CN116008443A - Method for detecting related substances in alpha 5-GABAA receptor modulator medicines - Google Patents
Method for detecting related substances in alpha 5-GABAA receptor modulator medicines Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The application relates to a detection method of related substances in alpha 5-GABAA receptor modulator drugs, which comprises the following steps: taking a reference substance of the alpha 5-GABAA receptor modulator drug, dissolving the reference substance in a first solvent, and preparing a reference substance solution; taking a sample to be measured, and dissolving the sample with a second solvent to prepare a sample solution to be measured; performing high performance liquid chromatography detection on the reference substance solution and the sample solution to be detected, wherein the conditions of the high performance liquid chromatography detection comprise: the mobile phase A is phosphoric acid aqueous solution with volume concentration of 0.08% -0.12%, and the mobile phase B is acetonitrile for gradient elution. According to the detection method, the high performance liquid chromatography detection is carried out on the raw material medicine under proper conditions, so that the raw material medicine and various impurities can be effectively separated, further, the detection of related substances of the raw material medicine is realized, and the detection method has important significance in the production quality control and research of the raw material medicine.
Description
Technical Field
The application relates to the technical field of quality detection, in particular to a detection method of related substances in alpha 5-GABAA receptor modulator medicines.
Background
The α5-containing GABAA receptor (α5-GABAA receptor) has a specific distribution in hippocampal tissues of the brain of mammals, and α5-GABAA receptor modulators have been studied to confirm that they are useful for the treatment of pain, particularly neuropathic pain (WO), and thus many studies on α5-GABAA receptor modulators are currently underway and a large number of compounds have been synthesized successively, for example, N-isopropyl-6- (((3- (5- (methoxymethyl) isoxazol-3-yl) - [1,2,4] triazol [3,4-a ] phthalazin-6-yl) oxy) methyl) nicotinamide (structure shown in the following formula (1)) disclosed in chinese patent application CN110256440 a.
At present, the synthetic route of the crude drug of N-isopropyl-6- (((3- (5- (methoxymethyl) isoxazol-3-yl) - [1,2,4] triazole [3,4-a ] phthalazin-6-yl) oxygen) methyl) nicotinamide is shown as follows:
in the industrial production of the above-mentioned synthetic routes of crude drugs, organic impurities inevitably remain, including raw materials, intermediates, byproducts, etc., collectively called "related substances", the presence of which affects the quality of the finished product and may even affect the efficacy and toxicity of the drug, for example, PA2 #
) The medicine has warning structure and may be genotoxic impurity, and features that it can damage genetic material of human body at very low concentration, and has mutagenicity and carcinogenicity, and this can seriously threaten human health during medicine application. Therefore, strict control of the substances is required in the drug substance. Generally, the total content of impurities in the raw material medicine needs to be strictly controlled to be less than 1.0%, and the content of single impurities needs to be less than 0.1%. Based on this, it is necessary to provide a method for detecting substances of interest in α5-GABAA receptor modulators.
Disclosure of Invention
Based on this, the present application provides a method for detecting substances of interest in alpha 5-GABAA receptor modulators.
The specific technical scheme is as follows:
a method for detecting related substances in alpha 5-GABAA receptor modulator drugs, wherein the alpha 5-GABAA receptor modulator drugs have the structural characteristics shown in the following formula (1):
the related substances comprise one or more of PA1-2, PA3, PA5, PA6, 1, 3-Tetramethylurea (TML) and 1-Hydroxybenzotriazole (HOBT), and the structures are shown as follows:
the detection method comprises the following steps:
taking a reference substance of the alpha 5-GABAA receptor modulator drug, dissolving the reference substance in a first solvent, and preparing a reference substance solution;
taking a sample to be measured, and dissolving the sample with a second solvent to prepare a sample solution to be measured;
performing high performance liquid chromatography detection on the reference substance solution and the sample solution to be detected, wherein the conditions of the high performance liquid chromatography detection comprise: the mobile phase A is phosphoric acid aqueous solution with volume concentration of 0.08% -0.12%, and the mobile phase B is acetonitrile for gradient elution.
In one embodiment, the gradient elution comprises the following elution procedure:
0-0.5 min, and keeping the volume percentage of the mobile phase A to be 95%;
0.5 min-13 min, wherein the volume percentage of the mobile phase A is reduced from 95% to 75%;
13-19 min, wherein the volume percentage of the mobile phase A is reduced from 75% to 53%;
19-24 min, and keeping the volume percentage of the mobile phase A to be 53%;
24-26 min, wherein the volume percentage of the mobile phase A is reduced from 53% to 10%;
26-28 min, and keeping the volume percentage of the mobile phase A to be 10%.
In one embodiment, the conditions of the high performance liquid chromatography detection further include one or more of the following (1) - (3):
(1) Chromatographic column: octadecylsilane chemically bonded silica is used as a filler;
(2) Flow rate: 0.8mL/min to 1.2mL/min;
(3) Column temperature: 25-35 ℃.
In one embodiment, the conditions of the high performance liquid chromatography detection further include one or more of the following (4) - (5):
(4) Detection wavelength: 225nm to 235nm;
(5) The sample injection amount is 8-12 mu L.
In one embodiment, the detection method further includes a step of constructing a standard curve, and a step of performing content calculation according to the high performance liquid chromatography detection of the sample solution to be detected and the standard curve;
wherein, the standard curve of the PA1-2 is as follows: y= 48049.5426x-48.4689;
the standard curve of the PA2 is as follows: y= 15235.5082x-154.2840;
the standard curve of the PA3 is as follows: y=35196.6552x+875.8689;
the standard curve of the PA5 is as follows: y= 46858.9092x-63.5186;
the standard curve of the PA6 is as follows: y= 49383.8328x +968.8785;
the standard curve of the 1, 3-tetramethylurea is as follows: y= 5721.4471x-37.4474;
the standard curve of the 1-hydroxybenzotriazole is as follows: y= 6929.5853x-150.3455;
the standard curve of the alpha 5-GABAA receptor modulator is as follows: y=44891.9390x+3496.5873.
It will be appreciated that x in each standard curve represents the concentration and y represents the response value of the peak area.
In one embodiment, the step of constructing a standard curve includes:
taking the related substances or the reference substances of alpha 5-GABAA receptor modulator drugs, dissolving the related substances or the reference substances of alpha 5-GABAA receptor modulator drugs in a first solvent, and preparing standard substance solutions with different concentrations;
and (3) carrying out the high performance liquid chromatography detection on the standard substance solutions with different concentrations, and constructing each standard curve according to detection results.
In one embodiment, the concentration distribution of the standard solution with different concentrations is 0.08-2.5 g/mL.
In one embodiment, the first solvent is an acetonitrile aqueous solution with a volume concentration of 45% -55%.
In one embodiment, the second solvent is an acetonitrile aqueous solution with a volume concentration of 45% -55%.
In one embodiment, the related substances are PA1-2, PA3, PA5, PA6, 1, 3-Tetramethylurea (TML) and 1-Hydroxybenzotriazole (HOBT).
According to the detection method for related substances in the alpha 5-GABAA receptor modulator drugs, the high performance liquid chromatography detection is carried out on the bulk drugs by adopting proper conditions, so that the bulk drugs and various impurities can be effectively separated, further the detection of the related substances of the bulk drugs is realized, and the detection method has important significance for the production quality control and research of the bulk drugs.
Drawings
FIG. 1 is a diagram showing the results of HPLC detection of a control solution and a sample solution to be tested of the drug substance in example 1, wherein (a) the control solution chromatogram of the drug substance and (b) the sample solution chromatogram to be tested;
FIG. 2 is a standard graph of each control;
FIG. 3 is a diagram of a specificity investigation result of impurity separation;
fig. 4 is a graph showing the results of separation of the respective impurities in comparative example 1.
Detailed Description
The method for detecting substances in the α5-GABAA receptor modulator of the present application will be described in further detail with reference to specific examples. This application may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other.
Herein, "one or more" refers to any one, any two, or any two or more of the listed items.
In this application, first, "second," etc. are for non-exhaustive list description purposes only, and it should be understood that no closed limitation on the number is made.
In the present application, the technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.
In the present application, reference is made to numerical intervals, where the numerical intervals are considered to be continuous unless specifically stated, and include the minimum and maximum values of the range, and each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The percentage content referred to in the present application refers to mass percent for both solid-liquid and solid-solid phase mixing and volume percent for liquid-liquid phase mixing unless otherwise specified.
The percentage concentrations referred to in this application, unless otherwise indicated, refer to the final concentrations. The final concentration refers to the ratio of the additive component in the system after the component is added.
The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
The room temperature in this application is generally 4 ℃ to 30 ℃, preferably 20+ -5 ℃.
Some examples of the present application provide a method for detecting substances of interest in an α5-GABAA receptor modulator, the α5-GABAA receptor modulator having structural features represented by the following formula (1):
the related substances comprise one or more of PA1-2, PA3, PA5, PA6, 1, 3-Tetramethylurea (TML) and 1-Hydroxybenzotriazole (HOBT), and the structures are shown as follows:
the detection method comprises the following steps:
taking a reference substance of the alpha 5-GABAA receptor modulator drug, dissolving the reference substance in a first solvent, and preparing a reference substance solution;
taking a sample to be measured, and dissolving the sample with a second solvent to prepare a sample solution to be measured;
performing high performance liquid chromatography detection on the reference substance solution and the sample solution to be detected, wherein the conditions of the high performance liquid chromatography detection comprise: the mobile phase A is phosphoric acid aqueous solution with volume concentration of 0.08% -0.12%, and the mobile phase B is acetonitrile for gradient elution.
Specifically, the volume concentration of the phosphoric acid aqueous solution includes, but is not limited to: 0.08%, 0.09%, 0.1%, 0.11%, 0.12% or any two of the foregoing values.
In some examples, the gradient elution comprises the following elution procedure:
0-0.5 min, and keeping the volume percentage of the mobile phase A to be 95%;
0.5 min-13 min, wherein the volume percentage of the mobile phase A is reduced from 95% to 75%;
13-19 min, wherein the volume percentage of the mobile phase A is reduced from 75% to 53%;
19-24 min, and keeping the volume percentage of the mobile phase A to be 53%;
24-26 min, wherein the volume percentage of the mobile phase A is reduced from 53% to 10%;
26-28 min, and keeping the volume percentage of the mobile phase A to be 10%.
It will be appreciated that the gradient elution also includes a mobile phase recovery procedure for the next sample injection, as follows:
28 min-28.1 min, wherein the volume percentage of the mobile phase A is increased from 10% to 95%;
28.1 min-35 min, and keeping the volume percentage of the mobile phase A to be 95%.
In some examples, the conditions of the high performance liquid chromatography detection further include one or more of the following (1) - (3):
(1) Chromatographic column: octadecylsilane chemically bonded silica is used as a filler;
(2) Flow rate: 0.8mL/min to 1.2mL/min; specifically, flow rates include, but are not limited to: 0.8mL/min, 0.9mL/min, 1mL/min, 1.1mL/min, 1.2mL/min, or a range formed by any two of the foregoing values;
(3) Column temperature: 25-35 ℃; specifically, column temperatures include, but are not limited to: 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃ or a range formed by any two of the foregoing values.
In some examples, the conditions of the high performance liquid chromatography detection further include one or more of the following (4) - (5):
(4) Detection wavelength: 225nm to 235nm; specifically, the detection wavelengths include, but are not limited to: 225nm, 226nm, 227nm, 228nm, 229nm, 230nm, 231nm, 232nm, 233nm, 234nm, 235nm or a range formed by any two of the foregoing values;
(5) The sample injection amount is 8-12 mu L; specifically, the sample loading includes, but is not limited to: 8. Mu.L, 9. Mu.L, 10. Mu.L, 11. Mu.L, 12. Mu.L, or any two of the foregoing values.
In some examples, the detection method further comprises a step of constructing a standard curve, and a step of performing content calculation according to a high performance liquid chromatography detection obtained chromatogram of the sample solution to be detected and the standard curve;
wherein, the standard curve of the PA1-2 is as follows: y= 48049.5426x-48.4689;
the standard curve of the PA2 is as follows: y= 15235.5082x-154.2840;
the standard curve of the PA3 is as follows: y=35196.6552x+875.8689;
the standard curve of the PA5 is as follows: y= 46858.9092x-63.5186;
the standard curve of the PA6 is as follows: y= 49383.8328x +968.8785;
the standard curve of the 1, 3-tetramethylurea is as follows: y= 5721.4471x-37.4474;
the standard curve of the 1-hydroxybenzotriazole is as follows: y= 6929.5853x-150.3455;
the standard curve of the alpha 5-GABAA receptor modulator is as follows: y=44891.9390x+3496.5873.
It will be appreciated that x in each standard curve represents the concentration of the corresponding standard and y represents the response of the peak area at that concentration.
In some examples, the step of constructing a standard curve includes:
taking the related substances or the reference substances of alpha 5-GABAA receptor modulator drugs, dissolving the related substances or the reference substances of alpha 5-GABAA receptor modulator drugs in a first solvent, and preparing standard substance solutions with different concentrations;
and (3) carrying out the high performance liquid chromatography detection on the standard substance solutions with different concentrations, and constructing each standard curve according to detection results.
In some examples, the concentration distribution of the standard solution with different concentrations is 0.08-2.5 g/mL.
In some examples, the first solvent is an aqueous acetonitrile solution with a volume concentration of 45% -55%. Specifically, the volume concentration of the acetonitrile aqueous solution includes, but is not limited to: 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55% or a range formed by any two of the foregoing values.
In some examples, the second solvent is an aqueous acetonitrile solution with a volume concentration of 45% -55%. Specifically, the volume concentration of the acetonitrile aqueous solution includes, but is not limited to: 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55% or a range formed by any two of the foregoing values.
In some examples, the related substances are PA1-2, PA3, PA5, PA6, 1, 3-Tetramethylurea (TML) and 1-Hydroxybenzotriazole (HOBT). The detection method can realize synchronous detection of related substances in alpha 5-GABAA receptor regulator medicines, greatly improve detection efficiency and further improve the efficiency of industrial production.
The following examples are given with the reagents used in the examples being commercially available unless otherwise specified.
The bulk drug in the examples is alpha 5-GABAA receptor modulator drug, N-isopropyl-6- (((3- (5- (methoxymethyl) isoxazol-3-yl) - [1,2,4] triazole [3,4-a ] phthalazin-6-yl) oxy) methyl) nicotinamide, the structure of which is shown in the following formula (1):
the structure of each impurity is as follows:
Examples
The instruments and reagents employed in the examples of the present invention are as follows:
1. instrument for measuring and controlling the intensity of light
High performance liquid chromatograph: waters e695, it is understood that other comparable chromatography systems, such as waters Arc, may be employed.
Chromatographic column: waters X-Bridge C18 (250 mm. Times.4.6 mm,5 μm).
2. Reagent
Acetonitrile and phosphoric acid are chromatographic purity, and water is ultrapure water; the rest reagents are all analytically pure;
the reference substance of the raw material medicine is derived from Shanghai Seiemerle biotechnology Co., ltd, and the purity is 99.7%; the synthesis can be performed with reference to the synthetic route disclosed in chinese patent application CN114773352 a.
The controls of PA1-2, PA3, PA5, PA6, 1, 3-tetramethylurea and 1-hydroxybenzotriazole were all obtained from Shanghai Sesamiro Biotech, inc. with purities of 87.12%, 94.57%, 99.14%, 88.17%, 93.26%, 99.97%, 99.98% in that order; can be synthesized by referring to the synthetic route disclosed in the Chinese patent application CN114773352A or obtained by enrichment of byproducts.
The raw material medicine to be tested is obtained from Shanghai Sesamero biotechnology Co., ltd; batch production was carried out with reference to the synthetic route disclosed in chinese patent application CN114773352 a.
3. Solution preparation
Mobile phase a (0.1% by volume phosphoric acid in water): transferring 1.0mL of phosphoric acid into 1000mL of water, and uniformly mixing;
a diluent: an aqueous acetonitrile solution having a volume fraction of 50%;
blank solution: a diluent;
control solution of drug substance: weighing about 20mg of reference substance of the crude drug, placing into a 100mL measuring flask, adding diluent for dissolution, diluting to scale, and shaking. Accurately transferring 1mL of the solution, placing the solution into a 200mL measuring flask, adding a diluent to dilute the solution to a scale, and shaking the solution uniformly.
Sample solution to be measured: taking 20mg of the raw material medicine to be measured, precisely weighing, placing into a 100mL volumetric flask, adding a diluent for dissolution, fixing the volume, and uniformly mixing.
4. Detection of
Performing high performance liquid chromatography detection on a reference substance solution and a sample solution to be detected of the crude drug, wherein the detection conditions are shown in the following table 1:
TABLE 1
As shown in fig. 1, as can be seen from fig. 1, the batch of bulk drugs contains PA 6.08%, unknown impurities of rt21.105min 0.06%, unknown impurities of rt23.360min 0.14%, and other known impurities which are not detected.
5. Linearity and range
Control solutions are prepared within a concentration range of 0.08-2.5 g/mL (specific concentrations are shown in tables 2-9, and the control solutions are prepared by diluents), and the linear relation and the range between the concentrations and the peak areas of each impurity and the raw material medicine are examined.
Acceptance criteria: in the concentration range of 0.08-2.5 g/mL, the response value and the concentration should be in good linearity, and the linear correlation coefficient (r) should not be less than 0.995. The Y-axis intercept value must not be greater than ±50% of the 0.05% horizontal response value.
Detecting the reference substance solutions according to the items of ' 4 and ' detection ', recording the chromatographic peak area, taking the peak area as an ordinate (y), taking the reference substance concentration as an abscissa (x), and drawing a standard curve, wherein the results are shown in fig. 2 and tables 2-9.
TABLE 2 PA1-2 Linear results
TABLE 3 PA2 Linear results
TABLE 4 PA3 Linear results
TABLE 5 PA5 Linear results
TABLE 6 PA6 Linear results
TABLE 7 HOBT Linear results
TABLE 8 TML Linear results
TABLE 9 Linear results of crude drugs
Referring to the results of each linear regression equation, correction factors of impurities PA1-2, PA3, PA5, PA6, HOBT, TML were calculated, and the test results are shown in Table 10.
TABLE 10 correction factor summary of crude drug related substances
6. Quantitative limit and detection limit
Acceptance criteria: the signal to noise ratio of the bulk drug and the quantitative limiting solution of each impurity is not lower than 10, and the RSD% of each peak area is not more than 5.0%. The signal to noise ratio of the crude drug and each impurity detection limit solution is not lower than 3.
The detection method comprises the following steps: taking each L1 level solution under the '5 and linear' item as a bulk drug and each impurity quantitative limiting solution (0.1 mug/mL), wherein the concentration is 0.05% of the concentration of a sample to be detected, taking the quantitative limiting solution for sample injection, continuously feeding 6 needles, and calculating the relative average deviation (RSD%) of the signal to noise ratio and the peak area. 3.0mL of each quantitative limiting solution is precisely measured, the solution is placed in a 10mL measuring flask, diluted to a scale by a diluent, and uniformly shaken, so that the crude drug and each impurity detection limiting solution (0.03 mug/mL) are obtained, and the concentration of the detected sample is 0.015%. Taking the solution for sample injection, and recording a chromatogram.
The test results are shown in Table 11:
TABLE 11 method for verifying quantitative limits and detection limit results for related substances of crude drugs
As can be seen from Table 11, the quantitative limit concentrations of the crude drugs, PA1-2, PA3, PA5, HOBT and TML are all 0.05% of the concentration of the sample to be measured (0.2 mg/mL); the detection limit concentrations are all 0.015% of the concentration (0.2 mg/mL) of the sample to be detected.
7. Precision investigation
(1) System precision
Acceptance criteria: the retention time RSD% of each component of the 6-needle control and the standard sample solution is not more than 1.0% and the peak area is not more than 5.0%.
The detection method comprises the following steps: preparing a reference substance solution, preparing a standard sample solution according to the method, enabling each impurity to be added to a concentration level of 0.3%, continuously feeding 6 samples for the reference substance and the standard sample solution, and recording the retention time and the peak area.
The detection results are shown in tables 12-14:
TABLE 12 results of retention time of components in sample solutions (min)
TABLE 13 Peak area results for each component in sample solutions
TABLE 14 results of retention time and peak area System precision in crude drug controls
(2) Repeatability of
Acceptance criteria: in 6 samples, the quantity X of the single related substances is less than or equal to 0.10 percent, and the RSD is less than or equal to 20 percent; 0.10 percent < X is less than or equal to 0.25 percent. RSD is less than or equal to 15 percent; x is more than 0.25%, and RSD is less than or equal to 5%.
The detection method comprises the following steps: impurity stock solutions were prepared in which the concentrations of PA1-2, PA3, PA5, PA6, 1, 3-tetramethylurea and 1-hydroxybenzotriazole were 20. Mu.g/mL, respectively. Adding an impurity stock solution into the raw material medicines to prepare a solution with the concentration level of 0.3%, preparing 6 parts of solutions in parallel, and taking each solution for sample injection.
The test results are shown in Table 15:
TABLE 15 method for verifying repeatability test results of crude drug related substances
(3) Intermediate precision
Acceptance criteria: another laboratory worker uses another instrument to prepare 6 samples, wherein the quantity X of the single related substance is less than or equal to 0.10 percent and the RSD is less than or equal to 20 percent; 0.10 percent of < X is less than or equal to 0.25 percent, and RSD is less than or equal to 15 percent; x is more than 0.25%, and RSD is less than or equal to 5%. In 12 samples of two experimenters, the quantity of single related substances is less than or equal to 0.10 percent, and the RSD is less than or equal to 25 percent; 0.10 percent of < X is less than or equal to 0.25 percent, and RSD is less than or equal to 20 percent; x is more than 0.25%, and RSD is less than or equal to 20%.
The detection method comprises the following steps: in the same laboratory, 6 parts of solution with the concentration level of 0.3% are prepared in parallel by another analyzer at different times, and the solution is taken and injected.
The detection results are shown in tables 16-17:
table 16 results of intermediate precision test of crude drug related substance method verification
Table 17 results of the test for verifying the precision by the method for preparing the crude drug
8. Accuracy investigation
Acceptance criteria: the accuracy of each impurity sample addition is 90% -108%, and RSD is not more than 5.0%.
The detection method comprises the following steps: impurity stock solutions were prepared in which the concentrations of PA1-2, PA3, PA5, 1, 3-tetramethylurea and 1-hydroxybenzotriazole were 20. Mu.g/mL, respectively. Adding an impurity stock solution into the crude drugs to prepare solutions with concentration levels of 0.1%, 0.3% and 0.5%, preparing three solutions in parallel for each level, and taking each solution for sample injection.
The test results are shown in Table 18:
TABLE 18 results of accuracy test for drug substance related substance method validation
9. Solution stability
Acceptance criteria: placing the reference substance solution of the raw material medicine and the sample solution to be detected at room temperature and 4 ℃, wherein the difference of the concentration of the reference substance solution of the raw material medicine is not more than 2.0%, and if X is less than or equal to 0.10%, the absolute value of the difference is not more than 0.05%; if 0.10 percent of < X is less than or equal to 0.25 percent, the deviation result of the X and the X is not more than 15 percent; x is more than 0.25%, the deviation result of the X and the X is not more than 5%, and no new impurity is generated, so that the solution is stable.
The reference substance solution and the sample solution to be tested of the raw material medicine are stable in 24 hours at the temperature of 4 ℃ and the room temperature.
10. Specialization of
Acceptance criteria: the main peak time of the sample is consistent with that of the reference substance; the blank solution should have no interference at the peak positions of the main peak and the impurity peak, if any, the blank solution should not be more than 0.05%; the degree of separation between the impurities and the main peak should not be less than 2.0, and the degree of separation between the impurities should not be less than 1.5.
The detection method comprises the following steps: preparing blank solution, sample solution to be tested and reference substance solution of crude drug according to the method of '3 and solution preparation'. And dissolving proper amounts of PA1-2, PA3, PA5, PA6, 1, 3-tetramethyl urea and 1-hydroxybenzotriazole respectively with a diluent, and uniformly mixing to prepare reference substance solutions with the concentration of 0.2mg/mL respectively. Taking the solutions and sampling.
The results of the test are shown in FIG. 3 and Table 19:
TABLE 19 method of verifying specificity results for drug substances
The blank solution has no interference, and each chromatographic peak can reach baseline separation.
11. Durability of
Acceptance criteria: the ratio of the impurity content of the sample solution after changing the detection condition to the initial condition is not more than 0.10% if the content X of the related substances is not more than 0.05% in absolute value; if 0.10 percent of < X is less than or equal to 0.25 percent, the deviation result of the X and the X is not more than 15 percent; x >0.25%, the deviation of the two results is not more than 10%, i.e. the method is considered to be good in durability under this condition.
Wherein the content I-the content of the component to be measured under the initial condition and the content C-the content of the component to be measured under the modified condition
The detection method comprises the following steps: preparing a sample solution according to the concentration level of 0.3% in the process of '8 and accuracy investigation', and preparing a reference substance solution of the bulk drug according to the process of '3 and solution preparation', so as to carry out investigation. The column temperature (25 ℃ C., 35 ℃) and the flow rate (0.9 mL/min, 1.1 mL/min) of the chromatographic column were changed, the detection wavelength (228 nm, 232 nm) was changed, and the chromatographic column was replaced, and samples were introduced according to the detection method in "4, detection".
The detection results are as follows:
table 20 column temperature change
TABLE 21 flow Rate Change
Table 22 wavelength change
Table 23 replacement chromatographic column
It can be seen that the results of each known and unknown impurity after changing the conditions meet the requirements, and the method has good durability.
Comparative example 1
This comparative example examined different elution procedures. Sample solutions (appropriate amounts of PA1-2, PA3, PA5, PA6, 1, 3-tetramethylurea, 1-hydroxybenzotriazole and the reference substances of the crude drugs were taken, respectively, dissolved in 50% acetonitrile aqueous solution, and then mixed, sample solutions containing PA1-2, PA3, PA5, PA6, 1, 3-tetramethylurea and 1-hydroxybenzotriazole control at a concentration of 20. Mu.g/mL and the drug substance control at a concentration of 0.2 mg/mL) were prepared and the elution procedure was adjusted to:
0-0.5 min, and keeping the volume percentage of the mobile phase A to be 95%;
0.5 min-13 min, wherein the volume percentage of the mobile phase A is reduced from 95% to 85%;
13-19 min, wherein the volume percentage of the mobile phase A is reduced from 85% to 63%;
19-24 min, keeping the volume percentage of the mobile phase A at 63%;
24-26 min, wherein the volume percentage of the mobile phase A is reduced from 63% to 10%;
26-28 min, keeping the volume percentage of the mobile phase A to be 10%;
28 min-28.1 min, wherein the volume percentage of the mobile phase A is increased from 10% to 95%;
28.1 min-30 min, and keeping the volume percentage of the mobile phase A to be 95%.
The test results are shown in fig. 4. It can be seen that both 1, 3-tetramethylurea and 1-hydroxybenzotriazole are not separated and PA1-2 is not completely separated from the unknown impurities.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. It should be understood that those skilled in the art, based on the technical solutions provided in the present application, can obtain technical solutions through logical analysis, reasoning or limited experiments, all fall within the protection scope of the claims attached in the present application. The scope of the patent application is therefore intended to be limited by the content of the appended claims, the description and drawings being presented to the extent that the claims are defined.
Claims (10)
1. The detection method of related substances in alpha 5-GABAA receptor modulator drugs is characterized in that the alpha 5-GABAA receptor modulator drugs have the structural characteristics shown in the following formula (1):
the related substances comprise one or more of PA1-2, PA3, PA5, PA6, 1, 3-tetramethylurea and 1-hydroxybenzotriazole, and the structures are shown as follows:
the detection method comprises the following steps:
taking a reference substance of the alpha 5-GABAA receptor modulator drug, dissolving the reference substance in a first solvent, and preparing a reference substance solution;
taking a sample to be measured, and dissolving the sample with a second solvent to prepare a sample solution to be measured;
performing high performance liquid chromatography detection on the reference substance solution and the sample solution to be detected, wherein the conditions of the high performance liquid chromatography detection comprise: the mobile phase A is phosphoric acid aqueous solution with volume concentration of 0.08% -0.12%, and the mobile phase B is acetonitrile for gradient elution.
2. The method for detecting substances of interest in an alpha 5-GABAA receptor modulator class of drugs according to claim 1, wherein said gradient elution comprises the following elution procedure:
0-0.5 min, and keeping the volume percentage of the mobile phase A to be 95%;
0.5 min-13 min, wherein the volume percentage of the mobile phase A is reduced from 95% to 75%;
13-19 min, wherein the volume percentage of the mobile phase A is reduced from 75% to 53%;
19-24 min, and keeping the volume percentage of the mobile phase A to be 53%;
24-26 min, wherein the volume percentage of the mobile phase A is reduced from 53% to 10%;
26-28 min, and keeping the volume percentage of the mobile phase A to be 10%.
3. The method for detecting substances related to alpha 5-GABAA receptor modulator according to claim 1, wherein the conditions for high performance liquid chromatography detection further comprise one or more of the following (1) to (3):
(1) Chromatographic column: octadecylsilane chemically bonded silica is used as a filler;
(2) Flow rate: 0.8mL/min to 1.2mL/min;
(3) Column temperature: 25-35 ℃.
4. The method for detecting substances related to alpha 5-GABAA receptor modulator according to claim 1, wherein the conditions for high performance liquid chromatography detection further comprise one or more of the following (4) - (5):
(4) Detection wavelength: 225nm to 235nm;
(5) The sample injection amount is 8-12 mu L.
5. The method for detecting substances related to a 5-GABAA receptor modulator according to claim 1, further comprising a step of constructing a standard curve, and a step of calculating the content of the standard curve and the chromatogram obtained by high performance liquid chromatography detection of the sample solution to be detected;
wherein, the standard curve of the PA1-2 is as follows: y= 48049.5426x-48.4689;
the standard curve of the PA2 is as follows: y= 15235.5082x-154.2840;
the standard curve of the PA3 is as follows: y=35196.6552x+875.8689;
the standard curve of the PA5 is as follows: y= 46858.9092x-63.5186;
the standard curve of the PA6 is as follows: y= 49383.8328 x+ 968.8785;
the standard curve of the 1, 3-tetramethylurea is as follows: y= 5721.4471x-37.4474;
the standard curve of the 1-hydroxybenzotriazole is as follows: y= 6929.5853x-150.3455;
the standard curve of the alpha 5-GABAA receptor modulator is as follows: y=44891.9390x+3496.5873.
6. The method for detecting substances involved in α5-GABAA receptor modulator according to claim 5, wherein the step of constructing a standard curve comprises:
taking the related substances or the reference substances of alpha 5-GABAA receptor modulator drugs, dissolving the related substances or the reference substances of alpha 5-GABAA receptor modulator drugs in a first solvent, and preparing standard substance solutions with different concentrations;
and (3) carrying out the high performance liquid chromatography detection on the standard substance solutions with different concentrations, and constructing each standard curve according to detection results.
7. The method for detecting substances related to alpha 5-GABAA receptor modulator according to claim 6, wherein the concentration distribution of the standard substance solutions with different concentrations is 0.08-2.5 g/mL.
8. The method for detecting substances related to a 5-GABAA receptor modulator according to claim 1, wherein the first solvent is an aqueous acetonitrile solution having a volume concentration of 45% -55%.
9. The method for detecting substances related to a 5-GABAA receptor modulator according to claim 1, wherein the second solvent is an aqueous acetonitrile solution having a volume concentration of 45% -55%.
10. The method for detecting related substances in an alpha 5-GABAA receptor modulator according to claim 1-9, wherein the related substances are PA1-2, PA3, PA5, PA6, 1, 3-tetramethylurea and 1-hydroxybenzotriazole.
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