A method for preparing three cis-isomer impurity reference substances of vitamin A palmitate

Abstract

The invention provides a method for preparing three cis-isomer impurity reference substances of vitamin A palmitate, wherein the three cis-isomers are 13-cis-isomer, 9,13-bis-cis-isomer and 9-cis-isomer respectively. Vitamin A palmitate is dissolved in n-hexane and oxidatively destroyed, and then a solution of three cis-isomer impurities is separated by isocratic elution using a preparative liquid chromatograph, and concentrated in a dark environment to obtain three cis-isomer impurity reference substances of vitamin A palmitate. The method of the invention is simple, and three cis-isomers of vitamin A palmitate can be prepared simultaneously in a short time, and can be used for the research of related substances of vitamin A palmitate bulk drug and related products, thereby realizing the quality control detection of cis-isomers of vitamin A palmitate, and further improving the quality standard of vitamin A palmitate and its preparation.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to a method for preparing a reference substance for three cis-isomer impurities of vitamin A palmitate. Background Art

[0002] Vitamin A palmitate has a long carbon chain, a low melting point, and good oil solubility. It has physiological functions such as maintaining vision, promoting growth and development, maintaining the integrity and soundness of epithelial structure, and enhancing immunity. Therefore, it is widely used in food, medicine, and cosmetics.

[0003] Vitamin A palmitate is the API for multivitamins for injection (12), 12 complex vitamins for injection, multivitamin injection (13) (10 / 3), multivitamins for injection (13), pediatric multivitamin injection (13), pediatric multivitamin injection (13), fat-soluble vitamins for injection (I), fat-soluble vitamins for injection (II). The biological potency of different cis- and trans-isomers of vitamin A palmitate varies greatly, among which the all-trans isomer is the most stable and effective active ingredient. The biological activity of the cis-isomer of vitamin A palmitate is lower than that of the all-trans, among which the relative activity of the 13-cis isomer is 76%, the relative activity of the 11-cis isomer is 31%, the relative activity of the 9-cis isomer is 19%, and the relative activity of the 9,13-bis-cis isomer is only 16% (Yu An. Study on the stability of vitamin A [D]. Hangzhou: Zhejiang University, 2012.). In addition, the cis-isomer of vitamin A palmitate is easily affected by factors such as light, heat, and solvents, and has poor stability. Therefore, the content of each cis-isomer in vitamin A palmitate API is unstable and the ratio is significantly different, which has a great impact on the research of vitamin A palmitate. Researching methods for separating and preparing cis-isomers of vitamin A palmitate is of great significance for the quality control of vitamin A palmitate API.

[0004] Common cis isomers of vitamin A palmitate include 13-cis isomer, 11-cis isomer, 9-cis isomer, and 9,13-bis-cis isomer. Among them, cis isomer 1: 13-cis vitamin A palmitate, with a molecular formula of C 36 H 60 O2, molecular weight is 524.87, its structural formula is:

[0005]

[0006] Cis isomer 2: 9,13-bis-cis vitamin A palmitate, molecular formula C 36 H 60 O2, molecular weight is 524.87, its structural formula is:

[0007]

[0008] Cis isomer 3: 9-cis vitamin A palmitate, molecular formula C 36 H 60 O2, molecular weight is 524.87, its structural formula is:

[0009]

[0010] The Chinese Pharmacopoeia, Part IV, General Rules, 0721, Method II of Vitamin A Assay, describes that the preparation of the system suitability solution is to take an appropriate amount of vitamin A reference substance (equivalent to 300 mg of vitamin A acetate), place it in a beaker, add 0.2 mL of iodine test solution, mix well, leave it for about 10 minutes, quantitatively transfer it to a 200 mL volumetric flask, dilute it to the mark with n-hexane, shake it well, accurately measure 1 mL, place it in a 100 mL volumetric flask, dilute it to the mark with n-hexane, and shake it well. However, the contents of the three cis isomers prepared using this system suitability solution are unstable and can only be used for qualitative tests of cis isomers, but not for quantitative studies of the impurity content of cis isomers.

[0011] Jiang Xiaoying et al. (Jiang Xiaoying, Ren Shengke. Rapid separation of five cis- and trans-isomers of vitamin A acetate and vitamin A palmitate by NP-HPLC [J]. Shanghai Chemical Industry, 2022, 47(06): 53-57.) established a method for separation and identification of cis- and trans-isomers of vitamin A acetate and vitamin A palmitate based on retinol using normal phase high performance liquid chromatography (NP-HPLC). Although this method can only separate 13-cis, 11-cis, 9-cis, 9,13-cis and all-trans isomers, and determine the elution order of each cis isomer, this method is not suitable for preparation of liquid phase, and the literature does not involve the preparation of 13-cis isomers, 9,13-di-cis isomers, and 9-cis isomers of vitamin A palmitate.

[0012] Tang Lei et al. used HPLC to simultaneously determine the concentrations of all-trans retinoic acid, 13-cis retinoic acid and 9-cis retinoic acid in human plasma (Tang Lei, Hu Junyong, Cui Yingpeng, et al. Monitoring the concentrations of all-trans retinoic acid, 13-cis retinoic acid and 9-cis retinoic acid in human plasma [J]. Chinese Practical Medicine, 2007, 2(31): 9-11.). Methods The chromatographic column was Kromasil C18 column (5μm, 4.6 mm*250 mm); the column temperature was room temperature. Mobile phase A: methanol; mobile phase B: 0.01 mol / L sodium acetate buffer (pH=5.7), gradient elution, flow rate 1 mL / min; detection wavelength 340 nm. Results: The blood concentrations of all-trans retinoic acid, 13-cis retinoic acid and 9-cis retinoic acid were in the range of 1-200 ng / mL, and there was a good linear relationship between the concentration and the peak area ratio, with the lowest detection concentration being 0.5 ng / mL. This method is only applicable to the detection of all-trans retinoic acid, 13-cis retinoic acid and 9-cis retinoic acid, and the detection conditions are not suitable for the separation and purification of the preparative liquid phase of 13-cis, 9-cis, and 9,13-cis.

[0013] US Patent Document US2676903A discloses a method for extracting vitamin A intermediates, which explores the use of existing aliphatic hydrocarbon solvents including pentane, hexane, and octane, such as n-pentane, n-heptane, isoheptane, n-hexane and isooctane, 2,2,4-trimethylpentane (isooctane), nonane, petroleum ether, and cycloalkanes (cyclohexane) for the light phase of the extraction process; acetonitrile, nitromethane, methanol, aniline, nitromethane and aniline mixed solvents, methanol and ethylene glycol mixed solvents, and ethylene glycol and ethanol monomethyl ether mixed solvents as the heavy phase solvent extraction system of the extraction process. The organic solvents used in the extraction system are expensive, highly toxic, and difficult to separate.

[0014] CN111004163A discloses a method for isomerization of vitamin A catalyzed by ruthenium catalyst, using a ruthenium compound as a catalyst, in the presence of an auxiliary agent, a plurality of vitamin A cis isomers with low biological activity can be converted into highly biologically active all-trans isomers in high proportion, wherein the cis isomers include 9-cis isomers, 11-cis isomers and 13-cis isomers. Ruthenium is a rare element with limited reserves, high price and unstable supply.

[0015] CN103936642A discloses a method for continuous extraction and purification of isomers 13 cis and all-trans in a vitamin A mother liquor, wherein the vitamin A mother liquor is continuously fed into a normal extraction tower, and non-polar impurities are removed by extraction and washing with a low-polarity solvent, and the obtained heavy phase is desolvated and fed into a reverse extraction tower, and polar impurities are removed by extraction and washing with a high-polarity mixed solvent of one or more alcohols and water. The method is to purify and prepare a mixed solution of 13 cis isomers and all-trans isomers, which requires the use of a large amount of organic reagents and is complicated to operate, and does not involve the preparation of 9,13-bis-cis isomers and 9-cis isomers.

[0016] Since the 13-cis isomer, 9,13-bis-cis isomer, and 9-cis isomer are easily affected by factors such as light, heat, solvents, and preparation methods, and have poor stability, it is currently difficult to purchase commercial cis-isomer reference substances of vitamin A palmitate, and there are no relevant patent documents and non-patent documents on the preparation method of cis-isomer impurity reference substances of vitamin A palmitate. Conventional preparation operations are time-consuming and cannot simultaneously prepare 13-cis-isomers, 9,13-bis-cis-isomers, and 9-cis-isomers with stable content and high purity. Therefore, it is necessary to explore a method for simultaneously preparing three cis-isomer impurity reference substances of vitamin A palmitate. Summary of the invention

[0017] In view of the shortcomings of the prior art, the present invention provides a method for simultaneously preparing three cis-isomers of vitamin A palmitate impurity reference substances. The method is simple, highly repeatable and operable. The prepared three cis-isomers of vitamin A palmitate can be used for the research of related substances of vitamin A palmitate raw materials and related products, thereby achieving quality control detection of cis-isomers of vitamin A palmitate and further improving the quality standard of vitamin A palmitate and its preparations.

[0018] The present invention studies the preparation of vitamin A palmitate cis isomer impurity reference substances. The structures of the three prepared cis impurity reference substances have been confirmed, and cis isomers 1, 2, and 3 are 13-cis vitamin A palmitate, 9,13-bis-cis vitamin A palmitate, and 9-cis vitamin A palmitate, respectively; the maximum absorption wavelengths of cis isomers 1, 2, and 3 are 328.0 nm, 324.0 nm, and 322.0 nm, respectively, as shown in the attached Figure 2 , Attachment Figures 5 to 10 .

[0019] The technical solution of the present invention is as follows:

[0020] Step 1: The destruction conditions for the production of the cis isomer are:

[0021] Add 50 mL~100 mL of n-hexane, 5.0 g~10.002 g of vitamin A palmitate, and 0.4 mL~2.0 mL of iodine test solution to the reaction bottle, and stir to react for 20~30 min. After the reaction is completed, add 0.5 g~3.012 g of sodium bisulfite and stir to quench the reaction.

[0022] Step 2: Separation and purification of three cis isomers of vitamin A palmitate, the chromatographic conditions are:

[0023] Preparative liquid chromatograph: SSI Prep-2100 preparative liquid chromatograph.

[0024] Chromatographic column: silica gel column;

[0025] Flow rate: 5.7~6.3 mL / min;

[0026] UV detector detection wavelength: 325 nm;

[0027] Test sample: the mixed solution of vitamin A palmitate obtained in step 1, diluted with n-hexane, and the concentration of vitamin A palmitate after dilution is 10 mg / mL based on the amount of vitamin A palmitate added in step 1;

[0028] Injection volume: 1.5 mL;

[0029] Mobile phase: n-hexane-methyl tert-butyl ether = 100: 0.16~0.22;

[0030] Step 3, preparation process: isocratic elution, according to the order of elution, the cis isomers are recorded as isomer 1, 2, 3 respectively; use a brown glass container to collect the isomer 1, 2, 3 solution, seal it and keep it in a dark place.

[0031] The concentration conditions for the three cis isomer solutions of vitamin A palmitate are as follows:

[0032] The prepared solutions of isomers 1, 2 and 3 were concentrated by rotary evaporation at 25-30°C and -0.095 MPa in the dark using a water pump and a rotary evaporator to obtain the impurity reference substances of isomers 1, 2 and 3. After the concentration, the solutions were nitrogen filled, sealed and frozen in the dark.

[0033] This method can be used for the preparation of vitamin A palmitate cis-isomer reference substances, facilitating better detection and utilization of vitamin A products.

[0034] The mixed solution of vitamin A palmitate was subjected to liquid phase monitoring and HPLC detection of isomers 1, 2, and 3. The chromatographic conditions were:

[0035] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0036] Flow rate: 1.0 mL / min; detection wavelength: 325 nm; column temperature: 30°C; isocratic elution;

[0037] Mobile phase: n-hexane-isopropanol (999.5:0.5).

[0038] Beneficial effects of the present invention:

[0039] 1) The preparation, separation and purification methods used in this technical solution are simple to operate, have good repeatability and strong operability. Three cis isomers of vitamin A palmitate were prepared: the purities of 13-cis isomer, 9,13-bis-cis isomer and 9-cis isomer were as high as 90.87%, 87.32% and 90.55% respectively, which solved the supply problem of vitamin A palmitate impurity reference substances, thereby realizing the quality control detection of cis isomers of vitamin A palmitate, and further improving the quality standards of vitamin A palmitate and its preparations.

[0040] 2) The maximum absorption wavelengths of the three cis isomers prepared by the present invention are consistent with the pharmacopoeia method; the pharmacopoeia method can only be used for qualitative tests. The present invention obtains three cis isomer impurity reference substances that are consistent with the pharmacopoeia method, which is convenient for quantitative detection of the content of vitamin A palmitate isomers.

[0041] 3) The present invention has a fast separation speed and a simple elution method. The purification method adopts a single mobile phase of n-hexane-methyl tert-butyl ether with a volume ratio of 100:0.16-0.22 and isocratic elution, and can quickly separate and obtain three cis isomers of vitamin A palmitate.

[0042] 4) The cis isomer of vitamin A palmitate is easily affected by factors such as light, heat, and solvents, and has poor stability. To ensure the stability of the impurity reference substance, the method of the present invention adds excess sodium bisulfite after the reaction in step 1 and stirs to quench the reaction; nitrogen stabilizer is added during the preparation process, and the prepared isomer is stored in a brown glass container and stored in a dark and frozen state. The three cis isomers 1, 2, and 3 of the prepared vitamin A palmitate have stable contents, and the purities are greater than or equal to 90.50%, 86.00%, and 90.30%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the HPLC monitoring spectrum of the mixed solution of vitamin A palmitate cis-isomers 1, 2, and 3 prepared in step 1 of Example 1;

[0044] Figure 2It is the maximum absorption wavelength HPLC spectrum of vitamin A palmitate cis isomers 1, 2, and 3 prepared in step 1 of Example 1, wherein Figure A is the liquid phase PDA spectrum of vitamin A palmitate cis isomer 1, Figure B is the liquid phase PDA spectrum of vitamin A palmitate cis isomer 2, Figure C is the liquid phase PDA spectrum of vitamin A palmitate cis isomer 3, Figure D is the liquid phase PDA spectrum of vitamin A palmitate, and Figure E is the maximum absorption wavelength HPLC spectrum of vitamin A palmitate cis isomers 1, 2, and 3 prepared in step 1;

[0045] Figure 3 is the preparation liquid phase spectrum of the test sample in step 2 of Example 1;

[0046] Figure 4 is the preparation liquid phase spectrum of the test sample in step 2 of Example 3;

[0047] Figure 5 is an HPLC spectrum of 13-cis vitamin A palmitate, wherein Figure A is a liquid phase PDA spectrum of 13-cis vitamin A palmitate, and Figure B is an HPLC spectrum of 13-cis vitamin A palmitate;

[0048] Figure 6 It is the HPLC peak result of 13-cis vitamin A palmitate;

[0049] Figure 7 It is the HPLC spectrum of 9,13-bis-cis vitamin A palmitate, wherein Figure A is the liquid phase PDA spectrum of 9,13-bis-cis vitamin A palmitate, and Figure B is the HPLC spectrum of 9,13-bis-cis vitamin A palmitate;

[0050] Figure 8 It is the HPLC peak result of 9,13-bis-cis vitamin A palmitate;

[0051] Fig. 9 is an HPLC spectrum of 9-cis vitamin A palmitate, wherein Figure A is a liquid phase PDA spectrum of 9-cis vitamin A palmitate, and Figure B is an HPLC spectrum of 9-cis vitamin A palmitate;

[0052] Fig.10 It is the HPLC peak result of 9-cis vitamin A palmitate;

[0053] Fig.11 is the MS spectrum of 13-cis vitamin A palmitate;

[0054] Fig.12 is the MS spectrum of 9,13-bis-cis vitamin A palmitate;

[0055] Fig.13is the MS spectrum of 9-cis vitamin A palmitate;

[0056] Fig.14 This is the HPLC spectrum of cis-isomer 1 prepared in the comparative example after five repeated injections. DETAILED DESCRIPTION

[0057] Example 1 Preparation of Cis-Isomers 1, 2 and 3 of Vitamin A Palmitate

[0058] Step 1: Destruction of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0059] To a 250 mL reaction bottle, add 100 mL of n-hexane, 10.002 g of vitamin A palmitate, and 2.0 mL of iodine test solution. Stir and react for 20 min. After the reaction is completed, add 3.012 g of sodium bisulfite and stir to quench the reaction to obtain a vitamin A palmitate mixed solution.

[0060] The mixed solution of vitamin A palmitate was monitored by liquid chromatography, and the chromatographic conditions were:

[0061] Chromatographic column: Luna 5 um silica (2) 100A, specification 250 mm*4.6 mm, 5 μm;

[0062] Flow rate: 1.0 mL / min; detection wavelength: 325 nm; column temperature: 30℃; mobile phase: n-hexane: methyl tert-butyl ether = 995:5; isocratic elution.

[0063] The monitoring results are attached. Figure 1 The HPLC monitoring spectrum of the mixed solution of vitamin A palmitate cis isomers 1, 2, and 3 shows that the maximum absorption wavelengths of vitamin A palmitate isomers 1, 2, and 3 are 328.0 nm, 324.0 nm, and 322.0 nm, respectively. Figure 1 and attached Figure 2 .

[0064] Step 2: Preparation of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0065] Chromatographic column: ACCHROM Silica, size 250 mm × 20 mm, 5 μm;

[0066] Flow rate: 6.0 mL / min;

[0067] UV detector detection wavelength: 325 nm;

[0068] Test sample concentration: 10 mg / mL;

[0069] Injection volume: 1.5 mL;

[0070] Mobile phase: n-hexane-methyl tert-butyl ether = 100: 0.18;

[0071] Test sample: the mixed solution of vitamin A palmitate obtained in step 1, diluted with n-hexane, and the concentration of vitamin A palmitate after dilution is 10 mg / mL based on the amount of vitamin A palmitate added in step 1;

[0072] Elution was performed isocratically, and isomers 1, 2, 3, and vitamin A palmitate were eluted in sequence. The isomers 1, 2, and 3 solutions were collected in a brown container, sealed, and stored in a dark place. The preparative liquid phase spectrum of the test sample is shown in the attached Figure 3 .

[0073] Step 3: Preparation and concentration of solutions of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0074] The solutions of cis isomers 1, 2, and 3 were concentrated by rotary evaporation at 26°C and -0.095 MPa in the dark using a water pump and a rotary evaporator. After concentration, a small amount of n-hexane was used to dissolve the fraction and then transferred into a brown cillin bottle. The solvent n-hexane was dried with nitrogen. After testing, the HPLC purity of cis isomers 1, 2, and 3 was 90.87%, 87.32%, and 90.55%, respectively. See the attached figure for details of the spectrum and peak results. Figure 5 ~Attached Fig.10 .

[0075] Example 2 Preparation of Cis-Isomers 1, 2, and 3 of Vitamin A Palmitate

[0076] Step 1: Destruction of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0077] Add 50 mL of n-hexane, 5.0 g of vitamin A palmitate, and 0.4 mL of iodine test solution to a 100 mL reaction bottle, and stir to react for 30 min. After the reaction is completed, add 0.5 g of sodium bisulfite and stir to quench the reaction to obtain a vitamin A palmitate mixed solution.

[0078] Step 2: Preparation of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0079] Chromatographic column: ACCHROM Silica, size 250 mm × 20 mm, 5 μm;

[0080] Flow rate: 6.0 mL / min;

[0081] UV detector detection wavelength: 325 nm;

[0082] Test sample concentration: 10 mg / mL;

[0083] Injection volume: 1.5 mL;

[0084] Mobile phase: n-hexane-methyl tert-butyl ether = 100: 0.18;

[0085] Test sample: the mixed solution of vitamin A palmitate obtained in step 1 is diluted with n-hexane, and the concentration of vitamin A palmitate after dilution is 10 mg / mL based on the amount of vitamin A palmitate added in step 1;

[0086] After isocratic elution, isomers 1, 2, 3 and vitamin A palmitate appear in sequence. Use a brown container to collect the isomers 1, 2 and 3 solutions, seal them and store them in a frozen state away from light.

[0087] Step 3: Preparation and concentration of solutions of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0088] The solutions of cis isomers 1, 2, and 3 were concentrated by rotary evaporation at 26°C and -0.095 MPa in the dark using a water pump and a rotary evaporator. After concentration, the fraction was dissolved in a small amount of n-hexane and transferred into a brown cillin bottle, and the solvent n-hexane was dried with nitrogen. After testing, the HPLC purities of cis isomers 1, 2, and 3 were 90.76%, 87.08%, and 90.53%, respectively.

[0089] Example 3 Preparation of Cis-Isomers 1, 2 and 3 of Vitamin A Palmitate

[0090] Step 1: Destruction of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0091] To a 250 mL reaction bottle, add 100 mL of n-hexane, 10.002 g of vitamin A palmitate, and 2.0 mL of iodine test solution. Stir and react for 20 min. After the reaction is completed, add 3.012 g of sodium bisulfite and stir to quench the reaction to obtain a vitamin A palmitate mixed solution.

[0092] Step 2: Preparation of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0093] Chromatographic column: ACCHROM Silica, size 250 mm × 20 mm, 5 μm;

[0094] Flow rate: 6.0 mL / min;

[0095] UV detector detection wavelength: 325 nm;

[0096] Test sample concentration: 10 mg / mL;

[0097] Injection volume: 1.5 mL;

[0098] Mobile phase: n-hexane-methyl tert-butyl ether = 100:0.2;

[0099] Test sample: the mixed solution of vitamin A palmitate obtained in step 1, diluted with n-hexane, and the concentration of vitamin A palmitate after dilution is 10 mg / mL based on the amount of vitamin A palmitate added in step 1;

[0100] Elution was performed isocratically, and isomers 1, 2, 3, and vitamin A palmitate were eluted in sequence. The isomers 1, 2, and 3 solutions were collected in brown containers, sealed and stored in a dark place. The preparative liquid phase spectrum of the test sample is shown in the attached Figure 4 .

[0101] Step 3: Preparation and concentration of solutions of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0102] The solutions of cis isomers 1, 2, and 3 were concentrated by rotary evaporation at 26°C and -0.095 MPa in the dark using a water pump and a rotary evaporator. After the concentration was completed, the fraction was dissolved in a small amount of n-hexane and transferred into a brown cillin bottle, and the solvent n-hexane was dried with nitrogen. After testing, the HPLC purity of cis isomers 1, 2, and 3 was 90.59%, 86.99%, and 90.49%, respectively.

[0103] Example 4 Preparation of Cis-Isomers 1, 2, and 3 of Vitamin A Palmitate

[0104] Step 1: Destruction of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0105] Add 50 mL of n-hexane, 5.0 g of vitamin A palmitate, and 0.4 mL of iodine test solution to a 100 mL reaction bottle, and stir to react for 30 min. After the reaction is completed, add 0.5 g of sodium bisulfite and stir to quench the reaction to obtain a vitamin A palmitate mixed solution.

[0106] Step 2: Preparation of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0107] Chromatographic column: ACCHROM Silica, size 250 mm × 20 mm, 5 μm;

[0108] Flow rate: 5.7~6.3 mL / min;

[0109] UV detector detection wavelength: 325 nm;

[0110] Test sample concentration: 10 mg / mL;

[0111] Injection volume: 1.5 mL;

[0112] Mobile phase: n-hexane-methyl tert-butyl ether = 100:0.2;

[0113] Test sample: the mixed solution of vitamin A palmitate obtained in step 1, diluted with n-hexane, and the concentration of vitamin A palmitate after dilution is 10 mg / mL;

[0114] After isocratic elution, isomers 1, 2, 3 and vitamin A palmitate appear in sequence. Use a brown container to collect the isomers 1, 2 and 3 solutions, seal them and store them in a frozen state away from light.

[0115] Step 3: Preparation and concentration of solutions of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0116] The solutions of cis isomers 1, 2, and 3 were concentrated by rotary evaporation at 26°C and -0.095 MPa in the dark using a water pump and a rotary evaporator. After concentration, the fraction was dissolved in a small amount of n-hexane and transferred into a brown cillin bottle, and the solvent n-hexane was dried with nitrogen. After testing, the HPLC purities of cis isomers 1, 2, and 3 were 90.56%, 86.76%, and 90.37%, respectively.

[0117] Comparative Example

[0118] Step 1: Destruction of cis-isomers 1, 2, and 3 of vitamin A palmitate

[0119] To a 250 mL reaction bottle, add 100 mL of n-hexane, 10.002 g of vitamin A palmitate, and 2.0 mL of iodine test solution. Stir and react for 20 min. After the reaction is completed, add 0.0105 g of sodium bisulfite and stir to quench the reaction to obtain a vitamin A palmitate mixed solution.

[0120] Step 2 and step 3 are the same as in Example 1.

[0121] The prepared vitamin A palmitate cis isomers 1, 2, and 3 were injected 5 times respectively. The injection results showed that the contents of vitamin A palmitate cis isomers 1, 2, and 3 in the three cis isomer samples were unstable, and isomers 1, 2, 3, and vitamin A palmitate were mutually converted. Fig.14 This is the HPLC spectrum of cis-isomer 1 obtained by five repeated injections.

Claims

1. A method for preparing a reference substance of three cis-isomer impurities of vitamin A palmitate, characterized in that: The following steps are involved: Step 1: Preparation of three cis isomers of vitamin A palmitate, the preparation conditions are: Add 50 mL to 100 mL of n-hexane, 5.0 g to 10.002 g of vitamin A palmitate, and 0.4 mL to 2.0 mL of iodine test solution to the reaction bottle, and stir to react for 20 to 30 min. After the reaction is completed, add 0.5 g to 3.012 g of sodium bisulfite and stir to quench the reaction to obtain a vitamin A palmitate mixed solution; The concentration of the iodine test solution is 0.05 mol / L, and the molar ratio of the sodium bisulfite to iodine is 240-290:1; Step 2: Separation and purification of three cis isomers of vitamin A palmitate. The preparative chromatographic conditions for the preparative liquid phase are: Preparative liquid chromatograph: SSI Prep-2100 preparative liquid chromatograph; Chromatographic column: silica gel column; Flow rate: 5.7~6.3mL / min; UV detector detection wavelength: 325nm; Injection volume: 1.5 mL; Mobile phase: n-hexane-methyl tert-butyl ether, volume ratio 100:0.16-0.22; Test sample: the mixed solution of vitamin A palmitate obtained in step 1 is diluted with n-hexane, and the concentration of vitamin A palmitate after dilution is 10 mg / mL based on the amount of vitamin A palmitate added in step 1; The mobile phase was used for isocratic elution, and the cis-isomer solutions were recorded as cis-isomer 1, 2, and 3 solutions in the order of peak elution time; the cis-isomer 1, 2, and 3 solutions were collected in a brown glass container, filled with nitrogen, and then sealed and stored in a dark and frozen state; Step 3: Concentration of the three cis isomer solutions of vitamin A palmitate, the concentration conditions are: The solutions of the cis isomers 1, 2, and 3 obtained in step 2 are respectively concentrated by rotary evaporation at 25-30°C and -0.095 MPa in the dark using a water pump and a rotary evaporator, filled with nitrogen, and then sealed and stored in a dark place and frozen to obtain; The cis isomer 1 is 13-cis vitamin A palmitate, the cis isomer 2 is 9,13-bis-cis vitamin A palmitate, and the cis isomer 3 is 9-cis vitamin A palmitate; The maximum absorption wavelengths of the cis-isomer 1, cis-isomer 2 and cis-isomer 3 are 328 nm, 324 nm and 322 nm respectively.

2. The method according to claim 1, characterized in that The chromatographic column in step 2 is ACCHROM Silica, 250 mm×20 mm, 5 μm, or a chromatographic column with equivalent performance to the chromatographic column.

3. The method according to claim 1, wherein the volume ratio of the mobile phase to n-hexane-methyl tert-butyl ether is 100:0.

18.

4. The method according to claim 1, wherein the volume ratio of the mobile phase to n-hexane-methyl tert-butyl ether is 100:0.

2.

5. The method according to claim 1, characterized in that: The flow rate was 6.0 mL / min.

6. The method according to claim 1, characterized in that The HPLC purity of the 13-cis vitamin A palmitate is greater than or equal to 90.50%; the HPLC purity of the 9,13-bis-cis vitamin A palmitate is greater than or equal to 86.00%; and the HPLC purity of the 9-cis vitamin A palmitate is greater than or equal to 90.30%.

7. The method according to claim 1, characterized in that The HPLC purity of the 13-cis vitamin A palmitate is 90.87%; the HPLC purity of the 9,13-bis-cis vitamin A palmitate is 87.32%; and the HPLC purity of the 9-cis vitamin A palmitate is 90.55%.

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