Method for detecting impurities in vitamin AD oily preparation and preparation of test solution

Abstract

The present invention discloses a method for detecting impurities in vitamin AD oily preparations and the preparation of test sample solutions, belonging to the technical field of detection and analysis. The preparation method of the test sample solution for detecting impurities in vitamin AD oily preparations provided by the present invention includes extraction, low-temperature treatment, concentration, and formulation steps. By combining the preparation method of the test sample solution with two-dimensional normal-phase high-performance liquid chromatography detection, a method for detecting impurities in vitamin AD oily preparations is obtained. Through specific extraction solvents and extraction steps, the test sample solution obtained has a high extraction rate of vitamin D3, good reproducibility, and few irrelevant impurities. Combining with the two-dimensional normal-phase high-performance liquid chromatography detection method provided by the present invention, it can achieve good detection effects on vitamin D3 and related impurities in vitamin AD oily preparations, with high detection sensitivity, good reproducibility, and strong specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis, and particularly relates to a method for detecting impurities in an oily preparation of vitamin AD and a preparation method of a test solution. Background Art

[0002] Vitamin A and vitamin D are essential substances for human growth and development, and play an important role especially in the development of infants and young children, the integrity of epithelial tissues, vision, reproductive organs, the constancy of blood calcium and phosphorus, bone, and tooth growth and development. Vitamin A and vitamin D can stably exist in an oily matrix. Therefore, in existing products, vitamin A and vitamin D are made into oily preparations, such as vitamin AD soft capsules or drops, which are made by dissolving vitamin A and vitamin D2 or vitamin D3, adding cod liver oil or refined edible oil (removing solid fat at about 0 °C) and adjusting the concentration. However, the difficulty in detecting vitamin D3 and related substances in such oily preparations of vitamin AD lies in that the proportion of oily excipients in such preparations is high, the content of vitamin D3 is low, and the interference of vitamin A is serious. Therefore, it is particularly urgent to invent a detection method for related substances of vitamin D3 with strong specificity, good reproducibility, high sensitivity and good resolution, and a preparation method of a supply solution with a high extraction rate, which can effectively control the quality of vitamin AD soft capsules and drops.

[0003] Vitamin D3 is a fat-soluble vitamin and is also regarded as a hormone precursor acting on calcium and phosphorus metabolism. It is often combined with calcium carbonate, vitamin A or a variety of vitamins and minerals to form a compound preparation. Vitamin D3 is liable to deteriorate when exposed to light or air, and is easily oxidized and photolyzed to generate impurities such as previtamin D3, trans-vitamin D3, lumisterol D3, tachysterol D3 and isotachysterol D3. Therefore, the storage method of vitamin D3 raw material specified in the Chinese Pharmacopoeia is to store it in a dark place, filled with nitrogen, sealed and stored in a cold place. At present, the pharmacopoeia method only publishes the detection method for related substances of vitamin D3 raw material, including chromatographic conditions and the preparation method of test solution. Some patents or literatures publish the preparation method of test sample for calcium carbonate D3 chewable tablets and the method for detecting related substances in a normal phase system. After being reproduced by the applicant, it is found that due to insufficient extraction during the preparation process of the test solution or significant interference of excipients during the detection process, the impurity content cannot be accurately detected. In addition, in some methods, the preparation method of the extraction solution is cumbersome, and a variety of toxic organic solvents are used, which poses a high operation risk to experimental personnel, and there are also problems that the active ingredients cannot be completely extracted and the interference of excipients is significant. And these methods are applicable to chewable tablets and are not applicable to oily preparations such as vitamin AD soft capsules and drops. Summary of the Invention

[0004] To overcome at least one of the above-mentioned problems existing in the prior art, one of the objectives of the present invention is to provide a method for preparing a test sample solution for detecting impurities in vitamin AD oily preparations, which method has a high extraction rate, good reproducibility, and few impurities in the extract.

[0005] Another objective of the present invention is to provide a method for detecting impurities in vitamin AD oily preparations.

[0006] To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0007] The first aspect of the present invention provides a method for preparing a test sample solution for detecting impurities in vitamin AD oily preparations, comprising the following steps:

[0008] S1: Extraction: Use an ethanol aqueous solution to extract the vitamin AD oily preparation, take the upper layer solution to obtain an extraction solution;

[0009] S2: Low-temperature treatment: Place the extraction solution obtained in step S1 in an environment at -10°C or below for low-temperature treatment, and take the separated solution after solid-liquid separation;

[0010] S3: Concentration: Remove the solvent of the separated solution obtained in step S2 to obtain a test sample residue;

[0011] S4: Preparation: Mix the test sample residue obtained in step S3 with n-hexane to obtain a test sample solution.

[0012] Through a large number of experiments, it is found in this application that by using the specific extraction solvent and extraction steps of the present invention, in the obtained test sample solution, the extraction rate of vitamin D3 can be close to 100%. However, referring to the extraction methods in the prior art, due to poor extraction conditions, the final extraction rate is low and the reproducibility is very poor. In this application, a comprehensive investigation is carried out on the extraction method of vitamin D3, and finally the selected extraction method has good reproducibility, high extraction rate, and few irrelevant impurities, and can achieve good detection effects for high-performance liquid chromatography.

[0013] Preferably, the vitamin AD oily preparation includes vitamin AD soft capsules, vitamin AD drops, or a combination thereof.

[0014] Preferably, the impurities in the vitamin AD oily preparation include at least one of provitamin D3, trans-vitamin D3, lumisterol D3, vitamin D3 precursor, tachysterol D3, or isotachysterol D3.

[0015] Preferably, in step S1, the volume concentration of ethanol in the ethanol aqueous solution is 80-98%; more preferably 85-97%; still more preferably 90-95%.

[0016] Preferably, in step S1, the number of extractions is 5 to 10 times; more preferably 6 to 8 times.

[0017] Preferably, in step S1, the extraction operation includes centrifugation; the centrifugation speed is preferably 2000 to 6000 rpm, more preferably 3000 to 5000 rpm; the centrifugation time is preferably 3 to 15 minutes, more preferably 8 to 12 minutes; the centrifugation temperature is preferably 0 to 5 °C, more preferably 0 to 1 °C.

[0018] Preferably, in step S1, the specification of vitamin D3 in the vitamin AD oily preparation is ≥ 400 IU; more preferably 400 to 700 IU. Non-limiting specific examples are 400 IU, 450 IU, 500 IU, 550 IU, 600 IU, 650 IU or 700 IU.

[0019] The method of the present invention can be used for the extraction of low-dose vitamin D3 and related impurities in vitamin AD oily preparations. It should be noted that those skilled in the art know that the extraction method and the related substance detection method of the present invention are also applicable to preparations with higher vitamin D3 content. Therefore, for the detection of specific impurities in vitamin D3-containing preparations, as long as the extraction method and / or detection method disclosed in the present invention are used, they are within the protection scope of the present invention.

[0020] Preferably, in step S1, the extraction operation includes shaking.

[0021] Preferably, the shaking method includes manual shaking or vortexing.

[0022] Preferably, the shaking time is ≥ 1 minute; more preferably 1 to 10 minutes; even more preferably 2 to 5 minutes.

[0023] Preferably, in step S2, the time for low-temperature treatment is 0.5 to 2.5 hours; more preferably 0.5 to 1.5 hours; even more preferably 0.8 to 1.2 hours.

[0024] Preferably, in step S2, the solid-liquid separation method is selected from centrifugation; in step S2, the centrifugation speed is preferably 3000 to 5000 rpm, more preferably 3500 to 4500 rpm; the centrifugation time is preferably 5 to 15 minutes, more preferably 8 to 12 minutes; the centrifugation temperature is preferably 0 to 5 °C, more preferably 0 to 1 °C.

[0025] Preferably, in step S3, the method for removing the solvent of the separation solution obtained in step S2 is selected from rotary evaporation to dryness.

[0026] Preferably, the rotary evaporation is carried out in a water bath at 40-60°C; more preferably, it is carried out in a water bath at 45-55°C; even more preferably, it is carried out in a water bath at 48-52°C.

[0027] The second aspect of the present invention provides a method for detecting impurities in a vitamin AD oily preparation, comprising the following steps:

[0028] Step 1, solution preparation: Prepare a test solution according to the preparation method described in the first aspect of the present invention;

[0029] Step 2, two-dimensional normal-phase high-performance liquid chromatography detection: Use normal-phase high-performance liquid chromatography to detect the test solution prepared in step S1, including the following steps carried out in sequence:

[0030] The first-dimensional liquid chromatography, mobile phase A is n-hexane, mobile phase B is n-hexane, n-pentanol and isopropanol, gradient elution;

[0031] The second-dimensional liquid chromatography, the mobile phase is n-hexane and n-pentanol, isocratic elution.

[0032] Through the comparative analysis of one-dimensional normal-phase high-performance liquid chromatography detection, one-dimensional reverse-phase high-performance liquid chromatography detection and two-dimensional normal-phase high-performance liquid chromatography detection, it is found that using the two-dimensional normal-phase high-performance liquid chromatography detection method for detection, the detected peak shape is more standard, the resolution between the main peak and adjacent peaks, and between known impurities and adjacent peaks is better, the excipients do not interfere with the detection of known impurities, and the detected impurity types are more and more comprehensive. Therefore, based on the test solution prepared by the preparation method described in the first aspect of the present invention and combined with the two-dimensional normal-phase high-performance liquid chromatography detection method, a good detection effect on related impurities can be achieved. First, use the first-dimensional liquid chromatography to separate some blank excipients, vitamin A and vitamin D, and then use the second-dimensional liquid chromatography to specifically detect vitamin D3 and related impurities. The separation effect of the first-dimensional liquid chromatography is good, thus avoiding the detection interference of excipients and vitamin A on vitamin D3 and related impurities. Using the extraction method and detection method of the present invention, the detection of vitamin D3 and related impurities in low-dose vitamin AD oily preparations can be realized, with high extraction rate, high sensitivity, good reproducibility and strong specificity.

[0033] Preferably, the chromatographic column used in the first-dimensional liquid chromatography is selected from urea-bonded silica columns; more preferably, the urea-bonded silica column is selected from Thermo Acclaim HILIC-10 or Nano ChromCore VD-ChP4, with an inner diameter × column length of (2-2.5) × (100-200) mm and a stationary phase particle size of 1-5 μm; even more preferably, the inner diameter × column length of the urea-bonded silica column is 2.1 × 150 mm and the stationary phase particle size is 3 μm.

[0034] Preferably, in mobile phase B of the first - dimension liquid chromatography, the volume ratio of n - hexane, n - pentanol, and isopropanol is (95 - 100):(0.5 - 2):1; more preferably (97 - 99):(0.8 - 1.2):1; and even more preferably 98:1:1.

[0035] Preferably, the gradient elution degree of the first - dimension liquid chromatography is as follows:

[0036] From 0 to 30 min, the volume of mobile phase A is maintained at 85 - 95%, and the volume of mobile phase B is maintained at 5 - 15%;

[0037] From 30 to 35 min, the volume of mobile phase A drops to 0 - 5%, and the volume of mobile phase B rises to 95 - 100%;

[0038] From 35 to 60 min, the volume of mobile phase A is maintained at 0 - 5%, and the volume of mobile phase B is maintained at 95 - 100%;

[0039] From 60 to 65 min, the volume of mobile phase A rises to 85 - 95%, and the volume of mobile phase B drops to 5 - 15%;

[0040] From 65 to 200 min, the volume of mobile phase A is maintained at 85 - 95%, and the volume of mobile phase B is maintained at 5 - 15%.

[0041] More preferably, the gradient elution degree of the first - dimension liquid chromatography is as follows:

[0042] From 0 to 30 min, the volume of mobile phase A is maintained at 88 - 92%, and the volume of mobile phase B is maintained at 8 - 12%;

[0043] From 30 to 35 min, the volume of mobile phase A drops to 0 - 2%, and the volume of mobile phase B rises to 98 - 100%;

[0044] From 35 to 60 min, the volume of mobile phase A is maintained at 0 - 2%, and the volume of mobile phase B is maintained at 98 - 100%;

[0045] From 60 to 65 min, the volume of mobile phase A rises to 88 - 92%, and the volume of mobile phase B drops to 8 - 12%;

[0046] From 65 to 200 min, the volume of mobile phase A is maintained at 88 - 92%, and the volume of mobile phase B is maintained at 8 - 12%.

[0047] Even more preferably, the gradient elution degree of the first - dimension liquid chromatography is, where % is volume percentage:

[0048] Time (min) Mobile Phase A % Mobile Phase B % 0 90 10 30 90 10 35 0 100 60 0 100 65 90 10 200 90 10

[0049] Preferably, the detection conditions of the first - dimension liquid chromatography satisfy at least one of the following conditions (1) to (5):

[0050] (1) The valve - switching time is 10 - 28 min;

[0051] (2) The detection wavelength is 260 - 270 nm; preferably 265 nm;

[0052] (3) The flow rate of the mobile phase is 0.1 - 1 mL / min; preferably 0.4 - 0.6 mL / min;

[0053] (4) The injection volume is 50 - 200 μL;

[0054] (5) The column temperature is 35 - 45 °C.

[0055] Preferably, the chromatographic column used in the second - dimension liquid chromatography is selected from silica columns; more preferably, the silica column is selected from Phenomenex Silica(2) or Welch Topsil silica, with an inner diameter × column length of (4 - 5)×(150 - 250) mm and a stationary - phase particle size of 3 - 5 μm; even more preferably, the inner diameter × column length of the silica column is 4.6×250 mm and the stationary - phase particle size is 5 μm.

[0056] Preferably, in the mobile phase of the second - dimension liquid chromatography, the volume ratio of n - hexane to n - pentanol is 1000:(2 - 5); more preferably 1000:(3 - 4); even more preferably 1000:3.5.

[0057] Preferably, the detection conditions of the second - dimension liquid chromatography satisfy at least one of the following conditions (1) to (3):

[0058] (1) The detection wavelength is 275 - 285 nm; preferably 280 nm;

[0059] (2) The flow rate of the mobile phase is 0.8 - 1.2 mL / min; preferably 1 - 1.2 mL / min;

[0060] (3) The column temperature is 35 - 45 °C.

[0061] The beneficial effects of the present invention are as follows: Through specific extraction solvents and extraction steps, the extraction rate of vitamin D3 in the test solution obtained by the present invention is high, the reproducibility is good, and there are few irrelevant impurities. Combining with the two - dimensional normal - phase high - performance liquid chromatography detection method provided by the present invention, it can achieve good detection effects on vitamin D3 and related impurities in vitamin AD oily preparations, with high detection sensitivity, good reproducibility, and strong specificity.

[0062] Specifically, compared with the prior art, the present invention has the following advantages:

[0063] 1. The preparation method of the test solution provided by the present invention has been fully investigated for the extraction rate. The results show that each extraction step has been completely extracted, with a high extraction rate and good reproducibility, and is suitable for popularization and application. Moreover, the types of organic solvents used in the preparation process of the test solution are few, and the toxicity is relatively low, further reducing the operation risk of experimental personnel.

[0064] 2. The present invention uses a two-dimensional high-performance liquid system to detect low-content vitamin D3 and related substances in vitamin AD soft capsules and drops. Compared with the one-dimensional detection system in the prior art, it has stronger specificity, better reproducibility, and higher sensitivity. In the two-dimensional method of the present invention, each main peak and impurity peak can be detected and well separated, with little interference from excipients, and the response degree of the main peak is high.

[0065] 3. The detection method for related substances of the present invention can effectively separate and determine compound vitamin AD soft capsules or drops with a low content of 500 IU, and can effectively control the quality of single and compound preparations containing vitamin D3. Description of the Drawings

[0066] Figure 1 It is the chromatogram of the system suitability solution for detecting Example 1.

[0067] Figure 2 It is the comparative chromatogram of each sample solution for detecting Example 1.

[0068] Figure 3 It is the linear relationship diagram of impurity A for detecting Example 2.

[0069] Figure 4 It is the linear relationship diagram of impurity C for detecting Example 2.

[0070] Figure 5 It is the linear relationship diagram of impurity D for detecting Example 2.

[0071] Figure 6 It is the comparative chromatogram of each sample solution for detecting Comparative Example 1.

[0072] Figure 7 It is the comparative chromatogram of each sample solution for detecting Comparative Example 2.

[0073] Figure 8 It is the comparative chromatogram of each sample solution for detecting Comparative Example 3.

[0074] Figure 9 It is the comparative chromatogram of each sample solution for detecting Comparative Example 4.

[0075] Figure 10 It is the comparative chromatogram of each sample solution for detecting Comparative Example 5.

[0076] Figure 11 To detect the comparative spectrograms of each sample solution in Comparative Example 6. Specific Embodiments

[0077] The content of the present invention will be further described in detail through specific embodiments below. It should also be understood that the following embodiments are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the principles described in the present invention all fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description in this article, rather than being limited to the specific data in the following examples. The raw materials, reagents or devices used in the following examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing known methods without special instructions.

[0078] Test Sample Example 1

[0079] This example provides a method for preparing a test sample solution for detecting impurities in vitamin AD oily preparations, including the following steps:

[0080] Take an appropriate amount of vitamin AD drops soft capsules, cut open the soft capsules, transfer the contents to a centrifuge tube, take an appropriate amount of the contents (equivalent to 100 μg of vitamin D3), weigh accurately, place in a stoppered centrifuge tube, add 5 mL of 95% ethanol (i.e., an ethanol aqueous solution with an ethanol volume concentration of 95%), stopper tightly, shake vigorously for 2 min, let stand, centrifuge for 5 min (5000 rpm, 0 °C), suck out the upper clear liquid, repeat the extraction operation with 95% ethanol, and extract a total of 5 times; combine the upper layer solutions and place them in a stoppered centrifuge tube, freeze in the refrigerator (-20 °C ± 5 °C) for 1 - 2 h, take out, centrifuge for 10 min (4000 rpm, 0 °C), immediately take out and suck out the upper clear liquid, place in a centrifuge tube, and let it stand to room temperature; accurately pipette 28 mL of the above solution into a pear-shaped flask, rotary evaporate to dryness in a 50 °C water bath, add 4 mL of n-hexane precisely to the residue, vortex for 2 min to dissolve, and use it as the test sample solution, denoted as sample 1.

[0081] Test Sample Example 2

[0082] This example provides a method for preparing a test sample solution for detecting impurities in vitamin AD oily preparations, which is different from Test Sample Example 1 in that the number of times of repeating the extraction operation with 95% ethanol in this example is 7 times, and other conditions remain unchanged. The obtained test sample solution is denoted as sample 2.

[0083] Test Sample Comparative Example 1

[0084] This example provides a method for preparing a test sample solution for detecting impurities in vitamin AD oily preparation. The difference from Test Sample Example 1 is that in this example, the extraction reagent 95% ethanol is replaced with absolute ethanol, and the extraction times are 7 times, with other conditions remaining unchanged. The obtained test sample solution is denoted as Comparative Sample 1.

[0085] Test Sample Comparative Example 2

[0086] This example provides a method for preparing a test sample solution for detecting impurities in vitamin AD oily preparation. The difference from Test Sample Example 1 is that in this example, the extraction reagent 95% ethanol is replaced with a 2,6-di-tert-butyl-p-cresol (BHT) ethanol solution with a volume concentration of 0.02%, and the extraction times are 7 times, with other conditions remaining unchanged. The obtained test sample solution is denoted as Comparative Sample 2.

[0087] Test Sample Comparative Example 3

[0088] This example provides a method for preparing a test sample solution for detecting impurities in vitamin AD oily preparation, including the following steps: Take an appropriate amount of vitamin AD drops soft capsules, cut open the soft capsules, transfer the contents to a centrifuge tube, take an appropriate amount of the contents, weigh accurately, add 30 mL of methanol, vortex for 15 min, take the upper clear liquid, repeat the extraction operation 2 times, combine the methanol layers of each time, place in a refrigerator freezer (-20°C ± 5°C) for 1 - 2 h, take out, rotary evaporate to dryness in a 50°C water bath, accurately add methanol to the residue, and vortex for 2 min to dissolve, as the test sample solution. The obtained test sample solution is denoted as Comparative Sample 3.

[0089] Measure and calculate the extraction rates of vitamin D3 in Test Sample Examples 1 - 2 and Test Sample Comparative Examples 1 - 3. The results are shown in Table 1.

[0090] Table 1 Extraction rate results of the test sample solutions of the present invention

[0091] Sample Name Investigation Conditions Extraction Rate (%) Sample 1 Extracted 5 times with 95% ethanol 92.3 Sample 2 Extracted 7 times with 95% ethanol 99.7 Control Sample 1 Absolute ethanol 93.3 Control Sample 2 0.02% BHT ethanol solution 94.1 Control Sample 3 Methanol 80.6

[0092] As can be seen from Table 1, the extraction rate of Sample 2 is significantly higher than that of Sample 1, and the extraction rate can reach about 100%, indicating that the preparation method of repeating centrifugation with 95% ethanol for 7 times for the test sample has basically achieved complete extraction. The extraction rates obtained by using absolute ethanol, 0.02% BHT ethanol solution, and methanol respectively are relatively low. The method of Test Sample Examples 1 - 2 of the present invention has a high extraction rate and good reproducibility. Moreover, the types of organic solvents used in this preparation process are less, and the toxicity is relatively low, reducing the operation risk of experimental personnel.

[0093] Detection Example 1

[0094] This example provides a method for detecting impurities in vitamin AD oily preparation, which is for the specificity investigation of the detection method of the present invention.

[0095] 1) Chromatographic conditions:

[0096] Instrument: Thermo Fisher two-dimensional liquid chromatograph.

[0097] Material: Commercially available vitamin AD drops.

[0098] Reference substance: The reference substance of vitamin D3 was purchased from the National Institutes for Food and Drug Control, and the batch number was 100061-201809.

[0099] Chromatographic column: Thermo Acclaim HILIC-10, 3μm, 2.1*150mm (First dimension); Phenomenex Silica(2) 5μm, 4.6*250mm (second dimension).

[0100] Mobile phase: Mobile phase A is n-hexane, and mobile phase B is n-hexane - n-pentanol - isopropanol (volume ratio 98:1:1) (first dimension); the mobile phase is n-hexane - n-pentanol (volume ratio 1000:3.5) (second dimension).

[0101] Detection wavelength: 265nm (first dimension); 280nm (second dimension).

[0102] Column temperature: 40°C.

[0103] Flow rate: 0.5mL / min (first dimension); 1.0mL / min (second dimension).

[0104] Valve switching time: 10min - 28min.

[0105] Gradient conditions:

[0106] First dimension: Gradient elution, specifically as follows, % is volume percentage:

[0107] Time (min) Mobile Phase A % Mobile Phase B % 0 90 10 30 90 10 35 0 100 60 0 100 65 90 10 200 90 10

[0108] Second dimension: Isocratic elution.

[0109] 2) Sample preparation

[0110] Preparation of impurity reference substance mixed solution: Respectively take the reference substances of vitamin D3, previtamin D3, trans-vitamin D3 (impurity A), lumisterol D3 (impurity C), isotachysterol D3 (impurity D), tachysterol D3 (impurity E) and provitamin D3 (impurity B), and prepare an impurity reference substance mixed solution with n-hexane.

[0111] Preparation of test solution: The same as Test Example 2 of the test article.

[0112] Preparation of blank excipient solution: Prepared according to the above method for preparing the test solution, with the only difference being that the content of vitamin AD drops is replaced by the excipient of the drops, and the rest of the method remains unchanged.

[0113] Preparation of blank solution: n - hexane.

[0114] Preparation of system suitability solution: Precisely pipette 8 mL of the reference stock solution, place it in a 20 - mL volumetric flask, dilute to the mark with isooctane, and shake well; measure 5 mL of this solution, place it in a stoppered glass container, seal it after purging with nitrogen, heat it in a 90 °C water bath for 1 h, take it out and cool it rapidly, add 5 mL of n - hexane, shake well, place it in a 1 - cm stoppered quartz absorption cell, under two 8 - W ultraviolet lamps with main wavelengths of 254 nm and 365 nm respectively, place the quartz absorption cell at a 45° angle and 5 - 6 cm away from the lamp tube, irradiate for 5 min to make the solution contain provitamin D3, trans - vitamin D3, lumisterol D3, vitamin D3, and tachysterol D3, which is used as the system suitability solution.

[0115] 3) Two - dimensional normal - phase high - performance liquid chromatography detection: Using the chromatographic conditions in 1), detect the samples in 2).

[0116] Experimental results

[0117] The results are as Figure 1 - 2 shown, among which Figure 1 is the chromatogram of the system suitability solution for detecting Example 1; Figure 2 is the comparative chromatogram of each sample solution for detecting Example 1. From top to bottom, they are the mixed solution of impurity reference substances, the test solution, the blank excipient solution, and the blank solution.

[0118] It can be seen from Figure 1 and Figure 2 that under the chromatographic conditions of this example, all known impurities can be detected. Compared with the prior art, the extraction method and detection method of the present invention can detect at least 6 known impurities, including provitamin D3, trans - vitamin D3 (impurity A), lumisterol D3 (impurity C), isotachysterol D3 (impurity D), tachysterol D3 (impurity E), and vitamin D3 precursor (impurity B), and the resolution of each peak is good. Under the chromatographic conditions of this example, the excipient peak does not interfere with the detection of the known impurities provitamin D3, trans - vitamin D3 (impurity A), lumisterol D3 (impurity C), isotachysterol D3 (impurity D), and tachysterol D3 (impurity E) in vitamin AD drops. Therefore, the detection method for related substances of the present invention can effectively separate and determine the impurities of vitamin D3 in vitamin AD drops, and can effectively control the quality of low - dose vitamin AD drops.

[0119] Detection Example 2

[0120] This example is for investigating the linear relationship of the detection method described in Detection Example 1 of the present invention.

[0121] Accurately weigh appropriate amounts of reference substances of trans-vitamin D3 (Impurity A), lumisterol D3 (Impurity C), and isotachysterol D3 (Impurity D), dissolve them in n-hexane, and dilute step by step with n-hexane to prepare a solution containing 1.0 μg / mL of trans-vitamin D3 (Impurity A), lumisterol D3 (Impurity C), and isotachysterol D3 (Impurity D). Shake well to obtain the linear stock solution. Accurately measure an appropriate amount of the linear stock solution and prepare the linear solutions of each impurity according to the following table.

[0122] Preparation of linear solutions:

[0123]

[0124] Operation: Take each linear solution respectively, inject 1 injection each, and record the chromatogram. Perform linear regression with the peak area against the concentration, and calculate the linear correlation coefficient r and the absolute value of the ratio of the Y-axis intercept to the Y-axis response value at the predicted limit concentration.

[0125] The results are shown in Figure 3 - 5 , Figure 3 which is the linear relationship diagram of Impurity A, Figure 4 which is the linear relationship diagram of Impurity C, Figure 5 which is the linear relationship diagram of Impurity D. The results show that for Impurity A (trans-vitamin D3) in the range of 0.0378 μg / mL to 0.3025 μg / mL, the linear equation is y = 145.7603x + 0.3484, r is 0.9998, and the linear relationship is good. For Impurity C (lumisterol D3) in the range of 0.0572 μg / mL to 0.3052 μg / mL, the linear equation is y = 31.3347x + 0.7676, r is 0.9886, and the linear relationship is good. For Impurity D (isotachysterol D3) in the range of 0.0384 μg / mL to 0.3072 μg / mL, the linear equation is y = 136.4834x + 0.7196, r is 0.9972, and the linear relationship is good.

[0126] Detection Example 3

[0127] This example is for investigating the detection limit of the detection method described in Detection Example 1 of the present invention.

[0128] Accurately weigh appropriate amounts of reference substances of trans-vitamin D3 (Impurity A), lumisterol D3 (Impurity C), and isotachysterol D3 (Impurity D), dissolve them in isooctane, and dilute step by step with n-hexane to prepare the test solution.

[0129] Chromatographic conditions: Refer to Detection Example 1.

[0130] The detection results are shown in Table 2, and the results indicate that: with a signal-to-noise ratio of 3:1 as the minimum detection limit, an injection volume of 80 μL, and using an ultraviolet-visible light detector, the minimum detection limits of trans-vitamin D3 and isotachysterol D3 are 0.03 μg / mL, that is, an injection volume of 2.4 ng, approximately 0.14% of the concentration of the test solution, and the minimum detection limit of lumisterol D3 is 0.06 μg / mL, that is, an injection volume of 4.8 ng, approximately 0.3% of the concentration of the test solution.

[0131] Table 2 Detection Results of Detection Limits

[0132] Detection of Comparative Example 1

[0133] In this example, one-dimensional normal-phase high-performance liquid chromatography conditions were used to investigate the related substances of vitamin AD drops. The specific method is as follows:

[0134] 1) Chromatographic conditions:

[0135] Instrument: High-performance liquid chromatograph;

[0136] Chromatographic column: Phenomenex Silica(2) 5 μm 4.6*250 mm;

[0137] Mobile phase: n-hexane - n-pentanol (997:3);

[0138] Injection volume: 100 μl;

[0139] Column temperature: 30 °C;

[0140] Elution conditions: Isocratic elution with n-hexane - n-pentanol (997:3);

[0141] 2) Sample preparation:

[0142] Preparation of test solution: Take an appropriate amount of vitamin AD drop soft capsules, cut open the soft capsules, transfer the contents to a centrifuge tube, take an appropriate amount of the contents (equivalent to 40 μg of vitamin D3), weigh accurately, place it in a 100 mL conical flask, add 50 mL of the mixed solution dimethyl sulfoxide: water (10:2), shake in a water bath at 50 °C for 10 min, accurately add 20 mL of the extraction solution, shake with a wrist-type oscillator at 200 rpm for 15 min, let it stand in the dark until it separates into layers, separate with a separating funnel, take the upper layer solution into a 50 mL centrifuge tube, centrifuge at 3000 rpm for 10 min, accurately measure 10 mL of the upper layer solution into a brown vial, dry it with nitrogen, and accurately add 1 mL of n-hexane to dissolve it to obtain the test solution.

[0143] System suitability solution preparation: Operate under light protection. Precisely measure 4 mL of the vitamin D reference stock solution, place it in a 20 mL brown volumetric flask, dilute it to the mark with n-hexane, shake well, heat it in a 60 °C water bath for 1 hour, take it out, quickly cool it, place it in a stoppered transparent volumetric flask, and under two 8 W ultraviolet lamps with main wavelengths of 254 nm and 365 nm respectively, tilt the bottle body at 45 °C and keep it 5 - 6 cm away from the lamp tube, irradiate for 30 minutes to obtain the system suitability solution.

[0144] Preparation of commercially available vitamin D agent solution: The preparation method refers to the above-mentioned test solution, with the only difference being that vitamin AD drops are replaced by vitamin D drops.

[0145] 3) One-dimensional normal-phase high-performance liquid chromatography detection: Using the chromatographic conditions in 1), detect the samples in 2).

[0146] Test results

[0147] The results are as Figure 6 shown. Figure 6 For the comparative spectrograms of each sample solution in Comparative Example 1, from top to bottom are the test solution, commercially available vitamin D agent solution, and system suitability solution. As can be seen from Figure 6 this, the baseline of the test solution is wavy, seriously interfering with the detection of impurities and the main peak. The method using one-dimensional liquid normal-phase chromatographic conditions is not applicable to the detection of this product.

[0148] Detection of Comparative Example 2

[0149] In this example, two-dimensional normal-phase high-performance liquid chromatography conditions were used to investigate the related substances of vitamin AD drops. The specific method is as follows:

[0150] 1) Chromatographic conditions:

[0151] Mobile phase:

[0152] First dimension: Mobile phase A is n-hexane, and mobile phase B is n-hexane - n-pentanol - isopropanol (98:1:1);

[0153] Second dimension: The mobile phase is n-hexane - n-pentanol - isopropanol (996:2:2).

[0154] Detection wavelength: 265 nm for both the first and second dimensions;

[0155] Flow rate: 0.5 mL / min for both the first and second dimensions, and the collection tube is a peek tube.

[0156] Chromatographic column:

[0157] First dimension: Thermo Acclaim HILIC-10, 3 μm 2.1*150 mm

[0158] Second dimension: Agilent ZORBAX RX-SIL, 3.0 * 100 mm, 1.8 μm

[0159] Elution program:

[0160] First dimension: isocratic elution;

[0161] Second dimension: gradient elution, specifically as follows:

[0162] Time (min) Mobile Phase A % Mobile Phase B % 0 95 5 30 95 5 35 0 100 60 0 100 65 95 5 80 95 5

[0163] Column temperature: 40 °C for both the first and second dimensions.

[0164] 2) Sample preparation:

[0165] Preparation of test solution: Take an appropriate amount of the test sample (equivalent to 500 units of total vitamin D), weigh accurately, place it in a 25 mL brown volumetric flask, dissolve with n-hexane and dilute to the mark, shake well to obtain the test solution.

[0166] Preparation of blank excipient solution: The same as the above test solution, except that the test sample is replaced with blank excipients.

[0167] Impurity mixed solution: The same as in Test Example 1, except that vitamin D3 is not added.

[0168] 3) Two-dimensional normal phase high performance liquid chromatography detection: Using the chromatographic conditions in 1), detect the samples in 2).

[0169] Test results

[0170] The results are as Figure 7 shown, Figure 7 For the comparative spectrum of each sample solution in Comparative Example 2, from top to bottom are the impurity mixed solution, blank excipient solution, and test solution. As Figure 7 can be seen, the separation effect between the main peak of the test solution and adjacent impurity peaks (or excipient peaks) is poor, and the interference of excipients on impurity detection is serious. It can be seen that the test solution, elution conditions, mobile phase ratio, and chromatographic column and other conditions have an important impact on the test results.

[0171] Detection of Comparative Example 3

[0172] This example provides a method for detecting vitamin D3 in vitamin AD drops, and the specific method is as follows:

[0173] 1) Chromatographic conditions:

[0174] Chromatographic column: Agilent Technologies ZORBAX RX-SIL, 5 μm, 4.6 * 250 mm.

[0175] Mobile phase: n-hexane: isopropanol (996:4) (V / V).

[0176] Elution program: isocratic elution.

[0177] Flow rate: 1.0 mL / min.

[0178] Run time: 60 min.

[0179] Sample injection volume: 100 μl.

[0180] Column temperature: 25 °C.

[0181] Detection wavelength: 265 nm.

[0182] 2) Sample preparation

[0183] Preparation of test solution: Take an appropriate amount of vitamin AD drops soft capsules, cut open the soft capsules, transfer the contents to a centrifuge tube, take an appropriate amount of the contents, weigh accurately, place in a 10 mL brown volumetric flask, dissolve with n-hexane and dilute to the mark, shake well to obtain the test solution (the concentration of vitamin D3 is about 4 μg / mL).

[0184] Blank solution: n-hexane.

[0185] 3) High performance liquid chromatography detection: Inject the test solution prepared in 2) into the column for detection according to the chromatographic conditions in 1).

[0186] Test results

[0187] The results are as Figure 8 shown, Figure 8 For the comparative spectrum of each sample solution in Comparative Example 3, from top to bottom are the test solution and the blank solution. It can be seen from Figure 8 that the retention time of the peak of vitamin D3 is about 25 min, the peak shape of the excipient peak (or impurity peak) is poor, and the separation effect is poor.

[0188] Detection of Comparative Example 4

[0189] In this example, the related substances of vitamin AD drops were investigated under the conditions of one-dimensional normal phase high performance liquid chromatography. The specific method is as follows:

[0190] 1) Chromatographic conditions:

[0191] Instrument: High performance liquid chromatograph;

[0192] Chromatographic column: Phenomenex Silica(2) 5 μm, 4.6 * 250 mm;

[0193] Mobile phase: n-hexane - n-pentanol (997:3);

[0194] Injection volume: 100 μL;

[0195] Column temperature: 30 °C;

[0196] Elution conditions: isocratic elution;

[0197] 2) Preparation of test solution: same as in Test Example 2 of the test substance.

[0198] Preparation of blank excipient solution: same as the above test solution, except that the test substance is replaced with blank excipient.

[0199] Preparation of vitamin D reference substance solution 1: Take vitamin D3 reference substance and prepare a reference substance solution 1 with n-hexane.

[0200] Preparation of vitamin D reference substance solution 2: Take vitamin D3 and previtamin D3 reference substances and prepare a reference substance solution 2 with n-hexane.

[0201] The preparation methods of the remaining solutions are the same as in Test Example 1.

[0202] 3) One-dimensional normal phase high performance liquid chromatography detection: Using the chromatographic conditions in 1), detect the samples in 2).

[0203] Test results

[0204] The results are as Figure 9 shown, Figure 9 For the comparative spectrum of each sample solution in Comparative Example 4, from top to bottom are the test solution, blank excipient solution, vitamin D reference substance solution 1, vitamin D reference substance solution 2, and blank solution. It can be seen from Figure 9 that under the detection method of Comparative Example 4, the blank excipient interferes at both the previtamin D3 peak and the vitamin D3 peak.

[0205] Comparative Example 5 for detection

[0206] This example provides a method for detecting impurities in vitamin AD oily preparation. The difference from Test Example 1 is that the chromatographic conditions in this example are: Phenomenex Silica(2) 5 μm 4.6 * 250 mm, mobile phase: n-hexane - isopropanol - n-pentanol (980:10:10), isocratic elution, and the remaining chromatographic conditions are the same as in Test Example 1.

[0207] Preparation of vitamin D reference substance solution: Take vitamin D3 and previtamin D3 reference substances and prepare a reference substance solution with n-hexane. The preparation of the remaining samples is the same as in Test Example 1.

[0208] Test results

[0209] The results are asFigure 10 As shown Figure 10 For detecting the comparative spectrograms of the sample solutions in Comparative Example 5, from top to bottom are the blank excipient solution, the vitamin D reference substance solution, and the blank solution. From Figure 10 It can be seen that under the detection method of Comparative Example 5, there is interference at the pre-vitamin D3 peak in the blank excipient solution and the resolution is poor, so the method is not applicable.

[0210] Detection of Comparative Example 6

[0211] This example provides a method for detecting impurities in vitamin AD oily preparations. The difference from Detection Example 1 is that in this example, the one-dimensional mobile phase A (n-hexane): mobile phase B (n-hexane - n-pentanol - isopropanol (98:1:1)) = 92:8, the two-dimensional mobile phase: n-hexane - n-pentanol = 1000:3, the two-dimensional chromatographic column: Agilent ZORBAX RX-SIL 3.0*100mm 1.8μm, and the other chromatographic conditions are the same as those in Detection Example 1.

[0212] Impurity mixed solution: The same as Detection Example 1, except that vitamin D3 is not added.

[0213] Preparation of vitamin D reference substance solution: Take vitamin D3 and pre-vitamin D3 reference substances and prepare a reference substance solution with n-hexane.

[0214] The preparation of the remaining samples is the same as that in Detection Example 1.

[0215] Test results

[0216] The results are as Figure 11 shown Figure 11 For detecting the comparative spectrograms of the sample solutions in Comparative Example 6, from top to bottom are the impurity mixed solution, the vitamin D reference substance solution, and the blank solution. From Figure 11 It can be seen that within the marked prescription box are impurity D and impurity E, and it can be seen that the impurities are not separated from each other.

[0217] As can be seen from the above examples and comparative examples, for the preparation method of the test solution provided in the embodiments of the present invention, sufficient extraction rate experiments have been conducted. The results show that each extraction step has been completely extracted, with a high extraction rate and good reproducibility, and it is suitable for popularization and application. Moreover, the types of organic solvents used in the preparation process of the test solution are less and the toxicity is relatively low, further reducing the operation risk of the experimental personnel.

[0218] Furthermore, the two-dimensional high-performance liquid system of the embodiments of the present invention is used to detect low-content vitamin D3 and related substances in vitamin AD soft capsules and drops. Compared with the one-dimensional detection system in the prior art, it has stronger specificity, better reproducibility, and higher sensitivity. In the two-dimensional manner of the present invention, each main peak and impurity peak can be detected and well separated, with little interference from excipients, and the main peak has a high response degree. The detection method for related substances of the present invention can effectively separate and determine compound vitamin AD soft capsules or drops with a low content of 500 IU, and can effectively control the quality of single-compound preparations containing vitamin D3.

[0219] In summary, through specific extraction solvents and extraction steps, the present invention obtains a test solution with a high extraction rate of vitamin D3, good reproducibility, and few irrelevant impurities. Combined with the two-dimensional normal-phase high-performance liquid chromatography detection method provided by the present invention, it can achieve good detection effects for vitamin D3 and related impurities in vitamin AD oily preparations, with high detection sensitivity, good reproducibility, and strong specificity.

Claims

1. A method for detecting impurities in vitamin AD oily preparation, characterized in that, It includes the following steps: Step 1, solution preparation: Prepare the test solution. Step 2, two-dimensional normal-phase high-performance liquid chromatography detection: Use normal-phase high-performance liquid chromatography to detect the test solution prepared in Step S1, including the following steps carried out in sequence: The first-dimensional liquid chromatography, with mobile phase A being n-hexane, mobile phase B being a mixture of n-hexane, n-pentanol and isopropanol, and gradient elution. The second-dimensional liquid chromatography, with the mobile phase being n-hexane and n-pentanol, and isocratic elution. The test solution is prepared by a method including the following steps: S1: Extraction: Use an ethanol aqueous solution to extract the vitamin AD oily preparation, take the upper-layer solution to obtain the extract. S2: Low-temperature treatment: Place the extract obtained in Step S1 in an environment at -10°C or below for low-temperature treatment, and take the separated solution after solid-liquid separation. S3: Concentration: Remove the solvent of the separated solution obtained in Step S2 to obtain the test sample residue. S4: Preparation: Mix the test sample residue obtained in Step S3 with n-hexane to obtain the test solution. The chromatographic column used in the first-dimensional liquid chromatography is selected from urea-bonded silica columns. In the mobile phase B of the first-dimensional liquid chromatography, the volume ratio of n-hexane, n-pentanol and isopropanol is (95 - 100):(0.5 - 2):

1. The gradient elution degree of the first-dimensional liquid chromatography is as follows: 0 - 30 min, the volume of mobile phase A is maintained at 85 - 95%, and the volume of mobile phase B is maintained at 5 - 15%. 30 - 35 min, the volume of mobile phase A drops to 0 - 5%, and the volume of mobile phase B rises to 95 - 100%. 35 - 60 min, the volume of mobile phase A is maintained at 0 - 5%, and the volume of mobile phase B is maintained at 95 - 100%. 60 - 65 min, the volume of mobile phase A rises to 85 - 95%, and the volume of mobile phase B drops to 5 - 15%. 65 - 200 min, the volume of mobile phase A is maintained at 85 - 95%, and the volume of mobile phase B is maintained at 5 - 15%. The chromatographic column used in the second-dimensional liquid chromatography is selected from silica columns; the silica column is selected from Phenomenex Luna® Silica (2) or Welch Topsil silica, with an inner diameter × column length of (4 - 5) × (150 - 250) mm and a stationary phase particle size of 3 - 5 μm; in the mobile phase of the second-dimensional liquid chromatography, the volume ratio of n-hexane and n-pentanol is 1000:(2 - 5). The impurities in the vitamin AD oily preparation include provitamin D3, trans-vitamin D3, lumisterol D3, vitamin D3 precursor, tachysterol D3 and isotachysterol D3.

2. The method according to claim 1, characterized in that, The vitamin AD oily preparation includes vitamin AD soft capsules, vitamin AD drops or a combination thereof.

3. The method according to claim 1, wherein In Step S1, the specification of vitamin D3 in the vitamin AD oily preparation is ≥400 IU.

4. The method according to any one of claims 1 to 3, characterized in that In Step S1, the volume concentration of ethanol in the ethanol aqueous solution is 90 - 95%. And / or, in Step S1, the number of extractions is 6 - 8 times. And / or, in Step S1, the extraction operation includes centrifugation.

5. The method according to claim 4, wherein In Step S1, the centrifugation speed is 2000 - 6000 rpm. And / or, in step S1, the centrifugation time is 3 to 10 min; And / or, in step S1, the centrifugation temperature is 0 to 5 °C.

6. The method according to any one of claims 1 to 3, characterized in that, In step S2, the time for low-temperature treatment is 0.5 to 2.5 h; And / or, in step S2, the method for solid-liquid separation is selected from centrifugation; And / or, in step S3, the method for removing the solvent of the separation solution obtained in step S2 is selected from rotary evaporation to dryness.

7. The method according to claim 6, characterized in that, In step S2, the centrifugation speed is 3000 to 5000 rpm; And / or, in step S2, the centrifugation time is 5 to 15 min; And / or, in step S2, the centrifugation temperature is 0 to 5 °C.

8. The method according to claim 6, characterized in that In step S3, the rotary evaporation to dryness is carried out in a water bath at 40 to 60 °C.

9. The method according to claim 1, wherein The ureido-bonded silica gel column is selected from Thermo Acclaim HILIC-10 or Nano ChromCore VD-ChP4, with an inner diameter × column length of (2 to 2.5) × (100 to 200) mm, and the particle size of the stationary phase is 1 to 5 μm.

10. The method according to claim 1, characterized in that, The detection conditions of the first-dimensional liquid chromatography satisfy at least one of the following conditions (1) to (5): (1) The valve switching time is 10 to 30 min; (2) The detection wavelength is 260 to 270 nm; (3) The flow rate of the mobile phase is 0.1 to 1 mL / min; (4) The injection volume is 50 to 200 μL; (5) The column temperature is 35 to 45 °C.

11. The method according to claim 1, wherein The detection conditions of the second-dimensional liquid chromatography satisfy at least one of the following conditions (1) to (3): (1) The detection wavelength is 270 to 280 nm; (2) The flow rate of the mobile phase is 0.8 to 1.2 mL / min; (3) The column temperature is 35 to 45 °C.

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