Method for detecting cyclosiloxane, phthalate plasticizer and nitrosamine in medicinal packaging material

Abstract

The invention relates to the technical field of detection of residues in medicine packaging materials, and discloses a method for detecting cyclosiloxane, a phthalate plasticizer and nitrosamine in a medicinal packaging material. Comprising the following steps: S1, preparing reference substance solutions: respectively preparing an epoxy silane mixed reference substance solution A, a phthalate plasticizer mixed reference substance solution B and a nitrosamine mixed reference substance solution C from corresponding residues; s2, preparing a sample solution: respectively extracting materials by using an organic solvent to obtain an epoxy silane sample solution A ', a phthalate plasticizer sample solution B' and a nitrosamine sample solution C '; s3, content detection: correspondingly performing gas chromatography-mass spectrometry detection on the reference substance solution and the sample solution, and calculating the content of the residues to be detected in the sample solution. According to the scheme, the contents of the three types of residues in the medicinal packaging material are detected, so that the safety of the medicinal packaging material is comprehensively evaluated, and the accuracy of an evaluation result is effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of residue detection in pharmaceutical packaging materials, and in particular to a method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in pharmaceutical packaging materials. Background Art

[0002] During the production process of pharmaceutical packaging materials (such as rubber plugs), a variety of reagents (including lubricants, plasticizers, etc.) need to be added, which may cause the content of residues such as cyclosiloxanes, phthalate plasticizers and nitrosamines in the rubber plug products to exceed the standard. Testing the content of the above-mentioned residues in the rubber plugs can effectively control the quality of the rubber plug products and improve their safety in use. However, the detection accuracy of existing detection technologies is limited, which significantly reduces the safety of rubber plugs in use.

[0003] For example, dimethicone is a widely used lubricant with excellent performance. It is made by catalytic polymerization of dimethylsiloxane ring body (DMC, i.e. cyclosiloxane), and belongs to the polymer of polydimethylsiloxane series. Among them, hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5) and dodecamethylcyclohexasiloxane (D6) are the main residues in polydimethylsiloxane polymers. According to the existing toxicological research results, D4, D5 and D6 have toxic effects on multiple organs or systems of mammals, especially D4, which is defined by the European Union as a Class 3 substance that is toxic to reproduction.

[0004] Nitrosamines are a class of highly toxic and carcinogenic chemicals with a nitroso structure, and are recognized as one of the three major carcinogens in the world. In 2009, the European Commission issued Directive 2009 / 48 / EEC, stipulating that the migration of total nitrosamines in elastomer or rubber nipples and pacifiers should be ≤0.05mg / kg; in 1999, the American Society for Testing and Materials re-approved the standard specification for nitrosamines in pacifiers (ASTM F1313-90), requiring the total migration to be ≤0.02mg / kg; in 2009, my country issued GB / T 24153-2009 "Method for the determination of nitrosamine content in rubber and elastomer materials and their products". However, in the detection process of this method, the compound is only cracked once to generate fragment ions, and one or several characteristic ions are scanned; due to the wide variety of nitrosamines and their strong carcinogens, specific trace detection is required, but the detection limit of this method is high and it cannot accurately characterize the nitrosamine content in packaging materials.

[0005] Plasticizers are widely used additives for polymer materials in industry. Adding this substance to plastic processing can enhance its flexibility and make it easier to process. However, plasticizers can affect the endocrine system and reproductive system, leading to diseases such as endocrine disorders. Especially for children in their developmental period, plasticizers may cause serious harm to their bodies and are not conducive to their growth and development. At the same time, they can cause problems with the immune system and induce cancer and other diseases, and their accumulation in the environment may also have long-term effects on the ecology. Therefore, the use of plasticizers needs to be strictly controlled.

[0006] As a plastic rubber product, rubber plugs use a large number of plasticizers to modify rubber during the production process. However, excessive abuse of plasticizers will cause the plasticizers in the material to enter the medicine and then enter the human body, causing serious effects on the body. Therefore, it is necessary to detect the plasticizers in rubber plugs. However, because there are many types of plasticizers, if only one or several can be detected according to some existing methods, it is easy to miss them.

[0007] In view of this, the development of a detection method for cyclosiloxanes, phthalate plasticizers and nitrosamines in pharmaceutical packaging materials can not only effectively make up for the shortcomings of the existing technology, but also effectively improve the detection accuracy of residues in pharmaceutical packaging materials, which is of great significance to improving the safety of drug use. Summary of the invention

[0008] The present invention aims to provide a method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in pharmaceutical packaging materials, so as to solve the technical problem that the prior art easily misses the above residues when detecting them, thereby reducing the detection accuracy.

[0009] To achieve the above object, the present invention adopts the following technical scheme: a method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in pharmaceutical packaging materials, comprising the following steps:

[0010] S1. Preparation of reference solution: Prepare epoxy silane mixed reference solution A, phthalate plasticizer mixed reference solution B and nitrosamine mixed reference solution C respectively according to the residues;

[0011] S2. Prepare sample solution: take the injection powder sample, add water and organic solvent to mix, vortex oscillate to extract the obtained supernatant, and dilute to volume with n-hexane to obtain epoxy silane sample solution;

[0012] The rubber plug is crushed with liquid nitrogen, extracted with an organic solvent and then filtered, and the filtrate is the nitrosamine sample solution;

[0013] Take the injection powder sample and add water to prepare the drug solution, take the rubber stopper and add the drug solution as the simulated extraction solution, after heating and extraction, take the simulated extraction solution and add n-hexane according to proportion to extract the supernatant by vortex oscillation to obtain the phthalate plasticizer sample solution;

[0014] S3. Content detection: The reference solution and the sample solution are subjected to gas chromatography-mass spectrometry detection to obtain the content of the residue to be detected in the sample solution.

[0015] The principles and advantages of this solution are:

[0016] 1. Compared with the existing technology that only detects one type of residue, or even can only detect 2-3 types of residues in one type of residue, which is prone to omissions and reduces the detection accuracy, this solution detects the content of three types of residues in pharmaceutical packaging materials, comprehensively evaluates the safety of pharmaceutical packaging materials based on the test results, effectively improves the accuracy of the evaluation results, and thus improves the safety of drug use.

[0017] 2. Compared with the existing technology for detecting pharmaceutical packaging materials with a high detection limit resulting in low detection accuracy, this solution extracts sample solutions from different samples separately to facilitate the full extraction of sample content in pharmaceutical packaging materials, and then uses gas chromatography-mass spectrometry equipment to separately detect the reference solution and sample solution of different samples, effectively improving the sample detection accuracy, and then accurately evaluating the content of cyclosiloxanes, phthalate plasticizers and nitrosamines in pharmaceutical packaging materials, thereby improving drug safety.

[0018] 3. This solution uses liquid nitrogen pulverization to extract nitrosamines, which effectively improves the extraction effect of nitrosamines. The applicant analyzed that the reason is that since nitrosamines are strong carcinogenic substances, trace amounts of nitrosamines can cause harm to the human body, so the sample needs to be pulverized and extracted with an organic solvent to completely extract the nitrosamines in the rubber plug; the sample must be pulverized at ultra-low temperature to avoid the high temperature generated during the pulverization causing the volatilization loss of nitrosamines in the rubber plug. And the applicant has found through long-term experiments that if the sample is not pulverized, there will be incomplete extraction during the extraction process, resulting in low and inaccurate test results. If the sample is pulverized without liquid nitrogen freezing, the sample temperature will reach a high temperature of nearly 100 degrees Celsius during the pulverization process, which is likely to cause some low-boiling nitrosamines to volatilize, resulting in low and inaccurate test results.

[0019] Preferably, as an improvement, the organic solvent is n-hexane or ethyl acetate.

[0020] Technical effect: This scheme adopts the above-mentioned setting, which facilitates the full extraction of the residues to be tested in the material, thereby effectively improving the accuracy of the test results.

[0021] Preferably, as an improvement, in S1, it also includes preparing reference substance working solutions with different concentration gradients.

[0022] Technical effect: This scheme adopts the above settings, which is convenient for on-machine testing, and linear regression calculation is performed with the injection concentration as the horizontal axis and the peak area as the vertical axis to obtain the linear regression equation and correlation coefficient of each reference substance component. After the sample is tested, it can be substituted into the regression equation to calculate the content and concentration of the residue in the sample solution, effectively ensuring the accuracy and reliability of the analysis. For example, after preparing the epoxy silane mixed reference substance solution A, it is diluted into reference substance working solutions with different gradient concentrations (such as each 1ml reference substance working solution contains about 1μg, 2μg, 5μg, 10μg, 15μg, 20μg, 25μg of reference substances respectively). After the on-machine test, it is convenient to fit the linear regression equation and correlation coefficient of each reference substance to ensure the accuracy and reliability of the analysis.

[0023] Preferably, as an improvement, in S2, it also includes preparing a sample spike solution, the method is as follows: adding a corresponding reference solution to each sample solution to obtain a sample spike solution.

[0024] Technical effect: This scheme adopts the above settings, and by testing the sample spike solution on the machine, the content of each residue in the sample can be calculated by comparing the known concentration of the reference substance in the sample spike solution with the linear regression equation. The applicant has found through long-term experiments that, especially for sample solutions with low residue content, by configuring the sample spike solution, the content of the residue to be tested in the solution can be effectively increased, thereby effectively avoiding the problem of large errors in the test results due to the fact that the residue is lower than the quantitative limit, thereby effectively improving the accuracy of the test results.

[0025] Preferably, as an improvement, S3 also includes the determination of the quantitative limit and the detection limit, including the following method: take a mixed reference solution of known concentration, dilute it step by step, and detect it until S / N=3 is the detection limit and S / N=10 is the quantitative limit.

[0026] Technical effect: This scheme adopts the above settings to facilitate the rapid determination of the detection limit and quantification limit of each residue to be tested in the sample solution, thereby effectively evaluating the accuracy, reliability and consistency of the detection method.

[0027] Preferably, as an improvement, the epoxysilane mixed reference solution A contains hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6) reference substances; the phthalate plasticizer mixed reference solution B contains 23 plasticizer reference substances, including dimethyl phthalate, diethyl phthalate, diisopropyl phthalate, diallyl phthalate, dipropyl phthalate, diisobutyl phthalate, dibutyl phthalate, dimethoxyethyl phthalate, diisoamyl phthalate, di(4-methyl-2-pentyl) phthalate, di(2-methyl-2-pentyl) phthalate, The nitrosamine mixed reference substance solution C contains 9 kinds of nitrosamine reference substances, including N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosodipropylamine, N-nitrosopiperidine, N-nitrosodibutylamine and N-nitrobenzylamine.

[0028] Technical effect: This scheme adopts the above settings to effectively cover the detection of multiple residues, integrate the detection results of multiple residues, and effectively comprehensively evaluate the safety of the use of pharmaceutical packaging materials.

[0029] Preferably, as an improvement, the gas chromatography conditions of the nitrosamines are: using a capillary chromatographic column, carrier gas: helium, flow rate: 1.0 ml / min, inlet temperature: 250°C, transfer tube temperature: 260°C, ion source temperature: 270°C; column temperature: initial temperature 50°C maintained for 4 min, increased to 100°C at 10°C / min and maintained for 2 min, then increased to 250°C at 20°C / min and maintained for 6 min.

[0030] Preferably, as an improvement, the gas chromatography conditions of the phthalate plasticizer are: using a capillary chromatographic column, an injection volume of 1 μL; the injection port temperature is 280°C, the split ratio is 50:1, the carrier gas is helium, and the column flow rate is 0.5 ml / min; the detector type is MS / MS, the detection mode is SRM mode, the transmission line temperature is 300°C, and the ion source temperature is 300°C.

[0031] Preferably, as an improvement, the gas chromatography conditions of the epoxy silane are: using a capillary chromatographic column, the chromatographic column temperature is programmed, the initial temperature is 33-37°C, maintained for 5 minutes, heated to 180°C at 10°C / min, maintained for 0 minutes, and then heated to 240°C at 60°C / min and maintained for 3 minutes; the injection port temperature is 250°C; the split ratio is 10:1; the carrier gas is nitrogen, and the column flow rate is 1.45-1.55 ml / min; the detector is FID, the temperature is 250°C; the injection volume is 1 μL.

[0032] Technical effect: This scheme adopts the above settings to improve the detection sensitivity and accuracy. Specifically, compared with the conventional 5% phenyl / 95% polydimethylsiloxane stationary phase chromatographic column, this scheme uses the TG-5MS gas phase mass spectrometry dedicated chromatographic column for inert treatment of the stationary phase, which can not only withstand higher temperature thermal shock and chemical corrosion, but also has a long life and high stability. Its column bleed will be much lower than that of conventional chromatographic columns, thereby greatly reducing the baseline noise caused by column bleed, improving the signal-to-noise ratio of the chromatographic peak, and improving the detection sensitivity, so that the detection limit reaches the ppb level. And this scheme adopts a chromatographic column with a specification of 60m×0.25mm and a film thickness of 0.25μm. Compared with conventional chromatographic columns, the longer chromatographic column length and smaller inner diameter are used to maximize the column efficiency, so as to improve the separation ability of the method, ensure that the target compound is not interfered by impurities, and improve the accuracy of the detection results.

[0033] Furthermore, the column temperature of this scheme adopts a lower initial temperature, such as an initial temperature 20°C lower than the boiling point of the solvent. When the sample is partially or completely condensed at the column head, the solvent begins to evaporate. The target substance with a volatility close to that of the solvent will be concentrated in the non-volatile solvent, thereby producing a very narrow initial spectrum band, making the peak shape sharper to improve the shape and separation of the chromatographic peak. The applicant has found through long-term experiments that if the initial temperature is close to the boiling point of the solvent or higher than the boiling point of the solvent, when the sample enters the chromatographic column, the solvent will evaporate rapidly, and the residual target substance will form a certain broadening at the column head, making the chromatographic peak shape worse and reducing the separation.

[0034] Finally, this scheme selects the AUTO SRM mode of the instrument to screen the parent ion, daughter ion, and collision energy of each compound, and selects the mass spectrometry parameters with the highest response, the largest signal-to-noise ratio, and the best specificity to improve the sensitivity of detection, reduce noise, and improve selectivity. The applicant found through long-term experiments that if the SIM mass spectrometry method is directly established based on the NIST spectral library of the compound, it is difficult to achieve the sensitivity of this method, and because the fragment ion specificity of the primary mass spectrometry is lower than that of the secondary mass spectrometry, it is easy to be interfered by impurities, there is a possibility of false positives, and the compound cannot be accurately identified and quantified. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The chromatograms of different solutions of cyclosiloxane in Example 1 of the present invention (a is blank solvent, b is reference solution, c is sample spiked solution, d is 0 month-0.25 g specification of batch A, e is 0 month-0.5 g specification of batch A, and f is 0 month-1.0 g specification of batch A).

[0036] Figure 2 This is the standard curve calibration diagram of the cyclosiloxane reference solution in Example 1 of the present invention (a is D3, b is D4, c is D5, and d is D6).

[0037] Figure 3 The chromatogram of the cyclosiloxane reference solution in Example 1 of the present invention (the upper part is the detection limit, and the lower part is the quantification limit).

[0038] Figure 4 This is the chromatogram of the phthalate plasticizer reference solution B' (500 ng / ml) in Example 2 of the present invention.

[0039] Figure 5 The chromatograms of different solutions of phthalate plasticizers in Example 2 of the present invention (a is blank solvent, b is reference solution, and c is simulated extract).

[0040] Figure 6 The chromatograms of different solutions of nitrosamine in Example 3 of the present invention (a is the reference solution, b is the sample solution).

[0041] Figure 7 These are chromatograms of typical nitrosamines in different batches of samples in Example 3 of the present invention (a is sample No. 2, b is sample No. 3, and c is sample No. 11). DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0043] Program Overview

[0044] This scheme provides a method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in pharmaceutical packaging materials, comprising the following steps:

[0045] S1. Prepare reference solution: prepare epoxy silane mixed reference solution A, phthalate plasticizer mixed reference solution B and nitrosamine mixed reference solution C respectively according to the residues; prepare reference working solutions with different concentration gradients as needed;

[0046] As a reference, epoxysilane mixed reference solution A contains hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6) reference substances;

[0047] Phthalate ester plasticizer mixed reference solution B contains 23 plasticizer reference substances, including dimethyl phthalate, diethyl phthalate, diisopropyl phthalate, diallyl phthalate, dipropyl phthalate, diisobutyl phthalate, dibutyl phthalate, dimethoxyethyl phthalate, diisoamyl phthalate, di(4-methyl-2-pentyl) phthalate , di(2-ethoxy)ethyl phthalate, dipentyl phthalate, dihexyl phthalate, butyl benzyl phthalate, dibutoxyethyl phthalate, diheptyl phthalate, di(2-ethyl)hexyl phthalate, dicyclohexyl phthalate, diphenyl phthalate, di-n-octyl phthalate, diisononyl phthalate, dinonyl phthalate, diisodecyl phthalate;

[0048] The nitrosamine mixed reference solution C contains 9 nitrosamine reference substances, including N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosodipropylamine, N-nitrosopiperidine, N-nitrosodibutylamine, and N-nitrosobibenzylamine.

[0049] S2. Prepare sample solutions: Use organic solvents to extract materials respectively to obtain epoxy silane sample solution A', phthalate plasticizer sample solution B' and nitrosamine sample solution C'; the sample solutions of the three types of residues are prepared as follows:

[0050] (1) adding water and an organic solvent to a sample of an injection powder and mixing them evenly, extracting the resulting supernatant by vortex oscillation, and fixing the volume with an organic solvent to obtain an epoxysilane sample solution;

[0051] (2) taking the rubber plug and crushing it with liquid nitrogen, ultrasonically extracting it with an organic solvent, and filtering it, and the filtrate is the nitrosamine sample solution;

[0052] (3) Take the injection powder sample and add water to prepare the drug solution, take the rubber stopper and add the drug solution as the simulated extraction solution, after heating and extraction, take the simulated extraction solution and add the organic solvent in proportion to vortex and oscillate the extracted supernatant to obtain the phthalate plasticizer sample solution. The organic solvent is n-hexane or ethyl acetate.

[0053] The method also includes preparing a sample spike solution as needed, by adding a corresponding reference solution to each sample solution to obtain a sample spike solution.

[0054] S3. Content detection: The above-mentioned reference solution and sample solution are subjected to gas chromatography-mass spectrometry detection, and the content of the residue to be detected in the above-mentioned sample solution is calculated; and a mixed reference solution of known concentration is taken, and the detection limit is when S / N=3 and the quantitative limit is when S / N=10. The gas chromatography conditions of the three types of residue solutions are as follows:

[0055] (1) The gas chromatography conditions for nitrosamines are as follows: using a capillary column, carrier gas: helium, flow rate: 1.0 ml / min, injection port temperature: 250°C, transfer tube temperature: 260°C, ion source temperature: 270°C; column temperature: initial temperature 50°C maintained for 4 min, increased to 100°C at 10°C / min, maintained for 2 min, and then increased to 250°C at 20°C / min, maintained for 6 min.

[0056] (2) The gas chromatography conditions for phthalate plasticizers are as follows: a capillary column with an injection volume of 1 μL; an injection port temperature of 280°C, a split ratio of 50:1, a carrier gas of helium, and a column flow rate of 0.5 ml / min; a detector type of MS / MS, a detection mode of SRM mode, a transfer line temperature of 300°C, and an ion source temperature of 300°C.

[0057] (3) The gas chromatography conditions of epoxysilane are as follows: a capillary column is used, the column temperature is programmed, the initial temperature is 33-37°C, maintained for 5 min, heated to 180°C at 10°C / min, maintained for 0 min, and then heated to 240°C at 60°C / min and maintained for 3 min; the injection port temperature is 250°C; the split ratio is 10:1; the carrier gas is nitrogen, and the column flow rate is 1.45-1.55 ml / min; the detector is FID, the temperature is 250°C; the injection volume is 1 μL.

[0058] Example 1

[0059] This embodiment provides a method for detecting cyclosiloxane in a pharmaceutical packaging material, comprising the following steps:

[0060] S1. Preparation of reference solution

[0061] Accurately weigh 0.02708g, 0.02619g, 0.02567g, and 0.02644g of D3, D4, D5, and D6 reference substances, respectively, and place them in a 50ml volumetric flask, add n-hexane to dissolve and dilute to the mark to prepare a mixed reference substance stock solution with a concentration of about 500μg / ml. After diluting the mixed reference substance stock solution 5 times, prepare a mixed reference substance intermediate stock solution (i.e., cyclosiloxane mixed reference substance solution A) with D3, D4, D5, and D6 contents of 106.2μg, 102.7μg, 101.7μg, and 102.6μg per 1ml.

[0062] Prepare reference substance working solution: dilute the mixed reference substance intermediate stock solution with n-hexane to obtain linear solutions with reference substance contents of approximately 1 μg, 2 μg, 5 μg, 10 μg, 15 μg, 20 μg, and 25 μg per 1 ml, respectively.

[0063] S2. Prepare sample solution

[0064] Take 1.0g of a certain injection powder sample (as a reference, latamoxef sodium, specification: 1.0g / 1 bottle), add 4ml of ultrapure water to dissolve and place in a centrifuge tube, add 9ml of n-hexane and vortex to extract for 1min, take the supernatant after centrifugation, add n-hexane to make up to 10ml, shake well, and obtain cyclosiloxane sample solution A'.

[0065] Prepare sample spike solution: take 1 bottle of a certain injection powder sample (specification: 1.0g), add 4ml of ultrapure water to dissolve it and place it in a centrifuge tube, add 0.2ml of the mixed reference stock solution with a concentration of about 500μg / ml prepared in S1, add 9ml of n-hexane, vortex and extract for 1min, take the supernatant after centrifugation, add n-hexane to make up to 10ml, shake well, and obtain a sample spike solution with a concentration of about 10μg / ml.

[0066] S3. Perform testing according to the method in “Overview of the Protocol”.

[0067] Example 2

[0068] This embodiment provides a method for detecting phthalate plasticizers in pharmaceutical packaging materials.

[0069] S1. Preparation of reference solution: Accurately measure 0.1 ml of 23 phthalate mixed reference substances (purchased from DIKMATECHNOLOGIES INC, batch number: 2596062. In the mixed reference substances, except for diisononyl phthalate and diisodecyl phthalate, the concentrations of the other reference substances are all 1000 μg / ml), place in a 10 ml volumetric flask, add n-hexane to dissolve and dilute to the mark as the reference substance stock solution. Dilute the reference substance stock solution 20 times to obtain a phthalate plasticizer reference substance solution B containing about 500 ng of phthalate per 1 ml.

[0070] S2. Prepare sample solution: take a sample of an injection powder (as a reference, latamoxef sodium, specification: 1.0g / 1 bottle), add purified water to prepare a 0.25g / ml drug solution; then take a rubber stopper, add the drug solution at a ratio of 0.2g / ml as a simulated extract, extract at 121°C for 1h, after the extraction is completed, take 16ml of the simulated extract, add 10ml of n-hexane and vortex oscillate to extract for 1min, take the upper solution to obtain the phthalate plasticizer sample solution B'.

[0071] Prepare sample spike solution; take 16 ml of the simulated extract in S2, add 0.5 ml of the reference substance stock solution, shake well, then accurately add 10 ml of n-hexane, shake thoroughly, let stand to separate layers, and take the upper layer solution to obtain phthalate plasticizer sample spike solution B.

[0072] S3. Perform testing according to the method in “Overview of the Protocol”.

[0073] Example 3

[0074] This embodiment provides a method for detecting nitrosamines in pharmaceutical packaging materials, comprising the following steps:

[0075] S1. Preparation of reference solution: Accurately measure 0.1 ml of the nitrosamine mixed reference substance, place it in a 200 ml volumetric flask, add ethyl acetate to dissolve and dilute to the mark to prepare a 50 ng / ml reference solution.

[0076] S2. Prepare sample solution: freeze an appropriate amount of rubber stopper (as a reference, chlorobutyl rubber stopper for sterile powder for injection) with liquid nitrogen, place it in a ZM200 homogenizer, add an appropriate amount of liquid nitrogen, and crush it; accurately weigh 2g of the crushed rubber stopper, place it in a 50ml conical flask, and add 20ml of ethyl acetate. Ultrasonic extraction for 1h, filter and transfer the filtrate to a 25ml volumetric flask after cooling to room temperature, repeatedly wash the filter flask and filter cake with ethyl acetate 3 times and combine them into a volumetric flask to make up the volume, filter the membrane, and obtain a nitrosamine sample solution.

[0077] S3. Perform testing according to the method in “Overview of the Protocol”.

[0078] Experimental Example 1: Methodological Validation of Cyclosiloxane Detection in Pharmaceutical Packaging Materials

[0079] 1. Specificity: By examining the spectra of blank solvent (n-hexane), reference solution, and sample spiked solution (see Table 1 for details), Figure 1 ) It was found that under the chromatographic conditions of this scheme, the blank solvent and sample matrix had no interference with the determination of D3, D4, D5, and D6, and the separation between the peaks was good, indicating that this method has good specificity.

[0080] Table 1 Specificity test results

[0081] name Retention time / min Peak area Theoretical plates Separation D3 3.333 0.0595 51102 Comply with regulations D4 6.462 0.0541 121677 Comply with regulations D5 9.802 0.0535 597114 Comply with regulations D6 12.35 0.0517 1126732 Comply with regulations

[0082] Take the reference substance working solution, test it on the machine according to the chromatographic conditions of this scheme, and record the chromatogram. Perform linear regression calculation with the injection concentration as the horizontal axis and the peak area as the vertical axis to obtain the linear regression equation and correlation coefficient of each component. The results are shown in Table 2 and Figure 2 Among them, the correlation coefficients R of the regression equations of D3, D4, D5, and D6 were 0.9998, 0.9999, 0.9998, and 0.9996, respectively, which met the requirement that the R value was greater than 0.995, indicating that D3, D4, D5, and D6 had good linearity within the linear range.

[0083] Table 2 Linearity and range

[0084] name Linear equations Linear range / μg / ml Correlation coefficient R D3 y=0.0059x-0.0001 1～25 0.9998 D4 y=0.0055x+0.0005 1～25 0.9999 D5 y=0.0056x+0.0001 1～25 0.9998 D6 y=0.0054x+0.0003 1～25 0.9996

[0085] 2. Detection limit and quantification limit: Take a mixed reference solution with a concentration of about 10μg / ml and dilute it step by step. The detection limit is about 3 when the S / N is about 3, and the quantification limit is about 10 when the S / N is about 10. The detection limit solution is injected continuously for 3 times, and the quantification limit solution is injected continuously for 6 times. According to the pretreatment of the sample, the method detection limit, method quantification limit and detection limit signal-to-noise ratio of this method are calculated. The results are shown in Table 3 and Figure 3 The experimental data showed that the detection limit and the quantitative limit of the method in this scheme were much lower than the 50% AET value of each substance to be tested, which met the requirements of the compatibility test for detection sensitivity.

[0086] Table 3 Detection limit and quantification limit

[0087]

[0088] Note: The detection limit of D3 method (μg / bottle) is calculated as follows: 0.5310μg / ml×10ml÷1bottle=5.310μg / bottle

[0089] 3. Precision

[0090] 3.1 Repeatability: According to the "Preparation of Sample Spiked Solution" mentioned above, 6 sample spiked solutions were prepared in parallel and injected separately. The repeatability results are shown in Table 4. According to the requirements of the 9101 "Guidelines for Validation of Analytical Methods" of the General Rules of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia, when the content of the component to be determined is 10μg / g (μg / ml), the RSD is 6%. The spiked concentration of the sample in this test is about 10μg / ml, and the RSD meets the requirements of the Pharmacopoeia, indicating that this method has good repeatability.

[0091] Table 4 Repeatability test results

[0092]

[0093] 3.2 Intermediate precision: According to the repeatability method, 6 sample spike solutions were re-prepared on different dates and then measured on the machine. The RSD of the 12 test results before and after was comprehensively calculated. The results are shown in Table 5. Referring to the requirements of the 9101 "Guidelines for Validation of Analytical Methods" of the General Rules of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia, the RSDs were all less than 6%, indicating that this method has good intermediate precision.

[0094] Table 5 Intermediate precision test results

[0095]

[0096] 3.3 Accuracy: Take 2 samples of a certain injection powder (specification: 1.0g) and test them according to the "Preparation of sample solution" mentioned above. Each sample is injected 3 times in parallel as the sample background. D3, D4, D5 and D6 were not detected in the samples.

[0097] Six samples of the repeatability test were spiked and measured on the machine. The results are shown in Table 6. According to the requirements of the 9101 "Guidelines for Validation of Analytical Methods" of the 2020 edition of the Chinese Pharmacopoeia, when the content of the component to be determined is 10μg / g (μg / ml), the recovery limit is 80% to 115%. The concentration level of the target substance in this test is about 10μg / ml, and the average recovery rate is between 91.24% and 92.18%, which meets the requirements of the pharmacopoeia, indicating that this method has good accuracy and can meet the requirements of compatibility determination.

[0098] Table 6 Spiked recovery results

[0099]

[0100] 3.4 Stability of the solution to be tested: Take one portion of the sample spiked solution in the repeatability test and inject it at 0h and 14h respectively. The calculated change rate results are shown in Table 7. The change rate in this test is less than 15%, indicating that the determination results of each target substance are stable within 14h.

[0101] Table 7 Stability test results

[0102] name 0h 14h Rate of change (%) name 0h 14h Rate of change (%) D3 0.0595 0.0647 8.7 D5 0.0535 0.0583 9 D4 0.0541 0.059 9.1 D6 0.0517 0.0569 10.1

[0103] In summary, the methodological study of D3, D4, D5, and D6 was conducted with reference to the "Technical Guidelines for Validation of Analytical Methods for Quality Control of Chemical Drugs" and the "Guidelines for Validation of Analytical Methods" of the General Rules of Part IV of the 2020 Edition of the Chinese Pharmacopoeia 9101. The results show that the analytical method has good specificity, linearity, and precision, the recovery rate is between 91.24% and 92.18%, and the test solution is stable within 14 hours. It shows that the established cyclosiloxane gas chromatography detection method can effectively detect the residual amount of cyclosiloxane in rubber stoppers.

[0104] 4. Determination of migration amount of D3, D4, D5 and D6

[0105] The difference from the sample solution in Example 1 is that the material is a rubber plug. The material extraction solution is obtained by the following steps:

[0106] Prepare material extraction solution: accurately weigh 1.0028 g of shredded rubber stoppers and place them in a graduated test tube, add n-hexane, and perform ultrasonic extraction for 30 min. During the ultrasonic process, control the temperature to room temperature. Transfer the ultrasonic solution to a 10 ml volumetric flask, wash it repeatedly with n-hexane for 3 times, combine the extracts and make up to the scale, shake well, and filter with a 0.45 μm organic filter membrane to obtain the solution.

[0107] Prepare material simulation extraction solution: take 1 rubber stopper, wash it with ultrapure water, dry it, add the injection powder solution (0.25g / ml) at a ratio of 0.2g / ml by weight of the rubber stopper, extract at 121℃ for 1h, after completion, accurately transfer 10ml of the simulated extraction solution into a centrifuge tube, add 9ml of n-hexane, vortex and extract for 1min, centrifuge to obtain the supernatant, add n-hexane to make up to volume, and shake well.

[0108] In October, migration tests were conducted on three batches of samples of three specifications of latamoxef sodium for injection, for a total of nine batches of samples, and the results are shown in Table 8. No cyclosiloxane was detected in any of the nine batches of rubber stopper samples.

[0109] Table 8 Sample 0 month migration test results

[0110]

[0111] Experimental Example 2: Detection results of phthalate plasticizers in pharmaceutical packaging materials

[0112] 1. Specificity: The SRM mode of the triple quadrupole mass detector is used to highly selectively scan the parent ion-daughter ion pairs of the sample, and the type of compound is determined by the relative abundance and retention time of the qualitative ions. The method has good specificity.

[0113] 2. Instrument sensitivity verification: dilute the reference stock solution in Example 2 and prepare a reference solution of 500ng / ml (DMEP with the lowest PDE among the 23 phthalates was selected to verify the sensitivity of the instrument, and its analysis threshold is 10μg / day÷(4g / day÷0.25g / ml)×16ml÷10ml×50% (uncertainty)=0.5μg / ml=500ng / ml). The S / N value is much greater than 10, indicating that the sensitivity of the instrument can meet the requirements of the compatibility test. See the chromatogram of the reference solution (500ng / ml) for details. Figure 4 .

[0114] 3. Repeatability: Take 6 portions of the sample spiked solution and inject and measure according to the above chromatographic conditions. The repeatability results are shown in Table 9. According to the requirements of the 2020 edition of the Chinese Pharmacopoeia, Part IV General 9101 "Guidelines for Validation of Analytical Methods", when the content of the component to be determined is 0.5μg / g (μg / ml), the RSD limit is 9%. In the analysis of complex matrices, component content less than 0.01% and multiple components, the precision limit can be appropriately relaxed to 2 times, that is, 18%. The spiked concentration of the sample in this test is about 500ng / ml, and the RSD result range is 0.51% to 12.29%, which meets the requirements of the pharmacopoeia, indicating that the method has good repeatability.

[0115] Table 9 Repeatability test results

[0116]

[0117] 4. Accuracy

[0118] Take 20 ml of simulated extract and inject it three times in parallel as the sample background according to the "Preparation of simulated drug extraction solution" under the previous item. Prepare 6 sample spike solutions in parallel according to the "Preparation of sample spike solution" under the previous item, test according to the law, calculate the recovery rate, and the results are shown in Table 10. According to the requirements of 9101 "Guidelines for Validation of Analytical Methods" in the General Rules of Part Four of the 2020 Edition of the "Chinese Pharmacopoeia", when the content of the component to be determined is 1μg / g and 0.01μg / g (μg / ml), the recovery limits are 75%-120% and 70%-125%, respectively. The recovery rate of this test is 80.27%-111.04%, which meets the requirements of the Pharmacopoeia, so this method has good accuracy.

[0119] Table 10 Spiked recovery results

[0120]

[0121] In summary, the established rapid screening method for gas quality detection of 23 phthalate plasticizers in rubber stoppers has good specificity, sensitivity, repeatability and accuracy, meets the requirements, and can be used for the detection of phthalate plasticizers in rubber stoppers.

[0122] 5. Practical application: Take the above-mentioned "drug solution simulation extraction solution" and test it on the machine. The results are shown in Table 11 and Figure 5 The simulated extract had responses at the peaks of DEP, DIBP, DBP, and DCHP reference substances, and the contents all met the requirement of no more than 50% limit.

[0123] Table 11 Screening results of phthalate plasticizers

[0124]

[0125]

[0126] Note: DMEP 50% limit = 10μg / day÷(4g / day÷0.25g / ml)×50%=0.3125μg / ml

[0127] Method sensitivity = 0.5 μg / ml × 10 ml ÷ 16 ml = 0.3125 μg / ml

[0128] Experimental Example 3: Detection results of nitrosamines in pharmaceutical packaging materials

[0129] 1. Methodology verification data: The sample solution and reference solution in Example 3 were tested on a machine. The results are shown in Tables 12 and Figure 6 . Methodological validation was carried out in accordance with the general rules 9101 of the fourth part of the 2020 edition of the Chinese Pharmacopoeia. The 9 nitrosamines were in the range of 5-35 ng / ml, and the correlation coefficient R ranged from 0.995 to 0.999, all meeting the requirement of ≥0.995, with good linearity.

[0130] Table 12 Linearity, detection limit and quantification limit

[0131]

[0132] 2. Accuracy, precision, and stability: Experiments were performed according to the aforementioned method, and the results are detailed in Tables 13 to 15. When the component content is 0.0001%, the recovery rate should be in the range of 70% to 125%. According to the requirements of 9101 "Guidelines for Validation of Analytical Methods" in Part IV of the 2020 edition of the Chinese Pharmacopoeia, the recovery rate limit can be appropriately relaxed in the analysis of component content below 0.01%.

[0133] Table 13 Accuracy

[0134]

[0135] Table 14 Precision

[0136]

[0137] Table 15 Stability

[0138] name 0h 48h Rate of change (%) N-Nitrosodimethylamine 22.885 22.06 3.6 N-Nitrosomethylethylamine 20.158 21.389 6.1 N-Nitrosodiethylamine 20.047 20.443 2 N-Nitrosopyrrolidine 22.617 20.341 10.1 N-Nitrosomorpholine 21.828 21.223 2.8 N-Nitrosodipropylamine 20.67 21.787 5.4 N-Nitrosopiperidine 23.741 22.234 6.3 N-Nitrosodibutylamine 23.299 23.183 0.5 N-Nitrobibenzylamine 22.678 23.936 5.5

[0139] 3. Application: Collect samples from different batches (see Table 16 for some sample information), obtain sample solutions according to the method in Example 3, and obtain test results from machine experiments, see Tables 17 and Figure 7 Among the 30 batches of samples collected, 7 batches were detected to contain N-nitrosodibenzylamine, 5 batches were detected to contain N-nitrosomorpholine, and 1 batch was detected to contain N-nitrosodibutylamine.

[0140] Table 16 Sample information table

[0141] serial number name Production Unit batch number 2 Halogenated butyl rubber stopper with polyester film for injection A M201250222-55 3 Halogenated butyl rubber stoppers for injection B 20116872 4 Bromobutyl rubber stoppers for freeze-dried sterile powder for injection C 210532281-61 5 Freeze-dried sterile powder for injection with halogenated butyl rubber stopper (chlorinated) D 21037701 6 Bromobutyl rubber stoppers for freeze-dried sterile powder for injection E 6006818 8 Halogenated butyl rubber stoppers for sterile powder for injection (brominated) F 21061214 11 Bromobutyl rubber pistons for pen syringes G 31902002 16 Chlorobutyl rubber stopper with ethylene-tetrafluoroethylene copolymer film for injection H A20200107 18 Halogenated butyl rubber stopper with polyester film for injection I 1908078

[0142] Table 17 Test results of 30 batches of samples from different sources (concentration in ng / g)

[0143]

[0144]

[0145] In summary, compared with the poor selectivity and low sensitivity of the existing technology using a single quadrupole mass spectrometer, the triple quadrupole mass spectrometer technology of this scheme significantly improves the selectivity and sensitivity of detection, and greatly improves the detection level of trace nitrosamines. Specifically, the chromatographic and mass spectrometric conditions of this scheme enable good resolution of ion pairs of 9 nitrosamines. The detection limit of each nitrosamine reaches 1ng / ml (ppb), which is significantly higher than the ppm level of the detection limit of the existing literature method.

[0146] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in pharmaceutical packaging materials, characterized in that: The steps include: S1. Preparation of reference solution: Prepare epoxy silane mixed reference solution A, phthalate plasticizer mixed reference solution B and nitrosamine mixed reference solution C respectively according to the residues; S2. Prepare sample solutions: extract the materials respectively using organic solvents to obtain epoxy silane sample solution A', phthalate plasticizer sample solution B' and nitrosamine sample solution C'; S3. Content detection: The reference solution and the sample solution are subjected to gas chromatography-mass spectrometry detection respectively, and the content of the residue to be detected in the sample solution is calculated.

2. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 1, characterized in that: In S2, sample solutions of three types of residues were prepared as follows: After adding water and an organic solvent to the injection powder sample and mixing evenly, vortex-extracting the obtained supernatant, and fixing the volume with the organic solvent to obtain an epoxysilane sample solution; The rubber plug is crushed with liquid nitrogen, extracted with an organic solvent and then filtered, and the filtrate is the nitrosamine sample solution; Take the injection powder sample and add water to prepare the drug solution, take the rubber stopper and add the drug solution as the simulated extraction solution, after heating and extraction, take the simulated extraction solution and add the organic solvent in proportion to vortex and oscillate the obtained supernatant to obtain the phthalate plasticizer sample solution.

3. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 2, characterized in that: The organic solvent is n-hexane or ethyl acetate.

4. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 1, characterized in that: In S1, it also includes preparing reference substance working solutions with different concentration gradients.

5. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 1, characterized in that: In S2, it also includes preparing a sample spike solution, the method is as follows: adding a corresponding reference solution to each sample solution to obtain a sample spike solution.

6. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 1, characterized in that: S3 also includes the determination of the limit of quantification and the limit of detection, including the following method: take a mixed reference solution of known concentration, dilute it step by step, and detect it until S / N=3 is the detection limit and S / N=10 is the limit of quantification.

7. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 1, characterized in that: The epoxysilane mixed reference solution A contains hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, and dodecamethylcyclohexasiloxane reference substances; the phthalate plasticizer mixed reference solution B contains 23 plasticizer reference substances, including dimethyl phthalate, diethyl phthalate, diisopropyl phthalate, diallyl phthalate, dipropyl phthalate, diisobutyl phthalate, dibutyl phthalate, dimethoxyethyl phthalate, diisoamyl phthalate, di(4-methyl-2-pentyl) phthalate, di(2-ethoxy)ethyl phthalate, di(2-ethoxy)ethyl phthalate, di(2-methyl-2-pentyl) phthalate, di(2-ethylhexyl) ...-2-pentyl) phthalate, di(2-ethyl-2-pentyl) phthalate, di(2-methyl-2-pentyl) phthalate, di(2-ethyl-2-pentyl) phthalate, di(2-ethyl-2-pentyl) phthalate, di(2-ethyl-2-pentyl) phthalate, di(2-ethyl-2-pentyl) phthalate, di(2-ethyl-2-pentyl) phthalate, di(2-ethyl-2-pentyl) phthalate, di(2-ethyl-2-pentyl) phthalate, di(2-ethyl The nitrosamine mixed reference substance solution C contains 9 kinds of nitrosamine reference substances, including N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosodipropylamine, N-nitrosopiperidine, N-nitrosodibutylamine and N-nitrosobibenzylamine.

8. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 1, characterized in that: The gas chromatography conditions of the nitrosamines are: using a capillary column, carrier gas: helium, flow rate: 1.0 ml / min, injection port temperature: 250° C., transfer tube temperature: 260° C., ion source temperature: 270° C.; Column temperature: initial temperature 50°C, maintained for 4 min, then increased to 100°C at 10°C / min, maintained for 2 min, then increased to 250°C at 20°C / min, maintained for 6 min.

9. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 8, characterized in that: The gas chromatography conditions for the phthalate plasticizers are: using a capillary column, an injection volume of 1 μL; the injection port temperature is 280°C, the split ratio is 50:1, the carrier gas is helium, and the column flow rate is 0.5 ml / min; the detector type is MS / MS, the detection mode is SRM mode, the transmission line temperature is 300°C, and the ion source temperature is 300°C.

10. The method for detecting cyclosiloxane, phthalate plasticizers and nitrosamines in a pharmaceutical packaging material according to claim 9, characterized in that: The gas chromatography conditions of the epoxy silane are as follows: a capillary column is used, the column temperature is programmed, the initial temperature is 33-37°C, maintained for 5 minutes, heated to 180°C at 10°C / min, maintained for 0 minutes, and then heated to 240°C at 60°C / min and maintained for 3 minutes; the injection port temperature is 250°C; the split ratio is 10:1; the carrier gas is nitrogen, the column flow rate is 1.45-1.55 ml / min; the detector is FID, the temperature is 250°C; the injection volume is 1 μL.

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