Method for determining leached substances in disposable hemoperfusion device

Abstract

The invention relates to the technical field of medical instrument inspection, in particular to a method for determining leached substances in a disposable hemoperfusion device, which establishes gas chromatography-mass spectrometry exclusive detection conditions for the leached substances such as styrene, divinylbenzene and naphthalene to realize precise determination of the leached substances. The leached substances in the determination method comprise styrene, divinylbenzene and naphthalene, according to the determination method provided by the invention, the leached substances such as styrene, divinylbenzene and naphthalene can be precisely determined through one-time detection by determining exclusive detection conditions, the detection limit can reach 1.02-2.38 ng / mL, and the detection precision is effectively improved while the detection efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical device inspection, in particular to a method for determining leachable matter in a disposable hemoperfusion device. Background Art

[0002] The commonly used adsorbent in disposable hemoperfusion devices is a macroporous neutral polystyrene adsorption resin. This resin can contain leachable residues of styrene, divinylbenzene, and naphthalene. Styrene is a monomer used in the polymerization reaction during the preparation of the polystyrene adsorption resin; divinylbenzene is a crosslinker in the polymerization reaction; and naphthalene is a byproduct of the divinylbenzene raw material preparation process. These residual leachables can pose a safety risk to patients. Therefore, the measurement of these leachables (styrene, divinylbenzene, and naphthalene) in disposable hemoperfusion devices is necessary to provide a basis for product safety evaluation.

[0003] Due to the different properties of styrene, divinylbenzene, and naphthalene, styrene and divinylbenzene are organic benzene series, while naphthalene is a polycyclic aromatic hydrocarbon (PAH). Existing technologies generally provide methods for determining either organic benzene series or PAHs separately, with few methods addressing both. For example, patent documents CN 104749283 A (application number 201510166744.5), CN 110161150 A (application number 201910560417.6), and the journal article "Determination of Styrene and Divinylbenzene Residues in L-Carnitine API by Gas Chromatography" all provide methods for determining benzene series. Furthermore, patent documents CN 108896674 A (application number 201810726684.1) and CN 116068079 A (application number 202211662328.0) both provide methods for determining PAHs.

[0004] Therefore, a method for the determination of styrene, divinylbenzene and naphthalene is urgently needed. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for determining leachable substances in a disposable hemoperfusion device. Specifically, gas chromatography-mass spectrometry-based detection conditions are established for the leachable substances styrene, divinylbenzene, and naphthalene, thereby achieving precise determination of the above-mentioned leachable substances.

[0006] In order to achieve the above technical effects, the present invention adopts the following technical solutions: A method for determining leachable substances in a disposable hemoperfusion device, wherein the leachable substances include styrene, divinylbenzene, and naphthalene. The testing method specifically comprises the following steps: S1. Prepare mixed standard stock solution Styrene, divinylbenzene and naphthalene are respectively taken, diluted to volume with ethanol and then shaken to obtain a styrene standard stock solution, a divinylbenzene standard stock solution and a naphthalene standard stock solution; the styrene standard stock solution, the divinylbenzene standard stock solution and the naphthalene standard stock solution are measured and placed in a volumetric flask, diluted to volume with ethanol and then shaken to obtain a mixed standard stock solution; S2. Prepare a series of standard solutions The mixed standard stock solution prepared in step S1 was configured into a mixed standard solution with a mixed solution of water and ethanol water, wherein the concentration range of styrene was 3.848-76.97 ng / mL, the concentration range of m-divinylbenzene was 5.947-118.9 ng / mL, the concentration range of p-divinylbenzene was 2.548-50.98 ng / mL, and the concentration range of naphthalene was 3.388-67.77 ng / mL; S3. Preparation of sample test solution Perform heparinization and pre-flushing treatments on the disposable hemoperfusion device to be tested in sequence; Using ethanol and water as the extraction solution, the sample to be tested was cyclically extracted at 37°C for 6 hours to obtain the sample extract; Use ethanol water as the extraction solution and circulate at 37°C for 6 hours under the same conditions as the sample to be tested to obtain a blank extraction solution; Pipette 1 mL of sample extract into a 20 mL headspace bottle, add 5 mL of pure water, mix well, and seal to obtain the sample test solution; Pipette 1 mL of blank extract into a 20 mL headspace bottle, add 5 mL of pure water, mix well, and seal to obtain a blank control solution; S4. Test conditions Chromatographic conditions: Column: DB-FFAP, 60m × 250µm, film thickness 0.5µm; temperature program, 40℃ for 7min, 10 Heat to 240℃ and maintain for 5min; Inlet temperature: 240℃; Carrier gas: high-purity helium, purity 99.999%, flow rate 1 ;Split injection, split ratio 20:1; Mass spectrometry conditions: electron impact ion source (EI); electron energy: 70 eV; ion source temperature: 230°C; quadrupole temperature: 150°C; scan mode: SCAN; scan range (m / z): 35-350.

[0007] Headspace conditions: equilibrium temperature 80°C; equilibrium time 30 minutes; S5. Test results analysis The mixed standard solution, the sample test solution, and the blank control solution are tested according to the test conditions provided in step S4, and a standard curve is drawn to obtain a linear regression equation, and the content of leachable matter in the sample test solution is determined.

[0008] Preferably, the operation mode of step S1 is specifically as follows: Weigh 23.18 mg of styrene, 31.86 mg of divinylbenzene, and 20.35 mg of naphthalene respectively into a 10 mL volumetric flask, dilute to the mark with ethanol, and shake well to obtain the styrene standard stock solution, divinylbenzene standard stock solution, and naphthalene standard stock solution; accurately measure 0.1 mL of the styrene standard stock solution, 0.2 mL of the divinylbenzene standard stock solution, and 0.1 mL of the naphthalene standard stock solution into a 10 mL volumetric flask, dilute to the mark with ethanol, and shake well to obtain the mixed standard stock solution.

[0009] Preferably, the operation mode of step S2 is specifically as follows: Accurately measure 1µL, 2µL, 5µL, 10µL, 15µL, and 20µL of the mixed standard stock solution prepared in step S1 into a mixed solution pre-added with 5mL of water and 1mL of ethanol water, and seal the container to obtain the mixed standard solution.

[0010] Preferably, the ethanol water has a density of (0.9373-0.9378) g / mL at 20°C.

[0011] Preferably, in step S3, the heparinization and priming treatment are specifically performed as follows: Take the disposable hemoperfusion device to be tested and perform static heparinization. After injecting 12,500 units of heparin injection into the disposable hemoperfusion device, shake it 180° up and down 8-12 times and let it stand for 30 minutes. After static heparinization is completed, the preservation solution in the disposable hemoperfusion device is completely emptied and 2000 mL of 0.9% (mass concentration) NaCl injection is used for pre-flushing at a flow rate of 200 mL / min and the pre-flushing solution is discarded.

[0012] Preferably, in step S3, when preparing the sample extract and the blank control solution, ethanol water is used to circulate at flow rates of 100 mL / min and 250 mL / min, respectively.

[0013] The beneficial effects of the present invention are as follows: The determination method provided by the present invention, by determining exclusive detection conditions, can accurately determine the leachable substances styrene, divinylbenzene and naphthalene in a single test, with a detection limit of 1.02-2.38 ng / mL, thereby improving detection efficiency and effectively enhancing detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a chromatogram of the blank control solution measured in Example 1 at a circulation flow rate of 100 mL / min; Figure 2 is a chromatogram of the blank control solution measured in Example 1 at a flow rate of 250 mL / min; Figure 3 is a chromatogram of the mixed standard solution obtained in Example 1; Figure 4 1 is a chromatogram of the sample test solution measured at a flow rate of 100 mL / min in Example 1; Figure 5 This is a chromatogram of the sample test solution measured in Example 1 at a flow rate of 250 mL / min. DETAILED DESCRIPTION

[0015] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0016] When preparing the sample extract and blank extract, they were circulated at flow rates of 100 mL / min and 250 mL / min, respectively. 100 mL / min and 250 mL / min are the minimum and maximum flow rates for clinical use of the sample, and the extraction time is the maximum contact time for clinical use, 6 hours.

[0017] The divinylbenzene used in the present invention is purchased from Sigma with a product number of 414565, and includes m-divinylbenzene and p-divinylbenzene. The purity of m-divinylbenzene is 56%, and the purity of p-divinylbenzene is 24%.

[0018] The styrene used in the present invention was purchased from Sinopharm Chemical Reagent Co., Ltd. with the product number 40057060.

[0019] The naphthalene used in the present invention is purchased from Sigma, and the product number is 147141.

[0020] Example 1 A method for determining leachable substances in a disposable hemoperfusion device, wherein the leachable substances include styrene, divinylbenzene, and naphthalene. The testing method specifically comprises the following steps: S1. Prepare mixed standard stock solution Accurately weigh 23.18 mg of styrene, 31.86 mg of divinylbenzene, and 20.35 mg of naphthalene into a 10 mL volumetric flask, dissolve and dilute to the mark with ethanol, and shake well to obtain the styrene standard stock solution, divinylbenzene standard stock solution, and naphthalene standard stock solution. Accurately weigh 0.1 mL of the styrene standard stock solution, 0.2 mL of the divinylbenzene standard stock solution, and 0.1 mL of the naphthalene standard stock solution into a 10 mL volumetric flask, dilute to the mark with ethanol, and shake well to obtain the mixed standard stock solution. S2. Prepare a series of standard solutions Accurately measure 1µL, 2µL, 5µL, 10µL, 15µL, and 20µL of the mixed standard stock solution prepared in step S1 into a mixed solution pre-added with 5mL of water and 1mL of ethanol water, and seal the container to obtain the mixed standard solution. S3. Preparation of sample test solution The disposable hemoperfusion device to be tested was heparinized and pre-flushed, and ethanol-water was used as the extraction solution. The sample extracts were circulated at flow rates of 100 mL / min and 250 mL / min at 37°C for 6 hours, respectively. Use ethanol water as the extraction solution and circulate at 37°C for 6 hours under the same conditions as the circular extraction to obtain a blank extract. Pipette 1 mL of sample extract into a 20 mL headspace bottle, add 5 mL of pure water, mix well, and seal to obtain the sample test solution; Pipette 1 mL of blank extract into a 20 mL headspace bottle, add 5 mL of pure water, mix well, and seal to obtain a blank control solution; S4. Test conditions Chromatographic conditions: Column: DB-FFAP, 60m × 250µm, film thickness 0.5µm; temperature program, 40℃ for 7min, 10 Heat to 240 °C and maintain for 5 min; Inlet temperature: 240 °C; Carrier gas: high-purity helium, purity 99.999%, flow rate 1 ;Split injection, split ratio 20:1; Mass spectrometry conditions: electron impact ion source (EI); electron energy: 70 eV; ion source temperature: 230°C; quadrupole temperature: 150°C; scan mode: SCAN; scan range (m / z): 35-350.

[0021] Headspace conditions: equilibrium temperature 80°C; equilibrium time 30 minutes; S5. Test results analysis The blank control solution, mixed standard solution and sample test solution were tested according to the test conditions provided in step S4, and the test results were as follows: Figure 1-Figure 5 As shown, Figure 1 and Figure 2 The chromatograms of blank control solution were measured at flow rates of 100 mL / min and 250 mL / min, respectively. Figure 3 is the measured chromatogram of the mixed standard solution, Figure 4 and Figure 5 The chromatograms of the sample solution measured at flow rates of 100 mL / min and 250 mL / min respectively show that the target compounds can be completely separated and there is no interference from the blank control solution, indicating that the method has good specificity.

[0022] The series of standard solutions prepared in step S2 were tested according to the test conditions provided in step S4, and linear regression was performed with the peak area of each target compound as the ordinate and the mass concentration of each target compound as the abscissa. A standard curve was drawn to obtain a regression equation. Mixed standard solutions of different mass concentrations were prepared and tested and analyzed according to the test conditions provided in step S4. The concentration of the standard solution corresponding to a signal-to-noise ratio (S / N) of approximately 3 was taken as the limit of detection (LOD), and the concentration of the standard solution with a good recovery rate at an S / N of approximately 10 was taken as the limit of quantification (LOQ). The data are shown in Table 1.

[0023] Table 1 Linear range and equation of target compound

[0024] In Table 1, retention time is expressed in minutes, linear range is expressed in ng / mL, and LOD (limit of detection) and LOQ (limit of quantification) are both expressed in ng / mL. As shown in Table 1, all target compounds exhibited good linearity.

[0025] Substitute the peak area of the target compound in the sample test solution into the linear regression equation to determine the content of the leachable matter in the sample test solution, as shown in Table 2; Table 2 Determination results of target substances in sample test solution

[0026] In Table 2, ND means not detected.

[0027] Experimental Example 2 Precision test Three mixed standard solutions of different concentrations, low, medium, and high, were selected, and the test conditions provided in step S4 of Example 1 were used. Each concentration was measured in parallel 6 times, and the precision was investigated by calculating the relative standard deviation (RSD) of the response values. The results are detailed in Table 3.

[0028] Table 3 Target precision determination results (n=6)

[0029] In Table 3, the spike concentration is in ng / mL. The data in Table 3 show that the precision of each target compound is good.

[0030] Experimental Example 3 Recovery test The accuracy of this method was evaluated by measuring the recovery rate using a matrix spike recovery test. 5 mL of pure water and 1 mL of the sample solution at a 250 mL / min circulation flow rate were precisely pipetted into a 20 mL headspace vial. Three mixed standard solutions of different concentrations (low, medium, and high) were added, sealed, mixed, and then injected and analyzed. Six replicates were performed. The results are shown in Table 4.

[0031] Table 4 Results of target compound recovery determination (n=6)

[0032] In Table 4, the unit of spiked concentration is ng / mL. The data in Table 4 show that the accuracy of this method meets the analytical requirements.

Claims

1. A method for determining leachable matter in a disposable hemoperfusion device, characterized in that: Leachables include styrene, divinylbenzene, and naphthalene. The test method specifically includes the following steps: S1. Prepare mixed standard stock solution Styrene, divinylbenzene and naphthalene are respectively taken, diluted to volume with ethanol and then shaken to obtain a styrene standard stock solution, a divinylbenzene standard stock solution and a naphthalene standard stock solution; the styrene standard stock solution, the divinylbenzene standard stock solution and the naphthalene standard stock solution are measured and placed in a volumetric flask, diluted to volume with ethanol and then shaken to obtain a mixed standard stock solution; S2. Prepare a series of standard solutions The mixed standard stock solution prepared in step S1 was configured into a mixed standard solution with a mixed solution of water and ethanol water, wherein the concentration range of styrene was 3.848-76.97 ng / mL, the concentration range of m-divinylbenzene was 5.947-118.9 ng / mL, the concentration range of p-divinylbenzene was 2.548-50.98 ng / mL, and the concentration range of naphthalene was 3.388-67.77 ng / mL; S3. Preparation of sample test solution Perform heparinization and pre-flushing treatments on the disposable hemoperfusion device to be tested in sequence; Using ethanol and water as the extraction solution, the sample to be tested was cyclically extracted at 37°C for 6 hours to obtain the sample extract; Use ethanol water as the extraction solution and circulate at 37°C for 6 hours under the same conditions as the sample to be tested to obtain a blank extraction solution; Pipette 1 mL of sample extract into a 20 mL headspace bottle, add 5 mL of pure water, mix well, and seal to obtain the sample test solution; Pipette 1 mL of blank extract into a 20 mL headspace bottle, add 5 mL of pure water, mix well, and seal to obtain a blank control solution; S4. Test conditions Chromatographic conditions: Column: DB-FFAP, 60m × 250µm, film thickness 0.5µm; Program temperature rise, maintain at 40℃ for 7min, then Heat to 240°C and maintain for 5 min; Inlet temperature: 240°C; Carrier gas: high purity helium, purity 99.999%, flow rate 1 ;Split injection, split ratio 20:1; Mass spectrometry conditions: electron impact ion source (EI); electron energy: 70 eV; ion source temperature: 230°C; quadrupole temperature: 150°C; scan mode: SCAN; scan range (m / z): 35-350; Headspace conditions: equilibrium temperature 80°C; equilibrium time 30 minutes; S5. Test results analysis The mixed standard solution, the sample test solution, and the blank control solution are tested according to the test conditions provided in step S4, and a standard curve is drawn to obtain a linear regression equation, and the content of leachable matter in the sample test solution is determined.

2. The assay method according to claim 1, wherein The specific operation mode of step S1 is: Weigh 23.18 mg of styrene, 31.86 mg of divinylbenzene, and 20.35 mg of naphthalene respectively into a 10 mL volumetric flask, dilute to the mark with ethanol, and shake well to obtain the styrene standard stock solution, divinylbenzene standard stock solution, and naphthalene standard stock solution; accurately measure 0.1 mL of the styrene standard stock solution, 0.2 mL of the divinylbenzene standard stock solution, and 0.1 mL of the naphthalene standard stock solution into a 10 mL volumetric flask, dilute to the mark with ethanol, and shake well to obtain the mixed standard stock solution.

3. The measuring method according to claim 1, wherein The specific operation mode of step S2 is: Accurately measure 1µL, 2µL, 5µL, 10µL, 15µL, and 20µL of the mixed standard stock solution prepared in step S1 into a mixed solution pre-added with 5mL of water and 1mL of ethanol water, and seal the container to obtain the mixed standard solution.

4. The measuring method according to claim 1, wherein The ethanol water has a density of (0.9373-0.9378) g / mL at 20°C.

5. The measuring method according to claim 1, wherein In step S3, the heparinization and priming treatment are specifically performed as follows: Take the disposable hemoperfusion device to be tested and perform static heparinization. After injecting 12,500 units of heparin injection into the disposable hemoperfusion device, shake it 180° up and down 8-12 times and let it stand for 30 minutes. After static heparinization is completed, the preservation solution in the disposable hemoperfusion device is completely emptied, and 2000 mL of 0.9% NaCl injection is used at a flow rate of 200 mL / min and the priming solution is discarded.

6. The assay method according to claim 1, wherein In step S3, when preparing the sample extract and the blank extract, ethanol water is circulated at flow rates of 100 mL / min and 250 mL / min, respectively.

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