Method for separating and determining calcitriol intermediate Z3 and its isomeric impurities

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a method for separating and determining a calcitriol intermediate Z3 and its isomeric impurities. The isomeric impurities are impurities Z 3b . The present invention adopts high performance liquid chromatography for determination, mobile phase: a mixed solution of methanol and acetonitrile is used as mobile phase A, and ethyl acetate is used as mobile phase B; stationary phase: the chromatographic column uses octadecylsilane bonded silica gel as a filler, linear gradient elution is performed, and enters the detector for detection; then, according to the chromatogram, the impurity content is calculated by the main component self-control method. This method creatively uses a reverse phase chromatographic column, elutes impurities with a normal phase mobile phase, and adopts a gradient elution method to improve the peak shape and separation effect of impurities. This method has the characteristics of good separation, good durability, high sensitivity, and good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for separating and determining a calcitriol intermediate Z3 and its isomer impurities. Background Art

[0002] Calcitriol is one of the most important metabolic products of vitamin D3 in the human body and has a wide range of clinical applications. It can be made into various preparations for postmenopausal and senile 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 the molecular formula is C 27 H 44 O3, 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 O3·H2O, molecular weight is 434.65, and the structural formula is shown in Formula II.

[0004]

[0005] During the synthesis of calcitriol API, intermediate B3 reacts with intermediate Z2 to obtain calcitriol intermediate Z3. In this process, its isomer impurity, impurity Z, is easily produced. 3b The structural formula of intermediate B3 is shown in Formula III, and the structural formula of intermediate Z2 is shown in Formula IV. The chemical name of calcitriol intermediate Z3 (abbreviated as CAT-Z3) is ((1R,3S,Z)-5-(2-((1R,3aS,7aR,E)-7a-methyl-1-((R)-6-methyl-6-((triethylsilyl)oxy)heptane-2-yl)octahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexane-1,3-diyl)bis(oxy))bis(tert-butyldimethylsilane), and its molecular formula is C 45 H 86 O3Si3, molecular weight is 759.44, and the structural formula is shown in Formula V. Impurity Z 3b The structural formula of is shown in Formula VI.

[0006]

[0007] The impurities generated or related substances introduced during the preparation of calcitriol need to be strictly controlled in the intermediate. However, there is no relevant patent or literature on the impurity Z in the calcitriol intermediate Z3. 3b The analytical methods of calcitriol have been reported, and only some of the prior art discloses analytical methods for other related substances in calcitriol. For example, patent CN116466016B discloses a method for determining the content of related substances in calcitriol oral solution, which uses high performance liquid chromatography to determine the calcitriol precursor and degradation impurities in calcitriol oral solution. The chromatographic column is a silica gel column; the mobile phase is ethanol, n-hexane and isopropanol in a volume ratio of 40:60:1, and the flow rate is 0.8-1.2ml / min. This method is difficult to achieve the impurity Z in the calcitriol intermediate Z3 3b effective separation.

[0008] Therefore, it is necessary to establish a new method to separate and determine the calcitriol intermediate Z3 and its related substances in order to control the quality of the final product calcitriol. Summary of the invention

[0009] In view of this, one of the objects of the present invention is to provide a method for separating the calcitriol intermediate Z3 and its isomeric impurities based on high performance liquid chromatography, which method can complete the separation of multiple substances in a relatively short time.

[0010] To achieve the above object, the technical solution of the present invention is:

[0011] A method for separating a calcitriol intermediate Z3 and its isomeric impurities based on high performance liquid chromatography, wherein the isomeric impurities are impurities Z 3b , the calcitriol intermediate Z3 and the impurity Z 3b together constitute a composition;

[0012] The structural formulas of the compounds are as follows:

[0013]

[0014] In the high performance liquid chromatography method, the mobile phase is a mixed solution of methanol and acetonitrile as mobile phase A, and ethyl acetate as mobile phase B, wherein the volume ratio of methanol to acetonitrile in the mobile phase A is 50-70:5-25; the stationary phase is a chromatographic column using octadecylsilane bonded silica gel as a filler, and linear gradient elution is performed.

[0015] After separation and testing, it is used for the next production step.

[0016] Preferably, the volume ratio of methanol to acetonitrile in the mobile phase A is 58-62:13-17, most preferably 60:15.

[0017] Further, the procedure of the linear gradient elution is as follows:

[0018] Time (minutes) Mobile phase A - volume Mobile phase B - volume 0 75±10 25±10 5±1 75±10 25±10 50±1 60±10 40±10 60±1 60±10 40±10 61±1 75±10 25±10 70±1 75±10 25±10 .

[0019] Preferably, the initial volume ratio of methanol, acetonitrile and ethyl acetate in the mobile phase is 58-62:13-17:23-27, and most preferably 60:15:25. The initial volume ratio here refers to the ratio when the elution time is 0, 5, 61 and 70 minutes.

[0020] Preferably, the procedure of the linear gradient elution is as follows:

[0021] Time (minutes) Mobile phase A - volume Mobile phase B - volume 0 73-77 23-27 5 73-77 23-27 50 60 40 60 60 40 61 73-77 23-27 70 73-77 23-27 .

[0022] Most preferably, the procedure of the linear gradient elution is as follows:

[0023] Time (minutes) Mobile phase A - volume Mobile phase B - volume 0 75 25 5 75 25 50 60 40 60 60 40 61 75 25 70 75 25 .

[0024] Furthermore, the flow rate is 1.0-2.0 mL / min; and the column temperature is 20-30°C.

[0025] Preferably, the flow rate is 1.4 to 1.6 mL / min; the column temperature is 23 to 27°C. For example, the flow rate is 1.4 mL / min ( Figure 8 ); or flow rate of 1.5mL / min ( Figure 4 ); or flow rate of 1.6 mL / min ( Fig. 9 ). For example, the column temperature is 23°C ( Fig.10 ); or column temperature is 24°C; or column temperature is 25°C ( Figure 4 ); or column temperature is 26°C; or column temperature is 27°C ( Fig.11 ).

[0026] Most preferably, the flow rate is 1.5 mL / min and the column temperature is 25°C.

[0027] Preferably, the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

[0028] Most preferably, the chromatographic column is Agilent ZORBAX Eclipse XDB-C18, 4.6 mm×250 mm, 5 μm.

[0029] Preferably, the injection volume is 50 μl.

[0030] Preferably, the running time is 70 minutes.

[0031] The second object of the present invention is to provide a method for identifying the calcitriol intermediate Z3 and its isomeric impurities, which method can complete the identification of multiple substances in a relatively short time.

[0032] To achieve the above object, the technical solution of the present invention is:

[0033] The method for identifying the calcitriol intermediate Z3 and its isomeric impurities comprises separating the composition by the aforementioned method, and entering a detector with a detection wavelength of 264±10 nm for detection to obtain a chromatogram; and determining whether the test sample contains the calcitriol intermediate Z3 and its isomeric impurities by comparing the chromatogram characteristics of the test sample with those of the reference sample.

[0034] About the detection wavelength: The setting range of ±10nm is based on the comprehensive consideration of many factors such as error considerations, method superiority and practical application requirements. This setting range helps to ensure the reliability of the test results, improve the repeatability and flexibility of the measurement, and meet the requirements of specific experiments.

[0035] Most preferably, the detection wavelength is 264 nm.

[0036] As a preferred solution, the components in the composition can be identified according to the order of relative retention time. The components of the composition are in ascending order: calcitriol intermediate Z3, impurity Z 3b .

[0037] 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).

[0038] As a preferred solution, the calcitriol intermediate Z3 is used as the reference peak, and the relative retention time is 1.05±0.02, which is determined to be the impurity Z 3b .

[0039] 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.

[0040] The third object of the present invention is to provide a method for determining the impurity content of the calcitriol intermediate Z3 and its isomers, which can complete the identification and content determination of multiple substances in a relatively short period of time.

[0041] To achieve the above object, the technical solution of the present invention is:

[0042] The method for determining the impurity content of the calcitriol intermediate Z3 and its isomers is to separate and identify the calcitriol intermediate Z3 and its isomers impurities by the above method to obtain a chromatogram; based on the obtained chromatogram, the impurity content is calculated by the main component self-reference method.

[0043] Through content determination, we can further determine whether the impurity content of calcitriol intermediate Z3 and its isomers is qualified. 3b The peak area of ​​the impurity Z is greater than the peak area of ​​the corresponding impurity in the control solution, indicating that the impurity content is unqualified; on the contrary, if the impurity Z 3b The peak area of ​​the impurity is not greater than the peak area of ​​the corresponding impurity in the control solution, indicating that the impurity content is qualified.

[0044] 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.

[0045] As a preferred solution, the sample preparation solvent is a mixed solution of ethyl acetate and acetonitrile.

[0046] As a preferred solution, in the sample preparation solvent, the volume ratio of ethyl acetate to acetonitrile is 70:30.

[0047] As a preferred solution, before separation, the sample preparation solution is used to prepare a test solution, and the test solution includes a sample solution, a control solution and / or a system suitability solution. The specific steps are as follows:

[0048] (1) Sample solution: Accurately weigh 10.41 mg of this product (calcitriol intermediate Z3), accurately weigh it, place it in a 10 ml volumetric bottle, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready. (Prepare before use)

[0049] (2) Control solution: Accurately pipette 0.5 ml of the sample solution into a 100 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0050] The beneficial effects of the present invention are:

[0051] 1. The present invention provides a proprietary and sensitive high performance liquid chromatography method for separating and determining the impurity Z in the calcitriol intermediate Z3. 3b This method can achieve the impurity Z in the calcitriol intermediate Z3 3b Effective separation and determination of impurity Z 3b Effective control can fundamentally determine the product quality, and it has the advantages of being exclusive, sensitive, simple, fast and highly accurate.

[0052] 2. The method of the present invention has the characteristics of good separation effect and short separation time (70 minutes). The method creatively uses a reverse phase chromatographic column to elute impurities with a normal phase mobile phase, and adopts a gradient elution method to improve the peak shape and separation effect of impurities. The method is of great significance for achieving quality control of the calcitriol intermediate Z3.

[0053] 3. The method of the present invention has the characteristic of high sensitivity. Under this chromatographic system, the quantitative limit concentration of the calcitriol intermediate Z3 is 0.3911μg / ml, and the detection limit concentration is 0.1173μg / ml; the impurity Z 3b The limit of quantification was 0.3897 μg / ml and the limit of detection was 0.1169 μg / ml.

[0054] 4. The method of the present invention has the characteristics of good durability. When the flow rate, column temperature and mobile phase ratio have slight fluctuations, the valley height ratio between the main peak and the adjacent impurity peaks and between the impurity peaks are all greater than 2.0. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is the HPLC chart of blank solution;

[0056] Figure 2 It is the HPLC chart of the test solution;

[0057] Figure 3 HPLC chart of self-control solution;

[0058] Figure 4 is the HPLC chart of the mixed solution;

[0059] Figure 5 HPLC chart of the quantitative limit solution;

[0060] Figure 6 HPLC graph of the detection limit solution;

[0061] Figure 7 This is the HPLC chart under normal conditions in the durability experiment;

[0062] Figure 8 This is the HPLC chart at a flow rate of 1.4 ml / min in the durability experiment;

[0063] Fig. 9 This is the HPLC chart at a flow rate of 1.6 ml / min in the durability experiment;

[0064] Fig.10 This is the HPLC chart with a column temperature of 23°C in the durability experiment;

[0065] Fig.11 This is the HPLC chart with the column temperature at 27°C in the durability experiment;

[0066] Fig.12 HPLC chart of the initial ratio of mobile phase in the durability experiment: methanol-acetonitrile-ethyl acetate (58:17:25);

[0067] Fig.13 HPLC chart of the initial ratio of mobile phase in the durability experiment: methanol-acetonitrile-ethyl acetate (62:13:25);

[0068] Fig.14 HPLC chart of the initial ratio of mobile phase in the durability experiment: methanol-acetonitrile-ethyl acetate (62:15:23);

[0069] Fig.15 HPLC chart of the initial ratio of mobile phase in the durability experiment: methanol-acetonitrile-ethyl acetate (58:15:27). DETAILED DESCRIPTION

[0070] 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.

[0071] Supplementary table to the drawings in the specification. Figure 1-Figure 15Visual 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 1-15) and data for more detailed information. Conversely, if any information that may cause misunderstanding or ambiguity is found in the process of reviewing Tables 1-15, 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 solution in the specification.

[0072] Table 1

[0073]

[0074] Table 2

[0075]

[0076] Table 3

[0077]

[0078] Table 4

[0079]

[0080] Table 5

[0081]

[0082] Table 6

[0083]

[0084] Table 7

[0085]

[0086] Table 8

[0087]

[0088] Table 9

[0089]

[0090] Table 10

[0091]

[0092] Table 11

[0093]

[0094] Table 12

[0095]

[0096] Table 13

[0097]

[0098] Table 14

[0099]

[0100] Table 15

[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 Huabang Shengkai Pharmaceutical Co., Ltd., the test sample batch number is CAT-Z3-231101, and the reference sample batch number is CAT-Z 3b -230501.

[0112] Example 1. Determination of related substances of calcitriol intermediate Z3

[0113] (1) Prepare the test solution

[0114] Diluent: a mixed solution of ethyl acetate and acetonitrile, wherein the volume ratio of ethyl acetate to acetonitrile is 70:30.

[0115] Test solution: accurately weigh 10.41 mg of the test sample, accurately weigh it, put it in a 10 ml volumetric bottle, add diluent to dissolve and dilute to the scale, shake well, and you have it. (Prepare before use)

[0116] Preparation of control solution: Accurately pipette 0.5 ml of the test solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0117] Impurity Z 3b Stock solution: weigh impurity Z 3b 12.96 mg of reference substance is accurately weighed and placed in a 25 ml volumetric flask. Add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0118] Mixed solution (system suitability solution): weigh 10.72 mg of the test sample, accurately weigh it, and place it in a 10 ml volumetric flask. 3b Place 1 ml of the stock solution in the same volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0119] (2) Chromatographic conditions

[0120] High performance liquid chromatograph: Shimadzu LC-2030 high performance liquid chromatograph (LC3001); chromatographic column: octadecylsilane bonded silica gel as filler (Agilent ZORBAX Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm or chromatographic column with equivalent performance); mobile phase A: a mixed solution of methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is 80:20, mobile phase B: ethyl acetate; elution mode: gradient elution, the gradient elution program is shown in Table A; wavelength: 264 nm; column temperature: 25 °C; flow rate: 1.5 ml / min; injection volume: 50 μl.

[0121] Table A. Gradient elution schedule

[0122] Time (minutes) Mobile phase A - volume Mobile phase B - volume 0 75 25 5 75 25 50 60 40 60 60 40 61 75 25 70 75 25

[0123] (3) Determination

[0124] Take the diluent, test solution, control solution and mixed solution respectively for HPLC analysis under the above chromatographic conditions and record the chromatogram.

[0125] System suitability results are shown in Table B and spectra are shown in Figure 1-Figure 4 The integration results are shown in Tables 1-4. From the results, it can be seen that the diluent does not interfere with the detection of the sample. The peak-to-valley ratio between the main peak and the adjacent impurity peaks and between each impurity peak is greater than 2.0. According to the main component self-control method, the impurity Z in the test solution 3b The content of calcitriol is 0.13%, and the content of other individual impurities is 0.39%, both of which are within the prescribed limits. Therefore, the related substances will be effectively removed during the synthesis and post-treatment of the calcitriol intermediate Z3.

[0126] Table B. System suitability test results

[0127]

[0128] Example 2. Selection of elution method

[0129] The separation effect of the calcitriol intermediate Z3 related substances was investigated by isocratic elution and gradient elution with methanol-ethyl acetate (70:30). The experimental results are shown in Table C. By isocratic elution, the main peak of Z3 and the known impurity Z 3b Between, impurities Z 3b The separation effect between Z3 and other single impurities is poor, almost no separation effect. When gradient elution is used, the main peak of Z3 is different from the known impurity Z 3b Between, impurities Z 3b The peak-to-valley ratios with other individual impurities were greater than 2.0, indicating a good separation effect. Finally, gradient elution was used to separate Z3-related substances.

[0130] Table C. Elution method selection results table

[0131]

[0132] Example 3. Basic research on the quantitative limit and detection limit of related substances of calcitriol intermediate Z3

[0133] (1) Prepare the test solution

[0134] Impurity Z 3b Stock solution: weigh impurity Z 3b Place 12.96 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.

[0135] Test sample stock solution: Accurately weigh 25.16 mg of the test sample, place it in a 25 ml volumetric flask, dissolve it with diluent and dilute to the scale, shake well, and obtain.

[0136] Linear stock solution: Take 2.5 ml of the test sample stock solution and impurity Z 3b Put 5.0ml of stock solution into the same 50ml volumetric flask, dilute to the mark with diluent, shake well, and you have it.

[0137] Quantitative limit solution: Accurately pipette 0.8 ml of the linear stock solution into a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, and the solution is ready.

[0138] Detection limit solution: Accurately pipette 3.0 ml of the quantitative limit solution into a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0139] (2) Determination

[0140] Take the above quantitative limit solution and inject it 6 times continuously, calculate the RSD and signal-to-noise ratio of each peak area; take the detection limit solution and inject it 3 times continuously, calculate the signal-to-noise ratio of each peak. Record the chromatogram.

[0141]

[0142] The test results are shown in Table D to Table E. Figure 5-6 , Table 5 to Table 6. From the experimental data in Table D and Table E, it can be seen that under this chromatographic system, the quantitative limit concentration of CAT-Z3 is 0.3911μg / ml, the RSD of the peak area is 3.2%, and the average signal-to-noise ratio is 25.1; impurity Z 3b The limit of quantification of impurity Z was 0.3897 μg / ml, which was expressed as 0.04% in the sample. The RSD of the peak area was 2.5%, and the average signal-to-noise ratio was 24.8. The limit of detection of impurity Z3 was 0.1173 μg / ml, and the average signal-to-noise ratio was 6.4. 3b The detection limit concentration is 0.1169 μg / ml, which is expressed as 0.01% in the test sample, and the signal-to-noise ratio is 7.3 on average. 3b The detection limit and quantification limit of the assay met the requirements of S / N=3(10):1 respectively.

[0143] Table D. Quantitation limit determination results

[0144]

[0145] Table E. Detection limit determination results

[0146]

[0147] Example 4. Basic study on durability of related substances of calcitriol intermediate Z3

[0148] (1) Prepare the test solution

[0149] Impurity Z 3b Stock solution: weigh impurity Z 3b Place 12.96 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.

[0150] Mixed solution: Accurately weigh 10.56 mg of the test sample and place it in a 10 ml volumetric flask. Then accurately measure the impurity Z 3b Place 100 μl of the stock solution in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0151] (2) Determination

[0152] Take the chromatographic conditions in Example 1 as normal conditions, adjust the experimental conditions (initial proportion of mobile phase ± 2%, flow rate ± 0.1 ml / min, column temperature ± 2°C), test them respectively after the instrument system is stable, record the chromatogram, and examine the valley height ratio between each peak.

[0153] The results are shown in Table F. Figure 7 to Figure 15 , Table 7 to Table 15, when the flow rate, column temperature, and mobile phase ratio have slight fluctuations, the valley height ratios between the main peak and the adjacent impurity peaks and between the impurity peaks are all greater than 2.0, which meets the requirements.

[0154] Table F. Durability test results

[0155]

[0156] Note: Under normal conditions, the initial ratio of the mobile phase is converted to methanol-acetonitrile-ethyl acetate, which is 60:15:25.

Claims

1. A method for separating calcitriol intermediate Z3 and its isomeric impurities based on high performance liquid chromatography, characterized in that: The isomeric impurity is impurity Z 3b , the calcitriol intermediate Z3 and the impurity Z 3b together constitute a composition; The structural formulas of the compounds are as follows: In the high performance liquid chromatography method, the mobile phase is a mixed solution of methanol and acetonitrile as mobile phase A, and ethyl acetate as mobile phase B, wherein the volume ratio of methanol to acetonitrile in the mobile phase A is 50-70:5-25; the stationary phase is a chromatographic column using octadecylsilane bonded silica gel as a filler, and linear gradient elution is performed.

2. The method according to claim 1, characterized in that The procedure of the linear gradient elution is as follows: 。 3. The method according to claim 2, characterized in that The procedure of the linear gradient elution is as follows: 。 4. The method according to claim 1, characterized in that The volume ratio of methanol to acetonitrile in the mobile phase A is 58-62:13-17.

5. The method according to claim 1, characterized in that The flow rate is 1.0-2.0 mL / min; the column temperature is 20-30°C.

6. A method for identifying calcitriol intermediate Z3 and its isomeric impurities, characterized in that: The composition is separated by the method described in any one of claims 1 to 5, and is detected by a detector with a detection wavelength of 264±10 nm to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample with those of the reference sample, it is determined whether the test sample contains the calcitriol intermediate Z3 and its isomer impurities.

7. The method according to claim 6, 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: calcitriol intermediate Z3, impurity Z 3b .

8. The method according to claim 7, characterized in that Taking the calcitriol intermediate Z3 as the reference peak, the relative retention time was 1.05±0.02, which was determined to be the impurity Z 3b .

9. A method for determining the impurity content of calcitriol intermediate Z3 and its isomers, characterized in that: The method described in any one of claims 6 to 8 is used to separate and identify the calcitriol intermediate Z3 and its isomeric impurities to obtain a chromatogram; and the impurity content is calculated based on the obtained chromatogram using the main component self-reference method.

10. The method according to claim 9, characterized in that The sample preparation solvent was a mixed solution of ethyl acetate and acetonitrile.

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