Detection method of maxacalcitol related substances

Abstract

The invention provides a high performance liquid chromatography analysis method of maxacalcitol related substances. By changing the mobile phase, changing the type of the chromatographic column and adjusting the column temperature, the separation degree of a main peak and a plurality of related impurities is improved, related substances in maxacalcitol can be accurately detected, and the separation effect is outstanding. The analysis method disclosed by the invention is stable in baseline, good in stability, good in specificity and high in sensitivity, and has very important significance for comprehensively, accurately and objectively evaluating the product quality.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting related substances of a maxacalcitol raw material drug. Background Art

[0002] Maxacalcitol, chemical name: (+)-(5Z,7E)-(1S,3R,20S)-20-(3-hydroxy-3-methylbutyloxy)-9,10-openedepgan-5,7,10(19)-triene-1,3-diol, is a new third-generation active vitamin D drug developed by Chugai Pharmaceutical Co., Ltd. of Japan. Its structural formula is as follows:

[0003]

[0004] Maxacalcitol injection was approved for marketing in 2000 under the trade name Oxarol, and is used to treat secondary hyperparathyroidism (SHPT) in patients undergoing renal dialysis. After its launch, it has shown good therapeutic effects in clinical applications, and adverse reactions such as hypercalcemia are not obvious, and patients have high tolerance, making it suitable for clinical promotion. Maxacalcitol ointment was approved for marketing in Japan in 2001 for the treatment of dry moss skin diseases such as psoriasis.

[0005] The chemical properties of maxacalcitol are relatively unstable. Some by-product impurities are inevitably produced in the synthesis process of maxacalcitol API, such as impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G. There are many impurities with similar structures, which are difficult to separate. Therefore, the detection of such related substances is more difficult than the detection of other drug impurities in the development and selection of analytical methods.

[0006] At present, this variety is not included in any pharmacopoeia, and no prior art has reported a detection method for substances related to maxacalcitol. The impurities produced in the synthesis process of the maxacalcitol API will directly affect the quality control of maxacalcitol, thereby affecting the therapeutic effect of the maxacalcitol preparation. Therefore, in order to ensure the safety and effectiveness of the maxacalcitol preparation, how to provide a method that can simultaneously detect impurities A, B, C, D, E, F, and G in the maxacalcitol API has become a problem to be solved. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for detecting maxacalcitol-related substances.

[0008] The present invention adopts the following technical solutions:

[0009] The present invention provides a high performance liquid chromatography analysis method for maxacalcitol-related substances, wherein the method can separate the related substances in maxacalcitol, and the separation degree between each chromatographic peak is not less than 1.5;

[0010] According to an embodiment of the present invention, the chromatographic conditions of the method are set to:

[0011] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0012] Mobile phase: acetonitrile as mobile phase A, methanol as mobile phase B, water as mobile phase C, gradient elution as shown in the table below;

[0013] Time (min) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 40 10 50 10 45 5 50 25 50 5 45 35 65 0 35 45 60 20 20 50 100 0 0 60 100 0 0 61 40 10 50 70 40 10 50

[0014] Flow rate: 0.5~1.0ml / min;

[0015] Column temperature: 30-45°C;

[0016] Detection wavelength: 260~270nm;

[0017] Injection volume: 10-30 μl;

[0018] Sample concentration: 0.1~1mg / ml.

[0019] According to an embodiment of the present invention, the chromatographic column C18 column is selected from SHIMADZU VP-ODS, with specifications of 4.6 mm×250 mm and 5 μm.

[0020] According to the embodiment of the present invention, the column temperature is 40°C; the flow rate is 0.8 ml / min; the injection volume is 20 μl; the wavelength is 265 nm; and the sample concentration is 0.4 mg / ml.

[0021] According to an embodiment of the present invention, the related substances include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, and the separation between each chromatographic peak is not less than 1.5;

[0022]

[0023]

[0024] According to an embodiment of the present invention, the present invention provides a high performance liquid chromatography analysis method for maxacalcitol-related substances, characterized in that it comprises the following steps:

[0025] (1) Sample preparation:

[0026] Diluent: 75% acetonitrile;

[0027] Test solution: Take an appropriate amount of this product, dissolve it with a diluent and dilute it to make a solution containing about 0.4 mg of maxacalcitol per 1 ml;

[0028] Self-control solution: accurately measure 1 ml of the test solution, place it in a 20 ml volumetric flask, dilute it to the mark with diluent, shake well, then take 1 ml, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well;

[0029] Stock solution of impurity reference substance: take about 6 mg of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and about 4 mg of impurity B, accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to scale; accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with diluent, and shake well;

[0030] System suitability solution: Take about 4 mg of maxacalcitol reference substance, place it in a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve it, accurately add 1 ml of impurity reference substance stock solution, dilute to the scale with diluent, shake well, and make a mixed solution containing about 0.4 mg of maxacalcitol, 0.4 μg of impurity B, and 0.6 μg of each other impurity per 1 ml;

[0031] (2) Chromatographic condition setting:

[0032] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0033] Mobile phase: acetonitrile as mobile phase A, methanol as mobile phase B, water as mobile phase C, gradient elution;

[0034] Flow rate: 0.5~1.0ml / min;

[0035] Column temperature: 30-45°C;

[0036] Detection wavelength: 260~270nm;

[0037] Injection volume: 10-30 μl;

[0038] Sample concentration: 0.1~1mg / ml;

[0039] (4) Detection and calculation:

[0040] Accurately measure 20μ1 of system suitability solution, 20μ1 of test solution, and 20μ1 of self-control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0041] According to an embodiment of the present invention, gradient elution is performed according to the following table:

[0042] Time (min) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 40 10 50 10 45 5 50 25 50 5 45 35 65 0 35 45 60 20 20 50 100 0 0 60 100 0 0 61 40 10 50 70 40 10 50

[0043] According to an embodiment of the present invention, the chromatographic column C18 column is selected from SHIMADZU VP-ODS, with specifications of 4.6 mm×250 mm and 5 μm.

[0044] According to the embodiment of the present invention, the column temperature is 40°C; the flow rate is 0.8 ml / min; the injection volume is 20 μl; the wavelength is 265 nm; and the sample concentration is 0.4 mg / ml.

[0045] According to an embodiment of the present invention, the degree of separation between the related substances and the main peak is not less than 1.5.

[0046] According to an embodiment of the present invention, the detection limit of the impurities is: if there are impurity peaks in the chromatogram of the test solution, except for the solvent peak, the peak areas of impurities A, impurity D and impurity G shall not be greater than 1.5 times the main peak area of ​​the control solution, i.e. 0.15%, the peak area of ​​impurity B shall not be greater than 1.0 times the main peak area of ​​the control solution, i.e. 0.10%, the corrected peak areas of impurities C and impurity F shall not be greater than 1.5 times the main peak area of ​​the control solution, i.e. 0.15%, the peak area of ​​other single impurities shall not be greater than 5 times the main peak area of ​​the control solution, i.e. 0.5%, and the sum of the corrected peak areas of each impurity shall not be greater than 20 times the main peak area of ​​the control solution, i.e. 2.0%.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The present invention provides, for the first time, an analytical method for simultaneously separating impurities A, B, C, D, E, F and G in the raw material drug of maxacalcitol without any prior art reference. The present invention improves the separation degree between the main peak and multiple related impurities by changing the mobile phase, changing the type of chromatographic column and adjusting the column temperature, and has an outstanding separation effect, which is of great significance.

[0049] (2) The present invention studies various impurities in the raw material of maxacalcitol, and controls the impurities of the product more comprehensively. The detection method of the present invention can accurately detect the related substances of maxacalcitol, which is of great significance for comprehensively, accurately and objectively evaluating the quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a chromatogram of the spiked test solution under the chromatographic conditions of Example 1 of the present invention.

[0051] Figure 2 It is a chromatogram of the spiked test sample solution under the chromatographic conditions of Example 2 of the present invention.

[0052] Figure 3 This is a chromatogram of the spiked test solution under chromatographic column screening condition 1 in Example 3 of the present invention.

[0053] Figure 4 This is a chromatogram of the spiked test solution under chromatographic column screening condition 2 in Example 3 of the present invention.

[0054] Figure 5 This is a chromatogram of the spiked test solution under column temperature screening condition 1 in Example 4 of the present invention.

[0055] Figure 6 This is a chromatogram of the spiked test sample solution under column temperature screening condition 2 in Example 4 of the present invention. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto. Unless otherwise specified, the raw materials used can be obtained from the market or homemade.

[0057] In the study of related substances of the finished product of maxacalcitol of the present invention, the information of organic impurities set into the standard is shown in Table 1. The impurities in the maxacalcitol raw material are mainly derived from the degradation impurities and process impurities of the raw material. The impurities are numerous and have similar structures, making separation difficult.

[0058] Table 1 Information on organic impurities in the finished product of Maxacalcitol

[0059]

[0060]

[0061]

[0062] The present invention optimizes the best analysis conditions after a large number of experiments. By changing the mobile phase components, screening the chromatographic column and changing the column temperature, the separation between the main peak and the related impurities and between the impurities is improved. The separation degree is greater than 1.5, and the separation effect is outstanding. The detection of related substances in this product mainly undergoes the following optimization steps:

[0063] Example 1

[0064] (1) Chromatographic condition setting:

[0065] Chromatographic column: octadecyl bonded silica as filler (Thermo Accucore XL C18, 4.6 mm × 250 mm, 4 μm);

[0066] Column temperature: 32°C;

[0067] Detection wavelength: 265nm;

[0068] Flow rate: 0.8ml / min;

[0069] Injection volume: 20 μl;

[0070] Sample concentration: 0.4 mg / ml;

[0071] Mobile phase: Use acetonitrile as mobile phase A and water as mobile phase B. Perform gradient elution according to the following table:

[0072] Time (min) Mobile phase A (%) Mobile phase B (%) 0 35 65 10 35 65 30 40 60 40 70 30 45 100 0 50 100 0 51 35 65 60 35 65

[0073] (2) Diluent: 75% acetonitrile.

[0074] (3) Test solution: Take an appropriate amount of the product, dissolve it in a diluent and dilute it to make a solution containing approximately 0.4 mg of maxacalcitol per 1 ml.

[0075] (4) Self-control solution: Accurately measure 1 ml of the test solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well. Then take another 1 ml and place it in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0076] (5) Stock solution of impurity reference substance: Take approximately 6 mg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and approximately 4 mg of impurity B, accurately weigh them, place them in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0077] (6) System suitability solution: Take about 4 mg of maxacalcitol reference substance and place it in a 10 ml volumetric flask. Add an appropriate amount of diluent to dissolve it. Accurately add 1 ml of the impurity reference substance stock solution and dilute to the mark with diluent. Shake well to prepare a mixed solution containing about 0.4 mg of maxacalcitol, 0.4 μg of impurity B, and 0.6 μg of each other impurity per 1 ml.

[0078] (7) Detection: Accurately measure 20 μl of system suitability solution, 20 μl of test solution, and 20 μl of self-control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0079] The results are as follows Figure 1 As shown, a conventional C18 chromatographic column and a conventional acetonitrile-water system were selected for development. The separation degrees did not meet the requirements and the separation effect was poor. Acetonitrile and water mobile phases were tried many times, but impurities B and E could not be separated. Therefore, this method was not applicable, and attempts were made to change the mobile phase for further optimization.

[0080] Example 2 (Changing the mobile phase)

[0081] (1) Chromatographic condition setting:

[0082] Chromatographic column: octadecyl bonded silica as filler (Thermo Accucore XL C18, 4.6 mm × 250 mm, 4 μm);

[0083] Column temperature: 32°C;

[0084] Detection wavelength: 265nm;

[0085] Flow rate: 0.8ml / min;

[0086] Injection volume: 20 μl;

[0087] Sample concentration: 0.4 mg / ml;

[0088] Mobile phase: Use acetonitrile as mobile phase A, methanol as mobile phase B, and water as mobile phase C. Perform gradient elution according to the following table:

[0089]

[0090]

[0091] (2) Diluent: 75% acetonitrile.

[0092] (3) Test solution: Take an appropriate amount of the product, dissolve it in a diluent and dilute it to make a solution containing approximately 0.4 mg of maxacalcitol per 1 ml.

[0093] (4) Self-control solution: Accurately measure 1 ml of the test solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well. Then take another 1 ml and place it in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0094] (5) Stock solution of impurity reference substance: Take approximately 6 mg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and approximately 4 mg of impurity B, accurately weigh them, place them in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0095] (6) System suitability solution: Take about 4 mg of maxacalcitol reference substance and place it in a 10 ml volumetric flask. Add an appropriate amount of diluent to dissolve it. Accurately add 1 ml of the impurity reference substance stock solution and dilute to the mark with diluent. Shake well to prepare a mixed solution containing about 0.4 mg of maxacalcitol, 0.4 μg of impurity B, and 0.6 μg of each other impurity per 1 ml.

[0096] (7) Detection: Accurately measure 20 μl of system suitability solution, 20 μl of test solution, and 20 μl of self-control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0097] The results are as follows Figure 2As shown in the figure, after changing the mobile phase, impurity B and impurity E can be well separated, proving that the mobile phase is critical to separating impurity B and impurity E. Therefore, the mobile phase system with acetonitrile as mobile phase A, methanol as mobile phase B, and water as mobile phase C is suitable for the detection of related substances. Other impurities are well separated, but impurity D is directly wrapped into the main peak, and the main peak and impurity D cannot be separated. Try to change the chromatographic column to continue optimization.

[0098] Example 3 (Screening of chromatographic columns)

[0099] (I) Chromatographic column screening conditions 1

[0100] (1) Chromatographic condition setting:

[0101] Chromatographic column: octadecyl bonded silica gel as filler (Phenomen Titank C18, 4.6 mm × 250 mm, 3 μm);

[0102] Column temperature: 32°C;

[0103] Detection wavelength: 265nm;

[0104] Flow rate: 0.8ml / min;

[0105] Injection volume: 20 μl;

[0106] Sample concentration: 0.4 mg / ml;

[0107] Mobile phase: Use acetonitrile as mobile phase A, methanol as mobile phase B, and water as mobile phase C. Perform gradient elution according to the following table:

[0108] Time (min) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 40 10 50 10 45 5 50 25 50 5 45 35 65 0 35 45 60 20 20 50 100 0 0 60 100 0 0 61 40 10 50 70 40 10 50

[0109] The rest is the same as in Example 1.

[0110] The results are as follows Figure 3 As shown, under the Pheromones Titank (C18, 4.6mm×250mm, 3μm) chromatographic column, the main peak and impurity D have a separation trend, but the separation degree between impurity B and impurity E is reduced and fails to reach above 1.5. The chromatographic column is not suitable and needs to be further optimized and replaced.

[0111] (II) Chromatographic column screening conditions 2

[0112] (1) Chromatographic condition setting:

[0113] Chromatographic column: Octadecyl bonded silica gel as filler (SHIMADZU VP-ODS, 4.6 mm × 250 mm, 5 μm);

[0114] Column temperature: 32°C;

[0115] Detection wavelength: 265nm;

[0116] Flow rate: 0.8ml / min;

[0117] Injection volume: 20 μl;

[0118] Sample concentration: 0.4 mg / ml;

[0119] Mobile phase: Use acetonitrile as mobile phase A, methanol as mobile phase B, and water as mobile phase C. Perform gradient elution according to the following table:

[0120]

[0121]

[0122] The rest is the same as in Example 1.

[0123] The results are as follows Figure 4 As shown in the figure, after using the SHIMADZU VP-ODS (4.6mm×250mm, 5μm) chromatographic column, the separation of other impurities became better, all greater than 1.5. Under the conditions of this chromatographic column, although the separation between the main peak and impurity D decreased, there was a separation trend, and the separation between impurities B and impurity E met the requirements. Therefore, SHIMADZU VP-ODS (4.6mm×250mm, 5μm) was subsequently used as the chromatographic column for the analysis method of related substances. Continue to try to change the column temperature to improve the separation between the main peak and impurity D.

[0124] Example 4 (screening column temperature)

[0125] (I) Column temperature screening conditions 1

[0126] (1) Chromatographic condition setting:

[0127] Chromatographic column: Octadecyl bonded silica gel as filler (SHIMADZU VP-ODS, 4.6 mm × 250 mm, 5 μm);

[0128] Column temperature: 35 °C;

[0129] Detection wavelength: 265nm;

[0130] Flow rate: 0.8ml / min;

[0131] Injection volume: 20 μl;

[0132] Sample concentration: 0.4 mg / ml;

[0133] Mobile phase: Use acetonitrile as mobile phase A, methanol as mobile phase B, and water as mobile phase C. Perform gradient elution according to the following table:

[0134]

[0135]

[0136] The rest is the same as in Example 1.

[0137] The results are as follows Figure 5 As shown in the figure, after increasing the column temperature, the separation between the main peak and impurity D is significantly improved, and the separation between other impurities is greater than 1.5. Therefore, increasing the column temperature is beneficial to improving the separation between the main peak and impurity D. Try to continue to increase the column temperature for optimization.

[0138] (II) Column temperature screening conditions 2

[0139] (1) Chromatographic condition setting:

[0140] Chromatographic column: Octadecyl bonded silica gel as filler (SHIMADZU VP-ODS, 4.6 mm × 250 mm, 5 μm);

[0141] Column temperature: 40°C;

[0142] Detection wavelength: 265nm;

[0143] Flow rate: 0.8ml / min;

[0144] Injection volume: 20 μl;

[0145] Sample concentration: 0.4 mg / ml;

[0146] Mobile phase: Use acetonitrile as mobile phase A, methanol as mobile phase B, and water as mobile phase C. Perform gradient elution according to the following table:

[0147] Time (min) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 40 10 50 10 45 5 50 25 50 5 45 35 65 0 35 45 60 20 20 50 100 0 0 60 100 0 0 61 40 10 50 70 40 10 50

[0148] The rest is the same as in Example 1.

[0149] The results are as follows Figure 6 As shown in the figure, after the column temperature is increased from 35°C to 40°C, the separation between the main peak and impurity D is greater than 1.5; the separation between other impurities is also greater than 1.5, all meeting the requirements. Impurities A, B, C, D, E, F, and G are completely separated from the main component under this method.

[0150] Test Example 1 Specificity

[0151] Solvent: acetonitrile-water (75:25)

[0152] Blank solution: Take 2 ml of methanol, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0153] Maxacalcitol reference solution: Take about 4 mg of maxacalcitol reference, place in a 10 ml volumetric flask, add about 2 ml of methanol, shake to dissolve, dilute to the scale with solvent, and shake well.

[0154] Stock solutions of each impurity: Take about 2 mg of each reference substance of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G, accurately weigh them, place them in 50 ml volumetric flasks, add about 2 ml of methanol, shake to dissolve, dilute to the scale with solvent, and shake well.

[0155] Each impurity location solution: accurately measure 0.15ml of impurity A stock solution, 0.1ml of impurity B stock solution, 0.15ml of impurity C stock solution, 0.15ml of impurity D stock solution, 0.15ml of impurity E stock solution, and 0.15ml of impurity F stock solution, place them in 10ml volumetric flasks respectively, dilute to the scale with solvent, and shake well.

[0156] Maxacalcitol reference solution: Accurately measure 1 ml of maxacalcitol reference solution, place in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0157] System suitability solution: Take about 6 mg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and about 4 mg of impurity B, accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale; accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to the scale with diluent, shake well, as the impurity reference stock solution; take about 4 mg of maxacalcitol reference, place in a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve, accurately add 1 ml of impurity reference stock solution, dilute to the scale with diluent, shake well, to make a mixed solution containing about 0.4 mg of maxacalcitol, 0.4 μg of impurity B and 0.6 μg of each other impurity per 1 ml.

[0158] Test solution: Take about 4 mg of maxacalcitol, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, dilute to the scale with solvent, and mix well.

[0159] The above solutions were respectively injected into the liquid chromatograph to examine whether the blank solvent interfered with the determination of the analyte, and to examine the separation between the known impurities and between the impurities and the main peak.

[0160] Test Example 2 Detection Limit and Quantification Limit

[0161] Solvent: acetonitrile-water (75:25)

[0162] Stock solutions of each impurity: Take about 2 mg of impurity A, B, C, D, E, F, and G reference substances, accurately weigh them, place them in 10 ml volumetric flasks respectively, add 2 ml of methanol and shake to dissolve, dilute to the scale with solvent, and shake well.

[0163] Maxacalcitol stock solution: Take about 4 mg of maxacalcitol reference substance, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, dilute to the scale with solvent, and shake well.

[0164] Quantitative limit solution: Accurately measure an appropriate amount of the stock solution of maxacalcitol and each impurity stock solution, and dilute them step by step with solvent until the S / N of each component peak is ≥10.

[0165] Detection limit solution: Take the quantitative limit solution, accurately measure 3 ml into a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0166] The above solutions were respectively injected into the liquid chromatograph to examine the quantitative limit and detection limit signal-to-noise ratio of each impurity, and the peak area RSD and retention time RSD of the quantitative limit solution were examined.

[0167] Test Example 3 Linearity and Range

[0168] Selection of linear solution concentration points: The impurity linear concentration points are LOQ, 20%, 50%, 80%, 100%, and 150% of the limit concentration.

[0169] Solvent: acetonitrile-water (75:25)

[0170] Blank solution: Take 2 ml of methanol, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0171] Maxacalcitol stock solution: Take about 4 mg of maxacalcitol reference substance, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, dilute to the scale with solvent, and shake well.

[0172] Stock solution of each impurity: Take about 2 mg of impurity A, B, C, D, F, and G reference substances, accurately weigh them, place them in 10 ml volumetric flasks respectively, add 2 ml of methanol and shake to dissolve, dilute to the scale with solvent, and shake well.

[0173] Linear mother solution: Accurately measure 5 ml of maxacalcitol, 3 ml each of impurity A, C, D, F, G stock solution and 2 ml of impurity B stock solution, place them in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0174] Preparation of linear solution: Accurately measure the linear stock solution according to the table below, place it in a volumetric bottle, and add solvent to dilute to the scale.

[0175]

[0176] The above linear solutions were respectively injected into the liquid chromatograph to examine the linear equation, correlation coefficient, response factor, etc. of each impurity.

[0177] Test Example 4 Accuracy

[0178] Solvent: acetonitrile-water (75:25)

[0179] Blank solution: Take 2 ml of methanol, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0180] Stock solution of each impurity: Take about 2 mg of impurity A, B, C, D, F, and G reference substances, accurately weigh them, place them in 10 ml volumetric flasks respectively, add 2 ml of methanol and shake to dissolve, dilute to the scale with solvent, and shake well.

[0181] Stock solution with quantitation limit accuracy: Accurately measure an appropriate amount of the stock solution of each impurity and quantitatively dilute it with a solvent to make a solution with a concentration 10 times the quantitation limit of each impurity.

[0182] Accuracy stock solution: Accurately measure 3 ml each of impurity A, C, D, F, G stock solution and 2 ml of impurity B stock solution, place in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0183] Test solution: Take about 4 mg of maxacalcitol, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, dilute to the scale with solvent, and shake well.

[0184] Quantitative limit accuracy solution: Take about 4 mg of Maxacalcitol, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, accurately add 1 ml of quantitative limit accuracy stock solution, dilute to the scale with solvent, shake well. Prepare 3 copies in parallel.

[0185] 50% accuracy solution: Take about 4 mg of Maxacalcitol, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, accurately add 0.5 ml of accuracy stock solution, dilute to scale with solvent, shake well. Prepare 3 copies in parallel.

[0186] 100% accuracy solution: Take about 4 mg of Maxacalcitol, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, accurately add 1 ml of the accuracy stock solution, dilute to the mark with solvent, shake well. Prepare 3 copies in parallel.

[0187] 150% accuracy solution: Take about 4 mg of Maxacalcitol, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, accurately add 1.5 ml of accuracy stock solution, dilute to scale with solvent, shake well. Prepare 3 copies in parallel.

[0188] Control solution: Accurately measure 1 ml of the test solution and each accuracy solution, place them in 50 ml volumetric flasks respectively, dilute to the scale with solvent, and shake well; accurately measure 1 ml, place them in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0189] The above solutions were respectively injected into the liquid chromatograph to examine the recovery range and RSD of each known impurity in the 9 accuracy solutions.

[0190] Test Example 5 Precision

[0191] Solvent: acetonitrile-water (75:25)

[0192] Blank solution: Take 2 ml of methanol, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0193] System suitability solution: Take about 6 mg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and about 4 mg of impurity B, accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale; accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to the scale with diluent, shake well, as the impurity reference stock solution; take about 4 mg of maxacalcitol reference, place in a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve, accurately add 1 ml of impurity reference stock solution, dilute to the scale with diluent, shake well, to make a mixed solution containing about 0.4 mg of maxacalcitol, 0.4 μg of impurity B and 0.6 μg of each other impurity per 1 ml.

[0194] Spiked test solution (prepared fresh for clinical use): about 4 mg of maxacalcitol, accurately weighed, placed in a 10 ml volumetric flask, added 2 ml of methanol and shaken to dissolve, accurately added 1 ml of accurate stock solution, diluted to the mark with solvent, shaken well. Prepare 6 portions in parallel.

[0195] Control solution: Accurately measure 1 ml of each test solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0196] The above solutions were respectively injected into the liquid chromatograph to examine the RSD of the content of each known impurity in 6 repeated solutions.

[0197] Test Example 6 Solution Stability

[0198] Solvent: acetonitrile-water (75:25)

[0199] Blank solution: Take 2 ml of methanol, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0200] Stock solution of impurity reference substance: Take approximately 6 mg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and approximately 4 mg of impurity B, accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale; accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0201] System suitability solution: Take about 4 mg of maxacalcitol reference substance and place it in a 10 ml volumetric flask. Add an appropriate amount of diluent to dissolve it. Accurately add 1 ml of impurity reference substance stock solution, dilute to the scale with diluent, shake well, and make a mixed solution containing about 0.4 mg of maxacalcitol, 0.4 μg of impurity B, and 0.6 μg of each other impurity per 1 ml.

[0202] Test solution: Take about 4 mg of maxacalcitol, weigh accurately, place in a 10 ml volumetric flask, add 2 ml of methanol and shake to dissolve, dilute to the scale with solvent, and mix well.

[0203] Control solution: Accurately measure an appropriate amount of the test sample solution and quantitatively dilute it with solvent to make a solution containing approximately 0.4 μg per 1 ml.

[0204] Spiked test solution: about 4 mg of Maxacalcitol, accurately weighed, placed in a 10 ml volumetric flask, added 2 ml of methanol and shaken to dissolve, accurately added 1 ml of accurate stock solution, diluted to the scale with solvent, shake well. Prepare 6 copies in parallel.

[0205] The spiked test solution and the control solution were placed at room temperature and injected at different time points to investigate the stability of the spiked test solution and the control solution.

[0206] Test Example 7 Durability

[0207] Solvent: acetonitrile-water (75:25)

[0208] Blank solution: Take 2 ml of methanol, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0209] System suitability solution: Take about 6 mg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and about 4 mg of impurity B, accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale; accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to the scale with diluent, shake well, as the impurity reference stock solution; take about 4 mg of maxacalcitol reference, place in a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve, accurately add 1 ml of impurity reference stock solution, dilute to the scale with diluent, shake well, to make a mixed solution containing about 0.4 mg of maxacalcitol, 0.4 μg of impurity B and 0.6 μg of each other impurity per 1 ml.

[0210] According to the table below (recommended range of variation), the flow rate (range: ±0.1 ml / min), column temperature (range: ±2°C), initial ratio of mobile phase (methanol, acetonitrile ±2%), detection wavelength (±5 nm) were fine-tuned to examine the durability of the method using different chromatographic columns and different instruments.

[0211]

[0212]

[0213] Under various durability conditions, the above solutions were respectively injected into the liquid chromatograph to examine the separation between each known impurity and the adjacent peaks, and the RSD of each impurity content.

[0214] The present invention verifies the detection method from the perspectives of specificity, detection limit and quantification limit, linearity and range, accuracy, precision, solution stability, durability, etc. The verification results are shown in the table below.

[0215]

[0216]

[0217]

Claims

1. A high performance liquid chromatography analysis method for maxacalcitol-related substances, characterized in that: The method can separate the related substances in maxacalcitol, and the separation degree between each chromatographic peak is not less than 1.5; The chromatographic conditions of the method are set as follows: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase: acetonitrile as mobile phase A, methanol as mobile phase B, water as mobile phase C, gradient elution as shown in the table below; Flow rate: 0.5~1.0ml / min; Column temperature: 30-45°C; Detection wavelength: 260~270nm; Injection volume: 10-30 μl; Sample concentration: 0.1~1mg / ml.

2. The method according to claim 1, characterized in that The chromatographic column C18 column is selected from SHIMADZU VP-ODS, with specifications of 4.6 mm×250 mm and 5 μm.

3. The method according to claim 1, characterized in that The column temperature is 40° C.; the flow rate is 0.8 ml / min; the injection volume is 20 μl; the wavelength is 265 nm; and the sample concentration is 0.4 mg / ml.

4. The method according to claim 1, characterized in that: The related substances include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, and the separation degree between each chromatographic peak is not less than 1.5; 。 5. A high performance liquid chromatography analysis method for maxacalcitol-related substances, characterized in that: The following steps are involved: (1) Sample preparation: Diluent: 75% acetonitrile; Test solution: Take an appropriate amount of this product, dissolve it with a diluent and dilute it to make a solution containing about 0.4 mg of maxacalcitol per 1 ml; Self-control solution: accurately measure 1 ml of the test solution, place it in a 20 ml volumetric flask, dilute it to the mark with diluent, shake well, then take 1 ml, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well; Stock solution of impurity reference substance: take about 6 mg of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G and about 4 mg of impurity B, accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to scale; accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with diluent, and shake well; System suitability solution: Take about 4 mg of maxacalcitol reference substance, place it in a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve it, accurately add 1 ml of impurity reference substance stock solution, dilute to the scale with diluent, shake well, and make a mixed solution containing about 0.4 mg of maxacalcitol, 0.4 μg of impurity B, and 0.6 μg of each other impurity per 1 ml; (2) Chromatographic condition setting: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase: acetonitrile as mobile phase A, methanol as mobile phase B, water as mobile phase C, gradient elution; Flow rate: 0.5~1.0ml / min; Column temperature: 30-45°C; Detection wavelength: 260~270nm; Injection volume: 10-30 μl; Sample concentration: 0.1~1mg / ml; (3) Detection and calculation: Accurately measure 20μ1 of system suitability solution, 20μ1 of test solution, and 20μ1 of self-control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

6. The method according to claim 5, characterized in that Perform gradient elution according to the following table: 。 7. The method according to claim 5, characterized in that The chromatographic column C18 column is selected from SHIMADZU VP-ODS, with specifications of 4.6 mm×250 mm and 5 μm.

8. The method according to claim 5, characterized in that The column temperature is 40° C.; the flow rate is 0.8 ml / min; the injection volume is 20 μl; the wavelength is 265 nm; and the sample concentration is 0.4 mg / ml.

9. The method according to claim 5, characterized in that The separation degree between the related substances and the main peak is not less than 1.

5.

10. The method according to claim 5, characterized in that The detection limit of the impurities is: if there are impurity peaks in the chromatogram of the test solution, except for the solvent peak, the peak areas of impurities A, impurity D and impurity G shall not be greater than 1.5 times the main peak area of ​​the control solution, i.e. 0.15%, the peak area of ​​impurity B shall not be greater than 1.0 times the main peak area of ​​the control solution, i.e. 0.10%, the corrected peak areas of impurities C and impurity F shall not be greater than 1.5 times the main peak area of ​​the control solution, i.e. 0.15%, the peak area of ​​other single impurities shall not be greater than 5 times the main peak area of ​​the control solution, i.e. 0.5%, and the sum of the corrected peak areas of all impurities shall not be greater than 20 times the main peak area of ​​the control solution, i.e. 2.0%.

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