Method for quantitative determination of ethylene oxide and 2-chloro ethanol as contaminants in food products

Abstract

The present disclosure reveals the method for the quantitative determination of ethylene oxide (EO) and 2-chloroethanol (2CE) as contaminants in food products. The method is based on concentrated hydrochloric acid-mediated derivatization of the processed samples under specific treatment conditions, which provides the quantitative conversion of EO to 2CE, followed by the quantitative determination of thus formed 2CE with gas chromatography coupled with mass spectrometry (GC / MS-MS). The method enables a highly accurate procedure for the analysis of EO and 2CE in food samples at the concentration range from 0.019-0.19 mg / kg.

Description

[0001] METHOD FOR QUANTITATIVE DETERMINATION OF ETHYLENE OXIDE AND 2 -CHLORO

[0002] ETHANOL AS CONTAMINANTS IN FOOD PRODUCTS

[0003] BACKGROUND

[0004] Field

[0005] The present disclosure refers to a novel method for the determination of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) as contaminants in food products, and the use of this method.

[0006] Description of the Related Art

[0007] Ethylene oxide (EO) is a chemical compound with the molecular formula C4H4O and a molar mass of 44.05 g / mol. Its CAS number is [75-21-8] . EO is considered a genotoxic and carcinogenic substance. An assessment was conducted based on the only available study of chronic oral toxicity, from which a BMDL10 of 0.37 mg / kg body weight per day for the development of tumors in the abdomen and stomach was derived. It is used in the industry for sterilization and disinfection. It may be present in some food products as a result of contamination during production or processing. Due to its small molecular size, EO exhibits high diffusivity and strong penetration properties, making it highly effective in the sterilization and disinfection of dry food products. Due to its high flammability, EO is always mixed in a 1:9 ratio with carbon dioxide (CO2) . Sterilization requires higher concentrations of EO and extended treatment times compared to insect control conditions. EO inactivates bacterial spores and viruses by reacting with DNA and proteins such as enzymes. Therefore, EO is an important food contaminant, see for instance literature reference 1:

[0008] 1) A. Dudkiewicz, P. Dutta, D. Kolozyn-Krajewska : Ethylene oxide in foods: current approach to the risk assessment and practical considerations based on the European food business operator perspective, Eur. Food Res. Technol . 248 (2022) 1951-1958.

[0009] Fortunately, EQ rapidly dissipates through aeration, so consumers' exposure to residues associated with EQ through food consumption is mainly related to the reaction products of EO, with 2-chloroethanol ( 2CE ) being the most prominent. Nevertheless, given the inconclusive toxicological profile of 2CE, the German Risk Assessment Body (BfR) has decided to adopt a precautionary approach and consider 2CE as equally toxic as EO while the European Food Safety Authority (EFSA) suggested further new in vitro gene mutation and in vitro micronucleus tests to clarify its genotoxic potential, see, e.g. , literature reference 2. In any case, 2CE is also an important food contaminant.

[0010] EO 2CE

[0011] 2) European Food Safety Authority (EFSA) : Statement on the BfR opinion regarding the toxicity of 2-chloroethanol, EFSA J. 20 (2022) 7147.

[0012] The primary sources of EO contaminants in food products are EO-spice treatment and plant contamination for other types of food products . Namely, EO is sometimes used for the sterilization or disinfection of spices to prevent microbial contamination. However, if not applied correctly or accordingly to prescribed guidelines, residue of EO may appear in the spices. In late August 2020. , Belgium initiated an RASFF notification regarding unauthorized residues of EO in various shipments of sesame seeds from India, at concentrations of up to 186 mg / kg. The notification was published on September 9, 2020., on the RASFF portal (2020.3678) , see literature reference 3:

[0013] 3) European Commission; RASFF Window; Notification 2020.4395:

[0014] Ethylene Oxide in sesame seeds (RASFF 2020.3678) - Products deliverance; available at the link below: https : / / webgate . ec . europa . eu / rasff- window / screen / notification / 445639

[0015] These products were delivered to several EU member states and used in the production of various processed food products. By November 20, 2020, approximately 140 notifications about EO in Indian sesame seeds had been reported on the EU Rapid Alert System for Food and Feed (RASFF) portal. These notifications came from 17 different EU member states and two EFTA countries. EO concentrations in the sesame samples mostly ranged from 0.1-10 mg / kg, all exceeding the EU-MRL (Maximum Residue Limit) of 0.05 mg / kg.

[0016] An additional way of introducing EQ into the food chain is via said plant contamination. EQ is used in the food industry for sterilization and disinfection of equipment and facilities. If the equipment is not properly cleaned or used incorrectly, there is a possibility that EO may be transferred to the food. According to the World Health Organization (WHO) , the most common sources of ethylene oxide in food are additives and auxiliary agents such as stabilizers, emulsifiers, and freezing agents, as well as contamination from the sterilization process. It is important to note that authorities and regulatory bodies are responsible for monitoring food safety and limiting the presence of harmful substances like EO. The food industry also has a responsibility to implement quality control measures to ensure the safety of food on the market.

[0017] The determination methods should ensure a qualification limit of at least 0.02 mg / kg (ppm) EO. They can be categorized based on: (i) sample preparation methods:

[0018] (a) direct determination of EO in the liquid phase (extraction) or gas phase (headspace) ;

[0019] (b) determination of EO and 2CE in the liquid phase (extraction) ; and

[0020] (c) determination of EO through conversion to the derivative 2CE or 2-bromoethanol, depending on the presence of chloride or bromide ions; as well as,

[0021] (ii) types of EQ detection: one of the most common methods is gas chromatography (GC) . This technique employs a gas chromatograph to separate ethylene oxide from other compounds in the food sample, followed by mass spectrometry (MS) for the identification and quantification of EO . This method is highly precise and sensitive but is also expensive and requires specialized personnel and complex equipment. Another method used for detecting EO in food is GC with flame ionization detection (GC-FID) . This technique also uses a GC to separate EO from other compounds in the food samples but employs a FID detector for EO detection. This method is less sensitive than GC-MS, but still precise and relatively fast. Finally, liquid chromatography (LC) with MS or UV detection is used less frequently .

[0022] Some typical analytical methods for the determination of EO in food samples are described in literature references 4-7 :

[0023] 4) F. Tateo, M. Bononi : Determination of ethylene chlorohydrin as marker of spices fumigation with ethylene oxide, J. Food Compost . Anal. 19 (2006) 83-87;

[0024] 5) Ethylene Oxide & 2-Chloroethanol analysis in Food using Triple Quadrupole GC / MS / MS; Agilent Technologies, Inc., USA (2022) ; available at the link below: https : / / gems . cz / labrule z -bucket -str api- h3hsga3 / br gems anaylsis ethylene oxide food samples 5994 4775en agilent e4863bd567 / br-gcms-anaylsis-ethylene-oxide-f ood- samples-5994-4775en-agilent . pdf

[0025] 6) X. Liu, P. Joza, A. Masters, B. Rickert: Determination of Ethylene Oxide (ETO) in Mainstream Cigarette Smoke Using Hydrobromic Acid Derivatization and Gas Chromatography-Mass Spectrometry Method; CORESTA Congress, October 12-16 (2014) Quebec, Canada; available at the link below: https : / / www .coresta.org / abstracts / de termination- ethyl ene- oxi domains tream- cigarette-smoke -usin -hydrobromic -acid

[0026] 7) I. Wenio, I. Bartosiewicz , D. Derewiaka, K. Dewiszek, K. Karnilowicz : A Fast Method for Determination of Ethylene Oxide Using Gas Chromatography Coupled with Mass Spectrometry GC-MS / MS, Appl. Sci. 13 (2023) 7480.

[0027] There are also several rapid tests that can be used for preliminary detection of EQ in food, such as detection strips, but these results are not as precise and reliable as more complex techniques like GC- MS.

[0028] The present invention solves the technical problem of accurate, precise, and effective determination of EQ in various food samples in a novel and inventive manner as is disclosed in the section Detailed Description .

[0029] SUMMARY

[0030] The present disclosure reveals a method for the quantitative determination of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) as contaminants in food products. The method includes the quantitative determination of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) as contaminants in food products where the said analytical process comprises the following steps:

[0031] (a) optionally, in the case of non-homogeneous food products, homogenization of a sample by cryogenic milling,

[0032] (b) weighing of the homogenized sample,

[0033] (c) adding 90% w / w acetonitrile (ACN) solution in analytical grade water, immediately closing the sample, and shortly shaking,

[0034] (d) placing the vessel with the sample to a laboratory shaker and subject to shaking at 1,000 periods / minute for 20 minutes at room temperature , (e) centrifugation of the sample at 5,000 revolutions per minute (rpm) at about -10°C for about 10 minutes for cooling the samples,

[0035] (f) transferring the supernatant to the dispersive solid phase extraction (dSPE) cuvette, closing, and shaking for about 1 minute,

[0036] (g) centrifugation of the sample at 5,000 rpm and -10°C for about 5 minutes ,

[0037] (h) transferring the supernatant to a clean cuvette, observing the obtained volume, and adding the derivatization reagent,

[0038] (i) derivatization of the eventually present trace amounts of EQ in the sample with added derivatization reagent,

[0039] (j) neutralization by stepwise addition of powderous sodium carbonate (Na2COs) in amounts required for the neutralization of employed HC1, with mild shaking for about 15 minutes,

[0040] (k) degassing of the cuvette content by opening it and allowing it to stay at rest for about 15 minutes,

[0041] (l) additional degassing by turning the closed cuvette for 180°, carefully opening, but not fully, and allowing it to stay at rest for an additional 15 minutes,

[0042] (m) centrifugation at 5,000 rpm at room temperature for about 5 minutes ,

[0043] (n) transferring the supernatant to a cuvette for evaporation,

[0044] (o) evaporation of the content to dryness,

[0045] (p) reconstitution of the residual material with ACN by ensuring the washing of all internal cuvette walls,

[0046] (q) transferring of thus obtained reconstituted sample solution in ACN to a gas chromatography (GC) vial,

[0047] (r) quantitative analysis of the sample by gas chromatography coupled with mass spectrometry (GC / MS-MS) , and

[0048] (s) calculation of the obtained result according to the following equation : c (EO+2CE) = c (2CE) -Fk-V / m where the constituents in the above-cited equation are: c (E0+2CE ) = a concentration of the sum of both analytes, EQ and 2CE in the sample, expressed in [mg / kg] , c ( 2CE ) = an obtained concentration of 2CE in the sample, expressed in [mg / kg] ,

[0049] Ek = a numerical conversion factor of 0.547, for the calculation of EQ to 2CE ,

[0050] V = a final volume of the sample after the reconstitution in step (p) , expressed in [mL] , and m = mass of the sample weighted in step (b) , expressed in

[0051] [g] , wherein, in the derivatization step (i) , the derivatization reagent is hydrochloric acid (HC1) of concentration about 30-38% w / w, in amounts of 0.1 mL per each 1 mL of supernatant from step (h) , where said HC1 provides quantitative conversion of eventually present EQ in the sample to 2CE via the epoxide ring-opening reaction with chloride ion (Cl-) as a nucleophile, catalyzed by hydronium ion (HsO+) , and said derivatization step (i) is performed for 15-20 minutes at a temperature of about 15-30 °C.

[0052] In a preferred embodiment of the method from the present disclosure, the analysis is conducted under the following conditions: in step (b) , the analysis typically starts with 2 ± 0.02 grams of the starting sample of food product being analyzed, which is placed in the cuvette of 15 mL volume, in step (f ) , the dSPE cuvette is for fatty samples, in step (c) , the volume of acetonitrile solution is 10 mL, in step (h) , the optimal cuvette volume is 15 mL, in step (j) , the amount of NakCOs is 0.15 g per each 1 mL of supernatant , in step (o) , evaporation is conducted at the bath temperature of about 35 °C, vacuum depot of about 1-100 Pa, at a rotation speed of 100-150 rpm, and in step (p) , the acetonitrile volume for the sample reconstitution is 1.0 mL . In an additional preferred embodiment of the method from the present disclosure, the GC / MS-MS analysis in step (s) involves the following conditions :

[0053] (i) injection volume: 1 pL,

[0054] (ii) GC conditions: temperature program: 50°C, hold 2 minutes; 20°C / minute to 230 °C; 1 minute, injector: 220 °C, flow: 1 mL / minute, split: splitless, 1 minute, purge flow control: 5.0 mL / minute, and

[0055] (iii) MS conditions: interface temperature: 230 °C, ion source temperature: 220 °C, acquisition mode: MRM, ionization mode: El, event time: 0.030 seconds, retention time ( 2CE ) : 2.4 minutes, MRM1: 80.0 > 31.0 ... 5 eV, and MRM3: 82.0 > 31.0 ... 5 eV.

[0056] The method according to the present invention is used for the quantitative determination of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) as contaminants in food products.

[0057] BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1. Depicts a typical gas chromatography (GC / MS-MS) chromatogram of 2-chloroethanol ( 2CE ) according to the method for the determination of ethylene oxide (EO) and 2- chloroethanol ( 2CE ) , as contaminants in food products, from the present disclosure, see Example 1.

[0059] Figure 2. Depicts a calibration curve for 2-chloroethanol ( 2CE ) obtained during the validation study of the method according to the present disclosure with a tested spice (oregano) sample. The calibration curve was obtained by injecting working solutions of 2CE in the concentration range defined in Table 1. Each concentration was injected 2 times, see Example 1.

[0060] Figure 3. Depicts a graph of residuals for 2-chloroethanol ( 2CE ) for a tested spice (oregano) sample, see Example 1. The calibration curve is linear for the employed concentration range. The determination coefficient is >0.99, and the graph shows that there is no systemic deviation of experimental points from the calibration line in the observed concentration range, see Example 1.

[0061] Figure 4. Depicts a calibration curve for 2-chloroethanol ( 2CE ) obtained during the validation study of the method according to the present disclosure with a tested sesame sample. The calibration curve was obtained by injecting working solutions of 2CE in the concentration range defined in Table 3. Each concentration was injected 2 times, see Example 1.

[0062] Figure 5. Depicts a graph of residuals for 2-chloroethanol ( 2CE ) for a tested sesame sample, see Example 1. The calibration curve is linear for the employed concentration range. The determination coefficient is >0.99, and the graph shows that there is no systemic deviation of experimental points from the calibration line in the observed concentration range, see Example 1.

[0063] Figure 6. Depicts a graph of recovery for 2-chloroethanol (2CE) , expressed in mg / L, for a tested spice (oregano) sample, see Example 1. All the results for the recovery were between the limits of 70-120%. The average value of 102% for recovery proved that the method is accurate, see Example 1. Figure 7. Depicts a graph of recovery for 2-chloroethanol (2CE) , expressed in mg / L, for a tested sesame sample, see Example 1. All the results for the recovery were between the limits of 70-120%. The average value of 102% for recovery proved that the method is accurate, see Example 1.

[0064] Figure 8. Depicts a typical gas chromatography (GC / MS-MS ) chromatogram of 2-chloroethanol ( 2CE ) according to the method for the determination of ethylene oxide (EO) and 2- chloroethanol ( 2CE ) , as contaminants in food products, from the present disclosure obtained during the testing of one typical sesame sample, see Example 2.

[0065] DETAILED DESCRIPTION

[0066] The present disclosure reveals a method for the quantitative determination of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) as contaminants in food products . The method according to the present disclosure is based on the conversion of the total ethylene oxide (EO) present into 2-chloroethanol ( 2CE ) .

[0067] The method includes the quantitative determination of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) as contaminants in food products where the said analytical process comprises the following steps:

[0068] (a) optionally, in the case of non-homogeneous food products, homogenization of a sample by cryogenic milling,

[0069] (b) weighing of the homogenized sample,

[0070] (c) adding 90% w / w acetonitrile (ACN) solution in analytical grade water, immediately closing the sample, and shortly shaking,

[0071] (d) placing the vessel with the sample to a laboratory shaker and subject to shaking at 1,000 periods / minute for 20 minutes at room temperature ,

[0072] (e) centrifugation of the sample at 5,000 revolutions per minute (rpm) at about -10°C for about 10 minutes for cooling the samples, (f) transferring the supernatant to the dispersive solid phase extraction (dSPE) cuvette, closing, and shaking for about 1 minute,

[0073] (g) centrifugation of the sample at 5,000 rpm and -10°C for about 5 minutes ,

[0074] (h) transferring the supernatant to a clean cuvette, observing the obtained volume, and adding the derivatization reagent,

[0075] (i) derivatization of the eventually present trace amounts of EQ in the sample with added derivatization reagent,

[0076] (j) neutralization by stepwise addition of powderous sodium carbonate (Na2COs) in amounts required for the neutralization of employed HC1, with mild shaking for about 15 minutes,

[0077] (k) degassing of the cuvette content by opening it and allowing it to stay at rest for about 15 minutes,

[0078] (l) additional degassing by turning the closed cuvette for 180°, carefully opening, but not fully, and allowing it to stay at rest for an additional 15 minutes,

[0079] (m) centrifugation at 5,000 rpm at room temperature for about 5 minutes ,

[0080] (n) transferring the supernatant to a cuvette for evaporation,

[0081] (o) evaporation of the content to dryness,

[0082] (p) reconstitution of the residual material with ACN by ensuring the washing of all internal cuvette walls,

[0083] (q) transferring of thus obtained reconstituted sample solution in ACN to a gas chromatography (GC) vial,

[0084] (r) quantitative analysis of the sample by gas chromatography coupled with mass spectrometry (GC / MS-MS) , and

[0085] (s) calculation of the obtained result according to the following equation : c (EO+2CE) = c (2CE) -Fk-V / m where the constituents in the above-cited equation are: c (EO+2CE ) = a concentration of the sum of both analytes, EQ and 2CE in the sample, expressed in [mg / kg] , c ( 2CE ) = an obtained concentration of 2CE in the sample, expressed in [mg / kg] ,

[0086] Ek = a numerical conversion factor of 0.547, for the calculation of EQ to 2CE ,

[0087] V = a final volume of the sample after the reconstitution in step (p) , expressed in [mL] , and m = mass of the sample weighted in step (b) , expressed in

[0088] [g] , wherein, in the derivatization step (i) , the derivatization reagent is hydrochloric acid (HC1) of concentration about 30-38% w / w, in amounts of 0.1 mL per each 1 mL of supernatant from step (h) , where said HC1 provides quantitative conversion of eventually present EQ in the sample to 2CE via the epoxide ring-opening reaction with chloride ion (Cl-) as a nucleophile, catalyzed by hydronium ion (HsO+) , and said derivatization step (i) is performed for 15-20 minutes at a temperature of about 15-30 °C.

[0089] In a preferred embodiment of the method from the present disclosure, the analysis is conducted under the following conditions: in step (b) , the analysis typically starts with 2 ± 0.02 grams of the starting sample of food product being analyzed, which is placed in the cuvette of 15 mL volume, in step (f ) , the dSPE cuvette is for fatty samples, in step (c) , the volume of acetonitrile solution is 10 mL, in step (h) , the optimal cuvette volume is 15 mL, in step (j) , the amount of NakCOs is 0.15 g per each 1 mL of supernatant , in step (o) , evaporation is conducted at the bath temperature of about 35 °C, vacuum depot of about 1-100 Pa, at a rotation speed of 100-150 rpm, and in step (p) , the acetonitrile volume for the sample reconstitution is 1.0 mL .

[0090] In an additional preferred embodiment of the method from the present disclosure, the GC / MS-MS analysis in step (s) involves the following conditions : (i) injection volume: 1 pL,

[0091] (ii) GC conditions: temperature program: 50°C, hold 2 minutes; 20°C / minute to 230 °C; 1 minute, injector: 220 °C, flow: 1 mL / minute, split: splitless, 1 minute, purge flow control: 5.0 mL / minute, and

[0092] (iii) MS conditions: interface temperature: 230 °C, ion source temperature: 220 °C, acquisition mode: MRM, ionization mode: El, event time: 0.030 seconds, retention time ( 2CE ) : 2.4 minutes, MRM1: 80.0 > 31.0 ... 5 eV, and MRM3: 82.0 > 31.0 ... 5 eV.

[0093] The final result is expressed as the sum of EQ and 2CE in [mg / kg] (ppm) .

[0094] The development and validation of the method according to the subject disclosure is described in Example 1. The corresponding GC chromatogram is given in Figure 1, and graphs presenting different aspects of the method's validation are given in Figures 2-7.

[0095] Use of the method according to the subject disclosure

[0096] The method according to the present invention is used for the quantitative determination of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) as contaminants in food products.

[0097] More precisely, the method according to the present disclosure is used for the determination of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) in food products selected from the group comprising dairy products, fats and oils, and fats and oil emulsions, fruits and fruit-based products, vegetables and vegetable-based products, confectionery products, cereals and cereal products, bakery wares, meat and meatbased products, fish and fisheries products, eggs and egg products, syrups, honey and other beehive products, spices, soups, sauces, salads, protein products, beverages, ready-to-eat savories and snacks, deserts, foods intended for particular nutritional uses including foods for infants and young children, and food supplements.

[0098] The typical example of performing the method according to the present disclosure is disclosed in Example 2. The corresponding GC chromatogram of this model food sample (sesame sample) is given in Figure 8.

[0099] Experimental Part

[0100] General information

[0101] The term "room temperature" refers to a temperature interval of 20-25 °C. The stirring speed during the homogenizations is expressed in the number of revolutions per minute (rpm) of the stirring element.

[0102] Example 1. Development and validation of the method for determination of ethylene oxide (EO) and 2-chloroethanol (2CE) in food samples according to the present disclosure

[0103] The method according to the present invention was developed for determining the sum of ethylene oxide (EO) and 2-chloroethanol ( 2CE ) in food samples using gas chromatography with a triple mass-selective detector. The validation protocol covers the following parameters: linearity, working range, sensitivity, limit of detection (LOD) , limit of quantification (LOQ) , accuracy, repeatability, and precision. For method validation, a sample of oregano spice and a sample of sesame without EO and 2CE were used. The measuring range is 0.019-0.19 mg / kg (ppm) . The literature that was consulted in this work is given below: 8) EURL-SRM - Analytical Observations Report: Analysis of Ethylene Oxide and its Metabolite 2-Chloroethanol by the QuOil or the QuEChERS Method and GC-MS / MS, Version 1.1 (December 2020) ;

[0104] 9) X. Liu, P. Joza, A. Masters, B. Rickert: Determination of

[0105] Ethylene Oxide (ETO) in Mainstream Cigarette Smoke Using Hydrobromic Acid Derivatization and Gas Chromatography-Mass Spectrometry Method. 2014 CORESTA CONGRESS, October 12-16, 2014., Quebec City, Canada;

[0106] 10) EURACHEM / CITAC Guide CG 4, Quantifying Uncertainty in Analytical Measurement (second edition) 2000; and

[0107] 11) EURACHEM, The Fitness for Purpose of Analytical Methods (ISBN 0-

[0108] 948926-12-0) 1998 ILAC G17:2002, Introducing the Concept of

[0109] Uncertainty of Measurement in Testing in Association with the Application of the Standard ISO / IEC 17025, November 2002.

[0110] Preparation of calibration curves

[0111] The following reference standards were used for the preparation of calibration standards:

[0112] (i) ethylene oxide (EQ) solution with c= 53.0 g / L, CAS No. [75-21- 8] , Sigma-Aldrich analytical standard, 99.5%; and

[0113] (ii) 2-chloroethanol ( 2CE ) solution with c= 2.0 g / L, CAS No. [107- 07-3] , Sigma-Aldrich analytical standard, 99.6%.

[0114] Preparation of solvent for the calibration curve and sample extraction (90% acetonitrile)

[0115] In a 1-liter volumetric flask, 100 mL of water was added, followed by up to 1, 000 mL with ACN. The mixture was transferred to a reagent bottle and shaken well.

[0116] Preparation of stock and working solution of 2CE Stock solution of 2CE standard of c= 50 mg / L: In a vial, 975 pL of ACN and 25 pL of the reference standard of 2CE (c= 2,000 mg / L) were pipetted .

[0117] Working solution of 2CE standard of c= 5 mg / L: In a vial, 900 pL of ACN and 100 pL of the stock solution of 2CE standard (c= 50 mg / L) were pipetted .

[0118] Preparation of stock and working solution of EQ standard

[0119] Stock solution of EQ standard of c= 530 mg / L: In a vial, 990 pL of ACN and 10 pL of the reference standard of EQ (c= 53.0 g / L) were pipetted .

[0120] Working solution of EQ standard of c= 53 mg / L: In a vial, 900 pL of ACN and 100 pL of the stock solution of EQ standard (c= 530 mg / L) were pipetted .

[0121] Preparation of working solutions for the calibration curve

[0122] The calibration curve was prepared in a matrix that went through all the preparation phases, similar to the sample. The mass of 2 g ± 0.2 g of the matrix was weighted, for which the absence of EQ and 2CE were determined through previous preparation and analysis, in a 15 mL centrifuge tube. Six (6) calibration levels were prepared according to the description below and Table 1:

[0123] Level 1 0.070 mg / L: 0.014 mL standard 2CE of c= 5 mg / L (7.2) and

[0124] 9.986 mL ACN ( 90%) ;

[0125] Level 2 0.145 mg / L: 0.029 mL standard 2CE of c= 5 mg / L (7.2) and

[0126] 9.971 mL ACN ( 90%) ;

[0127] Level 3 0.290 mg / L: 0.058 mL standard 2CE of c= 5 mg / L (7.2) and 9.942 mL ACN ( 90%) ;

[0128] Level 4 0.360 mg / L: 0.072 mL standard 2CE of c= 5 mg / L (7.2) and 9.928 mL ACN ( 90%) ;

[0129] Level 5 0.550 mg / L: 0.110 mL standard 2CE of c= 5 mg / L (7.2) and

[0130] 9.989 mL ACN (90%) ; and Level 6 - 0.700 mg / L: 0.140 mL standard 2CE of c= 5 mg / L (7.2) and

[0131] 9.860 mL ACN ( 90%) .

[0132] Table 1. Volumes of working standard 2CE solution and ACN for the preparation of the calibration curve.

[0133] L 1...6 = level 1...6

[0134] Acceptance criteria

[0135] Acceptance criteria are given in Table 2.

[0136] Table 2. Acceptance criteria applied during the development of the method according to the present invention.

[0137] RSD = relative standard deviation

[0138] Linearity

[0139] The calibration curve was constructed by injecting 2CE working solutions, within the concentration range specified in Table 1. Each concentration level was injected twice.

[0140] The parameters of the calibration curve for 2CE on the tested spice (oregano) are given in Table 3.

[0141] Table 3. The parameters of the calibration curve for 2CE obtained on the spice sample (oregano) .

[0142] CI = confidence interval

[0143] The analysis of variance is shown in Table 4 .

[0144] Table 4 . Analysis of variance for 2CE obtained on the spice sample

[0145] ( oregano ) .

[0146] Thus obtained calibration curve is presented in Figure 2 , while the graph of residuals for 2CE obtained on tested spice ( oregano ) is given in Figure 3 .

[0147] The calibration curve is linear within the specified concentration range , see Figure 2 . The coefficient of determination is greater than 0 . 99 , and the residual plot indicates that there is no systematic deviation of experimental points from the calibration line within the observed concentration range , see Figure 3 . Linearity is confirmed by both the t-test and the F-test , where texp. > ttabie and Fexp. > Ftabie for 2CE . The null hypothesis that the slope of the curve is 0 is rej ected by the t-test , and the null hypothesis that there is no linear correlation between analyte concentration and peak area on the chromatogram is rej ected by the F-test , both at a 95% level of statistical significance , see Table 5 .

[0148] Table 5. The results of the t-test and F-test for 2CE obtained with the tested spice ( oregano ) .

[0149] The parameters of the calibration curve for 2CE obtained on the sesame sample are given in Table 6 , while the variance analysis is shown in

[0150] Table 7 .

[0151] Table 6. The parameters of the calibration curve for 2CE obtained on the tested sesame sample .

[0152] CI = confidence interval

[0153] Table 7 . Analysis of variance for 2CE obtained on the tested sesame sample .

[0154] The calibration curve is linear within the specified concentration range , see Figure 4 . The coefficient of determination is greater than 0 . 99 , and the residual plot indicates that there is no systematic deviation of experimental points from the calibration line within the observed concentration range , see Figure 5 . Linearity is confirmed by both the t-test and the F-test , where texp. > ttabie and Fexp. > Ftabie for 2CE . The null hypothesis that the slope of the curve is 0 is rej ected by the t-test , and the null hypothesis that there is no linear correlation between analyte concentration and peak area on the chromatogram is rej ected by the F-test , both at a 95% level of statistical significance , see Table 8 . Table 8. The results of the t-test and F-test for 2CE obtained with the tested sesame sample.

[0155] Operating range

[0156] Based on the confirmed linearity for the working range for 2CE and EQ, the working range for the tested spice (oregano) is given in Table 9, and those obtained with the tested sesame sample in Table 10.

[0157] Table 9. The working range parameter value for 2CE and EQ obtained with the tested spice (oregano) .

[0158] Table 10. The working range for 2CE and EQ obtained with the tested sesame sample.

[0159] Sensitivity

[0160] Sensitivity is obtained from the slopes of the calibration curves, taking into account the confidence intervals (Cis) for the slope. The sensitivity parameters obtained with the tested spice (oregano) and sesame samples are given in Table 11.

[0161] Table 11. The sensitivity parameter value obtained with the tested spice (oregano) and sesame sample.

[0162] Limit of detection and limit of quantification

[0163] The limit of detection (LOD) is calculated to the following equation:

[0164] LOD = 3.3 a / K.

[0165] The limit of quantification (LOQ) is calculated according to the following equation:

[0166] LOQ = 19 CT / K, where : ct = standard deviation of ten measurements of the "blank" sample, and K = slope of the calibration curve.

[0167] The LOD and LOQ obtained with the tested spice (oregano) and sesame sample are presented in Table 12.

[0168] Table 12. The limit of detection (LOD) and limit of quantification (LOQ) parameter values obtained with the tested spice (oregano) and sesame samples.

[0169] Accuracy The accuracy of the method according to the present invention was determined by adding a known quantity of a reference standard to the samples at three concentration levels: 0.07 mg / L, 0.36 mg / L, and 0.70 mg / L. The recovery was determined from the results of the analysis of samples to which no standard was added and the analysis of samples to which a known quantity of the reference standard was added. A spice (oregano) sample without EQ and 2CE was used in the method validation. For spiking samples at multiple concentration levels, a working solution of EQ with a concentration of c= 53 mg / L was employed. The spiking of samples was performed according to Table 13 and the sample preparation procedure to demonstrate the efficiency of the conversion of EQ to 2CE.

[0170] Table 13. The spiking of the samples with the standard ethylene oxide (EO) solution. aEO concentration in the working standard solution.bVolume of standard EO solution added per 10 mL .

[0171] The obtained value for the accuracy parameter in experiments with the spice (oregano) was 98% (average) with an RSD of 7.28. All the results for recovery parameters are within the range of 70-120%. The mean value for the recovery parameter is 98%, which represents proof that the accuracy of the method according to the present disclosure is confirmed .

[0172] The obtained value for the accuracy parameter in experiments with the tested sesame sample was 102% (average) with an RSD of 7.70. All the results for recovery parameters are within the range of 70-120%. The mean value for the recovery parameter is 102%, which represents proof that the accuracy of the method according to the subject disclosure is confirmed. The graph for the recovery parameter values for 2CE obtained with the spice ( oregano ) sample is given in Figure 6 , while the analysis of the accuracy parameter is presented in Table 14 .

[0173] Table 14 . The analysis of the accuracy parameter for 2CE obtained on the tested spice sample ( oregano ) .

[0174] CI = confidence interval

[0175] The graph for the recovery parameter values for 2CE obtained with the tested sesame sample is given in Figure 7 , while the analysis of the accuracy parameter is presented in Table 15 .

[0176] Table 15 . The analysis of the accuracy parameter for 2CE obtained on the tested sesame sample .

[0177] CI = confidence interval

[0178] Precision

[0179] The repeatability of the method according to the present disclosure was determined at three concentration levels : LOQ, l OxLOQ, and 40xLOQ . Each concentration level was prepared ten times and inj ected . Precision was calculated based on all measurements : different concentration levels , multiple preparations , two analysts , and two days. On the first day of measurements, one analyst prepared the samples, and on the second day, another analyst did so.

[0180] The results of the validation of the method according to the present disclosure obtained with the tested spice (oregano) at three concentration levels are presented in Table 16.

[0181] Table 16. The results of the validation of the method according to the present disclosure obtained with the tested spice (oregano) at three concentration levels.

[0182] The relative standard deviation of the method for EQ and 2CE in the tested sesame sample under repeatability conditions (RSDr) is 2.6 ± 5.35%.

[0183] The relative standard deviation of the method for EQ and 2CE in the tested sesame sample under reproducibility conditions (RSDR) is 4.52 ± 7.60%.

[0184] The method's precision for EO and 2CE in sesame is 7.28%.

[0185] The results of the validation of the method according to the present disclosure obtained with the tested sesame sample at three concentration levels are presented in Table 17.

[0186] Table 17. The results of the validation of the method according to the present disclosure obtained with the tested sesame sample at three concentration levels.

[0187]

[0188] The relative standard deviation of the method for EQ and 2CE in the tested sesame sample under repeatability conditions (RSDr) is 2.6 ± 5.35%.

[0189] The relative standard deviation of the method for EQ and 2CE in the tested sesame sample under reproducibility conditions (RSDR) is 4.52 ± 7.60%.

[0190] The method's precision for EQ and 2CE in sesame is 7.70%.

[0191] Conclusion on the method validation

[0192] Based on the obtained validation results, it can be concluded that the method according to the subject disclosure has been successfully validated and can be used for the analysis of ethylene oxide (EO) and 2-chloroethanol ( 2CE; expressed as EO) in food samples. This method allows for the determination of the concentration of the sum of EO and 2CE (expressed as EO) in food samples at 0.019 [mg / kg] (ppm) .

[0193] Example 2. The typical analytical procedure for performing the method for the determination of ethylene oxide (EO) and 2-chloroethanol (2CE) in a model food sample - sesame

[0194] The sample of tested sesame was first homogenized, and then 2 g was weighed and mixed with 10 mL of a 90% sodium chloride (NaCl) solution. The sample underwent shaking for extraction and centrifugation at -10 °C. The entire volume of the supernatant was transferred to a dSPE (dispersive solid-phase extraction) for fatty samples and shaken for 1 minute. The sample was centrifuged again at -10 °C. The supernatant was rapidly transferred to an empty centrifuge tube, and hydrochloric acid ( 36% ) was added for the derivatization of EQ into 2CE . After shaking for 1 minute , the sample was allowed to stand for 15 minutes . Sodium carbonate (Na2COs ) was added for acid neutralization . The sample was centrifuged, and the supernatant was transferred to a vial for preparation . The sample was evaporated to dryness ( stopping evaporation as soon as possible as the last drop disappeared) . The sample was reconstituted with 1 mL of acetonitrile (ACN) and analyzed on a GC-MS / MS instrument .

[0195] A concentration of 0 . 073 mg / L was found in the sample , and upon conversion to sample preparation, it resulted in the concentration level in the tested sesame sample at 0 . 02 [mg / kg ] (ppm) of EQ .

[0196] Conclusion :

[0197] The method is based on concentrated hydrochloric acid-mediated derivatization of the processed samples under specific treatment conditions , which provides the quantitative conversion of ethylene oxide ( EO ) to 2-chloroethanol ( 2CE ) , followed by the quantitative determination of thus formed 2CE with gas chromatography coupled with mass spectrometry ( GC / MS-MS ) . The method was successfully validated and enables a highly accurate procedure for the analysis of EO and 2CE in food samples at the concentration range from 0 . 019-0 . 19 mg / kg .

[0198] INDUSTRIAL APPLICABILITY

[0199] The present invention discloses a method for the determination of ethylene oxide ( EO ) and 2-chloroethanol ( 2CE ; expressed as EO ) as contaminants in food products . Since the precise , accurate , and economic determination of contaminants in food products is a regular part of food product quality control , the industrial applicability of the present disclosure is obvious . ABBREVIATIONS

[0200] ACN = acetonitrile

[0201] BDML = Benchmark Dose Lower Confidence Level ; the "reference dose level" is the final point of the study associated with an additional 10% ris k of adverse effects in exposed test animals compared to background ris k levels .

[0202] 2CE = 2-chloroethanol

[0203] CI = confidence interval

[0204] El = electron impact ionization ( an ionization mode in the

[0205] MS ) eV = electron-volt

[0206] GC = gas chromatography

[0207] GC-FID = gas chromatography with flame ionization detection

[0208] GC / MS-MS = coupled system GC + MS

[0209] EQ = ethylene oxide

[0210] LCD = limit of detection

[0211] LOQ = limit of quantification

[0212] MRM = multiple reaction monitoring ; an acquisition mode in the

[0213] MS ; an MS-based technique for accurately measuring the relative or absolute concentration of particular molecules

[0214] MS = mass spectrometry

[0215] R2= R-squared coefficient

[0216] RSD = relative standard deviation

Claims

CLAIMS1. A method for quantitative determination of ethylene oxide <E0> and 2-chloroethanol <2CE> as contaminants in food products where the said analytical process comprises the following steps:(a) optionally, in the case of non-homogeneous food products, homogenization of a sample by cryogenic milling,(b) weighing of the homogenized sample,(c) adding 90% w / w acetonitrile <ACN> solution in analytical grade water, immediately closing the sample, and shortly shaking,(d) placing the vessel with the sample to a laboratory shaker and subject to shaking at 1,000 periods / minute for 20 minutes at room temperature,(e) centrifugation of the sample at 5,000 revolutions per minute <rpm> at about -10°C for about 10 minutes for cooling the samples ,(f) transferring the supernatant to the dispersive solid phase extraction <dSPE> cuvette, closing, and shaking for about 1 minute,(g) centrifugation of the sample at 5,000 rpm and -10°C for about 5 minutes,(h) transferring the supernatant to a clean cuvette, observing the obtained volume, and adding the derivatization reagent,(i) derivatization of the eventually present trace amounts of EQ in the sample with added derivatization reagent,(j) neutralization by stepwise addition of powderous sodium carbonate <Na2COs> in amounts required for the neutralization of employed HC1, with mild shaking for about 15 minutes,(k) degassing of the cuvette content by opening it and allowing it to stay at rest for about 15 minutes,(l) additional degassing by turning the closed cuvette for 180°, carefully opening, but not fully, and allowing it to stay at rest for an additional 15 minutes,(m) centrifugation at 5,000 rpm at room temperature for about 5 minutes ,(n) transferring the supernatant to a cuvette for evaporation,(o) evaporation of the content to dryness,(p) reconstitution of the residual material with ACN by ensuring the washing of all internal cuvette walls,(q) transferring of thus obtained reconstituted sample solution in ACN to a gas chromatography <GC> vial,(r) quantitative analysis of the sample by gas chromatography coupled with mass spectrometry <GC / MS-MS>, and(s) calculation of the obtained result according to the following equation : c<EO+2CE> = c<2CE> • Fk• V / m where the constituents in the above-cited equation are: c<EO+2CE> = a concentration of the sum of both analytes, EQ and 2CE in the sample, expressed in <mg / kg>, c<2CE> = an obtained concentration of 2CE in the sample, expressed in <mg / kg>,Ek = a numerical conversion factor of 0.547, for the calculation of EQ to 2CE ,V = a final volume of the sample after the reconstitution in step (p) , expressed in <mL>, and m = mass of the sample weighted in step (b) , expressed in <g>, wherein, in the derivatization step (i) , the derivatization reagent is hydrochloric acid <HC1> of concentration about 30-38% w / w, in amounts of 0.1 mL per each 1 mL of supernatant from step (h) , where said HC1 provides quantitative conversion of eventually present EQ in the sample to 2CE via the epoxide ring-opening reaction with chloride ion <C1~> as a nucleophile, catalyzed by hydronium ion <HsO+>, and said derivatization step (i) is performed for 15-20 minutes at a temperature of about 15-30 °C.

2. The method according to claim 1, where the analysis is conducted under the following conditions : in step (b) , the analysis typically starts with 2 ± 0.02 grams of the starting sample of food product being analyzed, which is placed in the cuvette of 15 mL volume, in step (f ) , the dSPE cuvette is for fatty samples, in step (c) , the volume of acetonitrile solution is 10 mL, in step (h) , the optimal cuvette volume is 15 mL, in step (j) , the amount of Na2COs is 0.15 g per each 1 mL of supernatant , in step (o) , evaporation is conducted at the bath temperature of about 35 °C, vacuum depot of about 1-100 Pa, at a rotation speed of 100-150 rpm, and in step (p) , the acetonitrile volume for the sample reconstitution is 1.0 mL.

3. The method according to claims 1 and 2, where the GC / MS-MS analysis in step (s) involves the following conditions:(i) injection volume: 1 pL,(ii) GC conditions: temperature program: 50°C, hold 2 minutes; 20°C / minute to 230 °C; 1 minute, injector: 220 °C, flow: 1 mL / minute, split: splitless, 1 minute, purge flow control: 5.0 mL / minute, and(iii) MS conditions: interface temperature: 230 °C, ion source temperature: 220 °C, acquisition mode: MRM, ionization mode: El, event time: 0.030 seconds, retention time <2CE> : 2.4 minutes, MRM1: 80.0 > 31.0 ... 5 eV, and MRM3: 82.0 > 31.0 ... 5 eV.determination of ethylene oxide <E0> and 2-chloroethanol <2CE> as contaminants in food products.

5. Use of the method according to claim 4, for the determination of ethylene oxide <E0> and 2-chloroethanol <2CE> in food products selected from the group comprising dairy products, fats and oils, and fats and oil emulsions, fruits and fruit-based products, vegetables and vegetable-based products, confectionery products, cereals and cereal products, bakery wares, meat and meat-based products, fish and fisheries products, eggs and egg products, syrups, honey and other beehive products, spices, soups, sauces, salads, protein products, beverages, ready-to-eat savories and snacks, deserts, foods intended for particular nutritional uses including foods for infants and young children, and food supplements .

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