Method for detecting paricalcitol related substances

Abstract

The invention provides a high performance liquid chromatography analysis method of paricalcitol related substances. By changing a mobile phase system, changing a reverse phase elution system into a normal phase elution system and adjusting the column temperature, the separation degree of a main peak and a plurality of related impurities is improved, related substances in paricalcitol 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 application belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method of paricalcitol related substances. BACKGROUND

[0002] Paricalcitol, chemical name: (7E, 22E)-19-demethyl-9,10-secoergosta-5,7,22-trien-1 alpha, 3 beta, 25-triol, also known as 19-demethyl-1, 25-dihydroxyvitamin D2, has a structural formula as follows:

[0003]

[0004] Paricalcitol is a synthetic, selective third-generation vitamin D receptor activator. Paricalcitol injection was first approved for marketing in the United States on April 17, 1998, and is used for the treatment of secondary hyperparathyroidism in adult patients with chronic renal failure receiving hemodialysis, mainly for hemodialysis patients with blood PTH >= 300 pg / mL. In 2005, the FDA approved paricalcitol capsules for the prevention and treatment of SHPT, and the capsules have a preventive and therapeutic effect on SHPT of Ш stage and IV stage chronic kidney disease (CKD) patients before dialysis and transplantation surgery.

[0005] The traditional chemical synthesis route of paricalcitol is relatively long, and impurities such as impurity K and impurity L are inevitably produced in the synthesis process. The structural formula is shown in the following figure. These impurities in paricalcitol will directly affect the quality of paricalcitol, thereby affecting the therapeutic effect of paricalcitol preparation.

[0006]

[0007] After searching, only USP43 of various pharmacopoeias collects the quality standard of paricalcitol. After reproducing the related substance method of USP, it is found that under the chromatographic conditions, process impurity L and photodegradation impurity K cannot achieve baseline separation, so the method is not suitable for the detection of paricalcitol related substances.

[0008] Patent CN105467021A provides a method for separating and determining paricalcitol related substances in paricalcitol bulk drug and preparation by HPLC. However, the impurity structure disclosed in the patent is different from impurity K and impurity L, and is not suitable for the detection of impurity K and impurity L in paricalcitol bulk drug. Patent CN109406695A provides a high performance liquid chromatography method for simultaneously separating and analyzing paricalcitol and isomer impurities in paricalcitol injection. However, this method is suitable for the detection of paricalcitol injection, and the detection method of impurity L is not disclosed, and is also not suitable for the detection of impurity K and impurity L in paricalcitol bulk drug.

[0009] Therefore, in order to ensure the safety and effectiveness of paricalcitol bulk drug and preparation, how to provide a method for simultaneously detecting impurities K and L in paricalcitol bulk drug has become an urgent problem to be solved. SUMMARY

[0010] In view of the deficiencies of the prior art, the purpose of the present application is to provide a method for detecting paricalcitol related substances.

[0011] The present application adopts the following technical solutions:

[0012] The present application provides a high-performance liquid chromatography analysis method for paricalcitol related substances, which can separate the related substances in paricalcitol, and the separation degree between each chromatographic peak is not less than 1.5.

[0013] The chromatographic conditions of the method are set as follows:

[0014] The chromatographic column is filled with silica gel bonded with surface covalent bonds and amylose-tris(3-chloro-5-methylphenylaminoformate).

[0015] The mobile phase is n-hexane-methanol-isopropyl alcohol (70:10:20), and isocratic elution.

[0016] The flow rate is 0.5-1 ml / min.

[0017] The column temperature is 30-40℃.

[0018] The detection wavelength is 250-260 nm.

[0019] The injection volume is 20-30 μl.

[0020] The sample concentration is 0.1-1 mg / ml.

[0021] According to the embodiments of the present application, the chromatographic column is selected from CHIRALPAK IG-3, with a size of 4.6 mm x 250 mm and a particle size of 3 μm.

[0022] According to the embodiments of the present application, the column temperature is 35℃, the flow rate is 0.8 ml / min, the injection volume is 25 μl, the wavelength is 252 nm, and the sample concentration is 0.1 mg / ml.

[0023] According to the embodiments of the present application, the related substances include impurities K and L, and the separation degree between each chromatographic peak is not less than 1.5.

[0024]

[0025] According to the embodiments of the present application, the present application provides a high-performance liquid chromatography analysis method for paricalcitol related substances, which comprises the following steps:

[0026] (1) Sample preparation:

[0027] Test sample solution: Take about 1 mg of paricalcitol, add it into a 10-ml volumetric flask, shake to dissolve and dilute to the mark with anhydrous ethanol, and shake well;

[0028] Impurity K stock solution: Take about 1 mg of impurity K, add it into a 10-ml volumetric flask, shake to dissolve and dilute to the mark with anhydrous ethanol, and shake well;

[0029] Impurity L stock solution: Take about 1 mg of impurity L, add it into a 10-ml volumetric flask, shake to dissolve and dilute to the mark with anhydrous ethanol, and shake well;

[0030] Mixed solution: Take 0.5 ml of each of the impurity K and impurity L stock solutions, add them into 4 ml of the test sample solution, and mix well;

[0031] (2) Chromatographic condition setting:

[0032] The method chromatographic condition setting is as follows:

[0033] Chromatographic column: silica gel with surface covalent bond and amylose-tris(3-chloro-5-methylphenylcarbamate) bond as filler;

[0034] Mobile phase: n-hexane-methanol-isopropanol, isocratic elution;

[0035] Flow rate: 0.5-1 ml / min;

[0036] Column temperature: 30-40°C;

[0037] Detection wavelength: 250-260 nm;

[0038] Injection volume: 20-30 μl;

[0039] Sample concentration: 0.1-1 mg / ml;

[0040] (4) Detection and calculation:

[0041] Precisely take 25 μl of the mixed solution, inject it into the liquid chromatograph, and record the chromatogram.

[0042] According to the embodiment of the present application, the isocratic elution ratio of the mobile phase n-hexane-methanol-isopropanol is 70:10:20.

[0043] According to the embodiment of the present application, the chromatographic column is selected from CHIRALPAK IG-3, with a specification of 4.6 mm x 250 mm, 3 μm.

[0044] According to the embodiment of the present application, the column temperature is 35℃; the flow rate is 0.8ml / min; the injection volume is 25μl; the wavelength is 252nm; and the sample concentration is 0.1mg / ml.

[0045] According to the embodiment of the present application, the resolution between the related substance and the main peak is not less than 1.5.

[0046] According to the embodiment of the present application, the detection limit of the impurities is that, in the mixed solution chromatogram, if there is an impurity peak, the RSD of the peak area of the impurity K, the impurity L and the paricalcitol peak, except the solvent peak, is 3.0%, 2.9% and 3.2% respectively, all of which are not more than 10.0%.

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

[0048] (1) The present application provides a high performance liquid chromatography analysis method for the related substances of paricalcitol, by changing the mobile phase system, the reversed phase elution system becomes normal phase elution system, and the column temperature is adjusted, so that the resolution of the main peak and the related impurities is improved, and the separation effect is outstanding.

[0049] (2) The present application studies the impurities in paricalcitol, and the impurity control of the product is more comprehensive; the detection method of the present application can accurately detect the related substances in paricalcitol, so as to comprehensively, accurately and objectively evaluate the product quality, and can be used for the quality research and quality control of the product, which has very important significance. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The figure is the mixed solution chromatogram under the chromatographic conditions of the present application comparative example 1.

[0051] Figure 2 The figure is the mixed solution chromatogram under the chromatographic conditions of the present application example 1.

[0052] Figure 3 The figure is the mixed solution chromatogram under the chromatographic conditions of the present application example 2.

[0053] Figure 4 The figure is the mixed solution chromatogram under the chromatographic conditions of the present application example 3.

[0054] Figure 5 The figure is the mixed solution chromatogram under the chromatographic conditions of the present application example 4.

[0055] Figure 6 The figure is the mixed solution chromatogram under the chromatographic conditions of the present application example 5.

[0056] Figure 7 The figure is the mixed solution chromatogram under the chromatographic conditions of the present application example 6. DETAILED DESCRIPTION

[0057] The application will be further described in connection with the specific embodiments and the accompanying drawings, the following examples are only descriptive, not limiting, and the protection scope of the application cannot be limited thereto, and the raw materials used are commercially available or self-made, unless otherwise specified.

[0058] In the study of related substances of paricalcitol finished product, the information of organic impurities in the standard is shown in Table 1. The impurities in paricalcitol bulk drug mainly come from degradation impurities and process impurities of raw materials.

[0059] Table 1 Information of organic impurities in paricalcitol finished product

[0060]

[0061] Comparative Example 1

[0062] The liquid chromatography detection standard method for related substances of paricalcitol bulk drug in USP43 specifically includes the following steps:

[0063] (1) Chromatographic condition setting:

[0064] Chromatographic column: octadecylsilane bonded silica gel as filler (HALO C18, 4.6mm x 100mm, 2.7μm);

[0065] Diluent: anhydrous ethanol-water (50:50);

[0066] Column temperature: 30℃;

[0067] Detection wavelength: 252nm;

[0068] Flow rate: 0.9ml / min;

[0069] Injection volume: 25μl;

[0070] Mobile phase: water-acetonitrile (95:5) as mobile phase A, acetonitrile-methanol (75:25) as mobile phase B, gradient elution according to the following table:

[0071]

[0072] (2) Test solution: take about 1mg of paricalcitol, put it in a 10ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, shake well.

[0073] (3) Impurity K stock solution: take about 1mg of impurity K, put it in a 10ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, shake well.

[0074] (4) Impurity L stock solution: take about 1mg of impurity L, put it in a 10ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the mark, shake well.

[0075] (5) Mixed solution: take 0.5 ml of impurity K and impurity L stock solution, add 4 ml of test solution, mix well.

[0076] (6) Detection: accurately take 25 μl of the mixed solution, inject into the liquid chromatograph, and record the chromatogram.

[0077] The results are shown in Figure 1 After reproducing the USP43 related substance method, paricalcitol and impurity L and impurity K cannot be completely separated, the USP43 standard method cannot effectively separate the above impurities, and further optimization is required.

[0078] The best analysis condition is preferred through a large number of experiments, the separation degree of the main peak and the related impurities is improved by adjusting the column temperature and strictly controlling the proportion of the mobile phase components, setting reasonable gradient elution conditions, and the separation degree is greater than 1.5, and the separation effect is outstanding. The related substance detection of the product mainly experiences the following optimization steps:

[0079] Example 1 (change the flow rate and column temperature)

[0080] (1) Chromatographic condition setting:

[0081] Chromatographic column: octadecylsilane bonded silica gel as the filler (HALO C18, 4.6 mm x 100 mm, 2.7 μm);

[0082] Diluent: anhydrous ethanol-water (50:50);

[0083] Column temperature: 15℃;

[0084] Detection wavelength: 252 nm;

[0085] Flow rate: 0.7 ml / min;

[0086] Injection volume: 25 μl;

[0087] Mobile phase: water-acetonitrile (95:5) as mobile phase A, acetonitrile-methanol (75:25) as mobile phase B, gradient elution according to the following table:

[0088]

[0089] (2) Test solution: take about 1 mg of paricalcitol, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the mark, shake well.

[0090] (3) Impurity K stock solution: take about 1 mg of impurity K, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the mark, shake well.

[0091] (4) Impurity L stock solution: about 1 mg of Impurity L was taken in a 10-ml volumetric flask, dissolved and diluted to the mark with absolute ethanol, and shaken well.

[0092] (5) Mixed solution: 0.5 ml of Impurity K stock solution and 0.5 ml of Impurity L stock solution were taken, added to 4 ml of test sample solution, and mixed well.

[0093] (6) Detection: 25 μl of the mixed solution was precisely measured and injected into the liquid chromatograph, and the chromatogram was recorded.

[0094] The results are shown in Table 1. Figure 2 After adjusting the column temperature and flow rate based on the USP43 related substance method, paricalcitol and impurity K were directly wrapped into the main peak, and the main peak and impurities K and L still could not be effectively separated. The chromatographic column was changed for further optimization.

[0095] Example 2 (change of chromatographic column)

[0096] (1) Chromatographic condition setting:

[0097] Chromatographic column: octadecylsilane-bonded silica gel as the filler (Kromasil 100-5 C18, 4.6 mm x 250 mm, 5 μm);

[0098] Diluent: absolute ethanol-water (50:50);

[0099] Column temperature: 30°C;

[0100] Detection wavelength: 252 nm;

[0101] Flow rate: 0.4 ml / min;

[0102] Injection volume: 25 μl;

[0103] Mobile phase: water-acetonitrile (95:5) as mobile phase A, acetonitrile-methanol (75:25) as mobile phase B, gradient elution was performed according to the following table:

[0104]

[0105] (2) Test sample solution: about 1 mg of paricalcitol was taken in a 10-ml volumetric flask, dissolved and diluted to the mark with absolute ethanol, and shaken well.

[0106] (3) Impurity K stock solution: about 1 mg of Impurity K was taken in a 10-ml volumetric flask, dissolved and diluted to the mark with absolute ethanol, and shaken well.

[0107] (4) Impurity L stock solution: about 1 mg of Impurity L was taken in a 10-ml volumetric flask, dissolved and diluted to the mark with absolute ethanol, and shaken well.

[0108] (5) Mixed solution: Take 0.5 ml of impurity K and impurity L stock solutions respectively, add 4 ml of test solution, mix well.

[0109] (6) Detection: Accurately take 25 μl of mixed solution, inject into liquid chromatograph, and record chromatogram.

[0110] Results as shown in Table 1, on the basis of USP43 related substances method, after changing the chromatographic column, paricalcitol and impurity K, impurity L still cannot achieve baseline separation, try to change the elution gradient to continue optimization. Figure 3

[0111] Example 3 (change elution gradient)

[0112] (1) Chromatographic condition setting:

[0113] Chromatographic column: octadecylsilane bonded silica gel as filler (HALO C18, 4.6 mm x 100 mm, 2.7 μm);

[0114] Diluent: anhydrous ethanol-water (50:50);

[0115] Column temperature: 30℃;

[0116] Detection wavelength: 252 nm;

[0117] Flow rate: 0.4 ml / min;

[0118] Injection volume: 25 μl;

[0119] Mobile phase: water-acetonitrile (95:5) as mobile phase A, acetonitrile-methanol (75:25) as mobile phase B, gradient elution according to the following table:

[0120]

[0121] (2) Test solution: Take about 1 mg of paricalcitol, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the mark, shake well.

[0122] (3) Impurity K stock solution: Take about 1 mg of impurity K, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the mark, shake well.

[0123] (4) Impurity L stock solution: Take about 1 mg of impurity L, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the mark, shake well.

[0124] (5) Mixed solution: Take 0.5 ml of impurity K and impurity L stock solutions respectively, add 4 ml of test solution, mix well.

[0125] ​(6) Detection: precisely take 25 μl of the mixed solution, inject into the liquid chromatograph, and record the chromatogram.

[0126] The results are shown in Table 1. Figure 4 As shown in Table 1, after changing the elution gradient of the mobile phase on the basis of the USP43 related substance method, the baseline separation of paricalcitol and impurity K, impurity L still cannot be achieved, and considering that the analysis method is a reversed-phase system, subsequent attempts are made to continue optimization in a normal-phase system.

[0127] Example 4 (normal-phase system)

[0128] (1) Chromatographic condition setting:

[0129] Chromatographic column: silica gel with surface covalent bond and amylose-tris(3-chloro-5-methylphenylcarbamate) bonded as the filler (CHIRALPAK IG-3, 4.6 mm x 250 mm, 3 μm);

[0130] Diluent: anhydrous ethanol;

[0131] Column temperature: 30°C;

[0132] Detection wavelength: 252 nm;

[0133] Flow rate: 1.0 ml / min;

[0134] Injection volume: 25 μl;

[0135] Sample concentration: 0.1 mg / ml;

[0136] Mobile phase: n-hexane-methanol (80:20);

[0137] (2) Test solution: take about 1 mg of paricalcitol, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the mark, and shake well.

[0138] (3) Impurity K stock solution: take about 1 mg of impurity K, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the mark, and shake well.

[0139] (4) Impurity L stock solution: take about 1 mg of impurity L, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the mark, and shake well.

[0140] (5) Mixed solution: take 0.5 ml of each of the impurity K and impurity L stock solutions, add 4 ml of the test solution, and mix well.

[0141] (6) Detection: precisely take 25 μl of the mixed solution, inject into the liquid chromatograph, and record the chromatogram.

[0142] The results are shown in Table 1. Figure 5As shown in the figure, the mobile phase system becomes normal phase, impurity K and impurity L are completely separated, but the separation degree between impurity L and the main peak fails to reach above 1.5, which proves that the normal phase mobile phase system is conducive to the separation of impurities K and L from the main peak. The subsequent optimization will be continued based on this normal phase mobile phase system.

[0143] Example 5 (Normal Phase System-Changing Mobile Phase)

[0144] (1) Chromatographic condition setting:

[0145] Chromatographic column: Surface covalent bonded and amylose-tris(3-chloro-5-methylphenylcarbamate) bonded silica gel was used as filler (CHIRALPAK IG-3, 4.6 mm × 250 mm, 3 μm);

[0146] Diluent: anhydrous ethanol;

[0147] Column temperature: 30°C;

[0148] Detection wavelength: 252nm;

[0149] Flow rate: 1.0 ml / min;

[0150] Injection volume: 25 μl;

[0151] Sample concentration: 0.1 mg / ml;

[0152] Mobile phase: n-hexane-methanol-isopropanol (70:10:20);

[0153] (2) Test solution: Take about 1 mg of paricalcitol and place it in a 10 ml volumetric flask. Add anhydrous ethanol and shake to dissolve and dilute to the mark. Shake well.

[0154] (3) Impurity K stock solution: Take about 1 mg of impurity K and place it in a 10 ml volumetric flask. Add anhydrous ethanol and shake to dissolve and dilute to the scale. Shake well.

[0155] (4) Stock solution of impurity L: Take about 1 mg of impurity L and place it in a 10 ml volumetric flask. Add anhydrous ethanol and shake to dissolve and dilute to the scale. Shake well.

[0156] (5) Mixed solution: Take 0.5 ml of each of the stock solutions of impurity K and impurity L, add 4 ml of the test solution, and mix well.

[0157] (6) Detection: Accurately measure 25 μl of the mixed solution, inject it into the liquid chromatograph, and record the chromatogram.

[0158] The results are as follows Figure 6 As shown in the figure, after changing the mobile phase, impurity K and impurity L are completely separated, and the separation between impurity L and the main peak is significantly improved, but the peak shape is not good. Based on this mobile phase, try to change the column temperature to continue optimization.

[0159] Example 6 (normal phase system - changing flow rate and column temperature)

[0160] (1) Chromatographic condition setting:

[0161] Chromatographic column: silica gel with surface covalent bond and amylose-tris(3-chloro-5-methylphenylcarbamate) bonded as packing agent (CHIRALPAK IG-3, 4.6 mm x 250 mm, 3 μm);

[0162] Diluent: anhydrous ethanol;

[0163] Column temperature: 35°C;

[0164] Detection wavelength: 252 nm;

[0165] Flow rate: 0.8 ml / min;

[0166] Injection volume: 25 μl;

[0167] Sample concentration: 0.1 mg / ml;

[0168] Mobile phase: n-hexane-methanol-isopropanol (70:10:20);

[0169] (2) Test sample solution: about 1 mg of paricalcitol was taken and placed in a 10-ml volumetric flask, anhydrous ethanol was added to dissolve and dilute to the mark, and shaken well.

[0170] (3) Impurity K stock solution: about 1 mg of impurity K was taken and placed in a 10-ml volumetric flask, anhydrous ethanol was added to dissolve and dilute to the mark, and shaken well.

[0171] (4) Impurity L stock solution: about 1 mg of impurity L was taken and placed in a 10-ml volumetric flask, anhydrous ethanol was added to dissolve and dilute to the mark, and shaken well.

[0172] (5) Mixed solution: 0.5 ml of the impurity K stock solution and 0.5 ml of the impurity L stock solution were taken and added to 4 ml of the test sample solution, and mixed well.

[0173] (6) Detection: 25 μl of the mixed solution was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded.

[0174] The results are shown in Table 1. Figure 7 After increasing the column temperature and decreasing the flow rate, impurity K and impurity L were completely separated, the main peak and impurity L were also completely separated, the resolution was greater than 1.5, and the peak shape was good, all meeting the requirements. Impurity K, impurity L and the main component were completely separated under this method.

[0175] Test Example 1 Specificity

[0176] Diluent: n-hexane-methanol-isopropanol (70:10:20).

[0177] Blank solvent: same as diluent.

[0178] Test solution: about 2 mg of paricalcitol was accurately weighed into a 10-ml volumetric flask, dissolved with a small amount of anhydrous ethanol, and diluted to the mark with the diluent, and shaken well.

[0179] Control solution: 1 ml of the test solution was accurately transferred into a 50-ml volumetric flask, diluted to the mark with the diluent, and shaken well; 1 ml was accurately measured into a 20-ml volumetric flask, diluted to the mark with the diluent, and shaken well.

[0180] Impurity K control stock solution: about 2 mg of the impurity K control was accurately weighed into a 20-ml volumetric flask, dissolved and diluted to the mark with anhydrous ethanol, and shaken well.

[0181] Impurity L control stock solution: about 2 mg of the impurity L control was accurately weighed into a 20-ml volumetric flask, dissolved and diluted to the mark with anhydrous ethanol, and shaken well.

[0182] Impurity K positioning solution: 1 ml of the impurity K control stock solution was accurately measured into a 50-ml volumetric flask, diluted to the mark with the diluent, and shaken well.

[0183] Impurity L positioning solution: 1 ml of the impurity L control stock solution was accurately measured into a 50-ml volumetric flask, diluted to the mark with the diluent, and shaken well.

[0184] Mixed impurity solution: 1 ml of each of the control stock solutions was taken into the same 50-ml volumetric flask, diluted to the mark with the diluent, and shaken well.

[0185] Test sample spiked solution: about 2 mg of the paricalcitol control was accurately weighed into a 10-ml volumetric flask, dissolved with a small amount of anhydrous ethanol, 1 ml of the mixed impurity solution was added, diluted to the mark with the diluent, and shaken well.

[0186] The above solutions were taken for sampling, respectively, and the chromatograms were recorded. The separation between each known impurity and between the impurities and the main peak was investigated.

[0187] Solution stability of test example 2

[0188] Diluent: n-hexane-methanol-isopropanol (70:10:20).

[0189] Blank solvent: same as diluent.

[0190] Test solution: about 2 mg of paricalcitol was accurately weighed into a 10-ml volumetric flask, dissolved with a small amount of anhydrous ethanol, and diluted to the mark with the diluent, and shaken well.

[0191] Control solution: Pipette 1 ml of the test solution into a 50-ml volumetric flask, dilute to the mark with diluent, and shake to mix; pipette 1 ml into a 20-ml volumetric flask, dilute to the mark with solvent, and shake to mix.

[0192] Impurity K control stock solution: Accurately weigh about 2 mg of impurity K control, place into a 20-ml volumetric flask, dissolve and dilute to the mark with absolute ethanol, and shake to mix.

[0193] Impurity L control stock solution: Accurately weigh about 2 mg of impurity L control, place into a 20-ml volumetric flask, dissolve and dilute to the mark with absolute ethanol, and shake to mix.

[0194] Spiked test solution: Accurately weigh about 2 mg of paricalcitol control, place into a 10-ml volumetric flask, add 20 μl of each of the impurity K and impurity L control stock solutions, add a small amount of absolute ethanol to dissolve, dilute to the mark with diluent, and shake to mix.

[0195] Take the above solution at different time points, record the chromatogram. Investigate the stability of the spiked test solution and the control solution.

[0196] Test Example 3 Quantitative limit and detection limit

[0197] Diluent: n-hexane-methanol-isopropyl alcohol (70:10:20).

[0198] Blank solvent: same as diluent.

[0199] Paricalcitol stock solution: Accurately weigh about 2 mg of paricalcitol control, place into a 10-ml volumetric flask, dissolve and dilute to the mark with absolute ethanol, and shake to mix.

[0200] Impurity K stock solution: Accurately weigh about 2 mg of impurity K control, place into a 10-ml volumetric flask, dissolve and dilute to the mark with absolute ethanol, and shake to mix.

[0201] Impurity L stock solution: Accurately weigh about 2 mg of impurity L control, place into a 10-ml volumetric flask, dissolve and dilute to the mark with absolute ethanol, and shake to mix.

[0202] Quantitative limit solution: Accurately pipette 1 ml each of the paricalcitol stock solution, impurity K stock solution, and impurity L stock solution into the same 50-ml volumetric flask, dilute to the mark with solvent, accurately pipette 1 ml into a 100-ml volumetric flask, dilute to the mark with diluent, and shake to mix. Prepare 6 portions in parallel.

[0203] Detection limit solution: Accurately pipette 3 ml of the quantitative limit solution into a 10-ml volumetric flask, dilute to the mark with diluent, and shake to mix.

[0204] Inject the above solution into the chromatograph, respectively, and record the chromatogram. The quantitative limit and detection limit signal-to-noise ratio of each impurity were investigated, and the peak area RSD and retention time RSD of the quantitative limit solution were investigated.

[0205] Test Example 4 Linearity and Range (Correction Factor)

[0206] Diluent: n-hexane-methanol-isopropanol (70:10:20).

[0207] Blank solvent: same as diluent.

[0208] Control stock solution: about 2 mg of impurity K, impurity L and paricalcitol control were accurately weighed into the same 10 ml volumetric flask, added with anhydrous ethanol, shaken to dissolve and diluted to the mark, and shaken uniformly; 1 ml was accurately measured and placed in a 50 ml volumetric flask, diluted to the mark with diluent, and shaken uniformly.

[0209] Linear concentration solution: prepared according to Table 1:

[0210] Table 1 Analysis method verification of related substance II - preparation of linear concentration solution

[0211]

[0212] The above different concentration linear solutions were injected into the liquid chromatograph, respectively, to investigate the linear relationship between concentration and peak response.

[0213] Test Example 5 Precision

[0214] Diluent: n-hexane-methanol-isopropanol (70:10:20).

[0215] Blank solvent: same as diluent.

[0216] Impurity control stock solution: about 2 mg of impurity K and impurity L control were accurately weighed into a 10 ml volumetric flask, added with anhydrous ethanol, shaken to dissolve and diluted to the mark, and shaken uniformly; 1 ml was accurately measured and placed in a 100 ml volumetric flask, diluted to the mark with diluent, and shaken uniformly.

[0217] Test solution: about 2 mg of the product was accurately weighed into a 10 ml volumetric flask, a small amount of anhydrous ethanol was added to shake and dissolve, 1 ml of impurity control stock solution was added, and diluted to the mark with diluent, and shaken uniformly. Six were prepared in parallel.

[0218] Control solution: 1 ml of test solution was accurately measured and placed in a 50 ml volumetric flask, diluted to the mark with diluent, and shaken uniformly; 1 ml was accurately measured and placed in a 20 ml volumetric flask, diluted to the mark with diluent, and shaken uniformly.

[0219] The above blank solvent, control solution and test solution were injected into the chromatograph, respectively, and the chromatogram was recorded. The repeatability of the method was investigated.

[0220] Accuracy of Test Example 6

[0221] Diluent: n-hexane-methanol-isopropanol (70:10:20).

[0222] Blank solvent: same as diluent.

[0223] Test solution: about 2 mg of paricalcitol was accurately weighed into a 10-ml volumetric flask, dissolved with a small amount of anhydrous ethanol, and diluted to the mark with the diluent, and shaken well.

[0224] Control solution: 1 ml of the test solution was accurately measured into a 50-ml volumetric flask, diluted to the mark with the diluent, and shaken well; 1 ml was accurately measured into a 20-ml volumetric flask, diluted to the mark with the diluent, and shaken well.

[0225] Impurity control stock solution: about 2 mg of each of the impurity K and impurity L controls was accurately weighed into a 10-ml volumetric flask, dissolved with anhydrous ethanol, and diluted to the mark, and shaken well; 1 ml was accurately measured into a 100-ml volumetric flask, diluted to the mark with the diluent, and shaken well.

[0226] 50% accuracy solution: about 2 mg of the product was accurately weighed into a 10-ml volumetric flask, dissolved with a small amount of anhydrous ethanol, and diluted to the mark with the diluent, and shaken well. Three were prepared in parallel.

[0227] 100% accuracy solution: about 2 mg of the product was accurately weighed into a 10-ml volumetric flask, dissolved with a small amount of anhydrous ethanol, and diluted to the mark with the diluent, and shaken well. Three were prepared in parallel.

[0228] 150% accuracy solution: about 2 mg of the product was accurately weighed into a 10-ml volumetric flask, dissolved with a small amount of anhydrous ethanol, and diluted to the mark with the diluent, and shaken well. Three were prepared in parallel.

[0229] The above solutions were injected into the sample, respectively, and the chromatogram was recorded. The recovery range and RSD of each known impurity were investigated.

[0230] Durability of Test Example 7

[0231] Diluent: n-hexane-methanol-isopropanol (70:10:20).

[0232] Blank solvent: same as diluent.

[0233] Impurity K control stock solution: about 2 mg of the impurity K control was accurately weighed into a 20-ml volumetric flask, dissolved with anhydrous ethanol, and diluted to the mark, and shaken well.

[0234] Impurity L reference stock solution: about 2 mg of Impurity L reference was accurately weighed into a 20-ml volumetric flask, dissolved in anhydrous ethanol and diluted to the mark, and shaken well.

[0235] Test sample spiked solution: about 2 mg of the sample was taken into a 10-ml volumetric flask, a small amount of anhydrous ethanol was added to dissolve the sample, 20 μl of Impurity K and Impurity L stock solutions were added, and diluted to the mark with diluent, and shaken well.

[0236] Reference solution: 1 ml of the test sample spiked solution was accurately measured into a 50-ml volumetric flask, diluted to the mark with diluent, and shaken well; 1 ml was accurately measured into a 20-ml volumetric flask, diluted to the mark with diluent, and shaken well.

[0237] Durability conditions: see Table 2

[0238] Table 2 Analysis method validation of related substance II - durability conditions

[0239]

[0240] Under each durability condition, the above solution was injected into a liquid chromatograph, and the separation degree of each known impurity from adjacent peaks and the RSD of the content of each impurity were investigated.

[0241] The detection method was verified from the aspects of specificity, solution stability, detection limit and quantitative limit, linearity and range, accuracy, precision, durability, and the verification results are shown in the following table.

[0242]

[0243]

[0244]

[0245]

Claims

1. A high performance liquid chromatography analysis method for paricalcitol-related substances, characterized in that: The method can separate related substances in paricalcitol, 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: Surface covalent bond and amylose-tris (3-chloro-5-methylphenylcarbamate) bonded silica gel as filler; Mobile phase: n-hexane-methanol-isopropanol (70:10:20), isocratic elution; Flow rate: 0.5-1 ml / min; Column temperature: 30-40°C; Detection wavelength: 250-260nm; Injection volume: 20-30 μl; Sample concentration: 0.1~1mg / ml.

2. The method according to claim 1, characterized in that The chromatographic column is selected from CHIRALPAK IG-3, with specifications of 4.6 mm×250 mm and 3 μm.

3. The method according to claim 1, characterized in that The column temperature was 35° C.; the flow rate was 0.8 ml / min; the injection volume was 25 μl; the wavelength was 252 nm; and the sample concentration was 0.1 mg / ml.

4. The method according to claim 1, wherein The related substances include impurity K and impurity L, and the separation degree between each chromatographic peak is not less than 1.5; 5. A high performance liquid chromatography analysis method for paricalcitol-related substances, characterized in that: The following steps are involved: (1) Sample preparation: Test solution: Take about 1 mg of paricalcitol and place it in a 10 ml volumetric flask. Add anhydrous ethanol and shake to dissolve and dilute to the mark. Shake well. Impurity K stock solution: Take about 1 mg of impurity K, place it in a 10 ml volumetric flask, add anhydrous ethanol, shake to dissolve and dilute to the scale, shake well; Impurity L stock solution: Take about 1 mg of impurity L and place it in a 10 ml volumetric flask. Add anhydrous ethanol and shake to dissolve and dilute to the scale. Shake well. Mixed solution: Take 0.5 ml of each of the stock solutions of impurity K and impurity L, add 4 ml of the test solution, and mix well; (2) Chromatographic condition setting: The chromatographic conditions of the method are set as follows: Chromatographic column: Surface covalent bond and amylose-tris (3-chloro-5-methylphenylcarbamate) bonded silica gel as filler; Mobile phase: n-hexane-methanol-isopropanol, isocratic elution; Flow rate: 0.5-1 ml / min; Column temperature: 30-40°C; Detection wavelength: 250-260nm; Injection volume: 20-30 μl; Sample concentration: 0.1-1 mg / ml; (3) Detection and calculation: Accurately measure 25 μl of the mixed solution, inject it into the liquid chromatograph, and record the chromatogram.

6. The method according to claim 5, characterized in that The isocratic elution ratio of the mobile phase was 70:10:20:n-hexane-methanol-isopropanol.

7. The method according to claim 5, characterized in that The chromatographic column is selected from CHIRALPAK IG-3, with specifications of 4.6 mm×250 mm and 3 μm.

8. The method according to claim 5, characterized in that The column temperature was 35° C.; the flow rate was 0.8 ml / min; the injection volume was 25 μl; the wavelength was 252 nm; and the sample concentration was 0.1 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 mixed solution, excluding the solvent peak, the RSDs of the peak areas of impurity K, impurity L and paricalcitol are 3.0%, 2.9% and 3.2%, respectively, all of which are no more than 10.0%.

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