Method for detecting antioxidant degradation impurities in injection

Abstract

The invention provides a method for detecting degradation impurities of an antioxidant in an injection, relates to the technical field of analytical chemistry, and adopts high performance liquid chromatography to detect a pharmaceutical preparation. Chromatographic conditions are as follows: a chromatographic column is an octadecyl silane bonded silica gel column; the mobile phase is composed of a mobile phase A and a mobile phase B; the mobile phase A is a 0.01% phosphoric acid solution; the mobile phase B is acetonitrile. Compared with the prior art, the method disclosed by the invention has the advantages that the degradation impurity contents of the injection and the antioxidant can be simultaneously determined under the same condition, the mobile phase composition is simple, and the determination result is relatively accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, relates to drug analysis technology, and specifically relates to a method for detecting antioxidant degradation impurities in an injection. Background Art

[0002] Butylated hydroxytoluene (BHT, 2,6-di-tert-butylphenol) is a common phenolic antioxidant that can react with free radicals, thereby interrupting the chain reaction of free radicals and exerting its antioxidant effect. It is widely used in food, medicine and cosmetics because it can reduce the oxidation rate of product ingredients, has no specific odor, is inexpensive, and can prevent changes in taste or color.

[0003] In injection preparations, when the main component is easily oxidized by oxygen in the air, the antioxidant BHT can inhibit the oxidation of the main component, thereby degrading multiple impurities with benzene rings; when the degraded impurities are attributed to the impurities of the main component, the related substances of the preparation will be unqualified. Therefore, there is an urgent need for a detection method for simultaneously determining the degradation impurity content of the main component and butylated hydroxytoluene under the same conditions.

[0004] The prior art provides several methods for detecting impurities from the degradation of the antioxidant BHT. For example, the patent document "Recommendations for Inspection of Relevant Substances in the Quality Standard of Butylated Hydroxytoluene, a Pharmaceutical Excipient, in the 2020 Edition of the Chinese Pharmacopoeia" provides a method for detecting nine impurities, including m-cresol, p-cresol, 2-tert-butyl-4-methylphenol, 2-tert-butyl-5-methylphenol, 6-di-tert-butylphenol, 5-di-tert-butyl-4-hydroxybenzoic acid, 5-di-tert-butyl-4-hydroxybenzaldehyde, 4-hydroxy-3-tert-butylanisole and 6-di-tert-butyl-m-cresol, produced by the degradation of BHT by high performance liquid chromatography; Patent CN202010384972 provides a method for detecting 2,6-di-tert-butyl-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one, 2,6-di-tert-butylbenzoquinone and 3,3',5,5'-tetra-tert-butyl-4,4'-stilbenequinone, which are degradation products of the antioxidant BHT in food.

[0005] Although these methods can detect BHT degradation impurities, they are unable to simultaneously determine the contents of the main component and BHT degradation impurities under the same conditions. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art and provides a method for detecting degradation impurities of an antioxidant in an injection. The method can simultaneously determine the degradation impurity contents of a main component and an auxiliary material BHT under the same conditions, has a simple mobile phase composition, and has relatively accurate determination results.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The invention provides a method for detecting degradation impurities of an antioxidant in an injection. The method adopts high performance liquid chromatography to detect a drug preparation, and the chromatographic conditions are as follows: the chromatographic column is an octadecylsilane bonded silica gel column; the mobile phase consists of a mobile phase A and a mobile phase B; the mobile phase A is a 0.01% phosphoric acid solution; and the mobile phase B is acetonitrile.

[0009] Specifically, the present invention adopts gradient elution, and the elution procedure is:

[0010] a: 0min-15min: 75% mobile phase A decreases to 23% mobile phase A, and the rest is mobile phase B;

[0011] b: 15min-45min: 23% mobile phase A is gradually reduced to 10% mobile phase A, and the rest is mobile phase B;

[0012] c: 45min-45.1min: 10% mobile phase A increased to 75% mobile phase A, the rest was mobile phase B;

[0013] d: 45.1min-60min: 75% mobile phase A + 25% mobile phase B.

[0014] Specifically, the length of the chromatographic column is 250 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of the filler of the chromatographic column is 4 μm.

[0015] More specifically, the chromatographic column is Poroshell 120EC-C18.

[0016] Specifically, the column temperature of the chromatographic column is 30-40°C.

[0017] Preferably, the column temperature is preferably 35°C.

[0018] Specifically, the injection volume is 800 μL-1200 μL

[0019] Preferably, the injection volume is preferably 1000 μL.

[0020] Specifically, the detection wavelength is 260nm-270nm

[0021] Preferably, the detection wavelength is preferably 265 nm.

[0022] In the present invention, the flow rate of the mobile phase is 1.5 mL / min-2.5 mL / min

[0023] Preferably, the flow rate is 2.0 mL / min.

[0024] Specifically, the degradation impurities of the BHT include: m-cresol, p-methylphenol, butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxybenzoic acid, 2-tert-butyl-p-cresol, 6-tert-butyl-m-cresol, 3,5-di-tert-butyl-4-hydroxybenzaldehyde, and 2,4-bis(1,1-dimethylethyl)-5-methylphenol.

[0025] In the examples of the present application, the injection used is doxorcalciferol injection.

[0026] Specifically, the present invention calculates the content of butylated hydroxytoluene (BHT) degradation impurities by using an external standard method according to the chromatogram of the test solution and the chromatogram of the reference solution.

[0027] Further specifically, the present invention adopts the formula: target substance concentration in sample = (ru / rs)×(Cs / Cu)×100; ru: peak area of ​​a certain analyte in the test solution, rs: peak area of ​​doxercalciferol in the reference solution; Cs: concentration of doxercalciferol reference in the reference solution (mg / mL), Cu: concentration of a certain analyte in the test solution (mg / mL).

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention adopts the above-mentioned chromatographic conditions to remove the interference of auxiliary materials in doxorcalciferol injection, and can accurately determine the content of butylated hydroxytoluene (BHT, 2,6-di-tert-butylphenol) degradation impurities, and can simultaneously determine the content of the main component and BHT degradation impurities under the same conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the HPLC chromatogram of the mixed impurity solution 1 in Example 1.

[0031] Figure 2 This is the high performance liquid chromatogram of the mixed impurity solution 2 in Example 1.

[0032] Figure 3 This is the HPLC chromatogram of the mixed impurity solution 3 in Example 1. DETAILED DESCRIPTION

[0033] 1. As used herein, the articles "a", "an" and "the" include plural referents unless expressly limited to one or more referents otherwise.

[0034] 2. As used herein, numerical range: Unless otherwise expressly indicated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, a range or ratio of 1 to 30 stated should be considered to be included between a minimum value of 1 and a maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio consisting of integers or decimals including the end points.

[0035] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0036] The following sources of raw materials are provided for illustrative purposes:

[0037] Instruments: Agilent 1260 Infinity HPLC; Shimadzu AUW120D 1 / 100,000 electronic balance;

[0038] Reagents: Injection drug preparation, doxorcalciferol reference substance (source: QCC; item number: QH323420; purity: 99.26%), m-cresol reference substance (source: China Food and Drug Research and Inspection Institute; item number: 101154; purity: 99.5%), p-cresol reference substance (source: China Food and Drug Research and Inspection Institute; item number: 101153; purity: 99.3%), butylated hydroxyanisole (BHA) reference substance (source: SINCO; item number: S13336; purity: 95.45%), 3,5-di-tert-butyl-4-hydroxybenzoic acid reference substance (source: SINCO; item number: D16488; purity: 98.23%), 2-tert-butyl-p-cresol reference substance (source: SINC O; Article No.: B17336; Purity: 99.03%), 6-tert-butyl-m-cresol (Source: SINCO; Article No.: B17337; Purity: 95.17%), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (Source: SINCO; Article No.: D14996; Purity: 99.73%), 2,4-bis(1,1-dimethylethyl)-5-methylphenol reference substance (Source: SINCO; Article No.: B17338; Purity: 98.31%), butylated hydroxytoluene (BHT, 2,6-di-tert-butylphenol) reference substance (Source: SINCO; Article No.: B17339; Purity: 99.15%); phosphoric acid (Tianjin Damao), acetonitrile (MREDA), methanol (MREDA).

[0039] Example 1 Determination of HPLC Conditions

[0040] Preparation of doxorcalciferol injection: doxorcalciferol 0.02 mg, BHT 0.2 mg, edetate disodium 0.11 mg, anhydrous ethanol 0.5 mL, polysorbate 20 100 mg, sodium chloride 15 mg, anhydrous disodium hydrogen phosphate 76 mg, sodium dihydrogen phosphate monohydrate 78 mg, dilute with water to 10 mL.

[0041] Preparation of spiked test solution: Take appropriate amount of m-cresol reference substance, p-cresol reference substance, butylated hydroxyanisole (BHA) reference substance, 3,5-di-tert-butyl-4-hydroxybenzoic acid reference substance, 2-tert-butyl-p-cresol reference substance, 6-tert-butyl-m-cresol reference substance, 3,5-di-tert-butyl-4-hydroxybenzaldehyde reference substance, 2,4-bis(1,1-dimethylethyl)-5-methylphenol reference substance, and 2,6-di-tert-butylphenol reference substance, add appropriate amount of doxorubicin injection solution to dissolve and dilute to make a solution containing approximately 0.02 μg of each in each mL.

[0042] 1. Based on the related substance determination method of the antioxidant butylated hydroxytoluene USP quality standard (USP2023), the injection volume was increased to 50 μL and the spiked test solution was tested.

[0043] The chromatographic conditions were as follows: the chromatographic column was an octadecylsilane bonded silica gel column (Finome Luna C18, 150×4.6 mm, 3 μm);

[0044] The mobile phase was 5% acetic acid solution-acetonitrile (35:65); the detection wavelength was 275 nm; the injection volume was 50 μL; the column temperature was 40°C; the flow rate was 0.7 mL / min; and the elution mode was isocratic elution.

[0045] The chromatogram of the spiked test solution is shown in Table 1. Only three known impurities (3,5-di-tert-butyl-4-hydroxybenzoic acid, 3,5-di-tert-butyl-4-hydroxybenzaldehyde and 2,6-di-tert-butylphenol) were detected. Three of the known impurities were included in the excipient peak of doxorcalciferol injection. The remaining three impurities were not detected. Therefore, the specificity of this method cannot meet the detection requirements.

[0046] Table 1

[0047] Component name Retention time (min) 3,5-Di-tert-butyl-4-hydroxybenzoic acid 6.723 3,5-Di-tert-butyl-4-hydroxybenzaldehyde 11.245 2,6-Di-tert-butylphenol 22.735

[0048] 2. Based on the related substance determination method of the USP quality standard (USP2023) of doxorcalciferol raw material, increase the injection volume to 1000μL and detect the spiked test solution.

[0049] The chromatographic conditions were as follows:

[0050] The chromatographic column was an octadecylsilane bonded silica column (Poroshell 120EC-C18, 150 × 4.6 mm, 4 μm);

[0051] Mobile phase A is water; mobile phase B is acetonitrile; detection wavelength is: 265nm; injection volume is 1000μL; column temperature is 35℃; flow rate is 1.7mL / min; elution mode is gradient elution, and the elution program is shown in Table 2.

[0052] Table 2

[0053] Time (min) Mobile phase A (%) Mobile phase B (%) 0 25 75 15 23 77 20 10 90 30 10 90 36 0 100 50 0 100 50.1 25 75 60 25 75

[0054] The results of the chromatograms of the spiked test solution are shown in Tables 3 and 4:

[0055] Table 3

[0056] Component name Retention time (min) m-Cresol 2.916 p-Toluene 2.915 3,5-Di-tert-butyl-4-hydroxybenzoic acid 3.274 Butylated Hydroxyanisole 3.436 2-tert-Butyl-p-cresol 3.890 6-tert-Butyl-m-cresol 3.910 3,5-Di-tert-butyl-4-hydroxybenzaldehyde 4.328 2,4-Bis(1,1-dimethylethyl)-5-methylphenol 5.648 2,6-Di-tert-butylphenol 6.167

[0057] Table 4

[0058]

[0059]

[0060] According to the above results, 6 known impurities of BHT and doxorcalciferol were detected in the spectrum of the spiked test sample, and the separation between the impurities was good. However, 3 known impurities of BHT (m-cresol, p-cresol and 3,5-di-tert-butyl-4-hydroxybenzoic acid) were wrapped by the excipient peaks and could not be detected. In addition, the impurity peaks that eluted earlier were affected by the excipient peaks. The specificity of this method could not meet the detection requirements, so the method will be optimized based on this method.

[0061] 3. Based on 2, the chromatographic conditions are optimized as follows:

[0062] The chromatographic column was an octadecylsilane bonded silica column (Poroshell 120EC-C18, 150 × 4.6 mm, 4 μm);

[0063] Mobile phase A was 0.01% phosphoric acid solution; mobile phase B was acetonitrile; detection wavelength was 265 nm; injection volume was 1000 μL; column temperature was 35° C.; flow rate was 2.0 mL / min; elution mode was gradient elution, and the elution conditions were shown in Table 5.

[0064] Table 5

[0065] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 15 23 77 45 10 90 45.1 75 25 60 75 25

[0066] Impurity mixed solution 1: Take m-cresol reference substance, butylated hydroxyanisole (BHA) reference substance, 2-tert-butyl-p-cresol reference substance, 3,5-di-tert-butyl-4-hydroxybenzaldehyde reference substance, and 2,4-bis(1,1-dimethylethyl)-5-methylphenol reference substance, add doxorcalciferol injection to dissolve and dilute to make a solution containing 0.02 μg of each in each 1 mL.

[0067] Impurity mixed solution 2: Take p-methylphenol reference substance, 3,5-di-tert-butyl-4-hydroxybenzoic acid reference substance, and 6-tert-butyl-meta-cresol reference substance, add doxorcalciferol injection to dissolve and dilute to make a solution containing 0.02 μg of each in each 1 mL.

[0068] Impurity mixed solution 3: Take p-methylphenol reference substance, 6-tert-butyl-m-cresol reference substance, and 2,6-di-tert-butylphenol reference substance, add doxorcalciferol injection to dissolve and dilute to make a solution containing 0.02 μg of each in each 1 mL.

[0069] The samples are impurity mixed solutions 1, 2, and 3. The chromatogram results are as follows: Figure 1-Figure 3 As shown in Table 6:

[0070] Table 6

[0071]

[0072] According to the above results, all 9 known impurities of BHT can be detected in the mixed impurity spectrum, and the separation between each impurity is good; among them, m-cresol and p-cresol, 2-tert-butyl-p-cresol and 6-tert-butyl-m-cresol are isomers that elute at the same retention time, which is consistent with the USP standard of BHT, and both impurities are controlled together; this method meets the detection requirements.

[0073] Example 2 System suitability test

[0074] Preparation method of reference solution: take about 10 mg of docalciferol reference substance, accurately weigh it, place it in a 100 mL volumetric flask, dissolve it and dilute it to the scale with methanol, shake it well; accurately measure 1 mL, place it in a 100 mL volumetric flask, dilute it to the scale with methanol, shake it well, as the reference substance stock solution; accurately measure 1 mL of the reference substance stock solution, place it in a 100 mL volumetric flask, dilute it to the scale with 50% methanol, shake it well, and obtain it.

[0075] Preparation method of sensitivity solution: Accurately measure 1 mL of reference solution, place in a 10 mL volumetric flask, dilute to the mark with 50% methanol, and shake well.

[0076] The reference solution was injected 6 times and the sensitivity solution was injected 1 time to investigate the injection precision and sensitivity of the titration method. The results are shown in Table 7.

[0077] Table 7

[0078]

[0079] Conclusion: According to the above data, the injection precision and system applicability of this method meet the requirements.

[0080] Example 3 Specificity Detection

[0081] Blank solution: 50% methanol

[0082] Blank excipients: Take appropriate amounts of docalciferol, edetate disodium, ethanol, polysorbate 20, sodium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate, dissolve in water and dilute to make solutions containing 2 μg, 1.1 mg, 50 mg, 10 mg, 1.5 mg, 14.4 mg, and 1.8 mg respectively per 1 mL.

[0083] Preparation method of impurity localization solution:

[0084] (1) Preparation of impurity stock solution: Take about 10 mg of each impurity reference substance, accurately weigh it, place it in a 100 mL volumetric flask, dissolve it with 50% methanol and dilute it to the mark, shake well; accurately measure 1 mL, place it in a 100 mL volumetric flask, dilute it to the mark with methanol, and shake well.

[0085] (2) Impurity localization solution: Take 1 mL of each impurity stock solution, place it in a 50 mL volumetric flask, dilute to the mark with 50% methanol, and shake well.

[0086] The blank solution, blank excipient and each impurity location solution were sampled and the results are shown in Table 8.

[0087] Table 8

[0088]

[0089]

[0090] Conclusion: According to the above data, blank solvents and blank excipients do not interfere with the detection of various impurities; among them, m-cresol and p-cresol, 2-tert-butyl-p-cresol and 6-tert-butyl-m-cresol are isomers that elute at the same retention time, which is consistent with the USP standard of BHT, and both impurities are controlled together; therefore, the specificity of this method meets the requirements.

[0091] Example 4 Quantitative Limit Detection

[0092] Quantitation limit solution: take m-cresol reference substance, p-methylphenol reference substance, butylated hydroxyanisole (BHA) reference substance, 3,5-di-tert-butyl-4-hydroxybenzoic acid reference substance, 2-tert-butyl-p-cresol reference substance, 6-tert-butyl-m-cresol reference substance, 3,5-di-tert-butyl-4-hydroxybenzaldehyde reference substance, 2,4-bis(1,1-dimethylethyl)-5-methylphenol reference substance, and 2,6-di-tert-butylphenol reference substance, and add methanol to quantitatively prepare solutions containing approximately 0.002 mg, 0.002 mg, 0.002 mg, 0.0004 mg, 0.001 mg, 0.001 mg, 0.0004 mg, 0.002 mg, and 0.002 mg respectively per 1 mL.

[0093] The quantitative limit solutions of the known impurities of each BHT were sampled and the results are shown in Table 9:

[0094] Table 9

[0095]

[0096] Note: Equivalent to detection amount (%) = degradation impurity concentration / main component concentration (2mg / mL)

[0097] Conclusion: Based on the above data, the sensitivity of this method meets the requirements.

[0098] Example 5 Linearity and range detection

[0099] Doxorcalciferol linear solutions 1-6: Take a doxorcalciferol reference substance, add methanol to dissolve it into a 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.040 μg, 0.010 μg, 0.016 μg, 0.020 μg, 0.030 μg, and 0.040 μg per 1 mL.

[0100] Meta-cresol linear solutions 1-6: Take meta-cresol reference substance, add methanol to dissolve it into 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.10μg, and 0.20μg per 1mL.

[0101] p-Cresol linear solutions 1-6: Take p-Cresol reference substance, dissolve it in methanol to make a 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.016 μg, 0.032 μg, 0.049 μg, 0.065 μg, 0.16 μg, and 0.32 μg per 1 mL.

[0102] Butylated hydroxyanisole (BHA) linear solutions 1-6: Take butylated hydroxyanisole (BHA) reference substance, add methanol to dissolve it into 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.10 μg, and 0.20 μg per 1 mL.

[0103] 3,5-di-tert-butyl-4-hydroxybenzoic acid linear solutions 1-6: Take 3,5-di-tert-butyl-4-hydroxybenzoic acid reference substance, add methanol to dissolve it into 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.10μg, and 0.20μg per 1mL.

[0104] 6-tert-Butyl-meta-cresol linear solutions 1-6: Take 6-tert-Butyl-meta-cresol reference substance, add methanol to dissolve it into 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.015μg, 0.030μg, 0.04μg, 0.06μg, 0.15μg, and 0.30μg per 1mL.

[0105] 2-tert-Butyl-p-cresol linear solutions 1-6: Take 2-tert-butyl-p-cresol reference substance, add methanol to dissolve it into 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.10μg, and 0.20μg per 1mL.

[0106] 3,5-di-tert-butyl-4-hydroxybenzaldehyde linear solutions 1-6: Take 3,5-di-tert-butyl-4-hydroxybenzaldehyde reference substance, dissolve it in methanol to make a 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.10μg, and 0.20μg per 1mL.

[0107] 2,4-Bis(1,1-dimethylethyl)-5-methylphenol linear solutions 1-6: Take 2,4-bis(1,1-dimethylethyl)-5-methylphenol reference substance, dissolve it in methanol to make a 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.10μg, and 0.20μg per 1mL.

[0108] 2,6-Di-tert-butylphenol linear solutions 1-6: Take a 2,6-di-tert-butylphenol reference substance, dissolve it in methanol to make a 0.1 mg / mL solution, and then quantitatively dilute it with 35% methanol to make solutions containing approximately 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.10μg, and 0.20μg per 1mL.

[0109] The linear solutions of each BHT known impurity were sampled and the results are shown in Table 10.

[0110] Table 10

[0111] Components Linear regression equation Correlation coefficient Doxorcalciferol y=1142086.9574x-234.2768 0.9999 m-Cresol y=289312.7652x-245.4557 1.0000 p-Toluene y=244545.3425x-150.3544 1.0000 Butylated Hydroxyanisole (BHA) y=100008.8729x-33.5127 1.0000 3,5-Di-tert-butyl-4-hydroxybenzoic acid y=1335393.8880x-245.7848 1.0000 6-tert-Butyl-m-cresol y=219140.4290x-27.2089 1.0000 2-tert-Butyl-p-cresol y=164419.1126x-8.4937 0.9926 3,5-Di-tert-butyl-4-hydroxybenzaldehyde y=936141.5817x-354.3608 1.0000 2,4-Bis(1,1-dimethylethyl)-5-methylphenol y=122340.8495x+37.1329 0.9999 2,6-Di-tert-butylphenol y=155533.7291x-445.8861 1.0000

[0112] Conclusion: According to the above data, the linear correlation coefficients between each known impurity and doxorcalciferol are greater than 0.99, so the linearity of this method meets the requirements.

[0113] Example 6 Accuracy Detection

[0114] Recovery rates of m-cresol, butylated hydroxyanisole (BHA), 2-tert-butyl-p-cresol, 3,5-di-tert-butyl-4-hydroxybenzaldehyde and 2,4-bis(1,1-dimethylethyl)-5-methylphenol: Precision impurity mixed solution 1 0.5mL (3 portions), 1mL (3 portions), 2mL (3 portions) were placed in 9 10mL volumetric bottles and diluted to the mark with doxorubicin injection.

[0115] Recovery rates of p-methylphenol, 3,5-di-tert-butyl-4-hydroxybenzoic acid and 6-tert-butyl-m-cresol: Precision impurity mixed solution 2 0.5mL (3 portions), 1mL (3 portions), 2mL (3 portions) were placed in 9 10mL volumetric bottles and diluted to the scale with doxorubicin injection.

[0116] Recovery rate of 6-tert-butyl-meta-cresol and 2,6-di-tert-butylphenol: Precision impurity mixed solution 3 0.5mL (3 portions), 1mL (3 portions), 2mL (3 portions) were placed in 9 10mL volumetric bottles, and diluted to the scale with doxorcalciferol injection.

[0117] The test results of each solution are shown in Table 11.

[0118] Table 11

[0119]

[0120]

[0121] Conclusion: According to the above data, the average recovery rate of each known impurity is between 95.0% and 105.0%, so the accuracy of this method meets the requirements.

[0122] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting antioxidant degradation impurities in an injection, characterized in that: The antioxidant is butylated hydroxytoluene, and the detection method adopts high performance liquid chromatography. The high performance liquid chromatography detection conditions include: the chromatographic column is an octadecylsilane bonded silica gel column; the mobile phase is composed of mobile phase A and mobile phase B, the mobile phase A is a 0.01% phosphoric acid solution, and the mobile phase B is acetonitrile; gradient elution, and the gradient elution program is: a: 0min-15min: 75% mobile phase A decreases to 23% mobile phase A, and the rest is mobile phase B; b: 15min-45min: 23% mobile phase A decreased to 10% mobile phase A, the rest was mobile phase B; c: 45min-45.1min: 10% mobile phase A gradually increased to 75% mobile phase A, the rest was mobile phase B; d: 45.1min-60min: 75% mobile phase A, the rest mobile phase B.

2. The detection method according to claim 1, characterized in that: The diameter of the chromatographic column is 2-10 mm, the length of the column is 150-250 mm, and the particle size of the filler is 2-10 μm.

3. The detection method according to claim 1, characterized in that: The high performance liquid chromatography detection conditions also include an injection volume of 800 μL-1200 μL.

4. The detection method according to claim 3, characterized in that: The injection volume was 1000 μL.

5. The detection method according to claim 1, characterized in that: The high performance liquid chromatography detection conditions also include a detection wavelength of 260nm-270nm.

6. The detection method according to claim 5, characterized in that: The detection wavelength is 265 nm.

7. The detection method according to claim 1, characterized in that: The high performance liquid chromatography detection conditions also include a mobile phase flow rate of 1.5 mL / min-2.5 mL / min.

8. The impurity detection method according to claim 7, characterized in that: The flow rate of the mobile phase was 2.0 mL / min.

9. The detection method according to claim 1, characterized in that: The high performance liquid chromatography detection conditions also include a column temperature of the chromatographic column of 30-40°C.

10. The detection method according to claim 1, characterized in that: The antioxidant degradation impurities include any one or more of m-cresol, p-methylphenol, butylated hydroxyanisole, 3,5-di-tert-butyl-4-hydroxybenzoic acid, 2-tert-butyl-p-cresol, 6-tert-butyl-m-cresol, 3,5-di-tert-butyl-4-hydroxybenzaldehyde, 2,4-bis(1,1-dimethylethyl)-5-methylphenol or 2,6-di-tert-butylphenol.

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