Method for qualitative and quantitative determination of microplastics as a contaminant in food products

Abstract

The present disclosure reveals the method for analysis of microplastics as a contaminant in food products based on nitric acid (HNO3)-based acidic, oxidative digestion of food samples at 65-69% w / w HNO3 in ultrasonic bath at 20 kHz, followed by dilution with demineralized water to the final concentration of 8-10 mol / L HNO3 and subsequent digestion at 100-110 °C during 30-100 minutes under microwave (MW) irradiation. The said method provides fast, chemoselective, and effective degradation of the food matrix, preserving the contained microplastic particles. The latter is analyzed by Raman microscopy (RM) which results in the qualitative identification of present microplastics as well as their quantitative composition and dimensions within the tested food sample. The method according to the present disclosure is used for qualitative and quantitative determination of microplastic particles as a contaminant in food products.

Description

[0001] METHOD FOR QUALITATIVE AND QUANTITATIVE DETERMINATION OF

[0002] MICROPLASTICS AS A CONTAMINANT IN FOOD PRODUCTS

[0003] BACKGROUND

[0004] 1. Field

[0005] The present disclosure refers to a novel method for qualitative and quantitative determination of microplastics as a contaminant in food products, their particle size analysis, and the use of this method.

[0006] 2. Description of the Related Art

[0007] Synthetic polymers have become an indispensable part of human life nowadays due to their numerous useful properties and applications . Unfortunately, many of these polymers are not biodegradable, and over time, they break into smaller particles through various processes, resulting in the formation of plastic debris . Particles with a diameter smaller than 5 mm are referred to as microplastics, while the study by Gigault and coworkers suggests that the lower limit for microplastics should be 1 pm, and particles ranging from 1-1,000 nm are called nanoplastics, see literature references 1 and 2:

[0008] 1) C. Artur, J. Baker, H. Bamford: Proceedings of the International Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris; National Oceanic and Atmospheric Administration (NOAA) ; Tacoma, WA, USA (2009) . NOAA Technical Memorandum NOS-OR&R-30.

[0009] 2) J. Gigault, A. T. Halle, M. Baudrimont, P.-I. Pascal, F. Gauffre, T . -L . Phi, H. El Hadri, B. Grassl, S. Reynaud: Current opinion: What is a nanoplastic? Environ. Pollut. 235 (2018) 1030-1034.

[0010] Polymer micro-particles can be classified based on their usage, thus dividing them into primary (e.g., found in personal care products) , and secondary (e.g., particles originating from packaging) microplastics, see literature reference 3:

[0011] 3) S. Sangkham, 0. Faikhaw, N. Munkong, P. Sakunkoo, C. Arunlertaree , M. Chavali, M. Mousazadeh, A. Tiwari: A review on microplastics and nanoplastics in the environment: Their occurrence, exposure routes, toxic studies, and potential effects on human health, Mar. Pollut. Bull. 181 (2022) 113832.

[0012] Considering the potential negative impact of microplastics on health after their ingestion into the human body through food or drinks, it is necessary to be able to detect and / or quantify them in food or beverage samples, in order to make an accurate risk assessment. To achieve this, it is first necessary to find a suitable extraction or separation method for polymeric micro-particles from the given sample.

[0013] One of the possible methods for extracting polymeric particles is flotation. This process starts with the digestion of the sample using a suitable reagent, most commonly hydrogen peroxide (H2O2) solution, followed by mixing the solution with a flotation medium that has a higher density than the polymers, but lower density than the remaining matrix of the sample from which the particles are separated, usually zinc chloride (ZnC12) or sodium iodide (Nal) solution. The aim is to achieve phase separation, and then the upper phase containing the polymers is decanted, see literature reference 4. An example of such a procedure is described in a study where the preparation of fish and crustacean samples for microplastic detection involved digestion with 20% w / V potassium hydroxide (KOH) solution or 35% w / V H2O2 solution, followed by flotation using 4.4 M Nal solution. In 57.5% of the analyzed fish samples, an average of 0.77 ± 1.25 particles were found in the gills and 0.52 ± 0.90 particles in the gastrointestinal (GI) tract, see literature reference 5:

[0014] 4) A. Sridhar, D. Kannan, A. Kapoor, S. Prabhakar: Extraction and detection methods of microplastics in food and marine systems : A critical review, Chemosphere 286 (2022) 131653. 5 ) Z . Fu, J . Wang : Current practices and future perspectives of microplastic pollution in freshwater ecosystems in China, Sol . Total . Environ . 691 ( 2019 ) 697 -712 .

[0015] Another possible method for extracting polymeric particles is through membrane filtration, where the pressure difference is utilized to allow the liquid to pass through membranes with pores of different sizes , while the particles are retained on the membrane with pore sizes smaller than that of the particles themselves . This approach has found its application in processes such as wastewater treatment and the extraction of microplastics from soil and food samples . The process of membrane filtration for extracting microplastics from food is often proceeded by various digestion procedures to sufficiently break down the food sample , allowing the solution to pass through the selected pore size filter , leaving the particles on the filter, see literature reference 4 .

[0016] Among the less commonly used methods for microplastic extraction are ultrasonic extraction, solid-phase microextraction ( SPME ) , and magnetic extraction . Although ultrasonic waves can separate microplastic particles from the biological matrix, this method can potentially damage more fragile particles , see literature reference 4 . SPME relies on high temperatures to thermally decompose microplastic polymers into volatile organic compounds , which are then adsorbed onto SPME fibers and analyzed using GC-MS technique , see literature reference 6 . Magnetic extraction involves the use of Fe- nanoparticles to magnetize and isolate microplastic particles , which can result in high extraction efficiency, but there is a possibility of damaging and fragmenting fragile particles , see literature reference 7 .

[0017] 6 ) F . Prosenc , P . Leban, U . Sunta , M . Bavcon Kral j : Extraction and Identification of a Wide Range of Microplastic Polymers in Soil and Compost , Polymers 13 ( 2021 ) 4069 . 1) J. Grbic, B. Nguyen, E. Guo, J. B. You, D. Sinton, C. M. Rochman: Magnetic Extraction of Microplastics from Environmental Samples, Environ. Sci . Technol. Lett. 6 (2019) 68-72.

[0018] Arnong the most efficient and therefore more commonly used methods for extracting microplastics from food samples are digestion procedures . Based on the type of reagents used, digestion procedures can generally be divided into chemical and enzymatic digestion, where chemical digestion further includes acid or alkaline digestion. Acid digestion involves the use of the acid solution, with nitric acid (HNO3) , hydrochloric acid (HC1) , and perchloric acid (HCIO4) being among the most commonly used acids, and some described procedures may include additional chemicals such as H2O2 and iron (II) sulfate (FeSCA) to enhance digestion efficacy, see literature reference 4. Alkaline digestion, on the other hand, typically employed sodium hydroxide (NaOH) or KOH solutions, with some exceptions.

[0019] The enzymatic digestion procedure offers an alternative to the chemical digestion of samples, which may degrade some of the polymers due to the often high concentrations of acids or alkalis required, potentially compromising the reliability of the analysis results. However, due to the high costs of individual enzymes in the quantities needed for the complete degradation of the food matrix and the small sample sizes they can handle, they are frequently used as an addition to some of the chemical digestion procedures, see literature reference 8. For instance, a study that aimed to determine the most suitable digestion protocol among ten observed protocols for the analysis of microplastics in seafood samples using infrared (IR) or Raman spectroscopy, and GC-Pyr-MS, found that the best digestion method involves a combination of rapid protein hydrolysis with pepsin, followed by subsequent alkaline hydrolysis with a 50% KOH solution, see literature reference 9:

[0020] 8) F. Bessa, J. Frias, T. Kbgel, A. Lusher, J. Andrade, J. Antunes, P. Sobral, E. Pagter, R. Nash, I. O'Connor, M. L. Pedrotti, E. Keros, V. Le6n, V. Tirelli, G. Suaria, C. Lopes, J. Raimundo, M. Gaetano, J. Gago, G. Gerdts: Harmonized protocol for monitoring microplastics in biota, Technical Report (2019)

[0021] 10.13140 / RG.2.2.28588.72321 / 1.

[0022] 9) J. Stissmann, T. Krause, D. Martin, E. Walz, R. Greiner, S. Rohn,

[0023] E. K. Fischer, J. Fritsche: Evaluation and optimisation of sample preparation protocols suitable for the analysis of plastic particles present in seafood, Food Control 125 (2021) 107969.

[0024] One of the major drawbacks of a large number of described digestion procedures for extracting microplastics from food is the relatively long incubation time, often exceeding ten hours, see literature reference 10, and sometimes even more than one day, see literature reference 11:

[0025] 10) M. Claessens, L. Van Cauwenberghe, M. B. Vandegehuchte, C. R. Janssen: New techniques for the detection of microplastics in sediments and field collected organisms, Mar. Pollut. Bull. 70 (2013) 227-233.

[0026] 11) A. Karami, A. Golieskardi, Y. B. Ho, V. Larat, B. Salamatinia: Microplastics in eviscerated flesh and excised organs of dried fish, Sci . Rep. 7 (2017) Article Number: 5473.

[0027] One of the newer and less explored approaches to the extraction of microplastics from food and biological tissues, which has the potential to significantly reduce the incubation time required for sample digestion, is microwave-assisted wet digestion (MAWD) . One of the described procedures involves the microwave digestion of tissue samples from marine organisms with 7 mol / L HNO3 in sealed Teflon (PTFE) containers for 45 minutes, followed by neutralization with NaOH, and treatment with 30% H2O2. Under the digestion conditions specified in this procedure, a mass loss occurs, and fragmentation or agglomeration of microplastic particles is observed, see literature reference 12 : 12) H. A. Leslie, S. H. Brandsma, M. J. M. van Velzen, A. D. Vethaak: Microplastics en route: Field measurements in the Dutch river delta and Amsterdam canals, wastewater treatment plants, North Sea sediments and biota, Environ. Int. 101 (2017) 133-142.

[0028] In another study, the first step towards optimizing the conditions of microwave digestion was taken, considering the influence of temperature, acid concentration, and program duration on the success of gravimetric determination of microplastics in fish tissue samples. An extraction efficiency of over 90% was achieved for all observed polymers except LDPE under conditions of 200 °C, 1 mol / L HNO3, and 20 minutes of incubation, see literature reference 13 :

[0029] 13) G. R. Bitencourt, P. A. Mello, E. M. M. Flores, C. Pirola, D. Carnaroglio, C. A. Bizzi: Determination of microplastic content in seafood: An integrated approach combined with the determination of elemental contaminants, Sci . Total. Environ. 749 (2020) 142301.

[0030] An innovative approach to optimizing the process of microplastic extraction from food and food waste was described in a recent study, where a factorial design with linear regression was used to find the optimal conditions for microwave acid digestion to extract microplastics, followed by subsequent filtration and gravimetric determination, see literature reference 14 :

[0031] 14) S. Lievens, T. Siegers, M. A. Mees, W. Thielemans, G. Poma, A. Covaci, M. Van Der Borght : A simple, rapid, and accurate method for the sample preparation and quantification of meso- and microplastics in food and food waste streams, Environ . Pollut. 307 (2022) 119511.

[0032] Despite the fact that HNO3 is currently described as a possible reagent for acid-catalyzed digestion of food samples in microplastic determination procedures, it is known to cause significant or complete degradation of microplastic particles. Among a range of potential reagents and 40 different digestion protocols for food sample digestion in microplastic determination procedures, HNO3 of different concentrations is not considered a good choice, while H2O2 is explicitly highlighted as the preferred reagent, see literature reference 15 for details :

[0033] 15) F. Pfeiffer, E. K. Fischer: Various Digestion Protocols Within Microplastic Sample Processing - Evaluating the Resistance of Different Synthetic Polymers and the Efficiency of Biogenic Organic Matter Destruction, Front. Enviro . Res. 8 (2020) Article 572424.

[0034] The present disclosure solves the technical problem of effective digestion of various food samples providing chemoselective destruction of the respective food matrixes in a relatively short incubation time, while completely preserving microplastic particles. After isolation through membrane filtration, microplastic particles can be precisely analyzed using Raman microscopy, as described in the Detailed Description section.

[0035] SUMMARY

[0036] The present disclosure reveals a method for the qualitative and quantitative determination of microplastics as a contaminant in food products where the said analytical process comprises the following steps :

[0037] (a) optionally, in the case of solid and non-homogeneous food products, homogenization of a sample by cryogenic milling,

[0038] (b) weighing of homogenized sample,

[0039] (c) drying the sample,

[0040] (d) adding 65-69% w / w nitric acid (HNO3) to the sample,

[0041] (e) placing the vessels with the sample in the ultrasonic bath at 20 kHz ,

[0042] (f) adding demineralized water to the mixture to form the final concentration of HNO3 within the formed mixture,

[0043] (g) microwave (MW) -assisted, HNOs-based, acidic, oxidative digestion of thus prepared sample, (h) quantitative transfer of the content into the glass volumetric flask with demineralized water, followed by dilution with demineralized water up to the total dilution volume,

[0044] (i) filtration of the final diluted solution through a silicon filter of 1 or 5 pm pores, yielding microplastic particles immobilized on the filter, and

[0045] (j) qualitative and quantitative analysis, as well as particles size analysis of immobilized microplastics on said silicon filter by Raman microscopy (RM) , wherein, in step (e) the ultrasonic treatment is conducted at 20-30 °C for 15-25 minutes, in step (f) the amount of demineralized water added to the sample corresponds to the targeted final concentration of HNO3 at the range of 8-10 mol / L, and where in step (g) said digestion is performed within an MW digestion system at 2.450 MHz under the following digestion regimen: from 0°C-to-about 110 °C for 10 minutes, at 100-110 °C for 30-100 minutes, and from 100-110 °C-to-0 °C for 10 minutes.

[0046] Preferably, the method of analysis is conducted under the following conditions : in step (b) , the analysis typically starts with 250 mg of starting sample of food product being analyzed, and in step (h) , the preferred total dilution volume is 50 mL.

[0047] Additionally, the analysis of the silicon filter with immobilized particles of microplastics in step (j) by Raman microscopy involves the following conditions : laser of wavelength 532 nm with corresponding filter and grid, spectral regions observed: 3400-1050, 900-600, 450-50 cm-1, using one or more Raman spectra databases selected from the group consisting of: 3M Polymers Raman, Polymers&AdditivesPackaging, Polymers &AdditivesPackaging (HR) , HR FT-Raman Polymer Library, HR FT-Raman Polymer Library Addendum. According to the present disclosure , the analysis of the sample in step ( j ) by Raman microscopy furnishes a tabular presentation of the results , which for each analyzed particle on the filter contains : corresponding Raman spectrum, the ordinal number of the particle , spatial dimensions (X and Y ) indicating the exact position of the observed particle on the silicon filter, qualitative identification of the type of polymer, matching value of the Raman spectrum of the particle with the associated spectrum in one of the Raman spectra databases used, expressed as a percentage ( 1-100% ) , whereby a matching value greater than or equal to 80% is used as a criterion for successful identification, the name of the Raman spectrum database from which the spectrum with the highest matching value was obtained, aspect ratio of the particle , circularity of the particle , length of the particle , and particle width .

[0048] The method according to the present disclosure is used for the qualitative and quantitative determination of microplastic particles as a contaminant in food products . Additionally, the method from the present disclosure is used for the determination of microplastics in food products selected from the group comprising pasta and pasta-based products , fruits and fruit-based products , vegetables , and vegetablebased products , marine products and marine-based products , meat and meat products , eggs and egg products , products based on edible mollus ks and insects , dairy products , cocoa, chocolate and other cocoa-based products , foods intended for particular nutritional uses including foods for infants and young children .

[0049] BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 . A typical Raman spectrum of polyethylene ( PE ) particles before the extraction procedure . Figure 2. A typical Raman spectrum of polypropylene (PP) particles before the extraction procedure.

[0051] Figure 3. A typical Raman spectrum of polyethylene terephthalate (PET) particles before the extraction procedure.

[0052] Figure 4. A typical Raman spectrum of polytetrafluoroethylene (PTFE) particles before the extraction procedure.

[0053] Figure 5. A typical Raman spectrum of poly (methyl methacrylate) (PMMA) particles before the extraction procedure.

[0054] Figure 6. A typical Raman spectrum of polyethylene (PET) particles after the extraction procedure from food matrix-1: bread, after the preparation.

[0055] Figure 7. A typical Raman spectrum of polypropylene (PP) particles after the extraction procedure from food matrix-2 : pepper, after the preparation.

[0056] Figure 8. A typical Raman spectrum of polyethylene terephthalate (PET) particles after the extraction procedure from food matrix-3: tuna, after the preparation.

[0057] Figure 9. A typical Raman spectrum of polytetrafluoroethylene (PTFE) particles after the extraction procedure from food matrix- 4: squid, after the preparation.

[0058] Figure 10. A typical Raman spectrum of poly (methyl methacrylate) (PMMA) particles after the extraction procedure from food matrix-1: bread, after the preparation. DETAILED DESCRIPTION

[0059] The present disclosure reveals a method for the qualitative and quantitative determination of microplastics as a contaminant in food products where the said analytical process comprises the following steps :

[0060] (a) optionally, in the case of solid and non-homogeneous food products, homogenization of a sample by cryogenic milling,

[0061] (b) weighing of homogenized sample,

[0062] (c) drying the sample,

[0063] (d) adding 65-69% w / w nitric acid (HNO3) to the sample,

[0064] (e) placing the vessels with the sample in the ultrasonic bath at 20 kHz ,

[0065] (f) adding demineralized water to the mixture to form the final concentration of HNO3 within the formed mixture,

[0066] (g) microwave (MW) -assisted, HNOs-based, acidic, oxidative digestion of thus prepared sample,

[0067] (h) quantitative transfer of the content into the glass volumetric flask with demineralized water, followed by dilution with demineralized water up to the total dilution volume,

[0068] (i) filtration of the final diluted solution through a silicon filter of 1 or 5 pm pores, yielding microplastic particles immobilized on the filter, and

[0069] (j) qualitative and quantitative analysis, as well as particles size analysis of immobilized microplastics on said silicon filter by Raman microscopy (RM) , wherein, in step (e) the ultrasonic treatment is conducted at 20-30 °C for 15-25 minutes, in step (f) the amount of demineralized water added to the sample corresponds to the targeted final concentration of HNO3 at the range of 8-10 mol / L, and where in step (g) said digestion is performed within an MW digestion system at 2.450 MHz under the following digestion regimen: from 0°C-to-about 110 °C for 10 minutes, at 100-110 °C for 30-100 minutes, and from 100-110 ° C-to-0 ° C for 10 minutes .

[0070] In a preferred embodiment of this disclosure , the method of analysis is conducted under the following conditions : in step (b ) , the analysis typically starts with 250 mg of starting sample of food product being analyzed, and in step (h ) , the preferred total dilution volume is 50 mL .

[0071] In an additional preferred embodiment of this disclosure , the analysis of the silicon filter with immobilized particles of microplastics in step ( j ) by Raman microscopy ( RM) involves the following conditions : laser of wavelength 532 nm with corresponding filter and grid, spectral regions observed : 3400-1050 , 900- 600 , 450-50 cm-1, using one or more Raman spectra databases selected from the group consisting of : 3M Polymers Raman, Polymers &AdditivesPackaging , Polymers &AdditivesPackaging ( HR) , HR FT-Raman Polymer Library, HR FT-Raman Polymer Library Addendum.

[0072] According to the present disclosure in any of its embodiments , the analysis of the sample in step ( j ) by Raman microscopy furnishes a tabular presentation of the results , which for each analyzed particle on the filter contains : corresponding Raman spectrum, the ordinal number of the particle , spatial dimensions <X and Y> indicating the exact position of the observed particle on the silicon filter, qualitative identification of the type of polymer, matching value of the Raman spectrum of the particle with the associated spectrum in one of the Raman spectra databases used, expressed as a percentage <l-100%> , whereby a matching value greater than or equal to 80% is used as a criterion for successful identification, the name of the Raman spectrum database from which the spectrum with the highest matching value was obtained, aspect ratio of the particle , circularity of the particle , length of the particle , and particle width .

[0073] This method enables simultaneous determination of the number, type ( chemical composition ) , and particle size of different synthetic polymers in various food matrices .

[0074] Homogenization of solid samples in step ( a ) using cryogenic grinding in a cryomill is important to create a finely powdered material that can be effectively degraded through acid digestion . Liquid samples , deemed clear enough to pass through 1 or 5 pm pore filters without additional preparation are directly filtered through silicon filters . However, liquid samples that are not clear enough to pass through the filters undergo the same preparation process as solid samples but without cryomill homogenization . A successfully prepared sample is one that , after microwave (MW) digestion and filtration, passes through the silicon filter without leaving a significant amount of undegraded organic material that could interfere with subsequent particle analysis on the Raman microscope . To eliminate the possibility of sample contamination with particles from the environment , sample handling is performed using a specific approach . This includes working with glass or metal utensils in a clean digester where the surface is first wiped, then covered with aluminum foil , and the openings of the crucibles containing the samples and silicon filters are covered during their transfer to avoid exposure to particles from the ambient air .

[0075] The analytical development and validation of the method according to the present disclosure is presented in Example 1 .

[0076] The method according to the present disclosure can be used for the determination of microplastics derived from a wide variety of synthetic polymers selected from the group comprising : polyvinyl chloride ( PVC ) ; polyvinylidene chloride ( PVDC ) ; polytetrafluoroethylene ( PTFE ) ; polyethylene ( PE ) ; polypropylene ( PP ) ; polyisobutylene ( PUB ) ; polymethyl pentene ( PMP ) ; polystyrene ( PS ) ; polyvinyl acetate ( PVA) ; polyethylene terephthalate ( PET ) ; poly (methyl methacrylate) (PMMA) ; copolymers of methyl or ethyl acrylate with styrene, acrylonitrile, vinyl acetate, vinyl chloride, vinylidene chloride, or butadiene; polyacrylonitrile (PAN) ; polyvinyl siloxane (PVS) ; poly (para-phenylene terephthalamide ) (para-aramid) ; poly (meta-phenylene isophthalamide) (meta-aramid) ; other synthetic polymers, and mixtures of these polymers.

[0077] Typical Raman spectra of five model polymers that most commonly occur in various food products, namely, polyethylene (PE) , polypropylene (PP) , polyethylene terephthalate (PET) , polytetrafluoroethylene (PTFE) , and poly(methyl methacrylate (PMMA) are given in Figures 1-5.

[0078] A typical procedure for performing a method for the determination of microplastics in food products is described in Example 2.

[0079] The method according to the present disclosure proved to be applicable for the determination of microplastics in a wide variety of different food matrixes contaminated with different polymers. To illustrate this wide applicability, four different model food matrixes have been tested with the simulated addition of five different model polymers microplastics :

[0080] (i) matrix-1: bread, with PET microplastic particles, see Figure

[0081] 6,

[0082] (ii) matrix-2: pepper, with PP microplastic particles, see Figure

[0083] 7,

[0084] (iii) matrix-3: tuna, with PET microplastic particles, see Figure

[0085] 8,

[0086] (iv) matrix-4: squid, with PTFE microplastic particles, see Figure

[0087] 9 , and

[0088] (v) matrix 1: bread, with PMMA microplastic particles, see Figure

[0089] 10,

[0090] Typical examples for performing a method for the determination of microplastics in food products are described in Examples 3 and 4.

[0091] Use of the method according to the present invention The method according to the present disclosure is used for the qualitative and quantitative determination of microplastic particles as a contaminant in food products.

[0092] Additionally, the method from the present disclosure is used for the determination of microplastics in food products selected from the group comprising pasta and pasta-based products, fruits and fruitbased products, vegetables, and vegetable-based products, marine products and marine-based products, meat and meat products, eggs and egg products, products based on edible mollusks and insects, dairy products, cocoa, chocolate and other cocoa-based products, foods intended for particular nutritional uses including foods for infants and young children.

[0093] Experimental Part

[0094] General information

[0095] The term "room temperature" refers to a temperature interval of 20-23 °C. The microwave (MW) digestions were performed using Ethos UP (Milestone Sri; Italy) , a high-performance microwave digestion system of a maximal power of 1, 900 W, with temperature control.

[0096] Example 1. Development and validation of the method for determination of microplastics in food samples according to the present disclosure

[0097] The method according to the present invention was developed for determining microplastics in food products using Raman microscopy (RM) on four food matrixes: (1) food matrix-1: bread, (2) food matrix-2: pepper; (3) food matrix-3: tuna, and (4) food matrix-4: squid. The validation protocol encompasses the following parameters: device performance, method applicability, method precision, and limit of detection (LOD) . Said four food matrixes, bread, pepper, tuna, and squid samples were used for method validation, as well as microparticles of five model polymers: PE, PP, PET, PTFE, and PMMA. The methodology of RM is well-known in the prior art, see literature references 16-18:

[0098] 16) DXRxi Raman Imaging Microscope with OMNICxi Raman Imaging Software User Guide, Thermo Fisher Scientific (2020) ; available at the link here: https : / / knowledgel . thermofisher . com / Molecular Spectroscopy / Dispe rsive Raman / Raman Operator Manuals / 269-300706 - Rev A -

[0099] DXRxi Raman Imaging Microscope with OMNICxi Raman Imaging Softw are User Guide

[0100] 17) D. Schymanski, B. E. OBmann, N. Benismail, K. Boukerma, G. Dallmann, E. von der Esch, D. Fischer, F. Fischer, D. Gilliland, K. Gias, T. Hofmann, A. Kappler, S. Lacorte, J. Marco, M. E. L. Rakwe, J. Weisser, C. Witzig, N. Zumbtilte, N. P. Ivleva: Analysis of microplastics in drinking water and other clean water samples with micro-Raman and micro-infrared spectroscopy: Minimum requirements and best practice guidelines. Anal. Bioanal. Chem. 413 (2021) 5969-5994, and

[0101] 18) Thermo Fischer Scientific: Technical Notice TN 53168: Use and development of the Microparticle Sample Preparation Kit; available at the link below: https: / / assets. thermofisher . com / TFS-Assets / MSD / Technical- Notes / TN53168-us e- development -mi croparticle- sample -preparation- kit . pdf

[0102] Preparation of microplastic standards

[0103] • PE standard (Sigma Aldrich, catalog number 434272-100 g) containing surface-modified microplastic particles of ultra-high molecular weight and a size range of 40-48 pm.

[0104] • PMMA standard containing PMMA microplastic particles with a size range of 250-300 pm (Cospheric) .

[0105] • For the preparation of PP, PTFE, and PET microplastic particles, cryomilling was used to reduce objects of different origins to a suitable size. RM instrument setting

[0106] Laser wavelength: 532 nm. Objective, numerical aperture: 20x, 0.40. Illumination mode: darkfield (DF) . Spectral range: 50-3, 400 cm-1. Laser power: 10.0 mW. Exposure time: 0.05 s. Number of scans: 30. EM gain: On. Observed spectral regions: 3,400-1,050 cm-1, 900-600 cm-1, 450-50 cm-1. Background intensity on the filter: 15-20%. Raman spectra libraries: 3M Polymers Raman, Polymers&AdditivesPackaging, Polymerrs&AdditivesPackaging (HR) , HR FT-Raman Polymer Library, FT- Raman Library Addendum.

[0107] Acceptance criteria are given in Table 1.

[0108] Table 1. Acceptance criteria applied during the development of the method according to the present invention.

[0109] Device performance

[0110] To demonstrate satisfactory device performance, a performance verification (PV) was conducted. It is based on determining the wavenumbers of four peaks in the spectrum of the polystyrene reference standard (at 620.9, 1001.4, 1031.8, 1602.3, and 3054.3 cm-1) and comparing them to specific predefined criteria for pass / fail. The PV was successfully performed.

[0111] Accuracy

[0112] To determine the method' s accuracy, it was necessary to establish the amount of obtained polymeric micro-particles after the extraction procedure from various food samples. Bread, pepper, tuna, and squid samples were used for method validation. For each food matrix, digestion conditions ( HNO3 concentration, temperature , program duration ) were utilized, which were previously determined to be sufficient for satisfactory extraction of microplastic particles from that particular matrix . All the conditions used are listed in Table 2 .

[0113] In each experiment , 50 mg of PE , PP, PET , PTFE , or PMMA micro-particles were added to 250 mg of food matrixes-1-4 : bread, pepper , tuna , or squid, respectively, and then covered with aluminum foil . The samplecontaining containers were placed in a drying oven and dried for 1 hour at 105 ° C . Subsequently, the containers were transferred to a digestion vessel , and using a glass pipette , a volume of 60% HNO3 ( 3 . 2 mL ) was added to achieve the desired concentration in the final volume of 10 mL ( see the Table above ) . The containers were closed, placed in a microwave digestion device Ethos UP (Milestone Sri ; IT ) , and subj ected to microwave digestion following the defined working program, see Table 2 . Upon completion of digestion, the containers were removed from the microwave , and the digested samples were transferred to a 50-mL glass measuring flas k and rinsed with 40 mL of ultra-pure water , followed by filtration through filter paper .

[0114] Table 2 . Conditions of digestion during validation .

[0115] The recovery was determined from the weight of the filter paper through which the samples without the added standard and samples with a known quantity of the reference standard were filtered .

[0116] The summarized results for the determination of the recovery on different food matrixes and various tested model polymers are presented in Table 3 . Table 3. Summarized results of determining the recovery parameter of the method according to the present invention for determining various model polymeric microparticles in different food matrixes .

[0117] All results for recovery were within the range of 70-120% , which demonstrates the method' s effectiveness .

[0118] Precision

[0119] The method' s repeatability was determined on ten weighings for each of the five model polymers , for each of the four types of food matrixes-1-4 ; a total of 200 preparations and measurements . The accuracy was calculated based on all measurements (multiple preparations , two days ) .

[0120] The relative standard deviation ( RSDr) of the method for determining microplastics in food products according to the present invention, under reproducibility conditions , ranges from 2 . 4569% to 14 . 6019% . The relative standard deviation (RSDR) of the method, under repeatability conditions, ranges from 2.6405% to 10.3447%.

[0121] The precision of the method is between 2.3063% and 9.8015%.

[0122] Limit of detection (LOD)

[0123] In the validation of this method, ten particle analyses were performed on ten different filters (labeled as Fl, F2,..., and F10) through which 10 mL of 10 M HNO3 (from the same sample preparation procedure) was filtered and diluted with 40 mL of ultrapure water (total volume of 50 mL) , representing a blank sample. The first five filtrations (through filters Fl-5) were conducted on the first day, and the remaining five filtrations (through filters F6-10) were performed on the second day.

[0124] The presence of microparticles and their chemical composition was determined using a Raman microscope (RM) . The presence of microparticles was identified with the OMNICxi software, which uses visual contrast of particles and substrate (in this case, filters) to determine the position of particles on the substrate (filter) . The chemical composition of the identified particles was determined spectroscopically by correlating (selected regions of) spectra from selected Raman spectra libraries (4.10) , obtaining a percentage match of the recorded spectrum with the associated spectrum from the Raman spectra library. The acceptance criterion for the identification of a polymer particle was set at an 80% matching percentage. Typical report with results contained, for each filter, the total number of analyzed particles, the total number of identified particles for each polymer, and the number of particles for each polymer within specified particle size ranges, 1-50 pm, 50-100 pm, 100-500 pm, and >500 pm.

[0125] The limit of detection (LOD) can be determined by adding the average number of particles found in 10 blank sample analyses (P) to three times the standard deviation (s) : LOD = P ( 10 blank sample analyses ) + 3x s ( 10 blank sample analyses )

[0126] The limit of detection (LOD ) calculated for each particular model polymer for each of the particle size ranges is given in Table 4 .

[0127] Table 4 . The limits of detection ( LOD) for each individual model polymer and for each particle size range .

[0128] The calculated limits of detection ( LOD) are 0 particles of PE , 10 particles of PP ( of which 4 are in the size range 1-50 pm, 2 in the size range 50-100 pm, and 4 in the size range of 100-500 pm) , 3 particles of PET ( of which 2 are in the size range of 1-50 pm and 1 in the size range of 100-500 pm) , 16 particles of PTFE ( of which 11 are in the size range of 1-50 pm and 5 in the size range of 100-500 pm) , and 0 particles of PMMA . Conclusion

[0129] The method according to the present invention enables the isolation of polymeric microparticles from the food samples and demonstrates their presence , chemical composition, number, and size . Based on the obtained validation results , it can be concluded that the method has been successfully validated and can be used for determining microplastics in food, with detection limits ( LCD) as shown in Table 4 .

[0130] Example 2 . A typical procedure for microplastic determination according to the present disclosure

[0131] Approximately 250 mg of homogenized sample is weighted on an analytical balance into a crucible for use with the microwave (MW) digestion device . The crucible with the sample is then placed in an oven, where the sample is dried at 105 ° C for 1 h . Afterward, 6 . 48 mL of 69% HNO3 is added to the crucible with the sample , covered with a lid, and placed in an ultrasonic bath for 20 minutes , where the digestion of the food matrix is accelerated using ultrasonic waves ( 20 kHz ) . Then, 3 . 52 mL of deionized water is added to the crucible to achieve a final HNO3 concentration of 10 mmol / L . The crucible is then sealed and placed in the microwave (MW ) digestion device , and the following program is set : temperature raised to 110 ° C, over 10 minutes , held at the same temperature for 100 minutes , and cooled to 0 ° C , over 10 minutes .

[0132] After completing the digestion, the solution is transferred to an appropriate glass container , e . g . , a 50 mL glass centrifuge tube with a Teflon ( PTFE ) cap ) , with the crucible used for digestion rinsed with 40 mL of deionized water, which is then transferred to the same glass container, resulting in a final volume of the diluted solution of 50 mL . This solution is then filtered through a silicon filter of pore size 1 or 5 pm, to yield a filter with immobilized microplastic particles . The latter is subj ected to analysis on a Raman microscope . The automated analysis of thus prepared sample using the Raman microscope is conducted under the following conditions:

[0133] Laser wavelength: 532 nm with corresponding filter and grid, Objective, numerical aperture of the objective: 20x, 0.40, Illumination mode: darkfield (DF) , Illuminator intensity: 50%, Aperture: 50 pm pinhole, Laser power at the sample: 10 mW, Exposure time to laste light: 0.05 s, Number of scans: 30, Background intensity on the filter: 15-20%, Observed spectral regions: 3400-1050 cm-1, 900-600 cm-1, 450-50 cm" i with the use of one or more Raman spectrum libraries selected from the group consisting of: 3M Polymers Raman,

[0134] Polymers &AdditivesPackaging, Polymers&AdditivesPackaging (HR) , HR FT- Raman Polymer Library, HR FT-Raman Polymer Library Addendum.

[0135] Through this procedure, a tabular presentation of data is obtained for each analyzed particle on the filter, which includes: corresponding Raman spectrum or Raman shift spectrum, the ordinal number of the particle, spatial dimensions (X and Y) indicating the exact position of the observed particle on the silicon filter, qualitative identification of the type of polymer (polymer name) , matching value of the Raman spectrum of the particle with the associated spectrum in one of the Raman spectra databases used, expressed as a percentage (1-100%) , whereby a matching value greater than or equal to 80% is used as a criterion for successful identification, the name of the Raman spectrum library (database) from which the spectrum with the highest matching value was obtained, aspect ratio of the particle, circularity of the particle, length of the particle, and particle width. Example 3 . Chocolate sample analysis using the method according to the present invention

[0136] A random sample of chocolate of a renowned manufacturer from a local market was taken . The sample was prepared and analyzed according to the procedure described in Example 2 . The results of the analysis regarding the number, size , and type of microplastic particles found are presented in Table 5 . A criterion for the successful identification of microplastic particles was used, which requires a spectrum match of the particle with a spectrum from the used spectral library equal to or greater than 80% .

[0137] Table 5. The results of the analysis - chocolate sample .

[0138] Example 4 . Pate sample analysis using the method according to the present invention

[0139] A random sample of pate of a renowned manufacturer from a local market was taken . The sample was prepared and analyzed according to the procedure described in Example 2 . There was no need to homogenize the sample in a cryomill as the sample was already in a satisfactory homogenous state . The results of the analysis regarding the number, size , and type of microplastic particles found are presented in Table 6 . A criterion for the successful identification of microplastic particles was used, which require a spectrum match of the particle with a spectrum from the used spectral library equal to or greater than 80% .

[0140] Table 6. The results of the analysis - pate sample .

[0141] INDUSTRIAL APPLICABILITY

[0142] The present invention discloses a method for the determination of microplastic as a contaminant 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 .

[0143] ABBREVIATIONS kHz = kiloHerz

[0144] LOD = limit of detection

[0145] MW = microwave

[0146] PE = polyethylene

[0147] PET = polyethylene terephthalate

[0148] PMMA = poly (methyl methacrylate )

[0149] PP = polypropylene

[0150] PTFE = polytetrafluoroethylene

[0151] PVS = polyvinyl siloxane

[0152] RM = Raman microscopy

[0153] RSD = relative standard deviation

Claims

CLAIMS1. A method for qualitative and quantitative determination of microplastics as a contaminant in food products where the said analytical process comprises the following steps :(a) optionally, in the case of solid and non-homogeneous food products, homogenization of a sample by cryogenic milling,(b) weighing of homogenized sample,(c) drying the sample,(d) adding 65-69% w / w nitric acid <HNOs> to the sample,(e) placing the vessels with the sample in the ultrasonic bath at 20 kHz,(f) adding demineralized water to the mixture to form the final concentration of HNO3 within the formed mixture,(g) microwave <MW>-assisted, HNOs-based, acidic, oxidative digestion of thus prepared sample,(h) quantitative transfer of the content into the glass volumetric flask with demineralized water, followed by dilution with demineralized water up to the total dilution volume ,(i) filtration of the final diluted solution through a silicon filter of 1 or 5 pm pores, yielding microplastic particles immobilized on the filter, and(j) qualitative and quantitative analysis, as well as particles size analysis of immobilized microplastics on said silicon filter by Raman microscopy <RM>, wherein, in step (e) the ultrasonic treatment is conducted at 20-30 °C for 15-25 minutes, in step (f) the amount of demineralized water added to the sample corresponds to the targeted final concentration of HNO3 at the range of 8-10 mol / L, and where in step (g) said digestion is performed within an MW digestion system at 2.450 MHz under the following digestion regimen: from 0°C to about 110 °C for 10 minutes, at 100-110 °C for 30-100 minutes, andfrom 100-110 ° C back to ° C for 10 minutes .2 . The method according to claim 1 , where the analysis is conducted under the following conditions : in step (b ) , the analysis typically starts with 250 mg of starting sample of food product being analyzed, and in step (h ) , the preferred total dilution volume is 50 mL .

3. The method according to claims 1 and 2 , where the analysis of the silicon filter with immobilized particles of microplastics in step ( j ) by Raman microscopy <RM> involves the following conditions : laser of wavelength 532 nm with corresponding filter and grid, spectral regions observed : 3400-1050 , 900- 600 , 450-50 cm-1, using one or more Raman spectra databases selected from the group consisting of : 3M Polymers Raman,Polymers & dditives Packaging, Polymers &Addi fives Packaging<HR> , HR FT-Raman Polymer Library, HR FT-Raman Polymer Library Addendum .4 . The method according to claims 1-3 , where the analysis of the sample in step ( j ) by Raman microscopy <RM> furnishes a tabular presentation of the results , which for each analyzed particle on the filter contains : corresponding Raman spectrum, the ordinal number of the particle , spatial dimensions <X and Y> indicating the exact position of the observed particle on the silicon filter , qualitative identification of the type of polymer, matching value of the Raman spectrum of the particle with the associated spectrum in one of the Raman spectra databases used, expressed as a percentage <l-100%>, whereby a matching value greater than or equal to 80% is used as a criterion for successful identification,the name of the Raman spectrum database from which the spectrum with the highest matching value was obtained, aspect ratio of the particle , circularity of the particle , and, length of the particle , and particle width .

5. Use of the method according to claims 1-4 , for the qualitative and quantitative determination of microplastic particles as a contaminant in food products .

6. Use of the method according to claim 5 , for the determination of microplastics in food products selected from the group comprising pasta and pasta-based products , fruits and fruit-based products , vegetables , and vegetable-based products , marine products and marine-based products , meat and meat products , eggs and egg products , products based on edible mollus ks and insects , dairy products , cocoa , chocolate and other cocoa-based products , foods intended for particular nutritional uses including foods for infants and young children .

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