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

Abstract

The invention discloses a method for detecting impurities in a vitamin AD oily preparation and preparation of a test solution, and belongs to the technical field of detection and analysis. The preparation method of the test solution for detecting the impurities in the vitamin AD oily preparation comprises the steps of extraction, low-temperature treatment, concentration and preparation, and the method for detecting the impurities in the vitamin AD oily preparation is obtained by combining the preparation method of the test solution with two-dimensional normal-phase high performance liquid chromatography detection. According to the method, through the specific extraction solvent and extraction steps, the extraction rate of the vitamin D3 in the obtained test solution is high, the reproducibility is good, irrelevant impurities are few, a good detection effect on the vitamin D3 and the relevant impurities in the vitamin AD oily preparation can be achieved in combination with the two-dimensional normal-phase high performance liquid chromatography detection method, and the method is suitable for popularization and application. The detection sensitivity is high, the reproducibility is good, and the specificity is strong.

Description

Technical Field

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

[0002] Vitamin A and vitamin D are essential substances for human growth and development, especially for the development of infants and young children, the integrity of epithelial tissue, vision, reproductive organs, the stability of blood calcium and phosphorus, the growth and development of bones and teeth, etc. Vitamin A and vitamin D can exist stably in an oily matrix, so 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 with cod liver oil or refined edible oil (solid fat removed at about 0°C) and adjusting the concentration. However, the difficulty in detecting vitamin D3 and related substances in such vitamin AD oily preparations is that the proportion of oily excipients in such preparations is high, the content of vitamin D3 is low, and vitamin A interferes seriously. Therefore, it is particularly urgent to invent a method for detecting related substances of vitamin D3 with strong specificity, good reproducibility, high sensitivity and good separation, and a method for preparing a supply solution with high extraction rate, which can effectively control the quality of vitamin AD soft capsules and drops.

[0003] Vitamin D3 is a fat-soluble vitamin, also regarded as a hormone precursor that acts on calcium and phosphorus metabolism. It is often combined with calcium carbonate, vitamin A or multiple vitamins and minerals to form a compound preparation. Vitamin D3 is easily deteriorated when exposed to light or air, and is easily oxidized and photolyzed to produce impurities such as provitamin D3, trans-vitamin D3, lumisterol D3, tachysterol D3 and isostachol D3. Therefore, the storage method of vitamin D3 raw materials specified in the Chinese Pharmacopoeia is to shield from light, fill with nitrogen, seal, and store in a cold place. At present, the pharmacopoeia method only publishes the detection method of related substances of vitamin D3 raw materials, including chromatographic conditions and the preparation method of test solution. Some patents or documents publish the preparation method of calcium carbonate D3 chewable tablets and the method of detecting related substances with a normal phase system. These methods have been reproduced by the applicant and it was found that the impurity content could not be accurately detected due to insufficient extraction during the preparation of the test solution or significant interference of auxiliary materials during the detection process. In addition, there are also methods that require complicated preparation of the extraction solution, use a variety of toxic organic solvents, have high operating risks for the experimenters, and have problems such as incomplete extraction of the effective ingredients and significant interference from the excipients. Moreover, these methods are suitable for chewable tablets, but not for oily preparations such as vitamin AD soft capsules and drops. Summary of the invention

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

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

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

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

[0008] S1: Extraction: Extract the vitamin AD oily preparation with ethanol aqueous solution, take the upper solution to obtain the extract;

[0009] S2: Low temperature treatment: The extract obtained in step S1 is placed in an environment of -10°C or below for low temperature treatment, and the separated solution is obtained after solid-liquid separation;

[0010] S3: Concentration: removing the solvent from 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, the present application found that the specific extraction solvent and extraction steps of the present invention can obtain a test solution with an extraction rate of vitamin D3 close to 100%, while the extraction method in the prior art has a low final extraction rate and very poor reproducibility due to poor extraction conditions. The present application conducted a comprehensive investigation on the extraction method of vitamin D3, and finally screened out an extraction method with good reproducibility, high extraction rate, and few irrelevant impurities, which can achieve good detection effect when used in 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, provitamin D3, tachysterol D3 or isostachystanol D3.

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

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

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

[0018] Preferably, in step S1, the specification of vitamin D3 in the vitamin AD oily preparation is ≥400 IU, more preferably 400-700 IU. Non-limiting specific examples include 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 related substance detection method of the present invention are also applicable to preparations with higher content of vitamin D3. Therefore, as long as the extraction method and / or detection method disclosed in the present invention are used, the detection of specific impurities in preparations containing vitamin D3 is 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 min; more preferably 1 to 10 min; and even more preferably 2 to 5 min.

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

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

[0025] Preferably, in step S3, the method for removing the solvent from 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; and 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: preparing 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: The test solution prepared in step S1 is detected by normal phase high performance liquid chromatography, including the following steps in sequence:

[0030] In the first dimension, the mobile phase A was n-hexane, and the mobile phase B was n-hexane, n-pentanol, and isopropanol, with gradient elution;

[0031] The second dimension liquid chromatography was performed with n-hexane and n-pentanol as the mobile phase and isocratic elution.

[0032] This application, through 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, finds that the peak shape detected by two-dimensional normal phase high performance liquid chromatography detection is more standard, the separation between the main peak and the adjacent peaks, and between the known impurities and the adjacent peaks is better, the auxiliary materials do not interfere with the detection of known impurities, and the types of impurities detected are more and more comprehensive. Therefore, on the basis of the test solution prepared by the preparation method described in the first aspect of the present invention, combined with the two-dimensional normal phase high performance liquid chromatography detection method, a good detection effect of relevant impurities can be achieved. First, the first dimension liquid chromatography is used to separate part of the blank auxiliary materials and vitamin A from vitamin D, and then the second dimension liquid chromatography is used to carry out targeted detection of vitamin D3 and related impurities. The separation effect of the first dimension liquid chromatography is good, thereby avoiding the interference of auxiliary materials and vitamin A on the detection of vitamin D3 and related impurities. The extraction method and detection method of the present invention can realize the detection of vitamin D3 and related impurities in low-dose vitamin AD oily preparations, 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 a urea-based bonded silica gel column; further preferably, the urea-based 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 a stationary phase particle size of 1 to 5 μm; further preferably, the inner diameter × column length of the urea-based bonded silica gel column is 2.1 × 150 mm, and the stationary phase particle size is 3 μm.

[0034] Preferably, 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; more preferably (97-99):(0.8-1.2):1; further preferably 98:1:1.

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

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

[0037] 30-35 min, the volume of mobile phase A is reduced to 0-5%, and the volume of mobile phase B is increased to 95-100%;

[0038] 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%;

[0039] At 60-65 min, the volume of mobile phase A increased to 85-95%, and the volume of mobile phase B decreased to 5-15%;

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

[0041] Further preferably, the gradient elution degree of the first dimension liquid chromatography is:

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

[0043] 30-35 min, the volume of mobile phase A is reduced to 0-2%, and the volume of mobile phase B is increased to 98-100%;

[0044] 35-60 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] At 60-65 min, the volume of mobile phase A increased to 88-92%, and the volume of mobile phase B decreased to 8-12%;

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

[0047] More preferably, the gradient elution degree of the first dimension liquid chromatography is, % 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-dimensional liquid chromatography satisfy at least one of the following conditions (1) to (5):

[0050] (1) Valve cutting time is 10 to 28 minutes;

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

[0052] (3) The flow rate of the mobile phase is 0.1 to 1 mL / min; preferably 0.4 to 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 a silica gel column; further preferably, the silica gel column is selected from Phenomenex Silica (2) or Welch Topsil silica, inner diameter × column length is (4-5) × (150-250) mm, and the particle size of the stationary phase is 3-5 μm; more preferably, the inner diameter × column length of the silica gel column is 4.6 × 250 mm, and the particle size of the stationary phase is 5 μm.

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

[0057] Preferably, the detection conditions of the second-dimensional 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 to 1.2 mL / min; preferably 1 to 1.2 mL / min;

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

[0061] The beneficial effects of the present invention are: through specific extraction solvents and extraction steps, the present invention can obtain a high extraction rate of vitamin D3 in the test solution with 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 a good detection effect of 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 by the extraction rate experiment. The results show that each extraction step has been completely extracted, the extraction rate is high, the reproducibility is good, and it is suitable for popularization and application. In addition, the types of organic solvents used in the preparation process of the test solution are relatively small, and the toxicity is relatively low, which further reduces the operating risks of the experimenters.

[0064] 2. The present invention adopts a two-dimensional high performance liquid phase system to detect low levels of 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. Using the two-dimensional method of the present invention, each main peak and impurity peak can be detected and well separated, the interference of auxiliary materials is small, and the responsiveness of the main peak is high.

[0065] 3. The detection method of the related substances of the present invention can effectively separate and determine the compound vitamin AD soft capsule or drops as low as 500 IU, and can effectively control the quality of single compound preparations containing vitamin D3. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a solution spectrum for testing the system suitability of Example 1.

[0067] Figure 2 This is a comparison spectrum of the sample solutions in Example 1.

[0068] Figure 3 This is a linear relationship diagram for detecting impurity A in Example 2.

[0069] Figure 4 This is a linear relationship diagram for detecting impurity C in Example 2.

[0070] Figure 5 This is a linear relationship diagram for detecting impurity D in Example 2.

[0071] Figure 6 This is a comparison spectrum of the sample solutions of Comparative Example 1.

[0072] Figure 7 This is a comparison spectrum of the sample solutions of Comparative Example 2.

[0073] Figure 8 This is a comparison spectrum of the sample solutions of Comparative Example 3.

[0074] Fig. 9 This is a comparison spectrum of the sample solutions of Comparative Example 4.

[0075] Fig.10 This is a comparison spectrum of the sample solutions of Comparative Example 5.

[0076] Fig.11 This is a comparison spectrum of the sample solutions of Comparative Example 6. DETAILED DESCRIPTION

[0077] The content of the present invention is further described in detail below through specific examples. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the principles set forth in the present invention all belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific data exemplified below. The raw materials, reagents or devices used in the following examples and comparative examples, unless otherwise specified, can all be obtained from conventional commercial sources, or can be obtained by existing known methods.

[0078] Test sample example 1

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

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

[0081] Test sample example 2

[0082] This example provides a method for preparing a test solution for detecting impurities in a vitamin AD oily preparation. The difference from the test example 1 is that the extraction operation with 95% ethanol is repeated 7 times in this example, and other conditions remain unchanged. The obtained test solution is recorded as sample 2.

[0083] Comparative Example 1

[0084] This example provides a method for preparing a test solution for detecting impurities in a vitamin AD oily preparation. The difference from the test sample example 1 is that the extraction reagent 95% ethanol is replaced with anhydrous ethanol, the number of extractions is 7, and other conditions remain unchanged. The obtained test solution is recorded as comparative sample 1.

[0085] Comparative Example 2

[0086] This example provides a method for preparing a test solution for detecting impurities in a vitamin AD oily preparation. The difference from the test sample example 1 is that the extraction reagent 95% ethanol is replaced with a 0.02% volume concentration of 2,6-di-tert-butyl-p-methylphenol (BHT) ethanol solution, the number of extractions is 7, and other conditions remain unchanged. The obtained test solution is recorded as comparative sample 2.

[0087] Test sample comparison 3

[0088] This example provides a method for preparing a test solution for detecting impurities in vitamin AD oily preparations, comprising the following steps: taking an appropriate amount of vitamin AD drop soft capsules, cutting the soft capsules, transferring the contents to a centrifuge tube, taking an appropriate amount of the contents, accurately weighing, adding 30 mL of methanol, vortexing for 15 minutes, taking the upper clear liquid, repeating the extraction operation twice, combining the methanol layers of each time, freezing in a refrigerator (-20°C ± 5°C) for 1 to 2 hours, taking out, and evaporating in a 50°C water bath until dry, accurately adding methanol to the residue, vortexing for 2 minutes to dissolve, and using it as the test solution. The obtained test solution is recorded as comparative sample 3.

[0089] The extraction rates of vitamin D3 of the test examples 1 to 2 and the test comparative examples 1 to 3 were measured and calculated, and the results are shown in Table 1.

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

[0091] Sample name Inspection conditions Extraction rate (%) Sample 1 Extract with 95% ethanol 5 times 92.3 Sample 2 Extracted 7 times with 95% ethanol 99.7 Comparative sample 1 Anhydrous ethanol 93.3 Comparative sample 2 0.02% BHT ethanol solution 94.1 Comparative sample 3 Methanol 80.6

[0092] As shown in 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 the test sample after repeated centrifugation for 7 times with 95% ethanol has basically achieved complete extraction. The extraction rates obtained by using anhydrous ethanol, 0.02% BHT ethanol solution and methanol are all relatively low. The extraction rate of the method of the test sample embodiments 1 to 2 of the present invention is high and reproducible. In addition, the types of organic solvents used in the preparation process are relatively small, and the toxicity is relatively low, which reduces the operating risk of the experimenter.

[0093] Detection Example 1

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

[0095] 1) Chromatographic conditions:

[0096] Instrument: Thermo Fisher 2D-LC.

[0097] Materials: Commercially available vitamin AD drops.

[0098] Reference substance: Vitamin D3 reference substance was purchased from China Food and Drug Inspection Institute with batch number 100061-201809.

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

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

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

[0102] Column temperature: 40℃.

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

[0104] Valve cutting time: 10min~28min.

[0105] Gradient conditions:

[0106] First dimension: gradient elution, 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: Take vitamin D3, provitamin D3, trans-vitamin D3 (impurity A), luminosterol D3 (impurity C), isostachystanol D3 (impurity D), tachysterol D3 (impurity E) and provitamin D3 (impurity B) reference substances respectively, and use n-hexane to make an impurity reference substance mixed solution.

[0111] Preparation of test solution: same as in Example 2.

[0112] Preparation of blank excipient solution: Prepare the solution according to the above method for preparing the test sample solution, except that the contents of vitamin AD drops are replaced with the drops excipient, and the rest of the method remains unchanged.

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

[0114] Preparation of system suitability solution: accurately pipette 8 mL of reference stock solution into a 20 mL volumetric flask, dilute to the mark with isooctane, and shake well; measure 5 mL of the solution, place in a stoppered glass container, seal after passing nitrogen, heat in a 90°C water bath for 1 h, take out and cool rapidly, add 5 mL of n-hexane, shake well, place in a 1 cm stoppered quartz absorption cell, and under two 8 W UV lamps with dominant wavelengths of 254 nm and 365 nm, tilt the quartz absorption cell at 45° and keep it 5 to 6 cm away from the lamp tube. Irradiate for 5 min so that the solution contains provitamin D3, trans-vitamin D3, luminol D3, vitamin D3 and tachysterol D3 as the system suitability solution.

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

[0116] Experimental Results

[0117] The results are as follows Figures 1-2 As shown, Figure 1 The solution spectrum for testing the system suitability of Example 1; Figure 2 To detect the comparative spectra of each sample solution in Example 1, from top to bottom are the impurity reference substance mixed solution, the test solution, the blank excipient solution, and the blank solution.

[0118] Depend on Figure 1 and Figure 2 It can be known that all known impurities can be detected under the chromatographic conditions of this example. Compared with the prior art, the extraction method of the present invention is combined with the detection method, and at least 6 known impurities can be detected, including previtamin D3, trans-vitamin D3 (impurity A), luminosterol D3 (impurity C), isostachol D3 (impurity D), tachysterol D3 (impurity E), vitamin D3 pro (impurity B), and each peak separation is good. Under the chromatographic conditions of this example, the auxiliary material peak does not interfere with the detection of known impurities previtamin D3, trans-vitamin D3 (impurity A), luminosterol D3 (impurity C), isostachol D3 (impurity D), and tachysterol D3 (impurity E) in vitamin AD drops. Therefore, the detection method of the present invention's related substances can effectively separate and measure 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 investigates the linear relationship of the detection method described in Detection Example 1 of the present invention.

[0121] Accurately weigh trans-vitamin D3 (impurity A), luminosterol D3 (impurity C), and isostanol D3 (impurity D) reference substances, dilute with n-hexane to prepare a solution containing 1.0 μg / mL of trans-vitamin D3 (impurity A), luminosterol D3 (impurity C), and isostanol D3 (impurity D), shake well, and use it as a linear stock solution. Accurately measure an appropriate amount of the linear stock solution and prepare the linear solutions of each impurity according to the table below.

[0122] Linear solution preparation:

[0123]

[0124] Operation: Take each linear solution, inject one needle each, and record the chromatogram. Perform linear regression on the peak area and 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] Results Figures 3 to 5 , Figure 3 is the linear relationship diagram of impurity A, Figure 4 is the linear relationship diagram of impurity C, Figure 5 The linear relationship diagram of impurity D. The results show that the linear equation of impurity A (trans vitamin D3) is y=145.7603x+0.3484 in the range of 0.0378μg / mL to 0.3025μg / mL, r is 0.9998, and the linear relationship is good. The linear equation of impurity C (luminosterol D3) is y=31.3347x+0.7676 in the range of 0.0572μg / mL to 0.3052μg / mL, r is 0.9886, and the linear relationship is good. The linear equation of impurity D (isosterol D3) is y=136.4834x+0.7196 in the range of 0.0384μg / mL to 0.3072μg / mL, r is 0.9972, and the linear relationship is good.

[0126] Detection Example 3

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

[0128] Accurately weigh appropriate amounts of trans-vitamin D3 (impurity A), luminosterol D3 (impurity C), and isostachyl D3 (impurity D) reference substances, dissolve them in isooctane, and dilute them stepwise with n-hexane to prepare the test solution.

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

[0130] The test results are shown in Table 2. The results show that: based on the signal-to-noise ratio of 3:1 as the minimum detection limit, the injection volume is 80 μL, and a UV-visible detector is used. The minimum detection limits of trans-vitamin D3 and isosterol D3 are 0.03 μg / mL, that is, the injection volume is 2.4 ng, which is approximately 0.14% of the concentration of the test solution. The minimum detection limit of isosterol D3 is 0.06 μg / mL, that is, the injection volume is 4.8 ng, which is approximately 0.3% of the concentration of the test solution.

[0131] Table 2 Detection results of detection limit

[0132] Detection Comparative Example 1

[0133] This example uses one-dimensional normal phase HPLC conditions to investigate the related substances of vitamin AD drops. The specific method is as follows:

[0134] 1) Chromatographic conditions:

[0135] Instrument: High performance liquid chromatography;

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

[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 drops soft capsules, cut the soft capsules, transfer the contents to a centrifuge tube, take an appropriate amount of the contents (equivalent to 40μg vitamin D3), accurately weigh, place in a 100mL conical flask, add 50mL of the mixed solution of dimethyl sulfoxide: water (10:2), shake in a water bath at 50℃ for 10min, accurately add 20mL of the extraction solution, shake at 200rpm for 15min on a wrist oscillator, keep away from light and allow to stand until stratification, separate the liquids with a separatory funnel, take the upper solution into a 50mL centrifuge tube, centrifuge at 3000rpm for 10min, accurately measure 10mL of the upper solution into a brown penicillin bottle, blow dry with nitrogen, accurately add 1mL of n-hexane to dissolve, and use as the test solution.

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

[0144] Preparation of commercially available vitamin D solution: The preparation method refers to the above-mentioned test solution, the only difference is that the 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), the sample in 2) is detected.

[0146] Test results

[0147] The results are as follows Figure 6 As shown, Figure 6 The comparison spectra of the sample solutions of Comparative Example 1 are as follows, from top to bottom: the test solution, the commercially available vitamin D solution, and the system suitability solution. Figure 6 It can be seen that the baseline of the test solution is wavy, which seriously interferes with the detection of impurities and main peaks. The one-dimensional liquid normal phase chromatography conditions are not suitable for the detection of this product.

[0148] Detection Comparative Example 2

[0149] In this case, two-dimensional normal phase HPLC 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 was n-hexane, and mobile phase B was n-hexane-n-pentanol-isopropanol (98:1:1);

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

[0154] Detection wavelength: 265nm 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 was a peek tube.

[0156] Chromatographic column:

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

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

[0159] Elution procedure:

[0160] First dimension: isocratic elution;

[0161] Second dimension: gradient elution, 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), accurately weigh it, place it in a 25mL brown volumetric flask, add n-hexane to dissolve and dilute to the scale, shake well, and use it as the test solution.

[0166] Preparation of blank excipient solution: Same as the above test sample solution, except that the test sample is replaced by blank excipient.

[0167] Impurity mixed solution: The only difference from Test Example 1 is that vitamin D3 is not added.

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

[0169] Test results

[0170] The results are as follows Figure 7 As shown, Figure 7 To detect the comparison spectra of each sample solution of Comparative Example 2, from top to bottom are impurity mixed solution, blank auxiliary material solution and test solution. Figure 7 It can be seen that the separation effect of the main peak of the test solution and the adjacent impurity peak (or auxiliary material peak) is poor, and the auxiliary material seriously interferes with the detection of impurities. It can be seen that the test solution, elution conditions, mobile phase ratio and chromatographic column conditions have an important influence on the test results.

[0171] Detection 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: 1.0mL / min.

[0178] Running time: 60min.

[0179] Injection volume: 100 μl.

[0180] Column temperature: 25℃.

[0181] Detection wavelength: 265nm.

[0182] 2) Sample preparation

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

[0184] Blank solution: n-hexane.

[0185] 3) HPLC detection: The test solution prepared in 2) is loaded for detection according to the chromatographic conditions in 1).

[0186] Test results

[0187] The results are as follows Figure 8 As shown, Figure 8 The comparison spectra of the sample solutions of Comparative Example 3 are as follows: the test solution and the blank solution from top to bottom. Figure 8 It can be seen that the retention time of the vitamin D3 peak is about 25 minutes, the peak shape of the excipient peak (or impurity peak) is poor, and the separation effect is poor.

[0188] Detection Comparative Example 4

[0189] This example uses one-dimensional normal phase HPLC conditions to investigate the related substances of vitamin AD drops. The specific method is as follows:

[0190] 1) Chromatographic conditions:

[0191] Instrument: High performance liquid chromatography;

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

[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 Example 2.

[0198] Preparation of blank excipient solution: Same as the above test sample solution, except that the test sample is replaced by blank excipient.

[0199] Preparation of vitamin D reference solution 1: Take vitamin D3 reference and use n-hexane to make reference solution 1.

[0200] Preparation of vitamin D reference solution 2: Take vitamin D3 and provitamin D3 reference substances and use n-hexane to make reference solution 2.

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

[0202] 3) One-dimensional normal phase high performance liquid chromatography detection: using the chromatographic conditions in 1), the sample in 2) is detected.

[0203] Test results

[0204] The results are as follows Fig. 9 As shown, Fig. 9 The comparison spectra of the sample solutions of comparative example 4 are as follows from top to bottom: test solution, blank excipient solution, vitamin D reference solution 1, vitamin D reference solution 2, and blank solution. Fig. 9 It can be seen that under the detection method of comparative example 4, the blank excipient interferes with both the previtamin D3 peak and the vitamin D3 peak.

[0205] Detection Comparative Example 5

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

[0207] Preparation of vitamin D reference solution: Vitamin D3 and previtamin D3 reference substances were taken and prepared into reference solution with n-hexane. The preparation of other samples was the same as that in detection example 1.

[0208] Test results

[0209] The results are as follows Fig.10 As shown, Fig.10 To test the comparative spectra of each sample solution of Comparative Example 5, from top to bottom are blank excipient solution, vitamin D reference solution, and blank solution. Fig.10 It can be seen that under the detection method of comparative example 5, there is interference and poor separation at the peak of previtamin D3 in the blank excipient solution, and the method is not applicable.

[0210] Detection Comparative Example 6

[0211] This example provides a method for detecting impurities in a vitamin AD oily preparation. The difference from Detection Example 1 is that, in this example, one-dimensional mobile phase A (n-hexane): mobile phase B (n-hexane-n-pentanol-isopropanol (98:1:1)) = 92:8, two-dimensional mobile phase: n-hexane-n-pentanol = 1000:3, two-dimensional chromatographic column: Agilent ZORBAX RX-SIL 3.0*100mm1.8μm, and other chromatographic conditions are the same as Detection Example 1.

[0212] Impurity mixed solution: The only difference from Test Example 1 is that vitamin D3 is not added.

[0213] Preparation of vitamin D reference solution: Take vitamin D3 and provitamin D3 reference substances and use n-hexane to make reference solution.

[0214] The remaining samples were prepared the same as in Example 1.

[0215] Test results

[0216] The results are as follows Fig.11 As shown, Fig.11 The comparison spectra of the sample solutions of Comparative Example 6 are as follows, from top to bottom: impurity mixed solution, vitamin D reference solution, and blank solution. Fig.11 It can be seen that the impurities D and E are marked in the prescription box, and it can be seen that the impurities are not separated.

[0217] From the above examples and comparative examples, it can be seen that the preparation method of the test solution provided in the examples of the present invention has been fully investigated by the extraction rate experiment. The results show that each extraction step has been completely extracted, the extraction rate is high, the reproducibility is good, and it is suitable for promotion and application. In addition, the types of organic solvents used in the preparation process of the test solution are relatively small, and the toxicity is relatively low, which further reduces the operating risk of the experimenter.

[0218] Furthermore, the two-dimensional high performance liquid phase system of the embodiment 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. Using the two-dimensional method of the present invention, each main peak and impurity peak can be detected and well separated, the auxiliary material interference is small, and the main peak has high responsiveness. The detection method of the related substances of the present invention can effectively separate and measure compound vitamin AD soft capsules or drops as low as 500IU, and can effectively control the quality of single compound preparations containing vitamin D3.

[0219] In summary, the present invention uses specific extraction solvents and extraction steps to obtain a high extraction rate of vitamin D3 in the test solution with 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 is possible to achieve a good detection effect of vitamin D3 and related impurities in vitamin AD oily preparations, with high detection sensitivity, good reproducibility and strong specificity.

Claims

1. A method for preparing a test solution for detecting impurities in a vitamin AD oily preparation, characterized in that: The following steps are involved: S1： Extraction: Use ethanol aqueous solution to extract the vitamin AD oily preparation, take the upper solution to obtain the extract; S2: Low temperature treatment: The extract obtained in step S1 is placed in an environment of -10°C or below for low temperature treatment, and the separated solution is obtained after solid-liquid separation; S3: Concentration: removing the solvent from the separated solution obtained in step S2 to obtain a test sample residue; S4: Preparation: Mix the test sample residue obtained in step S3 with n-hexane to obtain a test sample solution.

2. The preparation 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; And / or, the impurities in the vitamin AD oily preparation include at least one of provitamin D3, trans-vitamin D3, lumisterol D3, provitamin D3, tachysterol D3 or isostachystanol D3.

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

4. The preparation 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 to 8 times; And / or, in step S1, the extraction operation includes centrifugation, the centrifugal speed is preferably 2000-6000 rpm, the centrifugal time is preferably 3-10 min, and the centrifugal temperature is preferably 0-5°C.

5. The preparation method according to any one of claims 1 to 3, characterized in that: In step S2, the low temperature treatment time is 0.5 to 2.5 hours; And / or, in step S2, the solid-liquid separation method is selected from centrifugation, the centrifugal speed is preferably 3000-5000 rpm, the centrifugal time is preferably 5-15 min, and the centrifugal temperature is preferably 0-5°C; And / or, in step S3, the method for removing the solvent from the separated solution obtained in step S2 is selected from rotary evaporation; preferably, the rotary evaporation is performed in a water bath at 40-60°C.

6. A method for detecting impurities in a vitamin AD oily preparation, characterized in that: The following steps are involved: Step 1, solution preparation: prepare the test solution according to the preparation method according to any one of claims 1 to 5; Step 2, two-dimensional normal phase high performance liquid chromatography detection: The test solution prepared in step S1 is detected by normal phase high performance liquid chromatography, including the following steps in sequence: In the first dimension, the mobile phase A was n-hexane, and the mobile phase B was n-hexane, n-pentanol, and isopropanol, with gradient elution; The second dimension liquid chromatography was performed with n-hexane and n-pentanol as the mobile phase and isocratic elution.

7. The method according to claim 6, characterized in that The chromatographic column used in the first-dimensional liquid chromatography is selected from a urea-based bonded silica gel column; preferably, the urea-based bonded silica gel column is selected from Thermo Acclaim HILIC-10 or NanoChromCore 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; and / or, 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； And / or, the gradient elution degree of the first dimension liquid chromatography is: From 0 to 30 min, the volume of mobile phase A was maintained at 85 to 95%, and the volume of mobile phase B was maintained at 5 to 15%; 30-35 min, the volume of mobile phase A is reduced to 0-5%, and the volume of mobile phase B is increased 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%; At 60-65 min, the volume of mobile phase A increased to 85-95%, and the volume of mobile phase B decreased to 5-15%; From 65 to 200 min, the volume of mobile phase A is maintained at 85 to 95%, and the volume of mobile phase B is maintained at 5 to 15%.

8. The method according to claim 6 or 7, characterized in that: The detection conditions of the first-dimensional liquid chromatography meet at least one of the following conditions (1) to (5): (1) Valve cutting time is 10 to 30 minutes; (2) The detection wavelength is 260-270 nm; (3) The flow rate of the mobile phase is 0.1 to 1 mL / min; (4) The injection volume is 50-200 μL; (5) The column temperature is 35-45°C.

9. The method according to claim 6, characterized in that The chromatographic column used in the second dimension liquid chromatography is selected from a silica gel column; preferably, the silica gel column is selected from Phenomenex Silica (2) or Welch Topsil silica, inner diameter × column length is (4-5) × (150-250) mm, stationary phase particle size is 3-5 μm; And / or, in the mobile phase of the second-dimensional liquid chromatography, the volume ratio of n-hexane to n-pentanol is 1000:(2-5).

10. The method according to claim 6 or 9, characterized in that: The detection conditions of the second-dimensional liquid chromatography meet at least one of the following conditions (1) to (3): (1) The detection wavelength is 270-280nm; (2) The mobile phase flow rate is 0.8 to 1.2 mL / min; (3) The column temperature is 35-45°C.

Images