A Review of Methodology for the Analysis of Pyrethrin and Pyrethroid Residues in Food of Animal Origin

Food Additives & Contaminants: Part A

ISSN: 1944-0049 (Print) 1944-0057 (Online) Journal homepage: http://www.tandfonline.com/loi/tfac20

A review of methodology for the analysis of pyrethrin and pyrethroid residues in food of animal origin

S. Tuck, A. Furey, S.R.H. Crooks & M. Danaher

To cite this article: S. Tuck, A. Furey, S.R.H. Crooks & M. Danaher (2018): A review of methodology for the analysis of pyrethrin and pyrethroid residues in food of animal origin, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2017.1420919 To link to this article: https://doi.org/10.1080/19440049.2017.1420919

Accepted author version posted online: 16 Jan 2018.

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Journal: Food Additives & Contaminants: Part A

DOI: 10.1080/19440049.2017.1420919 A review of methodology for the analysis of pyrethrin and pyrethroid residues in food of animal origin

S. Tucka,b, A. Fureyb, S.R.H. Crooksc, M. Danahera*

aFood Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland bMass Spectrometry Research Group, Department of Physical Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland. cChemical Surveillance Branch, Veterinary Sciences Division, Agri-Food and Biosciences Institute, Belfast, UK.

Keywords: Pyrethrin; Pyrethroid; Insecticides; LC-MS/MS; GC-MS/MS; fat; meat; milk; honey.

Corresponding author: [email protected] Tele: +353 1 8059500 Fax: +353 1 8059550

Abstract Pyrethrin and pyrethroid pesticides are commonly used in crop protection and animal health, to control pests. As a result, they can potentially transfer into food if good agricultural practice is not followed or even due to accidental contamination. The analysis of these compounds has been widely reported in crops and the environment. However, the analysis of pyrethrin and pyrethroids has not been reported frequently in foods of animal origin, particularly animal tissues. The focus of this review is to report on pyrethrin and pyrethroid analysis including key aspects such as chemistry, choice of target matrix, sample preparation, chemicalAccepted analysis, legislation and method Manuscriptvalidation. This review shows that most methodologies for the analysis of these compounds are based on gas chromatography with the trend in recent years to move towards GC-MS or GC-MS/MS based platforms. This review shows that these compounds can also be satisfactorily analysed by LC-MS/MS, which can be advantageous because of shorter chromatographic run times. A wide range of sample preparation procedures have been applied in analytical methods and more complex protocols

are required for GC applications, whereas more crudely prepared extracts can be analysed by LC-MS/MS. This review demonstrates that pyrethrin and pyrethroid residues should be included as analytes in multi-class analytical methods for pesticides and veterinary drug residues in animal derived foods.

Introduction

Pyrethroids are synthetic pesticides, developed from the naturally occurring pyrethrum pesticide, produced from chrysanthemum flowers (Gullick et al., 2016). The use of more toxic pesticides, such as organochlorine, carbamate and organophosphorus insecticides, has been in decline over recent years due to growing concerns of the toxicity of these compounds towards humans (Feo et al., 2010). This has led the gradual replacement of such chemicals with pyrethroid pesticides, largely due to their low mammalian toxicity, lower environmental persistence and selective insecticidal activity (Gao et al., 2013). The availability and usage of pyrethroids has greatly increased in recent years for agricultural, residential and commercial pest control applications (Corcellas et al., 2012). Nowadays, pyrethroids are used in the home to control pests such as flies and ants with various gardening applications also possible (Feo et al., 2010). Pyrethroids have also been employed in numerous human and animal applications in the treatment of lice and diseases transmitted through mosquitos which act as vectors for the likes of malaria or dengue (Corcellas et al., 2012). The control of insect population on crops and plants has also benefitted from the application of pyrethroid insecticides both during harvest and storage (Panseri et al., 2013).

Although this migration towards the use of less toxic pyrethroids has been generally beneficial, this change has also brought about certain issues. The toxicity of these compounds to non-target organisms is an important aspect of their use. Although pyrethroids can be converted to non-toxic metabolites in mammals, through hydrolysis, and so their toxicity is believed to be low, the issue of carcinogenic and endocrine disrupting effects is an on-going issue (Gao et al., 2013). In addition, the widespread application of pyrethroids has caused contaminationAccepted of environmental compartments Manuscript such as aquatic ecosystems (Albaseer et al., 2010). Moreover, the various applications of these chemicals can lead to the ongoing possibility of food chain contamination leading to bioaccumulation of these insecticides in food products of animal origin such as meat, fish, fact, milk and honey (Bagheri et al., 2016). Therefore, the determination of pyrethroid pesticide residues is at the forefront in terms of preventative measures in food safety and public health. In the EU, multi-annual surveillance

programmes are carried out to monitor and prevent unacceptable contamination of food in order to protect consumers.

A number of review papers have been published on the analysis of pyrethrins and pyrethroids but have been limited mainly to gas chromatography methodologies in crops (Chen and Wang, 1996, Corcellas et al., 2013, LeDoux, 2011). The aim of this paper is to critically review different aspects of pyrethrin and pyrethroid analysis in foods of animal origin, which will provide a comprehensive reference source for scientists in this field of analysis. A comprehensive review of sample preparation strategies and detection methods has been provided. A considerable focus has been placed on the analysis of pyrethrin and pyrethroid residues using GC-MS(/MS) and LC-MS/(MS) because these are now the techniques of choice for analysing these compounds in biological matrices. The advantages and disadvantages of different analytical approaches are discussed with a focus on method performance and analytical throughput in the laboratory.

Structure and Chemistry

Pyrethrum is an extract found in the flowers of Tanacetum cinerariaefolium, also commonly known as Chrysanthemum cinerariaefolium (Ensley, 2007). Pyrethrum extract has been used as an insecticide for more than 150 years and contains six pyrethrin compounds, namely, pyrethrin I and II, jasmolin I and II and finally cinerin I and II ( Palmquist et al., 2012). However, as pyrethrins are derived from plants their supply has always been highly variable (Ensley, 2007, Schleier III and Peterson, 2011). The pyrethrin structure is based on a chrysanthemic acid, a cyclopropane ring bonded to a carboxylic acid moiety (cyclopropanecarboxylic acid), linked to an aromatic alcohol (cyclopentenolone alcohol) through a ester linkage, as presented in Figure 1 (Schleier III and Peterson, 2011). Although the naturally occurring pyrethrins make efficient natural pesticides and possess low mammalian toxicity, they are labile in sunlight, moisture and air, therefore dramatically reducing their commercial applicability. Consequently, pyrethroids were developed to yield moreAccepted environmentally stable and commercially Manuscript viable pesticides (Knaak et al., 2012).. The synthetic pyrethroids began with the replacement of the cyclopentenolone groups with various alcohol moieties, resulting in the production of more commercially useful products. Subsequently, the replacement of the cyclopropane ring with a number of acid functional groups was carried out, to produce pyrethroids more beneficial for outdoor and agricultural use, followed by modifications to the ester linkages (Knaak et al., 2012).

Insert Figure 1

An important aspect of the chemical and toxicological properties of pyrethroid is their overall stereochemical configuration. The cis- and trans- designation refers to the orientation of the substituent in carbon-3 of the cyclopropane ring, in relation to the carboxylic acid group, as depicted in Figure 2 (Clayden, 2001). Those pyrethroid structures with trans configuration and a primary alcohol (e.g. trans-permethrin) are hydrolysed more readily by esterases than those with the cis configuration (e.g. deltamethrin (DELTA)). In addition, those pyrethroids in the trans configuration are known to demonstrate reduced mammalian toxicity than those in the cis-configuration Schleier III and Peterson, 2011). For the first generation pyrethroids, the acid portion was based on chrysanthemic acid, and a range of halogenated and non- halogenated substituents. The alcohol portion was predominantly a primary or secondary alcohol, bound to a variety of heterocyclic structures. Several of the pyrethroid structures have a cyano substituent bound to the alcohol, producing improved compound toxicity (Palmquist et al., 2012, Schleier III and Peterson, 2011). Those pyrethroids lacking this α- cyano substituent are termed Type I compounds (e.g. permethrin), whilst those with the α- cyano substituent are termed as Type II compounds (e.g. cypermethrin (CYPER). Future pyrethroids, such as fenvalerate (FENVAL), were developed without the cyclopropane ring (Palmquist et al., 2012).

Insert Figure 2

Allethrin (ALLETH) was the first pyrethrin to be synthesized and was marketed for the control of household pests in 1952 (Palmquist et al., 2012). It was based upon the pyrethrin I structure, however, the pentadienyl side chain was replaced with an isosteric moiety, to improve metabolic and photochemical stability (Casida, 1980). The stability of ALLETH made it superior to the natural pyrethrins in both kill and knock-down effects against mosquitoes. Other early analogues involved replacing the side chain of the alcohol moieties, found in pyrethrins and ALLETH, with an aromatic substitute (Soderlund et al., 2002). ALLETH’sAccepted successful discovery was followed Manuscript by the development of other successful pyrethroids including tetramethrin (TETRA), synthesised by substituting the cyclopentenolone ring, with a 5-benzy-3-furylmethyl moiety (Palmquist et al., 2012).

Resmethrin (RES) was the first synthetic pyrethroid insecticide that possessed insecticidal activity approaching the potency of pyrethrin I. In RES, the furan ring replaced the cyclopentenone ring of pyrethrin I, and the benzene ring replaced the diene side chain of

pyrethrin I (Schleier III and Peterson, 2011). The success of RES led to the synthesis of a form that was less toxic to mammals, bioresmethrin (BIORES). Unfortunately, no improvement in photostability, in relation to the earlier synthetic pyrethroids was achieved, because both the furan ring and isobutenyl side chain were sites vulnerable to degradation Knaak et al., 2012).

Permethrin (PERM), was the first pyrethroid with sufficient photostability for agricultural use (Soderlund et al., 2002). It was synthesised through structural changes in the alcohol moiety and halogenation of the acid side chain, blocking degradation upon exposure to light (Soderlund et al., 2002). PERM proved to be over 100 times more photostable than RES, whilst maintaining high insecticidal activity and low mammalian toxicity (Schleier III and Peterson, 2011). The introduction of an α-cyano group led to the discovery of CYPER. The inclusion of an α-cyano group in the alcohol moiety significantly reduced the rate of hydrolysis, provided greater potency than PERM. This along with replacing a bromine for a chlorine atom in the acid side chain led to the discovery of DELTA (Palmquist et al., 2012). Following these developments, esters of the substituted phenyl acetic acids were introduced, replacing the cyclopropane pyrethroids. This led to the first commercial pyrethroid that was photostable, fenvalerate, and later to the synthesis of fluvalinate (FLUV) and CYPER (Ensley, 2007). Substitution of a chlorine at the 4’-position of CYPER produced the most active compound, cyfluthrin (CYFLU). Bifenthrin (BIFEN) is an ester with alcoholic components whose activity was not enhanced by an alpha-cyano substituent (Schleier III and Peterson, 2011). An overview of the pyrethroid and pyrethrin molecular weights, formulas and structures can be seen in Table 1.

Insert Table 1

Mode of Action

Pyrethroids interfere with the growth processes and feeding mechanisms in insect pest, disrupting important processes such as lipid synthesis. At a molecular level, pyrethroids exert their Acceptedeffects by modifying the kinetics of voltage-gated Manuscript sodium channels, which are a vital component of excitable nerve cell function in both mammals and invertebrates (Bradberry et al., 2005). The duration of the opening and closing of sodium channels is voltage dependant, which varies with the pyrethroid structure, as seen in Figure 3 (Bradberry et al., 2005). Type I pyrethroids (e.g. PERM), hold the voltage-gated channels open for a shorter time than the type II pyrethroids (e.g. FENVAL) (Schleier III and Peterson, 2011). The prolonged opening

of these channels leads to altered nerve function by the continuous discharge of nerve signals (nerve depolarisation). The type I pyrethroids display symptoms such as the development of body tremors and muscle spasms. While type II pyrethroids symptoms include spasms, salvation, paralysis and eventual death (Palmquist et al., 2012.

Insert Figure 3

Toxicity

While the development of synthetic pyrethroids was a major step forward in the control of pest invertebrates, the residual activity and high toxicity of these compounds was not selective to insects. These compounds have also displayed extreme toxicity to non-target organisms such as aquatic organisms (Palmquist et al., 2012). The widespread application of pyrethroids to agricultural fields can also result in the unintentional exposure to beneficial invertebrates, such as honeybee pollinators, impacting on their ability to forage, reproduce, grow and develop (Palmquist et al., 2012). Pyrethroids are far more toxic in insects than mammals due to the increased sodium channel sensitivity, lower body temperature and smaller body size of insects (Bradberry et al., 2005). In addition to higher body temperature mammals also display increased metabolism rates and reduced dermal absorption (Bradberry et al., 2005). However, acute toxicity through overexposure to these compounds, through foraging, spraying or ingestion, can result in toxicity in mammals. Despite pyrethroids displaying low to moderate toxicity in humans and mammals, exposure can still lead to neurotoxic symptoms (e.g. tremors), by affecting the sodium channels within the human body (Albert and Pombo-Willar., 1997). Chronic exposure to these pesticides can lead to the development of chronic intoxication, characterised by symptoms such as headaches, dizziness and nausea. Long-term pyrethroid exposure can also result in damage to the spleen and lymph nodes, with an increased risk of cancer developing. Pyrethroids such as BIFEN and CYPER have also been found to display immune toxicity, as these compounds to affect the functionalityAccepted of the human immune system (Wu Manuscript et al., 2011a).

A number of pyrethroids have also been shown to alter hormones (endocrine disruptors) in mammals and fish by interfering with endocrine signalling by blocking, mimicking or cooperating with endogenous hormones in vertebrates and invertebrates. The compounds can possess a similar structure to the hormone and competitively, non-competitively or

cooperatively bind to the receptor, mimicking the hormones behaviours. Endocrine effects are those which create an imbalance in normal hormonal signalling. This can adversely affect reproduction, sexual development and interfere with the immune systems (Mnif et al., 2011).

The neurotoxicity of pyrethroids in mammals is dependent upon the stereochemical configuration at cyclopropane C-1. Only esters of IR cyclopropanecarboxylates and isosteric 2S isomers of non-cyclopropane acids are neurotoxic. In mammals, the absolute configuration at cyclopropane C-3 of cyclopropanecarboxylate esters of primary alcohols (e.g. PERM) also strongly influences toxicity (Soderlund et al., 2002). Pyrethroids with the 1R, trans configuration, lack toxicity in mammals, however, those in the 1R, cis configuration and are both insecticidal and toxic to mammals. The presence of an α-cyano substituent in the S configuration at the 3-phenoxybenzyl alcohol moiety also greatly enhances acute neurotoxicity. The IR-trans cyclopropanecarboxylates of 3-phenoxybenyl alcohol (e.g. [IR- trans]-permethrin), exhibit low toxicity in mammals; however, the addition of an α-cyano substituent in the S configuration to these esters produces significant neurotoxicity (Soderlund et al., 2002).

Applications in food producing animals

Food producing animals can be infested by a range of external parasites including flies, lice mites and ticks that can affect animal health leading to a loss in production yields (Soderlund et al., 2012). In addition, parasites can often be vectors for diseases such as redwater (Zintl et al., 2014) and blue tongue (Bouwknegt et al., 2010). As a result, pyrethroids are administered to animals, to reduce and/or eliminate these pests (Soderlund et al., 2012). A range of formulations can be employed, including dips, pour-on, back rubbers, ear tags and body sprays. The pyrethroids are absorbed by the parasites which control the population of chewing and biting flies, lice and ticks. As the animals tend to be housed in large numbers these methods allow for quick administration with minimal stress (Constable et al., 2016).

Legislation The useAccepted of pesticides for the control and treatment Manuscript of external parasites on food producing animals and crop protection is essential to allow sustainable food production for a growing planet. Unfortunately, undesirable levels of residues can sometimes transfer into food following administration of animal health products or through exposure from contaminated water, bedding soil or feed.

Insert Table 2

The human food chain must to be protected to ensure that animal produce does not contain prohibited substances, pesticide residues or contaminants, and that they are not present at a level which may adversely affect human health (Anadón et al., 2009). Generally, licensed veterinary medicinal products are safe to use in food producing animals once correct doses and withdrawal periods are adhered to (Anadón et al., 2009). Within the European Union (EU) maximum residue limits (MRLs) for pharmacologically active substances in foodstuffs of animal origin are established under EC Regulation No. 470/2009 and listed in EU Regulation 37/2010 (European Commission., 2010). MRLs established for pyrethroids/pyrethrins under EC Regulation No. 470/2009 are listed in Table 2a. In addition, MRLs for pyrethroid and pyrethrin plant protection products are established under Commission Regulation 396/2005 (European Commission., 2005). Article 18(1)(b) of Regulation 396/2005 also lays down the criteria for establishing a default MRL of 0.01 mg kg-1. Table 2b provides a listing of MRLs for pyrethrin and pyrethroid under pesticide legislation. MRLs for pyrethroids/pyrethrin have also been approved in a number of other countries outside of the EU including Canada (Health Canada., 2002, Health Canada., 2016), The United States (National Pesticide Information Center., 2016), China (Ministry of Agriculture People's Republic of China., 2014), Australia (Australian Pesticides and Veterinary Medicines Authority., 2012) and Japan (The Japan Food Chemical Research Foundation., 2016).

The control of pyrethroid and pyrethrin pesticides in food of animal origin is complicated by the complex marker residue definition, which can be defined as different isomers of the active substance. In the case of some pyrethroids and all pyrethrins, the marker residue is not well defined in legislation. In such situations, it is advisable to have analytical standards available for different isomeric forms of each active ingredient to ensure accurate quantitation. The differences between MRLs established under veterinary and pesticide legislationAccepted can further complicate decision maki Manuscriptng, e.g. different MRLs exist for cyfluthrin, with higher tolerance levels established under CR 396/2005. In the case of α- cypermethrin/cypermethrin there is a ten-fold difference in MRLs. In the event of residues being identified in food products above either sets of legislation, it is advisable that on-farm investigations are carried out to identify the cause of residues.

Sample Preparation Methods

Selection of samples matrix

The majority of the methods for analysing pyrethrin and pyrethroid residues in animal tissue employ fat as the target matrix (Akre and MacNeil, 2006, Castillo et al., 2011, Zrostlíková et al., 2002). This is generally due to the fact that pyrethroids and pyrethrins are lipophilic in nature and so will tend to accumulate in the fat, hair and muscle tissues of animals. Some groups have developed methods for the analysis of these insecticides in other matrices such as fish (Bordet et al., 2002, Rawn et al., 2010) and beef muscle (Stefanelli et al., 2009). In addition, a number of groups have developed methods for the analysis of pyrethrins and pyrethroids in milk to ensure that approved withdrawal periods have been adhered to in dairy animals (Di Muccio et al., 1997, Fernandez-Alvarez et al., 2008). Honey is another matrix that is widely monitored for insecticide residues as honey bees may require treatment with a pesticide to eliminate the varroa mite (de Pinho et al., 2010, Jin et al., 2006, Li et al., 2013).

Extraction procedures

The sample preparation protocol employed for the isolation of pyrethrin and pyrethroids from foods of animal origin is generally dictated by the test matrix (LeDoux, 2011). For example, fat is usually extracted from the adipose tissue using liquid-liquid extraction (LLE) (Cunha and Fernandes, 2011, Shamsipur et al., 2016), soxhlet extraction (Carabias-Martı́nez et al., 2000) or simply by melting the fat and collecting the fat drippings (Bordet et al., 2002). In contrast, animal tissue samples (kidney, liver or muscle) are typically extracted directly using a dispersion tool (or homogeniser) in the presence of an organic solvent (Niewiadowska, 2010, Pang et al., 2006). In the case of liquid milk, LLE is the most widely used extraction procedure (Goulart et al., 2008, LeDoux, 2011, Martins et al., 2013), however a number of other techniques such as solid-matrix dispersion (Di Muccio et al., 1997) and QuEChERS (quick, easy, cheap, effective, rugged and safe) (Jeong et al., 2012) have also been applied to facilitate the precipitation of protein matrix components. The analysis of honey samples can be complicated based on their source of origin, including the flora that they pollinate. In general,Accepted honey is extracted using LLE (Paradis Manuscript et al., 2014, Rissato et al., 2007). Some groups have employed alternative extraction procedures such as supercritical fluid extraction (SFE) (Rissato et al., 2004), pressurised liquid extraction (PLE) (Wu et al., 2011b), automated solvent extraction (Chiesa et al., 2017), and matrix solid phase dispersion (MSPD) (Wiest et al., 2011). More novel methods have been employed for pyrethroid determination in various matrices including ionic liquid-linked dual magnetic micro-extraction (IL-DMME)

(Li et al., 2013) and solid phase microextraction (SPME) (Beltran et al., 2003, Campillo et al., 2007).

Sample clean-up

Sample extracts normally require some degree of clean-up prior to analysis to improve selectivity and sensitivity. Although, nowadays the requirement for more complex sample clean-up has been reduced through the use of more sophisticated detection techniques such as gas chromatography tandem mass spectrometry (GC-MS/MS) and particularly liquid chromatography tandem mass spectrometry (LC-MS/MS) (Majors, 2013a). Nevertheless, mass spectrometry (MS) based detection methods are prone to matrix effects that need to be negated through sample purification techniques, matrix matched calibration curves and/or the use of stable isotopically labelled internal standards.

Pyrethroids and pyrethrins are highly lipophilic in nature and so tend to accumulate in the fat, hair and edible animal tissues. These are often considered complex matrices due to the large composition of lipids and waxes, and so developed methods often include significant sample clean-up. Gel permeation chromatography (GPC) is one such technique that is widely used to isolate pesticide residues (typically ≤400 Da) from higher molecular weight matrix components (≥600 Da). It is attractive because it is automated and is applicable to a broad range of pesticides. Di Muccio et al. applied GPC in the determination of 14 pyrethroid residues in fatty materials, collecting analytes in a short 20 mL elution window. The authors used overlapping load and wash cycles to improve sample throughput. An Extelut-3 cartridge was used for the extraction of the pyrethroid residues with acetonitrile (MeCN) with fat removal carried out through SEC. This method suffered from matrix interferences in the final extract and so quantitation of cyhalothrin (CYHALO), esfenvalerate, FLUV, PERM, and tralomethrin (TRALO) was severely hampered. The poor sample clean-up and resulting inferences enabled quantification by GC-ECD of only nine of the included 14 pyrethroids with recoveries ranging from 63% to 113% (Di Muccio et al., 1999). Wu et al. utilised GPC clean-upAccepted in the multi-residue GC-MS analysis Manuscriptof pesticides in pork, poultry, fish and beef. The method included OP’s, OCP’s, carbamate and pyrethroid compounds. GPC clean-up was carried out on a column packed with Bio-Beads S-X3 using cyclohexane/ethyl acetate (EtOAc) as the mobile phase. Analytes were collected in a 60 mL window between 8 and 22 min (Wu et al., 2011b). Pang et al. similarly applied GPC for the multi-residue pesticide analysis in rabbit, pork, mutton, poultry and beef muscle. The GPC extract containing the

residues was then collected in the 22-40 min time window (90 mL of solvent) prior to concentration and LC-MS/MS analysis. Recoveries ranged from 53% to 124% for the pyrethroid residues (Pang et al., 2006). As can be seen from the above methods, an additional clean-up step using adsorption chromatography is generally required to produce sufficiently clean extracts for gas chromatography analysis but may be omitted when doing LC-MS/MS. A major drawback of GPC is that is uses large volumes of organic solvents to purify sample extracts.

Matrix solid phase dispersion (MSPD) can be an advantageous technique to employ in residue analysis because it combines analyte extraction and purification in a single step while eliminating tedious liquid handling steps. MSPD combined with GPC and SPE was employed by Hildmann et al. for the determination of multiple pesticide residues including 16 pyrethroids in chicken eggs (Hildmann et al., 2015). MSPD ensured the efficient of dispersion of samples on a non-retentive dispersant, namely, sand:sodium sulphate which resulted in high recovery of non-polar pyrethroid residues. A mixture of cyclohexane/EtOAc was used as the elution solvent due to the requirement to include polar and non-polar analytes in the analysis. This extraction solvent was also advantageous because it extracted low amounts of protein and was compatible with GPC clean-up. Samples were purified by GPC clean-up on a Bio-Beads S-X3 column, which significantly reduced high molecular weight lipids co-extractives, Although, SPE was needed for the removal of any remaining low

molecular weight lipids. It was proposed that C18 removed free cholesterol, tocopherol, triglycerides, and cholesterol esters, while PSA removed free fatty acids. Although the method produced poor analyte recoveries for some polar residues, pyrethroid recoveries were satisfactory ranging from 73% to 116 %. MSPD has also been applied to isolate CYPER and DELTA pyrethroid residues from porcine tissues. In this application samples were dispersed with neutral alumina, which was packed into a solid phase extraction (SPE) column and eluted with n-hexane. A number of solvents and mixtures were investigated including EtOAc, acetone and dichloromethane (DCM) but resulted in elution of unwanted peak components. As aAccepted consequence, n-hexane, was selected allowingManuscript for pyrethroid recoveries of 84% to 109%, to be achieved (Cheng et al., 2009).

The development of SPE streamlined sample preparation of samples in bioanalytical application by reducing the need for time consuming LLE, liquid-liquid partioning (LLP) and column chromatography clean-up. Sun et al. developed a multi-residue screening method for OP’s, OCP’s and 18 pyrethroid residues in beef fat through SPE. The fat was blended with

Florisil and packed into i empty SPE cartridges between two polyethylene frits. The column

was then coupled to a C18 SPE cartridge and the residues eluted using MeCN. The authors found that Florisil with 8% deactivation gave the best overall results. At higher deactivation levels, the recoveries of some pesticides, namely, aldrin, heptachlor, lindane and trifluralin

were impaired. This combination of Florisil and C18 sorbent clean-up resulted in few interferences in the final extract and provided sufficient pyrethroid recoveries ranging from 88% to 137% (Sun, 2003).

A number of other groups have employed multiple sorbent clean-up in pesticide methods.

Extrelut NT3 combined with a Sep-Pack C18 cartridge and a Florisil mini-cartridge was applied to beef meat allowing the multi-residue analysis of six pyrethroids and OCP residues. The mean recoveries for the pyrethroid residues were 84% to 99% (Stefanelli et al., 2009).

Bordet et al. applied sequential C18 and Florisil clean-ups for the multi-residue determination of 33 OCP’s including six pyrethroids in milk, fish, eggs and bovine fat (Bordet et al. 2002). The method was based on the European reference standard method NF-EN 1528 Parts 1-4 (European Union., 1996), but was modified to incorporate the analysis of pyrethroid residues

in fat. The C18 SPE step eliminated lipophilic components, while the Florisil clean-up reduced polar interferences. The recovery of the pyrethroid residues, however, varied from 85 to 127% with λ-CYHALO and DELTA showing poor recoveries at 14% and 32%, respectively (Bordet et al., 2002). Rissato et al. (2004) compared the traditional LLE approach to SFE with Florisil clean-up for pesticide analysis in honey, with the inclusion of three pyrethroid residues (CYFLU, CYPER and FENV). The group found that SFE combined with SPE clean-up significantly reduced sample handling, organic solvent usage and solvent evaporation and allowed for recoveries of 92% to 95% for the pyrethroid residues (Rissato et al., 2004).

Rissato et al. (2007) subsequently developed a method for the determination of 48 pesticide residues in honey using SPE with recoveries ranging from 85% to 199%. The method used a simplified extraction procedure based on dissolution in water and extraction into EtOAc prior to clean-upAccepted on a Florisil column (Rissato et al.Manuscript, 2007). The United States Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) developed a LLE method, employing Kuderna-Danish evaporation, to allow for the determination of five pyrethroids (FLUCY, PERM, CYPER, FENV and DELTA) in bovine fat ((USDA., 1991). Although this method was successful for pyrethroid determination it was extremely time consuming, required large amounts of space, copious amounts of glassware and was found to require two

to three days for completion of analysis of a batch of six samples. Therefore, Akre et al. (2006) modified the LLE approach to allow for a more efficient and economical method which allowed for the determination of the original five pyrethroid residues in addition to CYFLU, λ-CYHALO and FLUV in bovine fat. Glass funnels were replaced with disposable tubes and solvent usage in LLE was greatly reduced. The Kuderna-Danish evaporation step was replaced with evaporation under nitrogen at reduced temperature, while Florisil SPE cartridges were substituted in place of the large Florisil glass column clean-up. Results from the two methods were comparable, but the improved method increased sample throughput to 24 samples per day. Recoveries ranged from 80% to 123% (Akre and MacNeil, 2006). Kodba and Vončina developed a streamlined sample preparation procedure for isolating 26 OCP’s, three pyrethroid pesticides and six polychlorinated biphenyls (PCBs) from fatty foods (Kodba and Vončina, 2007). Sample clean-up was performed on a mini-glass column prepared by packing Celite into a Pasteur pipette, which was connected to a Florisil column. This modified method scaled down solvent usage approach in sample clean-up using only 5 mL of dimethyl sulphoxide extraction solvent. The method reported recoveries of >80% for pyrethroid residues and was applicable to milk, eggs and tissues (Kodba and Vončina, 2007).

The application of sorbent clean-up can be beneficial in complex matrices such as honey. The removal of interferences such as lipids and waxes, naturally found in honey, can significantly reduce interferences in MS analysis and improve analyte quantitation. Jin et al. employed a Florisil column for the clean-up of honey extracts, following the extraction of 23 pesticides residues, including six pyrethroids (Jin et al., 2006). The broad of analyte polarities included in the method required careful selection of the extraction solvent, with EtOAc providing the best overall extraction efficiencies with pyrethroid recoveries ranging from 86% to 112%. Chiesa et al. applied PLE coupled with in-line clean-up for the determination of 53 pesticide and pyrethroid residues in honey. The PLE method used MeCN as the extraction solvent and with primary-secondary amine (PSA) as the trapping material. The PLE in-line method was later applied to the evaluation of pesticide concentrations in organic honey samples (Chiesa et al., 2017).Accepted Manuscript QuEChERS and other high throughput methods

The QuEChERS sample preparation approach was reported originally by Anastassiades et al., for the extraction of multiple pesticide residues from fruit and vegetables prior to gas- chromatography analysis (Anastassiades et al., 2003). This technique offers a user and

environmentally friendly alternative to traditional LLE and SPE. The process involves two simple steps. First, the homogenized samples are extracted and partitioned using an organic solvent and salt solution. Then, the supernatant is further extracted and cleaned using a dispersive solid phase extraction (d-SPE) technique. It has been successfully applied to isolate non-polar and polar residues from complex biological matrices in numerous food analysis applications (Armenta et al., 2015). A range of modified versions have since been developed with many implementing the buffered approach using acetate (Lehotay et al., 2005) or citrate (Kovalczuk et al., 2008) whilst other have incorporated dry ice partitioning (Lee et al., 2011) and acidification (Shen et al., 2011). The QuEChERS approach has been used in a number of reference methods including the Official AOAC 2007.01 method entitled “Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate” (AOAC., 2007) and the European Standard EN 15662 method (British Standards., 2008).

A number of different d-SPE sorbents are now available for sample clean-up with the most popular of these being PSA, C18, Secondary Amine Exchanger (SAX), and Florisil (Kinsella et al., 2009). Various d-SPE sorbents were investigated by Castillo et al. to determine those which minimized matrix co-extractives and interferences for the determination of non-polar pesticide residues in fat samples. The d-SPE clean-up produced extracts than were cleaner (lower levels of interferences such as fats) than those obtained using GPC clean-up (Castillo et al., 2011). Jia et al. developed a modified QuEChERS method to enhance the extraction efficiency of pyrethroid residues from fish matrices (Jia et al. 2012). This group found that improved recoveries in the range 81% - 108% could be obtained by replacing acetonitrile with isopropanol as the extraction solvent. The authors proposed that the use of isopropanol slowed down the denaturisation of protein in samples allowing for improved release of bound residues (Jia et al., 2012).

Weist et al. evaluated the suitability of SPE and QuEChERS approaches for isolating residues of different polarities from honey (Wiest et al., 2011). It was found that most polar and most non-polarAccepted analyte recoveries varied depend ingManuscript on the SPE sorbent phase chosen. The modified QuEChERS approach provided overall better recoveries (49% to 115%) for all analytes, including eight pyrethroid compounds. Malhat et al. analysed honey samples using a QuEChERS approach in the study of OCP’s and three pyrethroid pesticides. The samples were extracted in water and MeCN acidified with acetic acid (HOAc). The removal of excess

water and sample clean-up were performed simultaneously with d-SPE using magnesium

sulphate (MgSO4) and PSA (Malhat et al., 2015). A modified QuEChERS approach was also adopted to analyse for 200 pesticides in honeybee wax comb, covering 50 different chemical classes. The method included a number of pyrethroids and employed a similar sample preparation protocol to the previous method with recoveries of 62% to 105%. Extracts were purified by d-SPE and concentrated under nitrogen evaporation prior to GC-MS analysis (Shendy et al., 2016). Chung et al. used a single step extraction and clean-up QuEChERS approach to analyse for pyrethroids and pyrethrins in dairy produce, fruit, vegetables, eggs,

seafood, meat and poultry. For analytes with >2% fat MgSO4, PSA and C18 were applied,

MgSO4 and PSA were used in matrices with less than 2% fat content, and finally for matrices

with a fat content of <2% and intense colouration MgSO4, PSA and graphitised carbon black (GCB) were applied. The modified QuEChERS approach was successfully applied to food samples and achieved recoveries in the range of 83% to 104% for the majority of analytes (Chung and Lam, 2012).

Over the last decade, the application of low-temperature clean-up has allowed for the development of multi-residue methods in fatty foods (Zhan et al., 2013a, Xie et al., 2015). Low temperature clean-up exploits the differences in melting points between analytes and lipophilic matrix components. It is proposed that the majority of lipids precipitate from solvent at -20°C. Thus low temperature clean-up can potentially be applicable to a broad range of compounds and can significantly reduce sample preparation costs. Goulart et al. carried out a low-temperature clean-up meat for the determination of pyrethroids in milk. The samples were extracted in MeCN to aid in the precipitation of undesirable water soluble proteins and carbohydrates. The addition of organic solvents such as MeCN to fatty matrices reduces the solubility of these interferences whilst allowing the residues of interest to dissolve in the solvent. The removal of fat was then carried out through low-temperature clean-up with pyrethroid to allow recoveries of 84% to 93%. More than 90% of fat could be easily separated from the extract after rapid freezing of the lipid filtrations (-40°C) without significant loss of analytes in the study (Goulart et al., 2008). Anagnostopoulos et al. applied low-temperatureAccepted clean-up in the development ofManuscript an easy multi-residue method for fat-soluble pesticides in animal products. The samples were extracted in MeCN, before being

precipitation at -20 °C for 12 h, followed by d-SPE with PSA and MgSO4, to remove any remaining water or fat. This approach produced satisfactory recoveries, 70% to 104% in meat and 83% to 128% in milk, for the 10 fat-soluble pyrethroids included in the study (Anagnostopoulos et al., 2014). Some groups have applied low-temperature clean-up in

conjunction with d-SPE clean-up sample extracts. This was the approach chosen by de Pinho et al. in the determination of pesticide and pyrethroid residues in honey samples (de Pinho et al., 2010). The method was based around LLE of water diluted honey samples and extracted with MeCN:EtOAc extraction solvent prior to low-temperature precipitation of lipophilic components. It was proposed that an additional Florisil clean-up step removed additional matrix components, which were made up of mainly sugars. Analyte recoveries ranged between 93% and 101% for the selected pyrethroids (de Pinho et al., 2010).

Novel sample preparation methods

In recent years, the focus in analytical chemistry has been on improving the quality analytical results, and the introduction of new technological developments involving miniaturization, simplifications and automation of entire analytical procedures (Al-Saidi and Emara, 2014). One of the newer techniques emerging to fulfil these objectives is dispersive liquid-liquid microextraction (DLLME), first introduced for the preconcentration of organic and inorganic analytes from aqueous matrices (Rezaee et al., 2006). Dispersive liquid–liquid microextraction is a miniaturized form of LLE, in which microliter volumes of extraction solvent are used. Although this approach has the benefits of being less time- and solvent- consuming, in comparison to the more traditional techniques it is limited to the extraction of liquid samples (Al-Saidi and Emara, 2014). First, a disperser solvent which has high miscibility in both organic and aqueous phases is chosen. Following this, an appropriate mixture of the extraction solvent and the disperser solvent is rapidly injected into the aqueous solution of sample. A cloudy solution is then formed as the extraction solvent forms fine droplets which disperse in the sample solution. Following centrifugation the analytes are then extracted into a small volume of the sedimented phase, which can be sampled for analysis (Rezaee et al., 2010a, Rezaee et al., 2010b).

Shamsipur et al. used a combination of SPE and DLLME to isolate pyrethroid residues from milk Acceptedand honey samples (Shamsipur et al. , Manuscript2016). SPE extracts in methanol (disperser solvent) were injected along with chlorobenzene (extraction solvent), into sodium chloride (NaCl) solution. Following centrifugation, the analytes were sedimented in the chlorobenzene phase. Recovery of pyrethroids ranged from honey and milk ranged from 80% to 102% and 66% to102%, respectively.

SPME is a relatively recent, simple and solvent-free technique for the extraction of analytes with gaseous, liquid, and solid matrices applications (Majors, 2013b). The underlying principle of the SPME methodology is based on the partitioning of analytes between a coated SPME fiber and a sample. The reusable fiber is coated with a stationary phase, which can be a liquid polymer, a solid sorbent, or a combination of both. The sampling period, which can be of considerable time (≥ 30 minutes), exposes the fibre to the vapour phase above a solution, known as headspace-SPME (HS-SPME), or direct immersion in the solution (DI- SPME). Once equilibrium is reached, the extracted compounds on the fiber are desorbed and inserted into the inlet system that then desorbs the solutes into a gas (GC) or liquid (LC) mobile phase (Majors, 2013b). Fernandez-Alvarez et al., developed one of the first applications of SPME in the determination of pyrethroids in milk. In this approach, milk samples could be extracted without the need for pre-treatment, solvent or extensive sample clean-up, however, the sampling period was time-consuming. The developed method employed DI-SPME (using a polydimethyl-siloxane (PDMS)/divinylbenzene (DVB) fibre

coating) for the extraction of analytes from milk samples diluted with H2O (1:10 ratio). The approach was applied to milk samples of varying fat content with sufficient recoveries for the 12 pyrethroids achieved (Fernandez-Alvarez et al., 2008). One of the main drawbacks of SPME techniques is the limited number of commercially available stationary phases (fiber materials). The fibers themselves can be expensive with limited polarity ranges, thereby restricting the type of analytes which can be included in such methods (Merkle et al., 2015). Further, relative to other techniques, obtaining equilibrium conditions can be a slow process in SPME. In addition as the fibre ages, its behaviour may change, resulting in a lower degree of reproducibility. The stability of the fibers can also be an issue as they are somewhat fragile, therefore, method robustness can be an issue (Merkle et al., 2015).

Insert Table 3

Detection of Pesticides in Food Gas chromatographyAccepted methods Manuscript Gas chromatography (GC) is generally the method choice for analysing pyrethrin and pyrethroid residues because these substances have sufficient volatility to be analysed directly without the need for chemical derivatisation. Most published methods report on the use GC coupled to electron-capture detection (ECD) for pyrethroid analysis as it provides excellent sensitivity for molecules containing halogen or nitrile groups (Akre and MacNeil, 2006,

Fernandez-Alvarez et al., 2008, Gelsomino et al., 1997, Goulart et al., 2008, Kodba and Vončina, 2007, Lentza-Rizos et al., 2001, Niewiadowska, 2010, Park et al., 2006, Rissato et al., 2004, Zeng et al., 2008, Zrostlíková et al., 2002). However, pyrethrin and some pyrethroids, namely, phenothrin (PHENO), resmethrin (RES) and tetramethrin (TETRA)) lack halogen and nitrile functional groups, resulting in poorer sensitivity by gas chromatography-electron capture detection GC-ECD (Chen and Wang, 1996). GC-ECD- flame photometric detection (GC-ECD-FPD) has been applied to the screening of pyrethrin, pyrethroid, organochlorine (OC) and organophosphate (OP) pesticides in beef fat. GC-ECD was employed for pyrethrins, pyrethroids and OCP’s, with GC-FPD applied to OPP’s. The detection capabilities of this method were hampered by the presence of matrix interference peaks (Sun, 2003). Pulsed flame photometric gas chromatography (GC-PFPD) with microelectron capture detection (µ-ECD) has also been applied to animal fat matrix for pesticide determination (Zrostlíková et al., 2002). PFPD was performed for OPP determination and µ- ECD in the detection of pesticides containing a halogen atom and nitrogen containing OCP’s.

The selective nature of GC-µECD has since enabled its application to high fat milk samples without pre-treatment (Fernandez-Alvarez et al., 2008). Nowadays, most pesticide laboratories have replaced ECD with mass spectrometric detectors. GC coupled to single quadrupole MS has become the technique for cost effective analysis of volatile and semi- volatile pesticide residues in food (Banerjee et al., 2013, Lesueur et al., 2008, Nguyen et al., 2008). Electron ionisation (EI) is the most widely used GC-MS ionisation technique because it can be applied to different classes of pesticides. Yoshida et al. reported that 18 pyrethrin/pyrethroid residues could be separated analysed in a 24 min run-time by GC-EI- MS, as presented in Figure 4 (Yoshida, 2009). It was found that more than one peak was observed for most pyrethroids in the total ion chromatogram, which is due to the separation of diastereoisomers.

Insert Table 4 CorcellasAccepted et al. provide a good overview on pyrethrinManuscript and pyrethroid isomers, along with the impact on GC separation (Corcellas et al., 2013). In general, most pyrethroids have between two and four enantiometric pairs, which gives rise to between two and four peaks on an achiral GC column. The separation of individual enantiometric pairs is generally carried out using chiral liquid chromatography (LC) because this type of separation is difficult by GC, where isomerisation can occur during the injector. In most cases, non-polar GC columns are

employed for the separation of pyrethrins and pyrethroids with DB5 (5% phenyl 95% methylpolysiloxane) being the most widely used column (Corcellas et al., 2013). In general, most reported separations employ 30 m or 15 m columns but shorter columns can be utilised to reduce separation cycle time. However, the separation of pyrethroid isomers must be prioritised especially when the maximum residue limits may apply to certain specific isomers e.g. cis-DELTA, λ-CYHALO, RS/SR FENVAL, RR/SS FENVAL, and τ-fluvalinate (FLUV) in products of plant origin in the EU (Portolés et al., 2012a). It has been shown that negative chemical ionisation (NCI) can be more sensitive for halogenated molecules, which makes NCI attractive for some pyrethroids (Gullick et al., 2016). Feo et al. reported that pyrethroids give low-mass ions with the same m/z ratios under EI conditions. In contrast, under softer NCI conditions fragmentation of pyrethroids is limited, resulting in the production of negative molecular ions. It was reported that GC-NCI-MS was at least one order of magnitude more sensitive than GC-ECD, largely due to reduced background matrix interference. Methane is the most commonly used moderating gas but ammonia also produces satisfactory results (Feo et al., 2010).

Insert Figure 4

Mass spectrometry (MS) coupled to GC is now widely used for the analysis of pyrethrins and pyrethroids because it is most suitable technique for identifying low levels of residues complex food samples, as can be seen from the methods outlined in Table 3. Although MS analysis can be complicated, knowledge of the isotopic abundance of analytes is important to allow the selection of appropriate precursor ions in tandem mass spectrometry (Table 4). Nowadays, GC coupled to triple quadrupole mass analysers (GC-MS/MS) is starting to be more widely employed in pesticide analysis laboratories. Garrido Frenich et al. carried out pesticide determination in complex matrices (high-fat matrix, egg, and the high water content matrix, cucumber) through a comparison of GC-MS/MS (QqQ) and GC-IT-MS (Garrido FrenichAccepted et al., 2008). In the end, each system displayedManuscript similar overall results in both types of matrices. However, GC-MS/MS was the more sensitive detection technique for high fat matrices employing a simpler sample preparation.

Anaceloto reported on the comparison of EI and NCI modes using a GC-MS/MS system for the analysis of pyrethroids (Anacleto, 2015). The results of this evaluation showed that calibration of the method could be altered from 0.1 – 50 μg/kg-1 to 0.001 - 5 μg/kg-1 for

19 several pyrethroids, when using GC-NCI-MS/MS. It was found that there was interference in the GC-EI-MS/MS analysis of the ocean sediment samples for ALLETH/pallethrin, CYPER isomer and DELTA at the transitions studied. However, when GC-NCI-MS/MS was employed, samples extracts gave comparable results to pure standards (Anacleto, 2015). In terms of pyrethroid analysis in food of animal origin, GC coupled to negative chemical ionisation (NCI) tends to be preferred to electron ionization (EI), due to softer ionisation conditions (Feo et al., 2010). Also, the presence of halogen atoms in many pyrethroid molecules gives higher sensitivity when working in NCI mode (Corcellas et al., 2012). As stated, under EI conditions, pyrethroid residues produce low-mass fragment ions, which can have similar m/z values. This can result in a loss of selectivity and, therefore, the need for good chromatographic separation is often required (Corcellas et al., 2012). The mass spectra of pyrethroid in NCI mode are often characterized by intense peaks formed through the loss of the ester substituents, resulting in the production of carboxylate ions. Feo et al., carried out a study which compared the performance of GC-MS and GC-MS/MS under both EI and NCI conditions for the simultaneous determination of twelve pyrethroid insecticides in food samples (Feo et al., 2011). This study found that NCI-MS/MS was more sensitive for the majority of compounds with the exception of TRALO, which was slightly more sensitive using EI-MS/MS. Using GC-EI-MS/MS the on-column LODs for CYFL, CYHAL, DELTA, FENVAl, TETRA, were 307, 187, 307, 1227 and 119 fg, respectively. While GC-NCI- MS/MS on-column LODs for the same the respective compounds were 1.99, 0.18, 8.1, 1.46 and 15.5 fg (Feo et al., 2011). In addition, it was found that matrix interferences were significantly reduced in water, sediment and milk samples using GC-NCI-MS/MS.

Paradis et al. reported a GC-EI-MS/MS method for the determination of 22 insecticides from three different groups, including pyrethroids, in honey (Paradis et al., 2014). Samples were prepared using a QuEChERS sample procedure and analysed in a 32 min GC-MS/MS separation on a non-polar DB 5 MS column; 30 m×0.25 mm. i.d.; 0.25 μm film thickness, preceded by a 1m pre-column. The limit of quantitation of the method ranged between 0.2 and 0.5Accepted µg kg-1 for the analytes (Paradis et al. , Manuscript2014). García et al. only recently reported the analysis of 160 pesticides (including 14 pyrethroids) in honey wax using GC-EI-MS/MS in single reaction monitoring (SRM) acquisition mode. Analysis was carried out on a shorter column (HP-5MS UI 15 m × 0.25 mm x 0.25 µm) and back-flushing was used to reduce the overall injection cycle time to 23 min (García et al., 2016).

Atmospheric pressure chemical ionisation (APCI) is an alternative ionisation technique that has been applied in LC-MS/MS to ionise compounds that are unsuitable for electrospray ionisation (ESI). It has been more recently been applied to enable triple quadrupole instruments that were developed for LC-MS/MS to be additionally interfaced with GC. APCI is a soft ionisation techniques similar to NCI but the ionisation process is different resulting in quasi-molecular ions, [M]+ or [M+H]+ [M]+ and [M+H]+, which are produced through charge transfer and proton transfer mechanisms, respectively (Portolés et al., 2012a, Portolés + et al., 2012b). The formation of the [M+H] is influenced by the presence of H2O, which can

be more favourably promoted by insertion of a vial containing H2O (so-called modifier) in the ionisation chamber, although it has been demonstrated that both [M]+ and [M+H]+ ions can be formed either with or without modifier (Portolés et al., 2012a, Portolés et al., 2012b). Portoles et al. made a comprehensive assessment of the performance of APCI and EI for the analysis of selected pyrethroids showing that GC-APCI-MS/MS was a more selective and sensitive technique largely due to the larger precursor ions produced (Portolés et al., 2012b). In addition, it was highlighted that many pyrethroids produce an unspecific GC-EI-MS/MS transition, 181 > 152, which is satisfactory to use when selective chromatographic separation is in place. However, this transition can present problems when analysing FENVAL and FLUV, which have overlapping isomers. It is illustrated in Figure 5 that this problem can be overcome through the application of GC-APCI-MS/MS, which generally uses more selective quasi-molecular precursor ions (Portolés et al., 2012b).

A number of researchers have reported the practical benefits of GC-APCI-MS/MS in pesticide analysis. Cherta et al. developed a multi-residue method for measuring 142 pesticides in fruit and vegetables using GC-APCI-MS/MS showing that a wider range of pesticide classes could be analysed compared to GC-NCI-MS/MS (Cherta et al., 2013). It was reported that enhancement was observed for many analytes by GC-APCI-MS/MS, whereas slight ion suppression was observed for the pyrethroid pesticides. Three different transitions were employed for each analyte and unlike previously, all analytes were examined in a singleAccepted run. The LOQ of this method for fruitManuscript and vegetable matrices was typically 10 µg kg-1 but LODs were 1 µg kg-1 or lower (Cherta et al., 2013).

Insert Figure 5

The application of GC coupled to high resolution mass spectrometry (HRMS) particularly time of flight (ToF) and quadrupole time of flight (QToF) is emerging as a powerful tool in

pesticide residue analysis. Using HRMS it is possible to carry out targeted analysis for known pesticides, while having the additional capability to search for unknown metabolites or degradants. Wiest et al. reported the application of GC-EI-ToF for analysing pesticide residues in honey samples reporting LOQs in the range of 10.7 µg/kg-1 (PERM) and 37.6 µg/kg-1 (CYPER) (Wiest et al., 2011). It was concluded that the selection of a mass extraction window of ± 75 ppm was important to reduce background noise, while maintaining satisfactory analyte signal. Portolés et al. reported the development and validation of a GC- APCI-QToF method for screening 132 pesticides in fruit and vegetables (Portolés et al., 2014). It was demonstrated that GC-APCI-QT-F MS in full scan mode was fit for purpose in the screening and identification of pesticides in fruit and vegetables, according to SANCO/12571/2013 (Portolés et al., 2014).

Liquid chromatography methods

As already mentioned in previous sections, pyrethrin and pyrethroid residues are most widely analysed in food samples by GC methodologies. This is because they can be analysed directly by GC without the need for derivatisation and probably display better sensitivity in this mode of chromatographic separation. Consequently, there are few LC-MS/MS methods reported in peer reviewed literature for the analysis pyrethroids. However, LC-MS/MS has the potential to negate the need for complex sample preparation procedures that are required to prolong the lifetime of GC columns and reduce GC-MS source maintenance. Additionally, LC-MS(/MS) is probably the most suitable technique for the analysis of certain pyrethroids e.g. TRALO and the lowly volatile flumethrin (FLUM). Valverde et al. reported one of the earliest LC-MS papers on pyrethroid analysis that compared the GC-ECD-MS and LC-API-MS analysis of TRALO and DELTA (Valverde et al., 2001). It was shown that the peaks for DELTA were also present in the GC-MS chromatographic trace of TRALO. This meant that two isomers of TRALO were transformed into DELTA in the injector port by the elimination of bromine. This was highly plausible because previous work reported that TRALO can be partially transformed in DELTA through the elimination of a bromine molecule. As a result, this group investigatedAccepted the analysis of these two pesticides Manuscript showing that it was not possible to distinguish between DELTA and TRALO by GC-MS. In contrast, both DELTA and TRALO could be selectively analysed by LC-MS/MS (Valverde et al., 2001).

Despite early research that showing that LC-MS was advantageous for some analytes, few groups have made the switch from GC. Soler et al. published work on the determination of

nine pesticides in fruit showing [M+H]+ and [M+Na]+ ions formed during electrospray ionisation (Soler et al., 2004). Product ions were identified for analytes with the exception of acrinathrin (ACRIN) and CYHALO, which formed sodium precursor ions that could not be fragmented in collision induced detection (CID) experiments (Soler et al., 2004). The LOQ of the method for orange samples was 0.06 and 0.4 mg kg-1 for ACRIN and CYHALO, respectively. Martínez et al. (2006) reported one of the first LC-MS (single quadrupole instrument) methods for the analysis of pyrethroid residues in vegetables (Martínez et al., 2006). The analytes were detected in single ion monitoring (SIM) mode using the most + + abundant ions which corresponded to [M+H] (fenproprathrin (FENPROP)) or [M+NH4] (CYHALO, DELTA and FENVAL). Other pyrethroids, namely, PERM, FLUV and BIFEN, + + were monitored using their fragment ions [CH2-C6H4-O-C6H5] , [CNCH-C6H4-O-C6H5] and

[CH2-C6H3CH3-C6H5], respectively. Optimal sensitivity was obtained using a binary gradient comprising of (A) 50 mM ammonium formate adjusted to pH 3.5 with formic acid and (B) MeCN (Martínez et al., 2006). The LOD of the method ranged between 3 and 5 ng mL-1 (20 µL injection volume) of solvent standards. The method was validated in different vegetables matrices at 10 and 30 µg kg-1 levels showing satisfactory results.

Insert Figure 6

LC-IT-MS has been successfully applied to analyse 10 pyrethrin and pyrethroid residues in vegetable matrices (Chen and Chen, 2007). The analytes were analysed using a binary gradient solvent system comprising of water and methanol (MeOH) without mobile phase additives. Both electrospray (ESI) and APCI ionisation probes were evaluated showing that ESI was the most suitable. Sodium adducts, [M+Na]+ were found to be the most abundant ions and were used for quantitation purposes. The authors proposed that the analytes favoured the formation of [M+Na]+ adduct ions due to the presence of the carbonyl group, which may donate a lone pair of electrons to form stable sodium adducts. The drawback with the [M+Na]+ adduct ions is that they are very stable and could not be fragmented in MS/MS. The method was validated for analysis in six different vegetable matrices including cabbage, carrot,Accepted cauliflower, legume, lettuce and green Manuscript pepper matrices. The LOQ of the method ranged between 30 and 100 µg kg-1 with method precision in the range of 5% to 14%. A drawback of this method was that the analytical runtime was long at >45 min. However, the separation of pyrethroids was very good even showing partial separation of four diastereoisomers for CYPER.

UHPLC-MS/MS (QqQ) was employed by Chung and Lam for the determination of 15 pyrethroids in various food matrices (Chung and Lam, 2012). During MS optimisation, it was found that the ionisation temperature had to be maintained at 120°C to prevent loss of sensitivity for the thermally labile tefluthrin. The pyrethroids were analysed as their [M+NH4]+ adducts, while pyrethrins were mostly monitored as [M+H]+. The pyrethrin cinerin I was detected using the less sensitive ammoniated adduct because matrix interference was observed in some samples when the pseudo-molecular ion was employed. A binary gradient separation was initially evaluated but resulted in condensation on the MS curtain plate. As a result, an isocratic mobile phase was developed comprising of 5 mM ammonium formate in MeOH:H2O (85:15, v/v). This allowed the separation of the 15 pyrethroids on a

C18 BEH column (100 × 2.1 mm) in a 5 min run, see Figure 6. The LOQ of the method was 10 µg kg-1. Giroud et al., reported an UHPLC-MS/MS application for the determination of five pyrethroid and eight neonicotinoid insecticides in beebread (Giroud et al., 2013). The residues were separated in a 12 min injection cycle, with the pyrethroids eluting between 8 and 8.5 minute. LODs ranged from 0.042 µg kg-1 (cypermethrin) to 0.825 µg kg-1 (λ- cyhalothrin). The application method identified pesticide contamination in several samples including bifenthrin and λ-cyhalothrin residues.

Insert Figure 7

While the application of LC-MS/MS methodology for the analysis of pyrethroids in food has not been widespread, many groups have developed methods for the determination of pyrethroid metabolites or transformation products (TPs), which are generally non-volatile and more suitable for LC-MS/MS analysis. Olsson et al. (2004) reported an early method for analysing pyrethroid TPs in urine samples using LC-ESI-MS/MS with a TurboIonSpray probe (Olsson et al., 2004). The methodology included 3-phenoxybenzoic acid (3-PBA) which was reported at the time as being the TP of 10 pyrethroids and 4-fluoro-3- phenoxybenzoic acid (4-F-3-PBA), a TP of CYFLU. The work also included cis- and trans-3- (2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acids (cis-/trans-DCCA), which is a TPAccepted of CYFLU, PERM and CYPER. A TP ofManuscript DELTA, namely, cis-3-(2,2-dibromovinyl)- 2,2-dimethylcyclopropane-1-carboxylic acid (DBCA) was included in the scope of the analysis (Olsson et al., 2004). Generally, these metabolites can occur as glucuronides in urine and require deconjugation prior to analysis, as depicted in Figure 7 (Arrebola et al., 1999). The pyrethroid TPs formed deprotonated molecular ions, [M-H]-, and thus were analysed in negative ionisation mode. Isotopically labelled internal standards were included in the

analysis to compensate for matrix effects and improve method accuracy. Chromatographic

separation was carried out in <9 min on a BetaSil C18 column (100 x 1.0 mm i.d.) using an isocratic mobile phase, 0.1% HOAc in MeCN:H2O (51/49, v/v), with LODs of 0.1 - 0.4 ng mL-1 (Arrebola et al., 1999). A number of groups have reported LC-MS/MS methods of similar scope for pyrethroid TPs in urine, see Figure 8, employing isotopically labelled standards to compensate for matrix effects (Davis et al., 2013, Le Grand et al., 2012, Roca et al., 2014). Yoshida et al. (2013) reported the analysis of eight metabolites, fluorine- containing pesticides, namely, metofluthrin, profluthrin and transfluthrin. The method was based on GC-MS (Yoshida, 2013), and highlighted the need to develop LC-MS/MS methods for the analysis of these pyrethroids in urine (Yoshida, 2013).

Insert Figure 8

Most methods only monitor for parent pesticides in food samples, as in most cases there is no requirement to monitor for environmental TPs because these are not marker residues as defined in the legislation. In some cases, however, the metabolites can be more toxic than the parent compounds and affect the endocrine system. It is proposed that we do not analyse for these compounds at present as the marker residues were developed based on available methodologies at the time. Li et al. (2013) reported the LC-MS/MS analysis of seven parent pesticides and five TPs in fruit and vegetables using three chromatographic separations (Li et al., 2016). Parent pyrethroids were analysed in two analytical runs, allowing for the analysis of positively ionised analytes and negatively ionised analytes (CYFLU and CYHALO). A mobile phase of 5 mM ammonium acetate in H2O:MeOH (2:98, v/v) was used for elution between 2.5 - 3.4 min. The pyrethroid isomers eluted as a single peak, simplifying integration and quantitation. All the TPs eluted between 2.2 and 3.0 min in a separate run. The method LODs and LODs were <0.5 µg kg-1 and <0.15 µg kg-1, respectively (Li et al., 2016). Carneiro et al. (2013) developed a method for the determination of 128 pesticides in bananas, including four pyrethroids (Carneiro et al., 2013). A binary gradient separation was employed with a 13 min run time. The LOQ of the method was 10 µg kg-1. A method for the rapid detectionAccepted of 220 residues in infant formula Manuscript using UHPLC-ESI-MS/MS has also been developed (Zhan et al., 2013b). The pesticides were analysed in two ESI + runs (87 and 94 analytes) and one ESI – run (39 analytes). The final method showed good linearity (R > 0.990) and LOQs of 0.01 to 5 µg kg-1 (Zhan et al., 2013b).

Future challenges

Some disagreement is evident in literature with respect to the analytical future of residue analysis. De Brabander et al., suggested that two possible strategies could be followed, the first being the classical approach of screening with inexpensive methods followed by mass spectrometric confirmation, while the second foresees the use of high-tech multi-residue (100-200 analytes) detection methods designed to include all B group substances (De Brabander et al., 2009). These authors suggest that the latter approach is more likely and sample analysis will therefore require increasingly sophisticated instrumentation which will, as a consequence, only be affordable to laboratories with sufficient financial resource. Situ et al., although agreeing the need for high-throughput analysis, suggest that, as food safety and public health continues to be a world-wide concern, future demand will be for rapid, simple, low cost screening tests which can be employed on-site in food production facilities (Situ et al., 2010).

Should the laboratory-based pathway be followed then clearly pyrethrin and pyrethroid analysis can readily be included within large multi-residue methods based on mass spectrometry. However, should the future require on-site analysis then this may provide a much greater challenge. To date, rapid, on-site methods for residue analysis have relied largely on antibody:antigen interaction, although other specific binders have also been employed. A number of workers have reported production of polyclonal and/or monoclonal antibodies for pyrethroids although to date, most antibodies have only been suitable for the detection of a single analyte or have been multi-analyte in nature but with limited sensitivity across the group. The production of a monoclonal antibody with broad spectrum detection capability and sensitivity has been reported (Wang et al., 2011). These workers utilised the antibody in a river water enzyme immunoassay but it seems likely that such an antibody could be used as the basis for a method for on-site food analysis. A more limiting factor in the development of such analytical methods is likely to be the absence of a simple and rapid sample preparation technique suitable for use on-site. Until this issue can be overcome it is unlikely that any demand for truly high-throughput, on-site analysis can be met.

ConclusionsAccepted Manuscript

The analysis of pyrethrin and pyrethroid residues is complicated by the differences in the chemical functional groups and the stereochemistry of these molecules. The conventional approach for analysing these molecules has involved the application of GC-ECD after complex sample clean-up procedures. However, this has generally limited the number of

26 analytes that can be satisfactorily analysed to MRLs. It has generally been recognised in most laboratories nowadays that GC-MS(/MS) is the most suitable analytical technique for the analysis of these compounds owing the universal nature of this detector. In addition, because of the exacting legal requirements of international legislation mass spectrometry is arguably the only suitable technique that can be used to confirm the identity of non-compliant residues at trace levels in complex food samples. This review paper highlights GC-APCI-MS/MS as an emerging detection technique that can be employed to detect pyrethrin and pyrethroid. In recent years, technique has been applied in a number of applications showing advantages over GC-EI-MS(/MS) and GC-CI-MS(/MS). LC-MS/MS has not been widely used to analyse pyrethrin and pyrethroid residues in food but is advantageous over GC-MS(/MS) methods because injection cycles times can be reduced by half, which allows greater numbers of samples to be injected in overnight batches. The application of LC-MS/MS in the analysis of foods of animal origin is particularly advantageous because it potentially allows the combining of pesticide and veterinary drug residues in one analyte test.

Sample preparation is arguably the greatest bottleneck in residue analysis but this obstacle has been partially addressed in recent years through new high throughput approaches such as QuEChERS, which can be applied to a wide range of food types such as meat, liver, kidney, milk and honey. Adipose tissue remains a challenging analytical matrix and requires more complex sample preparation processes to successfully extract target analytes. It is recommended that more focus should be paid to the development of methods for isolating pesticides residues from fat tissue. It is proposed that the overall goal should be to develop more-efficient miniaturised residue extraction protocols for fat that are more environmentally friendly and use low volumes of organic solvent. It is imperative that measurement uncertainty should not be compromised through miniaturisation of sample size and solvent volumes.

In summary developments in analytical instrumentation coupled with improvements in sample preparation should enable the analysis of a wider range of pyrethroids residues in food Acceptedof animal origin. The application of LC Manuscript-MS/MS is particularly interesting approach because it can allow the inclusion of pesticides and transformation products.

Acknowledgements

This research was funded by the Teagasc Walsh Fellowship programme (Project RMIS 6240).

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Accepted Manuscript

Table 1. Structurally related pyrethroids and pyrethrins

R1 = R1 = R1 = R1 = Acrinathrin Cyfluthrin Cyhalothrin Cypermethrin

R2 = H R3 = CN R2 = F R3 = CN R2 = H R3 = CN R2 = H R3 = CN

R1 = R1 = R1 = R1 =

Cyphenothrin Deltamethrin Fenpropathrin Fenvalerate

R2 = H R3 = CN R2 = H R3 = CN R2 = H R3 = CN R2 = H R3 = CN

R1 = R1 = R1 = R1 =

Flucythrinate Flumethrin Permethrin Phenothrin

R2 = F R3 = CN R2 = F R3 = CN R2 = H R3 = H R2 = H R3 = H

R1 = R1 =

Tau- Fluvalinate Tralomethrin

AcceptedR2 = H R3 = CN ManuscriptR2 = H R3 = CN (continued)

Table 1. (Continued)

R1 = R1 = R1 = R1 =

Allethrin I Bifenthrin Cinerin I Cinerin II

CH CH CH R2 = 3 R3 = 3 R2 = Cl R3 = CF 3 R2 = CH 3 R3 = 3 R2 = CH 3 R3 =

R1 = R1 = R1 = R1 =

Jasmolin I Jasmolin II Pyrethrin I Pyrethrin II

CH CH CH R2 = CH 3 R3 = CH R2 = 3 R3 = R2 = CH 3 R3 = 3 R2 = 3 R3 = 3

R1 = R1 =

Resmethrin Tetramethrin

R2 = CH R3 = CH R2 = CH R3 = CH Accepted3 3 Manuscript3 3

Table 2a. Maximum residue limits for pesticides as listed under Council Regulation 37/2010/EC (European Commission., 2010).

Substance Marker Species Maximum Residue Limit (mg kg-1) Residue Milk Eggs Muscle Liver Kidney Fat M/S Honey Cyfluthrin Cyfluthrin B, G 0.02 0.01 0.01 0.01 0.05 (sum of isomers) Cyhalothrin Cyhalothrin B 0.05 0.05 0.5 (sum of isomers)

Alphacypermethrin Cypermethrin B, Ov 0.02 0.02 0.02 0.02 0.2 (sum of isomers) Cypermethrin Cypermethrin All 0.02 0.02 0.02 0.02 0.2 (sum of ruminants isomers) Cypermethrin Cypermethrin Salmonidae 0.05 (sum of isomers) (sum of isomers) Deltamethrin Deltamethrin All 0.02 0.01 0.01 0.01 0.05 ruminants Fin Fish 0.01 Fenvalerate Fenvalerate B 0.04 0.025 0.025 0.025 0.25 (sum ofRR, SS, RS and SRisomers) Flumethrin Flumethrin B 0.03 0.01 0.02 0.01 0.15 (sum of Ov 0.01 0.02 0.01 0.15 trans-Z- isomers) Bees No MRL required Permethrin Permethrin B 0.05 0.05 0.05 0.05 0.5 (sum of isomers) Tau-Fluvalinate Not Bees No applicable MRL required Abbreviations:

B = Bovine, Ov = Ovine, G = Goat.

M/S = Muscle and skin Accepted Manuscript

Table 2b. Maximum residue limits for pyrethroids and pyrethrins under Council Regulation 396/2005 (European Commission 2005).

Substance Species Maximum Residue Limits (mg kg-1) Milk Eggs Muscle Liver Kidney Fat M/S Honey Acrinathrin B, Ov, G, 0.05 0.05 0.05 0.05 0.05 E, P 0.05 0.05 0.05 0.05 0.05 Py 0.05 0.05 0.05 0.05 0.05 Other 0.01 0.05 Allethrin B, Ov, G, 0.01 0.01 0.01 0.01 0.01 E P 0.01 0.01 0.01 0.01 Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.01 0.01 0.01 0.01 0.01 Bifenthrin B, Ov, G, 0.2 0.2 0.2 0.2 3 (sum of isomers) E P 0.2 0.2 0.2 3 Py 0.01 0.05 0.01 0.01 0.05 Other 0.01 0.05 Cyfluthrin/β-Cyfluthrin B, Ov, G, 0.02 0.05 0.05 0.05 0.2 E, (sum of isomers) P 0.05 0.05 0.05 0.2 Py 0.02 0.05 0.05 0.05 0.05 Other 0.01 0.05 Cyhalothrin/ γ- B, Ov, G, 0.01 0.01 0.01 0.01 0.01 Cyhalothrin E (sum of isomers) P 0.01 0.01 0.01 0.01 Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.01 0.01 0.01 0.01 0.01 λ-Cyhalothrin B, Ov, G, 0.05 0.5 0.5 0.5 0.5 E, (sum of isomers) Py 0.02 0.02 0.02 0.02 0.02 P 0.5 0.5 0.5 0.5 Other 0.01 0.05 Cyphenothrin B, Ov, G, 0.01 0.01 0.01 0.01 0.01 E P 0.01 0.01 0.01 0.01 Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.01 0.01 0.01 0.01 0.01 Alphacypermethrin/ B, Ov, G, 0.05 2 0.2 0.2 2 E Cypermethrin P 2 0.2 0.2 2 Py 0.05 0.1 0.05 0.05 0.1 Other 0.01 0.05 Deltamethrin B, Ov, G, 0.05 0.03 0.03 0.03 0.5 E (cis-isomer) Py 0.02 0.02 0.02 0.02 0.1 P 0.03 0.03 0.03 0.5 AcceptedOther Manuscript 0.01 0.05 Fenpropathrin B, Ov, G, 0.01 0.01 0.01 0.01 0.01 E P 0.01 0.01 0.01 0.01 Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.01 0.01 0.01 0.01 0.01 Esfenvalerate/Fenvalerate B 0.04 0.025 0.07 0.05 0.25 (any ratio of constituent Ov, G, E 0.02 0.02 0.07 0.05 0.2 isomers (RR, SS, RS & P 0.02 0.02 0.02 0.03 SR) including Py 0.02 0.02 0.02 0.02 0.02 esfenvalerate Other 0.01 0.05

Flucythrinate B, Ov, G, 0.01 0.01 0.01 0.01 0.01 (sum of isomers) E P 0.01 0.01 0.01 0.01 Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.01 0.01 0.01 0.01 0.05 Flumethrin B, Ov, G, 0.01 0.01 0.01 0.01 0.01 E P 0.01 0.01 0.01 0.01 Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.01 0.01 0.01 0.01 0.05 Permethrin B 0.05 0.05 0.05 0.05 0.5 (sum of isomers) Ov, G 0.05 0.01 0.05 0.05 0.05 E 0.05 0.01 0.01 0.01 0.01 P 0.05 0.05 0.05 0.05 Py 0.05/0.01* 0.05 0.05 0.05 0.05 Other 0.01 0.01 Phenothrin B, Ov, G, 0.05 0.05 0.05 0.05 0.05 E (sum of isomers) P 0.05 0.05 0.05 0.05 Py 0.05 0.05 0.05 0.05 0.05 Other 0.01 0.05 Pyrethrins B, Ov, G, 0.05 0.05 0.05 0.05 0.05 E P 0.05 0.05 0.05 0.05 Py 0.05 0.05 0.05 0.05 0.05 O 0.01 0.01 Resmethrin B, Ov, G, 0.02 0.02 0.02 0.02 0.02 E (sum of isomers) P 0.02 0.02 0.02 0.02 0.02 Py 0.02 0.02 0.02 0.02 O 0.01 0.05 Tau-Fluvalinate B, Ov, G, 0.05 0.05 0.01 0.02 0.3 E P 0.05 0.01 0.02 0.3 Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.05 Tetramethrin B, Ov, G, 0.01 0.01 0.01 0.01 0.01 E Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.01 0.01 0.01 0.01 0.01 Tralomethrin B, Ov, G, 0.01 0.01 0.01 0.01 0.01 E Py 0.01 0.01 0.01 0.01 0.01 Other 0.01 0.01 0.01 0.01 0.01 0.01 Abbreviations:

B = Bovine, E = Equine, Ov = Ovine, P = Porcine, Py = Poultry, G = Goat.

M/S = Muscle and skin. *MRLAccepted = 0.05 mg kg-1 for chicken eggs and 0.01 mg kg -1 Manuscriptfor eggs from other poultry.

Table 3. Summary of analytical methodologies applied for analysing pyrethroid residues in foods of animal origin.

Extraction Sample Recovery Analyte Matrix Clean-up Detection Quantification Ref Solvent extraction (%) LOD 7.7 - 14.4 μg kg-1 Fat MeCN/ SPE (Akre and 8 LLE GC-ECD 80-123 LOQ 25.8 - 47.9 μg kg- (0.5 g) n-hexane (Florisil) MacNeil, 2006) 1 a) Fat MeCN/ SPE LOD N.L. (Bordet et al., 6 Cryogenic GC-ECD 14-127 -1 (0.5 g) DCM (C18 + Florisil) LOQ 1.9 - 11.7 μg kg 2002) Fat SPE LOD 3-118 μg kg-1 18 MeCN MSPD GC-ECD 88-137 (Sun, 2003) (0.5 g) (C18) LOQ N.L. Fat SPE LOD N.L. (Kodba and 3 DMSO MSPD GC-ECD 84-96 (0.5 g) (Florisil) LOQ 30-50 μg kg-1 Vončina, 2007) Fat LOD N.L. (Zrostlíková et al., 2 EtOAc LLE GPC GC-ECD 52-72 (0.25 g) LOQ 13-40 μg kg-1 2002) Honey LOD 3.3 - 12.3 μg kg- (Wiest et al., 8 MeCN QuEChERS - a) GC-ToF 49-115 (5.0 g) LOQ 9.1 - 37.6 μg kg-1 2011) -1 Honey H2O/ SPE LOD 0.07 - 0.2 μg kg (Paradis et al., 9 QuEChERS GC-MS/MS 63 - 139 -1 (5.0 g) MeCN (PSA + MgSO4) LOQ 0.2 - 0.5 μg kg 2014) a) Honey (20.0 g) a) 80 - 102 LOD 0.5 - 1 ng kg-1 (Shamsipur et al., 4 Chlorobenzene SPE-DLLME - GC-MS b) Milk b) 66 - 102 LOQ N.L. 2016) (1.5 mL) Honey SPE (Rissato et al., 4 MeCN SFE GC-ECD 92 - 95 LOD 5.0 - 9.0 μg kg-1 (5.0 g) (Florisil) 2004) Honey MeCN/ Low temperature + SPE LOD 16 μg kg-1 (de Pinho et al., 3 LLE GC-ECD 93-101 (3.0 g) EtOAc (Florisil) LOQ 31 -33 μg kg-1 2010) Honey SPE LOD 0.3-9.5 μg kg-1 6 EtOAc LLE GC-MS 86-112 (Jin et al., 2006) (1.0 g) (Florisil) LOQ N.L. Honey d-SPE LOD 1 - 3 μg kg-1 (Shendy et al., 14 MeCN QuEChERS GC-MS/MS 62 - 105 -1 (5.0 g) (MgSO4 + PSA) LOQ 10 μg kg 2016)

Honey SPE LOD 1 - 2.5 μg L-1 (Rissato et al., 4 MeCN LLE GC-MS 85-119 (10.0 g) (Florisil) LOQ 4 - 8 μg L-1 2007) Honey SPE - LOD N.L. (Chiesa et al., 16 MeCN AcceptedPLE ManuscriptGC-MS/MS (5.0 g) (PSA) 1 - 10 μg kg-1 2017) Milk d-SPE LOD 0.6 - 3.4 μg kg-1 (Jeong et al., 4 MeCN QuEChERS GC-ECD 68 - 98 -1 (15.0 g) (MgSO4 + PSA + C18) LOQ 2.0 - 11.2 μg kg 2012)

Milk LOD 0.25 μg L-1 (Goulart et al., 2 MeCN LLE Low temperature GC-ECD 84 - 93 (4.0 mL) LOQ 0.75 μg L-1 2008) (continued)

Table 3. (Continued) Extraction Sample Recovery Analyte Matrix Clean-up Detection Quantification Ref Solvent extraction (%) Low temperature + Milk LOD 0.03 - 1.67 ng mL-1 (Chen et al., 6 MeCN LLE d-SPE GC-MS/MS 34 - 70 (1.0 mL) LOQ N.L. 2014) (GCB + PSA) a) Milk a) 83 - Low temperature + (5.0 g) 128 LOD 3 μg kg-1 (Anagnostopoulos 10 MeCN QuEChERS d-SPE GC-MS/MS b) Tissue b) 70 - LOQ 10 μg kg-1 et al., 2014) (MgSO + PSA) (5.0 g) 4 104 Milk MeCN/petroeleum LOD N.L. (Di Muccio et al., 13 LLE SEC GC-ECD 60-119 (10.0 mL) /EtOH LOQ 0.1 - 0.5 μg mL-1 1997) (Fernandez- Milk LOD 0.03 - 0.56 μg L-1 9 H O DI-SPME - GC-ECD 69 - 124 Alvarez et al., (1.0 mL) 2 LOQ 0.09 - 1.9 μg L-1 2008) Tissues Cyclohexane/ LOD N.L. 17 LLE GPC GC-ECD 53 - 124 (Pang et al., 2006) (10.0 g) EtOAc LOQ 25 - 300 μg kg-1 SPE Beef LOD -N.L. (Stefanelli et al., 6 Light petroleum MSPD (Extrelut NT3 + C + GC-MS 84 - 99 (10.0 g) 18 LOQ 10 - 100 μg kg-1 2009) Florisil) Tissue LOD 0.01 - 0.02 μg kg-1 (Cheng et al., 2 n-hexane MSPD - HPLC-UV 84-109 (0.5 g) LOQ 0.03 - 0.06 μg kg-1 2009) Tissues Acetone/ SPE LOD 5 μg kg-1 (Niewiadowska, 7 Cryogenic GC-ECD 64 - 97 (10.0 g) petroleum ether (Florisil) LOQ 10 μg kg-1 2010) Tissues LOD 0.2 - 11 μg kg-1 18 MeCN PLE GPC GC-MS 71 - 97 (Wu et al., 2011b) (10.0 g) LOQ 0.7 - 33 μg kg-1 Fish d-SPE LOD 0.01 - μg mL-1 6 Propan-2-ol QuEChERS GC-ECD 81 - 108 (Jia et al., 2012) (2.0g) Accepted(MgSO4 + PSA) Manuscript LOQ N.L. EtOAc = Ethyl acetate, H2O = Water, MeCN = Acetonitrile, NH2 = Amine, NH3 = ammonia, MgSO4 = Ammonium sulphate, GCB = Graphitized carbon black, PSA =

Primary secondary amine, CaCl2 = Calcium chloride, PLE = Pressurised liquid extraction, LLE = Liquid-liquid extraction, SPE = Solid phase extraction, d-SPE = Dispersive solid phase extraction, GPC = Gel permeation chromatography, MSPD = Matrix solid phase extraction, DI-SPME = Direct immersion solid-phase microextraction,

QuEChERS = Quick, easy, cheap, effective, rugged and safe, DLLME = Dispersive liquid–liquid microextraction GC = Gas chromatography, MS = Mass spectrometry, ECD, Electron capture detection, LC = Liquid chromatography, HPLC = High performance liquid chromatography, UV = Ultra-violet, LOD = Limit of detection, LOQ = Limit of quantitation, N.L. = Not listed

Table 4. Chemical composition and properties of pyrethroids and pyrethrins.

CAS Number Analyte Empirical Formula MWa (g/mol) Major Isotopes (% abundance) 01007-06-1 Acrinathrin C26H21F6NO5 541.45 541(100); 542(29); 584-79-2 Allethrin I C19H26O3 302.41 302(100);303(21.1); 82657-04-3 Bifenthrin C23H22ClF3O2 422.87 422(100); 424(35.8); 423(25.3); 68359-37-5 Cyfluthrin C22H18Cl2FNO3 434.29 433(100); 435(68.3); 434(24.6); 91465-08-6 Cyhalothrin C23H19ClF3NO3 449.85 449(100); 451(36.1); 450(25.7); 52315-07-8 Cypermethrin C22H19Cl2NO3 416.30 415(100); 417(68.3); 416(24.6); 39515-40-7 Cyphenothrin C24H25NO3 375.47 375(100); 376(26.8) 52918-63-5 Deltamethrin C22H19Br2NO3 505.21 505(100); 503(50.4); 507(51.3); 506(24.3); 39515-41-8 Fenpropathrin C22H23NO3 349.43 349(100); 350(24.6) 51630-58-1 Fenvalerate C25H22ClNO3 419.91 419(100); 421(36.7); 420(27.9); 70124-77-5 Flucythrinate C26H23F2NO4 451.47 451(100); 452(29) 69770-45-2 Flumethrin C28H22Cl2FNO3 510.39 509(100); 511(70); 510(31.1); 512(20.8) 52645-53-1 Permethrin C21H20Cl2O3 391.29 390(100); 391(23.2); 392(67.9) 26002-80-2 Phenothrin C23H26O3 350.46 350(100); 351(25.4) 10453-86-8 Resmethrin C22H26O3 338.45 338(100); 339(24.3) 102851-06-9 Tau-Fluvalinate C26H22ClF3N2O3 502.92 502(100); 504(37.1); 503(29.3); 7696-12-0 Tetramethrin C19H25NO4 331.41 331(100); 332(21.5) 66841-25-6 Tralomethrin C22H19Br4NO3 665.01 665(100); 663(67.4); 667(66.9); 666(24.2); 25402-06-6 Cinerin I C20H28O3 316.44 316(100); 317(22.2) 121-20-0 Cinerin II C21H28O5 360.45 360(100); 361(23.4) 4466-14-2 Jasmolin I C21H30O3 330.47 330(100); 331(23.3) 1172-63-0 Jasmolin II C22H30O5 374.48 374(100); 375(24.5) 121-21-1 Pyrethrin I Accepted C21H28O3 Manuscript328.45 328(100); 329(23.3) 121-29-9 Pyrethrin II C22H28O5 372.46 372(100); 373(24.4)

Figure 1. Chemical structures of (A) chrysanthemic acid, and (B) pyrethrin I. Source: (Corcellas et al., 2013). Reproduced with permission from Elsevier.

Figure 2. Stereochemical determinants of insecticidal activity in pyrethroid acids. Source: (Soderlund et al., 2002). Reproduced with permission from Elsevier.

Figure 3. Pyrethroid mode of action. Adapted from (Davies et al., 2007). Copyright permission from Wiley.

Figure 4. Separation of 18 pyrethrin/pyrethroid residues separated in a 24 min run-time through GC- EI-MS. Source: Yoshida et al. (Yoshida, 2009). Reposted with permission from Elssevier.

Accepted Manuscript

Figure 5. GC/MS/MS chromatograms for ?-fluvalinate (top) and fenvaleratte (bottom) in a standard in solvent (250 ng/mL). (A) EI; (B) APCI Source: Portolés et al. (Portolés et al., 2012b). Reposted with permission from ACS Publications

Figure 6. UPLC–MS–MS chromatogramms obtained for twenty target analyttes spiked at the MLOQ level. Source: Chung et al. (Chung and Lam, 2012). Reposted with permission from Springer.

Figure 7. General structures of synthetic pyrethroids and their main metabolic pathway in mammals. Source: Arrebola et al. (Arreboola et al., 1999). Reproduced with permission from Elsevier.

Accepted Manuscript

Figure 8. Chromatograms obtained from a urine sample: (A) cis- + trans-CCl2CA with blank (a) and LOQ (a?); (B) 3-PBA with blank (b) and LOQ (b?); (C) 4-FPBA with blank (c) and LOQ (c?); (D) Br2CA with blank (d) and LOQ (d?). Each chromatogram report both the quantification and confirmation transitions. Source: LeGrand et al. (Le Grand et al., 2012). Reposted with permission from Elsevier.

Accepted Manuscript