Method for determination of impurities in vitamin D preparations containing oily matrix by two-dimensional liquid chromatography

Abstract

The present application discloses a method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography, and relates to the technical field of drug detection. The detection method of the present application can effectively solve the problem of detecting related substances of vitamin D with extremely low content under the condition of interference from the oily matrix, and the method has the characteristics of good linear range, high sensitivity, high accuracy and good reproducibility.

Description

Technical Field

[0001] The present application relates to the technical field of drug detection, and in particular to a method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography. Background Art

[0002] From the synthesis of raw materials to the preparation of related preparations, drugs need to go through a relatively complex and lengthy process of storage, transportation, and use. During this period, each process may produce relevant impurities, such as starting materials, reagents, intermediates, by-products, and isomers that may be introduced during production; special impurities such as degradation products, polymers, or crystal transformations may be produced during storage and transportation. Impurities in drugs generally refer to process impurities or degradation products generated during the production, storage, and transportation of drugs. In addition to being related to the pharmacological activity of the main components, the adverse reactions of drugs in clinical use are also closely related to the impurities present in the drugs. The control of impurities in drugs is an important aspect of drug research and development and a guarantee of safety in clinical use. Therefore, in order to ensure the safety and effectiveness of drugs, and also to take into account the actual production situation, both domestic and foreign countries have used impurity detection as an important indicator for controlling drug quality during the drug research process. Drug impurity detection research is one of the weak points in my country's current drug research and development. In order to comprehensively improve the level of drug research and development in my country and effectively ensure the safety of public drug use, it is necessary to attach importance to and strengthen the research on relevant impurities in drugs.

[0003] The molecular formula of Vitamin D3 is shown below. Its chemical name is "9,10-secocholesterol-5,7,10(19)-triene-3β-ol". It is a fat-soluble vitamin and an oral drug used to prevent and treat vitamin D deficiency such as rickets.

[0004]

[0005] Vitamin D3 is converted into provitamin D3 (active ingredient) under heating conditions and generates impurity A (trans-vitamin D3) under light conditions; provitamin D3 can generate impurity E (tachysterol) under light conditions.

[0006] High performance liquid chromatography is used to determine the related substances in vitamin D3 in the European Pharmacopoeia and the Chinese Pharmacopoeia; there are fewer methods for determining related substances at home and abroad, especially for vitamin D preparations with smaller specifications.

[0007] For example, each tablet (250 mg) contains 400 IU (10 μg) of vitamin D3, which is only 40 parts per million, and the content of related substances (impurities) generally does not exceed 1.0% of the vitamin D3 content, which is equivalent to only 4 parts per million of impurities in the entire preparation. Since vitamin D3 is only stable in an oily matrix, which has a significant interference in the detection of vitamin D3 impurities, the detection of such impurities is more challenging than other drug impurity detection in terms of analytical method development and selection. Summary of the invention

[0008] In order to overcome the defects of the prior art, the present application provides a method for determining impurities in vitamin D preparations containing an oily matrix by two-dimensional liquid chromatography. The detection method provided in the present application can effectively solve the problem that the existing detection methods have large interference and cannot determine related substances (impurities) with extremely low content.

[0009] To achieve the above objectives, this application proposes the following technical solutions:

[0010] In a first aspect, the present application provides a method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography, the method comprising:

[0011] Inject the system suitability solution, reference solution, and test solution into the two-dimensional high performance liquid chromatograph respectively, record the chromatogram, and calculate the impurity content by the principal component self-reference method without adding a correction factor;

[0012] Wherein, in the two-dimensional high performance liquid chromatograph:

[0013] The first dimension HPLC conditions were:

[0014] Use n-hexane as mobile phase A and n-hexane-n-pentanol-isopropanol as mobile phase B for gradient elution;

[0015] The second dimension HPLC conditions were:

[0016] The mobile phase is n-hexane-n-pentanol-isopropanol; the switching procedure is: inject the system suitability solution into the first-dimension liquid chromatograph to locate the previtamin D3 peak and the vitamin D3 peak. The retention time of the main peak of vitamin D3 in the first-dimension liquid chromatograph is 22-25min, and the switching time is set to 0.8-1min after the previtamin D3 and impurity E peaks are completely eluted.

[0017] As a preferred technical solution of the present application, the detection conditions of the two-dimensional high performance liquid chromatography are:

[0018] Detection wavelength: 265 nm; column temperature: 40°C; flow rate: 0.5 mL / min; collection tube: polyetheretherketone tube.

[0019] More preferably, the collection tube is a polyetheretherketone (peek) tube with the following specifications: inner diameter 0.0762 cm (0.03 inches), 20 m, and a volume of about 9 mL.

[0020] As a preferred technical solution of the present application, the first-dimensional liquid chromatography uses a chromatographic column with urea-bonded silica gel as a filler;

[0021] The gradient elution program is: 0-30 min, the volume of mobile phase A is maintained at 94.5%, and the volume of mobile phase B is maintained at 5.5%;

[0022] 30-35 min, the volume of mobile phase B was increased from 5.5% to 100%;

[0023] 35-60 min, the volume of mobile phase B was maintained at 100%;

[0024] At 60-65 min, the volume of mobile phase B was reduced from 100% to 5.5% and maintained until 80 min.

[0025] The details are shown in Table 1.

[0026] Table 1:

[0027] Time (min) Mobile phase A (%) Mobile phase B (%) 0 94.5 5.5 30 94.5 5.5 35 0 100 60 0 100 65 94.5 5.5 80 94.5 5.5

[0028] As a preferred technical solution of the present application, in the mobile phase B of the first-dimensional liquid chromatography, the volume ratio of n-hexane, n-pentanol and isopropanol is 98:1:1.

[0029] As a preferred technical solution of the present application, the second-dimensional liquid chromatography uses a chromatographic column with silica gel as a filler.

[0030] As a preferred technical solution of the present application, in the mobile phase B of the second-dimensional liquid chromatography, the volume ratio of n-hexane, n-pentanol and isopropanol is 996:2:2.

[0031] As a more preferred technical solution of the present application, the chromatographic column of the first-dimensional liquid chromatography condition is: NanoChrom, Chromcore VD-chp4, 2.1×150 mm, 3 μm.

[0032] The chromatographic column of the second dimension liquid chromatography condition is: Agilent, ZORBAX RX-SIL, 3.0×100 mm, 1.8 μm.

[0033] As a preferred technical solution of the present application, the method for preparing the system suitability solution comprises:

[0034] Take the vitamin D3 reference substance, place it in a brown volumetric bottle, add isooctane to dissolve it, and make a stock solution containing 250 μg of vitamin D3 reference substance per 1 mL;

[0035] Take the stock solution of vitamin D3 reference substance, seal it after passing nitrogen, heat it in a water bath, add n-hexane after cooling, shake it well and place it in a quartz absorption cell, irradiate it under two 8W ultraviolet lamps with main wavelengths of 254nm and 365nm respectively. After irradiation, measure the solution into a brown volumetric bottle, add n-hexane to dilute to the scale, shake it well, and make a system suitability solution containing approximately 5μg vitamin D3 per 1mL.

[0036] More specifically, the preparation method of the system suitability solution is as follows: take an appropriate amount of vitamin D3 reference substance, accurately weigh it, place it in a brown volumetric bottle, add isooctane to dissolve it, and prepare a stock solution of vitamin D3 reference substance containing 250 μg per 1 mL; take 5 mL of the vitamin D3 reference substance stock solution, place it in a stoppered glass container, pass nitrogen through it and then seal it, heat it in a 90°C water bath for 1 hour, take it out, cool it quickly, add 5 mL of n-hexane, shake it well, place it in a 1 cm stoppered quartz absorption cell, and under 2 8W ultraviolet lamps with main wavelengths of 254nm and 365nm respectively, place the quartz absorption cell at an angle of 45° and 5-6 cm away from the lamp tube, irradiate it for 5 minutes, then accurately measure 1 mL, place it in a 25mL brown volumetric bottle, add n-hexane to dilute to the scale, shake it well, and prepare a system suitability solution containing approximately 5 μg of vitamin D3 per 1 mL.

[0037] As a preferred technical solution of the present application, the method for preparing the reference solution comprises:

[0038] Place the vitamin D3 reference substance in a brown bottle, add n-hexane to dissolve it, and prepare a reference substance solution containing 0.03 μg of vitamin D3 per 1 mL.

[0039] As a preferred technical solution of the present application, the method for preparing the test solution comprises:

[0040] Place the test sample in a brown volumetric flask, add n-hexane to dissolve it and dilute to the scale to prepare a test sample solution containing 3 μg of vitamin D3 per 1 mL.

[0041] In the prior art, the document "Analysis of Vitamin D3 Soft Capsules (Oily Matrix) by Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) System" studies the content detection method and the related substance analysis method, and the method is as follows: reverse-phase solvent extraction + rotary evaporation concentration + reverse-phase solvent dissolution quantification + reverse-phase injection analysis. However, the related substance analysis method still cannot eliminate the blank interference of the oily matrix, and the calculation is performed by deducting the interference peak area, and there is no clear impurity limit concentration.

[0042] The document "HPLC Rapid Determination of Vitamin D3 in Vitamin D3 Soft Capsules" uses reversed-phase high-performance liquid chromatography to determine vitamin D3 in vitamin D3 soft capsules, and the method uses vitamin D3 soft capsules containing 5000IU (125μg) of vitamin D3 per capsule. The method is as follows: reversed-phase solvent dissolution + strong shaking + ultrasound + reversed-phase solvent dilution quantification + reversed-phase injection analysis; this method is suitable for the determination of vitamin D3 content in varieties with high main component content, and the interference of oily matrix can be ignored at this time, but it is not suitable for the detection of related substances with low main component content.

[0043] The document "RP-HPLC Determination of Related Substances in Vitamin D3 Preparations" uses reversed-phase high performance liquid chromatography to determine related substances in vitamin D3 preparations. The method is as follows: reversed-phase solvent extraction + normal-phase solvent reconstitution + nitrogen blowing to dryness + reversed-phase solvent reconstitution quantification + reversed-phase injection analysis; this method is applicable to solid vitamin D preparations that do not contain an oily matrix, does not mention or analyze the interference of the oily matrix, and is not applicable to the detection of related substances in vitamin D preparations containing an oily matrix.

[0044] Compared with the prior art, this application has the following beneficial effects:

[0045] The detection method of the present application can effectively solve the problem of detecting extremely low levels of vitamin D-related substances in the presence of interference from an oily matrix. The method has the characteristics of a good linear range, high sensitivity, high accuracy, and good reproducibility.

[0046] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The drawings described herein are used to provide a further understanding of the present application, constitute a part of the present application, and do not constitute an improper limitation of the present application. In the drawings:

[0048] Figure 1 is the standard spectrum of the system suitability solution in Example 1;

[0049] Figure 2 is the standard spectrum of the blank auxiliary material solution (oily matrix) in Example 1;

[0050] Figure 3 It is the standard spectrum of the test solution in Example 1;

[0051] Figure 4 is the standard spectrum of the linear solution (25%) in Example 2;

[0052] Figure 5 is the standard spectrum of the linear solution (50%) in Example 2;

[0053] Figure 6 is the standard spectrum of the linear solution (100%) in Example 2;

[0054] Figure 7 is the standard spectrum of the linear solution (150%) in Example 2;

[0055] Figure 8 is the standard spectrum of the reproducible solution 1 in Example 3;

[0056] Fig. 9 is the standard spectrum of reproducible solution 2 in Example 3;

[0057] Fig.10 is the standard spectrum of reproducible solution 3 in Example 3;

[0058] Fig.11 is the standard spectrum of reproducible solution 4 in Example 3;

[0059] Fig.12 is the standard spectrum of the reproducible solution 5 in Example 3;

[0060] Fig.13 is the standard spectrum of reproducible solution 6 in Example 3;

[0061] Fig.14 is the standard spectrum of accuracy solution 1 (25% of the limit concentration) in Example 4;

[0062] Fig.15 is the standard spectrum of accuracy solution 2 (50% of the limit concentration) in Example 4;

[0063] Fig.16 is the standard spectrum of accuracy solution 3 (100% of the limit concentration) in Example 4;

[0064] Fig.17 is the standard spectrum of accuracy solution 4 (150% of the limit concentration) in Example 4;

[0065] Fig.18 This is a diagram of the experimental results of Example 7. DETAILED DESCRIPTION

[0066] The technical solutions in the embodiments will be described clearly and completely below in combination with the embodiments and drawings of the present application. Obviously, the embodiments described below are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0067] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0068] It should also be understood that the terms used in this specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0069] The materials required in the examples of the present application are all purchased from commercial channels; the two-dimensional liquid chromatograph is a 1260 Infinity II model produced by Agilent; the chromatographic column is a NanoChrom, Chromcore VD-chp4 2.1×150mm, 3μm produced by Nanospectroscopy Technology (Suzhou) Co., Ltd. and an Agilent, ZORBAXRX-SIL 3.0×100mm, 1.8μm produced by Agilent; the experimental methods not mentioned in the examples are conventional experimental methods and will not be described one by one here.

[0070] In the following examples, impurity A is trans-vitamin D3; impurity E is tachysterol.

[0071] The present application is further described below in conjunction with embodiments.

[0072] Example 1

[0073] Study on the specificity of the detection method

[0074] Chromatographic conditions:

[0075] Instrument: Two-dimensional high performance liquid chromatograph (first dimension with 10 mm path length detector, second dimension with 60 mm path length detector);

[0076] Detection wavelength: 265nm;

[0077] Column temperature: 40°C;

[0078] Flow rate: 0.5 mL / min;

[0079] Collection tube: polyetheretherketone (peek) tube, inner diameter 0.0762 cm (0.03 inches), 20 m, volume approximately 9 mL.

[0080] The first-dimension chromatographic conditions were as follows: NanoChrom, Chromcore VD-chp4 2.1×150 mm, 3 μm as the chromatographic column; n-hexane as the mobile phase A, n-hexane-n-pentanol-isopropanol (98:1:1) as the mobile phase B, and gradient elution according to the program in Table 2 below.

[0081] Table 2:

[0082]

[0083]

[0084] The second dimension chromatographic conditions were as follows: Agilent, ZORBAX RX-SIL 3.0×100mm, 1.8μm as the chromatographic column; n-hexane-n-pentanol-isopropanol (996:2:2) as the mobile phase; the system suitability solution was injected into the first dimension liquid chromatograph to locate the previtamin D3 peak and the vitamin D3 peak. The initial ratio of the first dimension liquid chromatographic mobile phase A and mobile phase B was adjusted to make the retention time of the vitamin D3 main peak about 23min, and the switching time was set to about 1min after the previtamin D3 peak was completed to the impurity E peak was completed.

[0085] Among them, the system suitability solution, reference solution, and test solution were prepared by the following method:

[0086] System suitability solution: Take an appropriate amount of vitamin D3 reference substance, weigh it accurately, place it in a brown volumetric bottle, add isooctane to dissolve it, and make a stock solution containing 250μg vitamin D3 reference substance per 1mL;

[0087] Take 5 mL of the stock solution of vitamin D3 reference substance, place it in a stoppered glass container, seal it after passing nitrogen, heat it in a 90°C water bath for 1 hour, take it out, cool it quickly, add 5 mL of n-hexane, shake it well, place it in a 1 cm stoppered quartz absorption cell, and under 2 8 W ultraviolet lamps with main wavelengths of 254 nm and 365 nm respectively, tilt the quartz absorption cell at 45° and 5-6 cm away from the lamp tube, irradiate it for 5 minutes, then accurately measure 1 mL, place it in a 25 mL brown volumetric flask, add n-hexane to dilute to the scale, shake it well, and make a system suitability solution containing approximately 5 μg of vitamin D3 per 1 mL.

[0088] Reference solution: Take an appropriate amount of vitamin D3 reference substance, weigh it accurately, place it in a brown volumetric flask, add n-hexane to dissolve it, and make a reference solution containing 0.03 μg of vitamin D3 per 1 mL.

[0089] Test solution: Take 1.5 g of the product, weigh accurately, place in a 20 mL brown volumetric flask, add n-hexane and vortex for 3 min to dissolve and dilute to the scale, shake well to make a test solution containing 3 μg of vitamin D3 per 1 mL.

[0090] Injection procedure: Take 100 μl of each of the above test solutions, inject into the two-dimensional liquid chromatograph, and record the chromatograms respectively. Figure 1 , Figure 2 and Figure 3 .

[0091] according to Figure 1-Figure 3 The test results show that the blank excipient solution (oily matrix) has no interference at the main peak positions of impurity A, impurity E and vitamin D3, and the specificity of the detection method is good.

[0092] Example 2

[0093] Linearity study of the detection method

[0094] Sample preparation:

[0095] Impurity A reference substance stock solution: Take an appropriate amount of Impurity A reference substance, weigh it accurately, place it in a brown volumetric flask, add n-hexane to dissolve it, and then add n-hexane to dilute it to make an Impurity A reference substance stock solution containing 0.5 μg Impurity A per 1 mL.

[0096] Each linear solution: Take a pipette and a volumetric bottle, accurately measure an appropriate amount of the impurity A reference substance stock solution (0.5 μg / mL) according to Table 3 below and place it in a volumetric bottle, add n-hexane to dissolve and dilute to the scale, shake well, and obtain.

[0097] Table 3:

[0098] Linear solution 1 2 3 4 Impurity A reference stock solution (mL) 1.5 1.5 3.0 9.0 Volumetric flask (mL) 100 50 50 100 Theoretical concentration (μg / mL) 0.0075 0.015 0.03 0.045 Impurity A concentration level 25％ 50％ 100％ 150％

[0099] Detection was performed according to the chromatographic conditions of Example 1.

[0100] Injection procedure: Take 100 μl of each of the above test solutions, inject into the two-dimensional liquid chromatograph, and record the chromatograms respectively. Figure 4 , Figure 5 , Figure 6 and Figure 7 .

[0101] Experimental conclusion: The linear research range is 8.7ng / mL-52.4ng / mL (equivalent to 25%-150% of the limit concentration), the linear regression equation is y=3505.5794x+1.4627, the y-axis ratio (y-axis intercept / 100% concentration peak area) is 1.2%, the relative response factors of all concentrations are between 0.99-1.04, the correlation coefficient r=1.0000, and the linear relationship between concentration and peak area is good, that is, the method has good linearity.

[0102] Example 3

[0103] Study on the precision of the detection method

[0104] Repeatability solution: Take 1.5g of vitamin D preparation containing oily matrix (specification: 400IU / capsule), weigh accurately, place in a 20mL brown volumetric bottle, accurately add 2.0mL of impurity A stock solution (0.3μg / mL), then add n-hexane and vortex for 1min to dissolve and dilute to the scale, shake well, and use as the test solution. Prepare 6 copies in parallel.

[0105] Detection was performed according to the chromatographic conditions of Example 1.

[0106] Injection procedure: Take 100 μl of each of the above test solutions, inject into the two-dimensional liquid chromatograph, and record the chromatograms respectively. Figure 8-Figure 13 .

[0107] Experimental conclusion: Six samples of the repeatability solution were prepared in parallel, the RSD (n=6) of the impurity A content was 0.9%, impurity E was not detected, and the RSD (n=6) of the total impurity content was 0.9%, with good repeatability.

[0108] Example 4

[0109] Study on the accuracy of detection method

[0110] Impurity A reference substance stock solution: Take an appropriate amount of the impurity reference substance, accurately weigh it, place it in a brown volumetric flask, add n-hexane to dissolve it, and make an impurity A reference substance stock solution containing 0.3 μg of impurity A per 1 mL.

[0111] Accuracy solution 1 (25% of the limit concentration): Take 1.5 g of blank oily matrix and put it into a 20 mL brown volumetric flask. Accurately measure 0.5 mL of impurity A reference substance stock solution and add it thereto. Add n-hexane to dissolve and dilute to the scale. Shake well to obtain.

[0112] Accuracy solution 2 (50% of the limit concentration): Take 1.5 g of blank oily matrix and put it into a 20 mL brown volumetric flask. Accurately measure 1.0 mL of impurity A reference substance stock solution and add it thereto. Add n-hexane to dissolve and dilute to the scale. Shake well to obtain.

[0113] Accuracy solution 3 (100% of limit concentration): Take 1.5 g of blank oily matrix and put it into a 20 mL brown volumetric flask. Accurately measure 2.0 mL of impurity A reference substance stock solution and add it thereto. Add n-hexane to dissolve and dilute to the scale. Shake well to obtain.

[0114] Accuracy solution 4 (150% of the limit concentration): Take 1.5 g of blank oily matrix and put it into a 20 mL brown volumetric flask. Accurately measure 3.0 mL of impurity A reference substance stock solution and add it thereto. Add n-hexane to dissolve and dilute to the scale. Shake well to obtain.

[0115] Detection was performed according to the chromatographic conditions of Example 1.

[0116] Injection procedure: Take 100 μl of each of the above test solutions, inject into the two-dimensional liquid chromatograph, and record the chromatograms respectively. Figures 14 to 17 ,The experimental results are shown in Table 4 below.

[0117] Table 4:

[0118]

[0119] according to Figures 14 to 17 From the test results in Table 3, it can be seen that the maximum accuracy research range of the test method of this application is 8.7ng / mL-52.4ng / mL (equivalent to 25%-150% of the limit concentration), the recovery rate of impurity A is between 80%-91%, and the method accuracy is good.

[0120] Example 5

[0121] Determined according to the National Drug Administration standard "Vitamin D Drops" YBH02542018 method

[0122] Study on the reproducibility and recovery of the detection method

[0123] Chromatographic conditions: silica gel as filler; n-hexane-n-pentanol (997:3) as mobile phase; column temperature 30°C, flow rate 1.5 ml per minute, detection wavelength 265 nm.

[0124] Impurity A reference substance stock solution: Take 5 mg of impurity A reference substance, accurately weigh it, place it in a 250 ml brown volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and use it as impurity A reference substance stock solution 1 (20 μg / ml); Accurately measure 5.0 ml of impurity A reference substance stock solution 1, place it in a 50 ml brown volumetric flask, add methanol to dilute to the scale, shake well, and use it as impurity A reference substance stock solution (2 μg / ml).

[0125] Impurity A reference solution: Accurately measure 2.5 ml of the impurity A reference stock solution, place it in a 20 ml brown volumetric flask, blow dry with nitrogen, add isooctane to dissolve and dilute to the scale, shake well, and use it as the impurity A reference positioning solution (0.25 μg / ml).

[0126] Preparation of test solution A: Take 10 g of the product content, place it in a 250 ml brown conical flask, add 50 ml of methanol, ultrasonicate for 3 min, and decant the methanol solution; repeat the extraction 3 times, collect the methanol solution, evaporate it at 30°C under reduced pressure to about 50 ml, remove the oil droplets in the methanol solution, continue to evaporate it to dryness under reduced pressure, accurately measure 4 ml of methanol, ultrasonicate for 1 min to dissolve, centrifuge for 3 min (10000r / min), and take the supernatant as the test solution A (100μg / ml).

[0127] Test solution B: Take appropriate amount of impurity A and vitamin D3 reference substance, weigh accurately, dissolve them in methanol to prepare a mixed reference solution containing impurity A and vitamin D3 at 0.4μg / ml and 35μg / ml respectively. Accurately measure 500μl of the mixed reference solution and test solution A respectively, inject according to the reverse phase chromatography conditions, accurately collect all the effluent (containing impurity A, vitamin D3 and impurity E) in the chromatogram of test solution A containing impurity A and vitamin D3, blow dry the effluent with nitrogen, accurately add 1ml of isooctane, and sonicate for 1min to dissolve, as test solution B (50μg / ml).

[0128] Preparation of spiked test solution A: Take 10 g of the product content, place it in a 250 ml brown conical flask, accurately measure 1.0 ml of impurity A reference stock solution and add it, then add 50 ml of methanol, ultrasonicate for 3 min, and decant the methanol solution; repeat the extraction 3 times, collect the methanol solution, evaporate it under reduced pressure at 30°C to about 50 ml, remove the oil droplets in the methanol solution, continue to evaporate it under reduced pressure to dryness, accurately measure 4 ml of methanol, ultrasonicate for 1 min to dissolve it, centrifuge for 3 min (10000 r / min), and take the supernatant as the test solution A (100 μg / ml).

[0129] Spiked test solution B: Accurately measure 500 μl of the mixed reference solution and spiked test solution A respectively, and inject according to the reverse phase chromatography conditions. Accurately collect all the effluent (including impurity A, vitamin D3 and impurity E) in the chromatogram of test solution A containing impurity A and vitamin D3. Blow dry the effluent with nitrogen, accurately add 1 ml of isooctane, and sonicate for 1 min to dissolve it as the spiked test solution B.

[0130] The experimental results are shown in Table 5 below.

[0131] Table 5

[0132]

[0133] According to the test results, the recoveries of the six test samples differed greatly, and the repeatability and recovery of the methods could not meet the requirements of general analytical methods. The operation was complicated and required two ordinary liquid phase preparation injections, which was very time-consuming and required very high operating requirements for analysts. The applicability and repeatability of the analytical method were not good, and it could not meet the increasingly stringent analytical technical standards, so this method was abandoned.

[0134] Example 6

[0135] Brief description of the method: dissolve the sample by ultrasonic treatment for 10 minutes + mechanical shaking for 10 minutes + extract with methanol 4 times + rotary evaporation without removing oil droplets + rotary evaporation to dryness + normal phase dissolution + two-dimensional normal phase injection analysis.

[0136] The chromatographic conditions are shown in Table 6.

[0137] Table 6:

[0138]

[0139]

[0140] System suitability solution: Take 25 mg of vitamin D3 reference substance, weigh it accurately, put it in a 100 ml brown volumetric flask, add 80 ml of isooctane, avoid heating, ultrasonicate for 1 minute to completely dissolve it, dilute it to the mark with isooctane, shake well, and make a stock solution of vitamin D3 reference substance containing 250 μg per 1 ml; measure 5 ml of vitamin D3 reference substance stock solution, put it in a stoppered glass container, seal it after nitrogen flow, heat it in a 90°C water bath for 1 hour, take it out, cool it quickly, add 5 ml of n-hexane, shake well, put it in a 1 cm stoppered quartz absorption cell, under 2 8 W ultraviolet lamps with dominant wavelengths of 254 nm and 365 nm respectively, tilt the quartz absorption cell at 45° and keep it 5 to 6 cm away from the lamp tube, irradiate it for 5 minutes, then accurately measure 1 ml, put it in a 25 ml brown volumetric flask, dilute it to the mark with n-hexane, shake well, and make a system suitability solution containing about 5 μg of vitamin D3 per 1 ml.

[0141] Reference solution (0.2μg / ml): Accurately measure 1.0ml of the above vitamin D3 reference stock solution (250μg / ml), place it in a 50ml brown volumetric flask, dilute to scale with n-hexane, shake well, then accurately measure 1.0ml, place it in a 25ml brown volumetric flask, dilute to scale with n-hexane, shake well, and make a reference solution containing 0.2μg vitamin D3 per 1ml (0.2μg / ml). It is also acceptable if it is already prepared.

[0142] Impurity A reference substance stock solution (2μg / ml): Take 2 mg of impurity A reference substance, accurately weigh it, place it in a 100ml brown volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and use it as impurity A stock solution 1 (20μg / ml); Accurately measure 5.0ml of impurity A reference substance stock solution 1, place it in a 50ml brown volumetric flask, add methanol to dilute to the scale, shake well, and use it as impurity A reference substance stock solution (2μg / ml).

[0143] Impurity A reference solution (0.2 μg / ml): Accurately measure 2.0 ml of the impurity A reference stock solution, place it in a 20 ml brown volumetric flask, blow dry with nitrogen, add n-hexane to dissolve and dilute to the scale, shake well, and use it as the impurity A reference solution (0.2 μg / ml).

[0144] Test solution: Take 10g of the content of this product, weigh it accurately, put it in a 250ml brown conical flask, add 50ml of methanol, ultrasonicate for 10min, and then mechanically shake for 10min (200r / min), centrifuge the mixed solution for 5min (10000r / min), and decant the upper methanol solution; repeat the extraction 4 times, collect the methanol solution, evaporate it under reduced pressure at 30℃ to dryness, dissolve it with an appropriate amount of n-hexane, rinse it with n-hexane, transfer it to a 20ml brown volumetric flask, add n-hexane to dilute to the scale, shake well, and use it as the test solution (20μg / ml).

[0145] Spiked test solution (impurity A + limit 1.0% + 0.2μg / ml): Take 10g of the content of this product, accurately weigh it, and place it in a 250ml brown conical flask, then accurately measure 2.0ml of impurity A reference substance stock solution (2μg / ml) and add it, add 50ml of methanol, ultrasonicate for 10min, and then mechanically shake for 10min (200r / min), centrifuge the mixed solution for 5min (10000r / min), and pour off the upper methanol liquid; repeat the extraction 4 times, collect the methanol liquid, evaporate it under reduced pressure at 30℃ to dryness, dissolve it with an appropriate amount of n-hexane by ultrasonication, rinse it and transfer it to a 20ml brown volumetric flask, then dilute it to the scale with n-hexane, shake it well, and use it as the spiked test solution.

[0146] The experimental results are shown in Table 7 below.

[0147] Table 7:

[0148]

[0149] According to the test results, the method has good repeatability, but the spiked recoveries of the three test samples are low, and the sample preparation operation method is more difficult than the general method. Therefore, this method has poor applicability and is abandoned.

[0150] Embodiment 7:

[0151] Determined according to the general rules of the fourth part of the 2020 edition of the Chinese Pharmacopoeia, "Vitamin D Assay - Method 4"

[0152] Method description: Adopt the general chromatographic conditions of the pharmacopoeia content determination method + increase the concentration of the test sample for sampling inspection.

[0153] The mobile phase is n-hexane-n-pentanol-isopropanol; the switching procedure is: inject the system suitability solution into the first-dimension liquid chromatograph to locate the previtamin D3 peak and the vitamin D3 peak. The retention time of the main peak of vitamin D3 in the first-dimension liquid chromatograph is 22-25min, and the switching time is set to 0.8-1min after the previtamin D3 and impurity E peaks are completely eluted.

[0154] The detection conditions of the two-dimensional high performance liquid chromatography are:

[0155] Detection wavelength: 265nm; column temperature: 40℃; flow rate: 0.5mL / min; collection tube: polyetheretherketone tube (inner diameter 0.0762cm (0.03 inches), 20m, volume about 9mL); one dimension is a chromatographic column filled with urea-bonded silica gel, mobile phase A is n-hexane, mobile phase B is n-hexane-n-pentanol-isopropanol (98:1:1), gradient elution; the second dimension is a chromatographic column filled with silica gel (3mm×100mm, 1.8μm), the mobile phase is n-hexane-n-pentanol-isopropanol (996:2:2), isocratic elution; the flow cell is a 10mm pathlength detector; the injection volume is 100μl.

[0156] The gradient elution program is: 0-30 min, the volume of mobile phase A is maintained at 93.5%, and the volume of mobile phase B is maintained at 6.5%;

[0157] 30-35 min, the volume of mobile phase B was increased from 6.5% to 100%;

[0158] 35-60 min, the volume of mobile phase B was maintained at 100%;

[0159] At 60-65 min, the volume of mobile phase B was reduced from 100% to 5.5% and maintained until 80 min.

[0160] The details are shown in Table 8.

[0161] Table 8:

[0162] Time (min) Mobile phase A (%) Mobile phase B (%) 0 93.5 6.5 30 93.5 6.5 35 0 100 60 0 100 65 93.5 6.5 80 93.5 6.5

[0163] System suitability solution: Take 25 mg of vitamin D3 reference substance, weigh it accurately, put it in a 100 ml brown volumetric flask, add 80 ml of isooctane, avoid heating, ultrasonicate for 1 minute to completely dissolve it, dilute it to the mark with isooctane, shake well, and make a stock solution of vitamin D3 reference substance containing 250 μg per 1 ml; measure 5 ml of vitamin D3 reference substance stock solution, put it in a stoppered glass container, seal it after nitrogen flow, heat it in a 90°C water bath for 1 hour, take it out, cool it quickly, add 5 ml of n-hexane, shake well, put it in a 1 cm stoppered quartz absorption cell, under 2 8 W ultraviolet lamps with dominant wavelengths of 254 nm and 365 nm respectively, tilt the quartz absorption cell at 45° and keep it 5 to 6 cm away from the lamp tube, irradiate it for 5 minutes, then accurately measure 1 ml, put it in a 25 ml brown volumetric flask, dilute it to the mark with n-hexane, shake well, and make a system suitability solution containing about 5 μg of vitamin D3 per 1 ml.

[0164] Reference solution: Take an appropriate amount of impurity A reference substance, weigh accurately, and dilute with n-hexane to make a solution containing 0.05 μg of vitamin D3 per 1 ml.

[0165] Test solution: Take 6.25 g of the product, weigh accurately, put it in a 25 ml brown volumetric flask, add n-hexane and vortex for 1 minute to dissolve and dilute to the scale, shake well, and use it as the test solution (10 μg / ml).

[0166] Blank excipient solution: Take 6.25 g of soybean oil, weigh it accurately, put it in a 25 ml brown volumetric flask, add n-hexane, vortex for 1 minute to dissolve and dilute to the scale, shake well, and use it as the blank excipient solution.

[0167] Experimental Results Fig.18 shown.

[0168] According to the spectrum, there is interference in the peak position of impurity A in the test sample. After accumulating a large number of experimental results, it can be known that the large baseline fluctuation in the spectrum is caused by the soybean oil in the sample. Therefore, this method is not suitable for the quality research of drug-related substances.

[0169] In summary, according to the experimental results of Examples 1 to 7, in order to ensure the accuracy and repeatability of the analytical method for the determination of related substances, the factors that must be considered are as follows: 1. Conventional general liquid phase equipment cannot accurately determine the impurity content in the vitamin D drop preparation containing an oily matrix, and a higher-level instrument must be used for detection; 2. In order to reduce the interference of the oily matrix, it can be considered to reduce the concentration of the oily matrix in the sample being tested, increase the instrument detection sensitivity by increasing the instrument hardware to increase the instrument detection sensitivity and thus reduce the oily matrix concentration, and select a suitable sample pretreatment method to reduce sample loss and increase the recovery rate of impurities while meeting the detection sensitivity.

[0170] The method provided in this application has solved the above two considerations: 1. The two-dimensional liquid phase is selected for detection, and the sample is processed and analyzed in two dimensions, reducing the interference of the oily matrix; 2. The preparation method is optimized, and n-hexane is directly used for dissolution, which is done in one step, eliminating the sample loss caused by complex and time-consuming pre-treatment, further improving the accuracy of sample impurity detection, and greatly reducing the cost of manpower and material resources; 3. A high-throughput detector (60mm optical path) is specially developed to improve the sample detection response. While meeting the sensitivity, it also reduces the concentration of the test sample (that is, reduces the concentration of the oily matrix), making the interference of the oily matrix on the sample detection almost negligible.

[0171] The technical solutions provided in the embodiments of the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for those skilled in the art, according to the embodiments of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present application.

Claims

1. A method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography, characterized in that: The method comprises: Inject the system suitability solution, reference solution, and test solution into the two-dimensional high performance liquid chromatograph respectively, record the chromatogram, and calculate the impurity content by the principal component self-reference method without adding a correction factor; The impurities include impurity A and impurity E; the impurity A is trans-vitamin D3, and the impurity E is tachysterol; Wherein, in the two-dimensional high performance liquid chromatograph: The first dimension HPLC conditions were: Use n-hexane as mobile phase A and n-hexane-n-pentanol-isopropanol as mobile phase B for gradient elution; The second dimension HPLC conditions were: The mobile phase is n-hexane-n-pentanol-isopropanol; the switching procedure is: inject the system suitability solution into the first-dimension liquid chromatograph to locate the peaks of previtamin D3 and vitamin D3. The retention time of the main peak of vitamin D3 in the first-dimension liquid chromatograph is 22-25 minutes, and the switching time is set to 0.8-1 minute after the peaks of previtamin D3 and impurity E are completely eluted; The detection conditions of the two-dimensional high performance liquid chromatography are: Detection wavelength: 265nm; column temperature: 40℃; flow rate: 0.5mL / min; collection tube: polyetheretherketone tube; The first dimension liquid chromatography uses a chromatographic column with urea-bonded silica gel as a filler; The gradient elution program is: 0-30min, the volume of mobile phase A is maintained at 94.5%, and the volume of mobile phase B is maintained at 5.5%; At 30-35 min, the volume of mobile phase B was increased from 5.5% to 100%; 35-60min, the volume of mobile phase B was maintained at 100%; At 60-65 min, the volume of mobile phase B was reduced from 100% to 5.5% and maintained until 80 min; In the mobile phase B of the first-dimensional liquid chromatography, the volume ratio of n-hexane, n-pentanol and isopropanol is 98:1:1; the second-dimensional liquid chromatography uses a chromatographic column with silica gel as a filler; In the mobile phase B of the second-dimensional liquid chromatography, the volume ratio of n-hexane, n-pentanol and isopropanol is 996:2:

2.

2. The method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography according to claim 1, characterized in that: The method for preparing the system suitability solution comprises: Take the vitamin D3 reference substance, place it in a brown volumetric bottle, add isooctane to dissolve it, and make a stock solution containing 250 μg of vitamin D3 reference substance per 1 mL; Take the stock solution of vitamin D3 reference substance, seal it after passing nitrogen, heat it in a water bath, add n-hexane after cooling, shake it well and place it in a quartz absorption cell, irradiate it under two 8W ultraviolet lamps with main wavelengths of 254nm and 365nm respectively. After irradiation, measure the solution into a brown volumetric bottle, add n-hexane to dilute to the scale, shake it well, and make a system suitability solution containing approximately 5μg vitamin D3 per 1mL.

3. The method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography according to claim 1, characterized in that: The preparation method of the reference solution comprises: Place the vitamin D3 reference substance in a brown bottle, add n-hexane to dissolve it, and prepare a reference substance solution containing 0.03 μg of vitamin D3 per 1 mL.

4. The method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography according to claim 1, characterized in that: The method for preparing the test solution comprises: Place the test sample in a brown volumetric flask, add n-hexane to dissolve it and dilute to the scale to prepare a test sample solution containing 3 μg of vitamin D3 per 1 mL.

5. The method for determining impurities in a vitamin D preparation containing an oily matrix by two-dimensional liquid chromatography according to claim 1, characterized in that: The columns for the first-dimension LC conditions were: NanoChrom, Chromcore VD-chp4, 2.1×150 mm, 3 µm; The chromatographic column of the second dimension liquid chromatography condition is: Agilent, ZORBAX RX-SIL, 3.0×100 mm, 1.8 μm.

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