Determination method for related substances in drops containing vitamin D3

Abstract

The invention provides a method for determining related substances in drops containing vitamin D3, and belongs to the technical field of medicine detection. The method comprises the following steps: mixing contents of drops, acetone and methanol, extracting, performing phase separation, and collecting an upper methanol extract; removing matrix components and a solvent, and redissolving with methanol to prepare a redissolved solution; carrying out saponification reaction on the mixed reconstitution fluid and a sodium methoxide reagent to prepare a test solution; detecting the test solution by adopting a reverse phase chromatography, and collecting an effluent component with the retention time of 11.5-15.5 minutes; detecting an effluent component by adopting normal phase chromatography; the related substances include (5E, 7E)-9, 10-open-loop cholest-5, 7, 10 (19)-triene-3 beta-alcohol, (6E)-9, 10-open-loop cholest-5 (10), 6, 8-triene-3 beta-alcohol, and / or 9, 10-open-loop cholest-6, 8, 10 (5)-triene-3 beta-alcohol. The method is accurate in detection and high in sensitivity.

Description

Technical Field

[0001] The present application belongs to the technical field of drug detection, and in particular relates to a method for determining related substances in drops containing vitamin D3. Background Art

[0002] Vitamin D3 is a fat-soluble vitamin also considered a hormone precursor that affects calcium and phosphorus metabolism. It has multiple physiological functions: improving the body's absorption of calcium and phosphorus, achieving saturation levels of plasma calcium and phosphorus; promoting growth and bone calcification, and promoting healthy teeth; increasing phosphorus absorption through the intestinal wall and phosphorus reabsorption through the renal tubules; maintaining normal blood citrate levels; and preventing amino acid loss through the kidneys. Numerous clinical trials and evidence-based medical studies have demonstrated that vitamin D3 also has the following functions: reducing the incidence of common cancers such as breast, lung, and colon cancer; preventing and treating autoimmune diseases such as hypertension and infectious diseases; regulating placental development and function, and preventing pregnancy complications such as miscarriage, preeclampsia, and premature birth. Furthermore, adequate vitamin D3 intake in utero and in infants and young children can reduce the incidence of type 1 diabetes, asthma, and schizophrenia.

[0003] Vitamin D3 drops (soft capsules) are used to prevent and treat rickets in children. They are susceptible to degradation under light and high temperature conditions, producing impurities (related substances). To ensure drug safety and efficacy, these related substances, as a key indicator of drug quality control, must be accurately measured.

[0004] However, the 2020 edition of the Chinese Pharmacopoeia only includes the content determination method of vitamin D3, but does not include the determination method of related substances of vitamin D3; in the current edition of the European Pharmacopoeia (EP11.6) and the United States Pharmacopoeia (NF-2024), although the related substances of vitamin D3 raw materials are controlled, the related substances of vitamin D3 drops are not controlled.

[0005] Because the matrix components of vitamin D3 drops are mostly fat-soluble vegetable oils (such as peanut oil, soybean oil, and olive oil), direct HPLC analysis of related substances will severely interfere with the determination of the main components and impurities of vitamin D3, resulting in low repeatability and accuracy, poor method operability, and difficulty in reproducibility. Therefore, vitamin D3 drop samples must undergo specific pretreatment before testing.

[0006] The current methods for determining related substances in vitamin D3 drops are:

[0007] 1. Methanol-water mixed liquid extraction method, such as Patent Document 1 (Zhao Qin, Wu Sisi, Zhao Zhirong, et al., High Performance Liquid Chromatography Detection Method for Related Substances in Preparations Containing Vitamin D3 and Method for Preparing a Test Solution [P], Patent Publication No. CN114324648A). In the method described in this patent document, vitamin D3 drops are first extracted with methanol and water to prepare a methanol-water extract, which is then extracted with n-hexane. The extract is rotary evaporated to dryness to obtain a residue, which is then re-dissolved in methanol and water to obtain a test solution. Finally, the test solution is subjected to C18 chromatographic column under reverse phase conditions for the determination of related substances.

[0008] 2. Two-dimensional liquid chromatography, such as that described in Patent Document 2 (Yang Min, Tang Jiahui, Chen Qiaowei, et al., A Method for Determining Related Substances in Vitamin D Drops [P], Patent Publication No. CN119291055A). This method uses a urea-bonded silica gel column as a packing material, with n-hexane-n-pentanol-isopropanol as the mobile phase for first-dimension gradient elution. This is followed by a silica gel column with n-hexane-n-pentanol-ethanol as the mobile phase for second-dimension isocratic elution. Impurity A, impurity D (referred to as impurity B in some literature), and impurity E (referred to as impurity C in some literature) are determined in vitamin D3 drops. The method described in Patent Document 2 is similar to that described in Patent Document 3 (Zhong Zengzhi, Rao Yinqi, Chen Huli, et al., Method for Determining Impurities in Vitamin D Preparations Containing an Oily Matrix by Two-Dimensional Liquid Chromatography [P], Patent Publication No. CN116297935A), except that the mobile phase composition is adjusted to include an additional impurity D (referred to as impurity B in some literature).

[0009] 3. Solid-phase extraction method, such as Patent Document 4 (Liu Tiecheng, Jiang Haitao, Yang Lei, et al., A method for detecting vitamin D3-related substances in vitamin D3 drops [P], Patent Publication No. CN115308338A). In the method described in this patent document, the first step is similar to that of Patent Document 1. The contents of the vitamin D3 drops are first extracted with an ethanol-water mixture, and then the ethanol-water extract is extracted with n-hexane. The extract is purified by SPE silica gel solid-phase extraction column and eluted with ethyl acetate. The eluate is blown dry with nitrogen and then redissolved with n-hexane to obtain a test solution, which is then determined by normal phase chromatography.

[0010] Since vitamin D3 is a fat-soluble vitamin and the matrix component of vitamin D3 drops is fat-soluble vegetable oil, the development of detection methods is very difficult in the determination of related substances due to the interference of the vegetable oil matrix.

[0011] When reproducing the assay method described in Patent Document 1 (Zhao Qin, Wu Sisi, Zhao Zhirong, et al., "High-Performance Liquid Chromatography Detection Method for Related Substances in Vitamin D3 Preparations and Method for Preparing Test Solution [P]," Patent Publication No. CN114324648A), it was found that the pretreatment process used to extract the matrix components of the vitamin D3 drops using a methanol-water mixture did not guarantee the complete extraction of impurities from the matrix. Furthermore, the method described in this patent document only examined the extraction rate of the main vitamin D3 component in vitamin D3 tablets and effervescent granules, not the extraction rate of impurities. Experimental replication revealed that when this method was used to extract impurities from vitamin D3 drops, the extraction rate was low, failing to reach above 80%, due to the weak polarity of the vegetable oil matrix component of the drops. Therefore, this method is not suitable for the determination of related substances in vitamin D3 drops.

[0012] Patent Document 2 (Yang Min, Tang Jiahui, Chen Qiaowei, et al., A Method for Determining Related Substances in Vitamin D Drops [P], Patent Publication No. CN119291055A) and Patent Document 3 (Zhong Zengzhi, Rao Yinqi, Chen Huli, et al., A Method for Determining Impurities in Vitamin D Preparations Containing Oily Matrix by Two-Dimensional Liquid Chromatography [P], Patent Publication No. CN116297935A) both use two-dimensional chromatography. In the above-mentioned patented methods, the vitamin D3 drop sample is dissolved in n-hexane without any pretreatment and directly enters the first-dimensional chromatography. Since the test solution contains a large amount of matrix components, when switching to the second-dimensional chromatography column, a large amount of peanut oil also enters the second-dimensional chromatography column, resulting in large baseline fluctuations in the detection spectrum and affecting the integral processing of the main component and impurity peaks.

[0013] Patent Document 4 (Liu Tiecheng, Jiang Haitao, Yang Lei, et al., "A Method for Detecting Vitamin D3-Related Substances in Vitamin D3 Drops [P], Patent Publication No. CN115308338A") describes a pretreatment method that uses an ethanol-water mixture to extract the matrix components of the vitamin D3 drops. This method suffers from the same drawbacks as Patent Document 1: it fails to fully extract the impurities from the vegetable oil matrix, resulting in a result that is lower than the true value when directly calculating the content of the related substances using the external standard method. Furthermore, the inventors did not examine the recovery rate of the impurities. When replicating this method, the recovery rate of the impurities extracted using the standard addition method was significantly lower, at only approximately 30%, failing to meet the accuracy requirements for determinations in the presence of impurities.

[0014] In view of this, this application is hereby filed. Summary of the Invention

[0015] Based on this, one or more embodiments of the present application provide a method for determining related substances in vitamin D3-containing drops, including the following technical solutions:

[0016] One or more embodiments of the present application provide a method for determining related substances in a vitamin D3-containing drop, the method comprising:

[0017] preparing a test sample solution from contents of the drop to be tested;

[0018] detecting the test sample solution by reverse phase chromatography, and collecting effluent components with retention times in the time period of 11.5 min to 15.5 min; and

[0019] detecting the effluent components by normal phase chromatography to determine the related substances in the contents;

[0020] the related substances include one or more of (5E, 7E)-9, 10-secocholesta-5, 7, 10(19)-triene-3β-ol, (6E)-9, 10-secocholesta-5(10), 6, 8-triene-3β-ol, and 9, 10-secocholesta-6, 8, 10(5)-triene-3β-ol;

[0021] The preparation of the test sample solution comprises:

[0022] mixing the contents, acetone, and methanol for extraction, phase separation, and collection of the upper methanol extract;

[0023] removing residual matrix components and solvents of the contents in the methanol extract, re-dissolving with methanol to prepare a re-dissolution; and

[0024] mixing the re-dissolution and a sodium methoxide reagent for saponification to prepare the test sample solution.

[0025] In some embodiments of the present application, the volume ratio of the contents, acetone, and methanol is (2-10):(2-10):(20-100); alternatively, the volume ratio of the contents, acetone, and methanol is (2-5):(2-5):(20-50).

[0026] In some embodiments of the present application, the concentration of sodium methoxide in the sodium methoxide reagent is 2-5 mol / L, and the volume of the sodium methoxide reagent corresponding to 2-10 mL of the contents is 0.5-1.5 mL.

[0027] In some embodiments of the present application, the contents and the acetone are mixed to prepare a mixture, and then the mixture is added with the methanol for extraction.

[0028] Alternatively, the mixing conditions include shaking or ultrasonic treatment for 5-10 min.

[0029] Optionally, the extraction conditions include: shaking or ultrasound, and the time is 5 minutes to 10 minutes.

[0030] In some embodiments of the present application, the step of preparing the methanol extract further comprises the following steps: collecting the lower layer after phase separation, adding methanol to repeat the extraction, and combining the methanol extract obtained in the second extraction with the methanol extract obtained in the previous extraction to prepare the test solution;

[0031] Optionally, the number of repeated extractions includes 1 to 3 times;

[0032] Optionally, the volume ratio of the contents to the methanol used in each repeated extraction is (2-10): (20-100); further optionally, the volume ratio of the contents to the methanol used in each repeated extraction is (2-5): (20-50).

[0033] In some embodiments of the present application, the upper methanol extract is collected by centrifugation;

[0034] Optionally, the centrifugal conditions include: a rotation speed of 5000 rpm to 8000 rpm and a time of 5 min to 10 min.

[0035] In some embodiments of the present application, the steps of preparing the reconstituted solution include:

[0036] The methanol extract is subjected to a first rotary evaporation to dryness, methanol is added for a first redissolution, the extract is allowed to stand, the matrix component remaining at the bottom is removed by suction, the remaining portion is subjected to a second rotary evaporation to dryness, and methanol is added for a second redissolution to prepare the reconstituted solution;

[0037] Optionally, the volume of methanol used for the first reconstitution corresponding to 2 mL to 10 mL of the contents is 150 mL to 250 mL;

[0038] Optionally, the conditions for the first rotary evaporation and the second rotary evaporation each independently include: reduced pressure, a temperature of 35°C to 45°C, and a rotation speed of 30 rpm to 100 rpm; further optionally, the conditions for the first rotary evaporation and the second rotary evaporation each independently include: reduced pressure, a temperature of 35°C to 40°C, and a rotation speed of 30 rpm to 90 rpm;

[0039] Optionally, the volume of methanol used for the second reconstitution corresponding to 2 mL to 10 mL of the content is 1.5 mL to 2.5 mL.

[0040] In some embodiments of the present application, the reverse phase chromatography method satisfies one or more of the following conditions:

[0041] (A) The detector is a UV detector;

[0042] (B) a liquid chromatography column with octadecylsilane-bonded silica gel as the filler, which is optionally Triart C18, 4.6 mm x 150 mm, 5.0 μm, or Welch Ultimate XB-C18, 150 mm x 4.6 mm, 5 μm;

[0043] (C) a detection wavelength of 260 nm to 270 nm, optionally 265 nm;

[0044] (D) a column temperature of 28°C to 32°C, optionally 30°C;

[0045] (E) an injection tray temperature of 2°C to 10°C, optionally 4°C to 10°C;

[0046] (F) a flow rate of 0.5 mL / min to 1.5 mL / min, optionally 1.0 mL / min;

[0047] (G) a mobile phase of methanol, acetonitrile and water in a volume ratio of (49-51):(49-51):(1-3), optionally, the volume ratio of methanol, acetonitrile and water in the mobile phase is 50:50:2;

[0048] (H) an injection volume of 100 μL to 500 μL; and,

[0049] (I) isocratic elution for 25 min to 35 min, optionally 30 min.

[0050] In some embodiments of the present application, the determination method comprises: performing a third rotary evaporation to dryness on the effluent component, and after redissolving with isooctane, performing normal phase chromatography detection;

[0051] Optionally, the third rotary evaporation conditions comprise: reduced pressure, a temperature of 35°C to 45°C, and a rotation speed of 30 rpm to 100 rpm; further optionally, the third rotary evaporation conditions comprise: reduced pressure, a temperature of 35°C to 40°C, and a rotation speed of 30 rpm to 90 rpm.

[0052] Optionally, the volume of isooctane corresponding to 2 mL to 10 mL of the content is 0.5 mL to 1.5 mL.

[0053] In some embodiments of the present application, the normal phase chromatography satisfies one or more of the following conditions:

[0054] A) the detector is an ultraviolet detector;

[0055] B) a liquid chromatography column with porous silica gel particles as the filler, which is optionally Topsil ®Silica, 4.6mm×250mm, 5.0μm, or Supersil SiO2, 4.6mm×250mm, 5.0μm;

[0056] C) The detection wavelength is 260nm~270nm, optionally 265nm;

[0057] D) Column temperature is 25-35°C, optionally 30°C;

[0058] E) The sample tray temperature is 2°C to 10°C, and can be set to 4°C.

[0059] F) Flow rate of 1.5 mL / min to 2.0 mL / min, optionally 1.7 mL / min;

[0060] G) The mobile phase is a mixture of n-pentanol and n-hexane in a volume ratio of (2-5): (998-995), or alternatively a mixture of n-pentanol and n-hexane in a volume ratio of (2-3): (998-997);

[0061] H) The injection volume is 150 μL to 250 μL, optionally 200 μL;

[0062] I) Isocratic elution for not less than 40 min, optionally 40 min to 50 min.

[0063] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0065] Figure 1 This is the detection spectrum of the reference solution in Example 1.

[0066] Figure 2 This is the test sample solution detection spectrum in Example 1.

[0067] Figure 3 This is the recovery investigation solution detection spectrum in Example 1.

[0068] Figure 4 This is the detection spectrum of the blank excipient solution in Example 1.

[0069] Figure 5This is the detection spectrum of the reference solution in Example 2.

[0070] Figure 6 This is the test sample solution detection spectrum in Example 2.

[0071] Figure 7 This is the recovery investigation solution detection spectrum in Example 2.

[0072] Figure 8 This is the detection spectrum of the blank excipient solution in Example 2.

[0073] Figure 9 This is the test pattern of the test solution in Comparative Example 2. DETAILED DESCRIPTION

[0074] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0076] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0077] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0078] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0079] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0080] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0081] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0082] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0083] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0084] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0085] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0086] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional values ​​within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two numerical endpoints. In this document, this is equivalent to directly listing each integer. For example, "t is an integer selected from 1 to 10" means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges may be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0087] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0088] In this application, %(w / w) and wt% both refer to weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0089] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0090] One or more embodiments of the present application provide a method for determining related substances in a vitamin D3-containing drop, the method comprising:

[0091] Take the contents of the drop to be tested and prepare the test solution;

[0092] The test solution was tested by reverse phase chromatography, and the effluent components with a retention time of 11.5 min to 15.5 min were collected; and

[0093] The effluent components are detected by normal phase chromatography to determine the related substances in the contents;

[0094] The related substances include one or more of (5E,7E)-9,10-opened cholesteryl-5,7,10(19)-triene-3β-ol, (6E)-9,10-opened cholesteryl-5(10),6,8-triene-3β-ol and 9,10-opened cholesteryl-6,8,10(5)-triene-3β-ol;

[0095] Wherein, the preparation steps of the test solution include:

[0096] The contents were mixed, extracted with acetone and methanol, phase separated, and the upper methanol extract was collected;

[0097] removing the matrix components and solvent of the contents remaining in the methanol extract, and re-dissolving the contents with methanol to prepare a reconstituted solution; and

[0098] The reconstituted solution and sodium methoxide reagent are mixed to carry out saponification reaction to prepare the test solution.

[0099] Drops are liquid preparations that are taken orally to supplement specific vitamins. They are primarily used to correct vitamin deficiencies or meet the nutritional needs of special populations (such as infants, pregnant women, and individuals with malabsorption disorders). The liquid portion of such a liquid preparation is referred to herein as the "content." For example, in the example of the vitamin D drops (capsules), the "content" is the liquid portion of the capsule.

[0100] The present application does not particularly limit the vitamin D3-containing drops. In the embodiments, the technical solution is explained using vitamin D drops (capsule type) as the drops to be tested. It can be understood that the vitamin D3-containing drops applicable to the method of the present application are not limited to the vitamin D drops of specific manufacturers and models mentioned in the embodiments.

[0101] The matrix component of the vitamin D3 drops is vegetable oil. When determining the relevant substances (impurity A, impurity B, and impurity C), the contents cannot be directly prepared into a solution and then subjected to a reverse-phase chromatography system or a normal-phase chromatography system to determine the impurity content, because vegetable oil will significantly interfere with the determination of each impurity component. First, the impurities in the vegetable oil matrix must be extracted, and after the extract is purified by a reverse-phase chromatography system, the effluent components are collected, and then the content of each impurity is determined under a normal-phase chromatography system.

[0102] Because methanol and vegetable oil have significantly different polarities, they are immiscible. However, methanol and acetone are miscible, and the distribution ratio of acetone in the methanol phase is much greater than that in the vegetable oil. Therefore, potential impurities extracted in acetone can be better transferred to the methanol extract. In this application, the addition of acetone during the extraction can increase the solubility of potential impurities A, B, and C. Furthermore, acetone is miscible with the vegetable oil contained in the vitamin D3 drops, making it easier to extract potential impurities from the vegetable oil.

[0103] Because trace amounts of residual vegetable oil remain in the reconstituted solution, they can affect the separation of potential impurities in a reversed-phase chromatography system and, in turn, interfere with the determination of the content of various impurity components in a normal-phase system. Therefore, the present application provides a technique for significantly reducing residual vegetable oil in the reconstituted solution. Specifically, sodium methoxide, a strong alkaline reagent, is precisely added to the reconstituted solution to hydrolyze and saponify the residual vegetable oil.

[0104] In some embodiments of the present application, the volume ratio of the contents, acetone and methanol is (2-10): (2-10): (20-100), for example, 2:2:20, 2:5:50, 2:10:100, 2:2:20, 2:2:50, 2:2:100, 2:5:20, 2:5:50, 2:5:100, 2:10:20, 2:10:50, 2:10:100, 5:2:20, 5:5:50, 5 ... :2:20, 5:2:50, 5:2:100, 5:5:20, 5:5:50, 5:5:100, 5:10:20, 5:10:50, 5:10:100, 10:2:20, 10:5:50, 10:10:100, 10:2:20, 10:2:50, 10:2:100, 10:5:20, 10:5:50, 10:5:100, 10:10:20, 10:10:50, 10:10:100. Optionally, the volume ratio of the contents, acetone and methanol is (2-5):(2-5):(20-50).

[0105] In some embodiments of the present application, the concentration of sodium methoxide in the sodium methoxide reagent is 2 mol / L~5 mol / L (for example, 2, 2.5, 3, 3.5, 4, 4.5 mol / L), and the volume of the sodium methoxide reagent corresponding to each 2 mL~10 mL of the content is 0.5 mL~1.5 mL (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mL).

[0106] In some embodiments of the present application, the contents and the acetone are first mixed to prepare a mixture, and then the methanol is added to the mixture for extraction;

[0107] Optionally, the mixing conditions include: shaking or ultrasonication for 5 min to 10 min, for example, 5, 6, 7, 8, 9, or 10 min;

[0108] Optionally, the extraction conditions include: shaking or ultrasound, and the time is 5 minutes to 10 minutes, for example, 5, 6, 7, 8, 9, or 10 minutes.

[0109] In some embodiments of the present application, the step of preparing the methanol extract further comprises the following steps: collecting the lower layer after phase separation, adding methanol to repeat the extraction, and combining the methanol extract obtained in the second extraction with the methanol extract obtained in the previous extraction to prepare the test solution;

[0110] Optionally, the number of repeated extractions includes 1 to 3 times, for example, 1, 2, or 3 times;

[0111] Optionally, the volume ratio of the contents to the methanol used in each repeated extraction is (2-10):(20-100), for example, 2:20, 2:50, 2:100, 5:20, 5:50, 5:100, 10:20, 10:50, 10:100. Further optionally, the volume ratio of the contents to the methanol used in each repeated extraction is (2-5):(20-50).

[0112] In some embodiments of the present application, the upper methanol extract is collected by centrifugation;

[0113] Optionally, the centrifugation conditions include: a rotation speed of 5000 rpm to 8000 rpm (for example, 5000, 5500, 6000, 6500, 7000, 7500, 8000 rpm), and a time of 5 min to 10 min (for example, 5, 6, 7, 8, 9, 10 min).

[0114] In some embodiments of the present application, the steps of preparing the reconstituted solution include:

[0115] The methanol extract is subjected to a first rotary evaporation to dryness, methanol is added for a first redissolution, the extract is allowed to stand, the matrix component remaining at the bottom is removed by suction, the remaining portion is subjected to a second rotary evaporation to dryness, and methanol is added for a second redissolution to prepare the reconstituted solution;

[0116] Optionally, the volume of methanol used for the first reconstitution corresponding to 2 mL to 10 mL of the content is 150 mL to 250 mL (for example, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 mL).

[0117] Optionally, the conditions for the first rotary evaporation and the second rotary evaporation each independently include: reduced pressure, a temperature of 35°C to 45°C (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C), and a rotation speed of 30 rpm to 100 rpm (for example, 30, 40, 50, 60, 70, 80, 90, or 100 rpm); further optionally, the conditions for the first rotary evaporation and the second rotary evaporation each independently include: reduced pressure, a temperature of 35°C to 40°C, and a rotation speed of 30 rpm to 90 rpm;

[0118] Optionally, the volume of methanol used for the second reconstitution corresponding to 2 mL to 10 mL of the content is 1.5 mL to 2.5 mL (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mL).

[0119] In some embodiments of the present application, the reverse phase chromatography method satisfies one or more of the following conditions:

[0120] (A) The detector is a UV detector;

[0121] (B) a liquid chromatography column filled with octadecylsilane bonded silica gel, optionally a Triart C18 column, 4.6 mm × 150 mm, 5.0 μm, or a Welch Ultimate XB-C18 column, 150 mm × 4.6 mm, 5 μm;

[0122] (C) The detection wavelength is 260 nm to 270 nm (for example, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270 nm), and optionally 265 nm;

[0123] (D) The column temperature is 28°C to 32°C (e.g., 28, 29, 30, 31, 32°C), optionally 30°C;

[0124] (E) The sample tray temperature is 2°C to 10°C (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10°C), optionally 4°C to 10°C;

[0125] (F) a flow rate of 0.5 mL / min to 1.5 mL / min (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mL / min), optionally 1.0 mL / min;

[0126] (G) The mobile phase is methanol, acetonitrile and water in a volume ratio of (49-51):(49-51):(1-3), for example, the volume ratio of methanol, acetonitrile and water is 49:49:1, 49:49:2, 49:49:3, 49:50:1, 49:50:2, 49:50:3, 49:51:1, 49:51:2, 49:51:3, 51:49:1, 51:49:2, 51:49:3, 51:50:1, 51:50:2, 51:50:3, 51:51:1, 51:51:2, 51:51:3. Optionally, the volume ratio of methanol, acetonitrile and water in the mobile phase is 50:50:2.

[0127] (H) injection volume of 100 μL to 500 μL (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500 μL); and,

[0128] (I) Isocratic elution for 25 min to 35 min (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 min), optionally for 30 min.

[0129] In some embodiments of the present application, the determination method comprises: performing a third rotary evaporation on the effluent component to dryness, adding isooctane for redissolution, and then loading the sample for normal phase chromatography detection;

[0130] Optionally, the conditions for the third rotary evaporation include: reduced pressure, a temperature of 35° C. to 45° C. (for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C.), and a rotation speed of 30 rpm to 100 rpm (for example, 30, 40, 50, 60, 70, 80, 90, or 100 rpm); further optionally, the conditions for the third rotary evaporation include: reduced pressure, a temperature of 35° C. to 40° C., and a rotation speed of 30 rpm to 90 rpm;

[0131] Optionally, the volume of isooctane corresponding to 2 mL to 10 mL of the content is 0.5 mL to 1.5 mL (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 mL).

[0132] In some embodiments of the present application, the normal phase chromatography satisfies one or more of the following conditions:

[0133] A) The detector is a UV detector;

[0134] B) Liquid chromatography column with porous silica particles as filler, optionally Topsil ® Silica, 4.6mm×250mm, 5.0μm, or Supersil SiO2, 4.6mm×250mm, 5.0μm;

[0135] C) the detection wavelength is 260 nm to 270 nm (e.g., 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270 nm), optionally 265 nm;

[0136] D) the column temperature is 25-35°C (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35°C), optionally 30°C;

[0137] E) The sample tray temperature is 2°C to 10°C (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10°C), optionally 4°C;

[0138] F) a flow rate of 1.5 mL / min to 2.0 mL / min (e.g., 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 mL / min), optionally 1.7 mL / min;

[0139] G) the mobile phase is n-pentanol and n-hexane in a volume ratio of (2-5): (998-995), for example, 2:998, 3:997, 4:996, or 5:995, and optionally n-pentanol-n-hexane in a volume ratio of (2-3): (998-997);

[0140] H) the injection volume is 150 μL to 250 μL (e.g., 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 μL), optionally 200 μL;

[0141] I) isocratic elution for not less than 40 min (e.g., 40, 45, 50, 55, or 60 min), optionally 40 to 50 min.

[0142] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0143] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods without specific conditions specified in the examples were carried out according to conventional conditions, such as those described in literature or books or methods recommended by manufacturers.

[0144] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0145] The reagents used in this application are:

[0146] Methanol: HPLC grade, Honeywell International;

[0147] Acetone: HPLC grade, Honeywell International;

[0148] Sodium methoxide: AR grade, Shanghai MacLean Biochemical Technology Co., Ltd.;

[0149] Isooctane: HPLC grade, Shanghai Yuanmu Biotechnology Co., Ltd.;

[0150] Acetonitrile: HPLC grade, Honeywell International;

[0151] n-Pentanol: HPLC grade, Honeywell International;

[0152] n-Hexane: HPLC grade, Honeywell International;

[0153] Vegetable oil: peanut oil, injection grade, Hubei Gedian Renfu Pharmaceutical Excipients Co., Ltd.; registration number: F20180000683; batch number: F125C240801;

[0154] Impurity A, Impurity B, and Impurity C: CATO Research Chemicals Inc.;

[0155] Vitamin D drops (capsule type): Shandong Dayin Marine Biopharmaceutical Co., Ltd.; Approval number: National Medicine Standard H20183354; Product batch number: 240401.

[0156] The structures of impurities A, B and C to be determined are as follows:

[0157] Impurity A:

[0158]

[0159] (5E,7E)-9,10-opened cholesteryl-5,7,10(19)-trien-3β-ol, C 27 H 44 O, 384.65.

[0160] Impurity B:

[0161]

[0162] (6E)-9,10-opened cholester-5(10),6,8-trien-3β-ol, C 27 H 44 O, 384.65.

[0163] Impurity C:

[0164] 9,10-opened cholesteryl-6,8,10(5)-trien-3β-ol, C 27 H 44 O, 384.65.

[0165] Example 1:

[0166] 1. Preparation of each solution

[0167] 1.1 Preparation of test solution for reversed-phase system determination

[0168] (1) Preparation of methanol extract of the test sample: Take 5 mL of the content of vitamin D drops, place it in a 250 mL conical flask, add 5 mL of acetone solvent, ultrasonicate for 10 minutes, mix thoroughly, then add 50 mL of methanol solvent to the conical flask, ultrasonicate for 10 minutes for extraction, transfer the entire amount of extract to a centrifuge tube, centrifuge at 8000 rpm for 10 minutes, carefully aspirate the upper methanol extract phase with a pipette, and repeat the extraction of the lower plant oil phase with 50 mL of methanol solvent twice, combine the methanol extracts, and obtain.

[0169] (2) Preparation of test solution for reverse phase system determination: Place the methanol extract in a round-bottom flask and evaporate it to dryness under reduced pressure at 40°C with the rotary evaporator speed controlled at 90 rpm. Then add 200 mL of methanol solvent to the round-bottom flask for re-dissolution. After the re-solution is allowed to stand, use a pipette to carefully remove the matrix components (vegetable oil) of the vitamin D drops remaining at the bottom of the flask. Evaporate it to dryness under reduced pressure again at 40°C with the rotary evaporator speed controlled at 90 rpm. Cool the flask to room temperature, accurately add 2 mL of methanol reagent for re-dissolution, and accurately add 1 mL of 5 mol / L sodium methoxide solution to the above methanol re-solution. Thoroughly saponify and mix.

[0170] 1.2 Preparation of blank excipient solution for reversed-phase system determination

[0171] Take 5 mL of vegetable oil, place it in a 250 mL conical flask, add 5 mL of acetone solvent, ultrasonicate for 10 minutes, mix thoroughly, then add 50 mL of methanol solvent to the conical flask, ultrasonicate for 10 minutes for extraction, transfer the entire extract to a centrifuge tube, centrifuge at 8000 rpm for 10 minutes, carefully aspirate the upper methanol extract phase with a pipette, and repeat the extraction twice with 50 mL of methanol solvent in the lower layer of vegetable oil, and combine the methanol extracts; place the methanol extract in a round-bottom flask, and evaporate it under reduced pressure at 40 ° C to dryness, and control the rotary evaporator speed to 90 rpm. Then, add 200 mL of methanol solvent to the round-bottom flask for re-dissolution. After the re-solution is allowed to stand, use a pipette to carefully remove the vegetable oil remaining at the bottom of the flask, and evaporate it under reduced pressure at 40 ° C again to dryness. Cool the flask to room temperature, accurately add 2 mL of methanol reagent for re-dissolution, and accurately add 1 mL of 5 mol / L sodium methoxide solution to the above methanol re-solution, fully saponify and mix.

[0172] 1.3 Preparation of impurity reference solution for reversed-phase system determination

[0173] (1) Preparation of impurity reference stock solution: Take appropriate amounts of impurity A, impurity B, and impurity C, dissolve them in acetone, and quantitatively dilute them to make a 10 μg / mL solution.

[0174] (2) Preparation of impurity reference solution for reverse phase system determination: Take 5 mL of vegetable oil and place it in a 250 mL conical flask. Then add an appropriate amount of impurity reference stock solution to make the concentration of the above impurities 1 μg / mL (0.5%). Add 5 mL of acetone solvent, sonicate for 10 minutes, mix thoroughly, then add 50 mL of methanol solvent to the conical flask and sonicate for 10 minutes for extraction. Transfer the entire amount of extract to a centrifuge tube and centrifuge at 8000 rpm for 10 minutes. Carefully aspirate the upper methanol extract phase with a pipette, and the lower vegetable oil phase with 50 The extraction was repeated twice with 1 mL of methanol solvent, and the methanol extracts were combined; the methanol extract was placed in a round-bottom flask, and vacuum-evaporated to dryness at 40°C with the rotary evaporator speed controlled at 90 rpm; then 200 mL of methanol solvent was added to the round-bottom flask for re-dissolution; after the reconstituted solution was allowed to stand, the vegetable oil remaining at the bottom of the flask was carefully aspirated with a pipette; the solution was again vacuum-evaporated to dryness at 40°C; the flask was cooled to room temperature, 2 mL of methanol reagent was accurately added for re-dissolution, and 1 mL of 5 mol / L sodium methoxide solution was accurately added to the above methanol reconstituted solution, and the mixture was fully saponified and mixed to obtain the product.

[0175] 1.4 Preparation of Recovery Evaluation Solution for Reversed-Phase System Determination

[0176] Take 5mL of the contents of vitamin D drops and place it in a 250mL conical flask. Then add an appropriate amount of impurity reference stock solution to make the concentration of the above impurities 1μg / mL (0.5%). Then add 5mL of acetone solvent, ultrasonicate for 10 minutes, mix thoroughly, then add 50mL of methanol solvent to the conical flask and ultrasonicate for 10 minutes for extraction. Transfer the entire amount of the extract to a centrifuge tube, centrifuge at 8000rpm for 10 minutes, carefully draw out the upper methanol extract phase with a pipette, and then add 50mL of methanol solvent to the lower plant oil phase. Repeat the extraction twice, combining the methanol extracts. Place the methanol extract in a round-bottom flask and evaporate to dryness under reduced pressure at 40°C (90 rpm). Then, add 200 mL of methanol to the round-bottom flask for reconstitution. After the reconstituted solution has settled, carefully remove any remaining vegetable oil from the bottom of the flask with a pipette. Evaporate again under reduced pressure at 40°C for reconstitution. Cool the flask to room temperature, then accurately add 2 mL of methanol to reconstitute the solution. Add 1 mL of 5 mol / L sodium methoxide solution to the methanol solution, saponify thoroughly, and mix thoroughly. Prepare six replicates.

[0177] 2. Reverse Phase System Assay

[0178] Take 500 μL each of the test solution for reverse phase system determination, blank excipient solution for reverse phase system determination, impurity reference solution for reverse phase system determination, and recovery rate investigation solution for reverse phase system determination, inject them into reverse phase chromatography for separation, and collect the eluting components with a retention time of 11.5 min to 15.5 min. The detection conditions of the reverse phase chromatography system are:

[0179] The detector is an ultraviolet detector;

[0180] Column: Triart C18, 4.6 mm × 150 mm, 5.0 μm;

[0181] The detection wavelength is 265nm;

[0182] The column temperature was 30°C;

[0183] Sample tray 4℃;

[0184] The flow rate was 1.0 mL / min;

[0185] The mobile phase ratio was methanol-acetonitrile-water (50:50:2 by volume);

[0186] The injection volume was 500 μL;

[0187] Isocratic elution was performed for 30 min.

[0188] 3. Normal Phase System Assay

[0189] The effluent components of the impurity reference solution, test solution, blank excipient solution, and recovery investigation solution were respectively evaporated to dryness under reduced pressure at 40°C, with the rotary evaporator speed at 90 rpm. The dried round-bottom flask was cooled to room temperature, and then 1 mL of isooctane was accurately added to dissolve it. The solution was dissolved in a 25°C water bath for more than 1 minute. 200 μL of each isooctane solution was accurately measured and injected into the normal phase chromatography system for determination. The detection conditions of the normal phase chromatography system were as follows:

[0190] The detector is an ultraviolet detector;

[0191] The chromatographic column is Topsil ® Silica, 4.6mm×250mm, 5.0μm;

[0192] The detection wavelength is 265nm;

[0193] Column temperature 30°C;

[0194] The injection plate temperature was 4°C;

[0195] The flow rate was 1.7 mL / min;

[0196] The mobile phase was n -pentanol-n -hexane (3:997) (v / v);

[0197] The injection volume was 200 μL;

[0198] Isocratic elution was performed for 40 min.

[0199] 4. Results

[0200] The determination spectrum of the reference solution is shown in Figure 1 As shown, the retention time of impurity A is 10.756 min, the retention time of impurity C is 11.561 min, and the retention time of impurity B is 21.529 min. The separation degrees between the three impurities are all greater than 1.5, achieving baseline separation.

[0201] The determination spectra of the test solution and the recovery investigation solution (spiked test solution) are shown in Figure 2 and Figure 3 As shown in the figure, impurity A, impurity C and impurity B peaks can be detected in the test solution, and the positions of the impurity peaks are consistent with those in the recovery investigation solution (spiked test solution). The three impurity peaks are not interfered by the main peak of vitamin D. Among them, the impurity B peak closest to the main peak of vitamin D has a separation degree of 1.8 from the main peak, achieving baseline separation.

[0202] The determination spectrum of blank excipient solution is shown in Figure 4 As shown in the blank excipient solution spectrum, there are no interfering peaks at the positions of impurity A, impurity C, impurity B and the main peak of vitamin D, indicating that the method has good specificity.

[0203] The recoveries of impurity A, impurity B and impurity C in 6 recovery investigation solutions were calculated according to the following formula. The results are shown in Table 1.

[0204] Table 1. Results of the recovery study solution in Example 1

[0205]

[0206] Where:

[0207] M 测 : Recovery rate refers to the measured amount of impurity A, impurity B and impurity C in the solution, μg;

[0208] M 本 : Background amount of impurity A, impurity B and impurity C in the test solution, μg;

[0209] M 理 : The recovery rate is based on the theoretical amount of impurity A, impurity B and impurity C added to the solution, μg.

[0210] As can be seen from the test results in Table 1, the recoveries of impurities A, B and C in the six recovery investigation solutions prepared in parallel ranged from 94.5% to 97.2%, 97.8% to 105.3% and 96.5% to 99.1%, respectively, all meeting the range requirement of 90% to 108% (requirements of the Guidelines for Validation of Analytical Methods of the Chinese Pharmacopoeia). The RSDs of the recoveries were 1.6%, 2.9% and 1.0%, respectively, all less than 5%, indicating that the accuracy and repeatability of the determination of impurities A, B and C were good.

[0211] Example 2:

[0212] 1. Preparation of each solution

[0213] 1.1 Preparation of test solution for reversed-phase system determination

[0214] (1) Preparation of methanol extract of the test sample: Take 2 mL of the content of vitamin D drops, place it in a 250 mL conical flask, add 2 mL of acetone solvent, ultrasonicate for 5 minutes, mix thoroughly, then add 20 mL of methanol solvent to the conical flask, ultrasonicate for 5 minutes for extraction, transfer the entire amount of the extract to a centrifuge tube, centrifuge at 5000 rpm for 5 minutes, carefully aspirate the upper methanol extract phase with a pipette, and repeat the extraction of the lower plant oil phase with 20 mL of methanol solvent twice, and combine the methanol extracts to obtain the product.

[0215] (2) Preparation of test solution for reverse phase system determination: Place the methanol extract in a round-bottom flask and evaporate it to dryness under reduced pressure at 35°C with the rotary evaporator speed controlled at 30 rpm. Then add 200 mL of methanol solvent to the round-bottom flask for redissolution. After the redissolution is allowed to stand, use a pipette to carefully remove the matrix components (vegetable oil) of the vitamin D drops remaining at the bottom of the flask. Evaporate it to dryness under reduced pressure at 35°C again with the rotary evaporator speed controlled at 30 rpm. Cool the flask to room temperature, accurately add 2 mL of methanol reagent for redissolution, and accurately add 1 mL of 5 mol / L sodium methoxide solution to the above methanol redissolution. Thoroughly saponify and mix.

[0216] 1.2 Preparation of blank excipient solution for reversed-phase system determination

[0217] Take 2 mL of vegetable oil, place it in a 250 mL conical flask, add 2 mL of acetone solvent, ultrasonicate for 5 minutes, mix thoroughly, then add 20 mL of methanol solvent to the conical flask, ultrasonicate for 5 minutes for extraction, transfer the entire extract to a centrifuge tube, centrifuge at 5000 rpm for 5 minutes, carefully aspirate the upper methanol extract phase with a pipette, and repeat the extraction twice with 20 mL of methanol solvent in the lower layer of vegetable oil, and combine the methanol extracts; place the methanol extract in a round-bottom flask, and evaporate it under reduced pressure at 35 ° C to dryness, and control the rotary evaporator speed to 30 rpm. Then, add 200 mL of methanol solvent to the round-bottom flask for re-dissolution. After the re-solution is allowed to stand, use a pipette to carefully remove the vegetable oil remaining at the bottom of the flask, and evaporate it under reduced pressure at 35 ° C again to dryness. Cool the flask to room temperature, accurately add 2 mL of methanol reagent for re-dissolution, and accurately add 1 mL of 5 mol / L sodium methoxide solution to the above methanol re-solution, fully saponify and mix.

[0218] 1.3 Preparation of impurity reference solution for reversed-phase system determination

[0219] (1) Preparation of impurity reference stock solution: Take appropriate amounts of impurity A, impurity B, and impurity C, dissolve them in acetone, and quantitatively dilute them to make a 10 μg / mL solution.

[0220] (2) Preparation of impurity reference solution for reverse phase system determination: Take 2 mL of vegetable oil and place it in a 250 mL conical flask. Then add an appropriate amount of impurity reference stock solution to make the concentration of the above impurities 1 μg / mL (0.5%). Add 2 mL of acetone solvent, sonicate for 5 minutes, mix thoroughly, then add 20 mL of methanol solvent to the conical flask and sonicate for 5 minutes for extraction. Transfer the entire amount of extract to a centrifuge tube and centrifuge at 5000 rpm for 5 minutes. Carefully aspirate the upper methanol extraction liquid phase with a pipette, and then use 20 mL of acetone solvent to extract the vegetable oil phase. The extraction was repeated twice with 1 L of methanol solvent, and the methanol extracts were combined; the methanol extract was placed in a round-bottom flask, and vacuum-evaporated at 35°C to dryness with the rotary evaporator speed controlled at 30 rpm, and then 200 mL of methanol solvent was added to the round-bottom flask for re-dissolution. After the re-solution was allowed to stand, the vegetable oil remaining at the bottom of the flask was carefully aspirated with a pipette, and vacuum-evaporated at 35°C again to dryness. The flask was cooled to room temperature, and 2 mL of methanol reagent was accurately added for re-dissolution. 1 mL of 5 mol / L sodium methoxide solution was accurately added to the above methanol re-solution, and the mixture was fully saponified and mixed to obtain the product.

[0221] 1.4 Preparation of Recovery Evaluation Solution for Reversed-Phase System Determination

[0222] Take 2mL of the contents of vitamin D drops and place it in a 250mL conical flask. Then add an appropriate amount of impurity reference stock solution to make the concentration of the above impurities 1μg / mL (0.5%). Then add 2mL of acetone solvent, ultrasonicate for 5min, mix thoroughly, then add 20mL of methanol solvent to the conical flask, ultrasonicate for 5min for extraction, transfer the entire amount of the extract to a centrifuge tube, centrifuge at 5000rpm for 5min, carefully aspirate the upper methanol extract phase with a pipette, and resuspend the lower plant oil phase with 20mL of methanol solvent. Repeat the extraction twice, combining the methanol extracts. Place the methanol extract in a round-bottom flask and evaporate to dryness under reduced pressure at 35°C using a rotary evaporator at 30 rpm. Then, add 200 mL of methanol to the round-bottom flask for reconstitution. After the reconstituted solution has settled, carefully remove any remaining vegetable oil from the bottom of the flask using a pipette. Evaporate again under reduced pressure at 35°C for reconstitution. Cool the flask to room temperature, then accurately add 2 mL of methanol to reconstitute the solution. Add 1 mL of 5 mol / L sodium methoxide solution to the methanol solution, saponify thoroughly, and mix thoroughly. Prepare six replicates.

[0223] 2. Inverted system detection

[0224] Take 500 μl each of the test solution for reverse phase system determination, blank excipient solution for reverse phase system determination, impurity reference solution for reverse phase system determination, and recovery rate investigation solution for reverse phase system determination, inject them into reverse phase chromatography for separation, and collect the eluting components with a retention time of 11.5 min to 15.5 min. The detection conditions of the reverse phase chromatography system are:

[0225] The detector is an ultraviolet detector;

[0226] Column: Triart C18, 4.6 mm × 150 mm, 5.0 μm;

[0227] The detection wavelength is 265nm;

[0228] The column temperature was 30°C;

[0229] Sample tray 10°C;

[0230] The flow rate was 1.0 mL / min;

[0231] The mobile phase ratio was methanol-acetonitrile-water (50:50:2 by volume);

[0232] The injection volume was 500 μL;

[0233] Isocratic elution was performed for 30 min.

[0234] 3. Normal Phase System Detection

[0235] The effluent components of the impurity reference solution, test solution, blank excipient solution, and recovery investigation solution were respectively evaporated to dryness under reduced pressure at 35°C, with the rotary evaporator speed at 30 rpm. The dried round-bottom flask was cooled to room temperature, and then 1 mL of isooctane was accurately added and shaken to reconstitute. 200 μL of each of the above isooctane solutions was accurately measured and injected into the normal phase chromatography system for determination. The detection conditions of the normal phase chromatography system were as follows:

[0236] The detector is an ultraviolet detector;

[0237] The chromatographic column is Topsil ® Silica, 4.6mm×250mm, 5.0μm;

[0238] The detection wavelength is 265nm;

[0239] Column temperature 25°C;

[0240] The injection plate temperature was 10°C;

[0241] The flow rate was 1.5 mL / min;

[0242] The mobile phase was n -pentanol-n -hexane (2:998) (v / v);

[0243] The injection volume was 200 μL;

[0244] Isocratic elution was performed for 50 min.

[0245] 4. Results

[0246] The determination spectrum of the reference solution is shown in Figure 5 As shown, the retention time of impurity A is 11.273 min, the retention time of impurity C is 12.158 min, and the retention time of impurity B is 22.882 min. The separation degrees between the three impurities are all greater than 1.5, achieving baseline separation.

[0247] The determination spectra of the test solution and the recovery investigation solution (spiked test solution) are shown in Figure 6 and Figure 7 As shown in the figure, impurity A, impurity C and impurity B peaks can be detected in the test solution, and the positions of the impurity peaks are consistent with those in the recovery investigation solution (spiked test solution). The three impurity peaks are not interfered by the main peak of vitamin D. Among them, the impurity B peak closest to the main peak of vitamin D has a separation degree of 1.9 from the main peak, achieving baseline separation.

[0248] The determination spectrum of blank excipient solution is shown in Figure 8 As shown in the blank excipient solution spectrum, there are no interfering peaks at the positions of impurity A, impurity C, impurity B and the main peak of vitamin D, indicating that the method has good specificity.

[0249] The recoveries of impurity A, impurity B, and impurity C in the six recovery investigation solutions were calculated according to the following formula. The results are shown in Table 2.

[0250]

[0251] Where:

[0252] M 测 : Recovery rate refers to the measured amount of impurity A, impurity B and impurity C in the solution, μg;

[0253] M 本 : Background amount of impurity A, impurity B and impurity C in the test solution, μg;

[0254] M 理 : The recovery rate is based on the theoretical amount of impurity A, impurity B and impurity C added to the solution, μg.

[0255] It can be seen from the test results in Table 2 that the recoveries of impurities A, B and C in the six recovery investigation solutions prepared in parallel ranged from 95.5% to 98.6%, 100.6% to 104.7% and 94.1% to 99.7%, respectively, all meeting the range requirement of 90% to 108% (requirements of the Guidelines for Validation of Analytical Methods of the Chinese Pharmacopoeia). The RSDs of the recoveries were 1.2%, 2.7% and 1.5%, respectively, all less than 5%, indicating that the accuracy and repeatability of the determination of impurities A, B and C were good.

[0256] Table 2. Recovery rate determination results of solution in Example 2

[0257]

[0258] Comparative Example 1:

[0259] This comparative example is a comparative example of Example 1, and mainly comprises the following steps:

[0260] 1. Preparation of each solution

[0261] 1.1 Preparation of test solution ①

[0262] (1) Preparation of methanol extract of the test sample ① (no acetone is added to the extract): Take 5 mL of the contents of vitamin D drops, place it in a 250 mL conical flask, add 50 mL of methanol solvent, and extract by ultrasonication for 10 minutes. Transfer the entire amount of the extract to a centrifuge tube, centrifuge at 8000 rpm for 10 minutes, carefully aspirate the upper methanol extract phase with a pipette, and repeat the extraction of the lower plant oil phase with 50 mL of methanol solvent twice. Combine the methanol extracts to obtain the product.

[0263] (2) Preparation of test solution ① for reverse phase system determination: Place the methanol extract in a round-bottom flask and evaporate it to dryness under reduced pressure at 40°C with the rotary evaporator speed controlled at 90 rpm. Then add 200 mL of methanol solvent to the round-bottom flask for redissolution. After the redissolution is allowed to stand, use a pipette to carefully remove the matrix components (vegetable oil) of the vitamin D drops remaining at the bottom of the flask. Evaporate it to dryness under reduced pressure again at 40°C with the rotary evaporator speed controlled at 90 rpm. Cool the flask to room temperature, accurately add 2 mL of methanol reagent for redissolution, and accurately add 1 mL of 5 mol / L sodium methoxide solution to the above methanol redissolution. Thoroughly saponify and mix.

[0264] 1.2 Preparation of test solution ②

[0265] (1) Preparation of methanol-water (90:10) extract ② of the test sample (the extract was changed to a methanol-water mixture, and acetone was not added): 5 mL of the vitamin D drops were placed in a 250 mL conical flask, and 50 mL of a methanol-water (90:10 by volume) mixed solvent was added. The extract was extracted by ultrasonication for 10 min. The entire amount of the extract was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 min. The upper methanol extract phase was carefully aspirated with a pipette, and the lower vegetable oil phase was extracted twice with 50 mL of a methanol-water (90:10 by volume) mixed solvent. The methanol-water (90:10 by volume) extracts were combined to obtain the product.

[0266] (2) Preparation of test solution ② for reverse phase system determination: Place the methanol-water (90:10) extract in a round-bottom flask, and evaporate it to dryness under reduced pressure at 40°C with the rotary evaporator speed controlled at 90 rpm. Then add 200 mL of methanol solvent to the round-bottom flask for re-dissolution. After the re-solution is allowed to stand, use a pipette to carefully remove the matrix components (vegetable oil) of the vitamin D drops remaining at the bottom of the flask. Evaporate it to dryness under reduced pressure again at 40°C with the rotary evaporator speed controlled at 90 rpm. Cool the flask to room temperature, accurately add 2 mL of methanol reagent for re-dissolution, and accurately add 1 mL of 5 mol / L sodium methoxide solution to the above methanol re-solution. Thoroughly saponify and mix.

[0267] 2. Inverted system detection

[0268] Take 500 μl of each of the test solution ① and the test solution ② for reverse phase system determination, inject them into reverse phase chromatography for separation, and collect the eluting components with a retention time of 11.5min~15.5min. The detection conditions of the reverse phase chromatography system are:

[0269] The detector is an ultraviolet detector;

[0270] Column: Triart C18, 4.6 mm × 150 mm, 5.0 μm;

[0271] The detection wavelength is 265nm;

[0272] The column temperature was 30°C;

[0273] Sample tray 4℃;

[0274] The flow rate was 1.0 mL / min;

[0275] The mobile phase ratio was methanol-acetonitrile-water (50:50:2);

[0276] The injection volume was 100 μL;

[0277] Isocratic elution was performed for 30 min.

[0278] 3. Normal Phase System Detection

[0279] The effluent fractions of the sample solution ① and the sample solution ② were respectively evaporated to dryness under reduced pressure at 40°C with a rotary evaporator speed of 90 rpm. The dried round-bottom flask was cooled to room temperature, and then 1 mL of isooctane was accurately added to dissolve the solution. The solution was dissolved in a 25°C water bath for more than 1 minute. 200 μl of each isooctane solution was accurately measured and injected into the normal phase chromatography system for determination. The detection conditions of the normal phase chromatography system were as follows:

[0280] The detector is an ultraviolet detector;

[0281] The chromatographic column is Topsil ® Silica, 4.6mm×250mm, 5.0μm;

[0282] The detection wavelength is 265nm;

[0283] Column temperature 30°C;

[0284] The injection plate temperature was 4°C;

[0285] The flow rate was 1.7 mL / min;

[0286] The mobile phase was n -pentanol-n -hexane (3:997) (v / v);

[0287] The injection volume was 200 μL;

[0288] Isocratic elution was performed for 50 min.

[0289] 4. Results

[0290] Table 3 compares the main peak areas of impurities A, B, C, and vitamin D extracted from the vitamin D drop sample using various extraction methods. The data in this table show that when acetone is omitted from the methanol extract, the peak areas of each component are only approximately 70% of those obtained with the methanol extract containing acetone. When acetone is omitted and the extraction is performed with a 9:1 methanol-water mixture, the peak areas of each component are only approximately 30% of those obtained with the methanol extract containing acetone. Therefore, adding acetone to the methanol extract significantly increases the extraction efficiency of each component, thereby improving the detection accuracy of impurities A, B, and C.

[0291] Table 3. Comparison of peak areas of components under several extraction methods in Comparative Example 1

[0292]

[0293] Comparative Example 2:

[0294] 1. Preparation of Solutions

[0295] Preparation of the methanol extract of the test sample: Take 5 mL of the contents of vitamin D drops, place it in a 250 mL conical flask, add 5 mL of acetone solvent, ultrasonicate for 10 minutes, mix thoroughly, then add 50 mL of methanol solvent to the conical flask, ultrasonicate for 10 minutes for extraction, transfer the entire amount of the extract to a centrifuge tube, centrifuge at 8000 rpm for 10 minutes, carefully aspirate the upper methanol extract phase with a pipette, and repeat the extraction of the lower plant oil phase with 50 mL of methanol solvent twice, combine the methanol extracts, and you are done.

[0296] Preparation of the test solution for reverse phase system determination (without adding sodium methoxide reagent): Place the methanol extract in a round-bottom flask and evaporate it to dryness under reduced pressure at 40°C with the rotary evaporator speed controlled at 90 rpm. Then add 200 mL of methanol solvent to the round-bottom flask for re-dissolution. After the re-solution has been allowed to stand, use a pipette to carefully remove the vitamin D drop matrix components (vegetable oil) remaining at the bottom of the flask. Evaporate it to dryness under reduced pressure at 40°C again. Cool the flask to room temperature and accurately add 2 mL of methanol reagent for re-dissolution.

[0297] 2. Reverse Phase System Assay

[0298] Take 500 μL of the test solution for reverse phase system determination, inject it into reverse phase chromatography for separation, and collect the elution components with a retention time of 11.5min~15.5min. The detection conditions of the reverse phase chromatography system are:

[0299] The detector is an ultraviolet detector;

[0300] Column: Triart C18, 4.6 mm × 150 mm, 5.0 μm;

[0301] The detection wavelength is 265nm;

[0302] The column temperature was 30°C;

[0303] Sample tray 4℃;

[0304] The flow rate was 1.0 mL / min;

[0305] The mobile phase ratio was methanol-acetonitrile-water (50:50:2);

[0306] The injection volume was 500 μL;

[0307] Isocratic elution was performed for 30 min.

[0308] 3. Normal Phase System Detection

[0309] The effluent fraction of the sample solution was evaporated to dryness under reduced pressure at 40°C with a rotary evaporator speed of 90 rpm. The dried round-bottom flask was cooled to room temperature, and then 1 mL of isooctane was accurately added for reconstitution. The solution was dissolved in a 25°C water bath for more than 1 minute. 200 μl of each isooctane solution was accurately measured and injected into the normal phase chromatography system for determination. The detection conditions of the normal phase chromatography system were as follows:

[0310] The detector is an ultraviolet detector;

[0311] The chromatographic column is Topsil ® Silica, 4.6mm×250mm, 5.0μm;

[0312] The detection wavelength is 265nm;

[0313] Column temperature 30°C;

[0314] The injection plate temperature was 4°C;

[0315] The flow rate was 1.7 mL / min;

[0316] The mobile phase was n -pentanol-n -hexane (3:997) (v / v);

[0317] The injection volume was 200 μL;

[0318] Isocratic elution was performed for 50 min.

[0319] 4. Results

[0320] The determination spectrum of the test solution is shown in Figure 9 As shown by Figure 9It can be seen that when sodium methoxide is not added to the methanol-extracted test solution for saponification, a large amount of plant oil matrix components will remain, which will seriously interfere with the determination of impurity A, impurity B, impurity C, and vitamin D component peaks in the normal phase system determination spectrum. As can be seen from the data in Table 4, when sodium methoxide is not added to the methanol-extracted test solution for saponification, the matrix components interfere with impurities A, impurity B, impurity C, and vitamin D, resulting in a low signal-to-noise ratio of the impurity peak height, and thus a significant reduction in detection sensitivity. Therefore, adding an appropriate amount of plant oil matrix components remaining in the sodium methoxide saponification extract to the methanol extract can significantly improve the specificity, sensitivity, and accuracy of the determination method.

[0321] Table 4. Comparative results of the effect of sodium methoxide reagent on the sensitivity of known impurities in Comparative Example 2

[0322]

[0323] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0324] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for determining related substances in a drop containing vitamin D3, characterized in that: The determination method comprises: Take the contents of the drop to be tested and prepare the test solution; The test solution was tested by reverse phase chromatography, and the effluent components with a retention time of 11.5 min to 15.5 min were collected; and The effluent components are detected by normal phase chromatography to determine the related substances in the contents; The related substances include one or more of (5E,7E)-9,10-opened cholesteryl-5,7,10(19)-triene-3β-ol, (6E)-9,10-opened cholesteryl-5(10),6,8-triene-3β-ol and 9,10-opened cholesteryl-6,8,10(5)-triene-3β-ol; Wherein, the preparation steps of the test solution include: The contents were mixed, extracted with acetone and methanol, phase separated, and the upper methanol extract was collected; removing the matrix components and solvent of the contents remaining in the methanol extract, and re-dissolving the contents with methanol to prepare a reconstituted solution; and The reconstituted solution and sodium methoxide reagent are mixed to carry out saponification reaction to prepare the test solution.

2. The method for determining related substances in the vitamin D3-containing drops according to claim 1, wherein: The volume ratio of the contents, acetone and methanol is (2-10): (2-10): (20-100); Optionally, the volume ratio of the contents, acetone and methanol is (2-5): (2-5): (20-50).

3. The method for determining related substances in the vitamin D3-containing drops according to claim 1, characterized in that: The concentration of sodium methoxide in the sodium methoxide reagent is 2 mol / L to 5 mol / L, and the volume of the sodium methoxide reagent corresponding to each 2 mL to 10 mL of the content is 0.5 mL to 1.5 mL.

4. The method for determining related substances in the vitamin D3-containing drops according to any one of claims 1 to 3, characterized in that: First, the contents and the acetone are mixed to prepare a mixture, and then the methanol is added to the mixture for extraction; Optionally, the mixing conditions include: shaking or ultrasonication for 5 min to 10 min; Optionally, the extraction conditions include: shaking or ultrasound, and the time is 5 minutes to 10 minutes.

5. The method for determining related substances in the vitamin D3-containing drops according to any one of claims 1 to 3, characterized in that: The preparation step of the methanol extract further comprises the following steps: collecting the lower layer after phase separation, adding methanol to repeat the extraction, and combining the methanol extract obtained in the second extraction with the methanol extract obtained in the previous extraction to prepare the test solution; Optionally, the number of repeated extractions includes 1 to 3 times; Optionally, the volume ratio of the contents to the methanol used in each repeated extraction is (2-10): (20-100); further optionally, the volume ratio of the contents to the methanol used in each repeated extraction is (2-5): (20-50).

6. The method for determining related substances in the vitamin D3-containing drops according to any one of claims 1 to 3, characterized in that: The upper methanol extract was collected by centrifugation; Optionally, the centrifugal conditions include: a rotation speed of 5000 rpm to 8000 rpm and a time of 5 min to 10 min.

7. The method for determining related substances in the vitamin D3-containing drops according to any one of claims 1 to 3, characterized in that: The preparation steps of the reconstitution solution include: The methanol extract is subjected to a first rotary evaporation to dryness, methanol is added for a first redissolution, the extract is allowed to stand, the matrix component remaining at the bottom is removed by suction, the remaining portion is subjected to a second rotary evaporation to dryness, and methanol is added for a second redissolution to prepare the reconstituted solution; Optionally, the volume of methanol used for the first reconstitution corresponding to 2 mL to 10 mL of the contents is 150 mL to 250 mL; Optionally, the conditions for the first rotary evaporation and the second rotary evaporation each independently include: reduced pressure, a temperature of 35°C to 45°C, and a rotation speed of 30 rpm to 100 rpm; further optionally, the conditions for the first rotary evaporation and the second rotary evaporation each independently include: reduced pressure, a temperature of 35°C to 40°C, and a rotation speed of 30 rpm to 90 rpm; Optionally, the volume of methanol used for the second reconstitution corresponding to 2 mL to 10 mL of the content is 1.5 mL to 2.5 mL.

8. The method for determining related substances in the vitamin D3-containing drops according to any one of claims 1 to 3, characterized in that: The reverse phase chromatography method satisfies one or more of the following conditions: (A) The detector is a UV detector; (B) a liquid chromatography column filled with octadecylsilane bonded silica gel, optionally a Triart C18 column, 4.6 mm × 150 mm, 5.0 μm, or a Welch Ultimate XB-C18 column, 150 mm × 4.6 mm, 5 μm; (C) Detection wavelength is 260nm~270nm; (D) Column temperature is 28℃~32℃; (E) The temperature of the sample tray is 2°C~10°C, and can be optionally 4°C~10°C; (F) Flow rate: 0.5 ml / min~1.5 mL / min; (G) The mobile phase consisted of methanol, acetonitrile, and water in a volume ratio of (49~51):(49~51):(1~3); (H) injection volume of 100 μL to 500 μL; and, (I) Isocratic elution for 25 min to 35 min.

9. The method for determining related substances in the vitamin D3-containing drops according to any one of claims 1 to 3, characterized in that: The determination method comprises: performing a third rotary evaporation on the effluent component to dryness, adding isooctane to redissolve it, and then loading the sample for normal phase chromatography detection; Optionally, the conditions for the third rotary evaporation include: reduced pressure, temperature of 35°C to 45°C, and rotation speed of 30 rpm to 100 rpm; further optionally, the conditions for the third rotary evaporation include: reduced pressure, temperature of 35°C to 40°C, and rotation speed of 30 rpm to 90 rpm; Optionally, the volume of isooctane corresponding to 2 mL to 10 mL of the content is 0.5 mL to 1.5 mL.

10. The method for determining related substances in the vitamin D3-containing drops according to any one of claims 1 to 3, characterized in that: The normal phase chromatography method satisfies one or more of the following conditions: A) The detector is a UV detector; B) Liquid chromatography column with porous silica particles as filler, optionally Topsil ® Silica, 4.6mm×250mm, 5.0μm, or Supersil SiO2, 4.6mm×250mm, 5.0μm; C) The detection wavelength is 260nm~270nm; D) Column temperature is 25~35℃; E) The temperature of the sample tray is 2℃~10℃; F) Flow rate is 1.5mL / min~2.0mL / min; G) The mobile phase is a mixture of n-pentanol and n-hexane in a volume ratio of (2-5): (998-995), or alternatively a mixture of n-pentanol and n-hexane in a volume ratio of (2-3): (998-997); H) injection volume of 150 μL to 250 μL; and, I) Isocratic elution for not less than 40 min, optionally 40 min to 50 min.

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