Method for detecting five hazardous substances in raincoat for children

Abstract

The invention discloses a method for detecting five hazardous substances in a child raincoat. The method comprises the following steps: (1) sample pretreatment: cutting a sample into pieces; (2) extracting a target substance; adding a solvent into the sample, performing ultrasonic extraction, and collecting and filtering supernate after ultrasonic extraction is completed; (3) analyzing by using a gas chromatography-high resolution time-of-flight mass spectrometer; the five harmful substances are isophorone, cyclohexanone, naphthalene, phenol and 2, 6-butylated hydroxytoluene. According to the method, five hazardous substances in multiple batches of children's raincoats made of multiple materials are quantified for the first time, and the method has important significance on safety risk monitoring, peculiar smell tracing and promotion of product quality improvement of raincoat products.

Description

Technical Field

[0001] The present invention relates to a method for detecting chemical substances, in particular to a method for detecting the residual amounts of five substances, namely isophorone, cyclohexanone, naphthalene, phenol, and 2,6-di-tert-butyl-p-cresol, in children's raincoat products. Background Art

[0002] Umbrellas and raincoats are the most commonly used rain-proof tools. For children and students, umbrellas can block the line of sight in crowded places and pose safety hazards. For example, the tip of the umbrella bone may cause eye injuries to others. In addition, umbrellas often cannot fully protect children's schoolbags and clothes from getting wet. Raincoats are more favored by children because of their high safety, portability, and practicality. However, as polymer products, harmful chemical substances may be intentionally / unintentionally introduced into raincoats during their raw material or production process. These substances can be absorbed and accumulated by children through inhalation or skin contact, causing potential harm. At present, there is no standard for children's raincoats in China. However, as textiles, it can refer to GB 31701-2015 "Technical Specifications for Safety of Infants' and Children's Textile Products" and GB / T 18885-2020 "Technical Requirements for Eco-Friendly Textiles", which limit substances such as formaldehyde, phthalates, carcinogenic aromatic amine dyes, perfluorinated compounds, and organotin. In recent years, among the 21 cases of children's raincoat recalls notified by the EU Rapid Alert System for Non-Food Products (RAPEX), 8 cases involve potential chemical risks. For example, a certain children's raincoat recalled on February 3, 2023, was detected to contain lead, and exposure to this substance may cause damage to children's central nervous system.

[0003] Existing literature on raincoats mainly focuses on the research of physical properties, such as abrasion resistance, color fastness, waterproofness, and breathability. The research on the chemical characteristics of raincoats focuses on new materials. There is very little research on odor substances or harmful chemical substances in raincoats. The literature that can be found only involves the determination of volatile substances such as toluene, ethylbenzene, and xylene. Most raincoats emit obvious odors, and there may be a large number of unknown volatile / semi-volatile hazardous substances among them. According to our non-target screening of 28 batches of children's raincoat samples using high-resolution gas chromatography-mass spectrometry, the detection frequencies and contents of five substances, namely isophorone, cyclohexanone, naphthalene, phenol, and 2,6-di-tert-butyl-p-cresol, are relatively high. According to the classification of the International Agency for Research on Cancer (IARC), among them, isophorone and naphthalene are Group 2B carcinogens, and cyclohexanone, phenol, and 2,6-di-tert-butyl-p-cresol are Group 3 carcinogens, all of which have relatively high hazards. Isophorone, phenol, and naphthalene are also typical odor substances, with odors presenting as camphor smell, burnt smell, earthy smell, plastic smell, and medicinal smell, which are the main sources of the odor of raincoat products. Therefore, it is very necessary to develop a quantitative detection method for these five substances, which helps to promote the improvement of the quality and safety of raincoats and protect children's health. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting five harmful substances in children's raincoats.

[0005] The method for detecting five harmful substances in children's raincoats of the present invention comprises the following steps:

[0006] (1) Sample pretreatment: Cut the sample into pieces;

[0007] (2) Target substance extraction; Add a solvent to the sample, perform ultrasonic extraction, and after completion, collect the supernatant and filter it;

[0008] (3) Analyze using a gas chromatography-high resolution time-of-flight mass spectrometer;

[0009] The five harmful substances are isophorone, cyclohexanone, naphthalene, phenol, and 2,6-di-tert-butyl-p-cresol.

[0010] For the method for detecting five harmful substances in children's raincoats of the present invention, the analysis conditions of the gas chromatography-high resolution time-of-flight mass spectrometer in step (3) are as follows:

[0011] The inlet temperature is 280 °C, the split injection mode is adopted, and the split ratio is set to 5:1; The carrier gas is high-purity helium with a purity of 99.999%, and the flow rate is 1 mL / min; Separation is carried out using a DB-5MS UI chromatographic column, 30 m × 0.25 mm × 0.25 μm, and the program temperature conditions are as follows: The initial temperature is 40 °C, held for 1 min, then raised to 280 °C at a rate of 15 °C / min and held for 5 minutes.

[0012] For the method for detecting five harmful substances in children's raincoats of the present invention, the analysis conditions of the gas chromatography-high resolution time-of-flight mass spectrometer further include:

[0013] Detection is carried out in the electron ionization EI mode, the electron energy is set to 70 eV; The full-scan mass spectrometry acquisition range is 40 to 450 m / z; The mass spectrometry transfer line temperature is set to 280 °C, the ion source emission current is set to 5 μA, the ion source temperature is 230 °C, the quadrupole temperature is 150 °C; The solvent delay is 3.2 min.

[0014] For the method for detecting five harmful substances in children's raincoats of the present invention, the analysis conditions further include the following chromatographic and mass spectrometric parameters:

[0015] Table 1 Chromatographic and mass spectrometric parameters of the substances determined in the present invention

[0016]

[0017] Note: The ones with * are the quantitative ions of the substances.

[0018] Detection method for five harmful substances in the children's raincoat of the present invention. Among them, the sample pretreatment process includes the following steps: Cut the children's raincoat sample into pieces smaller than 5mm×5mm. After each operation, wipe the scissors with anhydrous ethanol to avoid cross-contamination between samples.

[0019] Detection method for five harmful substances in the children's raincoat of the present invention. Among them, the target substance extraction process includes the following steps: Weigh 1 g of the treated sample, place it in a 25 mL stoppered colorimetric tube, add 10 mL of dichloromethane, and ultrasonically extract for 30 min at room temperature; After the extraction is completed, collect the supernatant, filter it through a 0.22 μm nylon filter membrane, and then measure it on a machine.

[0020] The difference between the detection method for five harmful substances in the children's raincoat of the present invention and the prior art lies in:

[0021] (1) The present invention is applicable to the detection of the residue amounts of five harmful substances, namely isophorone, cyclohexanone, naphthalene, phenol, and 2,6-di-tert-butyl-p-cresol, in children's raincoats made of three materials (EVA, PVC, TPU). In the past, there was no detection method for the residue amounts of these five substances in raincoats. There was only a detection method for the volatile amounts of individual substances such as phenol.

[0022] (2) By using samples of three material types, conditions such as extraction solvents and extraction solvent ratios are investigated respectively, so that the sample extraction method can adapt to the extraction of five substances in various types of children's raincoats. The instrument detection conditions such as split mode, ion source temperature, and emission current are investigated and optimized, so that the method has high sensitivity, and the substances have good chromatographic peaks, which is applicable to the quantitative detection of five substances.

[0023] (3) The present invention uses a gas chromatography-high resolution time-of-flight mass spectrometer to detect five harmful substances, and the monitored ions are accurate to the fourth decimal place. For example, the quantitative ion mass-to-charge ratio (m / z) of isophorone is 82.0413 (the conventional GC-MS quadrupole mass spectrometer has a low resolution and can only measure up to the first decimal place, that is, 82.0). The first advantage of this method is high sensitivity, and the second is more accurate detection (for example, the peak after extracting the 82.0413 ion is cleaner than the peak after extracting the 82.0 ion, that is, the selectivity is higher, and the ability to resist matrix background interference is stronger), significantly reducing the false positive detection results of the conventional low-resolution mass spectrometry.

[0024] (4) The present invention has quantitatively determined for the first time the odor substances isophorone, phenol, and naphthalene in multiple batches of children's raincoats, which is of great significance for the research on the odor source tracing of raincoat products and the promotion of product quality improvement.

[0025] In summary, the present invention is the first detection method for the residue amounts of five harmful substances, namely isophorone, cyclohexanone, naphthalene, phenol, and 2,6-di-tert-butyl-p-cresol, in children's raincoats.

[0026] The following further describes the detection method for the five harmful substances in children's raincoats according to the present invention with reference to the accompanying drawings. Description of the Drawings

[0027] Figure 1 It is an optimized comparison diagram of the sample extraction solvent in the method of the present invention;

[0028] Figure 2 It is an optimized comparison diagram of the solvent ratio of the sample extraction solvent in the method of the present invention;

[0029] Figure 3 It is an optimized comparison diagram of the instrument detection conditions in the method of the present invention; wherein: (a) optimization of the split mode; (b) optimization of the ion source temperature; (c) optimization of the emission current;

[0030] Figure 4 It is the chromatogram under the injection conditions with different split ratios in the method of the present invention;

[0031] Figure 5 It is the superimposed chromatogram of 9 representative raincoat samples in the embodiment of the present invention. Detailed Embodiments

[0032] I. Experimental Part

[0033] 1. Instruments, Reagents and Samples

[0034] 8890-7250 gas chromatography-high resolution time-of-flight mass spectrometry (Agilent Technologies, USA), Milli-Q Integral 5 water purification system (Millipore Corporation, USA). Pesticide-grade n-hexane was purchased from J&K Scientific Ltd. (Beijing, China). Chromatographic-grade acetone, dichloromethane, methanol, ethyl acetate, and n-hexane were purchased from ANPEL Laboratory Technologies Inc. (Shanghai, China).

[0035] A total of 28 batches of children's raincoat samples were randomly purchased from shopping malls in Beijing and e-commerce platforms such as Taobao and JD.com. Among them, 13 batches were made of ethylene-vinyl acetate copolymer (EVA), 9 batches were made of polyvinyl chloride (PVC), and 6 batches were made of thermoplastic polyurethane elastomer (TPU).

[0036] 2. Sample Preparation and Extraction of Target Substances

[0037] Cut the children's raincoat sample into pieces smaller than 5mm×5mm. After each operation, wipe the scissors with anhydrous ethanol to avoid cross-contamination between samples. Weigh 1g of the sample and place it in a 25mL stoppered colorimetric tube. Add 10mL of dichloromethane and ultrasonically extract for 30min at room temperature. After extraction, collect the supernatant, filter it through a 0.22μm nylon filter membrane, and then measure it on the machine.

[0038] 3. Instrument detection conditions

[0039] Analyze using a gas chromatography-high resolution time-of-flight mass spectrometer. The inlet temperature is 280℃, and the split injection mode is adopted with a split ratio set to 5:1; the carrier gas is high-purity helium with a purity of 99.999%, and the flow rate is 1mL / min; use a DB-5MSUI chromatographic column (30m×0.25mm×0.25μm) for separation, and the program temperature conditions are as follows: the initial temperature is 40℃, hold for 1min, then rise to 280℃ at a rate of 15℃ / min and hold for 5 minutes; detect in the electron ionization EI mode, and the electron energy is set to 70eV; the full scan mass spectrometry acquisition range is 40 to 450m / z; the mass spectrometry transfer line temperature is set to 280℃, the ion source emission current is set to 5μA, the ion source temperature is 230℃, and the quadrupole temperature is 150℃; the solvent delay is 3.2min.

[0040] Before analyzing the sample, the instrument was comprehensively maintained to minimize background interference, including cleaning the ion source, using a new chromatographic column, a new inlet liner, and a gasket, etc.

[0041] 4. The chromatographic and mass spectrometric parameter information of the 5 substances determined by the present invention is as follows.

[0042] Table 1 Chromatographic and mass spectrometric parameters of the substances determined by the present invention

[0043]

[0044] Note: The one with * is the quantitative ion of this substance.

[0045] II. Results and analysis

[0046] 1. Optimization of extraction solvent

[0047] Through the previous non-targeted screening of unknown substances using high-resolution gas chromatography-mass spectrometry (GC-MS), multiple batches of children's raincoat products were identified to contain the target substances isophorone, cyclohexanone, naphthalene, phenol, and 2,6-di-tert-butyl-p-cresol. Since there were no physical reference standards with known contents on the market, a positive sample was selected as a benchmark for each of the three types of products, and the sample extraction conditions were optimized. Among them, 5 substances were detected in both the selected EVA and TPU samples, and no PVC sample was found to contain all 5 substances simultaneously. Therefore, a sample that detected 4 substances (lacking 2,6-di-tert-butyl-p-cresol) was chosen as the positive sample.

[0048] First, the types of extraction solvents were investigated. 1 g of raincoat sample was extracted with 10 mL of acetone, dichloromethane, methanol, ethyl acetate, and n-hexane respectively, ultrasonically extracted at room temperature for 30 min, and the supernatant was collected after extraction. After filtration through a 0.22 μm nylon filter membrane, it was analyzed by the instrument. As Figure 1 shown, the peak areas of the quantitative ions for the extraction of each substance were compared horizontally. Three parallel tests were carried out for each detection, and the standard deviation was calculated. For the PVC material, methanol had the best extraction effect on isophorone and cyclohexanone, and dichloromethane had the best extraction effect on naphthalene. For the EVA material, dichloromethane had the best extraction effect on cyclohexanone, naphthalene, and 2,6-di-tert-butyl-p-cresol, and methanol had the best extraction effect on isophorone and phenol. For the TPU material, dichloromethane had the best extraction effect on cyclohexanone, naphthalene, and 2,6-di-tert-butyl-p-cresol, and ethyl acetate had the best extraction effect on isophorone and phenol. It can be seen that the extraction effect is significantly affected by the sample matrix. For different substances in different matrix samples, the most suitable extraction solvent is different, and there is no universal solvent that can have the best extraction effect on all 5 substances in the three materials. For the same substance, such as isophorone, methanol is the best for extraction in PVC and EVA, while ethyl acetate is the best for extraction in TPU. Considering comprehensively, n-hexane showed relatively poor extraction performance for almost all substances. Among the other 4 solvents, dichloromethane and methanol performed relatively well. They are not only suitable for samples of various materials but also have a good extraction rate for most substances. Therefore, dichloromethane and methanol were selected for further investigation.

[0049] 2. Optimization of the extraction solvent ratio

[0050] The positive sample was extracted with five different ratios of extraction solvents: methanol, dichloromethane, methanol:dichloromethane volume ratio of 1:1, methanol:dichloromethane volume ratio of 1:2, and methanol:dichloromethane volume ratio of 2:1 for further investigation and optimization. As Figure 2As shown, the peak areas of various substances were compared horizontally. Each test was conducted with three parallel trials, and the standard deviation was calculated. For PVC materials, the volume ratio of methanol:dichloromethane of 1:1 had the best extraction effect on isophorone and cyclohexanone, and dichloromethane had the best extraction effect on naphthalene. For EVA materials, the volume ratio of methanol:dichloromethane of 1:2 had the best extraction effect on cyclohexanone, phenol, and 2,6-di-tert-butyl-p-cresol, dichloromethane had the best extraction effect on naphthalene, and methanol had the best extraction effect on isophorone. For TPU materials, the volume ratio of methanol:dichloromethane of 1:1 had the best extraction effect on cyclohexanone and phenol, and dichloromethane had the best extraction effect on naphthalene, isophorone, and 2,6-di-tert-butyl-p-cresol. Thus, it can be seen that when the two solvents are mixed, the effect of 1+1 is not greater than 1. Different solvent ratios have a significant impact on the test results. Considering comprehensively, dichloromethane is at an upper-middle level in the extraction effect of most substances in the three materials, with little difference compared with other solvent ratios, and its extraction effect on naphthalene is far better than other solvents. Therefore, dichloromethane was finally selected as the extraction solvent for the raincoat samples.

[0051] 3. Optimization of Instrument Detection Conditions

[0052] The key conditions affecting the sensitivity and detection effect of gas chromatography-high resolution time-of-flight mass spectrometry were investigated and optimized, including chromatographic splitting mode, ion source temperature, and emission current. A standard solution of 5 substances with a concentration of 1 mg / L was injected for determination, and the peak areas obtained from the detection of each substance were compared horizontally.

[0053] As Figure 3 shown in Fig. a, five injection conditions of non-splitting, splitting ratio of 5:1, splitting ratio of 10:1, splitting ratio of 20:1, and splitting ratio of 30:1 were investigated respectively. The results showed that as the splitting ratio increased, the chromatographic peak areas of various substances gradually decreased, and this trend was consistent with expectations. The value of isophorone without splitting was relatively small, which might be an individual abnormal phenomenon due to the lack of parallel tests. When comparing the chromatographic peak shapes of various substances (see Figure 4 ), it was noted that in the non-splitting injection mode, the baseline of the chromatogram bulged abnormally, the chromatographic peaks were short and fat with burrs, and the peak shape was not as ideal as that in the splitting injection mode, which might affect the stability of the quantitative results. In order to ensure the maximum response of the target substance (i.e., the maximum sensitivity) while maintaining a good chromatographic peak shape, a splitting ratio of 5:1 was finally selected.

[0054] As Figure 3As shown in Figure b, five ion source temperature conditions of 190°C, 210°C, 230°C, 250°C, and 270°C were investigated respectively. It was found that the peak areas of all substances reached the maximum at 230°C. At this temperature, all substances could achieve the best ionization efficiency, thus obtaining the best detection sensitivity. The conditions at 250°C and 270°C were the second best, while those at 190°C and 210°C were the worst. The increase in temperature was helpful for ionization, but too high a temperature could not achieve the best ionization effect. Therefore, 230°C was determined as the ionization temperature of the ion source.

[0055] As Figure 3 shown in Figure c, five ion source emission current conditions of 1 μA, 2 μA, 3 μA, 4 μA, and 5 μA were investigated respectively. It was found that the greater the emission current, the better the detection response of all substances, and the peak areas showed an increasing trend. Although a higher emission current might bring a better response, too high a current might damage the ion source filament. Usually, the price of a filament for a high-resolution time-of-flight mass spectrometer was about 10,000 yuan. In order to ensure the best detection sensitivity while also protecting the life of the instrument filament, 5 μA was selected as the emission current.

[0056] For the finally optimized detection method, the quantitative limits of five harmful substances, namely isophorone, cyclohexanone, naphthalene, phenol, and 2,6-di-tert-butyl-p-cresol, in children's raincoats were 0.02 mg / kg, 0.02 mg / kg, 0.02 mg / kg, 0.1 mg / kg, and 0.1 mg / kg respectively, and the detection limits were 0.01 mg / kg, 0.005 mg / kg, 0.005 mg / kg, 0.05 mg / kg, and 0.05 mg / kg respectively. In the linear range of 0.02 - 20 mg / kg, the correlation coefficient R 2 was greater than 0.9991, and the method had good linearity. The specific parameters are shown in Table 1.

[0057] In this invention, a gas chromatography-high resolution time-of-flight mass spectrometer was used to detect five harmful substances in children's raincoats. The monitored ions were the exact mass numbers of the substances, accurate to the fourth decimal place. For example, the quantitative ion of isophorone was 82.0413 (the conventional GC-MS quadrupole mass spectrometer had a low resolution and could measure up to one decimal place after the decimal point, 82.0). The advantages of high-resolution mass spectrometry detection were high sensitivity on the one hand and more accurate detection on the other hand (for example, the peak after extracting the 82.0413 ion was definitely cleaner than the peak after extracting the 82.0 ion, that is, the selectivity was higher), significantly reducing the false positive detection results of the conventional low-resolution mass spectrometry.

[0058] 4. Detection of raincoat samples

[0059] Twenty-eight batches of children's raincoat samples were detected using the optimized extraction and detection methods. Figure 5The superimposed chromatograms of 9 representative raincoats (chromatograms in the full-scan mode) are shown incidentally. The different chromatograms correspond to PVC, EVA, and TPU samples respectively. As shown in the figure, various substances were detected in the raincoats, and there were differences in the presence of substances among different products. Some chromatographic peaks were unique to certain products, while some were common to multiple samples. The number and intensity of the chromatographic peaks detected in the PVC samples were significantly higher than those in the TPU and EVA samples. Thus, it can be seen that there are many substances present in the products, and it is very crucial to identify the potential unknown harmful substances among them. The present invention detects the residual amounts of 5 new types of harmful substances, providing a good reference for future detection of more potential harmful substances.

[0060] Table 2 summarizes the overall detection of children and raincoat samples. Taking isophorone as an example, isophorone was detected in 6 batches of PVC raincoats, 6 batches of TPU raincoats, and 11 batches of EVA raincoats, with average contents of 162.577 mg / kg, 11.806 mg / kg, and 6.567 mg / kg respectively. Table 3 gives the detailed detection results of 28 batches of children's raincoat samples. For the situation where the substance content exceeded the linear range, the solution was diluted to bring the concentration within the linear range.

[0061] Isophorone, phenol, and naphthalene are typical odor substances, with odors presenting as camphor smell, burnt smell, earthy smell, plastic smell, and medicinal smell. From the quantitative results in Table 2, it can be seen that the average content of isophorone in the samples is 6.567 mg / kg - 162.577 mg / kg, the average content of phenol in the samples is 10.558 mg / kg - 96.962 mg / kg, and the average content of naphthalene in the samples is 0.828 mg / kg - 17.004 mg / kg, and the content values are relatively high. After inquiry, the odor thresholds of isophorone, phenol, and naphthalene in the air are 0.0017 mg / m 3 、0.021 mg / m 3 、0.01 mg / m 3 respectively, and the thresholds are very low. Therefore, even at relatively low concentrations of these three substances, the odor is still very obvious. The high content of these three substances suggests that they may be the main sources of the odor in raincoat products. Further odor analysis and smelling work can be carried out accordingly in the future.

[0062] Table 2 Overall detection of children's raincoat samples

[0063]

[0064] Table 3 Detailed detection results of 28 batches of children's raincoat samples

[0065]

[0066] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A detection method for 5 harmful substances in children's raincoats, characterized in that: It includes the following steps: (1) Sample pretreatment: Cut the sample into pieces; (2) Target substance extraction: Add a solvent to the sample, perform ultrasonic extraction, and after completion, collect the supernatant and filter it; (3) Analyze using a gas chromatography-high resolution time-of-flight mass spectrometer; The 5 hazardous substances are isophorone, cyclohexanone, naphthalene, phenol, and 2,6-di-tert-butyl-p-cresol.

2. The detection method of five harmful substances in the children's raincoat according to claim 1, characterized in that: The analysis conditions of the gas chromatography-high resolution time-of-flight mass spectrometer in step (3) are as follows: The inlet temperature is 280 °C, the split injection mode is adopted, and the split ratio is set to 5:1; the carrier gas is high-purity helium with a purity of 99.999%, and the flow rate is 1 mL / min; separation is carried out using a DB-5MS UI chromatographic column, 30 m × 0.25 mm × 0.25 μm, and the program temperature conditions are as follows: the initial temperature is 40 °C, held for 1 min, then raised to 280 °C at a rate of 15 °C / min and held for 5 minutes.

3. The detection method of five hazardous substances in the children's raincoat according to claim 2, characterized in that: The analysis conditions of the gas chromatography-high resolution time-of-flight mass spectrometer also include: Detection is carried out in the electron ionization (EI) mode, the electron energy is set to 70 eV; the full scan mass spectrometry acquisition range is 40 to 450 m / z; the mass spectrometry transfer line temperature is set to 280 °C, the ion source emission current is set to 5 μA, the ion source temperature is 230 °C, the quadrupole temperature is 150 °C; the solvent delay is 3.2 min.

4. The detection method for five harmful substances in children's raincoats according to claim 3, characterized in that: The analysis conditions also include the following chromatographic and mass spectrometric parameters: Table 1 Chromatographic and mass spectrometric parameters of 5 substances Note: The ones with * are the quantitative ions of the substances.

5. The detection method of five harmful substances in the children's raincoat according to claim 1, characterized in that: The sample pretreatment process includes the following steps: Cut the children's raincoat sample into pieces smaller than 5 mm × 5 mm. After each operation, wipe the scissors with anhydrous ethanol to avoid cross-contamination between samples.

6. The detection method for five hazardous substances in the children's raincoat according to claim 5, characterized in that: The target substance extraction process includes the following steps: Weigh 1 g of the treated sample, place it in a 25 mL stoppered colorimetric tube, add 10 mL of dichloromethane, and perform ultrasonic extraction at room temperature for 30 min; after the extraction is completed, collect the supernatant, filter it through a 0.22 μm nylon filter membrane, and then measure it on the instrument.

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