the Compound Kushen Injection under these conditions, the chromatographic peak is slightly trailing and is not suitable for the determination of the Compound Kushen Injection.
Therefore, in order to effectively control the quality of the Compound Kushen Injection, it is necessary to provide a method that can simultaneously determine the content and fingerprint of multiple components of Compound Kushen Injection, so as to provide a fast and efficient technical method for quality control in Compound Kushen Injection, while reducing the workload for testing.
In view of the above technical status, the present application provides an improved method for detecting contents and fingerprints of active ingredients in Compound Kushen Injection. The method adopts a high-performance liquid chromatography for detection, in which conditions for the high-performance liquid chromatography include: a C18 column as the chromatographic column; and active ingredients, including matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine and sophoridine, or/and 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid (also named “piscidic acid”).
In the method of the present application, as one of the embodiments, the chromatographic column is preferably Waters XSelect CSH™ C18, TechMate C18-ST, Welch Ultimate AQ-C18, and Waters SunFire C18, more preferably Waters XSelect CSH™ C18, with a dimension of 5 μm and 4.6 mm×250 mm.
In the method of the present application, as one of the embodiments, the method further includes a mobile phase consisting of methanol in the organic phase and a phosphate buffer gradient elution in the aqueous phase; preferably 0.1%-0.34% potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid)-methanol gradient elution; more preferably, 0.2% potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid)-methanol gradient elution.
In the method of the present application, as one of the embodiments, the pH value of potassium dihydrogen phosphate solution is adjusted with phosphoric acid, preferably to 2.9-3.1, and more preferably to 3.0.
In the method of the present application, as one of the embodiments, the gradient elution conditions are as follow:
In the method of the present application, as one of the embodiments, the high performance liquid chromatography conditions in the method include a column temperature of 28-32° C., preferably 30° C.
In the method of the present application, as one of the embodiments, the high performance liquid chromatography conditions in the method include a flow rate of 0.58-0.62 ml/ml, preferably 0.6 ml/min.
In the method of the present application, as one of the embodiments, the high performance liquid chromatography conditions in the method include a detection wavelength of 209-213 nm, preferably 211 nm.
In the method of the present application, as one of the embodiments, the high performance liquid chromatography conditions in the method include an injection amount of 3-20 μl, preferred 5-15 μl, more preferred 8-12 μl, and most preferably 10 μl.
In the method of the present application, as one of the embodiments, the high performance liquid chromatography conditions in the method include preparation of a blank solution: adjusting a pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, preparing a mixed solution of 0.2% potassium dihydrogen phosphate solution-methanol=85:15, and filtering.
In the method of the present application, as one of the embodiments, the high performance liquid chromatography conditions in the method include:
In the method of the present application, as one of the embodiments, the content of the reference substance in the reference substance solution can be in the following range: preferably 0.28-0.40 mg and most preferably 0.33 mg for matrine; preferably 0.72-1.06 mg and most preferably 0.85 mg for oxymatrine; preferably 0.21-0.31 mg and most preferably 0.25 mg for oxysophocarpine; preferably 0.07-0.11 mg for sophocarpine, sophoridine, and macrozamin, and most preferably, 0.09 mg, 0.08 mg, and 0.08 mg, respectively.
In the method of the present application, as one of the embodiments, the high performance liquid chromatography conditions in the method include the preparation of the test substance solution: accurately weighing 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding a blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
In the method of the present application, as one of the embodiments, a method for detecting the content of active ingredients in Compound Kushen Injection is provided. The method includes performing detection by using a high-performance liquid chromatography method, in which the high-performance liquid chromatography conditions include:
In the method of the present application, as one of the embodiments, a method for detecting a fingerprint of a Compound Kushen Injection includes: constructing a fingerprint of the Compound Kushen Injection containing matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine, and sophoridine.
In the method of the present application, as one of the embodiments, the present application provides a method for detecting the fingerprint of the Compound Kushen Injection, which includes:
In the method of the present application, as one of the embodiments, the fingerprint in step (4) has 10 common characteristic peaks, in which, based on peak 7-oxymatrine as a reference, the relative retention time of peak 1-sophoramine is 0.442; the relative retention time of peak 2-macrozamin is 0.603; the relative retention time of peak 3-matrine is 0.693; the relative retention time of peak 4-sophocarpine is 0.816; the relative retention time of peak 5-sophoridine is 0.845; the relative retention time of peak 6-oxysophocarpine is 0.941; the relative retention time of peak 7-oxymatrine is 1.0; the relative retention time of peak 8-2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid is 1.149; the relative retention time of peak 9 is 1.639; and the relative retention time of peak 10-trifolirhizin is 1.888.
In the method of the present application, as one of the embodiments, in step (4), samples are injected into the liquid chromatograph in the following sequence, the chromatogram is recorded and the content is calculated by using an external standard method
In the method of the present application, as one of the embodiments, the method further includes continuously testing the reference substance solution 5 times, with a peak area RSD not exceeding 3.0% and a retention time RSD not exceeding 3.0%.
The present application further provides a high-performance liquid chromatography fingerprint of Compound Kushen Injection constructed according to any of the aforementioned methods. The fingerprint has 10 common characteristic peaks, in which, based on peak 7 as a reference, relative retention times of the common characteristic peaks are as follow: the relative retention time of peak 1 is 0.442; the relative retention time of peak 2 is 0.603; the relative retention time of peak 3 is 0.693; the relative retention time of peak 4 is 0.816; the relative retention time of peak 5 is 0.845; the relative retention time of peak 6 is 0.941; the relative retention time of peak 7 is 1.0; the relative retention time of peak 8 is 1.149; the relative retention time of peak 9 is 1.639; and the relative retention time of peak 10 is 1.888.
In the present application, as one of the embodiments, based on peak 7 as a reference, the relative peak areas of the common characteristic peaks are as follow: the relative peak areas of peak 1 is 0.039; the relative peak area of peak 2 is 0.068; the relative peak area of peak 3 is 0.468; the relative peak area of Peak 4 is 0.184; the relative peak area of peak 5 is 0.098; the relative peak area of peak 6 is 0.425; the relative peak area of Peak 7 is 1.0; the relative peak area of peak 8 is 0.224; the relative peak area of peak 9 is 0.049; and the relative peak area of peak 10 is 0.058.
In the present application, as one of the embodiments, the peak 1 represents sophoramine alkaloid, the peak 2 represents macrozamin, the peak 3 represents matrine, the peak 4 represents sophocarpine, the peak 5 represents sophoridine, the peak 6 represents oxysophocarpine, the peak 7 represents oxymatrine, the peak 8 represents 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid, the peak 9 represents unknown, and the peak 10 represents trifolirhizin.
Compared to the existing detection methods for Compound Kushen Injection, the present application adopts a high-performance liquid chromatography method, which can simultaneously determine 7 components in the Compound Kushen Injection, and construct a chromatographic fingerprint using this method, providing a fast and efficient technical method for quality control in the Compound Kushen Injection, while reducing the workload of testing. The method of the present application combines the three conditions in the standards for Compound Kushen Injection into one condition for testing, which saves time and effort.
The following examples and experimental examples are used to further elaborate on the present application, but will in no way limit the effective scope of the present application.
Apparatuses
Reference Substances
Test Substance in Example 1
Test Substance in Example 2
Test Substance in Example 3
Agents
1. Including Chromatographic Conditions, Sample Preparation, System Applicability Requirements, Calculation Formulas, and Limit Requirements
2. Verifying Specific Content
2.1 System Applicability
(1) Experimental Steps
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: accurately weighing an appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to prepare a mixed reference substance solution I containing 0.33 mg of matrine, 0.85 mg of oxymatrine, and 0.25 mg of oxysophocarpine per 1 ml, and shaking; accurately weighing an appropriate amount of sophocarpine reference substance, sophoridine reference substance, and macrozamin reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09 mg of sophocarpine, 0.08 mg of sophoridine, and 0.08 mg of macrozamin per 1 ml, and shaking; and accurately weighing 2 ml of mixed reference substance solution I and II, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
Preparation of test substance solution: accurately weighing 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
RSD values of peak area and retention time for 5 continuous injections of the reference substance solution.
(3) Conclusion
From the results, it can be seen that after 5 consecutive injections of the reference substance solution, the RSD of the peak area measurements of oxymatrine, matrine, and oxysophocarpine are all less than 2.0%, and the RSD of the retention time are all less than 2.0%. The RSD of the peak area measurements of sophocarpine, sophoridine, and macrozamin are all less than 3.0%, and the RSD of the retention time is less than 3.0%; the theoretical number of the six indicator components is greater than 3000, and the trailing factor is less than 2.0, meeting the requirements.
2.2 Specificity
(1) Experimental Steps
Preparation of Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of negative sample solution: accurately weighing 1 ml of Single Kusen Injection (wild and cultivated) and 1 ml of Single Heterosmilax yunnanensis Gagnep Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the negative sample solution.
Preparation of 0.25% Tween solution: weighing 0.25 g Tween 80, dissolving in water to 100 ml, shaking, filtering, and taking the subsequent filtrate as the 0.25% Tween solution.
Preparation of reference substance solution: preparing the reference substance solution according to a method under Section 2.1.
Preparation of test substance solution: accurately weighing 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, and reserving.
Preparation of filter membrane interference sample: centrifuging one portion of the test substance solution; and filtering one portion of the test substance solution, and discarding different volumes (1 ml, 3 ml, 5 ml, 7 ml, and 9 ml).
Requirements for Sampling Procedure
Injection Sequence
yunnanensis Gagnep alone)
(2) The Results are Reported in
(3) Conclusion
From the results, it can be seen that the blank solution, blank mobile phase, and 0.25% Tween 80 solution have no interfere with the sample. The negative sample solution of Sophora flavescens alone (wild Sophora flavescens and cultivated Sophora flavescens) have no interfere with macrozamin, and the negative sample solution of Heterosmilax yunnanensis Gagnep alone has no interfere with alkaloids.
After discarding different volumes, the relative content between the area of the indicator components of the test substance solution and the area of the centrifuged test substance solution is 95.0%-105.0%, and the adsorption can be ignored.
2.3 Linearity and Range
(1) Experimental Steps
Preparation of Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of linear stock solution: accurately weighing an appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to prepare a mixed reference substance solution I containing 0.33 mg of matrine, 0.85 mg of oxymatrine, and 0.25 mg of oxysophocarpine per 1 ml, and shaking; accurately weighing an appropriate amount of sophocarpine reference substance, sophoridine reference substance, and macrozamin reference substance, adding blank solution to prepare a mixed reference substance solution II containing 0.09 mg of sophocarpine, 0.08 mg of sophoridine, and 0.08 mg of macrozamin per 1 ml, and shaking.
25% reference substance solution: accurately weighing 0.5 ml of mixed reference substance solutions I and II, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
50% reference substance solution: accurately weighing 1 ml of mixed reference substance solutions I and II, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
100% reference substance solution: accurately weighing 2 ml of mixed reference substance solutions I and II, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
150% reference substance solution: accurately weighing 3 ml of mixed reference substance solutions I and II, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
200% reference substance solution: accurately weighing 4 ml of mixed reference substance solutions I and II, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
The regression equations, correlation coefficients, and linear graph results of individual indicator components are shown in
(3) Conclusion
Macrozamin shows linearity within the range of 0.00422 mg/ml-0.03374 mg/ml; matrine shows linearity within 0.01627 mg/ml-0.13013 mg/ml; sophorocarpine shows a linearity within the range of 0.0044 mg/ml-0.03517 mg/ml; sophoridine shows linearity within a range of 0.00438 mg/ml-0.03505 mg/ml; oxysophoridine shows linearity within the range of 0.01252 mg/ml-0.10016 mg/ml; and oxymatrine shows linearity within a range of 0.04228 mg/ml-0.33742 mg/ml. The linear correlation coefficients of individual components are greater than or equal to 0.999, meeting the standard.
2.4 Sensitivity
(1) Experimental Steps
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: accurately weighing an appropriate amount of macrozamin reference substance solution, and adding blank solution to prepare a reference substance solution containing 0.085 mg per 1 ml.
Quantitation limit of and detection limit solution: diluting the blank solution stepwise to a signal-to-noise ratio (S/N) of 10:1 as the limit of quantitation solution, and diluting the blank solution stepwise to a signal-to-noise ratio (S/N) of 2-3 as the limit of detection solution.
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
(3) Conclusion
From the results, it can be seen that, after continuous injection of the quantitative limit solution, the RSD value of peak retention time is less than 2.0%, and the peak area is less than 5.0%; the quantification limit is 8.09 ng and the detection limit is 2.43 ng.
2.5 Repeatability
(1) Experimental Steps
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: preparing reference substance solution according to the method under Section 2.1, and preparing two copies using the same method.
Preparation of test substance solution (6 copies): accurately weighing 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution; and preparing 6 copies in parallel.
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
(3) Conclusion
From the results, it can be seen that the RSD of the content of matrine, oxymatrine, and oxysophocarpine in the six test substances is less than 3.0%, while the RSD of the content of sophocarpine, sophoridine, and macrozamin is less than 4.0%, indicating good repeatability of the test substances.
2.6 Intermediate Precision
(1) Experimental Steps
According to the repeatability measurement method, six test substance solutions of the same batch were prepared in parallel by different analysts using different apparatuses and on different dates. The solution preparation and injection procedures were under the same repeatability item.
(2) Result Report
(3) Conclusion
From the results, it can be seen that, in the 12 test substances tested by different operators with different apparatus on different dates, the RSD of matrine content is 0.34%, the RSD of oxidized matrine content is 2.12%, and the RSD of oxidized sophocarpine content is 1.50%, all less than 3.0%. The RSD of sophocarpine content is 0.91%, the RSD of sophoridine content is 0.67%, and the RSD of macrozamin content is 1.18%, all less than 4.0%, indicating good intermediate precision of the test substance.
2.7 Solution Stability
(1) Experimental Steps
Preparation of blank solvent: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering, obtained.
Preparation of reference substance solution: preparing reference substance solution according to the method provided under Section 2.1, and preparing two copies using the same method.
Preparation of test substance solution: accurately weighing 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
(3) Conclusion
From the results, it can be seen that, 24 hours after standing the reference substance solution and the test substance solution, the RSD values of the content of matrine, oxymatrine, and oxysophocarpine in the test substance are less than 3.0%, while the RSD values of the content of sophocarpine, sophoridine, and macrozamin a less than 4.0%, indicating that the test substances are stable within 24 hours.
The percentages of the indicator component area at individual time points to the 0-hour indicator component area are calculated. Compared with the initial results, the relative content of the control and test substance solution at each time point is 98.0%-102.0%, indicating a good solution stability.
2.8 Accuracy
(1) Experimental Steps
Preparation of blank solution: Preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: preparing a reference substance solution by the method provided under Section 2.1, and preparing two copies using the same method.
Preparation of 50% recovery solution: accurately weighing 0.5 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding 2.5 ml of mixed reference substance solution I and II, respectively, adding blank solution to scale, shaking, and filtering to obtain a filtrate as a 5000 recovery solution (preparing 3 copies using the same method).
Preparation of 100% recovery solution: accurately weighing 0.5 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding 5 ml of mixed reference substance solution I and II respectively, adding blank solution to scale, shaking, and filtering to obtain a filtrate as a 100% recovery solution (preparing 3 copies using the same method).
Preparation of 150% recovery solution: accurately weighing 0.5 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding 7.5 ml of mixed reference substance solution I and II respectively, adding blank solution to scale, shaking, and filtering to obtain a filtrate as a 15000 recovery solution (preparing 3 copies using the same method).
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
Recovery Rate Calculation Formula:
(3) Conclusion
The recovery rates of matrine, oxymatrine, and oxysophocarpine in the test substance are ranged from 92% to 105%, with RSD values of 0.93%, 1.33%, and 1.01% for the nine recoveries, all less than 4%; and the recovery rates of sophocarpine, sophoridine, and macrozamin ranged from 90.0% to 108.0%, with RSDs of 2.01%, 1.26%, and 1.90% for the nine copies, all less than 5.0%, meeting the requirements.
2.9 Durability
(1) Experimental Steps
Different chromatographic conditions are shown in the table below.
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: preparing a reference substance solution by the method provided under Section 2.1.
Preparation of test substance solution: accurately weighing 1 ml of Compound Kushen Injection and adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution; and preparing two copies using the same method.
Requirements for injection procedure (samples are injected in this order under different inspection conditions)
Injection Sequence
Note: if the reference and test substance solution are stable within the detection time range, there is no need to prepare them again. If the solution is unstable and the chromatographic conditions are changed, it is necessary to prepare the reference and test substance solution again.
(2) Result Report
(3) Conclusion
The contents of the test substance solutions are basically the same under different conditions, and the content of individual indicator components are within 9000-110% relative to the standard conditions. This indicates that the content detection of this product has good durability under conditions such as column temperature, wavelength, mobile phase pH, and different chromatographic column models.
2.10 Sample Test
(1) Experimental Steps
Preparation of blank solvent: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: preparing a reference substance solution by the method provided under Section 2.1, and preparing two copies using the same method.
Preparation of test substance solution: accurately weighing 1 ml of individual batches of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
Requirements for Injection Procedure
Injection Sequence
(2) Result Report
1. Chromatographic Conditions, Sample Preparation, System Applicability Requirements, Calculation Formulas, Limit Requirements, Etc.
2. Verification Content
2.1 System Applicability
(1) Experimental Steps
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: accurately weighing an appropriate amount of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid reference substance solution, adding blank solution to prepare a reference substance stock solution containing 0.25 mg of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid per 1 ml, and shaking; and accurately weighing 2 ml of the reference substance stock solution, adding to a 10 ml volumetric flask, diluting the blank solution to scale, and shaking.
Preparation of test substance solution: accurately weighing 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
Requirements for Sampling Procedure
(2) Results Report
RSD values of peak area and retention time for 5 continuous injections of reference substance solution
(3) Conclusion
From the results, it can be seen that after 5 continuous injections of the reference substance solution, the RSD values of the peak area measurement values of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid are all less than 2.0%, the RSD values of the retention time are all less than 2.000, the numbers of theoretical plates are greater than 3000, and the tailing factors are all less than 1.5, meeting the requirements.
2.2 Specificity
(1) Experimental Steps
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering, obtained.
Preparation of negative sample solution: accurately weighing 1 ml of single Sophora flavescens injection (wild and cultivated) and 1 ml of single Heterosmilax yunnanensis Gagnep injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the negative sample solution.
Preparation of 0.25% Tween solution: weighing 0.25 g Tween 80, dissolving in water to 100 ml, shaking, filtering, and taking the subsequent filtrate as the 0.25% Tween solution.
Preparation of reference substance solution: preparing a reference substance solution by the method provided under Section 2.1.
Preparation of test substance solution: accurately weighing 1 ml of Compound Kushen Injection and adding to a 50 ml volumetric flask, adding blank solution to scale, and shaking.
Preparation of filter membrane interference sample: centrifuging one portion of the test substance solution; and filtering one portion of the test substance solution, discarding different volumes (1 ml, 3 ml, 5 ml, 7 ml, and 9 ml).
Requirements for Sampling Procedure
Injection Sequence
flavescens alone)
yunnanensis Gagnep alone)
(2) Result Report
Refer to
Table 2.2-1 Results of Filter Membrane Interference Experiment
(Area Percentage of Discarded Different Volumes Relative to the Centrifuged Samples)
(3) Conclusion
From the results, it can be seen that, the blank solution, the blank mobile phase, and 0.25% Tween-80 solution do not interfere with the sample, while the negative sample solution of single Heterosmilax yunnanensis Gagnep do not interfere with 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid.
After discarding different volumes, the relative content between the area of the indicator components of the test substance solution and the area of the centrifuged test substance solution is 98.0%-102.0%, and the adsorption can be ignored.
2.3 Linearity and Range
(1) Experimental Steps
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering, which is obtained.
Preparation of linear stock solution: accurately weighing an appropriate amount of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid reference substance, adding blank solution, preparing a stock solution containing 0.25 mg of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid per 1 ml of reference substance, and shaking.
25% linear solution: accurately measuring 0.5 ml of the reference stock solution, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
50% linear solution: accurately measuring 1 ml of the reference stock solution, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
100% linear solution: accurately measuring 2 ml of the reference stock solution, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
150% linear solution: accurately measuring 3 ml of the reference stock solution, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
200% linear solution: accurately measuring 4 ml of the reference stock solution, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking.
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
The regression equation, the correlation coefficient, and the linear graph results of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid are shown in
(3) Conclusion
2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid is linear within the range of 0.0122 mg/ml-0.0978 mg/ml; and the linear correlation coefficient is greater than or equal to 0.999, meeting the standard.
2.4 Repeatability
(1) Experimental Steps
Preparation of blank solution: preparing 0.21 potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering, which is obtained.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1, and preparing two portions using the same method.
Preparation of test substance solution (6 copies): accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution; and performing 6 operations in parallel.
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
(3) Conclusion
From the results, it can be seen that the RSD of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid content in the 6 test substances is less than 3.0%, indicating good repeatability of the test substances.
2.5 Intermediate Precision
(1) Experimental Steps
According to the repeatability measurement method, six test substance solutions of the same batch are prepared in parallel by different analysts using different apparatuses and on different dates. The solution preparation and injection procedures are the same as those under Section repeatability.
(2) Result Report
(3) Conclusion
From the results, it can be seen that, different personnel tested the samples with different apparatuses on different dates. The RSD of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid content in the 12 test substances is 0.65%, less than 3.0%, indicating good intermediate precision.
2.6 Solution Stability
(1) Experimental Steps
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering, which is obtained.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1, and preparing two portions using the same method.
Preparation of test substance solution: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
(3) Conclusion
From the results, it can be seen that after standing the test substance solution left for 24 hours, the RSD of the 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl] butanedioic acid content in the test substance is less than 3%, indicating that the test substance is stable within 24 hours.
The percentage of the indicator component area at individual time points to the 0-hour indicator component area is calculated. Compared with the initial results, the relative content of the control and test substance solution at individual time points is 98.0%-102.0%, indicating good solution stability.
2.7 Accuracy
(1) Experimental Steps
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1, and preparing two portions using the same method.
Preparation of 5000 recovery solution: accurately measuring 0.5 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding 2 ml of reference stock solution, adding blank solution to scale, shaking, filtering, and taking it as a 502 recovery solution (preparing 3 copies using the same method).
100% recovery solution: accurately measuring 0.5 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding 4 ml of the reference stock solution, adding a blank solution to scale, shaking, filtering, and taking it as a 100% recovery solution (preparing 3 copies using the same method).
150% recovery solution: accurately measuring 0.5 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding 6 ml of the reference stock solution, adding a blank solution to scale, shaking, filtering, and taking it as a 150% recovery solution (preparing 3 copies using the same method).
Requirements for Sampling Procedure
Injection Sequence
(2) Result Report
Recovery Rate Calculation Formula:
(3) Conclusion
The recovery rate of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid in the test substance ranges from 92% to 105%, with an RSD value of 1.75%, which is less than 4%, meeting the requirements.
2.8 Durability
(1) Experimental Steps
Different chromatographic conditions are shown in the table below.
Preparation of blank solution: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1.
Preparation of test substance solution: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution; and preparing two copies using the same method.
Requirements for injection procedure (samples are injected in this order under different inspection conditions)
Injection Sequence
Note: If the reference and test substance solution are stable within the detection time range, there is no need to prepare them again. If the solution is unstable and the chromatographic conditions are changed, it is necessary to prepare the reference and test substance solution again.
(2) Result Report
(3) Conclusion
The content of the test substance solution is basically the same under different conditions, and the content of each indicator component is between 90%-110% relative to the standard conditions. This indicates that the content detection of this product has good durability under conditions such as column temperature, wavelength, mobile phase pH, and different chromatographic column models.
2.9 Sample Test
(1) Experimental Steps
Preparation of blank solvent: preparing 0.2% potassium dihydrogen phosphate solution-methanol=85:15 mixed solution, and filtering.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1, and preparing two portions using the same method.
Preparation of test substance solution: accurately measuring 1 ml of individual batches of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
Requirements for Injection Procedure
Injection Sequence
(2) Result Report
1. Chromatographic Conditions, Elution Conditions, Sample Preparation, Etc.
Method Description
2. Verifying Specific Content
2.1 System Applicability
(1) Experimental Steps
Preparation of blank solution: preparing methanol 0.2% potassium dihydrogen phosphate=15:85, and filtering.
Preparation of reference substance solution: accurately weighing an appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding blank solution to prepare a mixed reference substance solution I containing 0.33 mg of matrine, 0.85 mg of oxymatrine, and 0.25 mg of oxysophocarpine per 1 ml, and shaking, which is obtained; accurately weighing an appropriate amount of sophocarpine reference substance, sophoridine reference substance, and macrozamin reference substance, and adding blank solution to prepare a mixed reference substance solution II containing 0.09 mg of sophocarpine, 0.08 mg of sophoridine, and 0.08 mg of macrozamin per 1 ml, and shaking, which is obtained; and accurately measuring 2 ml of mixed reference substance solution I and II, adding to a 10 ml volumetric flask, diluting with blank solution to scale, and shaking, which is obtained.
test substance solution: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
The injection sequence and requirements are shown in the table below.
Injection Sequence
(2) Result Report
RSD values of peak area and retention time for 5 consecutive injections of the reference substance solution.
(3) Conclusion
From the results, it can be seen that after 5 consecutive injections of the reference substance solution, the RSD of the peak areas of oxymatrine, matrine, and oxysophocarpine is less than 2.0%, and the RSD of the retention time is less than 2.0%. The RSD of the peak areas of sophocarpine, sophoridine, and methyloxyazomethanol primrose glycoside is less than 3.0%, and the RSD of the retention time is less than 3.0%; and the theoretical number of six indicator components is greater than 3000, and the trailing factor is less than 2.0, meeting the requirements.
2.2 Establish of Fingerprint
(1) Experimental Steps
Blank solution: preparing methanol-0.2% potassium dihydrogen phosphate=15:85 mixed solution, and filtering.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1.
Test substance solution for individual batches: accurately measuring 1 ml of Compound Kushen Injection from each of the batches, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
The injection sequence and requirements are shown in the table below.
Injection Sequence
(2) Result Report
Based on the chromatographic fingerprints of 10 batches of Compound Kushen Injection, data processing was carried out using the “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine” (2012 edition) recommended by the Pharmacopoeia Committee. The chromatographic peak of test substance 1 (S1) is used as the reference spectrum, and the median method is used, with a time window of 0.1. After multi-point correction, full peak matching is performed to generate a standard reference fingerprint and a common pattern. (Refer to
(3) Conclusion
After comparison with the reference substance, it can be concluded that, the first peak represents sophoramine, the second peak represents macrozamin, the third peak represents matrine, the fourth peak represents sophocarpine, the fifth peak represents sophoridine, the sixth peak represents oxysophocarpine, the seventh peak represents oxymatrine, the eighth peak represents 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid, and the tenth peak represents trifolirhizin.
From the results, it can be seen that the similarity between the 10 batches of samples and the control fingerprint is greater than 0.9, and the percentage of non-common peak areas is less than 5.0%.
2.3 Repeatability
(1) Experimental Steps
Preparation of blank solution: preparing methanol 0.2% potassium dihydrogen phosphate=15:85, and filtering.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1.
Preparation of test substance solution: accurately measuring 1 ml of 6 batches of Compound Kushen Injection of the same batch number, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
The injection sequence and requirements are shown in the table below.
Injection Sequence
(2) Result Report
Based on the repetitive chromatogram and using the same processing method as the sample, the similarity was calculated using the “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine”. The relative retention time and relative peak area are calculated using peak 7 (oxymatrine) as a reference. (Refer to
(3) Conclusion
From the results, it can be seen that, the similarity among the 6 test substances is greater than 0.99, and the RSD values of the relative retention time and relative peak area of each common peak are less than 3.0%, indicating good repeatability.
2.4 Intermediate Precision
(1) Experimental Steps
According to the repeatability measurement method, 6 test substance solutions were prepared in parallel on different dates, by different analysts, and using different apparatuses. The solution preparation and injection procedures are the same as the repeatability, and the precision of the determination results of 12 samples is evaluated.
(2) Result Report
Based on the intermediate precision chromatogram, using the same processing method as the sample, the similarity is calculated using the “Evaluation System for Chromatographic Fingerprint Similarity of Traditional Chinese Medicine”, and the relative retention time and relative peak area were calculated using peak 7 (oxymatrine) as a reference. (Refer to
(3) Conclusion
From the results, it can be seen that, the similarity of the 12 test substances is greater than 0.99, and the relative retention time RSD values of individual common peaks are all less than 5%; and the relative peak area RSD value of macrozamin is 5.72, and the relative peak area RSD values of other common peaks are less than 5%. Therefore, the peak area of macrozamin is greatly affected.
2.5 Solution Stability and Double Time Spectra
(1) Experimental Steps
Preparation of blank solution: preparing methanol 0.2% potassium dihydrogen phosphate=15:85, and filtering, which is obtained.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1.
Preparation of test substance solution: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
The injection sequence and requirements are shown in the table below.
Injection Sequence
(2) Result Report
Based on the stability chromatogram and using the same processing method as the sample, the similarity is calculated using the “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine”. The relative retention time and relative peak area are calculated using peak 7 (oxymatrine) as a reference (see
(3) Conclusion
From the results, it can be seen that the similarity of the test substance is greater than 0.99 within 24 hours, and the relative retention time and peak area RSD values of individual common peaks are less than 3.0%. Therefore, the test substance is stable within 24 hours. There is no peak in the chromatogram again within double time, showing good results.
2.6 Durability
(1) Experimental Steps
Preparation of blank solution: preparing methanol 0.2% potassium dihydrogen phosphate=15:85, and filtering, which is obtained.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1.
Preparation of test substance solution: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution; and preparing 2 copies using the same method.
10 μl of the above solutions are separately injected into HPLC under different conditions, and the injection sequence and requirements are shown in the table below (samples are injected according to this injection sequence under different investigation conditions).
Injection Sequence
(3) Result Report
(2) Conclusion
Based on the durability chromatogram (different chromatographic columns and apparatuses), using the same processing method as the sample, the similarity is calculated using the “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine”, and the relative retention time and relative peak area are calculated using peak 7 (oxymatrine) as a reference.
The durability results of different chromatographic columns are shown in
The durability results of different apparatuses are shown in
(3) Conclusion
From the results, it can be seen that, in the results of different chromatographic columns, the similarity of individual common peak is greater than 0.99, and the RSD values of relative retention time and relative peak area are less than 5.0%; and, in the inspection results of different apparatus, the similarity of common peaks is greater than 0.99, and the RSD values of relative retention time and relative peak area are less than 5.0%. Therefore, this method has good durability.
2.9 Inspection of Key Production Process Points
(1) Experimental Steps
Preparation of blank solution: preparing methanol 0.2% potassium dihydrogen phosphate=15:85, and filtering.
Preparation of reference substance solution: preparing the reference substance solution by the method under Section 2.1.
Preparation of test substance solution: based on the volume of individual key points, the sampling amount is calculated. For key point 1, 1 ml is added to 25 ml volumetric flask; for key points 2 and 6, 5 ml is added to 50 ml volumetric flask; for key point 4, 2 ml is added to 25 ml volumetric flask; for key point 5, 1 ml is added to 100 ml volumetric flask; and for other key points, 1 ml is added to 50 ml volumetric flasks. All samples are added with blank solution to scale, shaken, and filtered to obtain a filtrate as the test substance solution.
The injection sequence and requirements are shown in the table below.
Injection Sequence
(2) Result Report
(Refer to
(3) Conclusion
From the results, it can be seen that, the similarity between the key points of the production process is greater than 0.9, in which the similarity between the key points 8-22 (water precipitation solution sample after sterilization) is greater than 0.98, indicating a high similarity among the key points. However, from the figure, some components show slight losses.
1. Investigation of Different Chromatographic Columns
Experimental Steps:
(1) Chromatographic conditions: mobile phase 0.2% potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid) (A)-methanol (B) gradient elution, 0-10 min, 3% B; 10-15 min, 3%-5% B; 15-24 min 5%-15% B; 24-30 min, 15% B; 30-55 min, 15%-85% B; 55-75 min, 3%0B. The column temperature is 30° C., the flow rate is 0.6 ml/min, and the wavelength is 211 nm.
(2) Preparation of test substance: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution (0.2% potassium dihydrogen phosphate solution methanol=85:15) to scale, shaking, filtering, taking the subsequent filtrate as the test substance solution, injecting the sample according to the above chromatographic conditions and observing.
The chromatographic columns inspected are as follows:
(1) Waters XSelect CSH™ C18
The experimental results are shown in
(2) TechMate C18-ST
The experimental results are shown in
(3) Welch Ultimate AQ-C18
The experimental results are shown in
(4) Waters SunFire C18
The experimental results are shown in
In summary, the optimal chromatographic column is Waters XSelect CSH™ C18 (4.6 mm×250 mm, 5 μm).
2. Investigation of Different Mobile Phase Systems
Experimental Steps:
Preparation of test substance: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution (0.2% potassium dihydrogen phosphate solution methanol=85:15) to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution, injecting the sample according to the above chromatographic conditions and observing.
2.1 Adjusting of the Mobile Phase Gradient According to the Fingerprint Conditions in the Drug Standard of Compound Kushen Injection
Chromatographic conditions: column: Waters XSelect CSH™ C18 (4.6 mm×250 mm, 5 μm)
Detection wavelength: 225 nm injection volume: 10 μl Column temperature: 30° C. flow rate: 0.8 ml/min
Mobile phase: acetonitrile: 0.01M ammonium acetate (9:1), 0.01M ammonium acetate (adjusted to pH 8.0) gradient elution
This method is based on the inspection made after adjusting the gradient based on the fingerprint conditions in the injection drug standard
The experimental results are shown in
2.2 Other Methods
Chromatographic column: Waters XSelect CSH™ C18 (4.6 mm×250 mm, 5 μm)
Detection wavelength: 211 nm, column temperature: 30° C., flow rate: 0.6 ml/min, injection amount: 10 μl
The mobile phase systems inspected are as follow:
(1) Methanol-water: 10% methanol water to 90% methanol water, gradient elution for 120 minutes
The experimental results are shown in
(2) Methanol-0.1% formic acid water: 10% methanol 90% (0.1% formic acid water) to 90% methanol 10% (0.1% formic acid water), gradient elution for 120 minutes
The experimental results are shown in
(3) Methanol-0.1% acetic acid water: 10% methanol 90% (0.1% acetic acid water) to 90% methanol 10% (0.1% acetic acid water), gradient elution for 120 minutes
The experimental results are shown in
(4) Methanol-0.01% acetic acid water: 10% methanol 90% (0.01% acetic acid water) to 90% methanol 10% (0.01% acetic acid water), gradient elution for 120 minutes
The experimental results are shown in
(5) Methanol 0.4% phosphoric acid: 10% methanol 90% (0.1% phosphoric acid) to 90% methanol 10% (0.1% phosphoric acid), gradient elution for 120 minutes
The experimental results are shown in
(6) Acetonitrile-water: 10% acetonitrile water to 90% acetonitrile water, gradient elution for 120 minutes
The experimental results are shown in
(7) Methanol-0.01M ammonium acetate: 10% methanol 90% (0.01M ammonium acetate, adjusted to pH 4.0) to 90% methanol 10% (0.01M ammonium acetate, adjusted to pH 4.0), gradient elution for 120 minutes
The experimental results are shown in
(8) Methanol-0.2% potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid): this is the final chromatographic condition determined in this patent application, with gradient elution of 3% methanol 97% (0.2% potassium dihydrogen phosphate).
The experimental results are shown in
In summary, the optimal conditions include methanol 0.2% potassium dihydrogen phosphate, adjusting the pH value of phosphoric acid to 3, and gradient elution.
3. Investigation of Different pH Values
Experimental Steps:
Preparation of test substance: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding a blank solution (0.2% potassium dihydrogen phosphate solution methanol=85:15) to scale, shaking, filtering, taking the subsequent filtrate as the test substance solution, injecting the sample according to the above chromatographic conditions and observing.
3.1 Chromatographic Conditions:
Chromatographic column: Waters XSelect CSH™ C18 (4.6 mm×250 mm, 5 μm)
Detection wavelength: 211 nm, column temperature: 30° C., flow rate: 0.6 ml/min, injection amount: 10 μl
The pH values of potassium dihydrogen phosphate inspected are as follow:
(1) Methanol-0.1% potassium dihydrogen phosphate (adjusted to pH 5.0 with phosphoric acid)
The experimental results are shown in
(2) Methanol-0.1% potassium dihydrogen phosphate (adjusted to pH 4.0 with phosphoric acid)
The experimental results are shown in
(3) Methanol-0.1% potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid).
The experimental results are shown in
3.2 Experimental Results
Refer to
In summary, adjusting the pH value of potassium dihydrogen phosphate to 3 with phosphoric acid is the optimal condition.
4. Investigation of Potassium Dihydrogen Phosphate Concentration
Experimental Steps:
Preparation of test substance: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding a blank solution (0.2% potassium dihydrogen phosphate solution methanol=85:15) to scale, shaking, filtering, taking the subsequent filtrate as the test substance solution, injecting the sample according to the above chromatographic conditions and observing.
Chromatographic Conditions:
Chromatographic column: Waters XSelect CSH™ C18 (4.6 mm×250 mm, 5 μm)
Detection wavelength: 211 nm, column temperature: 30° C., flow rate: 0.6 ml/min, injection amount: 10 μl
The concentrations of potassium dihydrogen phosphate are investigated respectively, and as follows:
(1) Methanol-0.1% potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid)
The experimental results are shown in
(2) Methanol-0.34% potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid)
The experimental results are shown in
(3) Methanol-0.2% potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid)
The experimental results are shown in
The superposition diagram of different concentrations of potassium dihydrogen phosphate is shown in
In summary, 0.2% potassium dihydrogen phosphate as the mobile phase is the optimal condition.
5. Gradient Optimization
Experimental Steps:
Preparation of test substance: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding a blank solution (0.2% potassium dihydrogen phosphate solution methanol=85:15) to scale, shaking, filtering, taking the subsequent filtrate as the test substance solution, injecting the sample according to the above chromatographic conditions and observing.
Chromatographic column: Waters XSelect CSH™ C18 (4.6 mm×250 mm, 5 μm); mobile phase 0.2% potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid)-methanol (B) gradient adjustment; detection wavelength: 211 nm, column temperature: 30° C., flow rate: 0.6 ml/min, injection amount: 10 μl.
Experimental Methods
The elution gradients investigated are as follow:
Method 1:
Elution Conditions
The experimental results are shown in
Method 2:
Elution Conditions
The experimental results are shown in
Method 3:
Elution Conditions
The experimental results are shown in
In summary, the peak resolution in Method 3 (
6. Confirmation of Detection Wavelength
Experimental Steps:
Preparation of test substance: accurately measuring 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding blank solution (0.2% potassium dihydrogen phosphate solution methanol=85:15) to scale, shaking, filtering, taking the subsequent filtrate as the test substance solution, injecting the sample according to the above chromatographic conditions and observing.
Chromatographic column: Waters XSelect CSH™ C18 (4.6 mm×250 mm, 5 μm); Mobile phase 0.2% potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid) (A)-methanol (B) gradient elution; full wavelength scanning, column temperature: 30° C., flow rate: 0.6 ml/min, injection volume: 10 μl.
The experimental results are shown in
In summary, from the wavelength scanning results, it can be seen that under this condition, the Compound Kushen Injection has terminal absorption. Based on various absorption peaks and references, 211 nm was selected as the detection wavelength for the Compound Kushen Injection.
7. Determination of Chromatographic Conditions
(1) Chromatographic Conditions
Chromatographic column: Waters XSelect CSH™ C18 (5 μm, 4.6 mm×250 mm)
Mobile phase: 0.2% potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid)-methanol gradient elution
Column temperature: 30° C.
Flow rate: 0.6 ml/min
Detection wavelength: 211 nm
Injection volume: 10 μl
(2) Determination Method
According to the determined chromatographic conditions, 10 μl of the reference substance solution and the test substance solution is separately injected into a liquid chromatograph and record the chromatogram.
8. Optimization and Confirmation of the Preparation Method for the Test Substance Solution
Experimental method: same as in step 7 of Example 4, respectively investigating:
(1) Test substance (prepared from water) and blank solution
The experimental results are shown in
(2) Preparation of reference substance with methanol and test substance with purified water
The experimental results are shown in
(3) Preparation of blank solution for test and control samples (blank solution: 0.2% potassium dihydrogen phosphate solution methanol mixed solution)
The experimental results are shown in
In summary, compared to methanol preparation, the blank solution was used to prepare the reference substance, with symmetrical peak shapes and smooth baseline. Therefore, the blank solution was used to prepare the test substance and the reference substance.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011391813.X | Dec 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/134477 | 11/30/2021 | WO |
