Using Chromatography to Study Microplastics in Food: An Interview with Jose Bernal

Chromatography plays a critical role in analyzing microplastics in food, primarily by identifying the polymer types that constitute the microplastics and detecting the chemical compounds associated with these materials (1). The issue for analysts is that microplastic contamination can come from numerous sources, both in the environment and in the laboratory. This poses a challenge for scientists as they conduct research in laboratories, so the main question is how to prevent microplastics from contaminating food products that are distributed to grocery stores globally.

Dr. Jose Bernal of the University of Valladolid is exploring this issue. Bernal began his scientific career in 2003 during his PhD at the University of Valladolid. Postdoctoral work followed at CSIC’s Institute of Industrial Fermentations and the University of Helsinki. He joined the University of Valladolid as an Assistant Professor in 2010, advancing to Full Professor in 2024 (2). His expertise spans advanced analytical techniques like LC-MS, GC-MS, capillary electrophoresis, and green chemistry (2). Bernal’s research focuses on food safety, contaminants, and bioactive compounds, particularly in apiculture. He is member of the Editorial Board of several JCR journals, he has authored 115+ publications, supervised 4 PhD theses (and four others in development), and contributed to multiple research projects worth over €700,000 (2).

LCGC International recently sat downBernal to talk about the latest review article he and his team published, which highlights the latest trends and developments in using chromatographic techniques to analyze microplastics and related compounds in food products (3).

Dr. Jose Bernal of the University of Valladolid. | Photo Credit: © Jose Bernal

Your review highlights the growing interest in studying microplastics (MPs) in food using chromatographic techniques. What do you see are the key advantages of chromatography over spectroscopic techniques for MP analysis?

First, it is important to consider that traditional chromatographic techniques, such as gas chromatography (GC) or liquid chromatography (LC), are not the preferred options because MPs have high molecular weights and limited solubility in most solvents. In fact, many studies do not aim to quantify MPs, but rather focus on their detection and characterization using microscopic and spectroscopic techniques, such as Fourier-transform infrared spectroscopy (FT-IR) and Raman spectroscopy. Although these techniques are easy to perform and provide valuable information regarding the number, chemical composition, shape, and size distribution of MPs, they only yield an approximate estimation of their quantities. If the objective is precise quantification, alternative approaches combining thermal decomposition with mass spectrometry (MS) are more suitable.

However, a promising and increasingly employed technique in this context is gas chromatography– mass spectrometry with pyrolysis (Py–GC–MS). This technique uses heat in an inert environment to thermally decompose polymeric materials in a predictable manner. The resulting pyrolysis products are subsequently separated using GC based on their size and polarity, providing a chromatogram with MS data throughout the chromatogram. These data can be compared with a known reference library to identify the specific class of polymers being analyzed. It is crucial to consider that pyrolysis temperature can significantly affect the generated pyrolysis products, which may differ from those found in the reference library.

Because many libraries are based on pyrolysis at 600 ºC, this can pose challenges for the identification of polymers at different temperatures. However, by considering this factor, it is possible to create a customized database with other pyrolysis temperatures that enables accurate polymer identification. Nevertheless, Py–GC–MS has certain limitations. These include the requirement of a limited sample mass, the simultaneous determination of different types of MPs not being possible, the challenge of manually transferring handpicked particles into the pyrolysis cup, the lengthy analysis time, and its destructive nature. Despite these limitations, a key advantage of Py–GC–MS is the sensitivity and selectivity of MS, which enables simplification of sample treatment in many cases. Pyrolysis is usually performed online; however, in some cases, off-line pyrolysis is employed as an alternative technique. Because of the above-mentioned disadvantages, new variations have been developed, such as thermal extraction and desorption, which combines thermal extraction and thermal desorption with thermogravimetric (TGA) analysis, allowing heavier samples to be analyzed as well as the identification and quantification of heterogeneous matrices. Nonetheless, its main disadvantages include the high cost and handling complexity. Despite those two drawbacks, both techniques are highly reliable and will continue to be used more and more in this field.

The study mentions that Py-GC–MS is the preferred technique for MP analysis, while LC–MS/MS and GC–MS are used for bisphenols (BPs) and phthalates (PAEs). Could you explain why these specific chromatographic methods are more suited to these compounds?

In recent years, the use of Py-GC–MS has increased to identify MPs composition and simultaneously determine the presence of plastic additives with high safety and minimal pretreatment. In fact, it is becoming an indispensable tool in the identification and characterization of MPs in the environment. This technique provides improved sensitivity and selectivity, requiring small amounts of sample, and Py-GC–MS allows the simultaneous identification (in a single analytical run) of both the polymer and organic plastic additives.

Current methods for determining BPs rely predominantly on chromatographic techniques such as LC with fluorescence (LC–FLD), ultraviolet (LC–UV), diode-array (LC–DAD), and MS detection (LC–MS or LC–MS/MS), as well as GC–MS, GC–MS/MS, and GC with flame ionization detection (GC-FID). Ultrahigh pressure liquid chromatography (UHPLC) has been selected in some studies because this might obtain better resolutions, sensitivities, and shorter running times, implying lower solvent consumption, but the systems must be able to withstand the high pressures that will be required when using columns with such a small internal diameter. Because of the low volatility of these compounds, GC analysis requires some pre-treatment derivatization steps, such as alkylation, silylation, or acylation, prior to chromatographic separation. These additional manipulations not only increase the analysis time, but they also diminish the reproducibility of the method and introduce a potential source of contamination. So, considering the above, the LC(UHPLC)-MS/MS combination is the one that provides the best results in terms of analysis time, selectivity and sensitivity.

Finally, GC and LC are the most widely employed techniques since they provide high sensitivity and selectivity. However, the most employed separation technique is GC. This preference stems from the thermal stability and volatile characteristics exhibited. Although the use of GC–FID is more common than for MPs and BPs, the specificity and high sensitivity of MS make it the preferred detection technique when analyzing PAEs at low concentration level.

Sample treatment is a critical step in the analysis of MPs, BPs, and PAEs in food. Could you elaborate on the most effective extraction techniques for each type of compound, and what challenges researchers face during this process?

In this case, the answer is a bit complex because there is no common or standard procedure, especially for the analysis of BPs and PAEs. In fact, the matrix under study has a very important weight in this choice; hence, it is not possible to generalize. For the analysis of MPs, when using Py–GC–MS, the sample treatments are not as complex as with other techniques despite the complexity of the food matrices. They are generally based on a basic digestion with potassium hydroxide followed by filtration. This option is the simplest, but it usually requires the use of large quantities of solvents and long times, so the use of pressurized liquid extraction (PLE) and microwave assisted extraction (MAE) can serve as alternatives. They are more expensive and complex, but they shorten the analysis time and reduce the solvent consumption. In relation to sample treatments for BPs analysis in food, it is true that although solvent extraction and solid-phase extraction (SPE) predominated, the new trend is to use more environmentally friendly sample treatments, when possible, that allow a reduction in costs, stages, and reagents. This is why techniques with dispersive liquid-liquid microextraction (DLLME) or quick, easy, cheap, effective, rugged and safe (QuEChERS), or the use of molecularly imprinted polymers-SPE (MIP-SPE) or fabric phase sorptive extraction (FPSE) among others, are emerging as green alternatives to the abovementioned treatments. Finally, the most employed sample treatments when determining PAEs in food are solvent extraction and SPE, followed by QuEChERS and DLLME, although other recently introduced sample treatments that reduced not only the length of the procedure, but it also reduced the consumption of solvents that have been employed.

As for the challenges faced by researchers in the sample treatment stage, it must be said that they are not very different from those that could be found when determining other contaminants, with the (very important) exception of trying to avoid (control) contamination because of the ubiquitous presence of these compounds in the laboratory environment. However, I would recommend that the methods for each matrix being studied be optimized and validated, and that methodologies previously optimized for other matrices not be simply applied.

Contamination control is emphasized as a crucial factor in the analysis of MPs, BPs, and PAEs. Could you discuss the contamination risks associated with this type of analysis and the best practices for avoiding them in the laboratory?

MPs, BPs, and PAEs have become pervasive environmental contaminants, and their presence in the laboratory is a growing concern, especially for studies involving trace analysis, environmental monitoring, toxicology, and food safety. Their specific physico-chemical characteristics (small size, chemical stability, and widespread distribution) make them difficult to avoid and can lead to significant contamination risks during laboratory analyses.

For example, those compounds are commonly present in the laboratory environment, especially in settings that involve plastics or synthetic materials. They can contaminate samples during collection, preparation, or processing because they can be released from plastic equipment, clothing, and even from researchers themselves. Even the air can carry some of those particles. During the handling of samples, plastic contaminants from previous experiments can unintentionally mix with the new samples. Cross-contamination is particularly problematic when working with environmental samples or biological tissues, where trace amounts of those compounds can skew results and lead to inaccurate findings. In addition, MPs can interfere with various laboratory techniques, such as microscopy, spectrometry, and chromatography. Their small size and similar characteristics to other particulate matter may make it challenging to distinguish MPs from natural debris or other sample components. Therefore, if plastic contaminants are not properly identified or excluded during sample preparation or analysis, they could be mistaken for other compounds, resulting in misinterpretation of data.

This makes it clear that it is crucial to control the presence of plastic contaminants and there are several alternatives to do so:

• Use non-plastic equipment. Where possible, opt for glass, stainless steel, or other non-plastic materials for laboratory equipment.
• Establish "clean" areas where the handling of sensitive samples takes place. These areas should have controlled airflow and minimized exposure to plastic sources.
• Use laboratory coats, gloves, and masks made from non-synthetic fibers.
• Use HEPA filters in the laboratory ventilation system to capture airborne MPs and other particulates. Consider using laminar flow hoods when possible.
• Clean all laboratory surfaces and tools thoroughly before working. This includes wiping down surfaces with lint-free cloths or using microfiber towels, which are less likely to shed those contaminants.
• Reserve specific pipettes, tweezers, and spatulas exclusively for these analyses. Label and sterilize these tools to prevent cross-contamination. Consider using disposable pipette tips and laboratory tools that are pre-cleaned and certified free of plastic debris.
• Procedural blanks, in which ultrapure water replaced samples, must be systematically run between sets of samples to monitor potential abnormal background values.

Your review spans research from 2018 to 2022. In this period, how have advances in chromatographic techniques improved the detection sensitivity and accuracy for microplastic-related compounds in food?

Advances in chromatographic techniques have significantly improved the detection sensitivity and accuracy for MP-related compounds in food over the past few years. These improvements are primarily because of enhancements in chromatographic resolution, detection methods, sample preparation, and data analysis. These improvements can be summarized in the following points:

• New stationary phases and column technologies have led to faster and more efficient separation of complex mixtures, including MP additives, degradation products, and other contaminants in food.
• Coupling chromatography with mass spectrometry (for example, GC–MS or LC–MS) has greatly enhanced detection sensitivity. Mass spectrometric techniques provide excellent compound identification through high-resolution mass spectra, which is crucial for detecting trace amounts of MP-related contaminants. Advances in ionization techniques have improved the sensitivity for polar compounds often found in MPs. In addition, the use of HRMS, including Orbitrap and time-of-flight (TOF) instruments, has enabled ultra-sensitive detection of low-abundance compounds in complex food matrices. These systems can detect MP-related contaminants at parts per trillion (ppt) levels, which is essential for monitoring food safety.
• Advanced sample preparation techniques, such as SPME, FPSE, DLLME, and MIP-SPE, have allowed for the extraction of low-concentration MP-related compounds from complex food matrices with high efficiency and low solvent use. These techniques can concentrate analytes from food samples, improving sensitivity. Moreover, for food matrices with high moisture content, cryogenic grinding helps break down the samples into smaller particles, facilitating the extraction and detection of MP-related compounds.
• Finally, new automation techniques have streamlined the sample preparation process, reducing the potential for contamination and operator error. This leads to more reproducible results and better sensitivity in trace analyses.

One of the challenges mentioned is the difficulty in avoiding plastic contamination during food production and processing. How might chromatographic techniques help in identifying the sources of plastic contamination along the food chain?

Chromatographic techniques, particularly when coupled with advanced detection methods, especially MS and MS/MS, can be powerful tools for tracing the sources of plastic contamination along the food chain. As plastics are pervasive in the environment and in food production, processing, packaging, and storage, pinpointing the origin of contamination requires highly sensitive, selective, and sophisticated methods. Here's how chromatographic techniques can help identify the sources of plastic contamination throughout the food chain:

• Polymer characterization. MPs in food often come from various sources such as packaging materials, food processing equipment, and environmental contamination. Chromatographic methods such as LC-FTIR or GC-MS can help identify the specific type of polymer present in a food sample.
• Polymer fingerprinting. By comparing the chemical "fingerprints" (for example, FT-IR spectra or mass spectra) of MPs found in food with known standards of various plastics, chromatographic methods can help link specific contamination events to known sources along the food chain
• Detection of plasticizers, additives, and degradation products. Many plastics are treated with plasticizers, flame retardants, stabilizers, or colorants, which can leach into food. Using LC-MS/MS or GC-MS/MS, researchers can detect these specific chemical additives, which can serve as markers for identifying the type of plastic and its source. Over time, plastics degrade into smaller particles, releasing specific chemical byproducts. GC–MS/MS can detect plastic degradation products, which can indicate the source of contamination—whether it's because of aging packaging materials, exposure to heat during food processing, or degradation of plastic particles in the environment.
• Trace contaminant detection. Food production and processing often involve exposure to plastics at various stages, including packaging, storage, and cooking. By employing SPME-GC-MS or SPME-LC-MS, it is possible to extract trace amounts of MP particles or their chemical contaminants from food at different production stages. These can be analyzed to determine the timing and location of contamination.
• Packaging materials. Packaging is a major source of plastic contamination, and chromatographic methods can help identify the presence of MPs originating from food packaging. For example, GC-MS can detect the characteristic polymer components or additives of packaging materials in food samples.
• Food processing equipment. Many food processing operations involve equipment that might shed MPs into food products. Using chromatographic techniques, researchers can identify plastic particles and related additives.
• Environmental contamination. In addition to packaging and processing, MPs and related compounds can be introduced through environmental sources, such as water, air, or soil. Chromatographic analysis of water used in food processing, or air samples in food production facilities, can help identify if plastic contamination is coming from external environmental sources.
• The food matrix can influence the nature and quantity of plastic contamination. By analyzing raw ingredients (for example, flour, meat, or milk), processing stages (such as grinding, cooking, or mixing), and final food products using advanced chromatographic techniques (for example, LC–MS or GC–MS), researchers can trace the pathway of contamination through the food chain. Moreover, chromatographic analysis of raw ingredients (which may contain plastic residues from soil, water, or packaging) compared to processed food products can help identify at which point in the supply chain contamination is most significant. For instance, food products like canned vegetables might show a signature of contamination from both packaging and processing equipment, while packaged dairy products might show contamination primarily from packaging plastics.
• By comparing contamination levels at various stages (from farm to fork), researchers can assess which part of the supply chain is contributing the most to plastic contamination. This approach can help pinpoint key sources (for example, processing steps, storage, and packaging) and guide efforts to reduce contamination. To sum up, chromatographic techniques provide powerful tools for identifying and tracing the sources of plastic contamination throughout the food chain. By analyzing polymer types, plasticizers, degradation products, and other additives, these methods can reveal whether contamination is originating from packaging, processing equipment, or environmental sources. This level of specificity and sensitivity allows researchers to not only identify the plastic contaminants but also to trace them back to their origins, helping food producers, regulators, and consumers better understand and mitigate plastic contamination in the food supply.

Looking forward, the review suggests that residue or migration limits for these compounds may be established soon. How do you think this will affect the role of chromatographic techniques in food safety regulations?

Given the growing awareness of the presence of MPs and related products in all environments of daily life, and especially in those that affect health, the foreseeable trend is that the limits will be lowered. It is also possible at some point their presence will be prohibited, as is the case with bisphenol A in baby bottles and pacifiers. However, here I would like to differentiate MPs from BPs and PAEs. The limits are defined mainly for some BPs and PAEs, and not so much for MPs, since until now most of the analysis is qualitative rather than quantitative, with a predominance of spectroscopic techniques. However, in all cases, selective and sensitive analysis methods will be necessary to allow the identification of these plastic contaminants, and this is where chromatographic techniques would come into play. It is true that nowadays, it is possible to find publications where MS-based detectors are not used to determine BPs and PAEs because the limits are relatively high, but in the future, if the limits are lowered significantly or even banned, the use of MS or MS/MS will be necessary. Therefore, the role of chromatographic techniques when controlling food safety will be much more relevant than it is today, since quantitative methods are needed, and the option of spectroscopic techniques will no longer be the majority option as it is today for MPs.

For researchers new to this field, what key considerations should be considered when designing studies involving the chromatographic analysis of MPs, BPs, and PAEs in food?

From my point of view, the most important thing when analyzing these types of compounds is to try and avoid (or at least control) contamination of the work environment, since MPs, BPs, and PAEs are found in nearly all laboratory equipment for one reason or another. It is not directly related to chromatography, but it can significantly affect the results. Another important factor to consider is the sample treatment, which must be optimized for each matrix to determine BPs and MPs since unfortunately, there is no unique method, and the matrix has a lot of influence. In the case of MPs, this is simpler because when using Py-GC-MS, basic digestions are usually used in most cases. Finally, we come to the chromatographic separation, which from my point of view is the simplest of this process. It is true that in many cases because of the use of MS or MS/MS, not much attention is paid to the separation of the baseline, but I think it is convenient to try to achieve it because not everyone has these detectors, and on the other hand, it minimizes possible interferences when determining them.

References

1. HKwon, J.-H.; Kim, J.-W.; Pham, T. D. Microplastics in Food: A Review on Analytical Methods and Challenges. Int. J. Environ. Res. Public Health 2020, 17 (18), 6710. DOI: 10.3390/ijerph17186710
2. ORCID, Jose Bernal Del Nozal. ORCID. Available at: https://orcid.org/0000-0002-8618-3543 (accessed 2024-11-07).
3. Martin-Gomez, B.; Elmore, J. S.; Valverde, S.; et al. Recent Applications of Chromatography for Determining Microplastics and Related Compounds (Bisphenols and Phthalate Esters) in Food. Microchem J. 2024, 197, 109903. DOI: 10.1016/j.microc.2024.109903