A method for simultaneously and quantitatively detecting multiple phenolic antioxidants and related intermediates and degradation products

Abstract

The present invention discloses a method for simultaneously and quantitatively detecting a plurality of phenolic antioxidants and related intermediates and degradation products, using UPLC analysis, wherein the UPLC conditions are: chromatographic column C18 liquid chromatography column, 2.1×50mm, 1.8μm; injection volume 1-5μl; column temperature 30-40°C; detection wavelength 280nm; mobile phase is a three-phase mixed system, phase A is water, phase B is acetonitrile, phase C is isopropanol; gradient elution. The method for simultaneously and quantitatively detecting a plurality of phenolic antioxidants and related intermediates and degradation products provided by the present invention has a simple pretreatment method, is easy and quick to operate, has high sensitivity, and can simultaneously and quantitatively analyze a plurality of phenolic antioxidants and related intermediates and degradation products.

Description

Technical Field

[0001] The invention relates to the technical field of detection of antioxidants containing phenolic structures, and in particular to a method for simultaneously and quantitatively detecting a plurality of antioxidants containing phenolic structures and related intermediates and degradation products. Background Art

[0002] Antioxidants are a common type of additives used in a variety of pharmaceutical packaging materials. In plastic and rubber pharmaceutical packaging materials, common antioxidants mainly include antioxidants containing phenolic structures, such as tert-butyl-4-hydroxyanisole (CAS25013-16-5, antioxidant BHA), 2,6-di-tert-butyl-4-hydroxymethylphenol (CAS88-26-6, antioxidant 100), triethylene glycol ether-di(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate (CAS36443-68-2, antioxidant 245), 2,6-di-tert-butyl-p-methylphenol (CAS128-37-0, antioxidant BHT), divinyl [3,3-bis[3-(1,1-dimethylethyl)-4-hydroxyphenyl] butyrate (CAS32509-66-3, antioxidant O3), 1,3,5-tris(3,5-di-tert-butyl-4-hydroxy ... The main functions of these materials are to prevent or delay the oxidative degradation of polymers, thereby extending the service life of the materials.

[0003] During the sterilization and storage process of pharmaceutical packaging materials and drugs, antioxidants play a protective role by self-degradation, but antioxidants and their related intermediates and degradation products may migrate into drugs and affect the safety, effectiveness and quality control of drugs. Possible intermediates and degradation products include 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid (CAS20170-32-5, degradation product of antioxidant 1010), 4-tert-octylphenol (CAS140-66-9, tert-octylphenol), 2,4-di-tert-butylphenol (CAS96-76-4, degradation product of antioxidant 168), 2,6-di-tert-butylphenol (CAS128-39-2, degradation product of antioxidant 330), sulfur (CAS7704-34-9, degradation product of sulfur-containing antioxidants), etc. Therefore, it is necessary to test antioxidants and related intermediates and degradation products in pharmaceutical packaging materials and drugs.

[0004] Among the existing analytical methods, the GC method and the GC-MS method require that the antioxidant to be tested has good thermal stability and is volatile, and are suitable for antioxidants with a smaller molecular weight, but the types of antioxidants that can be analyzed are relatively limited; the HPLC method is suitable for antioxidants within a certain polarity range, and for antioxidants with large polarity differences, multiple HPLC methods need to be used for separate testing. For the various phenolic antioxidants and related intermediates and degradation products that may be used in pharmaceutical packaging materials, the existing test methods cannot simultaneously determine them due to large differences in boiling point or polarity. Therefore, it is urgent to provide a new analytical method for the simultaneous determination of various phenolic antioxidants and related intermediates and degradation products in pharmaceutical packaging materials and related drugs. Summary of the invention

[0005] The purpose of the present invention is to provide a method for simultaneously and quantitatively detecting a plurality of phenolic antioxidants and related intermediates and degradation products in view of the deficiencies in the prior art.

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

[0007] A method for simultaneously and quantitatively detecting a plurality of phenolic antioxidants and related intermediates and degradation products is provided, comprising the following steps:

[0008] Step 1: immerse the drug packaging material in the extraction medium and extract in a closed container to obtain an extraction solution; or dissolve or dilute the drug preparation in contact with the packaging material in the drug packaging to obtain an extraction solution;

[0009] Step 2, remove the extraction solution, add an organic solvent and an internal standard solution, extract, and stand to separate layers; remove the organic solution layer, and after concentration and dilution, use it as a test solution for the test sample;

[0010] Step 3, injecting the test solution into the UPLC system, recording the peak area of ​​each chromatographic peak, and performing quantitative analysis on the phenolic antioxidant and related intermediates and degradation products;

[0011] The UPLC conditions are:

[0012] Chromatographic column: C18 liquid chromatography column, 2.1×50mm, 1.8μm; injection volume: 1-5μl; column temperature: 30-40℃; detection wavelength: 280nm; mobile phase: three-phase mixed system, phase A: water, phase B: acetonitrile, phase C: isopropanol; gradient elution.

[0013] Furthermore, the phenolic structure-containing antioxidants and related intermediates and degradation products include: antioxidant BHA, antioxidant 100, antioxidant 1010 degradation products, tert-butyl phenol, antioxidant 168d degradation products, antioxidant 330 degradation products, antioxidant 245, antioxidant BHT, internal standard, antioxidant O3, sulfur, antioxidant 3114, antioxidant 1010, antioxidant 330, antioxidant 1076, and antioxidant 168.

[0014] Furthermore, the extraction medium is one of water, 0.9% sodium chloride solution, 5-100% methanol or ethanol or isopropanol solution, acidic buffer solution with pH=2.5-3.5, and alkaline buffer solution with pH=8.0-11.0.

[0015] Furthermore, the solvent used to dissolve or dilute the drug preparation in contact with the packaging material in the drug packaging is one of water for injection, 0.9% sodium chloride solution, and 5% glucose solution.

[0016] Furthermore, the organic solvent selected for the extraction is dichloromethane, n-hexane or a mixed solvent of the two.

[0017] Furthermore, the volume ratio of dichloromethane to n-hexane in the mixed solvent of dichloromethane and n-hexane is 1:(1-5).

[0018] Furthermore, the internal standard solution is a 4-bromo-2,6-di-tert-butylphenol solution.

[0019] Furthermore, the gradient elution is specifically:

[0020]

[0021] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0022] The method for simultaneously and quantitatively detecting a plurality of phenolic antioxidants and related intermediates and degradation products provided by the present invention has a simple pretreatment method, is easy and quick to operate, has high sensitivity, and can simultaneously and quantitatively analyze a plurality of phenolic antioxidants and related intermediates and degradation products.

[0023] Therefore, the method for simultaneously and quantitatively detecting a variety of phenolic antioxidants and related intermediates and degradation products is suitable for detecting and analyzing their extraction amounts and migration amounts in drug packaging materials and drug compatibility studies, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The UPLC chromatograms for detecting various antioxidants and related intermediates and degradation products in the embodiments of the present invention; wherein the chromatographic peaks are marked in the figure as antioxidant BHA, antioxidant 100, antioxidant 1010 degradation product, tert-octylphenol, antioxidant 168d degradation product, antioxidant 330 degradation product, antioxidant 245, antioxidant BHT, internal standard, antioxidant O3, sulfur, antioxidant 3114, antioxidant 1010, antioxidant 330, antioxidant 1076, and antioxidant 168. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0026] The present invention provides a method for simultaneously and quantitatively detecting a plurality of phenolic structure-containing antioxidants and related intermediates and degradation products, comprising the following steps:

[0027] Step 1: immerse the drug packaging material in the extraction medium and extract in a closed container to obtain an extraction solution; or dissolve or dilute the drug preparation in contact with the packaging material in the drug packaging to obtain an extraction solution;

[0028] Step 2, remove the extraction solution, adjust the pH value to acidic if necessary, add organic solvent and internal standard solution, extract, and stand to separate layers; remove the organic solution layer, add appropriate amount of methanol and then concentrate under reduced pressure, dilute with diluent and filter, as the test solution for the test sample;

[0029] Step 3, injecting the test solution into the UPLC system, recording the peak area of ​​each chromatographic peak, and performing quantitative analysis on the phenolic antioxidant and related intermediates and degradation products;

[0030] The UPLC conditions are:

[0031] Chromatographic column: C18 liquid chromatography column, 2.1×50mm, 1.8μm; injection volume: 1-5μl; column temperature: 30-40℃; detection wavelength: 280nm; mobile phase: three-phase mixed system, phase A: water, phase B: acetonitrile, phase C: isopropanol; gradient elution.

[0032] The above-mentioned phenolic structure antioxidants and related intermediates and degradation products include: antioxidant BHA, antioxidant 100, antioxidant 1010 degradation products, tert-butyl phenol, antioxidant 168d degradation products, antioxidant 330 degradation products, antioxidant 245, antioxidant BHT, internal standard, antioxidant O3, sulfur, antioxidant 3114, antioxidant 1010, antioxidant 330, antioxidant 1076, and antioxidant 168.

[0033] As a preferred example, the extraction medium is one of water, 0.9% sodium chloride solution, 5-100% methanol or ethanol or isopropanol solution, pH=2.5-3.5 acidic buffer solution, pH=8.0-11.0 alkaline buffer solution. The solvent used to dissolve or dilute the drug preparation in contact with the packaging material in the drug packaging is one of water for injection, 0.9% sodium chloride solution, 5-100% methanol or ethanol or isopropanol solution, pH=2.5-3.5 acidic buffer solution, pH=8.0-11.0 alkaline buffer solution.

[0034] As a preferred example, the organic solvent selected for extraction is dichloromethane, n-hexane or a mixed solvent of the two.

[0035] As a preferred example, the volume ratio of dichloromethane to n-hexane in the mixed solvent of dichloromethane and n-hexane is 1:(1-5), more preferably 1:1.

[0036] As a preferred example, the internal standard solution is 4-bromo-2,6-di-tert-butylphenol (CAS1139-52-2, bromophenol) solution.

[0037] Example 1

[0038] UPLC conditions included:

[0039] Chromatographic column EclipseplusC18, 2.1×50mm, 1.8μm; injection volume 3μl; column temperature 40℃; detection wavelength 280nm; mobile phase is a three-phase mixed system, phase A is water, phase B is acetonitrile, phase C is isopropanol, gradient elution; flow rate 0.4ml / min. The gradient program is as follows Table 1:

[0040] Table 1

[0041] Time (min) Phase A (%) Phase B (%) Phase C (%) 0.00 56.00 36.00 8.00 0.20 56.00 36.00 8.00 3.60 42.00 46.00 12.00 4.40 36.00 48.00 16.00 5.50 19.00 55.00 26.00 8.50 9.00 51.00 40.00 12.00 2.00 44.00 54.00 13.50 56.00 36.00 8.00

[0042] Accurately weigh an appropriate amount of bromophenol, dissolve it in dichloromethane, and dilute it to volume with acetone to make the internal standard working solution.

[0043] Take 30 ml of the extract, add an appropriate amount of 4% phosphoric acid solution if necessary, add 5 ml of a mixed solvent (the volume ratio of dichloromethane to n-hexane is 1:1), add an appropriate amount of internal standard working solution, shake vigorously, let stand to separate layers, take 1 ml of the upper solution, add 0.5 ml of methanol, mix well, place in a vacuum drying device to evaporate to a volume of about 0.4 ml, add 5% dichloromethane in methanol solution to make up the volume to 1 ml, mix well, and use as the test solution for the test sample.

[0044] Accurately weigh appropriate amounts of antioxidant BHA, antioxidant 100, antioxidant 1010 degradation products, tert-octyl phenol, antioxidant 168 degradation products, antioxidant 330 degradation products, antioxidant 245, antioxidant BHT, sulfur, antioxidant O3, antioxidant 3114, antioxidant 1010, antioxidant 330, antioxidant 1076, and antioxidant 168, dissolve them in dichloromethane, and dilute to volume with acetone to obtain a control working solution.

[0045] The method was investigated for the determination of the content of antioxidants and related degradation products in five media: water, 0.9% sodium chloride injection, pH2.5 buffer solution, pH8.0 buffer solution, and 15% ethanol. Take 30 ml of each different extraction medium, of which the pH8.0 buffer solution was adjusted to acidic by adding an appropriate amount of 4% phosphoric acid solution. Add 5 ml of mixed solvent (the volume ratio of dichloromethane to n-hexane is 1:1), add an appropriate amount of internal standard working solution, add low, medium and high concentration control working solutions, shake vigorously, let stand and separate, take 1 ml of the upper solution, add 0.5 ml of methanol, mix well, place in a vacuum drying device to evaporate to a volume of about 0.4 ml, add 5% dichloromethane methanol solution to make up the volume to 1 ml, and mix well. Take 3 μl of the above test solution and inject it into the UPLC system, determine each analyte in the sample solution according to the specified chromatographic conditions, and use the internal standard correction method for quantitative calculation.

[0046] In the ultra-high performance liquid chromatography method established by the present invention:

[0047] The detection limit of the antioxidant BHA is 0.08μg / ml, the quantification limit is 0.26μg / ml, the linearity is good in the range of 0.26μg / ml to 5.13μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 101% and 104%.

[0048] The detection limit of antioxidant 100 is 0.16μg / ml, the quantification limit is 0.55μg / ml, the linearity is good in the range of 0.55μg / ml to 10.98μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 109% and 117%.

[0049] The detection limit of 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid is 0.16μg / ml and the quantification limit is 0.53μg / ml. It has good linearity in the range of 0.53μg / ml to 10.69μg / ml and good stability within 4 days. The recoveries in the five medium extracts are all between 109% and 121%.

[0050] The detection limit of tert-octylphenol is 0.12μg / ml, the quantification limit is 0.39μg / ml, the linearity is good in the range of 0.39μg / ml to 7.70μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 105% and 110%.

[0051] The detection limit of 2,4-di-tert-butylphenol is 0.08μg / ml, the quantification limit is 0.26μg / ml, the linearity is good in the range of 0.26μg / ml to 5.20μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 95% and 107%.

[0052] The detection limit of 2,6-di-tert-butylphenol is 0.12μg / ml, the quantification limit is 0.41μg / ml, the linearity is good in the range of 0.41μg / ml to 8.24μg / ml, the stability is good within 4 days, and the recoveries in the five medium extracts are between 96% and 103%.

[0053] The detection limit of antioxidant 245 is 0.10μg / ml, the quantification limit is 0.33μg / ml, the linearity is good in the range of 0.33μg / ml to 6.69μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 108% and 118%.

[0054] The detection limit of the antioxidant BHT is 0.08μg / ml, the quantification limit is 0.27μg / ml, the linearity is good in the range of 0.27μg / ml to 5.42μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 94% and 100%.

[0055] The detection limit of antioxidant O3 is 0.06μg / ml, the quantification limit is 0.20μg / ml, the linearity is good in the range of 0.20μg / ml to 4.04μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 107% and 120%.

[0056] The detection limit of sulfur was 0.03 μg / ml, the quantification limit was 0.11 μg / ml, the linearity was good in the range of 0.11 μg / ml to 2.20 μg / ml, the stability was good within 4 days, and the recoveries in the five medium extracts were all between 97% and 105%.

[0057] The detection limit of antioxidant 3114 is 0.16μg / ml, the quantification limit is 0.53μg / ml, the linearity is good in the range of 0.53μg / ml to 10.50μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 110% and 121%.

[0058] The detection limit of antioxidant 1010 is 0.13μg / ml, the quantification limit is 0.43μg / ml, the linearity is good in the range of 0.43μg / ml to 8.50μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 111% and 121%.

[0059] The detection limit of antioxidant 330 is 0.09μg / ml, the quantification limit is 0.30μg / ml, the linearity is good in the range of 0.30μg / ml to 5.92μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 111% and 121%.

[0060] The detection limit of antioxidant 1076 is 0.23μg / ml, the quantification limit is 0.75μg / ml, the linearity is good in the range of 0.75μg / ml to 15.07μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 110% and 120%.

[0061] The detection limit of antioxidant 168 is 0.27μg / ml, the quantification limit is 0.89μg / ml, the linearity is good in the range of 0.89μg / ml to 17.72μg / ml, the stability is good within 4 days, and the recovery rates in five medium extracts are between 110% and 118%.

[0062] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously and quantitatively detecting a plurality of phenolic antioxidants and related intermediates and degradation products, characterized in that: The steps include: Step 1: immerse the drug packaging material in the extraction medium and extract in a closed container to obtain an extraction solution; or dissolve or dilute the drug preparation in contact with the packaging material in the drug packaging to obtain an extraction solution; The extraction medium is one of water, 0.9% sodium chloride solution, 5-100% methanol or ethanol or isopropanol solution, acidic buffer solution with pH=2.5-3.5, and alkaline buffer solution with pH=8.0-11.0; Step 2, remove the extraction solution, add an organic solvent and an internal standard solution, extract, and stand to separate layers; remove the organic solution layer, and after concentration and dilution, use it as a test solution for the test sample; the organic solvent is a mixed solvent of dichloromethane and n-hexane; Step 3, injecting the test solution of the test sample into the UPLC system, recording the peak area of ​​each chromatographic peak, and performing quantitative analysis on the phenolic antioxidant and related intermediates and degradation products; The phenolic structure-containing antioxidants and related intermediates and degradation products include: antioxidant BHA, antioxidant 100, antioxidant 1010 degradation products, tert-octylphenol, antioxidant 168d degradation products, antioxidant 330 degradation products, antioxidant 245, antioxidant BHT, internal standard, antioxidant O3, sulfur, antioxidant 3114, antioxidant 1010, antioxidant 330, antioxidant 1076, antioxidant 168; The UPLC conditions are: Chromatographic column: C18 liquid chromatography column, 2.1×50mm, 1.8μm; injection volume: 1~5μl; column temperature: 30~40℃; detection wavelength: 280nm; mobile phase: three-phase mixed system, phase A: water, phase B: acetonitrile, phase C: isopropanol; gradient elution; The gradient elution is specifically: Time min Phase A % Phase B % Phase C % 0.00 56.00 36.00 8.00 0.20 56.00 36.00 8.00 3.60 42.00 46.00 12.00 4.40 36.00 48.00 16.00 5.50 19.00 55.00 26.00 8.50 9.00 51.00 40.00 12.00 2.00 44.00 54.00 13.50 56.00 36.00 8.00。 2. The method according to claim 1, characterized in that: The solvent used to dissolve or dilute the drug preparation in contact with the packaging material in the drug packaging is one of water for injection, 0.9% sodium chloride solution, and 5% glucose solution.

3. The method according to claim 1, characterized in that: The volume ratio of dichloromethane to n-hexane in the mixed solvent of dichloromethane and n-hexane is 1:(1-5).

4. The method according to claim 1, characterized in that: The internal standard solution is 4-bromo-2,6-di-tert-butylphenol solution.

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