Method for detecting related substances in alfacalcidol soft capsule

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting related substances in alfacalcidol soft capsules, which comprises the following steps of: determining by adopting high performance liquid chromatography under the chromatographic conditions that: a chromatographic column adopts octadecylsilane chemically bonded silica as a packed column; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and a gradient elution mode is adopted; the detection wavelength is full wavelength scanning of 263 to 267 nm or 200 to 400 nm; the flow velocity is 1.0 to 2.0 mL / min. The method is high in precision, the recovery rate can reach 90% or below, the detection time is only 70-80 minutes, and guarantees and technical supports are provided for the quality reliability, stability and safety of the alfacalcidol soft capsules.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting related substances in alfacalcidol soft capsules. Background Art

[0002] Medicines are special substances that people use to prevent and treat diseases, and for rehabilitation and health care. They mainly have the functions of prevention, treatment, diagnosis, etc., and can effectively regulate human physiological functions and have specified indications or functional indications, usage and dosage. They include Chinese medicinal materials, important pieces of medicine, Chinese patent medicines, chemical raw materials and their preparations, antibiotics, biochemical drugs, radioactive drugs, serums, vaccines, blood products and diagnostic drugs. The quality of these drugs directly affects people's health and life. Therefore, drug quality supervision and inspection is of great significance to ensure that people use drugs safely, effectively and reasonably, and maintain people's physical health. The chemical name of alfacalcidol is (5Z, 7E)-9, 10-opencholester-5, 7, 10(19)-triene-1α, 3β-diol. Alfacalcidol can increase the reabsorption of calcium by the small intestine and renal tubules, inhibit parathyroid hyperplasia, reduce the synthesis and release of parathyroid hormone, inhibit bone resorption, increase the synthesis of transforming growth factor-B (TGF-B) and insulin-like growth factor-I (1GF-I), promote the synthesis of collagen and bone matrix protein, regulate muscle calcium metabolism, promote muscle cell differentiation, enhance muscle strength, increase neuromuscular coordination, and reduce the tendency to fall.

[0003] In the research of chemical drugs, the study of related substances is of great significance and helps to understand the quality and stability of products. However, the existing pharmacopoeias of various countries and existing literature lack the research on related substances of alfacalcidol soft capsules. The common sample preparation method has a large amount of soft capsule matrix and a large matrix effect, which affects the peak time and peak shape of the main peak and each impurity peak. Therefore, it is urgent to provide a detection method of related substances in alfacalcidol soft capsules with high reliability and accuracy. Summary of the invention

[0004] The present invention aims to provide a reverse high performance liquid chromatography analysis method for related substances of alfacalcidol soft capsules, so as to overcome the lack of related substance inspection methods of alfacalcidol soft capsules in the prior art. The common sample preparation method has a large amount of soft capsule matrix and a large matrix effect, which affects the peak time and peak shape of the main peak and each impurity peak. In order to better meet the quality control requirements of various specifications of alfacalcidol soft capsules, it is ensured that the quality of alfacalcidol soft capsules is controllable, safe and effective.

[0005] In view of the lack of research on related substances of alfacalcidol soft capsules in existing pharmacopoeias of various countries and existing literature, the reliability and accuracy of the method of the present invention are determined through exploration, optimization and methodological verification.

[0006] A method for detecting related substances of alfacalcidol soft capsules, the detection method comprising the following steps: The HPLC method was used for determination, and the chromatographic conditions were as follows: Chromatographic column: Octadecylsilane bonded silica gel is used as the filling column; Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode; Detection wavelength: 263-267nm or 200-400nm full wavelength scan; The flow rate is 1.0~2.0mL / min.

[0007] Furthermore, a gradient elution method is used, wherein the gradient elution method is:

[0008] Furthermore, the quantitative method of the related substances is area normalization method or area normalization method with correction factor added or self-control method or self-control method with correction factor added; the correction factor of alfacalcidol impurity A trans-alfacalcidol is 1.2, and the correction factor of alfacalcidol impurity B alfacalcidol-β isomer is 1.1.

[0009] Furthermore, the detection wavelength is 265 nm.

[0010] Furthermore, during the HPLC determination, the column temperature was controlled at 25-35°C.

[0011] Furthermore, in the HPLC determination, the chromatographic column is AQ-C18, 250 mm×4.6 mm, 5 μm.

[0012] Furthermore, the preparation method of the test solution comprises the following steps: Accurately weigh the contents of alfacalcidol soft capsules and mix them with an equal volume of n-hexane, add the mixture to a pre-activated cyano solid phase extraction column for extraction, then rinse and elute, collect the eluate, evaporate to dryness at 25°C under low-temperature vacuum rotary evaporation, dissolve in methanol and water, and finally filter, and use the filtrate as the test solution.

[0013] Furthermore, the preparation method of the control solution comprises the following steps: Accurately weigh 0.1 mL of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with methanol and water, shake well, and the solution is ready.

[0014] Further, the method for preparing the system suitability solution comprises the following steps: The trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol were accurately weighed, dissolved in a mixed solution of methanol and water, and diluted to prepare a solution containing 15 μg of alfacalcidol, 0.15 μg of the trans isomer of alfacalcidol and 0.15 μg of the 1β isomer of alfacalcidol per 1 mL, as the system suitability solution.

[0015] Further, the preparation method of the blank auxiliary material solution comprises the following steps: Accurately weigh the sum of the auxiliary materials of each component of alfacalcidol soft capsule except the main component, mix it with an equal volume of n-hexane, and add the mixture to the pre-activated solid phase extraction column for extraction, then rinse and elute, collect the eluate, evaporate to dryness at low temperature, dissolve it with methanol water, and finally filter it, and the filtrate is used as the blank auxiliary material solution.

[0016] The object of the present invention is to provide a method for detecting related substances of alfacalcidol soft capsules, the steps of which are as follows: (1) Take an appropriate amount of this product, remove the contents, combine the contents, take an appropriate amount of the contents (equivalent to 15 μg of alfacalcidol), mix with 5 mL~8 mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, pre-activate it with 5 mL~15 mL of n-hexane, pass the sample solution through the solid phase extraction column at a flow rate of about 1~3 mL / min, and discard the receiving solution; after the sample solution is completely filtered, wash the matrix on the solid phase extraction column with 5 mL~15 mL of n-hexane; take another 5 mL~15 mL of methanol to elute the column and collect the receiving solution; the receiving solution is vacuum-dried to dryness, and 1 mL of methanol-water mixed solution is taken to re-dissolve, filter, and take the filtrate to obtain the test solution; accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it with a methanol-water mixed solution and make up to the mark, shake well, and use it as the control solution.

[0017] (2) Take an appropriate amount of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol, and alfacalcidol, accurately weigh them, dissolve them in a mixed solution of methanol and water, and dilute them to make a solution containing about 15 μg of alfacalcidol, about 0.15 μg of the trans isomer of alfacalcidol, and about 0.15 μg of the 1β isomer of alfacalcidol per 1 mL, as the system practical solution; determine the solution by high performance liquid chromatography in accordance with the general rule 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, using octadecylsilane bonded silica gel as the filler of the chromatographic column, a mixed solution of acetonitrile-water as the mobile phase, the volume ratio of acetonitrile-water being 75:25, the column temperature being 25-35°C, and the detection range of the ultraviolet detector being 20°C. The measuring wavelength is 265nm or a diode array detector (200nm~400nm), and the flow rate is 1.0~2.0mL / min; 200~500μl of the system suitability solution and the control solution are accurately measured and injected into the liquid chromatograph, and the pre-alfacalcidol, the trans isomer of alfacalcidol, alfacalcidol, and the 1β isomer of alfacalcidol appear in sequence. The relative retention times of the pre-alfacalcidol, the trans isomer of alfacalcidol, and the 1β isomer of alfacalcidol relative to alfacalcidol are 0.88, 0.94, and 1.1, respectively. The separation between each impurity peak and the alfacalcidol peak meets the requirements, and the signal-to-noise ratio of the control solution should be greater than 100.

[0018] Beneficial Effects

[0019] The present invention can achieve complete separation of alfacalcidol and impurities before and after its main peak, with a minimum separation degree greater than 1.5; a blank matrix and an operation process will not interfere with the detection of impurities in alfacalcidol, and can ensure that the detection results of related substances are accurate and reliable.

[0020] This method has the following advantages: simple and convenient: the solid phase extraction device is easy to operate, does not require complex equipment and technology, and almost any laboratory can perform solid phase extraction operations; efficient and accurate: solid phase extraction can improve the separation and enrichment efficiency of samples and obtain more accurate analysis results. The solid phase extraction device can realize large-scale solid phase extraction operations, effectively saving experimental time; good repeatability: the operating steps of solid phase extraction are simple, easy to control, and can obtain good repeatability; wide scope of application: this method can also be applied to the inspection of related substances in similar soft capsule varieties.

[0021] The present invention adopts a unique method for preparing a test solution, so that low-concentration alfacalcidol soft capsules can be tested for related substances at a suitable concentration, and the impurity sensitivity can reach less than 5% of the impurity limit concentration, and the detection sensitivity is improved, so that the known impurities and unknown impurities of alfacalcidol can be effectively detected and controlled. The linear range of the method includes the quantitative limit concentration of impurities to 2 times of the limit concentration, and the detection range is wide and the applicability is strong. In addition, the method has high precision, the recovery rate can reach less than 90%, and the detection time only needs 70 to 80 minutes, which provides guarantee and technical support for the quality reliability, stability and safety of alfacalcidol soft capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the location map of impurity A of related substances in alfacalcidol soft capsules;

[0023] Figure 2 This is the location map of impurity B of related substances in alfacalcidol soft capsules;

[0024] Figure 3 This is the location map of alfacalcidol before the related substances of alfacalcidol soft capsules;

[0025] Figure 4 This is the system applicability map of related substances of Alfacalcidol Soft Capsules;

[0026] Figure 5 This is the linear regression diagram of related substances of Alfacalcidol Soft Capsules;

[0027] Figure 6 This is a comparison chart of related substances of Alfacalcidol Soft Capsules;

[0028] Figure 7 This is the spectrum of the test sample solution of related substances of Alfacalcidol Soft Capsules;

[0029] Figure 8 This is the spectrum of blank excipient solution of related substances of Alfacalcidol Soft Capsules;

[0030] Fig. 9 This is a comparative spectrum of Alfacalcidol Soft Capsule Related Substance Comparative Example 1;

[0031] Fig.10 This is a comparison chart of comparative example 2 of related substances of alfacalcidol soft capsules. DETAILED DESCRIPTION

[0032] Example 1

[0033] 1. Preparation of test solution

[0034] Take an appropriate amount of this product, remove the contents, combine the contents, take an appropriate amount of the contents (equivalent to about 15μg of alfacalcidol), mix with 5mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, rinse and activate it with 10mL of n-hexane in advance, pass the sample solution through the solid phase extraction column at a flow rate of about 1mL / min, and discard the receiving solution; after the sample solution is completely filtered, take another 10mL of methanol to elute the column and collect the receiving solution; the receiving solution is vacuum-rotated and dried to dryness, and another 1mL of methanol-water mixed solution is taken to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0035] 2. Control solution

[0036] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0037] 3. System suitability solution

[0038] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0039] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0040] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0041] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode;

[0042] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0043] Flow rate: 1.0 mL / min;

[0044] Detection time: 70 minutes;

[0045] Column temperature: 30°C;

[0046] The gradient elution method is:

[0047]

[0048] Example 2

[0049] 1. Preparation of test solution

[0050] Take an appropriate amount of this product, remove the contents, combine the contents, take an appropriate amount of the contents (equivalent to about 15μg of alfacalcidol), mix with 8mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, pre-activate it with 5mL of n-hexane, pass the sample solution through the solid phase extraction column at a flow rate of about 2mL / min, and discard the receiving solution; after the sample solution is completely filtered, take another 10mL of methanol to elute the column and collect the receiving solution; the receiving solution is vacuum-rotated and dried to dryness, and another 1mL of methanol-water mixed solution is taken to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0051] 2. Control solution

[0052] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0053] 3. System suitability solution

[0054] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0055] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0056] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0057] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, isocratic elution mode;

[0058] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0059] Flow rate: 1.0 mL / min;

[0060] Detection time: 70 minutes;

[0061] Column temperature: 30°C;

[0062] Example 3

[0063] 1. Preparation of test solution

[0064] Take an appropriate amount of this product, remove the contents, combine the contents, take an appropriate amount of the contents (equivalent to about 15μg of alfacalcidol), mix with 6mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, activate it with 8mL of n-hexane in advance, pass the sample solution through the solid phase extraction column, control the pressure of the column to be about 20MPa, and discard the receiving solution; after the sample solution is completely filtered, take another 8mL of n-hexane to clean the matrix in the solid phase column, and then elute the column with 8mL of methanol to collect the receiving solution; the receiving solution is vacuum-rotated and dried to dryness, and 1mL of methanol-water mixed solution is taken to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0065] 2. Control solution

[0066] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0067] 3. System suitability solution

[0068] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0069] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0070] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0071] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode;

[0072] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0073] Flow rate: 1.0 mL / min;

[0074] Detection time: 70 minutes;

[0075] Column temperature: 30°C;

[0076] The gradient elution method is:

[0077]

[0078] Example 4

[0079] 1. Preparation of test solution

[0080] Take an appropriate amount of this product, remove the contents, combine the contents, take an appropriate amount of the contents (equivalent to 7.5 μg of alfacalcidol), mix with 3 mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, activate it with 10 mL of n-hexane in advance, pass the sample solution through the solid phase extraction column, control the pressure of the column to be about 20 MPa, and discard the receiving solution; after the sample solution is completely filtered, take another 8 mL of n-hexane to wash the matrix in the solid phase column, and then elute the column with 8 mL of methanol to collect the receiving solution; take another cyano solid phase extraction column, repeat the above steps from "take an appropriate amount of this product, ... collect the receiving solution", combine the receiving solutions, dry the combined receiving solution by vacuum rotation until dry, take another 1 mL of methanol-water mixed solution to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0081] 2. Control solution

[0082] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0083] 3. System suitability solution

[0084] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0085] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0086] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0087] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode;

[0088] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0089] Flow rate: 1.0 mL / min;

[0090] Detection time: 70 minutes;

[0091] Column temperature: 30°C;

[0092] The gradient elution method is:

[0093]

[0094] Example 5

[0095] 1. Preparation of test solution

[0096] Take an appropriate amount of this product, remove the contents, combine the contents, take an appropriate amount of the contents (equivalent to 15μg of alfacalcidol), mix with 6mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, activate it with 5mL of n-hexane in advance, pass the sample solution through the solid phase extraction column, control the flow rate to 3 seconds / drop, and discard the receiving solution; after the sample solution is completely filtered, take another 8mL of n-hexane to clean the matrix in the solid phase column, and then elute the column with 8mL of methanol, collect the receiving solution, and dry the receiving solution by vacuum rotation until dry, take another 1mL of methanol-water mixed solution to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0097] 2. Control solution

[0098] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0099] 3. System suitability solution

[0100] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0101] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0102] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0103] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode;

[0104] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0105] Flow rate: 1.0 mL / min;

[0106] Detection time: 70 minutes;

[0107] Column temperature: 30°C;

[0108] The gradient elution method is:

[0109]

[0110] Example 6

[0111] 1. Preparation of test solution

[0112] Take an appropriate amount of this product, take out the contents, combine the contents, take an appropriate amount of the contents (equivalent to 15μg of alfacalcidol), mix with 6mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, pre-activate it with 10mL of n-hexane, pass the sample solution through the solid phase extraction column, control the flow rate to 3 seconds / drop, and discard the receiving solution; after the sample solution is completely filtered, take another 8mL of n-hexane to clean the matrix in the solid phase column, and control the flow rate to 3 drops / second during the cleaning process; then elute the column with 10mL of methanol, and control the flow rate to 2 seconds / drop during the elution process, collect the receiving solution, and dry the receiving solution by vacuum rotation until dry, and take another 1mL of methanol-water mixed solution to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0113] 2. Control solution

[0114] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0115] 3. System suitability solution

[0116] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0117] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0118] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0119] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode;

[0120] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0121] Flow rate: 1.0 mL / min;

[0122] Detection time: 70 minutes;

[0123] Column temperature: 30°C;

[0124] The gradient elution method is:

[0125]

[0126] Example 7

[0127] 1. Preparation of test solution

[0128] Take an appropriate amount of this product, remove the contents, combine the contents, and take an appropriate amount of the contents (equivalent to about 15μg of alfacalcidol) as the sample solution. Take a cyano solid phase extraction column, pre-activate it with 10mL of n-hexane, pass the sample solution through the solid phase extraction column, and discard the receiving solution; after the sample solution is completely filtered, take another 8mL of n-hexane to clean the matrix in the solid phase column, and control the flow rate during the cleaning process to 5 drops / second; then use 8mL of methanol to elute the column, and control the flow rate during the elution process to 2 seconds / drop, collect the receiving solution, and dry the receiving solution by vacuum rotation until dry, and take another 1mL of methanol-water mixed solution to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0129] 2. Control solution

[0130] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0131] 3. System suitability solution

[0132] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0133] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0134] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0135] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode;

[0136] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0137] Flow rate: 1.0 mL / min;

[0138] Detection time: 70 minutes;

[0139] Column temperature: 30°C;

[0140] The gradient elution method is:

[0141]

[0142] Example 8

[0143] 1. Preparation of test solution

[0144] Take an appropriate amount of this product, remove the contents, combine the contents, take an appropriate amount of the contents (equivalent to 15μg of alfacalcidol), mix with 6mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, pre-activate it with 5mL of n-hexane, pass the sample solution through the solid phase extraction column, control the flow rate to 2 seconds / drop, and discard the receiving solution; after the sample solution is completely filtered, take another 10mL of n-hexane to clean the matrix in the solid phase column, and control the flow rate to 3 drops / second during the cleaning process; then elute the column with 8mL of methanol, and control the flow rate to 1 second / drop during the elution process, collect the receiving solution, and dry the receiving solution by vacuum rotation until dry, and take another 1mL of methanol-water mixed solution to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0145] 2. Control solution

[0146] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0147] 3. System suitability solution

[0148] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0149] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0150] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0151] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode;

[0152] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0153] Flow rate: 1.0 mL / min;

[0154] Detection time: 70 minutes;

[0155] Column temperature: 30°C;

[0156] The gradient elution method is:

[0157]

[0158] Example 9

[0159] 1. Preparation of test solution

[0160] Take an appropriate amount of this product, remove the contents, combine the contents, take an appropriate amount of the contents (equivalent to 15μg of alfacalcidol), mix with 6mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, pre-activate it with 10mL of n-hexane, pass the sample solution through the solid phase extraction column, control the flow rate to 2 seconds / drop, and discard the receiving solution; after the sample solution is completely filtered, take another 10mL of n-hexane to clean the matrix in the solid phase column, and control the flow rate during the cleaning process to 5 drops / second; then elute the column with 10mL of methanol, and control the flow rate during the elution process to 2 seconds / drop, collect the receiving solution, and dry the receiving solution by vacuum rotation until dry, and take another 1mL of methanol-water mixed solution to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0161] 2. Control solution

[0162] Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0163] 3. System suitability solution

[0164] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0165] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 200 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0166] Chromatographic column: Welch AQ-C18 250mm*4.6mm, 5μm;

[0167] Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode;

[0168] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0169] Flow rate: 1.0 mL / min;

[0170] Detection time: 70 minutes;

[0171] Column temperature: 30°C;

[0172] The gradient elution method is:

[0173]

[0174] Example 10

[0175] Methodological research

[0176] 1. Instruments and test drugs

[0177] Instruments: Agilent 1260 high performance liquid chromatograph, Shimadzu LC-M20A high performance liquid chromatograph, EX225DZH electronic balance;

[0178] Test drugs: Alfacalcidol (batch number 100378-202104, content 99.8%, China Food and Drug Inspection Institute); Alfacalcidol trans isomer (content 98.5%, STD PHARM); Alfacalcidol alfacalcidol 1β isomer (content 94.6%, STD PHARM); the diluent is a mixed solution of methanol and water.

[0179] 2. Chromatographic conditions: Use octadecylsilane bonded silica gel as the filling column; model: YMC AQ-C18, 250mm×4.6mm, 5μm; acetonitrile as mobile phase A, water as mobile phase B; column temperature: 30℃; flow rate: 1.5mL / min; detection wavelength: 265nm, injection volume: 100μl. Perform gradient elution according to the table below.

[0180]

[0181] 3. Solution preparation

[0182] Alfacalcidol reference solution: Take an appropriate amount of alfacalcidol reference, weigh accurately, add a methanol-water mixture to dissolve and dilute to make a solution containing approximately 0.15 μg per 1 mL.

[0183] Pre-alfacalcidol localization solution: Take an appropriate amount of alfacalcidol reference substance, weigh it accurately, dissolve it in a methanol-water mixture and dilute it to make a solution containing about 15μg per 1mL. Accurately measure an appropriate amount and heat it in a 60℃ water bath for 3 hours to obtain the pre-alfacalcidol localization solution.

[0184] Alfacalcidol trans-isomer localization solution: Take an appropriate amount of alfacalcidol trans-isomer reference substance, weigh it accurately, add a methanol-water mixed solution to dissolve it and dilute it to make a solution containing about 0.15 μg per 1 mL.

[0185] Alfacalcidol 1β isomer localization solution: Take an appropriate amount of alfacalcidol 1β isomer reference substance, weigh accurately, add methanol-water mixed solution to dissolve and dilute to make a solution containing about 0.15 μg per 1 mL.

[0186] Preparation of the test solution: Take an appropriate amount of this product, take out the contents, combine the contents, take an appropriate amount of the contents (equivalent to 15μg of alfacalcidol), mix with 6mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, activate it with 10mL of n-hexane in advance, pass the sample solution through the solid phase extraction column, control the flow rate to 2~3 seconds / drop, and discard the receiving solution; after the sample solution is completely filtered, take another 10mL of n-hexane to clean the matrix in the solid phase column, and control the flow rate during the cleaning process to 3~5 drops / second; then use 10mL of methanol to elute the column, and control the flow rate during the elution process to 1~2 seconds / drop, collect the receiving solution, and dry the receiving solution by vacuum rotation. Take another 1mL of methanol-water mixed solution to re-dissolve, filter, and take the filtrate to obtain the test solution.

[0187] Blank auxiliary material solution: Take about 6g of the blank auxiliary material of this product and mix it with 6mL of n-hexane to form a uniform dispersion system as the sample solution. Take a cyano solid phase extraction column, pre-activate it with 10mL of n-hexane, pass the sample solution through the solid phase extraction column, control the flow rate to 2~3 seconds / drop, and discard the receiving solution; after the sample solution is completely filtered, take another 10mL of n-hexane to clean the matrix in the solid phase column, and control the flow rate during the cleaning process to 3~5 drops / second; then use 10mL of methanol to elute the column, and control the flow rate during the elution process to 1~2 seconds / drop, collect the receiving solution, and dry the receiving solution by vacuum rotation until dry, and take another 1mL of methanol-water mixed solution to re-dissolve, filter, and take the filtrate to obtain the blank auxiliary material solution.

[0188] Control solution: Accurately measure 100 μl of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with a methanol-water mixture, shake well, and use it as the control solution.

[0189] System suitability solution: Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute to prepare a solution containing approximately 15 μg of alfacalcidol, approximately 0.15 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.15 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0190] 4. Impurity location experiment

[0191] The trans-isomer positioning solution of alfacalcidol and the 1β-isomer positioning solution of alfacalcidol were injected into a high performance liquid chromatograph to obtain the positioning diagram of the trans-isomer of alfacalcidol and the 1β-isomer of alfacalcidol, see Figure 1 , Figure 2 and Figure 3 From the figure, the peaks of pro-alfacalcidol, trans-isomer of alfacalcidol and 1β-isomer of alfacalcidol and their retention times can be seen.

[0192] 5. System suitability experiment

[0193] Inject the system suitability solution into the high performance liquid chromatograph to obtain the system suitability spectrum of alfacalcidol related substances. Figure 4 .exist Figure 4 In the system suitability spectrum, the peak elution order is pre-alfacalcidol, trans-isomer of alfacalcidol, alfacalcidol and alfacalcidol 1β isomer, and the separation between each component meets the requirements.

[0194] 6. Linear relationship investigation

[0195] Take an appropriate amount of alfacalcidol reference substance, add methanol-water mixed solution to dissolve and dilute to prepare a series of concentration reference substance solutions, and determine according to the method. Linear regression is performed with concentration as the horizontal axis and peak area as the vertical axis. Alfacalcidol has a good linear relationship in the range of 0.0075μg / mL~0.3037μg / mL. The linear equation is y = 305,479.8013 x + 328.8970, and the correlation coefficient r is 0.9995, which meets the requirements of method validation. The results are shown in Tables 1 and Figure 5 .

[0196] Table 1 Linearity test results of related substances in Alfacalcidol soft capsules

[0197]

[0198] 7. Investigation of detection limit and quantification limit

[0199] Accurately measure the standard curve 1 solution as the quantitative limit solution, at this time the signal-to-noise ratio of the alfacalcidol peak is 10; dilute the solution until the signal-to-noise ratio of the alfacalcidol peak in the solution is 3, which is used as the detection limit concentration; the results show that the detection limit is 0.0025μg / mL and the quantitative limit is 0.0075μg / mL.

[0200] 8. Stability test

[0201] The test solution, system suitability solution and control solution were placed at room temperature to investigate the solution stability of the samples within 28 hours. The results are shown in Tables 2 and 3. The alfacalcidol test solution, system suitability solution and control solution were all stable within 28 hours at room temperature. The RSD of the peak area change of the sum of the alfacalcidol peak and the alfacalcidol peak in the test solution and the system suitability solution was less than 2% within 28 hours, the RSD of the peak area change of the trans isomer of alfacalcidol within 28 hours was less than 2%, and the RSD of the peak area change of the 1β isomer of alfacalcidol within 28 hours was less than 1%.

[0202] Table 2 Stability test results - system suitability solution

[0203]

[0204] Table 3 Stability test results - test solution and control solution

[0205]

[0206] 9. Durability

[0207] The system suitability solution was injected under the chromatographic conditions with slight changes, and the separation results between the components are shown in Table 4. The results show that under the chromatographic conditions with slight changes, the separation between the components in the system suitability solution meets the specifications, and the durability of this method is good.

[0208] Table 4 Durability test results

[0209]

[0210] Note: Column 2 and Column 3 are of the same brand and model but different batch numbers.

[0211] 10. Calculation method of impurities in samples

[0212] In the chromatogram of the test solution, after deducting the excipient peak and the pro-alfacalcidol peak, if there are peaks of the trans-isomer of alfacalcidol and the 1β-isomer of alfacalcidol, their peak areas shall not be greater than the main peak area of ​​the control solution (1.0%), and other individual impurities shall not be greater than the main peak area of ​​the control solution (1.0%). The total amount of impurities shall not exceed 3.0%. Peaks in the chromatogram of the test solution that are less than 0.1 times (0.1%) the main peak area of ​​the control solution shall be ignored.

[0213] 11. Sample determination

[0214] According to the determination method of the present invention, see Examples 6 to 8 for details, all three batches of samples meet the requirements. The results are shown in Table 5. The reference solution spectrum is attached, see Figure 6 ; Blank excipient spectrum, see Figure 7; and the chromatogram of the test solution of batch number 2304041, see Figure 8 .

[0215] Table 5 Determination results of related substances in Alfacalcidol soft capsules

[0216]

[0217] Comparative test with other detection methods of Alfacalcidol soft capsules

[0218] According to the determination method of the present invention, 2304041 test solution was tested according to the determination method of Example 6. The test results are shown in Figure 8 , detected according to the method of comparative example 1, the detection spectrum is shown in Fig. 9 , detected according to the method of comparative example 2, the detection spectrum is shown in Fig.10 .

[0219] Comparative Example 1

[0220] 1. Preparation of test solution

[0221] Take an appropriate amount of alfacalcidol raw material, accurately determine it, add 50% acetonitrile water to dissolve it by ultrasonication and dilute it to make a solution containing about 0.1 mg per 1 mL, which is used as the test solution.

[0222] 2. Alfacalcidol trans isomer control solution

[0223] Take an appropriate amount of the trans-isomer reference substance of alfacalcidol, accurately determine the amount, add 50% acetonitrile water to dissolve by ultrasonication and dilute to make a solution containing about 0.1 mg per 1 mL, which is used as the trans-isomer reference solution of alfacalcidol.

[0224] 3. Alfacalcidol 1β isomer control solution

[0225] Take an appropriate amount of alfacalcidol 1β isomer reference substance, accurately determine the amount, add 50% acetonitrile water to dissolve by ultrasonication and dilute to make a solution containing about 0.1 mg per 1 mL, which is used as the alfacalcidol 1β isomer reference solution.

[0226] 3. System suitability solution

[0227] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute to prepare a solution containing approximately 100 μg of alfacalcidol, approximately 1 μg of the trans isomer of alfacalcidol (impurity A) and approximately 1 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0228] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 100 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0229] Chromatographic column: ZORBAX SB-C18 250mm*4.6mm, 5μm;

[0230] Mobile phase: methanol-water-acetonitrile (150:170:680) was used as the mobile phase, and isocratic elution was adopted;

[0231] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0232] Flow rate: 1.0 mL / min;

[0233] Detection time: 60 minutes;

[0234] Column temperature: 30°C;

[0235] Comparative Example 2

[0236] 1. Preparation of test solution

[0237] Take 2 g of the contents of alfacalcidol soft capsules, place them in a 20 mL volumetric flask, dissolve and dilute to the scale with mobile phase, shake well, and use this as the test solution.

[0238] 2. Alfacalcidol trans isomer control solution

[0239] Take an appropriate amount of the trans-isomer reference substance of alfacalcidol, accurately determine it, add the mobile phase to ultrasonically dissolve it and dilute it to make a solution containing about 0.005 μg per 1 mL, which is used as the trans-isomer reference solution of alfacalcidol.

[0240] 3. Alfacalcidol 1β isomer control solution

[0241] Take an appropriate amount of alfacalcidol 1β isomer reference substance, accurately determine the amount, add mobile phase to dissolve by ultrasound and dilute to make a solution containing about 0.005 μg per 1 mL, which is used as the alfacalcidol 1β isomer reference solution.

[0242] 3. System suitability solution

[0243] Take appropriate amounts of the trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol, weigh them accurately, add a mixed solution of methanol and water to dissolve and dilute them to prepare a solution containing approximately 0.5 μg of alfacalcidol, approximately 0.005 μg of the trans isomer of alfacalcidol (impurity A) and approximately 0.005 μg of the 1β isomer of alfacalcidol (impurity B) per 1 mL.

[0244] 4. The determination was carried out by high performance liquid chromatography, the injection volume was 500 μl, and the chromatographic conditions of the high performance liquid chromatography were:

[0245] Chromatographic column: Agilent ZORBAX SB-CN 250mm*4.6mm, 5μm;

[0246] Mobile phase: dimethyl acetal-n-hexane (150:850) was used as the mobile phase, and isocratic elution was adopted;

[0247] Detection wavelength: 265nm or diode array detector using full wavelength detection;

[0248] Flow rate: 1.0 mL / min;

[0249] Detection time: 60~80 minutes;

[0250] Column temperature: 30°C;

[0251] Figure 8 , Fig. 9 and Fig.10 By comparison, it can be seen that when the method and chromatographic system of Comparative Example 1 are used for the test, the pre-alfacalcidol and the trans-isomer of alfacalcidol cannot be separated; when the method and chromatographic system of Comparative Example 2 are used for the test, the 1β isomer of alfacalcidol and the main peak cannot be separated. Figure 8 Using acetonitrile as mobile phase A and water as mobile phase B, the gradient elution method can effectively separate the pre-alfacalcidol, the trans isomer of alfacalcidol and the 1β isomer of alfacalcidol. The solid phase extraction method is applied, the response of the main peak and each impurity peak is relatively large, the impurity sensitivity is high, and the known impurities and unknown impurities can be effectively detected.

Claims

1. A method for detecting related substances in alfacalcidol soft capsules, characterized in that: The detection method comprises the following steps: The HPLC method was used for determination, and the chromatographic conditions were as follows: Chromatographic column: Octadecylsilane bonded silica gel is used as the filling column; Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution mode; Detection wavelength: 263-267nm or 200-400nm full wavelength scan; The flow rate is 1.0~2.0mL / min.

2. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: Adopt gradient elution mode, the gradient elution mode is: 。 3. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: The quantitative method for the relevant substances is area normalization method, area normalization method with correction factor, self-control method or self-control method with correction factor; The correction factor for impurity A (trans-alfacalcidol) of alfacalcidol is 1.2, and the correction factor for impurity B (alfacalcidol-β isomer) of alfacalcidol is 1.

1.

4. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: The detection wavelength is 265nm.

5. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: During the high performance liquid phase determination, the column temperature is controlled at 25-35°C.

6. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: In the high performance liquid phase determination, the chromatographic column is AQ-C18, 250mm×4.6mm, 5μm.

7. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: The method for preparing the test solution comprises the following steps: Accurately weigh the contents of alfacalcidol soft capsules and mix them with an equal volume of n-hexane, add the mixture to a pre-activated cyano solid phase extraction column for extraction, then rinse and elute, collect the eluate, evaporate to dryness at 25°C under low-temperature vacuum rotary evaporation, dissolve in methanol and water, and finally filter, and use the filtrate as the test solution.

8. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: The preparation method of the control solution comprises the following steps: Accurately weigh 0.1 mL of the test solution, place it in a 10 mL volumetric flask, dilute it to the mark with methanol and water, shake well, and the solution is ready.

9. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: The method for preparing the system suitability solution comprises the following steps: The trans isomer of alfacalcidol, the 1β isomer of alfacalcidol and alfacalcidol were accurately weighed, dissolved in a mixed solution of methanol and water, and diluted to prepare a solution containing 15 μg of alfacalcidol, 0.15 μg of the trans isomer of alfacalcidol and 0.15 μg of the 1β isomer of alfacalcidol per 1 mL, as the system suitability solution.

10. The method for determining related substances in alfacalcidol soft capsules according to claim 1, characterized in that: The preparation method of blank excipient solution comprises the following steps: Accurately weigh the sum of the auxiliary materials of each component of alfacalcidol soft capsule except the main component, mix it with an equal volume of n-hexane, and add the mixture to the pre-activated solid phase extraction column for extraction, then rinse and elute, collect the eluate, evaporate to dryness at low temperature, dissolve it with methanol water, and finally filter it, and the filtrate is used as the blank auxiliary material solution.

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