Jianru Stahl-Zeng

Modern eating habits have led to the further diversification of an already complex food supply chain. The public confidence in the food supply is not only impacted by publicized crises involving contaminations, but also through the misreporting of nutritional information. Food analysis is integral to the whole supply chain, be it through a rapid response to a food crisis, such as the fipronil egg scandal, the continued monitoring of pesticides that could harm an unsuspecting public, or accurately reporting nutritional information to provide the information the public needs to make an informed decision about the food they eat. Each event is fraught with difficulties, but by developing new methods of analysis, crises in the food industry can be avoided, or their effects mitigated. This article highlights three events that require new method development to meet various detection needs, ranging from the detection of pesticides (such as fipronil and glyphosate), to the detection and quantification of fat-soluble vitamins.

	Modern diets have resulted in unprecedented growth and diversification of the food supply chain. The necessity of a continual supply of food means crops are commonly being treated with pesticides that are essential to reduce the risk of failed harvests. These chemicals are toxic to both insects and humans, and as such the public must be protected from unsafe concentrations of pesticides. Regulations imposed by food standard agencies dictate pesticide maximum residue limits (MRLs), which ensure the food on supermarket shelves is safe for consumption. In addition to ensuring food is free from contaminants, it is also essential to provide the public with accurate nutritional information to help enable the healthy growth of the global population.

	To meet these goals, emphasis is placed on food content analysis to ensure quality and consistency between batches. Characterization and detection techniques, such as mass spectrometry (MS), offer manufacturers and producers the ability to screen large quantities of samples in a timely fashion, while guaranteeing reliable, repeatable measurements. Approaches to analysis are generally twofold-proactive monitoring of essential nutrients and possible contaminants, and crisis response, where widespread contamination requires the rapid development and deployment of analytical methods and equipment.

Rapid Response: Fipronil Egg Contamination

	One recent example highlighting the need for a crisis response is the fipronil contamination of eggs, which resulted in the recall of millions of eggs. Fipronil is an insecticide, belonging to the phenylpyrazole family of chemicals, developed in the 1980s (1). Its high toxicity makes fipronil useful for controlling levels of insects including fleas, mites, and cockroaches, and is even useful against pests resistant to various insecticides (2). However, in July 2017, fipronil made news headlines after it was found to be present in eggs across Europe. This crisis was so widespread that, by the end of August, fipronil-contaminated eggs were detected in 15 European countries and as far afield as Hong Kong and China.

	Fipronil treatment for crops used in the food chain is prohibited, and the US Environmental Protection Agency (EPA) has labeled fipronil as possibly carcinogenic, prohibiting its presence in the food chain (3). Furthermore, the European Food Safety Authority (EFSA) dictates fipronil concentrations of under 5 µg/kg to be safe for human consumption (4). Complying with concentrations dictated by regulation is therefore vital for manufacturers and producers to ensure the continued safety and confidence in food supplied to the public. Detection methods form one essential part of this process, identifying and quantifying potential contaminations prior to distribution in the food chain. There are several plausible methods of detecting contaminants such as fipronil in samples of food. The most common detection methods include liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS), with both requiring different methods of sample preparation prior to analysis.

	The challenge arises in detecting fipronil concentrations at the low level stipulated by government regulation, but novel methods capable of detecting fipronil and its associated metabolite, fipronil sulfone, have been developed. Since the fipronil contamination scandal began, demand for a fast, sensitive detection method has only continued to grow. One such detection method involves a modified Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) sample preparation technique prior to detection, using a triple quadrupole instrument and electrospray ionization (ESI). The high sensitivity of detection and ability to analyze two compounds in the same run enable regulation-compliant detection in a timely manner. This developed method can detect fipronil, as well as its major metabolite fipronil sulfone, to the MRL level of 5 µg/kg, as demonstrated in Figure 1 (5).


	Figure 1: A chromatogram showing the clear separation of analytes, identifying fipronil and amitraz (another insecticide) and their related metabolites. 1. DPMF, 2. DMA, 3. DMF, 4. fipronil, 5. fipronil sulfone, 6. amitraz.

	In responding to a food contamination crises, it is also prudent to develop methods that can screen for multiple contaminants at once, potentially preventing contamination from other, unexpected sources. Using nontargeted approaches, such as sequential window acquisition of all theoretical mass spectra (SWATH-MS) acquisition, contaminant detection is not limited to the chosen molecule, in this case enabling the analysis of fipronil and other contaminants such as pesticides and polyaromatic hydrocarbons (6). Combining instrumentation that provides linear, reproducible contaminant detection to regulation-specific concentrations, with novel sample preparation methods, is essential to avoid repeated events, and helps improve public confidence in the integrity of the food supply chain.

Continual Monitoring: Glyphosate Pesticide

	While fipronil is known to be hazardous for health, regulatory advice on other pesticides, such as glyphosate, is conflicted. Glyphosate is a widely used broad-spectrum systemic herbicide and crop desiccant. While it has recently made headlines for its potentially hazardous nature to humans as a possible carcinogen, its impact on human health is contested, and, therefore, the use of glyphosate as a farming pesticide is still permitted (7). In cases where potentially harmful pesticides are being used, continual monitoring is required to ensure chemical concentrations in foods are safe. Glyphosate is used globally and has been detected at trace levels, along with related metabolites, in 45% of European topsoils (8), and in samples of milk (9).

	The controversy surrounding the use and potential contamination of glyphosate has placed greater emphasis on data collection and analysis methods to ensure levels of glyphosate in food samples fall below the safe maximum residue level (MRL) (reported by the EFSA as 50 µg/kg) (10). However, while the analysis of glyphosate and its associated metabolites is essential, its detection presents different sample preparation and analysis challenges that must be overcome. The high polarity of glyphosate and its related metabolites previously made sample extraction and LC analysis difficult. To overcome retention issues, derivatization is employed using a method with fluorenylmethyloxycarbonyl chloride (FMOC-Cl) as the derivatization reagent to convert glyphosate into an analogue that can then be analyzed. However, while this approach enables detection, it is both complicated and time-consuming, and fails to detect pure glyphosate and its metabolites.

	Currently, the detection of underivatized glyphosate can be achieved using new methods of extraction coupled with instrumentation. One such underivatized method of detection starts by using the Quick Pesticide Preparation (QuPPe) extraction method to prepare samples (11). Using a combination of this approach with sensitive MS instruments, accurate quantification of glyphosate and its metabolites can be achieved. By combining the LC–MS/MS method with differential mobility separation (DMS) technology, interferences can be removed from analyses to improve the signal-to-noise ratio and, consequently, increase confidence in quantification results. These samples are then analyzed using LC–DMS–MS/MS to quantify and identify those contaminants present, as shown in Figure 2.


	Figure 2: MS spectra provide the ability to clearly identify and distinguish glyphosate and its metabolite AMPA and other pesticides, such as glufosinate.

	General food composition monitoring is not limited to the detection of harmful contaminants. It also forms an essential component of the accurate reporting of nutritional information for packaging labels. Vitamins are vital nutrients that are essential for an individual's growth and development. Deficiency in any vitamin is detrimental to health, and is linked to a multitude of health issues. For instance, deficiency in vitamin D leads to the bone disorder known as rickets, and has been associated with other health problems, including heart disease and cancer (12). While most vitamins can be obtained through natural means-exposure to sunlight is the best source of vitamin D-this is not always possible for some. Vitamin supplements are one option, and are commonly incorporated into food (for example, infant formula). It is therefore imperative to report accurate nutritional information on food packages to ensure an individual's vitamin needs are met, and for manufacturers to correctly advertise the benefits of their products.

	Vitamins broadly separate into two categories: water-soluble (vitamins B and C), and fat-soluble (vitamins A, D, E, and K). Detection of water-soluble vitamins is relatively easy, with analysis possible using MS. Conversely, analysis of fat-soluble vitamins is difficult, owing to the challenges associated with MS detection. These problems originate from the presence of lipids in fat-soluble vitamin samples that cause an effect known as 

 (13), negatively affecting the detection, precision, and capability of a mass spectrometer. The ease in the detection of water-soluble vitamin samples is a direct result of the absence of lipids, enabling clean detection of vitamin B and C in samples (14). No uniform solution to ion-suppression exists, but its effects can be circumvented by the removal of lipids.

	The detection of vitamins provides a specific detection challenge and, until recently, it was difficult to detect fat-soluble vitamins using LC–MS methods. Food samples contain various concentrations of vitamins, ranging from parts per billion to parts per million. To obtain clean, analyzable detection of fat-soluble vitamins, the lipid content should be removed from food samples. This reduces the ion suppression, and enables robust MS analysis within a single chromatogram that identifies multiple vitamins (see Figure 3). Combining sensitive MS methods with associated expertise in methodology allows universal application of the sample preparation and a simple analysis of vitamin concentrations in various food samples, meeting the specific requirements of the customer.


	Figure 3: A single chromatogram identifying the various vitamins that can be identified within a single mass spectrum run.

	Identification and quantification of potential contaminants and nutrients are essential to maintaining the integrity of the food supply chain. By avoiding unnecessary contamination of pesticides and providing clear labeling information, food manufacturers can be confident that food is safe for public consumption, while also delivering accurate nutritional information. Continual methodological and instrumentation development will enable more sensitive and timely detection to continue for years to come.

	(1) C.C. Tingle, J.A. Rother, C.F. Dewhurst, S. Lauer, and W.J. King, 

 (Royal Society of Chemistry, Information Services, Cambridge, United Kingdom, 3rd ed., 1991).

	(3) U.S. Environmental Protection Agency, New Pesticide Fact Sheet - Fipronil; EPA 737-F-96-005 (U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances (Office of Pesticide Programs, U.S. Government Printing Office Washington, DC, USA, 1996), pp 1–10.

	(4) European Commission Pesticide Database, 

ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public/?event=pesticide.residue.selection&language=EN

	(5) Analysis of fipronil and amitraz insecticides, 

sciex.com/Documents/tech%20notes/Rapid-LC-MS-MS-Method-for-the-Analysis-of-Fipronil-and-Amitraz-Insecticides.pdf

	(6) Screening residual pesticides in fruit and vegetables, 

https://sciex.com/Documents/tech%20notes/X500R-QTOF-System-with-SWATH-Acquisition-for-Pesticide-Residue-Screening-in-Fruits-and-Vegetables.pdf

	(7) EFSA: Conclusion on the peer review of pesticide risk assessment of the active substance 

glyphosate,https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/j.efsa.2015.4302

	(8) V. Silva, L. Montanarella, A. Jones, O. Fernández-Ugalde, H.G. Mol, C.J. Ritsema, and V. Geissen, 

	(9) N. Chamkasem, C. Morris, and T. Harmon, 

	(11) LC–MS/MS Analysis of Emerging Food Contaminants, 

https://sciex.com/Documents/tech%20notes/food_contaminants6500_QuPPe.pdf

	(12) D. Feldman, A.V. Krishnan, S. Swami, E. Giovannucci, and B.J. Feldman, 

	(14) Analysis of Vitamin B complex in infant formula samples, 

https://sciex.com/Documents/brochures/vitB_infant_formula_QTRAP6500_09340214.pdf

 is the Senior Manager, Applied Markets Development at Sciex, in Warrington, UK. 

 is the Applications Manager and Global Technical Leader, Food & Environmental at Sciex, in Darmstadt, Germany. 

 is the Global Marketing Manager, Food, Environment & Forensics at Sciex, in Warrington, UK. Direct correspondence to: 

Articles

In the human food supply, public confidence is affected by contaminants and misreporting of nutritional information. This article
highlights three events that required development of new mass spectrometry methods, including the detection of pesticides (such as fipronil and glyphosate), and the detection and quantification of fat-soluble vitamins.

Novel Methods Using Mass Spectrometry for Food Safety—From Contamination to Nutrition

Monitoring a highly polar, small organic pesticide such as glyphosate in food and water sources presents a significant challenge. Polar pesticides are not amenable to standard extraction procedures, are frequently poor ionizers, and do not separate well. Current analysis methods rely on labor-intensive derivatization and cleanup steps. This study provides details of a robust high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for the detection of glyphosate and its metabolites in complex food and water matrices, without the need for derivatization of samples prior to analysis. Polar pesticides are water soluble, and the extraction was therefore aqueous with methanol and formic acid added to improve efficiency. The large variety of interfering particulates in environmental and drinking water samples requires further sample preparation, and a simple filtration step was performed here before the MS/MS analysis. The food analytes were well separated from matrix interferences in most of the foods tested, and all water sample analytes were well retained after removal of interferences. This new method largely eliminated matrix effects and achieved unambiguous identification, reproducible retention times, and sensitivity; and attains the targeted limits of detection.

	Despite its association with various health risks, glyphosate remains the most commonly used herbicide worldwide. Glyphosate-based pesticide formulations are used as a simple solution to improve yields by reducing weed growth around glyphosate resistant crops. Almost inevitably, some level of glyphosate residue ends up in the food chain, and it has been detected in the urine of a range of animal species, including humans (1). Polar pesticides are frequently poor ionizers, may suffer from low extraction from the sample matrix, and demonstrate poor chromatographic separation. These pesticides, therefore, have historically required complex single residue methods to make them amenable to analysis, involving time-consuming derivatization steps and considerable clean up procedures.

	The analysis of glyphosate using high performance liquid chromatography with fluorescence detection (HPLC–FLD) in water is well known, with or without derivatization. Usually, glyphosate undergoes derivatization by the reaction of the native glyphosate with fluorenylmethyloxycarbonyl chloride (FMOC-Cl) before separation on a reversed-phase column. If the separation is performed on a polar chromatographic column, post-column derivatization before detection is required. The tedious derivatization step complicates the analysis, and the reproducibility is poor. Thus, there is a growing need for a method that can detect not only glyphosate and its major metabolite aminomethylphosphonic acid (AMPA), but also glufosinate and similar highly polar compounds, in their underivatized states.

	Modern triple quadrupole mass spectrometers that allow fragmentation analysis offer very low detection limits and high detection selectivity. This approach enables laboratories to rapidly screen samples for a variety of regulated pesticides using the European Union Reference Laboratories' (EURL) quick, easy, cheap, efficient, rugged, and safe (QuEChERS) sample extraction and cleanup method. Owing to this, multi-residue liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses have become the minimum standard for the quantification of many pesticides in food and water samples. Nevertheless, glyphosate and other highly polar pesticides are not suitable for this type of analysis, and remain challenging for routine screening.

	The EURL quick polar pesticides (QuPPe) method allows the simultaneous analysis of a number of highly polar pesticides that are not amenable to common multiresidue methods. However, in practice, the separation is not robust, and requires intense system maintenance. The method involves extraction with acidified methanol and LC–MS/MS measurement, using isotope labeled internal standards for accurate quantification. Ion chromatography is the preferred separation technique for polar ionic analytes, such as anions, cations or small polar analytes (metabolites), and sugars. To use an ion chromatographic approach for the analysis of polar pesticides offers the ability to include multiple analytes in a single injection, without derivatization or the use of an ion suppressor. Food testing laboratories would benefit greatly, in terms of both time and cost, from a methodology that avoids these complex analysis procedures. Here, we present a robust and sensitive method for the direct analysis of polar pesticides in food and environmental samples without derivatization (2).

	The EURL QuPPe method for extraction of polar pesticides from food samples of plant and animal origin is well developed (3). In this experiment, the QuPPe extraction was adjusted by using a little less methanol and more formic acid. Because polar pesticides are water soluble, the extraction was aqueous with methanol and formic acid added to improve efficiency. The addition of internal standards to matrices is considered essential in order to compensate for matrix effects and the shifting retention times observed in many chromatographic separation procedures. Dispersive solid-phase extraction (SPE) cleanup using C18 was also carried out as described in the QuPPe-AO3 method. A push-through method was finalized using two sorbents and SPE filters, because the extracts obtained from the QuPPe method require extensive cleanup for more stable chromatography and less pollution of the source.

	The eluents' incompatibility with electrospray ionization sources requires the use of a suppressor, so as to lower the ion load for the electrospray ionization (ESI) source, and gain sensitivity. By employing a polyvinyl alcohol based column with quaternary ammonium groups, and using an ammonium bicarbonate buffer prior to detection by a highly sensitive quadrupole MS system, the need for a suppressor was removed.

	Environmental and drinking water samples vary widely in the degree of particle content, causing difficulties for LC injection and the reproducibility. SPE-type cleanup would add significant time, as well as financial cost, in a high throughput laboratory situation where minimal sample preparation is desirable. To overcome these challenges, and to be able to swiftly change between food and environmental samples, the same setup for food samples (except the 500 µL injection volume) was carried out. A simple filtration step, using Chromacol 17-SF-02 (RC) from 17-mm syringe filters, was performed when transferring the samples to the LC vials. Internal standards to a final concentration of 1 ppb were added to samples and standard solutions, and QC samples in tap water were prepared in a similar fashion. To minimize matrix interferences, standard additions to the samples were used.

	Separation was achieved using a Shimadzu Nexera ultrahigh-pressure liquid chromatography (UHPLC) system comprising LC-30AD pumps, a SIL-30AC autosampler fitted with a 500 µL loop, and a CTO-20A column oven. For chromatography, a polyvinyl alcohol column, with quaternary ammonium groups using a bicarbonate eluent between pH 8 and 10, was used.

	The finalized chromatographic method used 10 µL injections onto a 150 x 4 mm column, employing a 20 mm guard column of the same material, and a 0.5 µm filter, with a flow rate of 0.6 mL/min. Both columns were replaced every 250 samples to maintain performance, and to keep the MS source clean. Performance was shown to be robust and reproducible on a variety of food matrices used for method verification laid out in Table I, all subject to the cleanup method as described above.

	An injection volume of 500 µL was employed in a chromatographic method similar to that used for the food samples.

	Analyses were performed using a triple quadrupole linear ion trap mass spectrometer (Sciex QTRAP 6500+) in negative electrospray ionization mode (Table II, source parameters).

	At least two multiple reaction monitoring (MRM) transitions were optimized for each analyte, in order to quantify and confirm their concentration in all samples (Table III, analytes with MRM transitions employed).

	Data were acquired using Analyst 1.6.3 (Sciex) and processed for quantification and confirmation with reference to internal standards (IS), using MultiQuant 3.0.2 software (Sciex) (Table IV, summary for limits of detection).

	The stability and reproducibility of the method, in terms of retention time, peak width, peak area, and tailing factor, were found to be excellent, and over one thousand food samples from a variety of commodities were analyzed without system maintenance (Figure 1, reproducibility data for glyphosate internal standard [IS]).


	Figure 1: Reproducibility data for glyphosate internal standard (IS). The modified method for food samples tested over one hundred injections of extracts from the variety of food matrices presented in Table I. 

	The sensitivity of the method was demonstrated by considerably lower limit of detection (LOD) for every analyte and commodity combination than is required in most cases (Figure 3, example chromatography from surface water samples). Regulations are not expected to change significantly within the lifetime of the current generation of the most sensitive MS instruments. The linearity of the calibration curve was established in the concentration range of 15.6 to 1000 ng/L of glyphosate. By plotting the ratio of the peak area of the standard to that of the IS against the ratio of their concentrations at seven calibration levels, calibration curves were obtained (Figure 2, method sensitivity and linearity of glyphosate).


	Figure 2: Method sensitivity and linearity of glyphosate. Several measures of reproducibility based on the glyphosate internal standard (IS). Calibration standards were achieved using the modified method for water samples. Ion ratios were all within the specified ±20% tolerance.

	The method described is highly robust and sensitive, achieving the target limits of detection required to meet current and proposed regulations. The separation has also been found to minimize any matrix interferences in all but the most complex samples. Through incorporation of this high throughput method into the workflows of food testing laboratories, complex sample preparation can be eliminated, running costs can be kept low, and valuable time can be saved.


	Figure 3: Example chromatography from surface water samples using the modified water method. The graphs highlight that even at concentrations five times (5x) lower than the European Union (EU) water regulations requirement, the presence of glyphosate in real environmental samples was detected and confirmed by the ion ratio between primary and secondary multiple reaction monitoring (MRM) transitions.

	Chromatographic performance using these modified methods for food and water samples achieved good separation between the analytes and from matrix interferences, and excellent repeatability in terms of peak profile and retention time. This cleanup is performed on all samples, not only for food from animal origin. The ion source was clean even after more than one hundred injections.

	In order to maintain peak shape and retention times, various chromatographic methods were investigated. Ion chromatography gave the most promising results, but the resulting eluents were not compatible with electrospray ionization sources and required a suppressor, which is detrimental to peak width. The need for a suppressor was removed by the use of ammonium buffers, a deviation from traditional LC buffers that enables detection of polar pesticides by MS/MS. The setup in this study allowed switching between polar and normal pesticide (reverse phase) suites without changing inlet, giving sharper, more intense peaks, and shorter run times. In busy food testing labs that primarily work with reversed-phase LC, this setup saves valuable time, eliminating the need to change inlet systems between analyses.

	Food samples analyzed using the modified method for QuPPe extracts achieved unambiguous separation between the analytes as well as excellent repeatability in terms of peak profile and retention times. Analytes were also well separated from matrix interferences in most of the foods tested. The EU maximum residue limits (MRLs) for pesticides in food commodities vary widely, depending on the pesticide and commodity in question. Although some matrix interference was present in the most complex food samples, LODs in both food and water samples were well below the concentration limits laid out in EU regulation. Over one thousand food samples from a variety of commodities were analyzed without maintenance of the system, and the stability in terms of retention time, peak width, peak area, and tailing factor was found to be excellent.

	This method largely eliminated matrix interferences in food sample extracts, further enhancing the reproducibility and robustness of the method. However, in the most complex food matrices, MRM ion ratios were observed outside of the standard ±20% tolerance. In future work, the QTRAP will be used to confirm positive results by their full scan MS/MS spectra, and the sample cleanup procedure will be further developed in order to remove background interferences.

	It is commonly known that LC–MS/MS, especially when working in ESI mode, is susceptible to matrix effects, affecting quantitative accuracy. This new methodology overcomes the necessity of pesticide derivatization. This was achieved by combining an ion chromatography separation with the high sensitivity of the mass spectrometer for analyte detection. Analytes are successfully separated using an LC column in a method switching reversed-phase system, with MS-amenable mobile phases at around pH 9. All analytes are well retained with reproducible retention times, allowing detection of the majority of background components. The method meets the Directorate-General for Health and Food Safety (DG SANTE) requirements of reproducibility (<20%) and recovery (80 to 110%), and the limit of detection (LOD) of the method is below 0.01 mg/kg. Excellent long-term stability and robustness were achieved throughout the validation of this method for food samples extracted by the QuPPe procedure.

	This modified LC–MS/MS analysis offers the possibility to analyze multiple polar pesticides in a single injection without derivatization. The ion chromatography based approach uses ammonium buffers, a deviation from traditional LC buffers that enables detection of polar pesticides by MS/MS. A sufficiently sensitive mass spectrometer allows the analysis to be performed without the need for an ion suppressor using a standard reversed-phase LC based system. This eliminates the need to change inlets between typical pesticide analyses.

	By removing the need for derivatization, sample preparation is greatly simplified, and the time required to analyze polar pesticides is reduced. As an additional advantage, the column lifetime is well over one thousand matrix samples, allowing high-throughput polar pesticide analysis with less system maintenance. This method was found to be considerably more robust and sensitive than other approaches, and has achieved the target limit of detection required to meet existing and proposed future regulations of glyphosate and similar polar pesticides.

	(1) M. Krüger, P. Schledron, W; Schrödl, H.W. Hoppe, W. Lutz, and A.A. Shehata, 

210 (2014). doi: 10.4172/2161-0525.1000210.

	(2) W. Broer, U. Chiuminato, J. Stahl-Zeng, D. McMillan and P. Taylor, "A Robust and Sensitive Method for the Direct Analysis of Polar Pesticides in Food and Environmental Samples Without Derivatization" (Nofalab Laboratories, Schiedam, the Netherlands; Sciex, Darmstadt, Denmark; Sciex, Warrington, United Kingdom, 2018).

	(3) M. Anastassiades, D.I. Kolberg, A. Benkenstein, E. Eichhorn, S. Zechmann, D. Mack, C. Wildgrube, I. Sigalov, D. Dörk, and A. Barth, "Quick Method for the Analysis of numerous Highly Polar Pesticides in Foods of Plant Origin via LC–MS/MS involving Simultaneous Extraction with Methanol (QuPPe-Method)" Version 9.3 (EU Reference Laboratory for Pesticides Requiring Single Residue Methods [EURL-SRM], Fellbach, Germany, August 2017).

 are with Nofalab Laboratories in Schiedam, the Netherlands. 

 is with Sciex in Warrington, UK. Direct correspondence to: 

A new high-throughput LC–MS/MS method meets the challenge of eliminating matrix effects for monitoring, with high specificity,
polar organic pesticides such as glyphosate in food and water, while meeting targeted limits of detection.

A Robust and Sensitive Method for Detecting Glyphosate and Other Polar Pesticides in Food and Water: Multiple Analytes in a Single Injection without Derivatization

In order to ensure the continued use of plastic packaging in food, the packaging used must be safe for contact with food products, and not cause contact contamination. Of rising concern is the role of non-intentionally added substances (NIAS). Therefore, this study assesses whether two commonly used plastic packaging materials, polyethylene and low-density polyethylene plus nylon, are safe for use. Their potential migrants are analyzed using data-independent acquisition and liquid chromatography coupled with high-resolution accurate mass spectrometry (HRAMS). Using automatic detection capabilities and filtering procedures, MS and tandem mass spectrometry (MS/MS) data were processed to find chemical formulae, and structures and toxicity information for the most abundant analytes in a given sample. Through this method, 26 migrating compounds, mainly cyclic oligomers, were identified. Using the toxicity rules set out by Cramer, 19 of these 26 compounds were identified as having moderate or high toxicity.


	(PHOTO CREDIT: SYDA PRODUCTIONS/STOCK.ADOBE.COM)

	Food packaging is a major market segment in the global food industry, often compared in size to that of the pharmaceutical industry (1). Over one third of all food packaging comprises materials made from plastic (1,2), which over the past several decades has extended to different plastic materials, copolymers, and additional ingredients.

	Packaging itself is essential to our modern society. As our food supply chain has grown to become more global, plastic packaging has been instrumental in ensuring that the food reaching consumers is safe to eat. Packaging helps to preserve food by protecting it from light, humidity, oxygen, foreign compounds, mechanical influences, and microbial contamination, all of which help to improve shelf life (1). Two polymers used frequently in food packaging include polypropylene and polyethylene, with the latter being used in various forms including low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), and high-density polyethylene (HDPE) (3).

	However, while food packaging protects from external contaminant introduction, there is increasing concern about the potential of chemical contamination from the plastic packaging itself. In fact, food contact migration is reported as one of the largest sources of food contamination, and is approximately 100–1000 times higher than that caused by pesticide residues (3,4). This migration can occur from multiple sources, including from contact with the internal face of the packaging, from diffusion and partition processes within multi-layered plastics, or even from adhesives.

	The potential health implications that plastic contamination poses to the consumer have resulted in the control and supervision of plastic materials in multiple countries. For instance, the European Union has imposed Regulation EU 10/2011, which has established specific rules for what plastic materials can be applied safely to food packaging, and lists specific compounds that are authorized for use in plastic formulations and manufacturing (5). But, this regulation does not take into account unintentional substances, often referred to as 

 (NIAS), meaning they frequently do not appear in lists of permitted ingredients. NIAS may be introduced through a variety of processes: a result of interaction between ingredients, degradation of material components, or from impurities within the raw material (6). As a result, the migration of non-authorized substances must not exceed a level of 0.01 mg/kg of food or simulant.

	To meet these strict criteria, highly sensitive and advanced analytical instruments and techniques are needed to test samples for the presence of NIAS. For nonvolatile chemicals in particular, liquid chromatography coupled to high-resolution accurate mass spectrometry (LC–HRAMS) has a proven history identifying nontarget compounds (7). In addition, non-targeted screening approaches assist in identifying potentially unknown contaminants compared to other acquisition methods. This technology has already been successfully applied to proteomics and metabolomics (8), as well as clinical and forensic toxicology (9).

	The present study investigates the migration of compounds from two different multilayer plastic packaging materials commonly used in fruit puree and juices. Using an LC–QTOF instrument, coupled with data-independent acquisition, non-targeted peaks were identified and concentrations were compared against those dictated by EU regulation. Following the successful identification of molecular formulae and structures, the toxicity of these structures was assessed to determine the potential harm such NIAS pose to consumers.

	Two commercially available multilayer plastic materials, used for the packaging of purees and juices from fruits and vegetables, were purchased over the internet for testing. The two multilayered materials used were: a polyethylene based material, and an LDPE plus nylon material.

	The migration test was performed according to the procedures established by EU Regulation 10/2011. The standardized test conditions, that simulate the long-term storage of food at or below room temperature, involve subjecting the material to 10 days of contact time with a contact temperature of 40 °C. Migration tests were performed using 1 dm

 surface area. This surface area was chosen as the regulation has previously established that materials can now be manufactured in such a way that the material is not releasing more than 10 mg per 1 dm

	Two simulants were used to measure the impact of material contact: simulant B, which consists of a solution of 3% acetic acid in water (w/v), and simulant C, which is a solution of 20% ethanol in water (v/v). For each material, 10 × 10 cm of plastic material was added to a 50 mL PTFE tube with 35 mL of the relevant simulant, in which carbendazim-d3 and malathion-d10 were added to the simulants as internal standards for quality control purposes. Three replicates and one control were performed. Each tube was placed in an incubator for 10 d at 40 °C. Afterwards, one aliquot of each sample was directly injected and analyzed by LC-HRAMS along with a calibration curve of ε-caprolactam.

	The liquid chromatography separation was carried out using an Exion LC (Sciex). Mobile phase A was made up from 98% water and 2% methanol, and mobile phase B was made up from 98% methanol and 2% water. Both phases contained 5 mM of ammonium formate and 0.1% formic acid. Separation was carried out using a Zorbax Eclipse Plus C8 column (Agilent) with a length, diameter and particle size of 100 mm, 2.1 mm and 1.8 µm, respectively. The column was thermostatted at 35 °C. The mobile phase gradient started from 80% of mobile phase A, which was maintained for 2 min. From 2 to 15 min, the amount of mobile phase B increased linearly to 100%, which was then maintained for 2 min. The mobile phase was then changed to 80% A, maintained for 3 min for re-equilibration, leading to a total running time of 20 min. An X500R (SCIEX) mass spectrometer equipped with Turbo V Source with a Twin Sprayer probe was used for acquisition. The data-independent acquisition tool SWATH Acquisition was used to analyze the data. A mixture containing 10 compounds with masses in the range of 132.9049–2034.6255 g/mol was used for calibration. SCIEX OS 1.3 was used for qualitative and quantitative analysis. The source parameters for the mass spectrometer are reported in Tables I–III.

	Control samples were run in similar conditions, which are left to stand for 10 days at 40 °C in the PTFE tube. Control samples were then compared with the three samples and used to filter out chemical peaks where the difference in intensity of the peaks was deemed to be negligible.

	A data-independent non-targeted acquisition mode was used to analyze the analytes. This approach segments the full scanned mass range into smaller segments for simpler MS/MS analysis, reducing the complexity of the fragmentation spectra. This allows for a retrospective analysis of the data, while ensuring that overlapping analyte peaks are not missed. This analysis proved vital in this study, owing to the complex overlap of spectra.

	Using the strategy outlined in the experimental section, a total of 3149 spectral features were identified in the polyethylene-based material. Using the filtering strategy, the number of elucidating ions decreased from 3149 to 57 (which is characteristic of polyethylene packaging material), which then further decreased to only 21 ions after focusing on the most abundant compounds (those with a signal intensity above 1 × 10

). However, to date, there is no library of NIAS substances to screen against the identified compounds, and none of the relevant ions were present in the commercial MS/MS spectral library used (SCIEX). MS/MS libraries are constantly evolving but still only cover a limited number of analytes. For those compounds whose MS and MS/MS had the lowest associated error, probable structures were constructed using ChemSpider and then modelled in silico to compare fragmentation patterns against those observed. Owing to the numerous possible structures, chemical intuition and prior knowledge of the substance was essential to rule out many database structures.


	Figure 1: Spectra of the two materials obtained using simulant C. The most abundant peaks are identified using compound ID numbers, the identities of which are reported in Table IV.

	From the two packaging materials, a total of 26 migrating compounds were identified from the screening method. Of these, 21 were assigned a plausible structure, which was later confirmed by fragmentation and bibliography agreement. A further 3 migrants were assigned a plausible structure based on fragmentation data, but these could not be confirmed. The final two structures could only be assigned molecular formulae, with not enough information collected to accurately assign structures. Spectra for the two materials using simulant C are reported in Figure 1. The summarized identity of those compounds identified is summarized in Table IV.

	Most migrants were identified in both materials. The only compound listed in Regulation EU 10/2011 was caprolactam, a monomer of nylon (compound 2). It was found that caprolactam was present at 42 mg/kg in the polyethylene material and 9.33 mg/kg in the LDPE + nylon material. As European legislation permits concentrations of only 15 mg/kg, the migrant compound caprolactam is present at more than two-fold higher concentrations in the polyethylene-based material (5). In addition, four cyclic oligomers of caprolactam were detected in both materials corresponding to the dimer, trimer, tetramer, and pentamer (compounds 4, 3, 5, and 6 respectively). This phenomenon is not surprising as oligomers up to the octamer have previously been identified in other migration studies (3, 9, 10).

	Two other substances were identified: the plasticizer bis (2-methoxyethyl) adipate (compound 9) and diethyl 5-({[(2,4,5-trimethoxybenzoyl)oxy]acetyl}amino) isophthalate (compound 15). These were the results of the best matches based on identified chemical structures and fragmentation patterns.

	All other compounds identified were classified as NIAS, with the majority being oligomers. The main migrants were oligomers from polyurethane adhesives. Cyclic ester oligomers were found to be made up of the monomers adipic acid (AA), phthalic acid (PA), diethylene glycol (DEG), monoethylene glycol (MEG) and neopentilglycol (NPG) in the combination 1:1 (AA-DEG, PA-DEG), 1:2 (PA-DEG-DEG), 2:2 (AA-MEG-AA-MEG, AA-DEG-AA-DEG, PA-DEG-PA-DEG), 1:1:2 (AA-MEG-AA-DEG, AA-DEG-PA-DEG) or 1:1:1:1 (PA-MEG-AA-DEG, PA-DEG-AA-NPG). However, once again most of the oligomers were not present in any library database consulted, and therefore identification was made based on a bibliographic search of their molecular masses. All oligomers were identified in the literature except for 3,6,9,12,15-Pentaoxabicyclo(15.3.1)henicosa-1(21),17,19-triene-2,16-dione, an ester whose appearance was attributed to the conceivable combination of monomers present in the materials.

	Only four substances were present only in the LDPE and nylon material. These are labelled as peaks 20–24 in Figure 1. Compounds 21, 22 and 24 had not been fully described in the literature, and there is currently no thorough hypothesis or explanation for the formation of these compounds in the present study. However, the peaks displayed similar characteristics to other oligomers already identified in the simulants. Peak 20 was identified as 1/6-dioxacyclodecane-7,12-dione and is attributed in the literature to the presence of polyurethane adhesives used to laminate packaging multilayer materials (11). Compound 23 was identified as 1,6,13,18-tetraoxacyclotetracosane-2,5,14,17-tetrone, also found in compostable adhesives (12).

	The two peaks that were not identified are those labelled as 17 and 19 in Figure 1. It was only possible to assign the most probable chemical formula, identified as C

. Due to the number of potential structures in the databases (22 and 83 possibilities respectively), it was not possible to identify a specific structure.

	The chromatographs for the migration of compounds for simulants B and C are overall very similar. The overlapping chromatographs are shown in Figure 2. Only two peaks were identified as exclusively present in simulant B, labelled as compounds 25 and 26. Based on individual masses, these molecules were identified as the cyclic oligomers AA-DEG and AA-DEG-PA-DEG with a molecule of water.


	Figure 2: Overlapping spectra of the two materials using simulants B and C. Compounds 20–24 are only observed in simulant B and are identified in Table IV. 

	The toxicity of individual NIAS was then assessed to determine whether those compounds identified in the analysis are hazardous to public health. A bibliographic search for their toxicity found that the majority have not been registered in EU regulation. Thus, a theoretical assessment was performed using the Threshold of Toxicological Concern (TTC) approach (13–15). TTC is based on Cramer rules, which assigns the toxicity of compounds based on the molecular structure into the following categories: low (class I), moderate (class II), and high (class III). Cramer has also devised a maximum recommended intake value for each compound class of 1.8, 0.54, and 0.09 mg/person/day, respectively. Using these rules, only 7 of the identified compounds were identified as low toxicity, with the results for all compounds summarized in Table IV.

	Due to the potential toxicity these compounds pose, their presence in plastic materials intended for food could generate a risk for consumers. Given the peak intensity, and the high presence of caprolactam, the polyethylene-based material could pose a higher risk to consumers compared to the LDPE material.

	This study sought to identify the potential migrating substances from food packaging contact. Two packaging materials were tested, polyethylene and low-density polyethylene plus nylon. A total of 26 substances were identified in this study, combining those identified in the LDPE and polyethylene materials: 21 have been assigned a confirmed structure, three have a tentative assigned structure and the final two only have assigned molecular formulae. The majority of the compounds are considered by Cramer's rules to be of moderate or high toxicity meaning their presence should be restricted to low concentrations. Only one compound identified is listed in the Regulation (EU) 10/2011: caprolactam, which was found to be above suggested concentrations in the sample of polyethylene tested. This work has shown the relevance and importance of evaluating NIAS in food contact materials to ensure consumer and food safety.

https://doi.org/10.1016/j.scitotenv.2009.05.006

	(2) B. Athenstädt, M. Fünfrocken, and T.C. Schmidt, 

	(3) M. Hoppe, P. de Voogt, and R. Franz, 

https://doi.org/10.1016/j.tifs.2016.01.018

https://doi.org/10.1016/j.foodcont.2014.05.044

	(5) Commission Regulation (EU) No 10/2011 on plastic materials and articles intended to come into contact with food, European Commission, The Official Journal of the European Union, 2011, 15.1.2011. 

https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32011R0010&from=EN

	(6) C. Bignardi, A. Cavazza, C. Laganà, P. Salvadeo, and C. Corradini, 

https://doi.org/10.1016/j.foodcont.2016.07.013

	(7) A.E. Lewandowska K. Macur, P. Czaplewska, J. Liss, K. Lukaszuk, and S. Oldziej, 

https://doi.org/10.1021/acs.jproteome.7b00366

	(8) M.P. Elmiger, M. Poetzsch, A.E. Steuer, and T. Kraemer, 

https://doi.org/10.1007/s00216-017-0594-1

	(9) D. Jenke, M. Poss, S. Sadain, J. Story, W. Smith, and D. Reiber, 

	(10) S. Úbeda, M. Aznar, P. Vera, C. Nerín, L. Henríquez, L. Taborda, and C. Restrepo, 

https://doi.org/10.1080/19440049.2017.1346390

	(11) J.S. Félix, F. Isella, O. Bosetti, and C. Nerín, 

https://doi.org/10.1007/s00216-012-5965-z

	(12) E. Canellas, P. Vera , and C. Nerín, 

https://doi.org/10.1007/s00216-015-8848-2

	(13) G.M. Cramer, R.A. Ford, and R.L. Hall, 

https://doi.org/10.1016/S0015-6264(76)80522-6

	(14) I.C. Munro, A.G. Renwick, and B. Danielewska-Nikiel, 

https://doi.org/10.1016/j.toxlet.2008.05.006

	(15) C. Nerin, P. Alfaro, M. Aznar, and C. Domeño, 

https://doi.org/10.1016/j.aca.2013.02.028

is the Senior Manager of Applied Markets Development at Sciex, in Warrington, UK. 

is the Applications Manager and Global Technical Leader for Food & Environmental at Sciex, in Darmstadt, Germany. 

 is a Professor of Analytical Chemistry at the University of Almeria, Spain. 

 is a postdoctoral researcher at the University of Almeria, Spain. 

 is a postdoctoral researcher at the University of Almeria, Spain. Direct correspondence to: 

This study assesses whether two commonly used plastic packaging materials are safe for use. Their potential migrants are analyzed using data-independent acquisition and liquid chromatography coupled with high-resolution, accurate-mass mass spectrometry.

Jianru Stahl-Zeng

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