A method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles

Abstract

This invention discloses a method for simultaneously detecting six phenolic antioxidants and their degradation products in polyethylene bottles, belonging to the field of analytical chemistry. The disclosed detection method enables the simultaneous determination of six phenolic antioxidants and their degradation products in pharmaceutical low-density polyethylene bottles. The sample solution is prepared using ultrasonic extraction, and the six phenolic antioxidants and their degradation products are separated using high-performance liquid chromatography (HPLC), achieving excellent separation. The content of the six phenolic antioxidants and their degradation products in pharmaceutical low-density polyethylene bottles is calculated based on peak area using the external standard method, achieving accurate quantification. This method is simple, environmentally friendly, and efficient, filling the gap in the domestic and international markets for the simultaneous quantitative determination of six antioxidants and their degradation products.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles. Background Art

[0002] In the pharmaceutical industry, plastics are often used in the form of sheets, films, tubes, bottles, bags, blisters, etc., which are used to package drugs alone or in combination with components made of other materials to protect drugs from external environments such as air, moisture, and microbial contamination. Low-density polyethylene (LDPE) is often used to produce bottles, bags, tubes, and inner and outer layers of multi-layer composite bags for packaging drugs.

[0003] In actual production, antioxidants are usually added to low-density polyethylene to prevent degradation. During the storage and transportation of drugs, antioxidants may migrate into the drug solution, affecting the quality of the drug. At the same time, antioxidants also have the risk of self-degradation, and their degradation products also have the risk of migrating into the drug, affecting the efficacy and even causing toxicity. Therefore, in the study of packaging material compatibility, it is necessary to analyze and detect antioxidant degradation products.

[0004] Common antioxidant degradation products in low-density polyethylene bottles for pharmaceutical packaging include 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid (degradation product of antioxidant 1076), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (degradation product of antioxidant 3114), and 2,4-di-tert-butylphenol (degradation product of antioxidant 168). In addition, triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate] (antioxidant 245), 2,6-di-tert-butyl-4-methylphenol (antioxidant 264), and 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester (EP plastic additive 08) are often added to pharmaceutical low-density polyethylene bottles to improve performance. EP plastic additive 08 has a phenolic hydroxyl group in its chemical structure, which can play a good antioxidant role and is a common additive in plastics. However, the existing technology does not meet the separation requirements when determining antioxidant degradation products, antioxidants, and EP plastic additive 08, and it is impossible to achieve the simultaneous determination of antioxidant degradation products, antioxidants, and EP plastic additive 08. Multiple tests are required to complete the determination of all compounds.

[0005] At present, there are few relevant standards and regulations for the detection methods of antioxidant degradation products in pharmaceutical low-density polyethylene bottles. The National Standard for Pharmaceutical Packaging Materials contains the quality standards for "low-density polyethylene infusion bottles" (National Standard for Pharmaceutical Packaging Materials YBB00012002-2015 Low-density polyethylene infusion bottles) and "low-density polyethylene medicinal eye drop bottles" (National Standard for Pharmaceutical Packaging Materials YBB00062002-2015 Low-density polyethylene medicinal eye drop bottles), but does not stipulate the limits and test methods for antioxidant degradation products. In the quality standard for "low-density polyethylene infusion bottles", thin-layer chromatography was used to test di(octadecyl) disulfide and 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester in low-density polyethylene infusion bottles, but it was impossible to accurately quantify.

[0006] USP43 version <661.1> (USP43 version <661> Plastic packaging systems and their materials of construction.) also did not set limits and test methods for antioxidant degradation products in pharmaceutical low-density polyethylene bottles. High-performance liquid chromatography was used to quantitatively determine 2,6-di-tert-butyl-4-methylphenol (antioxidant 264), USP plastic additive 1 (3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester), USP plastic additive 2 (antioxidant 1010), USP plastic additive 3 (antioxidant 330), USP plastic additive 4 (antioxidant 1076), USP plastic additive 5 (antioxidant 168), and USP plastic additive 6 (antioxidant 3114). Thin layer chromatography was used to perform limit checks on USP Plastic Additive 8 (dioctadecyl disulfide), USP Plastic Additive 9 (antioxidant DLTP), and USP Plastic Additive 10 (antioxidant DSTP), but accurate quantification was not possible.

[0007] EP10.0 version 3.1.5 (EP10.0 version 3.1.5 Polyethylene with additives for containers for parenteral preparations and for ophthalmic preparations.) EP plastic additive 07 (antioxidant 264), EP plastic additive 08, EP plastic additive 09 (antioxidant 1010), EP plastic additive 10 (antioxidant 330), EP plastic additive 11 (antioxidant 1076), EP plastic additive 12 (antioxidant 168), EP plastic additive 13 (antioxidant 3114) in polyethylene bottles were determined by high performance liquid chromatography. EP plastic additive 14 (antioxidant 618), EP plastic additive 15 (dioctadecyl disulfide), EP plastic additive 16 (antioxidant DLTP), EP plastic additive 17 (antioxidant DSTP) were tested by thin layer chromatography, but accurate quantification was not possible. However, antioxidant degradation products were not determined. See Table 1 for details.

[0008] Table 1 Comparison of pharmacopoeias of various countries

[0009] .

[0010] Patent document CN111595995A discloses a method for simultaneously detecting 12 antioxidants and free sulfur using ultra-high performance liquid chromatography, but does not provide a method for extracting antioxidants and antioxidant degradation products from drug packaging materials, and is unable to determine the content of antioxidants and antioxidant degradation products in packaging materials. Moreover, the invention uses ultra-high performance liquid chromatography for detection, and a chromatographic column with a particle size of 3 μm is used in the method, and an ultra-high performance liquid chromatograph is required, which has high requirements for instruments and chromatographic columns, and high detection costs.

[0011] Patent document CN109470784A discloses a method for screening antioxidants in pharmaceutical packaging elastomer seals by liquid chromatography-mass spectrometry, but does not provide a method for extracting antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles, and is unable to determine the content of antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles. The invention uses liquid chromatography-mass spectrometry for quantitative analysis, which has high requirements for instruments and equipment, high detection costs, and is not suitable for large-scale technology promotion. The invention uses microwave digestion for sample pretreatment, which requires the use of a microwave digester, which has high instrument requirements. The subsequent sample processing method is complicated, resulting in low detection efficiency.

[0012] Patent document CN109521130A discloses a method for detecting antioxidants in elastomeric seals for pharmaceutical packaging, but does not provide an extraction method for antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles, and is unable to detect antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles. The invention uses microwave digestion for extraction, which requires the use of a microwave digestion instrument. The subsequent processing method is complex, has high requirements for instruments, and has low processing efficiency. This method cannot determine the content of triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-tolyl) propionate] (antioxidant 245) and 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester (EP plastic additive 08).

[0013] Patent document CN114441691B discloses the detection of additives 08 and 15 in low-density polyethylene bottles. For the extraction of additive 08, the extractants used are toluene and methanol, tetrahydrofuran and acetonitrile. However, the invention does not provide an extraction method for antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles, and it is impossible to detect antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles. The invention uses toluene and methanol, tetrahydrofuran and acetonitrile to extract additive 08, and the extraction reagents are highly toxic and cause greater environmental pollution. This method also cannot meet the requirements of separation degree, and it is impossible to simultaneously determine the degradation products of additive 08 and antioxidant 245, antioxidant 264, and antioxidants 1076, 3114, and 168.

[0014] In summary, there is an urgent need to develop a method that can simultaneously determine antioxidant degradation products, antioxidants, and EP plastic additives 08 to accurately quantify additives in medicinal low-density polyethylene bottles. Summary of the invention

[0015] In view of the above problems, the present invention discloses a method for extracting and detecting six phenolic antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles. The method adopts ultrasonic extraction to prepare sample solution, and adopts high performance liquid chromatography to separate the six phenolic antioxidants and antioxidant degradation products, thereby achieving good separation requirements and accurate quantification.

[0016] In order to achieve the above object, the present invention provides the following technical solutions:

[0017] The present invention discloses a method for detecting six phenolic antioxidants and antioxidant degradation products in a medicinal low-density polyethylene bottle, wherein the specific steps of the detection method include:

[0018] Step 1 Preparation of the test solution: Take the shredded polyethylene bottle, add the extraction reagent, perform ultrasonic extraction, filter after ultrasonication, and take the filtrate as the test solution;

[0019] Step 2 Preparation of mixed reference solution: taking the six phenolic antioxidants and antioxidant degradation product reference substances respectively, diluting them to a fixed volume with anhydrous ethanol, mixing them, and preparing a series of concentrations of mixed reference substance solutions;

[0020] Step 3: Detection by high performance liquid chromatography: Using high performance liquid chromatography, accurately measure the blank extraction solution, the reference solution and the test solution, respectively inject them into the liquid chromatograph, perform gradient elution on each solution according to the high performance liquid chromatography detection conditions, and record the chromatogram;

[0021] Step 4: Determination of the contents of six phenolic antioxidants and antioxidant degradation products: According to the external standard method, the contents of six phenolic antioxidants and antioxidant degradation products are calculated using the peak areas.

[0022] Furthermore, the extraction reagent in step 1 is anhydrous ethanol.

[0023] Furthermore, step 1 is specifically to cut the low-density polyethylene bottle to be tested into strips of 0.5 cm×2 cm~1 cm×3 cm, take about 1 g of the cut medicinal low-density polyethylene bottle, accurately weigh it, put it in a stoppered test tube, accurately measure 10 mL of anhydrous ethanol and add it to the stoppered test tube, cover and seal it, sonicate it, filter it with a 0.22µm filter membrane, and take the filtrate as the test solution.

[0024] Furthermore, the ultrasonic extraction conditions are: at 20-40° C., for 20-60 minutes.

[0025] Preferably, the ultrasonic extraction conditions in step 1 are: ultrasonication for 30 minutes at 30°C.

[0026] Furthermore, the preparation of the mixed reference solution in step 2 is specifically to take the six phenolic antioxidants and antioxidant degradation products of the reference substances respectively, accurately weigh them and place them in different volumetric bottles, add anhydrous ethanol to dissolve them respectively and make up to the scale, shake them well, and use them as the reference substance stock solutions of each compound; accurately measure the reference substance stock solutions of the above six compounds in the same volumetric flask, add anhydrous ethanol to dilute and make up to the scale, shake them well, and prepare mixed reference substance solutions with concentrations of 9.6µg / mL, 1.6µg / mL, 6.4µg / mL, 12.8µg / mL, 9.6µg / mL, and 6.4µg / mL, respectively.

[0027] Furthermore, the HPLC detection conditions in step 3 are: C 18 Reverse phase chromatography column; mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution; column temperature is 30℃; flow rate: 1.0mL / min; injection volume: 10µL.

[0028] Furthermore, the HPLC detection wavelength in step 3 is 275 nm.

[0029] Furthermore, the gradient elution conditions in step 3 are:

[0030] .

[0031] Furthermore, the six phenolic antioxidants and antioxidant degradation products mentioned above are: 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid; 3,5-di-tert-butyl-4-hydroxybenzaldehyde; 2,4-di-tert-butylphenol; triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate]; 2,6-di-tert-butyl-4-methylphenol; 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester.

[0032] Furthermore, the detection limits of the above-mentioned six phenolic antioxidants and antioxidant degradation products are as follows: the detection limit of 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid is 0.058µg / mL; the detection limit of 3,5-di-tert-butyl-4-hydroxybenzaldehyde is 0.009µg / mL; the detection limit of 2,4-di-tert-butylphenol is 0.039µg / mL; the detection limit of triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate] is 0.076µg / mL; the detection limit of 2,6-di-tert-butyl-4-methylphenol is 0.056µg / mL; the detection limit of 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester is 0.037µg / mL. Application of any of the above methods for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles in detecting six phenolic antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles.

[0033] The invention also discloses an extraction method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles, which is characterized in that the method adopts anhydrous ethanol ultrasonic extraction method to pre-treat the sample to be tested.

[0034] Furthermore, the specific steps include: cutting the medicinal low-density polyethylene bottle to be tested into strips of 0.5cm×2cm~1cm×3cm, taking about 1g of the cut medicinal low-density polyethylene bottle, accurately weighing it, placing it in a stoppered test tube, accurately measuring 10mL of anhydrous ethanol and adding it to the stoppered test tube, covering and sealing it, ultrasonicating it, filtering it with a 0.22µm filter membrane, and taking the filtrate.

[0035] Furthermore, the ultrasonic extraction conditions are: at 20-40° C., for 20-60 minutes.

[0036] Preferably, the ultrasonic extraction conditions in step 1 are: ultrasonication for 30 minutes at 30°C.

[0037] Furthermore, the six phenolic antioxidants and antioxidant degradation products are: 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid; 3,5-di-tert-butyl-4-hydroxybenzaldehyde; 2,4-di-tert-butylphenol; triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate]; 2,6-di-tert-butyl-4-methylphenol; 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester.

[0038] Compared with the prior art, the present invention has the following beneficial effects.

[0039] 1. The present invention establishes a high-sensitivity, easy-to-operate HPLC method that can accurately determine the contents of six phenolic antioxidants and their degradation products in medicinal low-density polyethylene bottles, filling the gap in the lack of quantitative determination methods for antioxidant degradation products in USP and EP.

[0040] 2. The present invention establishes a method for extracting six phenolic antioxidants and antioxidant degradation products from medicinal low-density polyethylene bottles, which is simple to operate, environmentally friendly and efficient. It is used to prepare samples for the determination of six phenolic antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles. The method is simple, environmentally friendly and efficient. The extraction test data of different solvents and different methods show that the anhydrous ethanol ultrasonic extraction method of the present invention has better repeatability and recovery rate.

[0041] 3. The existing technologies mostly focus on the determination of antioxidants themselves, ignoring the determination of degradation products produced by antioxidant degradation. The detection method disclosed in the present invention can accurately and simultaneously determine the contents of 6 phenolic antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles.

[0042] 4. Triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate] (Antioxidant 245) and 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester (EP plastic additive 08) are often added to medicinal low-density polyethylene bottles to improve performance. EP plastic additive 08 has phenolic hydroxyl groups in its chemical structure and has antioxidant effects. It is a common additive in plastics. When the existing method is used to simultaneously determine antioxidant degradation products, antioxidants, and EP plastic additive 08, the separation degree does not meet the requirements, and other methods are needed to determine triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate] (antioxidant 245) and 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester (EP plastic additive 08). The present invention can meet the simultaneous determination and improve the inspection efficiency.

[0043] 5. The detection method disclosed in the present invention not only realizes the simultaneous determination of six phenolic antioxidants and antioxidant degradation products, thereby greatly improving the detection efficiency, but also, after a large number of creative experiments, creatively adopts chromatographic acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, and adjusts the gradient elution ratio of different mobile phases at different times to adjust the polarity of the mobile phase, so that antioxidants of different polarities can be better separated, so that the separation degree meets the requirements, and finally uses a liquid chromatograph at a wavelength of 275nm to realize the simultaneous determination of six phenolic antioxidants and antioxidant degradation products. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 UV full wavelength scan of 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid.

[0045] Figure 2 UV full wavelength scan of 3,5-di-tert-butyl-4-hydroxybenzaldehyde.

[0046] Figure 3 UV full wavelength scan of 2,4-di-tert-butylphenol.

[0047] Figure 4 UV full wavelength scan of triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate].

[0048] Figure 5 UV full wavelength scan of 2,6-di-tert-butyl-4-methylphenol.

[0049] Figure 6UV full wavelength scan of 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester.

[0050] Figure 7 Chromatogram of the separation of each compound in the single-label reference substance. Among them, A is 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid; B is 3,5-di-tert-butyl-4-hydroxybenzaldehyde; C is 2,4-di-tert-butylphenol; D is antioxidant 245; E is antioxidant 264; and F is EP plastic additive 08.

[0051] Figure 8 Comparison of the separation of each compound in the single-label reference substance.

[0052] Fig. 9 Chromatogram of mixed reference sample screened under gradient conditions.

[0053] Fig.10 Chromatograms of six phenolic antioxidants and antioxidant degradation products of the present invention.

[0054] Fig.11 Chromatogram of EP plastic additive 08 under three chromatographic conditions of the prior art. DETAILED DESCRIPTION

[0055] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will contribute to the understanding of the present invention, but these embodiments are only for the purpose of illustrating the present invention, and the present invention is not limited to these contents. The operating methods in the embodiments are all conventional operating methods in the art.

[0056] The codes of the six phenolic antioxidants and antioxidant degradation products described in the present invention and their corresponding chemical names, Chinese names, and codes in pharmacopoeias of various countries are shown in Table 2.

[0057] Table 2 Correspondence table of the names of the six compounds in the present invention

[0058]

[0059] Example 1. Comparison of key conditions and screening tests.

[0060] 1. Experimental comparison of different extraction methods.

[0061] The prior art mostly uses microwave digestion or toluene reflux for pretreatment, which has complicated processing steps and high requirements for instruments. The present invention provides an anhydrous ethanol ultrasonic extraction method for sample pretreatment, which is simple and environmentally friendly, has low requirements for instruments, can reduce the introduction of impurities, has good repeatability and recovery rate, and is convenient for liquid chromatography detection.

[0062] The specific operating steps of the ultrasonic extraction method are as follows: cut the medicinal low-density polyethylene bottle into strips of 0.5cm×2cm~1cm×3cm, take about 1g of the cut medicinal low-density polyethylene bottle, accurately weigh it, and put it in a stoppered test tube, accurately measure 10mL of anhydrous ethanol, toluene and water respectively, add them to the stoppered test tube, cover and seal it, ultrasonicate for 30 minutes at 30℃, filter it with a 0.22µm filter membrane, and take the filtrate as the test solution.

[0063] Specific operation steps of anhydrous ethanol microwave: cut the medicinal low-density polyethylene bottle into strips of 0.5cm×2cm~1cm×3cm, take about 1g of the cut medicinal low-density polyethylene bottle, accurately weigh it, put it in a microwave extraction tank, accurately add 10mL of anhydrous ethanol, cover and seal it, put it in a microwave digester at 80℃ for 30min, let it cool to room temperature, filter the extract with a 0.22µm filter membrane, and take the filtrate as the test solution control solution 1.

[0064] Specific operation steps for reflux of anhydrous ethanol: cut the medicinal low-density polyethylene bottle into strips of 0.5cm×2cm~1cm×3cm, take about 5g of the cut medicinal low-density polyethylene bottle, accurately weigh it, put it in a round-bottom flask, add 40mL of anhydrous ethanol, heat and reflux to extract for 30min, cool to room temperature, transfer the extract to a 50mL volumetric flask, add an appropriate amount of anhydrous ethanol to wash the round-bottom flask, transfer the washing solution to the same 50mL volumetric flask, add anhydrous ethanol to the scale, shake well, filter with a 0.22µm filter membrane, and take the filtrate as the test solution control solution 2.

[0065] The comparison of different extraction methods is shown in Table 3.

[0066] Table 3 Comparison of data from different extraction methods

[0067] .

[0068] From the data in the above table, it can be seen that when using anhydrous ethanol ultrasonic extraction, the repeatability and recovery rate of the sample are good; when using toluene ultrasonic extraction, the relative standard deviation of the repeatability of the sample is large, and the parallelism of the extraction is not good; when using water ultrasonic extraction, the recovery rate result is good, but the test compound was not detected in the repeatability sample, so the water ultrasound cannot achieve the purpose of extraction; when using anhydrous ethanol microwave digestion for pretreatment, the recovery rate is low; when using anhydrous ethanol for reflux extraction, the relative standard deviation of the repeatability is large, and the parallelism is not good. In summary, the use of anhydrous ethanol ultrasonic extraction of six phenolic antioxidants and degradation products in medicinal low-density polyethylene bottles has better repeatability and recovery rate, and achieves the best effect.

[0069] 2. Screening of chromatographic conditions.

[0070] 1. Screening of detection wavelength.

[0071] Take the six phenolic antioxidants and antioxidant degradation products of the reference substances, accurately weigh them and place them in different volumetric bottles, add anhydrous ethanol to dissolve them and make up to the scale, shake well, and use them as the reference substance stock solutions of each compound; accurately measure the reference substance stock solutions of the above six compounds and put them in the same volumetric flask, add anhydrous ethanol to dilute and make up to the scale, shake well, and prepare mixed reference substance solutions with concentrations of 9.6µg / mL, 1.6µg / mL, 6.4µg / mL, 12.8µg / mL, 9.6µg / mL, and 6.4µg / mL, respectively.

[0072] In the process of detecting wavelength screening, six phenolic antioxidants and antioxidant degradation products were scanned at full wavelength. Figure 1 to Figure 6 .

[0073] The maximum absorption wavelength of each compound was determined based on the ultraviolet full-wavelength scanning diagram of six phenolic antioxidants and antioxidant degradation products. The ultraviolet absorption of the six compounds in the present invention was at a peak position at 275nm, with good ultraviolet response and no interference from the solvent peak. Finally, 275nm was determined as the detection wavelength of this method.

[0074] 2. Screening of organic phase ratio.

[0075] The preparation of the test solution was carried out according to the steps described in the experimental comparison of different extraction methods in Example 1, and the preparation of the mixed control solution was carried out according to the steps described in the detection wavelength screening experiment in Example 1.

[0076] When screening chromatograms, we first examined the separation of each compound under isocratic conditions and selected C 18 Reversed phase column; acetonitrile: 0.1% formic acid water ratio 90:10, isocratic elution; column temperature 30℃; detection wavelength 275nm; flow rate: 1.0mL / min; injection volume: 10µL. The separation of each compound is shown in Figure 7~Figure 8 The results showed that 3,5-di-tert-butyl-4-hydroxybenzaldehyde and triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate] (antioxidant 245) could not be separated under these conditions.

[0077] 3. Gradient condition screening.

[0078] The preparation of the test solution was carried out according to the steps described in the experimental comparison of different extraction methods in Example 1, and the preparation of the mixed control solution was carried out according to the steps described in the detection wavelength screening experiment in Example 1.

[0079] According to the test results of the above 2 organic phase ratio screening, consider reducing the organic phase concentration, but reducing the organic phase ratio will inevitably increase the total retention time, so consider using gradient elution conditions. Select C 18Reverse phase chromatography column, mobile phase A is chromatographic acetonitrile, mobile phase B is 0.1% formic acid aqueous solution; gradient elution, elution conditions are shown in Table 4 below; column temperature 30°C; detection wavelength 275nm; flow rate: 1.0mL / min; injection volume: 10µL.

[0080] Table 4 HPLC elution conditions

[0081] .

[0082] The results show that 2,6-di-tert-butyl-4-methylphenol (antioxidant 264) and EP plastic additive 08 cannot be separated under this condition. See the chromatogram Fig. 9 .

[0083] To separate the six compounds, continue to adjust the gradient elution conditions and select C 18 Reverse phase chromatography column, mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution; gradient elution, elution conditions are shown in Table 5 below; column temperature 30°C; detection wavelength 275nm; flow rate: 1.0mL / min; injection volume: 10µL.

[0084] Table 5 HPLC elution conditions

[0085] .

[0086] Under this condition, the six phenolic antioxidants and antioxidant degradation products can be well separated. The separation degree is shown in Table 6 below, which meets the separation degree requirements. Fig.10 .

[0087] Table 6 Separation of six phenolic antioxidants and antioxidant degradation products of the present invention

[0088] .

[0089] Example 2 The method for determining six phenolic antioxidants and antioxidant degradation products in a medicinal low-density polyethylene bottle disclosed in the present invention comprises the following steps.

[0090] 1. Preparation of test solution: Cut the medicinal low-density polyethylene bottle into strips of 0.5cm×2cm~1cm×3cm. Take about 1g of the cut medicinal low-density polyethylene bottle, accurately weigh it, and put it in a stoppered test tube. Accurately measure 10mL of anhydrous ethanol and add it to the stoppered test tube. Cover and seal it. Ultrasonicate at 30℃ for 30 minutes, filter it with a 0.22µm filter membrane, and take the filtrate as the test solution.

[0091] 2. Preparation of mixed reference solution: Take about 20 mg of each reference substance of compound 1 to 6 (see Table 2), weigh accurately, place in different 25 mL volumetric flasks, add anhydrous ethanol to dissolve and dilute to scale, shake well, and use as reference substance stock solution of each compound; accurately measure 3.0 mL, 0.5 mL, 2.0 mL, 4.0 mL, 3.0 mL, and 2.0 mL of reference substance stock solution of the above compounds 1 to 6 in the same 25 mL volumetric flask, dilute with anhydrous ethanol and dilute to scale, shake well, and use as mixed reference substance stock solution 1. Prepare mixed reference substance stock solution 2 in the same way. Accurately measure 1.0 mL of mixed reference substance stock solutions 1 and 2 in different 10 mL volumetric flasks, dilute with anhydrous ethanol and dilute to scale, shake well, and use as mixed reference substance solutions 1 and 2. The concentrations of the prepared mixed reference solutions were 9.6µg / mL, 1.6µg / mL, 6.4µg / mL, 12.8µg / mL, 9.6µg / mL, and 6.4µg / mL, respectively.

[0092] 3. The HPLC conditions for detecting six phenolic antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles: C 18 Reverse phase chromatographic column; mobile phase A is chromatographic acetonitrile, mobile phase B is 0.1% formic acid aqueous solution; gradient elution, elution conditions are shown in Table 7 below; column temperature 30°C; detection wavelength 275nm; flow rate: 1.0mL / min; injection volume: 10µL.

[0093] Table 7 HPLC elution conditions

[0094] .

[0095] 4. Determination method: According to the HPLC method (General Rule 0512) of Part IV of the 2020 edition of the Chinese Pharmacopoeia, accurately measure 10 µL of each of the blank extraction solution, reference solution, and test solution, inject them into the liquid chromatograph, and record the chromatogram. According to the external standard method, calculate the contents of the six phenolic antioxidants and antioxidant degradation products by peak area.

[0096] Example 3 Detection limit of the detection method disclosed in the present invention.

[0097] The present invention uses a conventional C18 chromatographic column and a common high performance liquid chromatograph for testing, but obtains a lower detection limit than the ultra-high performance liquid chromatography disclosed in patent document CN111595995A, has a higher detection sensitivity, and the instrument is generally available, which greatly reduces the testing cost. The comparison of detection limits is shown in Table 8.

[0098] Table 8 Detection limit comparison table

[0099] .

[0100] Example 4 The method disclosed in the present invention is compared with the prior art.

[0101] 1. Compared with patent document CN109521130A, the method disclosed in the document cannot determine the content of triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate] (antioxidant 245) and 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester (EP plastic additive 08). Triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate] (antioxidant 245) and 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester (EP plastic additive 08) are often added in the production process of low-density polyethylene bottles for medicinal use to improve performance. EP plastic additive 08 has phenolic hydroxyl groups in its chemical structure and has antioxidant effects. It is a common additive in plastics. When this method is used to simultaneously determine antioxidant degradation products, antioxidants, and EP plastic additive 08, the separation does not meet the requirements. The chromatographic peak of EP plastic additive 08 is located on the gradient peak and cannot be accurately quantified. Fig.11 . It is necessary to separately determine triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate] (Antioxidant 245) and 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester (EP Plastic Additive 08), which is time-consuming and labor-intensive.

[0102] 2. The comparison between the present invention and the prior art is shown in Table 9.

[0103] Table 9 Comparison between the present invention and the prior art

[0104] .

[0105] In summary, the present invention establishes an extraction and detection method for six phenolic antioxidants and antioxidant degradation products in a medicinal low-density polyethylene bottle, adopts an ultrasonic extraction method to prepare a sample solution, and adopts a high-performance liquid chromatography method to separate the six phenolic antioxidants and antioxidant degradation products, thereby achieving a good separation requirement and accurate quantification. Compared with the prior art, the method for determining the six phenolic antioxidants and antioxidant degradation products in a medicinal low-density polyethylene bottle provided by the present invention can accurately and simultaneously determine the contents of the six phenolic antioxidants and antioxidant degradation products in the medicinal low-density polyethylene bottle, and the prior art does not have a method for simultaneously determining the contents of these six additives, which can fill the technical gap in this field.

[0106] The existing technology focuses on the determination of antioxidants themselves, ignoring the determination of degradation products produced by antioxidant degradation. The invention can achieve the simultaneous determination of six phenolic antioxidants and antioxidant degradation products in medicinal low-density polyethylene bottles.

[0107] The prior art mostly uses microwave digestion or toluene reflux for pretreatment, which has complicated processing steps and high requirements for instruments. The present invention provides an anhydrous ethanol ultrasonic extraction method for sample pretreatment, which is simple and environmentally friendly, has low requirements for instruments, can reduce the introduction of impurities, has good repeatability and recovery rate, and is convenient for liquid chromatography detection.

[0108] The prior art uses expensive ultra-high performance liquid chromatography for testing, while the present invention uses a conventional C18 chromatographic column and an ordinary high performance liquid chromatograph for testing, which achieves a lower detection limit than the technical ultra-high performance liquid chromatography method, a higher detection sensitivity, and the instrument is widely available, which greatly reduces the testing cost.

Claims

1. A method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles, characterized in that: The specific steps of the method include: Step 1 Preparation of the test solution: Take the shredded polyethylene bottle, add the extraction reagent, perform ultrasonic extraction, filter after ultrasonication, and take the filtrate as the test solution; Step 2 Preparation of mixed reference solution: taking the six phenolic antioxidants and antioxidant degradation product reference substances respectively, diluting them to a fixed volume with anhydrous ethanol, mixing them, and preparing a series of concentrations of mixed reference substance solutions; Step 3: Detection by high performance liquid chromatography: Using high performance liquid chromatography, accurately measure the blank extraction solution, the reference solution and the test solution, respectively inject them into the liquid chromatograph, perform gradient elution on each solution according to the high performance liquid chromatography detection conditions, and record the chromatogram; The HPLC detection conditions are: 18 Reversed phase chromatographic column; mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid aqueous solution; column temperature 30℃; flow rate: 1.0mL / min; injection volume: 10µL; detection wavelength is 275nm; Gradient elution conditions: ； Step 4: Determination of the contents of six phenolic antioxidants and antioxidant degradation products: by using the external standard method, the contents of six phenolic antioxidants and antioxidant degradation products are calculated by peak areas; The six phenolic antioxidants and antioxidant degradation products are: 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid; 3,5-di-tert-butyl-4-hydroxybenzaldehyde; 2,4-di-tert-butylphenol; triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl) propionate]; 2,6-di-tert-butyl-4-methylphenol; 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester.

2. The method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles according to claim 1, characterized in that: The extraction reagent in step 1 is anhydrous ethanol.

3. The method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles according to claim 1, characterized in that: Step 1 is to cut the low-density polyethylene bottle to be tested into strips of 0.5 cm × 2 cm ~ 1 cm × 3 cm, take about 1 g of the cut medicinal low-density polyethylene bottle, accurately weigh it, put it in a stoppered test tube, accurately measure 10 mL of anhydrous ethanol and add it to the stoppered test tube, cover and seal it, sonicate it, filter it with a 0.22µm filter membrane, and take the filtrate as the test solution.

4. The method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles according to any one of claims 1 to 3, characterized in that: The ultrasonic extraction conditions are: at 20-40° C., and for 20-60 minutes.

5. The method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles according to claim 4, characterized in that: The ultrasonic extraction conditions are: ultrasonication for 30 minutes at 30°C.

6. The method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles according to claim 1, characterized in that: Step 2 is the preparation of mixed control solutions, specifically, taking the six phenolic antioxidants and antioxidant degradation products of the reference substances, accurately weighing them and placing them in different volumetric bottles, adding anhydrous ethanol to dissolve them and dilute them to the scale, shaking them well, and using them as the reference substance stock solutions of each compound; accurately measuring the reference substance stock solutions of the above six compounds in the same volumetric flask, adding anhydrous ethanol to dilute them and dilute them to the scale, shaking them well, and preparing mixed reference substance solutions with concentrations of 9.6µg / mL, 1.6µg / mL, 6.4µg / mL, 12.8µg / mL, 9.6µg / mL, and 6.4µg / mL, respectively.

7. The method for simultaneously detecting six phenolic antioxidants and antioxidant degradation products in polyethylene bottles according to claim 1, characterized in that: Among them, the detection limit of 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid is 0.058µg / mL; the detection limit of 3,5-di-tert-butyl-4-hydroxybenzaldehyde is 0.009µg / mL; the detection limit of 2,4-di-tert-butylphenol is 0.039µg / mL; the detection limit of triethylene glycol bis[3-(3-tert-butyl-4-hydroxy-5-methylphenyl)propionate] is 0.076µg / mL; the detection limit of 2,6-di-tert-butyl-4-methylphenol is 0.056µg / mL; the detection limit of 3-(1,1-dimethylethyl)-β-[3-(1,1-dimethylethyl)-4-hydroxyphenyl]-4-hydroxy-β-methylbenzoic acid-1,2-ethylene ester is 0.037µg / mL.

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