Method for separating and detecting calcitriol starting material B1 and its impurities

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a method for separating and detecting calcitriol starting material B1 and its impurities. The impurities include impurities B 1a 、Impurity B 1b 、Impurity B 1c One or more of. The self-built high performance liquid chromatography method of the present invention is used for the separation and detection of the calcitriol starting material B1 and its impurities. The chromatographic column uses silica gel covalently bonded to cellulose tris (3,5-dimethylphenylcarbamate) as a filler, and a mixed solution of n-hexane, tert-butyl methyl ether and methanol as a mobile phase for isocratic elution; then enters the detector for detection, and the main component self-control method is used to calculate the content of each impurity according to the measured chromatogram. The method has the characteristics of good separation, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and specifically relates to separation and detection of calcitriol starting material B 1 and its impurities. Background Art

[0002] Calcitriol is the active form of vitamin D, which has physiological effects such as promoting intestinal calcium absorption and regulating bone calcification. Calcitriol can be made into various preparations for the treatment of postmenopausal osteoporosis, chronic renal failure, especially renal osteodystrophy in patients receiving hemodialysis, postoperative hypoparathyroidism, idiopathic hypoparathyroidism, pseudohypoparathyroidism, vitamin D-dependent rickets and hypophosphatemic vitamin D-resistant rickets.

[0003] Calcitriol exists in two forms: anhydrous and monohydrate. The chemical name of the anhydrous form is (5Z,7E)-9,10-opencholester-5,7,10(19)-triene-1α,3β,25-triol, and its molecular formula is C 27 H 44 O 3 , molecular weight is 416.64, structural formula is shown in formula I; chemical name of monohydrate is (5Z,7E)-9,10-opened cholesteryl-5,7,10(19)-triene-1α,3β,25-triol monohydrate, molecular formula is C 27 H 44 O 3 ·H 2 O, with a molecular weight of 434.65, and its structural formula is shown in Formula II.

[0004]

[0005] Calcitriol starting material B 1 (CALO-B 1 ) is the key starting material for the synthesis of calcitriol. Its chemical name is (1R,3S,Z)-5-(2-hydroxy-ethylidene)-4-methylene-1,3-di(tert-butyldimethylsilyloxy)cyclohexane, and its molecular formula is C 21 H 42 O 3 Si 2 , molecular weight is 398.73, and the structural formula is shown in Formula III.

[0006]

[0007] The study found that the calcitriol starting material B 1 Three isomeric impurities are easily introduced during the preparation process: Impurity B 1a 、Impurity B1b and impurity B 1c . Impurity B 1a 、Impurity B 1b and impurity B 1c The structural formulas are shown in Formula IV to Formula VI respectively.

[0008]

[0009] For the preparation of calcitriol starting material B 1 Impurities generated during the process or related substances introduced need to be strictly controlled in the starting materials. 1a 、Impurity B 1b 、Impurity B 1c Calcitriol starting material B 1 The isomeric impurities are not included in the pharmacopoeia and are difficult to separate using conventional liquid phase reverse method. In addition, there is no public method for quickly and simultaneously separating the above three impurities. Only some existing technologies disclose analytical methods for calcitriol or its related substances. For example, patent CN109752471A discloses a method for detecting calcitriol, which detects calcitriol by HPLC. The chromatographic conditions include: the chromatographic column is a C18 column; mobile phase A is methanol; mobile phase B is methanol and acetonitrile; the elution method is gradient elution; the flow rate is 1.5-2.0 mL / min; the column temperature is 30-40 ° C; the detection wavelength is 255 nm. For example, patent CN117630194A discloses a high performance liquid chromatography analysis method for calcipotriol related substances. The chromatographic column of the invention is octylsilane bonded silica gel Kromasil100-5-C8, with a specification of 4.6mm×250mm, 5μm; water is used as mobile phase A and acetonitrile is used as mobile phase B for gradient elution. These methods are not suitable for calcipotriol starting material B 1 Separation and detection of its three isomeric impurities.

[0010] Therefore, in order to ensure that the starting material B of calcitriol 1 In order to improve the quality reliability and process stability of calcitriol synthesis, a method for rapid and effective separation and determination of calcitriol starting material B was developed. 1 The analytical methods of its three isomeric impurities are very meaningful for drug quality control and degradation pathway research. Summary of the invention

[0011] In view of this, one of the objects of the present invention is to provide a method for separating the calcitriol starting material B based on high performance liquid chromatography. 1 The method can separate a variety of substances in a shorter time.

[0012] To achieve the above object, the technical solution of the present invention is:

[0013] Separation of calcitriol starting material B based on HPLC 1 and its impurities, the calcitriol starting material B 1 and the impurities together form a composition, wherein the impurities include impurity B 1a 、Impurity B 1b 、Impurity B 1c Any one or more of; the structural formula of each component in the composition is as follows:

[0014]

[0015] In the high performance liquid chromatography method, the mobile phase is a mixed solution of n-hexane, tert-butyl methyl ether and methanol, and the volume ratio of the n-hexane, the tert-butyl methyl ether and the methanol is 92-98:2-8:0.08-0.12; the stationary phase is a chromatographic column using silica gel with cellulose-tris (3,5-dimethylphenylcarbamate) covalently bonded on the surface as a filler, and the calcitriol starting material B is eluted by isocratic elution. 1 and its impurities are separated.

[0016] After separation and testing, it is used for the next production step.

[0017] The above impurities can be arranged and combined in many ways. For example, combination 1: impurity B 1a 、Impurity B 1b For example, combination 2: impurity B 1b 、Impurity B 1c For example, combination 3: impurity B 1a 、Impurity B 1b 、Impurity B 1c .

[0018] Theoretically, when the upper limit of the substances to be separated, identified and / or detected by the method is n (number), 1-n substances can be detected naturally.

[0019] Preferably, the volume ratio of n-hexane, tert-butyl methyl ether and methanol in the mobile phase is 93-97:3-7:0.1, and most preferably is 95:5:0.1.

[0020] The present invention has found that adding a trace amount of methanol to the mobile phase can improve the impurity separation effect, which provides a new idea for the development of other subsequent analytical methods.

[0021] Preferably, the specifications of the chromatographic column are 4.6 mm×250 mm, 3 μm.

[0022] Most preferably, the chromatographic column is CHIRALPAK IB N-3, 4.6 mm×250 mm, 3 μm.

[0023] Furthermore, the flow rate is 0.5-1.2 mL / min; and the column temperature is 20-30°C.

[0024] As a preferred solution, the flow rate is 0.7-0.9 mL / min; the column temperature is 23-27°C. For example, the flow rate is 0.7 mL / min ( Fig. 9 ), or a flow rate of 0.8 mL / min ( Figure 8 ); or flow rate of 0.9 mL / min ( Fig.10 ). For example, the column temperature is 23°C ( Fig.11 ), or the column temperature is 24°C, or the column temperature is 25°C ( Figure 8 ), or column temperature is 26℃, or column temperature is 27℃( Fig.12 ).

[0025] Most preferably, the flow rate is 0.8 mL / min and the column temperature is 25°C.

[0026] Preferably, the injection volume is 10 μl.

[0027] Preferably, the running time is 30 minutes.

[0028] Furthermore, the sample preparation solvent is a mixed solution of n-hexane and tert-butyl methyl ether, and the volume ratio of the n-hexane to the tert-butyl methyl ether is 90-97:3-10.

[0029] Preferably, in the sample preparation solvent, the volume ratio of n-hexane to tert-butyl methyl ether is 95:5.

[0030] Preferably, the sample preparation solvent is used to prepare a sample solution, a control solution and / or a system suitability solution.

[0031] As a preferred solution, preparation of the sample solution: accurately weigh the test sample, such as 250.68 mg, and place it in a 50 ml volumetric flask, add the sample preparation solvent to dissolve and dilute to the scale, shake well, and obtain the test sample stock solution; accurately pipette the test sample stock solution, such as 2 ml, and place it in a 10 ml volumetric flask, add the sample preparation solvent to dilute to the scale, shake well, and obtain the sample solution.

[0032] As a preferred solution, the control solution is prepared by accurately transferring the sample solution, such as 5 ml, into a 100 ml volumetric flask, adding the sample preparation solvent to dissolve and dilute to the scale, and shaking well; accurately transferring 2 ml of the solution into a 100 ml volumetric flask, adding the sample preparation solvent to dissolve and dilute to the scale, and shaking well to obtain a control solution.

[0033] As a preferred solution, the system suitability solution is prepared by accurately weighing CALO-B 1 2.610mg, impurity B 1a 2.794mg, Impurity B 1b 2.450mg, impurity B 1c 2.437 mg, put it in the same 50ml volumetric flask, add the sample preparation solvent to dissolve and dilute to the scale, shake well; accurately pipette 10ml, put it in a 50ml volumetric flask, add the sample preparation solvent to dilute to the scale, shake well, and obtain the reference substance stock solution; accurately pipette 1.0ml of the reference substance stock solution and 2ml of the test substance stock solution, put them in the same 10ml volumetric flask, add the sample preparation solvent to dilute to the scale, shake well, and obtain.

[0034] The second object of the present invention is to provide a method for identifying the calcitriol starting material B 1 The method can complete the identification of multiple substances in a shorter time.

[0035] To achieve the above object, the technical solution of the present invention is:

[0036] Identification of calcitriol starting material B 1 The method for separating the composition and its impurities is to separate the composition by the aforementioned separation method, and enter the composition into a detector for detection to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample and the reference sample, it is determined whether the test sample contains the calcitriol starting material B. 1 and its impurities.

[0037] As a preferred solution, the detection wavelength of the detector is 220±10nm. The setting range of ±10nm is a comprehensive consideration based on multiple factors such as error considerations, method superiority, and practical application requirements. This setting range helps to ensure the reliability of the detection results, improve the repeatability and flexibility of the measurement, and meet the requirements of specific experiments.

[0038] Preferably, the detection wavelength of the detector is 220 nm.

[0039] As a preferred solution, the components in the composition can be identified in the order of relative retention time. The components of the composition are in ascending order: impurity B 1a , calcitriol starting material B 1 、Impurity B 1b 、Impurity B 1c .

[0040] Generally, relative retention time is used to describe the relative retention of different components in a mixture on a chromatographic column. It refers to the ratio of the retention time of a component to the retention time of a reference component (usually the main peak or a known component). This ratio reflects the relative retention performance of different components on a chromatographic column and is an important parameter for positioning, qualitative and quantitative analysis in chromatographic analysis. The relative retention time is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0041] As a preferred solution, the calcitriol starting material B 1 The reference peak has a relative retention time of 0.93±0.02 and is identified as impurity B. 1a ; The relative retention time is 1.13±0.06, which is determined to be impurity B 1b ; The relative retention time is 1.38±0.10, which is determined to be impurity B 1c .

[0042] In addition to relative retention time, retention time can also be used to determine components. Retention time refers to the time required for a sample to enter the chromatographic column and be detected by the detector. This time is calculated based on the movement speed of the component on the chromatographic column, that is, the time interval from the start of injection to the chromatographic apex (maximum concentration) of a certain component. It is mainly used to determine the peak order and position of each component in the sample and is one of the basic data in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0043] The third object of the present invention is to provide a method for determining the starting material B of calcitriol 1 The method can complete the identification and content determination of multiple substances in a relatively short time.

[0044] To achieve the above object, the technical solution of the present invention is:

[0045] Determination of calcitriol starting material B 1 and its impurity content, using the aforementioned identification method to separate and identify the calcitriol starting material B 1 and its impurities to obtain a chromatogram; based on the obtained chromatogram, the content of each impurity is calculated using the main component self-comparison method.

[0046] Through content determination, we can further determine the calcitriol starting material B 1 And whether the impurity content is qualified. 1a 、Impurity B 1b 、Impurity B 1c If the peak area of ​​any one or more impurities in the solution is greater than the peak area of ​​the corresponding impurities in the control solution, it indicates that the impurity content is unqualified; conversely, if impurity B1a 、Impurity B 1b 、Impurity B 1c Any one or more peak areas in the test solution are not greater than the peak areas of the corresponding impurities in the control solution, indicating that the impurity content is qualified.

[0047] The above determination method can be used as a drug quality determination model, and further as an indispensable key module in the intelligent production process. This model can achieve accurate separation and quantitative analysis of active ingredients, impurities and degradation products in drugs by accurately controlling parameters such as mobile phase composition, flow rate, column temperature, etc., providing a scientific basis for the comprehensive evaluation of drug quality. In the intelligent production system, this model is seamlessly integrated, able to receive raw data from the production line in real time, automatically perform analysis tasks, and quickly feedback the determination results based on preset quality standards.

[0048] The fourth object of the present invention is to provide a method for separating and determining the starting material B of calcitriol. 1 Reagent compositions and their impurities.

[0049] To achieve the above object, the technical solution of the present invention is:

[0050] Used for separation and determination of calcitriol starting material B 1 and a reagent composition containing impurities thereof, wherein the reagent composition comprises the following components:

[0051] 1) Mobile phase: a mixed solution of n-hexane, tert-butyl methyl ether and methanol, wherein the volume ratio of the n-hexane, the tert-butyl methyl ether and the methanol is 92-98:2-8:0.08-0.12;

[0052] and / or 2) sample preparation solvent: a mixed solution of n-hexane and tert-butyl methyl ether, wherein the volume ratio of the n-hexane to the tert-butyl methyl ether is 90-97:3-10.

[0053] The beneficial effects of the present invention are:

[0054] 1. The separation and detection of calcitriol starting material B provided by the present invention 1 The method for removing impurities from the calcitriol starting material B1 can achieve 1a 、Impurity B 1b and / or impurity B 1c The effective separation and determination of impurities achieves effective control and fundamentally determines the quality of the product. It has the advantages of being exclusive, sensitive, simple, fast and highly accurate.

[0055] 2. The high performance liquid chromatography method built by the present invention can simultaneously separate the calcitriol starting material B within 30 minutes 1The three isomeric impurities in the calcitriol have the characteristics of short analysis time, simple operation, strong practicality, and accurate and reliable detection results. 1 Quality control is of great significance.

[0056] 3. The method of the present invention has the characteristic of high sensitivity. CALO-B 1 The detection limit concentration is 0.1462μg / ml, and the quantification limit concentration is 0.4176μg / ml; impurity B 1a The detection limit concentration is 0.1561μg / ml, and the quantification limit concentration is 0.4461μg / ml; impurity B 1b The detection limit concentration is 0.1357μg / ml, and the quantification limit concentration is 0.3877μg / ml; impurity B 1c The detection limit concentration was 0.1320μg / ml, and the quantification limit concentration was 0.3771μg / ml.

[0057] 4. Separation and Detection of Calcitriol Starting Material B of the Present Invention 1 The method of separating the degradation products of the test sample under sunlight and its impurities is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is the chromatogram of the blank solution;

[0059] Figure 2 is the chromatogram of the system suitability solution;

[0060] Figure 3 is the chromatogram of the test solution;

[0061] Figure 4 is the chromatogram of the self-control solution;

[0062] Figure 5 is the chromatogram of the quantitation limit solution;

[0063] Figure 6 is the chromatogram of the detection limit solution;

[0064] Figure 7 Starting material B 1 Chromatogram of sunlight degradation;

[0065] Figure 8 This is the HPLC chart under normal conditions of the durability experiment;

[0066] Fig. 9 This is the HPLC chart of the durability experiment under the condition of flow rate 0.7ml / min;

[0067] Fig.10 This is the HPLC chart of the durability experiment under the condition of flow rate 0.9ml / min;

[0068] Fig.11 This is the HPLC chart of the durability experiment under the condition of column temperature of 23℃;

[0069] Fig.12 This is the HPLC chart of the durability experiment under the condition of column temperature of 27℃;

[0070] Fig.13 This is the HPLC chart under the conditions of the mobile phase ratio (n-hexane: tert-butyl methyl ether: methanol = 97:3:0.1) in the durability experiment;

[0071] Fig.14 This is the HPLC chart under the conditions of the mobile phase ratio (n-hexane: tert-butyl methyl ether: methanol = 93:7:0.1) in the durability experiment. DETAILED DESCRIPTION

[0072] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0073] Supplementary table to the drawings in the specification. Figure 1-Figure 14 Visual aids are provided for the purpose of understanding and interpretation. In the event of any ambiguity, the user should refer to the corresponding numbered tables (Tables 1-14) and data for more detailed information. Conversely, if any information that may cause misunderstanding or ambiguity is found in the process of reviewing Tables 1-14, the content in the corresponding numbered figures shall prevail. The above guidelines are intended to ensure the correct interpretation of this document and the consistency of its information. Although some of the handwriting in the spectrum of this application document overlaps, it can still be viewed clearly, and the integration results of each figure are recorded in detail in the specification. In addition, the numbers in the spectrum have no effect on the scope of protection of the claims and the full disclosure of the technical solutions in the specification.

[0074] Table 1

[0075]

[0076] Table 2

[0077]

[0078] Table 3

[0079]

[0080] Table 4

[0081]

[0082] Table 5

[0083]

[0084] Table 6

[0085]

[0086] Table 7

[0087]

[0088] Table 8

[0089]

[0090] Table 9

[0091]

[0092] Table 10

[0093]

[0094] Table 11

[0095]

[0096] Table 12

[0097]

[0098] Table 13

[0099]

[0100] Table 14

[0101]

[0102] In order to enhance the understanding of the present invention, some key technologies and scientific terms are clearly defined below. Unless specifically defined herein, all other technical and scientific terms follow the meanings generally accepted and understood in the field to which the present invention belongs. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference substances, and test substances described, but reasonable flexibility and adjustment in these aspects are allowed. At the same time, please understand that the terms used in this article are intended to illustrate specific embodiments rather than to be interpreted restrictively.

[0103] In addition, all literature cited herein, including but not limited to patents, patent applications, academic papers, textbooks and further citations therein, are deemed to be incorporated herein by reference as a whole to the extent not directly cited. If there is any inconsistency or conflict between the contents of these cited documents or similar materials and this application, especially in terms of term definitions, term usage, technical descriptions, etc., the contents of this application shall prevail.

[0104] If there are any unmentioned conditions in the chromatographic conditions, they can be determined by referring to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part 4 0512).

[0105] the term

[0106] The limit of quantitation refers to the lowest amount of the substance to be measured in the sample, and the measurement result should have a certain degree of accuracy and precision. In other words, the limit of quantitation is the lowest level at which the analytical method can accurately and reliably measure the concentration of the substance to be measured in the sample. In HPLC, the determination of the limit of quantitation usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the substance to be measured corresponding to a certain level of signal-to-noise ratio is used as the limit of quantitation. The determination of the limit of quantitation is crucial to ensure the accuracy and reliability of the analysis results.

[0107] The durability of chromatographic conditions refers to the ability of the chromatographic analysis system to maintain stable analytical performance and not significantly affect the measurement results when there is a slight change in the measurement conditions. This durability is crucial to ensure the reliability, repeatability and stability of the analysis results.

[0108] The limit of detection (LOD) refers to the lowest concentration or amount of the substance in the sample that can be detected. It reflects the sensitivity and noise of the analytical method and instrument, and also indicates the level of the blank (background) value after the sample is processed.

[0109] The correction factor is a coefficient or parameter used to correct the results during the analysis process. It is intended to improve data accuracy and reliability. In HPLC analysis, since the same detector has different response values ​​for different substances, when different substances of the same mass pass through the detector, the peak areas generated are not necessarily equal. In order to make the peak area accurately reflect the content of the component to be measured, standard substances are used for correction, the correction factor is calculated, and it is applied to the measurement results of the sample to be measured.

[0110] The ratio of peak height to noise (S / N, i.e. signal-to-noise ratio) is used in HPLC to evaluate the detection sensitivity and resolution of the instrument and is an important indicator for measuring instrument performance. Among them: peak height refers to the signal value output by the detector when the component to be tested is eluted from the column; and noise refers to the fluctuation of the baseline signal, that is, the signal value measured by the blank sample. The signal-to-noise ratio is the ratio of the signal measured by a sample of known concentration to the signal measured by a blank sample. The larger the signal-to-noise ratio, the more accurately the instrument can separate and identify the target component when detecting the sample, while also reducing the interference of background noise.

[0111] In the embodiments of the present invention, the test sample and the reference sample are from Gansu Haotian Pharmaceutical Technology Co., Ltd., and the raw material B 1 The batch number is HT056-01-14-2307006, impurity B 1a The batch number is HT056B-09-20230701, impurity B 1b The batch number is HT056B-12-20230701, impurity B 1c The batch number is HT056C-18-20230701.

[0112] In the embodiment of the present invention, the high performance liquid chromatograph is a Shimadzu LC-2030 high performance liquid chromatograph.

[0113] In the embodiment of the present invention, “——” in the table means none.

[0114] Example 1. Separation and Detection of Calcitriol Starting Material B 1 Medium impurity B 1a 、Impurity B 1b 、Impurity B 1c Methods

[0115] (1) Prepare the test solution

[0116] Diluent: a mixed solution of n-hexane and tert-butyl methyl ether, wherein the volume ratio of n-hexane to tert-butyl methyl ether is 95:5.

[0117] Reference stock solution: Accurately weigh CALO-B 1 2.610mg, impurity B 1a 2.794mg, Impurity B 1b 2.450mg, impurity B 1c 2.437mg, put it in the same 50ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well; accurately transfer 10ml, put it in a 50ml volumetric flask, add diluent to dilute to the scale, shake well, and you have it.

[0118] Test sample stock solution: Accurately weigh 250.68 mg of the test sample, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0119] Test sample solution: Accurately pipette 2 ml of the test sample stock solution into a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0120] Control solution: Accurately pipette 5 ml of the test solution into a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well; accurately pipette 2 ml of the solution into a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well.

[0121] System suitability solution: Accurately pipette 1.0 ml of reference stock solution and 2 ml of test stock solution into the same 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0122] (2) Chromatographic conditions

[0123] The chromatographic column uses silica gel with cellulose-tris(3,5-dimethylphenylcarbamate) covalently bonded on the surface as a filler (CHIRALPAK IB N-3, 4.6mm×250mm, 3μm or a chromatographic column with equivalent performance); the mobile phase is a mixed solution of n-hexane, tert-butyl methyl ether and methanol, wherein the volume ratio of n-hexane, tert-butyl methyl ether and methanol is 95:5:0.1; the elution mode is isocratic elution; the wavelength is 220nm; the column temperature is 25℃; the flow rate is 0.8ml / min; and the injection volume is 10μl.

[0124] (3) Detection

[0125] Take the diluent, test solution, control solution, and system suitability solution respectively, perform HPLC analysis according to the above chromatographic conditions, record the chromatogram; and quantify each impurity according to the main component self-reference method.

[0126] The test results are shown in Table A. Figure 1 to Figure 4 , as shown in Tables 1 to 4. The results show that the diluent does not interfere with the detection of the sample; the separation between the main peak and the adjacent impurity peaks and between the known impurity peaks is greater than 1.5, and the system applicability meets the requirements. According to the main component self-control method, the impurity B in the test solution 1a Content less than 0.05%, impurity B 1b 、Impurity B 1c None of them were detected, so in the calcitriol starting material B 1 Impurity B during synthesis and post-treatment 1a 、Impurity B 1b 、Impurity B 1c will be effectively removed.

[0127] Table A. System suitability test results

[0128]

[0129] Example 2. Selection of Chromatographic System and Chromatographic Column

[0130] This example investigates different chromatographic systems and chromatographic columns, as shown in Tables B to C. As shown in Table B, in the reverse phase system, CHIRALCEL OD-RH column, InfinityLab poroshell 120EC-C18 column, and XDB-C18 column were used for detection with different mobile phases, and the separation effect of isomeric impurities and the main peak was not good. Therefore, it is considered to use a normal phase system to detect isomeric impurities. As shown in Table C, CHIRALCEL IA column and CHIRALCEL IB N-3 column were used for method screening and optimization. The chromatographic column selected was CHIRALCEL IB N-3 column (4.6×250mm, 3μm), which can make impurity B 1a 、Impurity B 1b 、Impurity B 1c and calcitriol starting material B 1 The separation was good. Therefore, the normal phase system was finally adopted.

[0131] Table B. Results of the selection of chromatographic system and chromatographic column

[0132]

[0133] Table C. Results of the selection of chromatographic system and chromatographic column

[0134]

[0135]

[0136] Example 3. Selection of mobile phase

[0137] This example further investigated different mobile phases. A CHIRALCEL IB N-3 column (4.6×250 mm, 3 μm) was used with n-hexane-methanol, n-hexane-anhydrous ethanol, n-hexane-tert-butyl methyl ether, n-hexane-tert-butyl methyl ether-methanol, n-hexane-tert-butyl methyl ether-anhydrous ethanol, and n-hexane-tert-butyl methyl ether-isopropanol as mobile phases to determine the impurity B. 1a 、Impurity B 1b 、Impurity B 1c and calcitriol intermediate Z 3 The separation was performed, and the screening process is shown in Table D. After screening, the mobile phase selected by the present invention is n-hexane-tert-butyl methyl ether-methanol (95:5:0.1), and the measurement results are shown in Table D.

[0138] Table D. Mobile phase selection results

[0139]

[0140] Example 4. Limit of Quantitation and Limit of Detection Test

[0141] Quantitative limit stock solution: Accurately weigh CALO-B 1 2.610mg, impurity B 1a 2.794mg, Impurity B 1b 2.450mg, impurity B 1c 2.437mg, put it in the same 50ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well; accurately transfer 10ml, put it in a 50ml volumetric flask, add diluent to dilute to the scale, shake well, and you have it.

[0142] Quantitation limit solution: Accurately pipette 1 ml of the quantitation limit stock solution into a 25 ml volumetric flask, add diluent to dilute to the scale, shake well, and the solution is ready.

[0143] Detection limit solution: Accurately pipette 3.5 ml of the quantitative limit solution into a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0144] The above quantitative limit solution was sampled continuously for 6 times, and the detection limit solution was sampled continuously for 3 times, the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated, and the chromatogram was recorded.

[0145]

[0146] The results of the quantitative limit test are shown in Table E. Figure 5 , as shown in Table 5. From Table E, we can see that CALO-B 1 The quantitative limit concentration was 0.4176 μg / ml, the RSD of the peak area was 2.7%, and the average signal-to-noise ratio was 31.5; impurity B 1a The quantitative limit concentration of impurity B is 0.4461μg / ml, which is expressed as 0.045% in the test sample, with an RSD of 2.9% for the peak area and an average signal-to-noise ratio of 38.0. 1b The quantitative limit concentration is 0.3877μg / ml, which is expressed as 0.039% in the test sample, the RSD of the peak area is 3.7%, and the average signal-to-noise ratio is 24.9; impurity B 1c The limit of quantitation concentration is 0.3771 μg / ml, which is expressed as 0.038% in the test sample, the RSD of the peak area is 4.4%, and the average signal-to-noise ratio is 20.1. The detection limit test results are shown in Table F. Figure 6 , as shown in Table 6. From Table F, we can see that CALO-B 1 The detection limit concentration was 0.1462 μg / ml, and the average signal-to-noise ratio was 10.3; impurity B 1aThe detection limit concentration is 0.1561μg / ml, which is expressed as 0.014% in the test sample, and the average signal-to-noise ratio is 11.4; impurity B 1b The detection limit concentration is 0.1357μg / ml, which is expressed as 0.014% in the test sample, and the average signal-to-noise ratio is 7.5; impurity B 1c The detection limit concentration is 0.1320 μg / ml, which is expressed as 0.013% in the test sample, and the average signal-to-noise ratio is 6.1. 1 And impurity B 1a 、Impurity B 1b 、Impurity B 1c The detection limit and quantification limit of the assay met the requirements of S / N=3(10):1 respectively.

[0147] Table E. Quantitation limit determination results

[0148]

[0149]

[0150] Table F. Detection limit determination results

[0151]

[0152] Example 5. Sunlight degradation test

[0153] To confirm the raw material CALO-B 1 The sensitivity to acid, alkali, oxidation, water, high temperature and light is finally confirmed, and the storage conditions are further carried out in this example.

[0154] Test solution: Accurately weigh 10.83 mg of the product, place in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0155] Take about 1 ml of the test solution, place it in a transparent injection vial, place it under sunlight for 6 hours, inject it according to the above chromatographic conditions, and record the chromatogram. Calculate the content of each impurity by the main component self-reference method, and calculate the main peak content by the external standard method.

[0156] The test results are shown in Table G. Figure 7 This product has no new impurities under sunlight degradation conditions, impurity B 1a The increase was 0.16%, and the other impurities had no significant change, and the mass was conserved. The separation degree between the main peak and the adjacent impurity peak was greater than 1.5, and the purity of the main peak was greater than 990. This shows that the chromatographic conditions are good for separating the degradation products under sunlight.

[0157] Table G. Results of sunlight degradation test

[0158]

[0159] Example 6 Durability

[0160] (1) Prepare the test solution

[0161] Impurity B 1a Stock solution: weigh impurity B 1a Place 2.80 mg of reference substance in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0162] Impurity B 1b Stock solution: weigh impurity B 1b Place 2.92 mg of reference substance in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0163] Impurity B 1c Stock solution: weigh impurity B 1c Place 2.54 mg of reference substance in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0164] Mixed solution: Take CALO-B 1 25.38mg, accurately weighed, placed in a 25ml volumetric flask, and then measured impurity B 1a Stock solution, impurity B 1b Stock solution, impurity B 1c Place 250 μl of each stock solution in the same volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0165] (2) Detection

[0166] Take the mixed solution and adjust the experimental conditions (ratio of n-hexane in mobile phase ±2%, flow rate ±0.1 ml / min, column temperature ±2°C) respectively. After the instrument system is stable, test them respectively to examine the separation between the peaks.

[0167] The test results are shown in Table H. Figure 8 -Table 14, Table 8 - As shown in Table 14, when there are slight fluctuations in flow rate, column temperature, and mobile phase ratio, the separation between the main peak and the adjacent impurity peaks and between each known impurity peak is greater than 1.5, which meets the verification requirements.

[0168] Table H. Chromatographic Condition Change Durability Test Results (Degree of Separation)

[0169]

[0170]

Claims

1. A method for separating calcitriol starting material B1 and its impurities based on high performance liquid chromatography, characterized in that: The calcitriol starting material B1 and the impurities together form a composition, and the impurities include impurities B 1a 、Impurity B 1b 、Impurity B 1c Any one or more of; the structural formula of each component in the composition is as follows: In the high performance liquid chromatography method, the mobile phase is a mixed solution of n-hexane, tert-butyl methyl ether and methanol, and the volume ratio of the n-hexane, the tert-butyl methyl ether and the methanol is 92-98:2-8:0.08-0.12; the stationary phase is a chromatographic column using silica gel with cellulose-tris (3,5-dimethylphenylcarbamate) covalently bonded on the surface as a filler, and the calcitriol starting material B1 and its impurities are separated by isocratic elution.

2. The method according to claim 1, characterized in that: The volume ratio of n-hexane, tert-butyl methyl ether and methanol in the mobile phase is 93-97:3-7:0.

1.

3. The method according to claim 1, characterized in that The flow rate was 0.5-1.2 mL / min; the column temperature was 20-30°C.

4. The method according to claim 1, characterized in that The sample preparation solvent is a mixed solution of n-hexane and tert-butyl methyl ether, and the volume ratio of the n-hexane to the tert-butyl methyl ether is 90-97:3-10.

5. A method for identifying calcitriol starting material B1 and its impurities, characterized in that: The composition is separated by the method described in any one of claims 1 to 4, and enters a detector for detection to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample and the reference sample, it is determined whether the test sample contains the calcitriol starting material B1 and its impurities.

6. The method according to claim 5, characterized in that The detection wavelength of the detector is 220±10 nm.

7. The method according to claim 5, characterized in that According to the order of relative retention time, the components in the composition can be identified. The components of the composition are in ascending order: Impurity B 1a , calcitriol starting material B1, impurity B 1b 、Impurity B 1c .

8. The method according to claim 7, characterized in that Taking calcitriol starting material B1 as the reference peak, the relative retention time was 0.93±0.02, which was determined to be impurity B 1a ; The relative retention time is 1.13±0.06, which is determined to be impurity B 1b ; The relative retention time is 1.38±0.10, which is determined to be impurity B 1c .

9. A method for determining the calcitriol starting material B1 and its impurity content, characterized in that: The method according to any one of claims 5 to 8 is used to separate and identify the calcitriol starting material B1 and its impurities to obtain a chromatogram; and the content of each impurity is calculated according to the obtained chromatogram using the main component self-reference method.

10. A reagent composition for separating and determining calcitriol starting material B1 and its impurities, characterized in that: The reagent composition comprises the following components: 1) Mobile phase: a mixed solution of n-hexane, tert-butyl methyl ether and methanol, wherein the volume ratio of the n-hexane, the tert-butyl methyl ether and the methanol is 92-98:2-8:0.08-0.12; and / or 2) sample preparation solvent: a mixed solution of n-hexane and tert-butyl methyl ether, wherein the volume ratio of the n-hexane to the tert-butyl methyl ether is 90-97:3-10.

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