CN116466016B

CN116466016B - Method for measuring content of related substances in calcitriol oral liquid

Info

Publication number: CN116466016B
Application number: CN202310438626.XA
Authority: CN
Inventors: 张春利; 张丽敏; 李洁; 孙青华; 张佳鸿; 王贤贤; 王志; 刘振飞; 吕仁雪; 臧云龙
Original assignee: CP Pharmaceutical Qingdao Co Ltd
Current assignee: CP Pharmaceutical Qingdao Co Ltd
Priority date: 2023-04-21
Filing date: 2023-04-21
Publication date: 2023-09-15
Anticipated expiration: 2043-04-21
Also published as: CN116466016A

Abstract

The invention relates to the technical field of medicine analysis, and discloses a method for measuring the content of related substances in calcitriol oral liquid, wherein the method is carried out by combining a high performance liquid chromatography; the chromatographic column is a silica gel column; the mobile phase is ethanol, n-hexane and isopropanol with the volume ratio of 40:60:1, and the flow rate is 0.8-1.2ml/min. According to the invention, the content of related substances in calcitriol oral liquid can be effectively measured under specific high performance liquid chromatography conditions, and the accuracy and precision of the measurement result are good.

Description

Method for measuring content of related substances in calcitriol oral liquid

Technical Field

The invention relates to the technical field of medicine analysis, in particular to a method for measuring the content of related substances in calcitriol oral liquid.

Background

Calcitriol (Calcitriol) is also known as: 1 alpha, 25-dihydroxyvitamin D3, calcitriol, is one of the most important active metabolites of vitamin D3, is usually converted in the kidney from its precursor 25-hydroxyvitamin D3, is produced in a daily amount of 0.5-1.0 microgram in normal physiology, and is produced in a slightly increased period of bone synthesis. Calcitriol can promote the absorption of calcium by intestinal tracts, regulate the mineralization of bones, inhibit the proliferation of keratinocytes, promote the differentiation of keratinocytes, inhibit the proliferation of T-lymphocytes, and regulate the secretion of different inflammatory response factors to normal level.

Calcitriol products generally comprise calcitriol capsules and calcitriol oral liquid, but in the existing calcitriol product determination method, except for the calcitriol precursor determination method in the calcitriol capsule quality standard, no other relevant quality standard exists in the calcitriol product determination method, and in particular, the calcitriol oral liquid related substance determination method is used.

Disclosure of Invention

In view of the above, the invention provides a method for measuring the content of related substances in calcitriol oral liquid.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a method for measuring the content of related substances in calcitriol oral liquid is carried out by combining high performance liquid chromatography;

the chromatographic column is a silica gel column;

the mobile phase is ethanol, n-hexane and isopropanol with the volume ratio of 40:60:1, and the flow rate is 0.8-1.2ml/min.

Preferably, in the method for measuring the content of related substances in calcitriol oral liquid, the diameter of the chromatographic column is 2-10mm, the column length is 150-250mm, and the diameter of the filler in the chromatographic column is 2-10 mu m; further preferably, the diameter of the chromatographic column is 4.6mm, the column length is 250mm, and the diameter of the packing in the chromatographic column is 5 mu m.

Preferably, in the method for determining the content of the related substances in calcitriol oral liquid, the elution mode in the high performance liquid chromatography is isocratic elution.

Preferably, in the method for determining the content of related substances in calcitriol oral liquid, the column temperature in the high performance liquid chromatography is 25-35 ℃.

Preferably, in the method for measuring the content of related substances in calcitriol oral liquid, the detection wavelength in the high performance liquid chromatography is 260-270nm.

Preferably, in the method for determining the content of the related substances in the calcitriol oral liquid, the sample injection amount in the high performance liquid chromatography is 500 μl.

Preferably, in the method for determining the content of the related substances in the calcitriol oral liquid, the related substances comprise calcitriol precursors and degradation impurities.

Preferably, in the method for determining the content of related substances in calcitriol oral liquid, the degradation impurities include an impurity A and an impurity B;

the impurity A is shown in a chemical formula I, and the impurity B is shown in a chemical formula II:

Ⅰ Ⅱ。

preferably, in the method for measuring the content of related substances in calcitriol oral liquid, the method comprises the following steps:

(1) Preparation of test solution: adding calcitriol and an appropriate amount of impurity A, impurity B and calcitriol precursor into the solution with calcitriol concentration of 1 mug/ml, impurity A of 0.01 mug/ml, impurity B of 0.01 mug/ml and calcitriol precursor of 0.1 mug/ml, and obtaining a sample solution;

(2) Preparation of stock solution: dissolving calcitriol and impurity A, impurity B and calcitriol precursor in mobile phase, and diluting to obtain calcitriol stock solution, calcitriol precursor stock solution, impurity A stock solution and impurity B stock solution;

(3) Precisely measuring 500 mu L of the sample solution, the calcitriol stock solution, the calcitriol precursor stock solution, the impurity A stock solution and the impurity B stock solution, injecting into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of related substances by a peak area normalization method.

Preferably, in the method for determining the content of related substances in calcitriol oral liquid, the preparation method of the calcitriol stock solution comprises the following steps: precisely weighing calcitriol reference substance, adding mobile phase to dissolve and dilute to obtain 5 μg/ml solution as calcitriol stock solution;

the preparation method of the calcitriol precursor stock solution comprises the following steps: precisely weighing a proper amount of calcitriol precursor reference substance, adding a mobile phase to dissolve and dilute the calcitriol precursor reference substance to prepare a solution of 1 mug/ml, and taking the solution as a calcitriol precursor stock solution;

the preparation method of the impurity A stock solution comprises the following steps: precisely weighing a proper amount of impurity A reference substance, adding a mobile phase to dissolve and dilute the impurity A reference substance to prepare a solution with the concentration of 0.1 mug/ml, and taking the solution as an impurity A stock solution;

the preparation method of the impurity B stock solution comprises the following steps: accurately weighing a proper amount of impurity B reference substance, adding a mobile phase to dissolve and dilute the impurity B reference substance to prepare a solution with the concentration of 0.1 mug/ml serving as an impurity B stock solution.

Preferably, in the method for measuring the content of related substances in calcitriol oral liquid, the limit of each impurity in calcitriol oral liquid is as follows: impurity A is less than or equal to 1.0%; impurity B is less than or equal to 1.0%; calcitriol precursor is less than or equal to 5.0 percent; unknown impurities are less than or equal to 1.0 percent; the total impurity is less than or equal to 7.0 percent.

The invention provides a method for measuring the content of related substances in calcitriol oral liquid, which has the beneficial effects that compared with the prior art:

according to the invention, the content of related substances in calcitriol oral liquid can be effectively measured under specific high performance liquid chromatography conditions, and the accuracy and precision of the measurement result are good.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a chromatogram of a related substance in calcitriol oral liquid of test example 1;

FIG. 2 is a chromatogram of related substances in calcitriol oral liquid of test example 2;

FIG. 3 is a spectral diagram of calcitriol;

FIG. 4 is a spectral diagram of calcitriol precursors;

FIG. 5 is a spectrum of impurity A;

FIG. 6 is a spectrum of impurity B;

FIG. 7 is a spectrum of impurity D;

FIG. 8 is a chromatogram of a white solvent related substance in a blank interference test;

FIG. 9 is a chromatogram of a blank adjuvant related substance in a blank interference test;

FIG. 10 is a chromatogram of BHA related materials in a blank interference test;

FIG. 11 is a chromatogram of a BHT-related substance in a blank interference test;

FIG. 12 is a chromatogram of MCT-related material in a blank interference test;

FIG. 13 is a chromatogram of calcitriol-related substance in a blank interference test;

FIG. 14 is a chromatogram of an impurity A related substance in a blank interference test;

FIG. 15 is a chromatogram of an impurity B-related substance in a blank interference test;

FIG. 16 is a chromatogram of an impurity C related substance in a blank interference test;

FIG. 17 is a chromatogram of an impurity D-related substance in a blank interference test;

FIG. 18 is a chromatogram of calcitriol precursor related substance in a blank interference assay;

FIG. 19 is a chromatogram of a substance involved in system adaptation in a blank interference test;

FIG. 20 is a chromatogram of a substance associated with a self-made sample (lot 1908021) in a blank interference test;

FIG. 21 is a chromatogram of a reference formulation (lot RV 1711B) for a blank interference test;

FIG. 22 is a standard graph of impurity A;

FIG. 23 is a standard graph of impurity B;

FIG. 24 is a standard graph of calcitriol precursors;

FIG. 25 is a graph showing the effect of different flow rate changes on separation;

FIG. 26 is a graph showing the effect of different column temperature changes on the degree of separation;

FIG. 27 is a graph showing the effect of different wavelength variations on the degree of separation;

FIG. 28 is a graph comparing the effect of column variation of different brands on separation;

FIG. 29 is a graph showing the effect of different batches of columns on separation.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

1. Solution preparation

Blank solvent: ethyl acetate-n-hexane-methanol (60:60:1).

Test solution: calcitriol oral liquid is used as a test solution.

Blank auxiliary material solution: and weighing medium chain triglyceride, BHA and BHT according to the prescription, preparing a solution serving as a blank auxiliary material, and shaking uniformly to obtain the compound.

System adaptation solution: mixing calcitriol reference substance with impurity A, impurity B and calcitriol precursor, and adding mobile phase to obtain calcitriol solution with calcitriol concentration of 1 μg/ml, impurity A of 0.01 μg/ml, impurity B of 0.01 μg/ml and calcitriol precursor of 0.1 μg/ml.

Calcitriol stock solution: precisely weighing calcitriol reference substance, adding mobile phase to dissolve and dilute to obtain solution containing about 5 μg per 1ml, and using as calcitriol stock solution.

Calcitriol precursor stock solution: precisely weighing a proper amount of calcitriol precursor reference substance, adding a mobile phase to dissolve and dilute to prepare a solution containing about 1 mug of calcitriol precursor per 1ml, and taking the solution as a calcitriol precursor stock solution.

Impurity a stock solution: accurately weighing a proper amount of the impurity A reference substance, adding a mobile phase to dissolve and diluting to prepare a solution containing about 0.1 mug of the impurity A reference substance per 1ml, and taking the solution as an impurity A stock solution.

Impurity B stock solution: accurately weighing a proper amount of the reference substance of the impurity B, adding a mobile phase to dissolve and dilute the reference substance to prepare a solution containing about 0.1 mug of the reference substance of the impurity B in 1ml, and taking the solution as a stock solution of the impurity B.

Impurity C stock solution: accurately weighing a proper amount of impurity D reference substance, adding mobile phase to dissolve and dilute to obtain a solution containing about 0.1 mug of impurity D reference substance per 1ml, and taking the solution as an impurity A stock solution.

Impurity D stock solution: accurately weighing a proper amount of the impurity D reference substance, adding a mobile phase to dissolve and dilute the impurity D reference substance to prepare a solution with about 0.1 mug of each 1ml serving as an impurity B stock solution.

Wherein, the specification of calcitriol reference substance is 10 mg/branch, 99.9%, the batch number is 11.0, and the source is EP; the calcitriol precursor has the specification of 25 mg/branch, 99.48 percent, the lot number of HT013-45-20200401 and the source of Gansu Haoda medical technology; the specification of the impurity A reference substance is 10mg,99.51%, the batch number is 01-MAY-1726, and the source is Shenzhen Yuanyang medical science and technology Co., ltd; the specification of the impurity B reference substance is 10mg,97.25%, the batch number is 02-Apr-1714, and the source is Shenzhen Yuanyang medical science and technology Co., ltd; the specification of the impurity C reference substance is 10mg,97.89%, the batch number is 03-APR-1913, and the source is Shenzhen Yuanyang medical science and technology Co., ltd; the specification of the impurity D reference substance is 10mg,97.60%, the batch number is 04-APR-1910, and the impurity D reference substance is Shenzhen Yuan Yangzhi medicine technology Co.

2. Determination of conditions for high performance liquid chromatography

Test example 1

The conditions of the high performance liquid chromatography are as follows: an insertsustein C8 (150×4.6mm,3 μm) chromatographic column is adopted, a mobile phase A is a trimethylol aminomethane solution, a mobile phase B is acetonitrile, the column temperature is 40 ℃, the flow rate is 1.0ml/min, the detection wavelength is 265nm, the elution mode is gradient elution, and the specific process of gradient elution is shown in Table 1:

preparation of mobile phase A: weighing 1g of tris (hydroxymethyl) aminomethane solution, adding 1L of purified water for dissolving and filtering, and adding a phosphoric acid solution to adjust the pH value to 7.0-7.5;

adding known impurity A and impurity B into the solution of the sample, recording the chromatograms as shown in figure 1, and respectively and sequentially making peaks of calcitriol precursor, impurity A, calcitriol and impurity B at 15-17min, wherein the separation degree of the impurity A from calcitriol is about 1.7, but the separation degree of the impurity B from calcitriol is about 1.2, which does not meet the separation requirement.

After gradient optimization is continued, the separation degree of the impurity B and calcitriol still does not reach the ideal requirement, so that the separation degree is not further optimized.

Test example 2

The conditions of the high performance liquid chromatography are as follows: an OSAKA SG120 (250 multiplied by 4.6mm,5 mu m) chromatographic column with silica gel as a filling agent is adopted, the mobile phase is a mixed solution of ethyl acetate, normal hexane and methanol with the volume ratio of 60:60:1, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the detection wavelength is 265nm, the sample injection amount is 100 mu L, and the elution mode is isocratic elution.

As shown in fig. 2, calcitriol has a peak time of about 24min, calcitriol precursor has a peak time of about 36min, impurity a has a peak time of about 25min, impurity B has a peak time of about 20min, impurity C cannot be detected, impurity D has a peak time of about 24min, and impurity D is not overlapped with calcitriol under the chromatographic condition and is not separated; the impurity C and the impurity D are process impurities, the impurity C and the impurity D are not generated in the synthesis process by the synthesis process, and the calcitriol raw material method is used for controlling the impurity C and the impurity D, so that further research is not performed under the relevant material item of the preparation, and the analysis of relevant substances in the sample is not interfered by a blank solvent, a blank auxiliary material and the like.

Test example 3

The conditions of the high performance liquid chromatography are as follows: an OSAKA SG120 (250 multiplied by 4.6mm,5 mu m) chromatographic column with silica gel as a filling agent is adopted, the mobile phase is a mixed solution of ethyl acetate, normal hexane and methanol with the volume ratio of 60:60:1, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the detection wavelength is 265nm, the sample injection amount is 500 mu L, and the elution mode is isocratic elution.

The sample injection amount of the sample to be tested is increased from 100 mu L to 500 mu L, so that the detection sensitivity can be improved, and the method has good applicability.

3. Analytical method validation

1. Specialization of

1.1 full wavelength scanning

Precisely measuring calcitriol control, calcitriol precursor, impurity A, impurity B, impurity C and impurity D control solution, injecting into liquid chromatograph, and recording chromatogram with DAD detector, wherein the results are shown in Table 2 and figures 3-7:

note that: the impurity C does not show a peak in the system, and because the impurity C is the addition product of calcitriol, the impurity D is methylated calcitriol and is not degradation product, and the impurity C is not generated in the storage process, only positioning is carried out, and no further intensive research is carried out in the invention.

The results show that calcitriol, calcitriol precursor and impurities A, B and D have a maximum absorption substantially in the range 249-265nm, so that the detection wavelength is chosen to be 265nm for detection.

1.2 Blank interference test

Respectively precisely measuring a blank solvent, a blank auxiliary material solution, each impurity reference substance solution and a test sample solution, respectively injecting into a liquid chromatograph, recording a chromatogram, and referring to table 3 and fig. 8-21:

as can be seen from table 3 and fig. 8 to 21, the blank solvent and the blank auxiliary material have no interference to the analysis of calcitriol related substances in the product, the impurity C has no peak under the condition, the impurity D has a peak overlapping with calcitriol, and the impurity C and the impurity D are neither degraded nor detected in the raw materials, so that no further study is performed in the study of the calcitriol related substances in the product. Therefore, under the condition, the solvent and each auxiliary material do not interfere with the determination of calcitriol precursor, impurity A and impurity B.

2. System applicability

Taking a system applicability solution, continuously injecting 6 needles according to a method, recording a chromatogram, and referring to tables 4-5 for results:

as shown in tables 4-5, after the system adaptive solution is continuously injected for 6 times, the separation degree of calcitriol and adjacent impurity B is more than 1.5, the theoretical plate number is more than 2000, and the tailing factors meet the requirements. Indicating that the system has good adaptability.

3. Detection limit

Taking each impurity reference substance solution, sequentially diluting and preparing standard solutions with serial concentrations, sequentially sampling, calculating according to signal/noise=3:1, namely, the detection limit concentration, continuously sampling for 5 times, wherein the concentration of the sample solution is 1 mug/ml, and the results are shown in Table 6:

4. quantitative limit

Taking each impurity reference substance solution, sequentially diluting and preparing standard solutions with serial concentrations, sequentially sampling, calculating according to signal/noise=10:1 to obtain quantitative limiting concentration, preparing 6 parts of quantitative limiting concentration solutions in parallel, and sequentially sampling, wherein the results are shown in Table 7:

as is clear from Table 7, the retention time RSD% of each substance in the six quantitative limiting solutions was less than 1%, and the RSD% of the peak area was less than 16%, which meets the regulations.

5. Linearity and range

Taking a proper amount of impurity A, impurity B and calcitriol precursor reference substance stock solution, and establishing quantitative limit-200% linear investigation on the basis of the limit. Preparing a series of impurity reference substance linear solutions, precisely measuring 500 mu l of each linear solution, injecting the solutions into a liquid chromatograph, recording a chromatogram, taking the sample injection concentration (mu g/ml) as an X axis, taking the peak area as a Y axis, and performing linear regression, wherein the results are shown in Table 8 and FIGS. 22-24:

as can be seen from table 8 and fig. 22 to 24, the concentration of impurity a in the range of 0.00376 μg/ml to 0.02508 μg/ml (corresponding to 30 to 200% of the concentration of the sample solution), the concentration of impurity B in the range of 0.00593 μg/ml to 0.02372 μg/ml (corresponding to 50 to 200% of the concentration of the sample solution), and the concentration of calcitriol precursor in the range of 0.01682 μg/ml to 0.2242 μg/ml (corresponding to 15 to 200% of the concentration of the sample solution) have a significant linear relationship with the peak area, and the linear equations are y=1550766.06x+310.28, respectively, and γ=1; y=702693x+545.74, γ= 0.9932; and y= 510343x-2756.1, γ= 0.9986.

6. Precision of

Precisely measuring the impurity A, the impurity B and a calcitriol precursor stock solution with proper amounts, preparing into impurity A with the concentration of 0.01 mug/ml, impurity B with the concentration of 0.01 mug/ml and calcitriol precursor reference substance solution with the concentration of 0.1 mug/ml, continuously injecting the sample for 6 times, recording a chromatogram, and obtaining a result shown in Table 9:

as shown in Table 9, the sample was continuously fed 6 times, the retention time RSD in the control solution was less than 1.0%, the peak area RSD was less than 8.0%, and the precision was good.

7. Sample reproducibility

Calcitriol oral liquid is taken, the calcitriol oral liquid is measured by high performance liquid chromatography, related substances are calculated according to a normalization method, and the results are shown in table 10:

as is clear from Table 10, the analytical method according to the present invention was excellent in reproducibility.

8. Solution stability

The impurity A, the impurity B reference substance solution and the sample solution to be tested are respectively taken and placed in a sample injector (15 ℃), and are respectively sampled and measured at 0, 3, 6, 9, 12, 20, 24 and 40 hours, and the stability of the solutions is examined, and the results are shown in Table 11.

As shown in Table 11, the peak areas RDS of the reference solutions of the impurity A and the impurity B are less than 6% after the reference solutions are placed for 40 hours at 15 ℃, the impurity deviation in the reference solutions is not more than +/-0.2%, and the stability of the solutions is good within 40 hours;

the calcitriol precursor has a main peak area obviously exceeding 5% after being placed for 6 hours at 15 ℃, and the peak area is in a descending trend along with the time, so that new preparation is needed, and the calcitriol precursor is used within 2 hours.

9. Durability of

Taking system adaptability and sample solution, properly adjusting the flow rate, wavelength, column temperature of a mobile phase, replacing different chromatographic columns and the like, and examining the separation condition of the system adaptability solution after the chromatographic conditions are changed, wherein the result profile is shown in table 12:

(1) Flow rate variation

The solution flow rates were varied and the retention times and separation of each solution at different flow rates were recorded, see table 13 for results:

as is clear from Table 13, the calcitriol was separated from impurity A in a substantially uniform manner after the flow rate was changed, and the calcitriol was satisfactory.

(2) Column temperature variation

The column temperature was varied and the retention time and separation of each solution at different column temperatures were recorded, see table 14 for results:

as shown in Table 14, the column temperature was 25-35℃and the separation of calcitriol from impurity A was substantially consistent, and all of them were satisfactory.

(3) Wavelength variation

The retention times and the degree of separation of the solutions at the different column temperatures were recorded by changing the wavelength, see table 15 for results:

as shown in Table 15, the separation of calcitriol from impurity A was substantially uniform over the wavelength range of 260-270nm, and was satisfactory.

10. Sample measurement

The relevant substances were detected by high performance liquid chromatography from prepared samples and reference preparations, see table 16 for results:

as can be seen from Table 16, the self-made samples and the reference formulation were substantially identical, and all met the limit specifications.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the solution disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple, and the relevant points refer to the description of the method section.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The method for measuring the content of related substances in calcitriol oral liquid is characterized by combining high performance liquid chromatography;

the chromatographic column is an OSAKA silica gel chromatographic column with the diameter of 4.6mm, the column length of 250mm and the diameter of the packing in the chromatographic column of 5 μm;

the mobile phase is ethanol, normal hexane and methanol with the volume ratio of 40:60:1, the flow rate is 0.8-1.2ml/min, and the elution mode is isocratic elution;

the related substances comprise calcitriol precursors and degradation impurities; the degradation impurities comprise an impurity A and an impurity B, wherein the impurity A is shown in a chemical formula I, and the impurity B is shown in a chemical formula II:

Ⅰ Ⅱ。

2. the method for determining the content of related substances in calcitriol oral liquid according to claim 1, wherein the column temperature in the high performance liquid chromatography is 25-35 ℃.

3. The method for determining the content of related substances in calcitriol oral liquid according to claim 1, wherein the detection wavelength in the high performance liquid chromatography is 260-270nm.

4. The method for determining the content of related substances in calcitriol oral liquid according to claim 1, wherein the sample injection amount in the high performance liquid chromatography is 500 μl.

5. The method for measuring the content of related substances in calcitriol oral liquid according to any one of claims 1 to 4, which is characterized by comprising the following steps:

6. The method for determining the content of related substances in calcitriol oral liquid according to claim 5, wherein the preparation method of calcitriol stock solution is as follows: precisely weighing calcitriol reference substance, adding mobile phase to dissolve and dilute to obtain 5 μg/ml solution as calcitriol stock solution;