Method for detecting related substances of vitamin D drops

Abstract

The invention provides a high performance liquid chromatography analysis method of vitamin D drop related substances. A two-dimensional liquid chromatography detection method is adopted, interference of oily matrixes in the vitamin D drops is avoided, and related substances in the vitamin D drops can be accurately detected. The analysis method disclosed by the invention is stable in baseline, good in stability, good in specificity and high in sensitivity, so that the product quality is comprehensively, accurately and objectively evaluated, and the analysis method has very important significance.

Description

Technical Field

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

[0002] Vitamins are a type of trace organic substances that humans and animals must obtain from food to maintain normal physiological functions. The content of vitamins in the body is very small, but they are indispensable and play an important role in the growth, metabolism and development of the human body. Among them, fat-soluble vitamins are a type of vitamins that are insoluble in water but soluble in fat and non-polar organic solvents (such as benzene, ether and chloroform), including vitamin A, vitamin D, vitamin E, vitamin K, etc.

[0003] Vitamin D 3 (VitaminD 3 )The molecular formula is shown below, and its chemical name is "9,10-openocholesterol-5,7,10(19)-triene-3β-ol". It is a fat-soluble vitamin used to prevent and treat vitamin D deficiency rickets and prevent osteoporosis.

[0004]

[0005] Vitamin preparations mainly consist of oral and injectable vitamins. my country's oral vitamin preparation market is dominated by regular-release dosage forms (including tablets, soft capsules, hard capsules, drops, etc.). In the oral vitamin market, sales of vitamin D drops have grown rapidly, and in 2017, it has become the product with the largest sales volume in the oral vitamin market.

[0006] Vitamin D is listed in the European Pharmacopoeia (EP11.6). 3 The method for related substances of raw materials clearly stipulates that vitamin D 3 Impurities A, B, C, D, E and F need to be studied in the raw materials. Among them, impurities A, D and E are degradation impurities, and are also the impurities that need to be studied and controlled in vitamin D drops. According to the requirements of the ICH guidelines, the limit of a single impurity in vitamin D drops must be controlled below 1.0%.

[0007] The 2020 edition of the Chinese Pharmacopoeia only records vitamin D in vitamin D drops. 3The method for determining the content of vitamin D drops does not record the method for determining related substances. The pharmacopoeias of various countries, such as the European Pharmacopoeia (EP11.6) and the United States Pharmacopoeia (NF-2024), do not record the method for determining related substances in vitamin D drops. In addition, vitamin D drops contain a large amount of oily matrix, which has a great interference with the detection of related substances. Therefore, the detection of such related substances is more difficult than other drug impurity detection in the development and selection of analytical methods.

[0008] Chinese patent CN 116297935 A discloses a method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography. However, the patent only discloses the detection methods of impurities A and E, and is not suitable for the detection of impurity D in vitamin D drops.

[0009] Impurity D is isosterol vitamin D 3 , vitamin D 3 The results of the destruction experiment showed that when 1% trifluoroacetic acid was used to destroy vitamin D 3 After 0.5 hours of destruction, impurity D is produced at a content of about 1.3%, which exceeds the limit requirements of the ICH guidelines. This shows that impurity D is more easily produced under acidic conditions. The auxiliary material soybean oil of vitamin D drops contains a large amount of free fatty acids, which may lead to the production of impurity D, thus seriously affecting the quality of the product.

[0010]

[0011] Therefore, in order to ensure the safety and effectiveness of vitamin D drops, how to provide a method that can simultaneously detect impurities A, D, and E in vitamin D drops has become an urgent problem to be solved. Summary of the invention

[0012] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for detecting related substances in vitamin D drops.

[0013] The present invention adopts the following technical solutions:

[0014] The present invention provides a high performance liquid chromatography analysis method for related substances in vitamin D drops, characterized in that the method can separate related substances in vitamin D drops, wherein the related substances include impurity A, impurity D, and impurity E, and the separation degree between each chromatographic peak is not less than 1.5;

[0015] The first dimension HPLC conditions were set as follows:

[0016] Chromatographic column: a chromatographic column with urea-bonded silica gel as filler or a filler with similar functions;

[0017] Mobile phase: n-hexane as mobile phase A, isopropanol as mobile phase B, n-hexane-n-pentanol-isopropanol = 98:1:1 as mobile phase C, for gradient elution;

[0018] Flow rate: 0.5~1.0ml / min;

[0019] Detection wavelength: 260~270nm;

[0020] Column temperature: 25-45°C;

[0021] Injection volume: 50-150 μl;

[0022] Collection tube: polyetheretherketone tube, inner diameter 0.0762cm, 20m, volume approximately 9ml.

[0023] The second dimension HPLC conditions were set as follows:

[0024] Chromatographic column: silica gel is used as filler;

[0025] Mobile phase: n-hexane-n-pentanol-ethanol, isocratic elution;

[0026] Flow rate: 0.5~1.0ml / min;

[0027] Detection wavelength: 260~270nm;

[0028] Column temperature: 20-40°C;

[0029] Injection volume: 50-150 μl;

[0030] Collection tube: polyetheretherketone tube, inner diameter 0.0762cm, 20m, volume about 9ml;

[0031] The switching procedure is as follows: a cut-valve positioning solution (1) is injected into the first-dimension liquid chromatograph to locate impurities A and E. The switching time of impurities A and E in the first-dimension liquid chromatograph is set to 2 to 3 minutes before and after their respective retention times; a cut-valve positioning solution (2) is injected into the first-dimension liquid chromatograph to locate vitamin D 3 and impurity D, vitamin D in the first dimension liquid chromatography 3 The switching time of impurity D is set to vitamin D 3 2 to 3 minutes before the peak starts and 2 to 3 minutes after the peak ends;

[0032] In the first-dimensional liquid chromatography, the retention time of impurity A is 19 min to 23 min; the retention time of impurity D is 24.5 min to 29 min; and the retention time of impurity E is 29.5 min to 35 min.

[0033] According to an embodiment of the present invention, the related substances include impurity A, impurity D, and impurity E, and the structural formula is:

[0034]

[0035]

[0037] According to an embodiment of the present invention, the chromatographic column under the first-dimensional liquid chromatography conditions is ChromCore VD-ChP41, 2.1 mm×150 mm, 3 μm; the chromatographic column under the second-dimensional liquid chromatography conditions is ShimNex HE Sil, 4.6 mm×150 mm, 3 μm.

[0038] According to an embodiment of the present invention, the column temperature under the first-dimensional liquid chromatography condition is 40°C; and the column temperature under the second-dimensional liquid chromatography condition is 30°C.

[0039] According to an embodiment of the present invention, the mobile phase gradient elution procedure under the first dimension liquid chromatography conditions is:

[0040] Time (minutes) A(％) B(％) C(％) 0 95 0 5 40 95 0 5 45 0 0 100 50 0 0 100 55 0 20 80 63 0 20 80 65 95 0 5 120 95 0 5

[0041] According to an embodiment of the present invention, the elution ratio of the mobile phase n-hexane-n-pentanol-ethanol under the second dimension liquid chromatography conditions is 998:1:1.

[0042] According to an embodiment of the present invention, the flow rate under the first-dimensional liquid chromatography conditions and the second-dimensional liquid chromatography conditions is 0.5 ml / min.

[0043] According to an embodiment of the present invention, the wavelength under the first-dimensional liquid chromatography conditions and the second-dimensional liquid chromatography conditions is 265 nm.

[0044] According to an embodiment of the present invention, the injection volume under the first-dimensional liquid chromatography conditions and the second-dimensional liquid chromatography conditions is 100 μL.

[0045] According to an embodiment of the present invention, the present invention provides a method for detecting related substances in vitamin D drops, comprising the following steps:

[0046] First dimension liquid chromatography:

[0047] (1) Chromatographic condition setting:

[0048] Chromatographic column: urea-bonded silica gel as filler or filler with similar functions, ChromCore VD-ChP41, 2.1mm×150mm, 3μm;

[0049] Mobile phase: n-hexane as mobile phase A, isopropanol as mobile phase B, n-hexane-n-pentanol-isopropanol (98:1:1) as mobile phase C, and gradient elution as shown in the following table;

[0050] Flow rate: 0.5ml / min;

[0051] Detection wavelength: 265nm;

[0052] Column temperature: 40°C;

[0053] Injection volume: 100 μl;

[0054] Collection tube: polyetheretherketone tube, inner diameter 0.0762cm, 20m, volume approximately 9ml.

[0055] Time (minutes) A(％) B(％) C(％) 0 95 0 5 40 95 0 5 45 0 0 100 50 0 0 100 55 0 20 80 63 0 20 80 65 95 0 5 120 95 0 5

[0056] Second Dimension Liquid Chromatography:

[0057] (1) Chromatographic condition setting:

[0058] Chromatographic column: silica gel as filler, ShimNex HE Sil, 4.6 mm × 150 mm, 3 μm;

[0059] Mobile phase: n-hexane-n-pentanol-ethanol ratio 998:1:1, isocratic elution;

[0060] Flow rate: 0.5ml / min;

[0061] Detection wavelength: 265nm;

[0062] Column temperature: 30°C;

[0063] Injection volume: 100 μl;

[0064] Collection tube: polyetheretherketone tube, inner diameter 0.0762cm, 20m, volume approximately 9ml.

[0065] Vitamin D 3 Reference stock solution: Take vitamin D 3 About 5 mg of the reference substance is accurately weighed and placed in a 20 ml volumetric flask. Add isooctane to dissolve and dilute to the mark.

[0066] Cut valve positioning solution (1) Measure vitamin D 35 ml of the reference stock solution is placed in a stoppered glass container, sealed after nitrogen is passed through it, and heated in a 90°C water bath for 1 hour. Take it out, cool it quickly, add 5 ml of n-hexane, shake it well, measure 1 ml of the solution, place it in a 25 ml transparent volumetric bottle, add n-hexane to dilute to the scale, shake it well, and place the volumetric bottle 5 to 6 cm away from the lamp under two 8W ultraviolet lamps with main wavelengths of 254nm and 365nm respectively, and irradiate it for 40 minutes to make the solution contain impurities A and vitamin D 3 and impurity E, as a valve positioning solution for impurity A and impurity E.

[0067] Cut valve positioning solution (2) Measure vitamin D 3 An appropriate amount of reference stock solution was diluted with n-hexane to make a solution containing about 0.05 μg per 1 ml as vitamin D 3 Cut valve positioning solution with impurity D.

[0068] The cut-valve positioning solution (1) was injected into the first-dimension liquid chromatograph to locate impurities A and E. The switching time of impurities A and E in the first-dimension liquid chromatograph was set to 2 minutes before and after their respective retention times. The cut-valve positioning solution (2) was injected into the first-dimension liquid chromatograph to locate vitamin D. 3 and impurity D, vitamin D in the first dimension liquid chromatography 3 The switching time of impurity D is set to vitamin D 3 2.5 minutes before the peak start time and 2 minutes after the peak end time.

[0069] (2) Preparation of test solution: Take about 2.5 g of the product, weigh accurately, place in a 20 ml brown volumetric flask, add n-hexane to dissolve and dilute to the mark, and shake well.

[0070] (3) Preparation of reference solution: Accurately measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with n-hexane and shake well.

[0071] (4) Preparation of system suitability solution: Take the cut-off valve positioning solution (1).

[0072] (5) Detection: Accurately measure 100 μl of the system suitability solution and inject it into the liquid chromatograph. Accurately measure 100 μl of the test solution and the control solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0073] Compared with the prior art, the present invention has the following beneficial effects:

[0074] 1. The present invention provides a method for making vitamin D in vitamin D drops 3The analytical method in which the vitamin D and its related impurities A, D, and E are detected simultaneously has high separation and good detection effect, avoids the interference of the oily matrix in the vitamin D drops, can better meet the needs of vitamin D drops determination, and can be used for quality research and quality control of vitamin D drops products.

[0075] 2. Compared with conventional chromatographic analysis methods, the present invention adopts a two-dimensional liquid chromatography detection method. This analysis method has a stable baseline, good stability, good specificity, and high sensitivity. It can accurately detect related substances in vitamin D drops, thereby comprehensively, accurately, and objectively evaluating product quality, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It is a one-dimensional liquid chromatogram under the chromatographic conditions of Comparative Example 1 of the present invention.

[0077] Figure 2 It is a one-dimensional liquid chromatogram under the chromatographic conditions of Example 1 of the present invention.

[0078] Figure 3 It is a two-dimensional liquid chromatogram under the chromatographic conditions of Example 2 of the present invention.

[0079] Figure 4 This is a two-dimensional liquid chromatogram of chromatographic column screening condition 1 in Example 3 of the present invention.

[0080] Figure 5 This is a two-dimensional liquid chromatogram of chromatographic column screening condition 2 of Example 3 of the present invention.

[0081] Figure 6 It is a two-dimensional liquid chromatogram under the chromatographic conditions of Example 4 of the present invention.

[0082] Figure 7 It is a two-dimensional liquid chromatogram under the chromatographic conditions of Example 5 of the present invention.

[0083] Figure 8 It is a two-dimensional liquid chromatogram under the chromatographic conditions of Example 6 of the present invention. DETAILED DESCRIPTION

[0084] 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.

[0085] The prescription of vitamin D drops is the raw material vitamin D 3 and solvent soybean oil. The impurities in the preparations are mainly derived from the degradation impurities of the raw materials. For reference, vitamin D 3 The relevant statutory standards for vitamin D 3 Analysis of forced degradation test results of raw material vitamin D3 The possible degradation impurities in the product include impurity A (trans-vitamin D 3 , light degradation impurities), impurity E (tachysterol vitamin D 3 , which may be an impurity degraded under combined conditions of light and heat) and impurity D (isotachysterol vitamin D 3 , acid degradation impurities), impurities A and E will be generated under improper storage conditions, such as not shielded from light or high temperature conditions. In order to ensure the quality of the product, impurities A and E are studied and controlled in the drop product. Impurity D is generated under acidic conditions. The soybean oil in the vitamin D drops contains a large amount of free fatty acids, which will lead to the generation of impurity D, thus seriously affecting the quality of the product. Therefore, impurity D is also studied and controlled in the drops product. The specific impurity information in this product is shown in the following table.

[0086] Table 1 Information on organic impurities in vitamin D drops

[0087]

[0088] Comparative Example 1

[0089] Patent CN 116297935 A discloses a method for detecting related substances in vitamin D drops, which specifically includes the following steps:

[0090] First dimension liquid chromatography:

[0091] (1) Chromatographic condition setting:

[0092] Chromatographic column: a column with urea-bonded silica gel as filler or a filler with similar functions (ChromCore VD-ChP41, 2.1 mm × 150 mm, 3 μm);

[0093] Mobile phase: n-hexane as mobile phase A, n-hexane-n-pentanol-isopropanol (98:1:1) as mobile phase B, gradient elution as shown in the following table;

[0094] Flow rate: 0.5ml / min;

[0095] Detection wavelength: 265nm;

[0096] Column temperature: 40°C;

[0097] Injection volume: 100 μl;

[0098] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0099] Time (minutes) A(％) B(％) 0 94.5 5.5 30 94.5 5.5 35 0 100 60 0 100 65 94.5 5.5 80 94.5 5.5

[0100] Vitamin D 3Reference stock solution: Take vitamin D 3 About 5 mg of the reference substance is accurately weighed and placed in a 20 ml volumetric flask. Add isooctane to dissolve and dilute to the mark.

[0101] Cut valve positioning solution (2) Measure vitamin D 3 An appropriate amount of reference stock solution was diluted with n-hexane to make a solution containing about 0.05 μg per 1 ml as vitamin D 3 Cut valve positioning solution with impurity D.

[0102] The cutting valve positioning solution (2) was injected into the first dimension liquid chromatograph to detect vitamin D 3 and impurity D, vitamin D in the first dimension liquid chromatography 3 The switching time of impurity D is set to vitamin D 3 2.5 minutes before the peak start time and 2 minutes after the peak end time (in the first dimension liquid chromatography system, vitamin D 3 The peak time coincides with that of impurity D).

[0103] The one-dimensional chromatogram is shown in Figure 1 As shown, under this one-dimensional liquid chromatography condition, vitamin D 3 The elution time of the chromatographic peak is greatly affected by the oily matrix. 3 The large deviation in the peak elution time makes it impossible to locate the chromatographic peak to be measured and determine the valve cutting time. Therefore, it is necessary to optimize the method based on the original one-dimensional chromatographic conditions to stabilize the peak elution time of the chromatographic peak to be measured.

[0104] Because vitamin D drops contain a large amount of soybean oil, when the cleaning ability of the mobile phase is poor or the running time is short, soybean oil may remain in the chromatographic column. The residue of multiple needles may cause abnormal conditions such as ghost peaks in the chromatogram and unstable elution time of the chromatographic peak. Therefore, the one-dimensional mobile phase was adjusted and optimized. First, isopropanol with stronger cleaning ability was added, and secondly, the running time of the mobile phase was extended.

[0105] Example 1 (Changing one-dimensional chromatography mobile phase and running time)

[0106] First dimension liquid chromatography:

[0107] (1) Chromatographic condition setting:

[0108] Chromatographic column: a column with urea-bonded silica gel as filler or a filler with similar functions (ChromCore VD-ChP41, 2.1 mm × 150 mm, 3 μm);

[0109] Mobile phase: n-hexane as mobile phase A, isopropanol as mobile phase B, n-hexane-n-pentanol-isopropanol (98:1:1) as mobile phase C, and gradient elution as shown in the following table;

[0110] Flow rate: 0.5ml / min;

[0111] Detection wavelength: 265nm;

[0112] Column temperature: 40°C;

[0113] Injection volume: 100 μl;

[0114] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0115] Time (minutes) A(％) B(％) C(％) 0 95 0 5 40 95 0 5 45 0 0 100 50 0 0 100 55 0 20 80 63 0 20 80 65 95 0 5 120 95 0 5

[0116] Vitamin D 3 Reference stock solution: Take vitamin D 3 About 5 mg of the reference substance is accurately weighed and placed in a 20 ml volumetric flask. Add isooctane to dissolve and dilute to the mark.

[0117] Cut valve positioning solution (2) Measure vitamin D 3 An appropriate amount of reference stock solution was diluted with n-hexane to make a solution containing about 0.05 μg per 1 ml as vitamin D 3 Cut valve positioning solution with impurity D.

[0118] The cutting valve positioning solution (2) was injected into the first dimension liquid chromatograph to detect vitamin D 3 and impurity D, vitamin D in the first dimension liquid chromatography 3 The switching time of impurity D is set to vitamin D 3 2.5 minutes before the peak start time and 2 minutes after the peak end time (in the first dimension liquid chromatography system, vitamin D 3 The peak time coincides with that of impurity D).

[0119] The one-dimensional chromatogram is shown in Figure 2 As shown in the figure, under this one-dimensional chromatographic condition, the one-dimensional spectrum is significantly improved and is less affected by the oily matrix. 3 The chromatographic peak has a stable elution time, which is convenient for chromatographic peak positioning and valve cutting.

[0120] Example 2 (cut valve into two dimensions)

[0121] First dimension liquid chromatography:

[0122] (1) Chromatographic condition setting:

[0123] Chromatographic column: a column with urea-bonded silica gel as filler or a filler with similar functions (ChromCore VD-ChP41, 2.1 mm × 150 mm, 3 μm);

[0124] Mobile phase: n-hexane as mobile phase A, isopropanol as mobile phase B, n-hexane-n-pentanol-isopropanol (98:1:1) as mobile phase C, and gradient elution as shown in the following table;

[0125] Flow rate: 0.5ml / min;

[0126] Detection wavelength: 265nm;

[0127] Column temperature: 40°C;

[0128] Injection volume: 100 μl;

[0129] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0130] Time (minutes) A(％) B(％) C(％) 0 95 0 5 40 95 0 5 45 0 0 100 50 0 0 100 55 0 20 80 63 0 20 80 65 95 0 5 120 95 0 5

[0131] Second Dimension Liquid Chromatography:

[0132] (1) Chromatographic condition setting:

[0133] Chromatographic column: Silica gel as filler (Agilent Zorbax Silica, 3.0 mm × 100 mm, 1.8 μm);

[0134] Mobile phase: n-hexane-n-pentanol-isopropanol (996:2:2), isocratic elution;

[0135] Flow rate: 0.5ml / min;

[0136] Detection wavelength: 265nm;

[0137] Column temperature: 40°C;

[0138] Injection volume: 100 μl;

[0139] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0140] Vitamin D 3 Reference stock solution: Take vitamin D 3 About 5 mg of the reference substance is accurately weighed and placed in a 20 ml volumetric flask. Add isooctane to dissolve and dilute to the mark.

[0141] Cut valve positioning solution (1) Measure vitamin D 35 ml of the reference stock solution is placed in a stoppered glass container, sealed after nitrogen is passed through it, and heated in a 90°C water bath for 1 hour. Take it out, cool it quickly, add 5 ml of n-hexane, shake it well, measure 1 ml of the solution, place it in a 25 ml transparent volumetric bottle, add n-hexane to dilute to the scale, shake it well, and place the volumetric bottle 5 to 6 cm away from the lamp under two 8W ultraviolet lamps with main wavelengths of 254nm and 365nm respectively, and irradiate it for 40 minutes to make the solution contain impurities A and vitamin D 3 and impurity E, as a valve positioning solution for impurity A and impurity E.

[0142] Cut valve positioning solution (2) Measure vitamin D 3 An appropriate amount of reference stock solution was diluted with n-hexane to make a solution containing about 0.05 μg per 1 ml as vitamin D 3 Cut valve positioning solution with impurity D.

[0143] The cut-valve positioning solution (1) was injected into the first-dimension liquid chromatograph to locate impurities A and E. The switching time of impurities A and E in the first-dimension liquid chromatograph was set to 2 minutes before and after their respective retention times. The cut-valve positioning solution (2) was injected into the first-dimension liquid chromatograph to locate vitamin D. 3 and impurity D, vitamin D in the first dimension liquid chromatography 3 The switching time of impurity D is set to vitamin D 3 2.5 minutes before the peak start time and 2 minutes after the peak end time.

[0144] (2) Preparation of test solution: Take about 2.5 g of the product, weigh accurately, place in a 20 ml brown volumetric flask, add n-hexane to dissolve and dilute to the mark, and shake well.

[0145] (3) Preparation of reference solution: Accurately measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with n-hexane and shake well.

[0146] (4) Preparation of system suitability solution: Take the cut-off valve positioning solution (1).

[0147] (5) Detection: Accurately measure 100 μl of the system suitability solution and inject it into the liquid chromatograph. Accurately measure 100 μl of the test solution and the control solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0148] Under the one-dimensional chromatography conditions, the retention time of each analyte is determined and set: the retention time of impurity A is about 21 minutes; the retention time of impurity D is about 27 minutes; the retention time of impurity E is about 32 minutes. After the valve is cut into the second dimension, under the two-dimensional chromatography conditions, the two-dimensional spectrum is as follows Figure 3 As shown, it can be seen that the baselines of the impurities A, D, and E peak positions are uneven, the peak shapes are poor, and the peak separation is also poor. Figure 2 This indicates that under this one-dimensional chromatographic condition, vitamin D 3 The chromatographic peak has a stable elution time, impurities A, D, E and vitamin D 3 can be cut into two dimensions, but Figure 3 This indicates that the related substances cannot be effectively separated under the two-dimensional chromatography conditions, so subsequent optimization only needs to be performed based on the two-dimensional chromatography conditions. The optimization of the two-dimensional chromatography conditions of this product mainly involves the following steps:

[0149] Example 3 (Screening of chromatographic columns)

[0150] (I) Chromatographic column screening conditions 1

[0151] Second Dimension Liquid Chromatography:

[0152] (1) Chromatographic condition setting:

[0153] Chromatographic column: filled with silica gel (Agilent Polaris Si-A, 3.0 mm × 150 mm, 3 μm);

[0154] Mobile phase: n-hexane-n-pentanol-isopropanol (996:2:2), isocratic elution;

[0155] Flow rate: 0.5ml / min;

[0156] Detection wavelength: 265nm;

[0157] Column temperature: 40°C;

[0158] Injection volume: 100 μl;

[0159] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0160] The one-dimensional chromatography conditions are the same as those in Example 2. The retention time of each analyte is determined and set: the retention time of impurity A is about 22 minutes; the retention time of impurity D is about 26 minutes; the retention time of impurity E is about 32 minutes. After the valve is cut into the second dimension, under the two-dimensional chromatography conditions, the two-dimensional spectrum is as follows Figure 4 As shown, after replacing the chromatographic column, there was no obvious improvement in the spectrum, the baseline was uneven, the peak shape of impurity A was poor, impurity D peaked near the inverted peak, and impurity E was not completely separated from the main peak. Further optimization is needed, and replacement of the chromatographic column should be considered.

[0161] (II) Column screening conditions 2

[0162] Second Dimension Liquid Chromatography:

[0163] (1) Chromatographic condition setting:

[0164] Chromatographic column: silica gel as filler (ShimNex HE Sil, 4.6 mm × 150 mm, 3 μm);

[0165] Mobile phase: n-hexane-n-pentanol-isopropanol (996:2:2), isocratic elution;

[0166] Flow rate: 0.5ml / min;

[0167] Detection wavelength: 265nm;

[0168] Column temperature: 40°C;

[0169] Injection volume: 100 μl;

[0170] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0171] The one-dimensional chromatography conditions are the same as those in Example 2. The retention time of each analyte is determined and set: the retention time of impurity A is about 21 minutes; the retention time of impurity D is about 26 minutes; the retention time of impurity E is about 32 minutes. After the valve is cut into the second dimension, under the two-dimensional chromatography conditions, the two-dimensional spectrum is as follows Figure 5 As shown in the figure, under the chromatographic column (ShimNex HE Sil, 4.6mm×150mm, 3μm), the baseline is relatively flat, the peak shapes of impurities A and D are significantly improved, and the separation of impurity D and the main peak is significantly improved. In the future, (ShimNex HESil, 4.6mm×150mm, 3μm) can be used as the chromatographic column for the analysis method of related substances and continue to be optimized. Impurity E cannot be completely separated from the main peak, and the column temperature can be changed.

[0172] Example 4 (Changing column temperature)

[0173] Second Dimension Liquid Chromatography:

[0174] (1) Chromatographic condition setting:

[0175] Chromatographic column: silica gel as filler (ShimNex HE Sil, 4.6 mm × 150 mm, 3 μm);

[0176] Mobile phase: n-hexane-n-pentanol-isopropanol (996:2:2), isocratic elution;

[0177] Flow rate: 0.5ml / min;

[0178] Detection wavelength: 265nm;

[0179] Column temperature: 30°C;

[0180] Injection volume: 100 μl;

[0181] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0182] The one-dimensional chromatography conditions are the same as those in Example 2. The retention time of each analyte is determined and set: the retention time of impurity A is about 22 minutes; the retention time of impurity D is about 27 minutes; and the retention time of impurity E is about 33 minutes. After the valve is cut into the second dimension, under the two-dimensional chromatography conditions, the two-dimensional spectrum is as follows Figure 6 As shown in the figure, by lowering the column temperature, the separation between the main peak and impurity E is improved, but it still does not meet the requirements of complete separation. The baseline at the peak of impurity A is poor, and there is an interfering peak on the right side of impurity D. Try to change the mobile phase and continue to optimize.

[0183] Example 5 (Changing the mobile phase)

[0184] Second Dimension Liquid Chromatography:

[0185] (1) Chromatographic condition setting:

[0186] Chromatographic column: silica gel as filler (ShimNex HE Sil, 4.6 mm × 150 mm, 3 μm);

[0187] Mobile phase: n-hexane-n-pentanol-ethanol (996:2:2), isocratic elution;

[0188] Flow rate: 0.5ml / min;

[0189] Detection wavelength: 265nm;

[0190] Column temperature: 30°C;

[0191] Injection volume: 100 μl;

[0192] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0193] The one-dimensional chromatography conditions are the same as those in Example 2. The retention time of each analyte is determined and set: the retention time of impurity A is about 21 minutes; the retention time of impurity D is about 27 minutes; the retention time of impurity E is about 33 minutes. After the valve is cut into the second dimension, under the two-dimensional chromatography conditions, the two-dimensional spectrum is as follows Figure 7 As shown, compared with Example 4, the mobile phase was changed. Under this condition, the baseline of the impurity A peak position was slightly uneven, but the separation between impurities D and impurity E and the main peak was significantly improved, and the separation requirements were met. Try to continue changing the mobile phase ratio to improve the peak shape.

[0194] Example 6 (Changing the mobile phase ratio)

[0195] Second Dimension Liquid Chromatography:

[0196] (1) Chromatographic condition setting:

[0197] Chromatographic column: silica gel as filler (ShimNex HE Sil, 4.6 mm × 150 mm, 3 μm);

[0198] Mobile phase: n-hexane-n-pentanol-ethanol (998:1:1), isocratic elution;

[0199] Flow rate: 0.5ml / min;

[0200] Detection wavelength: 265nm;

[0201] Column temperature: 30°C;

[0202] Injection volume: 100 μl;

[0203] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 ml.

[0204] The one-dimensional chromatography conditions are the same as those in Example 2. The retention time of each analyte is determined and set: the retention time of impurity A is about 21 minutes; the retention time of impurity D is about 26 minutes; and the retention time of impurity E is about 33 minutes. After the valve is cut into the two-dimensional chromatography conditions, the two-dimensional spectrum is as follows Figure 8 As shown, compared with Example 5, the mobile phase ratio was further adjusted, the baseline of the impurity A peak position was flat, the impurities D and E had good peak shapes, the separation between the main peak and the impurities was greater than 1.5, and the impurities A, D, E were completely separated from the main component under this method.

[0205] Test Example 1 Specificity

[0206] Diluent: n-hexane

[0207] Blank excipient solution: Take about 2.5 g of blank content, weigh accurately, place in a 20 ml volumetric flask, dissolve and dilute to the scale with n-hexane, and shake well.

[0208] Test solution: Take about 2.5g of vitamin D drops, weigh accurately, place in a 20ml volumetric flask, and add n-hexane to dissolve and make up to volume.

[0209] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with n-hexane, shake well, and obtain.

[0210] Vitamin D 3 Stock solution: Take vitamin D 3 About 5 mg of the reference substance is placed in a 20 ml volumetric flask and isooctane is added to dissolve it.

[0211] System suitability solution: Take vitamin D 35 ml of stock solution is placed in a stoppered glass container, sealed after nitrogen is passed through, heated in a 90°C water bath for 1 hour, taken out, cooled rapidly, added 5 ml of n-hexane, shaken well, 1 ml is transferred to a 25 ml transparent volumetric bottle, and diluted to the mark with n-hexane. Irradiate for 40 minutes under two 8W UV lamps with dominant wavelengths at 254 nm and 365 nm, 5 to 6 cm from the lamp tube.

[0212] Stock solutions of various impurities:

[0213] Impurity A stock solution ①: Take about 2 mg of impurity A, weigh accurately, place in a 20 ml volumetric flask, dissolve with n-hexane and dilute to the scale.

[0214] Impurity D stock solution①: Take about 2 mg of impurity D, weigh accurately, place in a 20 ml volumetric flask, dissolve with n-hexane and dilute to the scale.

[0215] Impurity E stock solution ①: Take about 2 mg of impurity E, weigh accurately, place in a 20 ml volumetric flask, dissolve with n-hexane and dilute to the scale.

[0216] Impurity stock solution ②: Accurately measure 0.5 ml of each impurity stock solution ① into different 50 ml volumetric bottles, add n-hexane to dilute to the scale.

[0217] Impurity location solutions: Accurately measure ②1ml of each impurity stock solution into a 20ml volumetric bottle, add n-hexane to dilute to the scale.

[0218] Spiked test solution: Take about 2.5g of vitamin D drops, weigh accurately, place in a 20ml volumetric flask, add 1ml each of impurity A, D, and E stock solutions②, and dilute to the scale with n-hexane.

[0219] Separately mix the above solutions (except for the impurity stock solution and vitamin D 3 The blank solution (outside the stock solution) was injected into the liquid chromatograph to examine whether the blank solvent and blank excipients interfered with the determination of the analyte, and to examine the separation between the known impurities and between the impurities and the main peak.

[0220] Test Example 2 Solution Stability

[0221] Diluent: n-hexane;

[0222] Test solution: Take about 2.5g of vitamin D drops, weigh accurately, place in a 20ml volumetric flask, and add n-hexane to dissolve and make up to volume.

[0223] System suitability solution: Take vitamin D 35 ml of stock solution is placed in a stoppered glass container, sealed after nitrogen is passed through, heated in a 90°C water bath for 1 hour, taken out, cooled rapidly, added 5 ml of n-hexane, shaken well, 1 ml is transferred to a 25 ml transparent volumetric bottle, and diluted to the mark with n-hexane. Irradiate for 40 minutes under two 8W UV lamps with dominant wavelengths at 254 nm and 365 nm, 5 to 6 cm from the lamp tube.

[0224] Spiked test solution: Take about 2.5g of vitamin D drops, weigh accurately, place in a 20ml volumetric flask, add 1ml each of impurity A, D, and E stock solutions②, and dilute to the scale with n-hexane.

[0225] Control solution: Accurately measure 1 ml of the spiked test solution, place it in a 100 ml volumetric flask, dilute to the mark with n-hexane, and shake well. Place the spiked test solution and the control solution at room temperature, and inject samples at different time points to examine the stability of the spiked test solution and the control solution.

[0226] Test Example 3 Detection Limit and Quantification Limit

[0227] Diluent: n-hexane;

[0228] LOQ solution: Take an appropriate amount of each impurity stock solution and the main component stock solution, dilute them with n-hexane to make a solution of a certain concentration, until the signal-to-noise ratio of each impurity peak and the main peak is ≥10:1.

[0229] LOD solution: Take an appropriate amount of each impurity stock solution and main component stock solution, dilute them with n-hexane to make a solution of a certain concentration, until the signal-to-noise ratio of each impurity peak and the main peak is ≥3:1.

[0230] 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.

[0231] Test Example 4 Linearity and Range

[0232] Diluent: n-hexane;

[0233] Selection of linear solution concentration points: The linear concentration points are 50%, 100%, 150%, and 200% of the LOQ and limit concentration (1.0%) of each component.

[0234] Preparation of linear stock solution: Accurately measure appropriate amounts of impurity A, impurity D, impurity E stock solution and main component stock solution, dilute with diluent to make a stock solution containing 1 μg / ml of each component, and shake well.

[0235] The linear solution preparation is shown in the following table:

[0236]

[0237] The above linear solutions were respectively injected into the liquid chromatograph to examine the linear equation, correlation coefficient, response factor, etc. of each impurity.

[0238] Test Example 5 Precision

[0239] Diluent: n-hexane

[0240] Repeatability solution: Take about 2.5g of this product, weigh accurately, place in a 20ml volumetric flask, add appropriate amount of diluent to dissolve, accurately measure 1ml of impurity mixed stock solution, add n-hexane to dilute to the scale, shake well, and prepare 6 copies in parallel.

[0241] Control solution: Accurately measure 1 ml of the repeatability solution, place it in a 100 ml volumetric flask, add n-hexane to dilute to the scale, shake well, and obtain.

[0242] 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.

[0243] Test Example 6 Accuracy

[0244] Diluent: n-hexane

[0245] Test solution: Take about 2.5g of vitamin D drops, weigh accurately, place in a 20ml volumetric flask, and add n-hexane to dissolve and make up to volume.

[0246] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with n-hexane, shake well, and obtain.

[0247] The accuracy solution preparation is shown in the following table:

[0248]

[0249]

[0250] Accuracy control solution: Accurately measure 1 ml of accuracy solution, place it in a 100 ml volumetric flask, add n-hexane to dilute to the scale, shake well, and obtain.

[0251] 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.

[0252] Test Example 7 Durability

[0253] Diluent: n-hexane

[0254] System suitability solution: Take 5 ml of vitamin D3 stock solution, place it in a stoppered glass container, fill it with nitrogen and then seal it tightly, heat it in a 90℃ water bath for 1 hour, take it out, cool it quickly, add 5 ml of n-hexane, shake it well, take 1 ml to a 25 ml transparent volumetric bottle, add n-hexane to dilute to the mark. Irradiate it for 40 minutes under two 8W UV lamps with dominant wavelengths at 254nm and 365nm, 5 to 6 cm away from the lamp tube.

[0255] Spiked test solution: Take about 2.5g of vitamin D drops, weigh accurately, place in a 20ml volumetric flask, add 1ml each of impurity A, D, and E stock solutions②, and dilute to the scale with n-hexane.

[0256] Control solution: Accurately measure 1 ml of the spiked test solution, place it in a 100 ml volumetric flask, dilute to the mark with n-hexane, shake well, and obtain.

[0257] According to the table below, fine-tune the second-dimension HPLC flow rate (range: ±0.1 ml / min), column temperature (range: ±1°C), chromatographic column and other conditions to examine the durability of the method.

[0258] Chromatographic conditions Flow rate (ml / min) Column temperature (℃) Original conditions 0.5 30 Column 1 0.6 30 Column 1 0.4 30 Column 1 0.5 29 Column 1 0.5 31 Column 1 0.5 30 Column 1 0.5 30 Column 2 0.5 30

[0259] 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.

[0260] The present invention verifies the detection method from the perspectives of specificity, solution stability, detection limit and quantification limit, linearity and range, precision, accuracy, durability, etc. The verification results are shown in the table below.

[0261]

[0262]

Claims

1. A high performance liquid chromatography analysis method for related substances of vitamin D drops, characterized in that: The method can separate related substances in vitamin D drops, wherein the related substances include impurity A, impurity D, and impurity E, and the separation degree between each chromatographic peak is not less than 1.5; The first dimension HPLC conditions were set as follows: Chromatographic column: a chromatographic column with urea-bonded silica gel as filler or a filler with similar functions; Mobile phase: n-hexane as mobile phase A, isopropanol as mobile phase B, n-hexane-n-pentanol-isopropanol = 98:1:1 as mobile phase C, for gradient elution; Flow rate: 0.5~1.0ml / min; Detection wavelength: 260~270nm; Column temperature: 25-45°C; Injection volume: 50-150 μl; The second dimension HPLC conditions were set as follows: Chromatographic column: silica gel is used as filler; Mobile phase: n-hexane-n-pentanol-ethanol, isocratic elution; Flow rate: 0.5~1.0ml / min; Detection wavelength: 260~270nm; Column temperature: 20-40°C; Injection volume: 50-150 μl; The switching procedure is as follows: taking a cut-valve positioning solution (1) and injecting it into a first-dimension liquid chromatograph to locate impurities A and impurity E, and the switching time of impurities A and impurity E in the first-dimension liquid chromatograph is set to 2 to 3 minutes before and after their respective retention times; taking a cut-valve positioning solution (2) and injecting it into the first-dimension liquid chromatograph to locate vitamin D3 and impurity D, and the switching time of vitamin D3 and impurity D in the first-dimension liquid chromatograph is set to 2 to 3 minutes before the start time of the vitamin D3 peak and 2 to 3 minutes after the end time of the vitamin D3 peak; In the first-dimensional liquid chromatography, the retention time of impurity A is 19 min to 23 min; the retention time of impurity D is 24.5 min to 29 min; and the retention time of impurity E is 29.5 min to 35 min.

2. The method according to claim 1, characterized in that The related substances include impurity A, impurity D, and impurity E, and the structural formula is:

3. The analysis method according to claim 1, characterized in that The chromatographic column under the first-dimensional liquid chromatography conditions is ChromCore VD-ChP41, 2.1 mm×150 mm, 3 μm; the chromatographic column under the second-dimensional liquid chromatography conditions is ShimNexHE Sil, 4.6 mm×150 mm, 3 μm.

4. The analysis method according to claim 1, characterized in that The column temperature under the first-dimension liquid chromatography condition is 40°C; the column temperature under the second-dimension liquid chromatography condition is 30°C.

5. The analysis method according to claim 1, characterized in that The mobile phase gradient elution program under the first dimension liquid chromatography conditions is:

6. The analysis method according to claim 1, characterized in that The elution ratio of the mobile phase under the second dimension liquid chromatography conditions is 998:1:1:

1.

7. The analysis method according to claim 1, characterized in that The flow rate under the first-dimension liquid chromatography condition and the second-dimension liquid chromatography condition was 0.5 ml / min.

8. The analysis method according to claim 1, characterized in that The wavelength under the first-dimension liquid chromatography condition and the second-dimension liquid chromatography condition is 265 nm.

9. The analysis method according to claim 1, characterized in that The injection volume under the first-dimension liquid chromatography conditions and the second-dimension liquid chromatography conditions was 100 μL.

10. A method for detecting related substances in vitamin D drops, comprising the following steps: First dimension liquid chromatography: (1) Chromatographic condition setting: Chromatographic column: urea-bonded silica gel as filler or filler with similar functions, ChromCore VD-ChP41, 2.1mm×150mm, 3μm; Mobile phase: n-hexane as mobile phase A, isopropanol as mobile phase B, n-hexane-n-pentanol-isopropanol (98:1:1) as mobile phase C, and gradient elution as shown in the following table; Flow rate: 0.5ml / min; Detection wavelength: 265nm; Column temperature: 40°C; Injection volume: 100 μl; Collection tube: polyetheretherketone tube, inner diameter 0.0762cm, 20m, volume about 9ml; Second Dimension Liquid Chromatography: (1) Chromatographic condition setting: Chromatographic column: silica gel as filler, ShimNex HE Sil, 4.6 mm × 150 mm, 3 μm; Mobile phase: n-hexane-n-pentanol-ethanol ratio 998:1:1, isocratic elution; Flow rate: 0.5ml / min; Detection wavelength: 265nm; Column temperature: 30°C; Injection volume: 100 μl; Collection tube: polyetheretherketone tube, inner diameter 0.0762cm, 20m, volume about 9ml; Vitamin D3 reference stock solution: Take about 5 mg of vitamin D3 reference, weigh accurately, place in a 20 ml volumetric flask, add isooctane to dissolve and dilute to scale; Valve positioning solution (1) Measure 5 ml of the vitamin D3 reference stock solution, place it in a stoppered glass container, pass nitrogen through it and then seal it, heat it in a 90°C water bath for 1 hour, take it out, cool it quickly, add 5 ml of n-hexane, shake it well, measure 1 ml of the solution, place it in a 25 ml transparent volumetric bottle, add n-hexane to dilute to the scale, shake it well, and place the volumetric bottle 5 to 6 cm away from the lamp tube under two 8 W ultraviolet lamps with main wavelengths of 254 nm and 365 nm respectively, and irradiate it for 40 minutes to make the solution contain impurity A, vitamin D3 and impurity E, which is used as the valve positioning solution of impurities A and impurity E; Valve cutting positioning solution (2) Take an appropriate amount of the vitamin D3 reference substance stock solution and dilute it with n-hexane to make a solution containing about 0.05 μg per 1 ml, which serves as the valve cutting positioning solution of vitamin D3 and impurity D; The cut-valve positioning solution (1) is injected into the first-dimension liquid chromatograph to locate impurities A and impurity E. The switching time between impurities A and impurity E in the first-dimension liquid chromatograph is set to 2 minutes before and after their respective retention times. The cut-valve positioning solution (2) is injected into the first-dimension liquid chromatograph to locate vitamin D3 and impurity D. The switching time between vitamin D3 and impurity D in the first-dimension liquid chromatograph is set to 2.5 minutes before the start time of the vitamin D3 peak and 2 minutes after the end time of the vitamin D3 peak. (2) Preparation of test solution: Take about 2.5 g of the product, weigh accurately, place in a 20 ml brown volumetric flask, add n-hexane to dissolve and dilute to the mark, shake well; (3) Preparation of reference solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with n-hexane, and shake well; (4) Preparation of system suitability solution: Take the valve positioning solution (1) to obtain; (5) Detection: Accurately measure 100 μl of the system suitability solution and inject it into the liquid chromatograph. Accurately measure 100 μl of the test solution and the control solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

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