CN114755346A - Method for measuring substances related to cyclosporine soft capsules - Google Patents
Method for measuring substances related to cyclosporine soft capsules Download PDFInfo
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Abstract
The invention discloses a method for measuring substances related to a ciclosporin soft capsule, belonging to the technical field of pharmaceutical analysis. The method adopts high performance liquid chromatography to measure related substances in the ciclosporin soft capsules, and the chromatographic conditions are as follows: the chromatographic column takes octadecylsilane chemically bonded silica as a filling agent, tetrahydrofuran as a mobile phase A, the mobile phase B comprises a mixed solution of tetrahydrofuran and a disodium hydrogen phosphate aqueous solution, the pH value of the mobile phase B is 1.9-2.1, gradient elution is carried out, the detection wavelength is 218-222 nm, the column temperature is 74-76 ℃, and the flow rate of the mobile phase is 0.95-1.05 ml/min. The determination method can ensure that the auxiliary material components are eluted in time by screening chromatographic conditions, avoids the interference of auxiliary material peaks on the detection of related substances, greatly advances the peak emergence time of the isocyclosporin A and the isocyclosporin H, effectively improves the detection sensitivity and the result accuracy, and further ensures the quality controllability of the cyclosporine soft capsule.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a method for determining related substances of a ciclosporin soft capsule.
Background
Cyclosporine A (CyA) is cyclic polypeptide consisting of 11 amino acids, is an active metabolite of fungi in soil, can be used as an immunosuppressant, can selectively act on the initial stage of T lymphocyte activation, and is mainly used for clinically preventing adverse immune reactions such as rejection during xenogenic organ or bone marrow transplantation. There are 6 known impurities in cyclosporine, and many impurities in cyclosporine process and degradation impurities are different along with different dosage forms, and the control of the content of the related substances has important significance for ensuring the quality of medicines and the safety of medication.
The current pharmacopoeia of various countries only controls the related substances of cyclosporine raw material medicines, but does not control the related substances of cyclosporine preparation products. For the ciclosporin soft capsule, on one hand, the ingredients of the auxiliary materials are complex, the ultraviolet absorption is strong, more auxiliary material peaks exist in a chromatogram map, and the risk of auxiliary material interference exists; on the other hand, because the production process, the interaction of raw materials and auxiliary materials, the stability in the storage process and other influences can generate some new impurities, the safety, the effectiveness and the controllable quality of a preparation product cannot be ensured by the existing standards. Regarding impurities contained in the ciclosporin soft capsules, the analysis method of related substances in the quality standard of the ciclosporin soft capsules is disclosed in 2015 by the national pharmacopoeia committee, and the related analysis method is introduced in the literature of HPLC method determination of the content and related substances of the ciclosporin capsules and the soft capsules published by Poncirus et al. However, the prior art only detects and controls three related substances, namely cyclosporine H, cyclosporine A and isocyclosporine A, and does not control other unknown impurities. Meanwhile, partial auxiliary material peaks cannot be eluted in time under the chromatographic conditions of the method, and the auxiliary material peaks are eluted in the subsequent second needle and third needle, so that the detection of related substances is interfered, the result is inaccurate, and the service life of the chromatographic column is influenced. In addition, in the analysis method in the prior art, chromatographic peaks of cyclosporine H, cyclosporine A and isocyclosporine A are retained for a relatively short time, and the chromatographic peaks of the three related substances are short and wide, and have relatively low detection sensitivity and relatively poor accuracy due to the fact that the diffusion effect of chromatographic separation is increased along with the increase of the retention time.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for measuring substances related to a cyclosporin soft capsule, which can ensure that auxiliary material components in a preparation are eluted in time, effectively avoid the interference of auxiliary material peaks and effectively improve the detection sensitivity and the result accuracy.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a method for measuring substances related to cyclosporin soft capsules adopts high performance liquid chromatography for measurement, and the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a chromatographic column filler;
mobile phase: and (3) taking tetrahydrofuran as a mobile phase A, taking a mixed solution of the tetrahydrofuran and disodium hydrogen phosphate as a mobile phase B, and carrying out gradient elution when the pH value of the mobile phase B is 1.9-2.1.
Preferably, the procedure of the gradient elution is as follows: the volume percentage of the mobile phase B is maintained at 100 percent in 0-40 min; in 40-55 min, the volume percent of the mobile phase A is changed from 0% to 70-90%, and the volume percent of the mobile phase B is changed from 100% to 10-30%; at 56min-65min, the volume percentage of the mobile phase A is 0%, and the volume percentage of the mobile phase B is maintained at 100%.
Preferably, the procedure of the gradient elution is as follows: the volume percentage of the mobile phase B is maintained at 100 percent in 0-40 min; in 40-55 min, the volume percent of the mobile phase A is changed from 0% to 75-85%, and the volume percent of the mobile phase B is changed from 100% to 15-25%; at 56min-65min, the volume percentage of the mobile phase A is 0%, and the volume percentage of the mobile phase B is maintained at 100%.
Preferably, the procedure of the gradient elution is as follows: the volume percentage of the mobile phase B is maintained at 100 percent in 0-40 min; in 40-55 min, the volume percent of the mobile phase A is changed from 0% to 78-82%, and the volume percent of the mobile phase B is changed from 100% to 18-22%; at 56min-65min, the volume percentage of the mobile phase A is 0%, and the volume percentage of the mobile phase B is maintained at 100%.
Further preferably, the elution procedure of the mobile phase is as follows:
in a preferred embodiment of the invention, the volume ratio of tetrahydrofuran to disodium hydrogen phosphate in the mobile phase B is 44-46: 54-56. Alternatively, the concentration of disodium hydrogen phosphate is 0.005 to 0.2mol/L, further 0.005 to 0.15mol/L, further 0.01 mol/L.
In a preferred embodiment of the present invention, the conditions for performing the hplc assay are as follows: the detection wavelength is 218-222 nm, the column temperature is 74-76 ℃, the flow rate is 0.95-1.05 ml/min, and the sample injection amount is 10 mu l.
Optionally, the chromatography column is of a type specification: kromasil 100-5, C18, 4.6 mm. times.250 mm, 5 μm.
Specifically, the method for measuring related substances of the ciclosporin soft capsule provided by the invention comprises the following steps:
s1, preparing a blank auxiliary material solution: taking appropriate amount of medicinal liquid adjuvant and capsule shell adjuvant, adding solvent, shaking strongly to dissolve, and diluting into blank adjuvant solution containing medicinal liquid adjuvant 19mg and capsule shell adjuvant 12mg per 1 ml;
s2, preparation of a test solution: taking a proper amount of the contents of the ciclosporin soft capsule, precisely weighing, adding a solvent, and shaking uniformly to prepare a test solution containing 2mg of ciclosporin per 1 ml;
s3, preparation of a control solution: precisely measuring 1ml of the test solution, adding a solvent, and shaking uniformly to obtain a control solution containing 10 microgram of cyclosporine per 1 ml;
s4, preparation of a separation degree solution:
taking appropriate amount of cyclosporin reference substance, cyclosporin H reference substance, isocyclosporin A reference substance, and isocyclosporin H reference substance, dissolving with solvent, and diluting to obtain a separation degree solution containing 25 μ g of cyclosporin, 25 μ g of cyclosporin H, 25 μ g of isocyclosporin A, and 25 μ g of isocyclosporin H per 1 ml;
s5, detection: respectively and precisely measuring 10 mu L of blank auxiliary material solution, resolution solution, test solution and control solution, respectively injecting into a liquid chromatograph, recording chromatogram, comparing the areas of impurity peaks of the test solution and main peaks of the control solution, and calculating the content of each related substance according to a self-control method with correction factors.
Optionally, the time of the strong shaking is 5-10 min.
Optionally, the solvent is a mixed solution of tetrahydrofuran and water in a volume ratio of 4: 1.
Specifically, the calculation formula of the self-comparison method is as follows:
the content of related substances is F X [ A ]U/(AS×200)]×100％
Wherein, AUIs the peak area of each impurity in the test sample solution, ASThe peak area of cyclosporine in the control solution is shown as F, the correction factors of cyclosporine and cyclosporine H are 1.0, the correction factor of isocyclosporine A is 1.2, and the correction factor of isocyclosporine H is 1.4.
Compared with the prior art, the beneficial effects of the scheme are as follows:
according to the method, the high performance liquid chromatography is adopted to measure the related substances of the ciclosporin soft capsules, and the chromatographic conditions are screened and optimized, so that the auxiliary material components in the ciclosporin soft capsules are timely eluted in a chromatographic column, the detection of the related substances is prevented from being interfered by auxiliary material peaks, and the service life of the chromatographic column is prolonged; compared with the prior art, the determination method has the advantages that the appearance time of the isocyclosporin A and the isocyclosporin H is greatly advanced, the chromatographic peak is obviously increased and narrowed, and the detection sensitivity and the result accuracy are greatly improved, so that the quality controllability and the product safety of the cyclosporine soft capsule are ensured.
Drawings
Fig. 1 is a graph of the pH 2.2 of the mobile phase in the chromatographic condition screening;
FIG. 2 is a diagram of the pH value of the mobile phase at 1.8 in the chromatographic condition screening;
FIG. 3 is a graph of the pH 2.5 of the mobile phase in the chromatographic condition screening;
FIG. 4 is a graph of the pH of the mobile phase at 1.95 in the chromatographic condition screening;
FIG. 5 is a graph of an auxiliary material peak interfering with a main peak of a control solution when no gradient elution is performed in chromatographic condition screening;
FIG. 6 is a chromatogram of an auxiliary material peak interfering cyclosporin H when no gradient elution is performed in chromatographic condition screening;
FIG. 7 is a graph of a blank adjuvant without an adjuvant peak on the next needle after increasing gradient elution in the chromatographic condition screening;
FIG. 8 is a chromatogram of the separation degree between a blank auxiliary material and each known impurity in the chromatographic condition optimization specificity study;
FIG. 9 is a diagram of a blank auxiliary material and a sample which are not strongly degraded in the chromatographic condition optimization specificity research;
FIG. 10 is a graph of oxidative damage of blank excipients and a test sample in chromatographic condition optimization specificity research;
FIG. 11 is a chromatogram of strong light damage of blank auxiliary materials and test samples in chromatographic condition optimization specificity research;
FIG. 12 is a graph of the destruction of a blank auxiliary material and a test sample by strong base in the chromatographic condition optimization specificity study;
FIG. 13 is a diagram showing the destruction of a blank auxiliary material and a test sample by a strong acid in the chromatographic condition optimization specificity study;
FIG. 14 is a diagram of blank excipients and a sample undergoing high temperature destruction in chromatographic condition optimization specificity study;
FIG. 15 is a comparison graph showing the chromatogram stack of strong degradation conditions of cyclosporin A.C. raw material drug during optimization of chromatographic conditions;
FIG. 16 is a specificity test profile for example 5;
fig. 17 is a specificity test spectrum of comparative example 1.
Detailed Description
The invention is described in further detail below with reference to the drawings and the detailed description.
A method for measuring substances related to a cyclosporin soft capsule, which adopts high performance liquid chromatography for measurement, comprises the following steps:
s1, preparation of a solvent and a blank auxiliary material solution:
mixing tetrahydrofuran and water according to a volume ratio of 4:1 to prepare a solvent;
taking a proper amount of liquid medicine auxiliary materials and capsule shell auxiliary materials, adding a solvent, strongly shaking for 5-10 min to dissolve the liquid medicine auxiliary materials and the capsule shell auxiliary materials, and diluting the mixture into blank auxiliary material solution containing 19mg of liquid medicine auxiliary materials and 12mg of capsule shell auxiliary materials per 1 ml;
s2, preparation of a test solution: taking a proper amount of the contents of the ciclosporin soft capsule, precisely weighing, adding a solvent, and shaking uniformly to prepare a test solution containing 2mg of ciclosporin per 1 ml;
s3, preparation of control solution: precisely measuring 1ml of the test solution, adding a solvent, and shaking uniformly to obtain a control solution containing 10 microgram of cyclosporine per 1 ml;
S4, preparation of a resolution solution:
taking appropriate amount of cyclosporin control, cyclosporin H control, isocyclosporin A control and isocyclosporin H control, adding solvent to dissolve and dilute into resolution solution containing 25 μ g of cyclosporin, 25 μ g of cyclosporin H, 25 μ g of isocyclosporin A and 25 μ g of isocyclosporin H per 1 ml;
s5, detection: respectively and precisely measuring 10 μ L of blank adjuvant solution, separation degree solution, sample solution and control solution, respectively, injecting into a liquid chromatograph, respectively, wherein the separation degree between each component peak should be not less than 1.5, the blank adjuvant does not interfere with detection of cyclosporine and known impurities, and recording chromatogram. The chromatographic conditions were as follows:
stationary phase: octadecylsilane chemically bonded silica is used as a chromatographic column filler, and the type specification of the chromatographic column is as follows: kromasil 100-5, C18, 4.6mm by 250mm, 5 μm or a column with performance equivalent thereto; a heat exchanger is arranged between the chromatographic column and the sample injection valve, and the heat exchanger can be a column incubator with a preheating module or a stainless steel tube with the thickness of 0.25mm multiplied by 1 m.
Mobile phase: taking tetrahydrofuran as a mobile phase A, taking a mixed solution of tetrahydrofuran and 0.01mol/L disodium hydrogen phosphate (the volume ratio is 44-46: 54-56) as a mobile phase B, adjusting the pH of the mobile phase B to be 1.95 +/-0.05 by using phosphoric acid, and carrying out gradient elution;
The elution procedure for the mobile phase was as follows:
the detection wavelength is 218-222 nm, the column temperature is 74-76 ℃, the flow rate is 0.95-1.05 ml/min, and the sample injection amount is 10 mu l.
Comparing the impurity peak of the sample solution with the main peak area of the reference solution, and calculating the content of each related substance according to a self-comparison method of adding a correction factor. Chromatographic peaks smaller than 0.1 times (0.05%) of the area of the main peak of the control solution in the chromatogram of the test solution are ignored.
The specific calculation formula is as follows:
the content of related substances is F x [ A ]U/(AS×200)]×100％
Wherein A isUIs the peak area of each impurity in the test solution, ASThe peak area of cyclosporin in the control solution was designated as F, the correction factors for cyclosporin and cyclosporin H were 1.0, the correction factor for cyclosporin A was 1.2, and the correction factor for cyclosporin H was 1.4.
Experimental example 1: chromatographic condition screening for measuring related substances of ciclosporin soft capsules
(1) Initial chromatographic conditions
Octadecylsilane chemically bonded silica is used as filler and filled in a chromatographic column (the specification of the chromatographic column is Kromasil 100-5, C18, 4.6mm multiplied by 250mm, 5 μm), and a stainless steel tube (or a column incubator with a preheating module) of 0.25mm multiplied by 1m is connected between the chromatographic column and a sample injection valve and is used as a heat exchanger; the column temperature was 75 ℃ and the detection wavelength was 220nm, and a mixed solution of tetrahydrofuran-0.01 mol/L disodium hydrogenphosphate (mixed volume ratio: 45:55) adjusted to pH 2.2 with phosphoric acid was used as the mobile phase. The peak appearance of each related substance was analyzed, and the chromatographic results are shown in FIG. 1. The results in FIG. 1 show that the peak appearance of the isocyclosporin H is disturbed by the solvent peak.
(2) Screening of the pH of the Mobile phase
Through comparative study on adjustment (such as column temperature, mobile phase pH, mobile phase proportion and the like) of chromatographic conditions, the mobile phase pH has a remarkable influence on the retention time of the isocyclosporin H and the isocyclosporin A, and has a small influence on the retention time of impurities such as the cyclosporine C, the cyclosporine B, the cyclosporine H and the like. Therefore, it is considered to avoid the solvent peak and the adjuvant peak from interfering with the detection of each impurity by adjusting the pH of the mobile phase. The pH of the mobile phase is adjusted to 1.8, 2.5 and 1.95 respectively, and the separation conditions of impurities, a solvent peak and an auxiliary material peak are shown in figures 2-4 respectively.
As can be seen from fig. 2 to 4, when the mobile phase pH is 1.8, both the isocyclosporin H and isocyclosporin a are interfered by the solvent peak, and the separation degree of both is poor; when the pH value of the mobile phase is 2.5, the isocyclosporin H is interfered by a solvent peak, and the isocyclosporin A is interfered by an auxiliary material peak; when the pH of the mobile phase is 1.95, other impurities except the cyclosporin B are not interfered by a solvent peak or an auxiliary material peak, and the separation degrees of the isocyclosporin H and the isocyclosporin A meet the specification. Cyclosporin B is interfered by auxiliary material peaks under different pH conditions of a mobile phase, the impurities are process impurities and are controlled in raw materials according to known impurities, and the preparation products do not need to be controlled. The mobile phase pH was thus determined to be 1.95.
(3) Gradient elution procedure
With the increase of the inspection amount, the applicant finds that partial auxiliary material peaks of the ciclosporin soft capsules can not be completely eluted within 40min, and peaks appear in a next injection chromatogram, the retention time is greatly influenced by the mobile phase batch and the column temperature, and the ciclosporin or the ciclosporin H can be detected in some cases, as shown in fig. 5 and fig. 6. In order to solve the problems, the applicant adds a gradient elution program for increasing the tetrahydrofuran proportion on the basis of collecting 40min under the initial chromatographic condition, and the elution program of a mobile phase is specifically as follows:
the results are shown in FIG. 7. The results in fig. 7 show that the adjuvant peak can be eluted efficiently using the gradient elution procedure as above, and the baseline is smooth.
(3) Specialization inspection
The method is characterized in that a cyclosporin soft capsule produced by a Lizhu pharmaceutical factory is used as a self-made preparation, a cyclosporin soft capsule produced by Nowa pharmaceutical is used as a reference preparation to carry out a special investigation experiment, and the specific operation is as follows: weighing a proper amount (equivalent to 100 granules) of blank auxiliary materials in the proportion of the formula, placing the blank auxiliary materials in a 250ml volumetric flask, adding 80% tetrahydrofuran for dissolving, fixing the volume to a scale, and shaking up to be used as a blank auxiliary material stock solution. The self-made preparation and a reference preparation are precisely weighed respectively and are respectively placed in 7 volumetric flasks of 50ml in proper amount (which is about equal to the amount of the cyclosporine of 100 mg), and the test solution under each strong degradation condition is prepared according to the method shown in the following table. Taking 5ml of blank auxiliary material stock solution, putting the blank auxiliary material stock solution into a 50ml volumetric flask, operating in parallel with the sample solution, and preparing the blank auxiliary material solution with various strong degradation conditions according to the method in the table 1. And respectively and precisely measuring 10 mu l of each strong degradation solution, injecting the solution into a liquid chromatograph, and recording a chromatogram, wherein the result is shown in figures 8-16.
TABLE 1 preparation method of strong degradation test
The results of the undamaged samples in fig. 8 show that the impurity peaks of the self-prepared preparation and the reference preparation are basically consistent, the degrees of separation of the main peak, the impurity peaks and the auxiliary material peaks are in accordance with the regulations, the purity of the main peak is in accordance with the regulations, and the specificity of the method is good. Fig. 9 shows that, under the conditions of the high-temperature destruction test, the main impurity generated by the degradation of the reference preparation and the self-made preparation is the isocyclosporin a, the auxiliary material peak is also obviously increased, the separation degrees of the main peak, the impurity peak and the auxiliary material peak meet the specification, the purity of the main peak meets the specification, and the method specificity is good. Fig. 10 shows that, under the acid destruction test conditions, the main impurity generated by the degradation of the reference preparation and the self-made preparation is the isocyclosporin a, the auxiliary material peak is also obviously increased, the separation degrees of the main peak, the impurity peak and the auxiliary material peak meet the specification, the purity of the main peak meets the specification, and the method specificity is good. Fig. 11 shows that under the conditions of the alkali destruction test, the reference preparation and the self-made preparation, i.e., the cyclosporin a, are slightly increased, and the auxiliary material peak is degraded into a plurality of peaks without affecting the determination of each known impurity, so that the purity of the main peak meets the requirements, and the specificity of the method is good. Fig. 12 shows that, under the conditions of the oxidative damage test, both the reference preparation and the self-made preparation are stable, the degrees of separation of the main peak, impurity peak and auxiliary peak meet the specifications, the purity of the main peak meets the specifications, and the method specificity is good. Fig. 13 shows that, under the conditions of the light damage test, both the reference preparation and the self-made preparation are stable, the degrees of separation of the main peak, impurity peak and auxiliary peak meet the specifications, the purity of the main peak meets the specifications, and the method specificity is good. Therefore, the method of the invention has good specificity.
Experimental example 2: methodology survey
(1) Limit of quantification and limit of detection
Taking tetrahydrofuran-water mixed solution mixed according to the volume ratio of 4:1 as a solvent, taking 950mg of liquid medicine auxiliary materials and 600mg of capsule shell auxiliary materials according to the proportion of a formula, placing the liquid medicine auxiliary materials and the capsule shell auxiliary materials into a 50ml measuring flask, diluting the liquid medicine auxiliary materials to a scale with the solvent, and shaking the liquid medicine auxiliary materials with strong force for about 5-10 minutes to obtain blank auxiliary material solution. Accurately measuring appropriate amounts of an isocyclosporine A, an isocyclosporine H and a cyclosporine H stock solution respectively, dissolving and diluting with a solvent, and preparing a mixed impurity reference solution; precisely measuring cyclosporine reference substance stock solution and mixed impurity reference substance solution, injecting 10 mul of each into a liquid chromatograph, recording a chromatogram, and observing the signal-to-noise ratio of cyclosporine and each impurity peak. And taking the reference substance solution, further diluting with a blank auxiliary material solution, and carrying out sample injection detection. And when the signal-to-noise ratio is larger than or equal to 10, taking the quantitative limiting solution as the quantitative limiting solution, carrying out continuous sample introduction 6 times, and inspecting the RSD and the signal-to-noise ratio of each sample introduction detection peak area of each component. Precisely measuring 3ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding the blank auxiliary material solution to dilute to a scale, and shaking up to be used as the detection limiting solution.
The results show that the quantitative limits of the isocyclosporin H, the isocyclosporin A, the cyclosporine and the cyclosporine H are respectively 1.24 mu g/ml, 1.43 mu g/ml, 1.48 mu g/ml and 2.21 mu g/ml, the RSD of the solution with the quantitative limit in continuous sampling for 6 times of peak areas is respectively 2.1 percent, 1.7 percent, 3.9 percent and 8.9 percent, and the average signal-to-noise ratio is respectively 23, 37, 21 and 18; the detection limit is 0.37 mug/ml, 0.43 mug/ml, 0.45 mug/ml and 0.66 mug/ml respectively, and the corresponding signal-to-noise ratio is 8, 12, 9 and 6 respectively.
(2) Line survey
Taking the cyclosporine reference substance stock solution and the mixed impurity reference substance stock solution, respectively placing the two solutions into corresponding measuring bottles according to the method shown in the table 2, adding a solvent to dilute the solutions to a scale, and preparing linear test solutions with series concentrations.
TABLE 2 method of formulating the Linear test solution
Respectively and precisely measuring 10 μ l, injecting into a liquid chromatograph, and recording chromatogram. And taking the concentrations of the prepared solution and the quantitative limiting solution as abscissa and the corresponding peak area as ordinate, and performing linear regression according to a least square method. The above investigation was carried out by two persons A and B, respectively, and the test results are shown in Table 3.
TABLE 3 results of linear test investigation
The results in Table 3 show that cyclosporine and each impurity have good linear relationship in the concentration range from the quantitative limit concentration to the limit 200%.
(3) Accuracy survey
10mg of an isocyclosporin A reference substance, 5mg of an isocyclosporin H reference substance and 5mg of a cyclosporine H reference substance are precisely weighed, placed in a same 50ml measuring flask, dissolved and diluted to a scale by adding a solvent, shaken up and used as a mixed impurity reference substance stock solution. Taking 20 granules of the product, precisely weighing, pouring out contents, uniformly mixing the contents, precisely weighing a proper amount of the contents (about 40mg equivalent to cyclosporine), placing the contents in a 20ml measuring flask, respectively adding 1ml, 2ml and 3ml of mixed impurity reference substance stock solutions, adding a solvent to dilute to a constant volume to scale, and shaking up to obtain accuracy test solutions with concentration levels of 50%, 100% and 150%. Repeat the preparation of 3 parts for each concentration. Precisely measuring 1ml of the accuracy test solution, placing the accuracy test solution in a 200ml measuring flask, diluting the accuracy test solution to a scale with a solvent, and shaking up to obtain a control solution.
Respectively and precisely measuring 10 mu l of the test solution, the reference solution and the accuracy test solution, injecting the solutions into a liquid chromatograph, recording a chromatogram, and respectively calculating the content and the recovery rate of each impurity, wherein the results are shown in Table 4.
TABLE 4 accuracy test results
The results in table 4 show that the recovery of each impurity meets the specifications and the process accuracy is good.
(4) Repeatability test
Taking a proper amount of the contents (about 50mg equivalent to the cyclosporine) of the cyclosporine soft capsule, precisely weighing, placing in a 50ml measuring flask, precisely adding 5ml of mixed impurity reference substance stock solution, adding a solvent to dissolve and dilute to a scale, and shaking up to be used as a test solution. Taking 1ml of the test solution, placing the test solution in a 200ml measuring flask, adding a solvent to dilute the test solution to a scale mark, and using the test solution as a control solution. 6 parts of test solution and control solution are prepared in parallel by the same method. Respectively and precisely measuring 10 mu l of the test solution and the reference solution, injecting the solutions into a liquid chromatograph, recording a chromatogram, and calculating RSD of each impurity result and 6 parts of results, wherein the results are detailed in a table 5.
TABLE 5 results of the repeatability tests
| Sample numbering | 1 | 2 | 3 | 4 | 5 | 6 | RSD(％) |
| Isocyclosporin H (%) | 0.41 | 0.43 | 0.42 | 0.42 | 0.42 | 0.42 | 1.6 |
| Isocyclosporin A (%) | 1.23 | 1.29 | 1.26 | 1.27 | 1.25 | 1.26 | 1.6 |
| Cyclosporin H (%) | 0.49 | 0.51 | 0.50 | 0.51 | 0.50 | 0.50 | 1.6 |
| Total impurities (%) | 0.19 | 0.20 | 0.19 | 0.18 | 0.19 | 0.21 | 5.4 |
As can be seen from Table 5, the method is excellent in reproducibility.
(5) Intermediate precision
Experiments were performed by different experimenters on different dates according to the method under the repeatability test item, and the measurement was performed by using different instruments. Test data and results are recorded, and intermediate precision is examined by combining 12 results of repeatability tests, and the results are shown in table 6.
TABLE 6 intermediate precision test results
As is clear from Table 6, the method of the present invention is excellent in intermediate accuracy.
(6) Stability of solution
Taking 1 part of the test solution and the control solution under the repeatability item, and respectively measuring at room temperature for 0 hour, 12 hours, 24 hours and 48 hours. Calculating the RSD of the main peak area of the control solution and the RSD of the impurity result of the test solution at different time points, and the results are shown in a table 7.
TABLE 7 solution stability test results
FIG. 7 shows that the test solution and the control solution are stable at room temperature for 48 hours.
(7) Durability
Taking a test solution and a reference solution under a repeatability test item, changing a pair of single parameters of flow rate, column temperature, buffer solution pH, detection wavelength and flow phase ratio one by one on the basis of standard parameters according to table 8, carrying out sample injection detection, recording a chromatogram, and inspecting the influence on detection after chromatographic condition change, wherein the results are shown in tables 9 and 10.
TABLE 8 durability test parameter variation chart
| Variable factors | Flow rate of flow | Column temperature | Buffer pH | Detection wavelength | Flow phase ratio |
| Standard parameter | 1.0mL/min | 75℃ | 1.95 | 220nm | 45:55 |
| |
0.95mL/min | 74℃ | 1.90 | 218nm | 44:56 |
| |
1.05mL/min | 76℃ | 2.00 | 222nm | 46:54 |
TABLE 9 durability test results I
TABLE 10 durability test results II
The results show that after each parameter is changed within a certain range, the separation degree of each impurity in the system adaptability test is in accordance with the specification, and the result of each impurity has no obvious difference with the result before the parameter is changed.
Examples 1 to 4:
a method for measuring substances related to ciclosporin soft capsules specifically comprises the following steps:
(1) preparation of a solvent: tetrahydrofuran and water are mixed according to the volume ratio of 4:1 to prepare the solvent.
(2) Preparing a blank auxiliary material solution: taking 950mg of the liquid medicine auxiliary material and 600mg of the capsule shell auxiliary material, placing the liquid medicine auxiliary material and the capsule shell auxiliary material into a 50ml measuring flask, adding a proper amount of solvent, shaking strongly for about 5-10 minutes, and diluting the liquid medicine auxiliary material and the capsule shell auxiliary material to the scale with the solvent to obtain a blank auxiliary material solution.
(3) Preparing a test solution: 20 pieces of the product of different production batches (V191201, V191202, V191203, and SRW 94) were taken as sample solutions in examples 1 to 4, and the contents were poured out, mixed uniformly, weighed precisely to an appropriate amount (about 100mg equivalent to cyclosporin), placed in a 50ml measuring flask, diluted to the scale with a solvent, and shaken to obtain a sample solution.
(4) Preparation of control solution: precisely measuring 1ml of the test solution, placing the test solution in a 200ml measuring flask, diluting the test solution to a scale with a solvent, and shaking up to obtain a control solution.
(5) Preparation of a resolution solution: 5mg of cyclosporine reference substance, 5mg of cyclosporine H reference substance, 5mg of isocyclosporine A reference substance and 5mg of isocyclosporine H reference substance are placed in the same 200ml measuring flask, dissolved by adding a solvent and fixed to the scale, and shaken up to be used as a resolution solution.
(6) Chromatographic conditions and system adaptability:
octadecylsilane chemically bonded silica is used as a filler of a chromatographic column (a chromatographic column with specification of Kromasil 100-5, C18, 4.6mm multiplied by 250mm, 5 mu m or equivalent performance) and a stainless steel tube with the thickness of 0.25mm multiplied by 1m (or a column incubator with a preheating module is used) is connected between the chromatographic column and a sample injection valve and is used as a heat exchanger; the column temperature is 75 ℃, the detection wavelength is 220nm, tetrahydrofuran is taken as a mobile phase A, tetrahydrofuran-0.01 mol/L disodium hydrogen phosphate mixed solution (the pH is adjusted to 1.95 +/-0.05 by adopting phosphoric acid) in a volume ratio of 45:55 is taken as a mobile phase B, and elution is carried out according to the following gradient. And (3) taking 10 mu l of each of the resolution determination reference substance solution, the blank auxiliary material solution and the reference solution, injecting the reference solution into a liquid chromatograph, repeatedly injecting the reference solution for 6 times, and adjusting a chromatographic system to ensure that the resolution of the isocyclosporin A and the isocyclosporin H is not less than 1.2, the resolution between other known impurity peaks is not less than 1.5, and the RSD of the reference solution in the area of 6 continuous injection peaks is not more than 5.0%. The blank auxiliary material peak can not interfere the main cyclosporine peak.
(7) And (3) determination: precisely measuring 10 μ l of each of the test solution and the control solution, injecting into a liquid chromatograph, and recording chromatogram. The respective impurity results were calculated as follows:
In the formula
F: correction factors (1.2 for isocyclosporin A, 1.4 for isocyclosporin H, 1.0 for other impurities)
AU: peak area of a single impurity in a test solution;
AS: area of cyclosporine peak in control solution.
(8) And (3) measuring results: the results are shown in Table 11.
TABLE 11 measurement results of cyclosporin soft capsules of examples 1 to 4
The control limit of the cyclosporin soft capsule, i.e. the isocyclosporin H, the cyclosporin H and the maximum unknown single impurity is 0.5%, and the control limit of the sum of the isocyclosporin A and the unknown impurity is 1.0%. As is clear from Table 10, the substances of each of the 4 batches of cyclosporin soft capsules of examples 1 to 4 were detected effectively within the control limits.
Example 5
S1, chromatographic conditions: octadecylsilane chemically bonded silica gel is used as filler of chromatographic column (specification of the chromatographic column is Kromasil 100-5, C18, 4.6mm × 250mm, 5 μm), and a stainless steel tube of 0.25mm × 1m (or column incubator with preheating module) is connected between the chromatographic column and the sample injection valve to serve as heat exchanger; the column temperature is 75 ℃, the detection wavelength is 220nm, tetrahydrofuran is used as a mobile phase A, the pH value is adjusted to 1.95 +/-0.05 by adopting phosphoric acid, tetrahydrofuran-0.01 mol/L disodium hydrogen phosphate mixed solution according to the volume ratio of 45:55 is used as a mobile phase B, and elution is carried out according to the following gradient, wherein the sample amount is 10 mu L.
S2, solution preparation: a) tetrahydrofuran and water are mixed according to the volume ratio of 4:1 to prepare the solvent. b) Taking 950mg of the liquid medicine auxiliary material and 600mg of the capsule shell auxiliary material according to the formula proportion, placing the liquid medicine auxiliary material and the capsule shell auxiliary material in a 50ml measuring flask, diluting the liquid medicine auxiliary material to a scale with a solvent, and shaking the liquid medicine auxiliary material and the capsule shell auxiliary material strongly for about 5-10 minutes to obtain a blank auxiliary material solution. c) Taking 5mg of cyclosporine reference substance, 5mg of cyclosporine H reference substance, 5mg of isocyclosporine A reference substance and 5mg of isocyclosporine H reference substance in the same 200ml measuring flask, adding a solvent to dissolve and dilute to a scale, shaking up to be used as a resolution solution.
S3, specificity: precisely measuring blank adjuvant solution and separation degree solution 10 μ l respectively, and sequentially sampling and detecting, wherein the result is shown in FIG. 16. The results in FIG. 16 show that all adjuvant components in the blank adjuvant solution can be eluted in time without interfering with the detection of the next injection.
S4, detection limit and quantification limit: accurately measuring appropriate amounts of an isocyclosporin A stock solution, an isocyclosporin H stock solution and a cyclosporine H stock solution respectively, dissolving and diluting the solutions by using a solvent, and preparing a mixed impurity reference solution; and diluting the solution with a blank auxiliary material step by step, and injecting a sample to detect and observe the signal to noise ratio of each impurity peak. And when the signal-to-noise ratio is larger than or equal to 10, taking the quantitative limiting solution as the quantitative limiting solution, carrying out continuous sample introduction 6 times, and inspecting the RSD and the signal-to-noise ratio of each sample introduction detection peak area of each component. Precisely measuring 3ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding the blank auxiliary material solution to dilute to a scale, and shaking up to be used as the detection limiting solution.
Comparative example 1:
the method disclosed in the 'HPLC method for measuring the content of cyclosporine capsules and soft capsules and related substances' published by Pongwen philosophy and the like is taken as a comparative example 1, and the quantitative limit and the detection limit concentration are compared with those in example 5.
The specific operation of comparative example 1 is as follows:
s1, chromatographic conditions: octadecylsilane chemically bonded silica gel is used as filler (250mm × 4.6mm, 5 μm), and a stainless steel tube with diameter of 0.25mm and length of 1m is connected between the chromatographic column and the sample injection valve as heat exchanger (or column oven with preheating module is used); taking tetrahydrofuran-0.05 mol/L phosphoric acid solution (45:55) as a mobile phase; the temperature of the stainless steel pipe and the column temperature are both 75 ℃; the detection wavelength is 220 nm; the sample volume was 10. mu.l.
S2, solution preparation:
a) tetrahydrofuran and water were mixed in a volume ratio of 4:1 as a solvent.
b) Taking 950mg of the liquid medicine auxiliary material and 600mg of the capsule shell auxiliary material according to the formula proportion, placing the liquid medicine auxiliary material and the capsule shell auxiliary material in a 50ml measuring flask, diluting the liquid medicine auxiliary material to a scale with a solvent, and shaking the liquid medicine auxiliary material and the capsule shell auxiliary material strongly for about 5-10 minutes to obtain a blank auxiliary material solution.
c) Taking 5mg of cyclosporine reference substance, 5mg of cyclosporine H reference substance, 5mg of isocyclosporine A reference substance and 5mg of isocyclosporine H reference substance in the same 200ml measuring flask, adding a solvent to dissolve and dilute to a scale, shaking up to be used as a resolution solution.
S3, specificity: precisely measuring blank adjuvant solution, solvent, and separation degree solution 10 μ l, and sequentially sampling to detect, the result is shown in FIG. 17. The result of fig. 17 shows that part of the adjuvant components in the blank adjuvant solution are not eluted in time and show peaks when the solvent is injected and run, and the adjuvant peaks which are not eluted in time can interfere the detection of cyclosporine H according to the position of the peaks appearing in the separation degree solution.
S4, detection limit and quantification limit: accurately measuring appropriate amounts of an isocyclosporin A stock solution, an isocyclosporin H stock solution and a cyclosporine H stock solution respectively, dissolving and diluting the solutions by using a solvent, and preparing a mixed impurity reference solution; and diluting the solution with a blank auxiliary material step by step, and injecting a sample to detect and observe the signal to noise ratio of each impurity peak. And when the signal-to-noise ratio is larger than or equal to 10, taking the quantitative limiting solution as the quantitative limiting solution, carrying out continuous sample introduction 6 times, and inspecting the RSD and the signal-to-noise ratio of each sample introduction detection peak area of each component. Precisely measuring 3ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding the blank auxiliary material solution to dilute to a scale, and shaking up to be used as the detection limiting solution.
The results are shown in Table 12.
TABLE 12 comparison of detection limits for comparative example 1 and example 5
The results in Table 12 show that the method of the present invention has significantly lower limits of quantitation and detection of isocyclosporin H and isocyclosporin A and more sensitive analytical method than the method of comparative example 1.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (10)
1. A method for measuring related substances of a ciclosporin soft capsule is characterized in that: the determination is carried out by adopting high performance liquid chromatography, and the high performance liquid chromatography comprises the following chromatographic conditions:
a chromatographic column: octadecylsilane chemically bonded silica is used as a chromatographic column filler;
mobile phase: and (3) taking tetrahydrofuran as a mobile phase A, taking a mixed solution containing tetrahydrofuran and a disodium hydrogen phosphate aqueous solution as a mobile phase B, and performing gradient elution when the pH value of the mobile phase B is 1.9-2.1.
2. The method for measuring substances related to cyclosporin soft capsules according to claim 1, wherein: the procedure for the gradient elution was as follows: the volume percentage of the mobile phase B is maintained at 100 percent in 0-40 min; in 40-55 min, the volume percent of the mobile phase A is changed from 0% to 70-90%, and the volume percent of the mobile phase B is changed from 100% to 10-30%; at 56min-65min, the volume percentage of the mobile phase A is 0%, and the volume percentage of the mobile phase B is maintained at 100%.
3. The method for measuring substances related to cyclosporin soft capsules according to claim 1, wherein: the elution procedure for the mobile phase was as follows:
4. the method for measuring substances related to cyclosporin soft capsules according to claim 1, wherein: the volume ratio of tetrahydrofuran to disodium hydrogen phosphate in the mobile phase B is 44-46: 54-56; the concentration of the disodium hydrogen phosphate is 0.01 mol/L.
5. The method for measuring substances related to cyclosporin soft capsules according to claim 1, wherein: the conditions for performing high performance liquid chromatography analysis and detection are as follows: the detection wavelength is 218-222 nm, the column temperature is 74-76 ℃, the flow rate is 0.95-1.05 ml/min, and the sample injection amount is 10 mu l.
6. The method for measuring substances related to cyclosporin soft capsules according to claim 1, wherein: the method comprises the following steps:
s1, preparing a blank auxiliary material solution: taking appropriate amount of medicinal liquid adjuvant and capsule shell adjuvant, adding solvent, shaking strongly to dissolve, and diluting to obtain blank adjuvant solution containing medicinal liquid adjuvant 19mg and capsule shell adjuvant 12mg per 1 ml;
s2, preparation of a test solution: taking a proper amount of the contents of the ciclosporin soft capsule, precisely weighing, adding a solvent, and shaking uniformly to prepare a test solution containing 2mg of ciclosporin per 1 ml;
S3, preparation of a control solution: precisely measuring 1ml of the test solution, adding a solvent, and shaking uniformly to prepare a control solution containing 10 micrograms of cyclosporine per 1 ml;
s4, preparation of a separation degree solution:
taking appropriate amount of cyclosporin reference substance, cyclosporin H reference substance, isocyclosporin A reference substance, and isocyclosporin H reference substance, dissolving with solvent, and diluting to obtain a separation degree solution containing 25 μ g of cyclosporin, 25 μ g of cyclosporin H, 25 μ g of isocyclosporin A, and 25 μ g of isocyclosporin H per 1 ml;
s5, detection: respectively and precisely measuring 10 mu L of the blank auxiliary material solution, the separation degree solution, the test solution and the comparison solution, respectively injecting into a liquid chromatograph, recording a chromatogram, comparing the areas of impurity peaks of the test solution and main peaks of the comparison solution, and calculating the content of each related substance according to a self comparison method of adding correction factors.
7. The method for measuring substances related to cyclosporin soft capsules according to claim 6, wherein: the strong shaking time is 5-10 min.
8. The method for measuring substances related to cyclosporin soft capsules according to claim 6, wherein: the solvent is a mixed solution formed by mixing tetrahydrofuran and water according to the volume ratio of 4: 1.
9. The method for measuring substances related to cyclosporin soft capsules according to claim 6, wherein: the calculation formula of the self-comparison method is as follows: the content of related substances is F x [ A ]U/(AS×200)]X 100% where AUIs the peak area of each impurity in the test solution, ASThe peak area of cyclosporine in the control solution is shown, and F is the correction factor.
10. The method for measuring substances related to cyclosporin soft capsules according to claim 9, wherein: the correction factors of the cyclosporine and the cyclosporine H are 1.0, the correction factor of the isocyclosporine A is 1.2, and the correction factor of the isocyclosporine H is 1.4.
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| CN115266996A (en) * | 2022-08-02 | 2022-11-01 | 兆科（广州）眼科药物有限公司 | Analysis method and application of related substances in cyclosporin A preparation |
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