A method for detecting ethylene oxide, 2-chloroethanol and acetaldehyde in frozen drinks by solid phase microextraction gas chromatography mass spectrometry

Abstract

The application belongs to the field of chemical detection, and particularly relates to a method for detecting ethylene oxide, 2-chloroethanol and acetaldehyde in frozen drinks by solid phase microextraction gas chromatography mass spectrometry. The method for detecting ethylene oxide, 2-chloroethanol and acetaldehyde in frozen drinks by solid phase microextraction gas chromatography mass spectrometry comprises the following steps: S.1) taking a frozen drink sample to be detected, and uniformly mixing; S.2) weighing a certain amount of the sample, and adding sodium chloride and hydrochloric acid; S.3) determining the sample by solid phase microextraction gas chromatography mass spectrometry; and S.4) quantitatively analyzing the content of ethylene oxide, 2-chloroethanol and acetaldehyde in the frozen drink sample to be detected by an external standard method. The application combines a derivatization reaction and a solid phase microextraction process into one, and overcomes the difficulties in traditional analysis technology, such as complex pretreatment, long time consumption, low accuracy, unstable results, high detection limit and the like. Meanwhile, the detection result has high precision, the influence of a complex matrix is removed, and the reproducibility is good.

Description

Technical Field

[0001] This invention belongs to the field of chemical detection, specifically relating to a method for detecting ethylene oxide, 2-chloroethanol and acetaldehyde in frozen beverages using solid-phase microextraction gas chromatography-mass spectrometry. Background Technology

[0002] Ethylene oxide (EO) is a flammable, colorless gas with a slightly sweet odor. Discovered in 1936 for its sterilizing properties, it has been widely used as a disinfectant. Due to its excellent sterilization effect, it is widely applied in various fields. However, prolonged exposure to ethylene oxide can cause vomiting, headaches, difficulty breathing, nausea, and skin damage, and may even affect fertility. Ethylene oxide in the blood can cause cardiovascular disease, and it is particularly genotoxic after ingestion; long-term intake can lead to cancer. On October 27, 2017, the International Agency for Research on Cancer (IARC) of the World Health Organization classified ethylene oxide as a Group 1 carcinogen. During sterilization, ethylene oxide may produce or transform into the derivative 2-chloroethanol, a more stable and difficult-to-remove toxic compound that can cause harm to the body through skin absorption or ingestion.

[0003] Acetaldehyde is a low-toxicity, polar organic compound and a volatile liquid. At low concentrations, it can irritate the eyes, skin, and upper respiratory tract mucosa; inhalation of high concentrations can have a paralyzing effect and cause acute poisoning. Acetaldehyde in food has a wide range of sources. Firstly, it can migrate from plastic or paper food packaging materials. For example, polyethylene terephthalate (PET), a widely produced and used plastic food packaging material, may decompose to generate acetaldehyde during its synthesis, processing, and use, and acetaldehyde may migrate from food packaging into the food. Secondly, acetaldehyde may be generated during food production processes, such as during the fermentation of wine and rice wine, or through the biochemical reactions of microorganisms during the production of fructose syrup.

[0004] Solid-phase microextraction (SPME) technology integrates sampling, separation, enrichment, and sample introduction into a single operation, offering simplicity and high efficiency. The principle involves using fused silica optical fibers or other materials as a matrix support, leveraging the "like dissolves like" principle. Thin layers of polymeric stationary phases with varying properties are coated onto the surface of the support. The analyte is extracted and enriched directly or via headspace methods. The enriched fiber is then transferred directly to the instrument for desorption (typically thermal or solvent desorption) followed by analysis.

[0005] Currently, there are no domestic standards for the detection of ethylene oxide and 2-chloroethanol in food, and a scientifically sound and comprehensive qualitative and quantitative detection method is lacking. The main reported methods for detecting ethylene oxide and 2-chloroethanol include QuEChERS, headspace sampling, and direct SPME. QuEChERS has relatively low accuracy because the high water content of matrices such as frozen drinks leads to exothermic pretreatment, and ethylene oxide, with its low boiling point and high volatility, results in significant losses and unstable results in practical applications. Headspace sampling has a relatively high detection limit but low sensitivity. Direct solid-phase microextraction (SPME) methods have been reported less frequently; existing techniques mainly target natural foods such as grains and spices, and no reports have been found for frozen drink matrices. This may be related to the complexity of frozen drink matrices, especially the potential presence of thickeners, emulsifiers, and flavorings, which can easily cause interference.

[0006] Currently reported methods for acetaldehyde detection mainly include headspace gas chromatography and liquid chromatography, which have high detection limits but low sensitivity. Solid-phase microextraction (SPE) is an alternative technique for acetaldehyde analysis, offering advantages such as ease of operation and low cost. Considering that acetaldehyde and ethylene oxide are isomers with extremely similar properties (ethylene oxide has a boiling point of 10.8℃, and acetaldehyde has a boiling point of 20.8℃), both being polar molecules, and both potentially coexisting in frozen beverages, successful separation of acetaldehyde and ethylene oxide, along with their separate qualitative and quantitative detection, is crucial.

[0007] Patent CN117929548A discloses a method for determining ethylene oxide, which utilizes acidic salt solution to treat the sample. This converts ethylene oxide in the solution into 2-chloroethanol. A series of standard solutions are prepared using ethylene oxide, and a standard curve is plotted. The treated sample is then extracted, centrifuged, and the supernatant is collected. This supernatant is detected using gas chromatography-triple quadrupole mass spectrometry (GC-MS / MS), and the ethylene oxide content is calculated based on the standard curve. However, this method may interfere with derivatization reactions or produce false positive signals in complex matrices (such as food or biological samples), requiring additional purification steps. Furthermore, triple quadrupole mass spectrometers are expensive, complex to maintain, require highly skilled operators, and necessitate regular calibration and optimization of mass spectrometry parameters, resulting in high operating costs. Patent CN 114487208 A proposes a method for determining the total amount of ethylene oxide and 2-chloroethanol in food based on GC-MSMS. This patent uses acidic sodium chloride solution for derivatization to completely convert ethylene oxide into 2-chloroethanol, followed by extraction with ethyl acetate. Quantification is performed using an isotope internal standard method, and analysis is conducted via a tandem gas chromatography-mass spectrometry system. However, this patent's derivatization reaction requires 180 minutes, and extraction requires 60 minutes, resulting in complex and time-consuming pretreatment. Furthermore, replicating the experimental procedure revealed unstable derivatization results. Summary of the Invention

[0008] In order to overcome the problems of complex pretreatment, long processing time, low accuracy, unstable results and high detection limit in the existing technology, this invention proposes a solid phase microextraction method for determining ethylene oxide, 2-chloroethanol and acetaldehyde in frozen beverages.

[0009] The present invention is implemented using the following technical solutions:

[0010] In a first aspect, the present invention provides a technical method for determining ethylene oxide, 2-chloroethanol, and acetaldehyde in frozen beverages by solid-phase microextraction, comprising the following steps:

[0011] S.1) Take the frozen beverage sample to be tested and homogenize it.

[0012] S.2) Weigh a certain amount of sample and add sodium chloride and hydrochloric acid;

[0013] S.3) Solid-phase microextraction gas chromatography-mass spectrometry was used to determine the sample;

[0014] S.4) The content of ethylene oxide, 2-chloroethanol and acetaldehyde in the frozen beverage samples was quantitatively analyzed using the external standard method.

[0015] Furthermore, the sample amount and the ratio of sodium chloride to hydrochloric acid in step S.2 are 4.5-5.5g: 1.5-2.5g: 5-15uL.

[0016] Furthermore, the solid-phase microextraction (SPE) conditions in step S.3 using gas chromatography-mass spectrometry are as follows: Agi lent CTC autosampler; extraction fiber: 80µm DVB / CAR / PDMS; extraction fiber aging temperature: 250℃.

[0017] Further, extract at 40℃-50℃ for 15-25 min with a shaking rate of 200-300 rpm.

[0018] Furthermore, the mass spectrometry conditions in the solid-phase microextraction gas chromatography-mass spectrometry method described in step S.3 are: voltage 70 eV; ion source temperature 230℃; interface temperature: 250℃; quadrupole temperature 150℃.

[0019] Furthermore, in step S.3, the temperature program used in solid-phase microextraction gas chromatography-mass spectrometry is as follows: heat the sample to 35°C and hold for 1.5-2.5 min, slowly increase the temperature to 50°C at a rate of 1.5°C / min and hold for 2 min, and then rapidly increase the temperature to 250°C at a rate of 40°C / min and hold for 5 min.

[0020] Furthermore, the carrier gas mentioned in step S.3 is helium.

[0021] Furthermore, the carrier gas is a split injection with a split ratio of 5:1.

[0022] Furthermore, the external standard method described in step S.4 includes the following steps:

[0023] (1) Preparation of standard solutions: Prepare standard solutions of 2-chloroethanol and acetaldehyde;

[0024] (2) Prepare a standard curve: Load the standard series working solutions into the instrument in order from low to high and measure the corresponding response values. Plot the standard curve with the mass concentration of the analyte in the standard series working solutions as the x-axis and the response value of the analyte in the standard series working solutions as the y-axis.

[0025] (3) Determination of frozen beverage samples: Substitute the peak area obtained from the detection into the standard curve of the corresponding substance, and analyze and calculate the content of ethylene oxide, 2-chloroethanol and acetaldehyde in the frozen beverage.

[0026] The present invention has the following beneficial effects:

[0027] (1) This invention develops and establishes a method for determining ethylene oxide, 2-chloroethanol and acetaldehyde in frozen drinks using solid phase microextraction gas chromatography-mass spectrometry. This method is simple to operate, combining the derivatization reaction and solid phase microextraction process into one, shortening the time to only 20 minutes. It overcomes the difficulties of traditional analytical techniques such as complex pretreatment, long time consumption, low accuracy, unstable results and high detection limit. At the same time, it provides a basic experimental idea for solving the determination of ethylene oxide, 2-chloroethanol and acetaldehyde in food with complex matrix.

[0028] (2) This invention enables the separation of three analytes—ethylene oxide, 2-chloroethanol, and acetaldehyde—within 21 minutes. 2-chloroethanol exhibits good linearity within the detection range of 0.02-1.00 μg, and acetaldehyde exhibits good linearity within the detection range of 0.5-25.0 μg. The linear correlation coefficient R0 is [value missing]. 2 For 2-chloroethanol, the detection limit is greater than 0.999 μg / kg, the quantitation limit is 16.0 μg / kg, with high precision and a recovery rate of 104.66-106.82%, indicating high accuracy. For ethylene oxide, the detection limit is 4.4 μg / kg, the quantitation limit is 8.8 μg / kg, with high precision and a recovery rate of 103.72-106.29%, indicating high accuracy. For acetaldehyde, the detection limit is 0.4 mg / kg, the quantitation limit is 1.6 mg / kg, with high precision and a recovery rate of 98.43-105.65%, indicating high accuracy.

[0029] (3) Ethylene oxide, 2-chloroethanol and acetaldehyde have good reproducibility. In the frozen beverage matrix, the relative standard deviation (RSD) of ethylene oxide is 2.08-3.51%, that of 2-chloroethanol is 0.94-3.67%, and that of acetaldehyde is 2.75-5.79%.

[0030] (4) This method is improved to eliminate the interference of matrix effect by using a blank sample matrix to prepare a standard curve. Attached Figure Description

[0031] Figure 1 This is the mass spectrum of a mixed standard solution of 2-chloroethanol and acetaldehyde. Detailed Implementation

[0032] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0033] Example 1: Determination of Anti-interference Ability of Complex Matrix (Ice Cream)

[0034] Ethylene oxide is characterized by its low boiling point and high volatility. Furthermore, gas chromatography-mass spectrometry (GC-MS) methods often result in early peak detection, but low response at low concentrations can lead to poor peak shape. Therefore, the proposed detection method considers converting ethylene oxide into stable 2-chloroethanol. The conversion and solid-phase microextraction steps are performed simultaneously, resulting in a simple method that saves pretreatment time, achieves high conversion efficiency, and provides stable conversion results.

[0035] Experiments were conducted on blank ice cream samples at ethylene oxide spiking levels of 4.4 μg / kg, 8.8 μg / kg, and 44.0 μg / kg. Recovery rate and RSD% were used as evaluation indicators. The obtained data were statistically analyzed, and the results are shown in Table 1.

[0036] Table 1 Recovery rate and RSD%

[0037]

[0038] The results showed that the average recovery rate of ethylene oxide in frozen beverage ice cream was 103.72-106.29%, and the relative standard deviation (RSD%) was 2.08-3.51%, which met the detection requirements.

[0039] Example 2:

[0040] I. Experimental Materials

[0041] 1. Main instruments: Agilent 8890-5977 gas chromatograph-mass spectrometer (with CTC autosampler); analytical balance: sensitivity 0.1 mg.

[0042] 2. Standard substances: 2-chloroethanol (1000mg / L), Tanmo Quality Inspection; acetaldehyde (color standard), 99.5%, Shanghai Anpu.

[0043] 3. Samples: Nine batches of frozen drinks were sampled from markets in Zhejiang Province.

[0044] II. Experimental Procedure

[0045] 1. Sample preparation: Take the edible portion of the sample and homogenize it for later use. Weigh 5g (accurate to 0.01g) of the homogenized sample into a 20mL headspace vial, add 2g of sodium chloride and 10uL of hydrochloric acid, and prepare for instrumentation.

[0046] 2. Prepare standard solutions: Prepare a series of standard working solutions according to Table 2.

[0047] Table 2. Standard Solution Concentration and Preparation Information

[0048]

[0049] 3. Solid-phase microextraction gas chromatography-mass spectrometry detection:

[0050] (1) Solid-phase microextraction conditions: The extraction fiber coating contains DVB, CAR, and PDMS, and the coating thickness is 70-90 μm. Aging temperature of the extraction fiber: 250℃. Extraction temperature: 45℃; extraction time: 20 min; shaking rate: 250 rpm.

[0051] (2) Chromatographic conditions: A DB-624UI capillary column (60m*0.25mm*1.40um) was used. The chromatographic process requires heating, which includes the following steps:

[0052] K.1) Heat the sample to 35°C and hold for 2 minutes;

[0053] K.2) Slowly increase the temperature to 50℃ at a rate of 1.5℃ / min and hold for 2 minutes;

[0054] K.3) Rapidly heat to 250℃ at a rate of 40℃ / min and hold for 5min.

[0055] The carrier gas is helium; the flow rate is 0.8 mL / min; the injection method is split injection with a split ratio of 5:1; the injection port temperature is 240℃. The liner is a liner without glass wool.

[0056] (3) Mass spectrometry conditions: Electron impact ionization (EI) source, voltage 70eV; ion source temperature 230℃; interface temperature: 250℃; quadrupole temperature 150℃.

[0057] The parameters of the sample to be tested are shown in Table 3.

[0058] Table 3 Mass spectrometry parameters of the analytes

[0059]

[0060] 3. Constructing a Standard Curve: Prepare a series of mixed standard solutions with varying concentrations as shown in Table 2 to construct a standard curve. Measure the corresponding response values ​​sequentially from low to high concentrations. Plot the standard curve with the mass concentration of the analyte in the standard working solutions as the x-axis and the response value of the analyte in the standard working solutions as the y-axis. The curve should be linear without forced zero-crossing. See Table 4 for details. The correlation coefficients of the standard curves are all ≥0.999. The mass spectrum of the mixed standard solution of 2-chloroethanol and acetaldehyde is shown below. Figure 1 As shown.

[0061] Table 4. Linear Range, Linear Equation, and Linear Correlation Coefficient

[0062]

[0063] III. Experimental Results

[0064] As shown in Table 3, the analytes were well separated within 21 minutes. Acetaldehyde showed good linearity in the detection range of 0.5-25.0 μg, and 2-chloroethanol showed good linearity in the detection range of 0.02-1.0 μg, with a correlation coefficient R2 > 0.999.

[0065] Quantification of ethylene oxide (calculated as 2-chloroethanol) and acetaldehyde in the sample: The prepared sample was tested, and the peak area obtained was substituted into the corresponding standard curve to analyze and calculate the content of ethylene oxide (calculated as 2-chloroethanol) and acetaldehyde in the frozen beverage. The results are shown in Table 5 below.

[0066] Table 5. Content of ethylene oxide (calculated as 2-chloroethanol) and acetaldehyde in the samples (unit: mg / kg)

[0067]

[0068] Note: ND indicates not detected (< limit of quantitation)

[0069] Frozen ice cream containing no ethylene oxide, 2-chloroethanol, or acetaldehyde was selected as the blank matrix. Six parallel experiments were conducted on each matrix at low, medium, and high concentrations to examine the accuracy and repeatability of the method. Recovery rate and RSD% were used as evaluation indicators. The obtained data were statistically analyzed, and the results are shown in Table 6.

[0070] Table 6 Recovery Rate and RSD%

[0071]

[0072] The results showed that the average recoveries of 2-chloroethanol in frozen drinks and ice cream ranged from 104.66% to 106.82%, with high accuracy and relative standard deviations (RSD%) ranging from 0.94% to 3.67%, indicating strong repeatability. The average recoveries of acetaldehyde ranged from 98.43% to 105.65%, with high accuracy and relative standard deviations (RSD%) ranging from 2.75% to 5.79%, also indicating strong repeatability.

Claims

1. A method for detecting ethylene oxide, 2-chloroethanol, and acetaldehyde in frozen beverages using solid-phase microextraction gas chromatography-mass spectrometry, characterized in that... Includes the following steps: S.1 Take a sample of the frozen beverage to be tested and homogenize it. S.2 Weigh a certain amount of sample and add sodium chloride and hydrochloric acid; S.3 The sample was determined by solid-phase microextraction gas chromatography-mass spectrometry (SCIMS). The mass spectrometry conditions for SCIMS were: voltage 70 eV; ion source temperature 230℃; interface temperature 250℃; quadrupole temperature 150℃. The chromatographic conditions for SCIMS were: DB-624UI capillary column was used, and the temperature program was as follows: heat the sample to 35℃ and hold for 1.5-2.5 min, slowly increase the temperature to 50℃ at a rate of 1.5℃ / min and hold for 2 min, and then rapidly increase the temperature to 250℃ at a rate of 40℃ / min and hold for 5 min. S.4 The external standard method was used to quantitatively analyze the contents of ethylene oxide, 2-chloroethanol and acetaldehyde in the frozen beverage samples to be tested.

2. The method for detecting ethylene oxide, 2-chloroethanol, and acetaldehyde in frozen beverages by solid-phase microextraction gas chromatography-mass spectrometry as described in claim 1, characterized in that, The sample amount and the ratio of sodium chloride to hydrochloric acid in step S.2 are 4.5-5.5g: 1.5-2.5g: 5-15uL.

3. The method for detecting ethylene oxide, 2-chloroethanol, and acetaldehyde in frozen beverages by solid-phase microextraction gas chromatography-mass spectrometry as described in claim 1, characterized in that... In step S.3, the solid-phase microextraction (SPE) conditions for gas chromatography-mass spectrometry (GC-MS) are as follows: Agilent CTC autosampler; extraction fiber: 80µm DVB / CAR / PDMS; extraction fiber aging temperature: 250℃.

4. The method for detecting ethylene oxide, 2-chloroethanol, and acetaldehyde in frozen beverages by solid-phase microextraction gas chromatography-mass spectrometry as described in claim 3, characterized in that... The solid-phase microextraction conditions are as follows: extraction at 40℃-50℃ for 15-25 min, with a shaking rate of 200-300 rpm.

5. The method for detecting ethylene oxide, 2-chloroethanol, and acetaldehyde in frozen beverages by solid-phase microextraction gas chromatography-mass spectrometry as described in claim 3, characterized in that... In step S.3, the carrier gas is helium.

6. The method for detecting ethylene oxide, 2-chloroethanol, and acetaldehyde in frozen beverages by solid-phase microextraction gas chromatography-mass spectrometry as described in claim 5, characterized in that... The carrier gas is injected in a split stream with a split ratio of 5:

1.

7. The method for detecting ethylene oxide, 2-chloroethanol, and acetaldehyde in frozen beverages by solid-phase microextraction gas chromatography-mass spectrometry as described in claim 1, characterized in that... Step S.4, the external standard method, includes the following steps: (1) Preparation of standard solutions: Prepare standard solutions of 2-chloroethanol and acetaldehyde; (2) Prepare a standard curve: Load the standard series working solutions into the instrument in order from low to high and measure the corresponding response values. Plot the standard curve with the mass concentration of the analyte in the standard series working solutions as the abscissa and the response value of the analyte in the standard series working solutions as the ordinate. (3) Determination of frozen beverage samples: Substitute the peak area obtained from the detection into the standard curve of the corresponding substance, and analyze and calculate the content of ethylene oxide, 2-chloroethanol and acetaldehyde in the frozen beverage.

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