Method for separating and determining calcitriol intermediate Z2 and impurity DMP

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining calcitriol intermediate Z2 and impurity DMP. According to the method, the calcitriol intermediate Z2 and the impurity DMP are separated and measured by adopting high performance liquid chromatography, an AQ column with octadecylsilane chemically bonded silica as a filler is adopted as a chromatographic column, a phosphoric acid aqueous solution is adopted as a mobile phase A, an organic solvent is adopted as a mobile phase B, and the calcitriol intermediate Z2 and the impurity DMP are separated through linear gradient elution. Then detecting in a detector to obtain a chromatogram; and finally quantifying by adopting a limit method. According to the method, the impurity DMP in the calcitriol intermediate Z2 is effectively separated and measured, effective control over the impurity is achieved, the product quality is fundamentally determined, and the method has the advantages of being simple, convenient, rapid, high in specificity, high in sensitivity and high in accuracy.

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 Z2 and an impurity DMP. Background Art

[0002] Calcitriol is one of the most important active metabolites of vitamin D3. It plays an important role in regulating blood calcium and phosphorus concentrations, preventing and treating osteoporosis and other physiological functions in the human body. Calcitriol can be made into various preparations for the treatment of diseases related to vitamin D3 deficiency, such as 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 its molecular formula is C 27 H 44 O3, molecular weight is 416.64, structural formula is shown in formula III; 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 IV.

[0004]

[0005] DMP, also known as Dess-Martin reagent, Dess-Martin oxidant, Dess-Martin Periodinane, has a chemical name of (1,1,1-triacetoxy)-1,1-dihydro-1,2-benzidoyl-3(1H)-one, and its structural formula is shown in Formula II. In the synthesis of calcitriol API, the starting material SM1 is usually used to undergo an oxidation reaction with DMP to obtain the calcitriol intermediate Z1. The main function of DMP is to oxidize SM1 as an oxidant to obtain the intermediate Z1, and it will be added in excess during the reaction. In order to prevent the residual DMP from affecting the subsequent reaction, a certain post-treatment method will be used to remove it after the first step of the reaction. In order to determine whether the impurity DMP is effectively removed, a method needs to be established to measure it. However, since the intermediate Z1 is unstable, generally only the intermediate Z1 reaction solution is taken for TLC control, and then a chromatographic method is established in the subsequent more stable calcitriol intermediate Z2 to separate and measure the impurity DMP to control the quality of the final product calcitriol.

[0006] The above starting material SM1 is the raw material for the synthesis of calcitriol, and its chemical name is (1R, 3aR, 7aR)-1-((R)-6-hydroxy-6-methylhept-2-yl)-7-methyloctahydro-1H-inden-4-ol, and its molecular formula is C 18 H 34 O2, molecular weight is 282.46, structural formula is shown in V. Calcitriol intermediate Z1 is a key intermediate in the synthesis of calcitriol, its chemical name is (1R, 3aR, 7aR)-1-((R)-6-hydroxy-6-methylhept-2-yl)-7a-methyloctahydro-4H-inden-4-one, molecular formula is C 18 H 32 O2, molecular weight 280.45, structural formula as shown in formula VI. Calcitriol intermediate Z2 is a key intermediate in the synthesis of calcitriol, its chemical name is (1R, 3aR, 7aR)-7a-methyl-1-((R)-6-methyl-6-((triethylsilyl)oxy)hept-2-yl)octahydro-4H-inden-4-one, molecular formula C 24 H 46 O2Si, molecular weight is 394.71, and the structural formula is shown in Formula Ⅰ.

[0007]

[0008] Currently, there is a lack of methods that can effectively detect the intermediate Z2 of calcitriol and the impurity DMP. In order to ensure the quality of the finished product of calcitriol, it is necessary to establish an efficient, exclusive and sensitive method to separate and determine the intermediate Z2 of calcitriol and the impurity DMP. 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 Z2 and the impurity DMP based on high performance liquid chromatography, which method can separate the calcitriol intermediate Z2 and the impurity DMP in a shorter time.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] A method for separating a calcitriol intermediate Z2 and an impurity DMP based on high performance liquid chromatography, wherein the calcitriol intermediate Z2 and the impurity DMP together constitute a composition to be tested; the method comprises: using an AQ column with octadecylsilane bonded silica gel as a filler as a chromatographic column, using aqueous phosphoric acid solution as a mobile phase A and acetonitrile as a mobile phase B, and separating the calcitriol intermediate Z2 and the impurity DMP by linear gradient elution; the structural formula of the calcitriol intermediate Z2 is shown in Formula I, and the structural formula of the impurity DMP is shown in Formula II:

[0012]

[0013] The separated substances can also be used in production.

[0014] Furthermore, the composition to be tested further comprises any one or more of triethylsilanol, 4-dimethylaminopyridine, imidazole, DMP byproduct and glacial acetic acid; the structural formula of the DMP byproduct is shown in Formula VII;

[0015]

[0016] Part of DMP will remove acetic acid during the reaction and generate DMP by-product. The chemical name of the DMP by-product is 1-acetoxy-1,2-benzyliodine-3-(1H)-one, the molecular formula is C9H7IO4, and the molecular weight is 306.05.

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

[0018] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 85-95:15-5;

[0019] At 4 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 85-95:15-5;

[0020] At 5 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0021] At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0022] At 31 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 85-95:15-5;

[0023] At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 85-95:15-5.

[0024] Preferably, the procedure of the linear gradient elution is as follows:

[0025] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 89-91:11-9;

[0026] At 4 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 89-91:11-9;

[0027] At 5 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0028] At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0029] At 31 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 89-91:11-9;

[0030] At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 89-91:11-9.

[0031] Most preferably, the procedure of the linear gradient elution is as follows:

[0032] At 0 minute, the volume ratio of the mobile phase A to the mobile phase B was set to 90:10;

[0033] At 4 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90:10;

[0034] At 5 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0035] At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0036] At 31 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90:10;

[0037] At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90:10.

[0038] Furthermore, the pH value of the phosphoric acid aqueous solution is 2.5-3.5; the flow rate is 0.5 mL / min-1.5 mL / min; the column temperature is 25° C.-35° C.; and the injection plate temperature is 3° C.-5° C.

[0039] Preferably, the mobile phase A is a phosphoric acid aqueous solution with a pH value of 2.8 to 3.2.

[0040] Most preferably, the mobile phase A is a phosphoric acid aqueous solution with a pH value of 3.0.

[0041] Furthermore, phosphoric acid is used to adjust the pH value of the mobile phase A.

[0042] Preferably, the flow rate is 0.9 mL / min to 1.1 mL / min, and most preferably 1.0 mL / min.

[0043] Preferably, the column temperature is 28°C to 32°C, most preferably 30°C.

[0044] Preferably, the injection plate temperature is 4°C.

[0045] Furthermore, the injection volume was 20 μl.

[0046] Furthermore, the running time is 40 min.

[0047] Preferably, the chromatographic column has a specification of 4.6 mm×250 mm and 5 μm.

[0048] Most preferably, the chromatographic column is ChromCoreAQ-C18 4.6 mm×250 mm, 5 μm or a chromatographic column with equivalent performance.

[0049] Furthermore, the method is a method for separating the impurity DMP in the calcitriol intermediate Z2 and its related substances; the related substances include any one or more of triethylsilanol, 4-dimethylaminopyridine, imidazole, DMP byproduct and glacial acetic acid.

[0050] The second object of the present invention is to provide a method for identifying the impurity DMP in the calcitriol intermediate Z2, which can complete the identification of the impurity DMP in the calcitriol intermediate Z2 in a relatively short time, and the impurity detection limit concentration is as low as 0.0574 μg / ml.

[0051] To achieve the above object, the present invention adopts the following technical solutions:

[0052] The method for identifying the impurity DMP in the calcitriol intermediate Z2 is to separate the composition by the aforementioned separation method, and enter the 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 impurity DMP.

[0053] Preferably, the detector is an ultraviolet detector.

[0054] Furthermore, the detection wavelength of the detector is 250±10 nm, preferably 250 nm.

[0055] Furthermore, the retention time was 2.9±0.5 min, which was determined to be the impurity DMP.

[0056] Preferably, the retention time is 3.2±0.5 min, and it is determined to be 4-dimethylaminopyridine.

[0057] The impurity DMP can be qualitatively identified based on the relative retention time and / or retention time.

[0058] The third object of the present invention is to provide a method for detecting whether the content of impurity DMP in calcitriol intermediate Z2 is qualified. The method can complete the determination of impurity DMP in calcitriol intermediate Z2 in a relatively short time, and the quantitative limit concentration of the impurity is as low as 0.1912 μg / ml.

[0059] To achieve the above object, the present invention adopts the following technical solutions:

[0060] The method for detecting whether the content of impurity DMP in calcitriol intermediate Z2 is qualified is to separate and identify the impurity DMP in calcitriol intermediate Z2 by the aforementioned identification method to obtain a chromatogram; based on the obtained chromatogram, the limit method is used to determine whether the impurity content in each test product is qualified.

[0061] Furthermore, if the peak area of ​​the impurity DMP in the test sample is not greater than the peak area of ​​the impurity DMP in the reference solution, it indicates that the impurity DMP content is qualified; conversely, if the peak area of ​​the impurity DMP in the test sample is greater than the peak area of ​​the impurity DMP in the reference solution, it indicates that the impurity DMP content is unqualified; the limit of the impurity DMP is 0.10%.

[0062] Furthermore, the sample preparation solvent is a phosphoric acid acetonitrile solution.

[0063] Preferably, the sample preparation solvent is a phosphoric acid acetonitrile solution with a concentration of 0.05%.

[0064] Furthermore, the concentration of the sample solution is preferably 2 mg / mL; the concentration of the reference solution is preferably 2 μg / mL.

[0065] Preferably, the determination method specifically comprises the following steps:

[0066] 1. Take an appropriate amount of this product, weigh it accurately, add the sample preparation solvent to dissolve it and quantitatively dilute it to make a solution with a concentration of about 2 mg / mL as the sample solution;

[0067] 2. Take an appropriate amount of impurity DMP reference substance, accurately weigh it, add the sample preparation solvent to dissolve it and quantitatively dilute it to make a solution with a concentration of about 2 μg / mL as the reference substance solution;

[0068] 3. Octadecylsilane bonded silica gel was used as the filler, the chromatographic column specifications were 4.6 mm × 250 mm, 5 μm, phosphoric acid aqueous solution with a pH value of 2.8 to 3.2 was used as the mobile phase A, acetonitrile was used as the mobile phase B, and linear gradient elution was performed; the flow rate was 0.9 ml / min to 1.1 ml / min; the detection wavelength was 250 nm; the column temperature was 28°C to 32°C; the injection volume was 20 μl; the injection plate temperature was 4°C; the gradient elution program was as follows:

[0069] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 89-91:11-9;

[0070] At 4 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 89-91:11-9;

[0071] At 5 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0072] At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0073] At 31 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 89-91:11-9;

[0074] At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 89-91:11-9;

[0075] 4. Take the sample solution and the reference solution, inject them for testing respectively, and obtain a chromatogram; and use the limit method to detect whether the impurity content is qualified.

[0076] The beneficial effects of the present invention are:

[0077] 1. The present invention proposes for the first time a method for separating and determining the impurity DMP in the calcitriol intermediate Z2. The method can effectively separate the impurity DMP in the calcitriol intermediate Z2, effectively control the impurities, fundamentally determine the product quality, and has the advantages of being exclusive, sensitive, simple, rapid, and highly accurate.

[0078] 2. The method for separating and determining the impurity DMP in the calcitriol intermediate Z2 proposed in the present invention can achieve effective separation of the impurity DMP in the calcitriol intermediate Z2 within 40 minutes, and is not interfered by other substances such as triethylsilanol, 4-dimethylaminopyridine, imidazole, DMP by-product, glacial acetic acid, etc., and has the characteristics of good separation and strong specificity.

[0079] 3. The method of the present invention has the characteristics of high sensitivity. The quantitative limit concentration of the impurity DMP is as low as 0.1912 μg / ml, the detection limit concentration is as low as 0.0574 μg / ml, and the S / N is ≥3:1.

[0080] 4. The method of the present invention is used to separate and determine the impurity DMP in the calcitriol intermediate Z2. When there are slight fluctuations in the column temperature, flow rate, initial ratio of the mobile phase, and pH value of the mobile phase A, there is no obvious difference in the relative retention time of the impurity; and the separation degree between the impurity DMP and the adjacent peaks under various conditions is greater than 1.5, indicating that the method has the characteristics of strong durability and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 is the chromatogram of the blank solution;

[0082] Figure 2 is the chromatogram of the mixed solution;

[0083] Figure 3 is the chromatogram of the reference solution;

[0084] Figure 4 is the chromatogram of the test solution;

[0085] Figure 5 is the chromatogram of the quantitative limit solution; Figure 5 -A is the full chromatogram of the quantitative limit solution; Figure 5 -B is a partial enlarged view of the quantitative limit solution;

[0086] Figure 6 is the chromatogram of the detection limit solution; Figure 6 -A is the full chromatogram of the detection limit solution; Figure 6 -B is a partial enlarged view of the detection limit solution;

[0087] Figure 7 This is the chromatogram of the spiked test solution in the standard addition experiment;

[0088] Figure 8 This is the chromatogram of the mixed solution when the pH value of the phosphoric acid aqueous solution is 2.8 in the durability experiment;

[0089] Fig. 9 This is the chromatogram of the mixed solution when the pH value of the phosphoric acid aqueous solution is 3.2 in the durability experiment;

[0090] Fig.10 This is the chromatogram of the mixed solution at a flow rate of 0.9 ml / min in the durability experiment;

[0091] Fig.11This is the chromatogram of the mixed solution at a flow rate of 1.1 ml / min in the durability experiment;

[0092] Fig.12 This is the chromatogram of the mixed solution when the column temperature is 28°C in the durability experiment;

[0093] Fig.13 This is the chromatogram of the mixed solution when the column temperature is 32°C in the durability experiment;

[0094] Fig.14 This is the chromatogram of the mixed solution when the initial ratio of the mobile phase in the durability experiment was phosphoric acid aqueous solution: acetonitrile = 89:11;

[0095] Fig.15 This is the chromatogram of the mixed solution when the initial ratio of the mobile phase in the durability experiment was phosphoric acid aqueous solution: acetonitrile = 91:9. DETAILED DESCRIPTION

[0096] 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.

[0097] In the present invention, the sample to be tested is a solution whose main component is the calcitriol intermediate Z2, and the sample to be tested contains either or both of the calcitriol intermediate Z2 and the impurity DMP.

[0098] The sample to be tested may also contain any one or more compounds of triethylsilanol, 4-dimethylaminopyridine, imidazole, DMP byproduct, glacial acetic acid, and other unspecified compounds, wherein the other unspecified compounds refer to other impurities that may be introduced when preparing the calcitriol intermediate Z2 using conventional methods known in the art.

[0099] Unless otherwise specified, all numerical values ​​and similar values ​​used in the present invention to express parameters are understood to be modified by the term "about". That is, these numerical values ​​are allowed to have certain reasonable deviations or error ranges in practical applications, and do not have to strictly conform to the exact numerical values ​​given. Unless otherwise indicated, the numerical parameters described in the present invention are approximate values ​​that can be changed according to the performance required to be obtained.

[0100] Unless otherwise stated, terms or phrases involved in the present invention should not be considered as uncertain or unclear if there is no special definition, but should be understood according to the ordinary meaning in the art.

[0101] In the present invention, the term "high performance liquid chromatography", also known as "high pressure liquid chromatography", "high speed liquid chromatography", "high resolution liquid chromatography", "modern column chromatography", etc., is a chromatographic analysis technology based on classical liquid chromatography and citing the theory of gas chromatography. High performance liquid chromatography (HPLC) uses liquid as the mobile phase and adopts a high-pressure infusion system to pump a single solvent with different polarities or a mixed solvent with different proportions, a buffer and other mobile phases into a chromatographic column equipped with a stationary phase. After the components in the column are separated, they enter the detector for detection, thereby realizing the analysis of the sample. This method can be used to separate, identify and quantitatively analyze various complex samples.

[0102] In the present invention, specificity, also known as selectivity or specificity, refers to the ability of the analytical method to accurately and selectively detect the component being measured when other components may be present in the sample. Specificity is a measure of mutual interference when the method is used for complex sample analysis. The present invention verifies whether the HPLC method has sufficient specificity to ensure accurate and unerring determination of the target analyte in complex samples.

[0103] In the present invention, the limit of detection (LOD) refers to the minimum concentration or amount of the target compound that the analytical method can reliably detect. The detection sensitivity of the HPLC method is determined, that is, the lowest concentration of the target analyte in the sample that can be identified. This helps to evaluate the reliability of the method at very low concentrations.

[0104] In the present invention, the limit of quantitation (LOQ) refers to the lowest concentration or content of the target compound that the analytical method can reliably quantify. The limit of quantitation reflects the ability of the analytical method to detect sensitive quantification and determines at what concentration the HPLC method can accurately and reliably quantify the target analyte.

[0105] In the present invention, robustness refers to the degree to which the measurement results are not affected when there are small changes in the measurement conditions. It is mainly used to detect the anti-interference ability of the analytical method to variable test factors.

[0106] In the present invention, the basic information of the calcitriol intermediate Z2 and related impurities is shown in Table 1.

[0107] Table 1. Compound structural information

[0108]

[0109] In the examples of the present invention, the reference substance is derived from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0110] In the embodiment of the present invention, the diluent is a phosphoric acid-acetonitrile solution with a concentration of 0.05%.

[0111] In the embodiment of the present invention, the preparation method of the test solution is: take an appropriate amount of the product, accurately weigh it, add a diluent to dissolve it and quantitatively dilute it to prepare a solution with a concentration of about 2 mg / mL as the test solution.

[0112] In the embodiment of the present invention, the preparation method of the reference solution is: take an appropriate amount of the impurity DMP reference substance, accurately weigh it, add a solvent to dissolve it and quantitatively dilute it to prepare a solution with a concentration of about 2 μg / mL as the reference solution.

[0113] In the embodiments of the present invention, the instruments and chromatographic conditions used are as follows:

[0114] The high performance liquid chromatograph was Thermo Scientific Ultimate 3000; octadecylsilane bonded silica gel was used as filler (ChromCoreAQ-C18 4.6 mm × 250 mm, 5 μm or chromatographic column of equivalent performance), phosphoric acid aqueous solution (pH adjusted to 3.0 with phosphoric acid) was used as mobile phase A, acetonitrile was used as mobile phase B, and linear gradient elution was performed according to Table 2; the flow rate was 1.0 ml per minute; the detection wavelength was 250 nm; the column temperature was 30°C; the injection volume was 20 μl; and the injection plate temperature was 4°C.

[0115] Table 2. Gradient elution program

[0116] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 4 90 10 5 0 100 30 0 100 31 90 10 40 90 10

[0117] In the embodiment of the present invention, the determination method is: accurately measure 20 μl of the test solution and the reference solution, respectively, inject and detect, obtain a chromatogram; and use the limit method for quantification.

[0118] In the embodiments of the present invention, the impurity limit is: if the impurity DMP is detected in the test solution, the presence level (peak area) of the impurity DMP in the test solution should not be greater than the amount of the impurity DMP in the reference solution; the limit of the impurity DMP is 0.10%.

[0119] In the embodiment of the present invention, since DMP has strong polarity and is substantially unretained by conventional C18, an AQ column is selected.

[0120] Example 1. Specificity experiment

[0121] 1. Prepare the test solution

[0122] Blank solution: 0.05% phosphoric acid acetonitrile solution (add 0.5 ml of phosphoric acid to 1000 ml of acetonitrile and shake well).

[0123] Test solution: Take an appropriate amount of the product, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing about 2 mg per 1 ml.

[0124] Reference solution: Take an appropriate amount of impurity DMP reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 2 μg per 1 ml.

[0125] Impurity localization solutions: (triethylsilanol, 4-dimethylaminopyridine, imidazole, DMP byproduct and glacial acetic acid may also be present in the calcitriol intermediate Z2), weigh appropriate amounts of each impurity, add diluent to dilute to make a 2 μg / ml solution as a single needle localization solution.

[0126] Mixed solution: A mixed solution containing the calcitriol intermediate Z2, the impurity DMP and the above-mentioned impurities (each 1 ml contains about 2 mg of the intermediate Z2 and about 2 μg of each impurity).

[0127] 2. Determination

[0128] Take the blank solution (diluent), test solution, reference solution, each impurity location solution and mixed solution respectively, perform HPLC analysis according to the above chromatographic conditions, and record the chromatogram.

[0129] The test results are shown in Table 3-5. Figure 1 to Figure 4 As shown, Figure 2 and Figure 3 The integration results are shown in Table 4 and Table 5. The results show that the diluent does not interfere with the detection of the sample; according to the limit method, the impurity DMP in the test solution is not detected, and its existence level is much lower than the amount of impurity DMP in the reference solution (0.10%), so the raw material DMP that is not completely reacted during the synthesis of calcitriol will be effectively removed.

[0130] Table 3. Specificity test results

[0131]

[0132] Table 4. Figure 2 The integral result table

[0133]

[0134] Table 5. Figure 3 The integral result table

[0135]

[0136] Example 2. Selection of diluent

[0137] This example further investigates the diluents, using ChromCoreAQ-C18 (250 mm×4.6 mm, 5 μm), and using acetonitrile and 0.05% phosphoric acid acetonitrile solution as diluents.

[0138] The results showed that the impurity DMP would degrade when pure acetonitrile was used as the diluent, and the spiked recovery rate of the impurity DMP was 32.5%. After using 0.05% phosphoric acid acetonitrile solution as the diluent, the spiked recovery rate of the impurity DMP was 92.4%, which was in the range of 90% to 108%, indicating that the degradation of the impurity DMP was improved.

[0139] Example 3. Sensitivity experiment

[0140] Quantitative limit solution: Weigh an appropriate amount of impurity DMP, add diluent to dissolve and dilute to make a solution containing 0.1912 μg of the impurity per 1 ml.

[0141] Detection limit solution: Take the quantitative limit solution and dilute it with diluent to make a solution containing 0.0574μg of the impurity per 1ml.

[0142] Take the above quantitative limit solution and detection limit solution, and inject them for detection respectively.

[0143] Results: The quantitative limit spectra and detection limit spectra are as follows: Figure 5 and Figure 6 The integration results are shown in Table 6 and Table 7. The quantitative limit concentration of the impurity DMP is 0.1912 μg / ml, which is 0.01% in the intermediate Z2, S / N is ≥10:1, and the peak area RSD is less than 10.0%. The detection limit concentration of the impurity DMP is 0.0574 μg / ml, which is 0.003% in the intermediate Z2, S / N is ≥3:1. This shows that this method has high sensitivity.

[0144] Table 6. Figure 5 The integral result table

[0145]

[0146] Table 7. Figure 6 The integral result table

[0147]

[0148] Example 4 Standard addition experiment

[0149] Reference solution: Take an appropriate amount of impurity DMP reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 2 μg per 1 ml.

[0150] Unspiked solution: Take an appropriate amount of this product, weigh accurately, dissolve it in diluent and quantitatively dilute it to make a solution containing about 2 mg per 1 ml.

[0151] Spiked test solution: Take appropriate amount of intermediate Z2 and impurity DMP, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing approximately 2 mg of intermediate Z2 and 2 μg of impurity DMP per 1 ml.

[0152] Take the reference solution, the unspiked test solution and the spiked test solution for injection. The chromatogram of the spiked test solution is as follows: Figure 7 As shown. Figure 3 ) was used as a benchmark to calculate the spiked recovery of impurity DMP. The test results are shown in Tables 8 and 9. The recovery of impurity DMP was in the range of 90% to 108%, indicating that the sample matrix had no effect on the determination of impurity DMP.

[0153] Table 8. Standard addition test results

[0154] name Peak area of ​​reference substance Peak area of ​​test product Peak area of ​​spiked test product Recovery rate (%) Impurity DMP 0.245 0.000 0.249 101.6

[0155] Table 9. Figure 7 The integral result table

[0156]

[0157] Example 5 Durability

[0158] Impurity DMP stock solution: weigh 20 mg of impurity DMP, place in a 100 ml volumetric flask, add 5 ml N, N-dimethylacetamide to dissolve, then add diluent to dilute to scale, shake well, and you have it. (Prepared fresh for use)

[0159] Triethylsilanol positioning solution: Weigh 12.5 mg of triethylsilanol, place in a 25 ml volumetric flask, add diluent to dilute to scale, shake well, and obtain triethylsilanol stock solution. Accurately pipette 0.5 ml, place in a 25 ml volumetric flask, add diluent to dilute to scale, shake well, and obtain.

[0160] 4-Dimethylaminopyridine positioning solution: Weigh 12.5 mg of 4-dimethylaminopyridine, place in a 25 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, and obtain 4-dimethylaminopyridine stock solution. Accurately pipette 0.5 ml, place in a 25 ml volumetric flask, add diluent to dilute to scale, shake well, and obtain.

[0161] Imidazole fixation solution: Weigh 12.5 mg of imidazole, place in a 25 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, and the imidazole stock solution is obtained. Accurately pipette 0.5 ml, place in a 25 ml volumetric flask, add diluent to dilute to scale, shake well, and the solution is obtained.

[0162] Glacial acetic acid fixation solution: Weigh 12.5 mg of glacial acetic acid, place in a 25 ml volumetric flask, add diluent to dilute to scale, shake well, and you will get glacial acetic acid stock solution. Accurately pipette 0.5 ml, place in a 25 ml volumetric flask, add diluent to dilute to scale, shake well, and you will get.

[0163] DMP byproduct localization solution: Weigh 10 mg of DMP byproduct, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, and obtain DMP byproduct stock solution. Accurately pipette 1.0 ml, place it in a 10 ml volumetric flask, add diluent to dilute to scale, shake well, and obtain.

[0164] Impurity mixed solution: Measure 0.1 ml each of the aforementioned triethylsilanol stock solution, 4-dimethylaminopyridine stock solution and imidazole stock solution, 0.5 ml each of the DMP byproduct stock solution and glacial acetic acid stock solution, and 0.25 ml of DMP stock solution, place them in the same 25 ml volumetric bottle, add diluent to dilute to the scale, shake well, and you have it. (Prepare for immediate use)

[0165] Mixed solution: Accurately weigh 10 mg of this product, place it in a 5 ml volumetric flask, add the impurity mixed solution to dissolve and dilute to the scale, shake well, and obtain.

[0166] Take the mixed solution and adjust the experimental conditions (flow rate ± 0.1 ml / min, column temperature ± 2 °C, initial proportion of mobile phase ± 1%, pH value of phosphoric acid aqueous solution ± 0.2). After the instrument system is stable, test them separately to examine the separation degree of impurity DMP and adjacent peaks.

[0167] Results: Figure 2 , Figure 8 to Figure 15 As shown in Tables 10 to 18, when there are slight fluctuations in column temperature, flow rate, initial ratio of mobile phase, and pH value of mobile phase A, there is no significant difference in the relative retention time of each impurity; and the separation degree between the impurity DMP and the adjacent peaks under each condition is greater than 1.5, indicating that the durability of this method meets the requirements.

[0168] Table 10. Durability test results

[0169] Chromatographic conditions Impurity DMP retention time (min) Separation degree of impurity DMP and adjacent peaks Normal conditions 2.943 2.93 Phosphoric acid aqueous solution pH 2.8 2.953 3.61 Phosphoric acid aqueous solution pH 3.2 2.987 2.89 Column flow rate 0.9ml / min 3.260 3.50 Column flow rate 1.1ml / min 2.673 3.03 Column temperature 28℃ 2.940 3.33 Column temperature 32℃ 2.937 3.36 Initial ratio of mobile phase A:B = 89:11 2.907 2.79 Initial ratio of mobile phase A:B = 91:9 2.970 3.13

[0170] Table 11. Figure 8 The integral result table

[0171]

[0172] Table 12. Fig. 9 The integral result table

[0173]

[0174] Table 13. Fig.10The integral result table

[0175]

[0176]

[0177] Table 14. Fig.11 The integral result table

[0178]

[0179] Table 15. Fig.12 The integral result table

[0180]

[0181] Table 16. Fig.13 The integral result table

[0182]

[0183] Table 17. Fig.14 The integral result table

[0184]

[0185] Table 18. Fig.15 The integral result table

[0186]

Claims

1. A method for separating a calcitriol intermediate Z2 and an impurity DMP based on high performance liquid chromatography, characterized in that: The calcitriol intermediate Z2 and the impurity DMP together constitute a composition to be tested; the method comprises: using an AQ column with octadecylsilane bonded silica gel as a filler as a chromatographic column, using phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and separating the calcitriol intermediate Z2 and the impurity DMP by linear gradient elution; the structural formula of the calcitriol intermediate Z2 is shown in Formula I, and the structural formula of the impurity DMP is shown in Formula II:

2. The method according to claim 1, characterized in that The composition to be tested further contains any one or more of triethylsilanol, 4-dimethylaminopyridine, imidazole, DMP byproduct and glacial acetic acid; the structural formula of the DMP byproduct is shown in Formula VII; 3. The method according to claim 1, characterized in that The linear gradient elution program was set up as follows: At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 85-95:15-5; At 4 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 85-95:15-5; At 5 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100; At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100; At 31 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 85-95:15-5; At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 85-95:15-5.

4. The method according to claim 1, characterized in that The pH value of the phosphoric acid aqueous solution is 2.5-3.5; the flow rate is 0.5mL / min-1.5mL / min; the column temperature is 25°C-35°C; and the injection plate temperature is 3°C-5°C.

5. A method for identifying the impurity DMP in the calcitriol intermediate Z2, characterized in that: The composition is separated by the method described in any one of claims 1 to 4, and is detected in a detector to obtain a chromatogram; and whether the test sample contains the impurity DMP is determined by comparing the chromatogram characteristics of the test sample with those of the reference sample.

6. The method according to claim 5, characterized in that The detection wavelength of the detector is 250±10 nm.

7. The method according to claim 5, characterized in that The retention time was 2.9±0.5 min, which was determined to be the impurity DMP.

8. A method for detecting whether the content of impurity DMP in calcitriol intermediate Z2 is qualified, characterized in that: The method described in any one of claims 5 to 7 is used to separate and identify the impurity DMP in the calcitriol intermediate Z2 to obtain a chromatogram; based on the obtained chromatogram, the limit method is used to determine whether the impurity content in each test product is qualified.

9. The method according to claim 8, characterized in that If the peak area of ​​the impurity DMP in the test sample is not greater than the peak area of ​​the impurity DMP in the reference solution, it indicates that the impurity DMP content is qualified; conversely, if the peak area of ​​the impurity DMP in the test sample is greater than the peak area of ​​the impurity DMP in the reference solution, it indicates that the impurity DMP content is unqualified; the limit of the impurity DMP is 0.1%.

10. The method according to claim 8, characterized in that The sample preparation solvent was phosphoric acid-acetonitrile solution.

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