Determination of pyrethrin and pyrethroid residues in animal fat using liquid chromatography coupled to tandem mass spectrometry

Accepted Manuscript Title: Determination of Pyrethrin and Pyrethroid Residues in Animal Fat using Liquid Chromatography coupled to Tandem Mass Spectrometry Authors: M. Moloney, S. Tuck, A. Ramkumar, A. Furey, M. Danaher PII: DOI: Reference:

S1570-0232(17)31236-9 https://doi.org/10.1016/j.jchromb.2017.12.022 CHROMB 20965

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

4-8-2017 19-11-2017 12-12-2017

Please cite this article as: M.Moloney, S.Tuck, A.Ramkumar, A.Furey, M.Danaher, Determination of Pyrethrin and Pyrethroid Residues in Animal Fat using Liquid Chromatography coupled to Tandem Mass Spectrometry, Journal of Chromatography B https://doi.org/10.1016/j.jchromb.2017.12.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Determination of Pyrethrin and Pyrethroid Residues in Animal Fat using Liquid Chromatography coupled to Tandem Mass Spectrometry

M. Moloneya, S. Tucka,b, A. Ramkumara, A. Fureyb, M. Danahera* Food Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland

b

Mass Spectrometry Research Group, Department of Physical Sciences, Cork Institute of

IP T

a

SC R

Technology, Bishopstown, Cork, Ireland

*

Corresponding author: [email protected]

U

Tele: +353 1 8059500 Fax: +353 1 8059550



A

M

 

Pyrethrin and pyrethroid residues can be effectively analysed in fat by LCMS/MS. Samples were prepared using an innovative protocol with low volumes of solvent. The use of isotope standards negated matrix effects and improved precision. The method includes difficult analytes, namely, flumethrin and tralomethrin.

ED



N

Highlights:

PT

Abstract

A method was developed for the confirmatory and quantitative analysis of one pyrethrin and

CC E

18 pyrethroid residues in animal fat. Fat was extracted was collected from adipose tissue melted in an oven at 65°C for 2 h. Fat samples (1 g) were dispersed with deactivated Florisil® sorbent and extracted with MeCN. Sample extracts were purified by cold

A

temperature precipitation at -30°C for 4 h and further purified using dispersive solid-phase extraction (d-SPE) clean-up in tubes 500 mg of Z-SEP+ and 125 mg of PSA bonded silica. Purified samples were analysed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) detection. Chromatographic separation was carried out on a Acquity C8 BEH column, using a binary gradient separation comprising of

mobile phase A, 5 mM ammonium formate in water:MeOH (80:20, v/v,) and mobile phase B, 5 mM ammonium formate in MeOH. The mass spectrometer was operated in the positive electrospray ionisation mode (ESI(+)). Validation was performed following the 2002/657/EC guidelines. Trueness ranged between 84% and 143% and precision ranged between 3.9% and 29%. The developed method is particularly advantageous because the sample preparation

IP T

procedure does not require complex sample extraction equipment and uses less solvent

SC R

compared to other sample preparation methods.

U

Keywords: Pyrethrin; Pyrethroid; Pesticides; UHPLC-MS/MS; Animal Fat

N

1. Introduction

A

The pyrethroid pesticides are synthetic analogues of naturally occurring pyrethrins,

M

insecticides contained in pyrethrum, an oleoresin extract obtained from dried chrysanthemum flowers

ED

[1]. However, since the discovery of pyrethrins new generations of more effective pyrethroids have been introduced with improved photo stability [2]. Both pyrethrins and synthetic pyrethroids possess

PT

closely related chemical structures, resulting in similar mode of actions, and toxicological properties and high insecticidal activity [3]. It has been demonstrated that pyrethroids, in particular those

CC E

compounds which lack an α-cyano substituent, exert their insecticidal effects on arthropods through interfering with voltage sensitive sodium, chloride and calcium channels [1]. However, this mode of action can also take place in mammals, along with the target insects, resulting in pyrethroids

A

presenting neurotoxicity behaviour in both mammals and insects [3]. The pyrethroids have found widespread use in the agriculture as crop protection agents, as

biocides to control insect infestations and as ectoparasiticides used for treating food producing animals [4]. Pyrethroids are potent against a broad range of external parasites, including horn flies and mosquitoes, at relatively low doses. There are multiple potential routes that food producing animals

can be exposed to pyrethroid and pyrethrin pesticides including direct treatment of animals with ectoparasiticides, fly treatments within intensive production units, spray drift during application of crop protection agents, consumption of contaminated feed, decontamination of transport trucks and contaminated bedding. In order to protect the health of consumers, maximum residue limits (MRLs) have been established for pyrethroid and pyrethrin pesticides under European Union (EU) legislation

IP T

for the licensing of veterinary medicinal products [5] and pesticide legislation [6]. Consequently, EU MRLs have been laid down for veterinary medicinal products and pesticides under Commission

Regulations 37/2010 [7] under 212/2013 [8], respectively. These insecticides are lipophilic in nature

SC R

and partition into fatty tissues, therefore, fat is often the target matrix of choice for surveillance

purposes, and is frequently extracted from other food commodities to allow analysis [9]. The MRLs

U

for pyrethroid and pyrethrin pesticides in animal fats are presented in Table 1 [7,10]. In a few cases,

N

MRL values can differ between the two pieces of legislations because veterinary drug MRLs are based on toxicological factors, which pesticides tolerances are based on good agriculture practice. For

A

example, in the case of cypermethrin where two different limits of 200 µg kg-1 and 2000 µg kg-1 in fat

M

listed under veterinary drug and pesticide legislation, respectively. A similar difference can be seen

ED

with deltamethrin, which has a veterinary drug MRL of 50 µg kg-1 and a pesticide limit of 500 µg kg-1 in fat. In the event of MRL breaches, follow up investigations have to be carried out on farm to

PT

identify the cause of residues. In addition, exposure assessments should be carried out to ensure that consumer health is not impaired. In order to ensure consumer protection, ongoing monitoring of

CC E

foodstuffs is carried out by designated laboratories to ensure compliance with these MRLs. The analytical tests applied in this monitoring should be comprehensive, suitably sensitive and most importantly should be fit for purpose. In order to address these requirements, there is an ongoing need

A

for the development and validation of multi-residue methods that will efficiently analyse pesticide residues.

Insert Table 1.

The majority of analytical methods for the determination of pyrethroid and pyrethrin pesticide residues have been reported for fruit and vegetable samples [3,11]. While methods for foods of animal origin mainly focus on fish, milk, meat and honey [2,9, 12-16], fewer methods have been reported in literature for the analysis of these pyrethroid or pyrethrin residues in animal fat and these applications

IP T

generally include a wider range of pesticides such as the organochlorine (OCPs) and

organophosphates (OPs) pesticides. Examples of such multi-residue methods include reference

SC R

methods for analysing non-polar pesticide residues in animal fat [17,18]. These standard methods are regarded as very rugged but generally require large volumes of organic solvent and are laborious, taking several days to process a batch of samples for analysis. Alternative methods have been reported

U

in the literature to streamline the analysis and reduce the volumes of solvent used in sample analysis.

N

Akre and MacNeil (2006) reported a more efficient method for the analysis of eight pyrethroids in

A

bovine fat based on Florisil-PR SPE clean-up that reduced solvent usage and reduced sample

M

processing time [19]. Kodba and Vončina (2007) developed a method for the analysis of pyrethroids and other pesticides in animal fat based dispersion with Celite and Florisil column clean-up [20]. Park

ED

et al. also implemented GC-ECD for the analysis of 22 OCP and OP pesticides in beef fat using hexane extraction, liquid-liquid partitioning clean-up and cold temperature precipitation of lipids at -

PT

70°C [21]. Sun et al. (2006) similarly reported an efficient method for OCPs, OPs and pyrethroids in melted fat based on dispersion with Florisil, MeCN extraction and C18 SPE clean-up prior to GC-ECD

CC E

analysis [22].

Pyrethroid and pyrethrin residues are most commonly analysed by gas chromatography and less

A

frequently by HPLC or TLC [23, 24]. Indeed, analysis of a recent EU proficiency test report shows that only 21% of laboratories who participated in the study used LC-MS/MS, while the remainder used GC methods [25]. Many of the synthetic pyrethroids possess halogen atoms or nitrile functional groups in their molecular structures, which allows them to be more sensitively detected using an electron-capture detector (ECD) [23]. Nitrogen-phosphorus detection (NPD) coupled to GC has also been applied for pyrethroid analysis but for a limited number of analytes [24]. Nowadays, GC coupled

to MS (single stage mass spectrometry detection) or MS/MS (tandem mass spectrometry) are more widely employed for pyrethroid and pyrethrin analysis [26,27]. The application of GC-MS or GCMS/MS enables greater levels of sensitivity, selectivity and qualitative identification of residues in complex matrices. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is an alternative technique that can potentially be applied for analysing pyrethroid or pyrethrin pesticides

IP T

[28-33]. However, there are few publications in the literature reporting on the application of LCMS/MS for these compounds, probably because GC instruments were more common in pesticide

analysis laboratories than LC-MS/MS systems. The limited number of published papers indicate that

SC R

LC-MS/MS is a viable alternative to GC and in some cases, it may be more suitable for less volatile or thermally stabile pyrethroids such as flumethrin and tralomethrin, respectively [33].

U

The objective of this research was to develop and validate an UHPLC-MS/MS comprehensive method

N

for the determination of pyrethroid and pyrethrin I,II residues in fat of different animal species. The

A

research investigates the suitability of different UHPLC-MS/MS conditions and evaluates different

2. Experimental

ED

M

samples preparation protocols.

PT

2.1 Reagents and chemicals

CC E

Acetonitrile (MeCN), methanol (MeOH) and 2-propano, all Super Purity grade were sourced from ROMIL (Cambridge, UK). Dimethyl sulphoxide puriss p.a. (DMSO), ethylene glycol (>98%), deuterated methanol-D (MeOH-D), ammonium formate puriss p.a., Florisil® adsorbent for

A

chromatography 60-100 mesh, Z-SEP+ and PSA bonded silica were all sourced from Sigma-Aldrich (Dublin, Ireland). Florisil® was prepared by heating at 550°C overnight in a furnace (Carbolite, Sheffield, UK) and stored in a desiccator until cool. Deactivated Florisil® was prepared by gradually adding 15 mL of water to 85 g of Florisil® and mixed manually to avoid clumping. The Florisil® was then shaken on a horizontal shaker (Gerhardt; Königswinter, Germany) at 200 oscillations min-1 for 20 min and allowed to equilibrate overnight. Ethanol (EtOH) EMPROVE® grade was obtained from

Merck (Darmstadt, Germany). Ultra-pure water (18.2 MΩ cm-1) was generated in house using a Millipore (Cork, Ireland) water purification system. Polypropylene tubes (15 and 50 mL) with screw caps were obtained from Sarstedt Ltd. (Wexford, Ireland) and, 0.2 µm 13 mm PTFE syringe filters were sourced from Agilent Technologies Ireland Ltd., (Cork, Ireland ). Acrinathrin (ACRIN), fenpropathrin (FENPROP), flucythrinate (FLUCYTH) and pyrethrins (PYR)

IP T

were purchased from Dr. Ehrensorfer GmBH (Augsburg, Germany). The standards, allethrin

(ALLETH), bifenthrin (BIFEN), cypermethrin (CYPER), cyphenothrin (CYPHENO), deltamethrin,

SC R

(DELTA), fenvalerate (FENVAL), flumethrin (FLUM), permethrin (PERM), phenothrin (PHENO),

resmethrin (RES), tetramethrin (TETRA), tralomethrin (TRALO), β-cyfluthrin (CYFLU), λcyhalothrin (CYHALO) and τ-fluvalinate, (FLUV) were sourced from Sigma Aldrich (Dublin,

U

Ireland).

N

Cyfluthrin-D6 (CYFLU-D6) and cypermethrin-D6 (CYPER-D6) both at concentrations of 0.1 mg

A

mL-1 were supplied by Qmx Laboratories (Essex, UK). Bifenthrin-D5 (BIFEN-D5), deltamethrin-D5

M

(DELTA-D5) and fenvalerate-D5 (FENVAL-D5) were obtained from Toronto Research Chemicals (Toronto, Canada). Stock solutions of 0.1 mg mL-1 were prepared for BIFEN-D5 and DELTA-D5 in

ED

DMSO and EtOH respectively. FENVAL-D5 stock solution was prepared at a concentration of 0.2

PT

mg mL-1 also in EtOH. A working internal standard solution was prepared at 2 µg mL -1 in MeOH-D. Standard stock solutions of 2 mg mL-1 were prepared by dissolving ACRIN, ALLETH, CYPER,

CC E

DELTA, FENVAL, PERM, PYR, RES and TRALO in EtOH. BIFEN and CYFLU were combined and prepared in DMSO with the remaining analytes prepared in MeOH. The primary standard stock solutions were stable for at least 12 months, when stored in the dark at -20°C. An intermediate mixed

A

100 µg mL-1 stock solution was prepared by diluting the stock solutions in MeCN. It was necessary to prepare two separate intermediate stock standard solutions because of the number of analytes. Intermediate stock standard solution (A) contained ACRIN, ALLETH, CYPER, BIFEN, CYFLU, CYHALO, CYPHENO, DELTA and FENPROP, with the remaining analytes contained in working standard solution (B). Working calibration standards containing all of the analytes were prepared in

MeCN at 20 (std.8), 10 (std.7), 5 (std.6), 2 (std.5), 1 (std.4) and 0.5 (std.3) µg mL -1. The two remaining standards, two standards at 0.2 (std.2) and 0.1 (std.1) µg mL-1 were prepared by diluting standard 7.

IP T

2.2 Quality control and calibrants Animal fat samples was prepared by finely chopping frozen tissue and transferring them into

SC R

glass funnels plugged with small amounts of glass wool, which were placed in 50 mL Sarstedt tubes. The chopped samples were placed in an oven at 65±5°C for two hours and the melted fat was collected. The melted fat were stored at -20°C prior to analysis and test samples containing no

U

detectable analyte peaks were selected as negative controls. Extracted matrix calibration curves were

N

prepared by fortifying 1 g of melted fat samples (1 g) with 50 µL of working standards and 50 µL of

A

working internal standard prior to extraction to give a calibration curve in the range 5 to 1000 µg kg1

M

. Calibration curves were prepared by plotting peak area or response factor against concentration with

a calibration line fitted using linear least-square regression with 1/X fit. The identification of analytes

ED

was performed through the use of retention time and ion ratio criteria as outlined in 2002/657/EC [29]. Analyte recovery was evaluated by spiking negative sample extracts with 25 µL of working

PT

standard two (n=2) and calibrant six (n=2), following sample extraction. Recovery was calculated by comparing the area counts of the fortified sample (pre-extraction) with that of a spiked extract matrix

CC E

sample (post-extraction).

A

2.3 Sample preparation Melted fat (1.0 ± 0.010 g) was weighed into 50 mL polypropylene tubes when still in liquid

form and were maintained in their liquid form by heating them to 50°C in a GLS Aqua Plus waterbath (Grant; Cambridgeshire, UK). A 5 g quantity of deactivated Florisil® was added to samples which were then blended with a stainless steel spatula until a free flowing powder was formed. A 20 mL

volume of MeCN was added to each blended fat sample and shaken at 80 oscillations min-1 for 5 mins on a horizontal shaker. Samples were centrifuged at 2842 ×g (10 min, 4°C) in a Mistral 3000i centrifuge (MSE; London, UK) and samples were placed in the freezer at -30°C for a minimum of 4 h. The supernatant was transferred to 50 mL polypropylene tubes containing 500 mg of Z-SEP+ and 125 mg of PSA bonded silica. The samples were inverted, end over end for 30 sec and centrifuged at

IP T

2842 × g (10 min, 4°C). A 10 mL portion of the supernatant was then transferred to a 15 mL polypropylene tube containing 0.5 mL of ethylene glycol and concentrated under nitrogen in a Turbovap LV evaporator (Caliper Life Sciences; Runcorn, UK) until only the ethylene glycol

SC R

remained. Finally, samples were vortexed at 1700 rpm for 30 seconds on a TALBOYS multi-vortex (Tromner, NJ, U.S.A.) and filtered through 0.2 µm PTFE 13mm Millex-FG syringe filter (Millipore,

N

U

Cork, Ireland) into an autosampler vial, before injection onto the UHPLC-MS/MS system.

M

A

2.4 Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis Chromatographic separation was carried out using an Agilent 1290 Infinity LC system

ED

(Agilent Technologies, Santa-Clara, California, U.S.A.) using a stainless steel Waters Acquity C8 BEH column (2.1 × 50 mm, particle size 1.7µm) fitted with a Vanguard pre-column packed with the

PT

same stationary phase, which were maintained at a temperature of 50°C (all Waters, Dublin, Ireland). A binary gradient was employed comprising of mobile phase A, 5 mM ammonium formate in

CC E

water:MeOH (80:20, v/v,) and mobile phase B, 5 mM ammonium formate in MeOH. The gradient profile was programmed as follows: (1) 0–2.50 min, 95.0% A, (2) 2.50–3.50 min, 95% A, (3) 3.50– 4.50 min, 50.0% A, (4) 4.50–15.00 min, 40% A, (5) 15.00–15.20 min, 20% A, (6) 15.20–18.00 min,

A

20% A, (7) 18.00–18.20 min, 95% A, (8) 18.20–21.00 min, 95% A. The pump flow rate was set at 0.3 mL min-1 and 1 µL of purified sample extract was injected on the analytical column. Analytes were detected using an Agilent 6460 triple quadrupole mass spectrometer equipped with a Jet Stream electrospray ionisation probe operating in positive ionisation. The MS conditions were established for each analyte using Agilent MassHunter Optimiser software by the direct injection of 1

µg ml-1 standard solutions. The most abundant ion transition was used as quantifier; second and in some cases a third ions were selected as qualifiers. The MS conditions were as follows: gas temperature 250°C, gas flow 8 L min-1, sheath gas temperature 250°C, sheath gas flow 10 L min-1, nebulizer pressure 50 psi, capillary voltage 5000 V, and nozzle voltage 1000 V and a dwell time of 0.05 sec. The optimised MS conditions for each analyte are summarized in Table 2. The experimental

SC R

MassHunter Workstation Software version B.06.00 (Agilent Technologies).

IP T

data was acquired in dynamic multiple-reaction monitoring (MRM) mode and processed using

Insert Table 2.

N

U

2.5 Method validation

A

The selectivity of the method was evaluated through the analysis of 40 different fat samples from 40 different animals (10 avian, 10 bovine, 10 ovine and 10 porcine). The trueness of analytical

M

methods is usually carried out through analysis certified reference materials (CRMs). However, in the

ED

case of this method no CRM was available and trueness was measured using fortified blank samples, which is acceptable according to 2002/657/EC guidelines [34]. Within laboratory repeatability (WLr)

PT

was carried for bovine fat out by a single analyst on three separate days using the same instrumentation. In the WLr study bovine fat samples from different animals (n = 24) were fortified at

CC E

0.5 (n=8), 1.0 (n=8) and 1.5 (n=8) times the validation level. This validation level was equal to the MRL or 10 µg kg-1 for substances with no listed MRLs. In addition, a within laboratory reproducibility (WLR) validation was carried out by three different analysts on three different days

A

for avian, bovine and ovine species. Equipment and lot numbers of reagents were varied as much as possible during WLR runs. Notably, a fresh batch of Florisil was dried and deactivated on each day of analysis. The decision limit (CCα), is the concentration at and above which it can be concluded, with an error probability of α, that the sample is non-compliant, and the detection limit (CCβ), the

concentration at which the method is able to detect permitted limit concentrations with a statistical certainty of 1-β, were determined as described in Commission Decision 2002/657/EC [34].

IP T

3 Results and discussion

SC R

3.1 Development of UHPLC-MS/MS conditions

During the MS optimisation experiments it was found that the pyrethrins and pyrethroids ionised more intensely in positive ESI to form [M+H]+ or [M+NH4]+. This in agreement with other

U

published work that found pyrethrin and pyrethroid compounds have a tendency to form [M+H]+ or

N

[M+NH4]+ adducts, respectively [29]. In accordance with 2002/657/EC requirements, a minimum of

A

three identification points are required to confirm the presence of a group B substance [34]. In order

M

to satisfy this criteria, one precursor (one point) and two daughter (three points) ions are generally selected for each analytes to give a total of four identification points. It was found that many of the

ED

pyrethroids fragmented poorly in low energy collision induced dissociation experiments or produced product ions of low intensity. Consequently, the different chlorine or bromine isotopes were selected

PT

in many cases as the precursor ion to improve the sensitivity of the method. For example, the isotopic

CC E

peaks at m/z 523 and m/z 525 were selected precursor for deltamethrin. The impact of different mobile phase additives including organic acids (formic or acetic acid)

and salts (ammonium formate or acetate) on MS sensitivity was assessed. Organic acids were found to

A

improve sensitivity for CYFLU by a factor of seven but resulted in lower peak intensity for other analytes. Whereas ammonium salts, in particular ammonium formate, gave better overall sensitivity for a wider range of analytes. The effect of organic mobile phase modifiers was also investigated, showing that MeOH was more suitable than MeCN for the analysis of pyrethroids and pyrethrin I, II. However, the inclusion of MeOH resulted in a significant increase in back column pressure, which limited the maximum mobile phase flow rate to 0.3 mL min-1. In the end it was determined that the

most satisfactory results for pyrethroid analysis were achieved using mobile phases composed of (A) 5 mM ammonium formate in H2O:MeOH (80:20, v/v) as the aqueous component and (B) 5mM ammonium formate in MeOH as the organic component. The optimised gradient conditions allowed the elution of the analytes from the column in approximately 15 mins with an additional 7 min equilibration time required to stabilise chromatography between injections. Under these conditions,

IP T

each pyrethroid or pyrethrin eluted as one single or as two peaks. It can be seen from the LC-MS/MS traces presented in Figure 1 that allethrin, cyphenothrin, permethrin, phenothrin, resmethrin, tetramethrin and tralomethrin elute as two peaks, which were well resolved. The chromatographic

SC R

traces of cyfluthrin, cypermethrin and flumethrin also showed two peaks, but these were not fully

chromatographically resolved in the final gradient separation. In practice, both isomer peaks are

U

summed by integrating as one for quantitation and confirmatory purposes, with the exception of

A

N

tralomethrin.

ED

M

Insert Figure 1.

PT

3.2 Development of the sample preparation procedure As stated in the introduction, few analytical methods are available for the determination of

CC E

pyrethroids and pyrethrins in animal fat samples. Most published methods report animal fat as a difficult matrix that requires laborious sample preparation protocols that sometimes use nonenvironmentally friendly solvents and large amounts of glassware [19,35]. Thus a major objective of

A

this work was to develop a more efficient sample preparation protocol and less time consuming procedure for isolating pyrethroid and pyrethrin I,II pesticides residues from animal fat. In the current work, extracted fat was obtained by melting adipose tissue samples in an oven and collecting the dripping in test tubes. This approach is advantageous because it avoids the need for complex solvent based extraction [36]. The melting points of the major lipid molecules, namely, free fatty acids, monoglycerides, diglycerides and triglycerides) varies between -40 and 70°C [37]. The melting point

of fat molecules can vary depending on the number of carbon atoms, whether chains are straight or branched in nature, the degree of unsaturation and position of the fatty acid in the glycerol molecule. A number of preliminary experiments were carried out in our laboratory to investigate the impact of melting temperature on fat collection. Avian fat can be easily extracted from adipose tissue by melting adipose tissue at ≤40°C for 1 h. In contrast, the extraction of fat from the adipose tissue of bovine,

IP T

ovine and particularly porcine species requires higher melting temperatures for longer time periods. In practical application, a melting temperature of ≥60°C is required to allow the extraction of melted fat from the adipose tissue from a wide range of animal fats. In order to test the suitability of this

SC R

procedure, analyte stability was assessed in fat samples from different species (avian, bovine, ovine

and porcine) incubated at two temperatures (60 and 70°C) for different extraction times (0, 2 and 4 h).

U

The results of the study indicate that the analytes could be effectively extracted at both temperatures

N

(Figure 2). Consequently, a protocol was developed using a melting conditions temperature of 65±5°C for 2±1 h. It was decided not to use higher temperature because this was unnecessary and it could

M

A

potentially result in degradation of some analytes.

PT

ED

Insert Figure 2.

MeCN is frequently the solvent of choice in sample preparation protocols for animal tissue because

CC E

the solubility of proteins and carbohydrates decreases dramatically in this organic solvent, whereas the residues are easily dissolved [38]. However, MeCN is unsuitable for the direct extraction of residues from fat because it does not completely dissolve the sample matrix. Alternative extraction

A

approaches were evaluated including sonication or vortexing of liquefied fat with different extraction solvents (MeCN, MeCN:Acetone (75:25, v/v) or ethyl acetate) but were found to be ineffective, while more non-polar solvents such as ethyl acetate resulted in the co-extraction of unwanted lipid matrix components. This is in agreement with previous research that showed pesticide recovery from highly lipophilic matrices was impaired by the choice of extraction solvent [39]. In particular, MeCN was

found to be unsuitable for the direct extraction of pesticides from fatty foods because for the poor solubility of lipids in this solvent. Alternative approaches have been adopted by other groups such as accelerated solvent extraction (ASE) [40], supercritical fluid extraction (SFE) [41] and microwave assisted extraction (MAE) [42]. However, these techniques require capital investment, potentially have low sample throughput and can use large volumes of solvent. A number of methods have been

IP T

reported to analyse pyrethroid and other pesticides in animal fat that use large volumes of organic solvent, typically >100 mL, generating large volumes of waste solvent [19,36,43]. The trend in recent

years has been to increase sample throughput by using simpler protocols such as QuEChERS, matrix

SC R

solid phase dispersion solid phase extraction and low temperature sample clean-up [44]. Our laboratory previously adapted a published method to analyse five pyrethroid residues in animal fat

U

with GC-ECD detection [22]. In this method pyrethroid residues were extracted from melted fat

N

samples dispersed on Florisil® using MeCN and purified by C18 SPE clean-up. As a result, of the good experiences with this method, it was used as the starting point for further sample preparation

A

development work. The extraction of analytes from melted fat samples was evaluated using Florisil®

M

with different levels of deactivation (0, 6, 7.5, 10.5, and 15%). The results from this study found that

ED

samples extracted with Florisil® deactivated with 15% water gave the highest overall recovery of analytes. Some other dispersing materials were substituted in place of Florisil®, namely, sodium

PT

sulphate and magnesium sulphate but were found to be unsuitable. A further method development objective was to develop a rapid simple low cost sample clean-up

CC E

procedure that could be employed to analyse ≥30 test samples in a single day and would not produce large volumes of organic solvent waste. Two different sample purification approaches were investigated for the removal of matrix co-extractives, namely, low-temperature clean-up and

A

dispersive solid phase extraction (d-SPE). Low-temperature clean-up has been developed to purify the solvent extracts of animal tissue samples by exploiting differences in melting points between the analytes and matrix components (namely, lipids). The use of low-temperature clean-up has been applied to analyse residues in several different test matrices such as animal tissue [29,38,45-47], cheese [48], milk [48-50], egg [49], infant formula [51], honey [52], fish [53] and olive oil [53]. It is

proposed that lipids are precipitated from sample extracts at temperatures of -20 °C and below [47,48]. In contrast, the analytes remain dissolved in the extraction solvent, which has a lower freezing point [38,48]. The simple and efficient nature of low temperature clean-up also means that it greatly reduces the amount of solvent required through elimination of LLE steps, in comparison to the more traditional methods and reduces sample preparation time [45]. In this work, low temperature

IP T

clean-up was evaluated for MeCN extracts post solid phase dispersion. MeCN extracts were incubated at -20oC for 2, 4, and 16 h with 0 h as the control. It was found that a low temperature incubation

period of 4 h provided the best overall. In addition, it was decided to implement a rapid d-SPE clean-

SC R

up to provide additional clean-up of samples in order to negate samples matrix effects. A range of dSPE clean-up were evaluated during method development including C8, C18, NH2, Florisil®, PSA,

U

PSA/C18, Z-SEP and Z-SEP+ sorbents. These studies showed that Z-Sep+ or PSA provided the best

N

overall clean-up and recovery of analytes. The d-SPE step was further optimised by examining the impact of sorbent quantity and compositions on clean-up. The Z-SEP+ material existed as a single

A

hybrid material made up of zirconia and C18 bonded onto single silica particles and retained both fat

M

and pigments, selectively, through Lewis acid-base interactions. The zirconia portion of the particle

ED

acts as a Lewis acid, attracting compounds with electron donating groups, such as the polar hydroxyl groups of lipids, whilst the hydrophobic group of the sorbent interacts with the hydrophobic chains

PT

present in the lipids [55]. This optimisation process investigated different quantities of PSA (0, 75, 125, 150 and 250 mg) and Z-Sep+ (0, 50, 150, 250 and 500 mg). The results from this study showed

CC E

that a mixture of Z-Sep+/PSA (500 mg/125 mg) gave best overall clean-up and recovery.

A

3.3 Method Validation

3.3.1 Selectivity Method selectivity is one of the most important parameters and should be assessed at the start of the method validation process. This approach allows intervention changes to be implemented into

the analytical method to improve selectivity e.g. by modifying chromatographic conditions, selection of alternative MS/MS transitions, improving the sample preparation method or by inclusion of isotopically labelled internal standards. The selectivity of the method was firstly evaluated through injecting individual analyte and internal standard solutions. Cross-talk and isobaric interference was assessed through inspection of chromatograms for peaks that could interfere with the quantitation or

IP T

confirmatory ion of each analyte. This aspect of the method development process is important because such interfering peaks could potentially impact on the precision and accuracy of the analytical

method. Portolés et al. reported that the pyrethroids can produce similar non-specific ions in GC-EI-

SC R

MS, which can complicate both quantitation and identification [27]. In order, to offset this problem long gas chromatographic runs are often required because some pyrethroids can have up to four

N

and the selectivity of the method was deemed satisfactory.

U

diastereoisomers. Fortunately, it was found in our work that no cross-talk interference was observed

A

In addition, endogenous matrix effects were assessed by analysing 40 fat samples from different

M

animal species (avian, bovine, ovine and porcine) collected at Irish slaughter plants to investigate potential endogenous matrix interference. The results from this study showed that for the majority of

ED

analytes that matrix interfering peaks were not present or at background levels less than the lowest calibration point. In some samples, interfering peaks were observed in samples but at differing

PT

retention times. A matrix peak was observed in several chromatograms of the allethrin quantitation ion (303.2>169.1) but differed in retention time by -0.2 min. In other cases, analyte peaks were

CC E

confirmed in samples satisfying retention time and ion ratio criteria. For example, acrinathrin was detected in one porcine sample at a concentration of 7.2 µg/kg. Additionally, several samples were found to contain cypermethrin (n = 7) and deltamethrin (n = 5), which were generally present at

A

concentrations less than the lowest calibration level. Deltamethrin was measured in one porcine sample at a concentration of 8.4 µg/kg. In all cases the pesticide concentrations were below the MRLs laid down for fat samples. It is not surprising to find residues of these insecticides because they are routinely used to control external parasites on food producing animals.

LC-MS/MS is susceptible to matrix effects, which can affect accuracy and precision. Thus it is important to assess matrix effects by analysing samples collected from different animals and species. Samples from different species spiked post-extraction at a concentration of 250 µg kg-1 were injected onto the UHPLC-MS/MS system along with solvent standards at the equivalent concentration. The matrix effect was calculated in each species individually (Table 3). In general, it

IP T

can be seen that the response for pyrethrin I,II and pyrethroid analytes is enhanced in the presence of sample matrix. The greatest signal enhancement was seen for acrinathrin and fluvalinate with

responses increasing by two and five times, respectively. The matrix effect was generally consistent

SC R

with species with RSD values of typically ≤15%. The matrix effects were more variable in ovine fat for fluvalinate, tetramethrin and tralomethrin, which had RSDs of 39.5, 35.5 and 36.3%, respectively.

U

The sample matrix effects were negated by the inclusion of stable isotopically labelled internal

N

standards, which were available for bifenthrin, cypermethrin, deltamethrin and fenvalerate and incorporated into the method as outlined in Table 2. Internal standards were not used for two of the

M

A

analytes (allethrin I and tetramethrin) because they did not improve accuracy and/or precision.

PT

ED

Insert Table 3.

For the validation work all requirements in terms of signal-to-noise (S/N), ion ratios (IR) and

CC E

retention times (RT) were examined for all samples and stock standards used in validation work, and found to be in line with the EU requirements for all analytes under investigation. The RT of analyte peaks in matrix samples and stock standards were examined, with analyte peaks in matrix samples

A

within the 2.5% tolerance when compared to standard solutions. In addition to this, two transitions were monitored for all 20 analytes, with the most intense ion signal being used as the quantification ion. All IR’s of the samples were within the required tolerance, in terms of EU criteria, when compared to standards analysed during validation work and all S/N ratios were determined to be greater than 10.

3.3.2 Analytical performance of the method The retention times, ion ratios and S/N values for each analytes were also monitored throughout the validation studies, to ensure the qualitative analytical criteria for each analyte was adhered to. During

IP T

each validation run the retention times for each analyte were consistent at <2.5%, relative to the standard chromatographic peaks, and S/N values were >10 for each transition. European Union legislation (2002/657/EC) requires three identification points (IP’s) to confirm the presence of a group

SC R

B substance [34]. This was achieved throughout the validation studies through the monitoring of one precursor and two product ions. The ion ratios for the analytes in positive control samples were then

compared to those in validation samples, to confirm the presence of analytes. Through the application

U

of these criteria, no false-negative results were identified and all analytes were successfully

A

N

confirmed.

M

The linearity of the method, measured as R2, was found to be satisfactory for all analytes, R2 > 0.99. The intra- and inter-assay trueness and precision ranges were evaluated on three different days

ED

by one analyst (WLr) and on three different days by three different analysts (WLR), with three runs carried out in total by each analyst. The results from the validation studies show that the method

PT

trueness was in the acceptable range for the majority of analytes (Table 4 and 5). According to Commission Decision 2002/657/EC, acceptable ranges of trueness are 70-110% and 80-110% when

CC E

measuring concentrations of >1 to 10 µg/kg and >10 µg/kg, respectively [34]. In general it can be seen that repeatability and reproducibility precision values were the in the ranges of 3.9 to 22% and 5.2% to 23%, respectively. In some cases, particularly for analytes validated around the default limit

A

of 10 µg kg-1, precision marginally exceeded the acceptable range. The method precision should be as low as possible for analytes validated at levels lower than 100 µg kg-1. The exceptions were bovine fat WLR precision results for fenvalerate and tralomethrin at 200 and 10 µg kg-1, which were 26.5% and 28.4%, respectively.

Insert Tables 4 and 5.

3.3.3 Decision limit (CCα) and detection limit (CCβ) The decision limit (CCα) and the detection capability (CCβ) values for each analyte were

IP T

calculated using precision data generated from the WLR studies. The CCα is the most important

parameter for quantitative methods because this is the value at and above which a sample is deemed to

SC R

be non-compliant. In the case of licensed substances, CCα is always greater than the MRL but it is desirable that the gap between the two values is as low as possible to prevent violative results being

A

N

U

declared compliant.

M

Conclusions

ED

This research has shown that pyrethrin I,II and pyrethroid residues can be analysed directly by LCMS/MS. The method developed in this work is advantageous because it allows the analysis of

PT

pyrethroids residues that are not easily analysed by gas chromatography, namely, flumethrin and tralomethrin. In addition, the method uses on 21 mL of organic solvent compared to other published

CC E

or standard methods of analysis, which can use more than 100 mL. The method has a reasonably high throughput, with a single analyst capable of processing 30 test samples in one day. Furthermore, the developed method is between two and ten times more sensitive that the most comprehensive method

A

that have been published for animal fat. We recommend that further research should be made in the area of chromatography to further reduce the chromatographic run time and improve sample throughput.

In addition, the analysis of

pyrethroids and pyrethrin residues in farmed fish was not in the scope of this funded research project but should be further investigated with other antiparasitic agents.

Acknowledgements

IP T

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

SC R

References

U

[1] L.M. He, J. Troiano, A. Wang, K. Goh,;1; Environmenatl Chemistry, ecotoxicity, and fate of lambda-cyhalothrin, Rev. Environ. Contam. Toxicol. 195 (2008) 71-91.

M

A

N

[2] D.M. Soderlund, J.M. Clark, L.P. Sheets, L.S. Mullin, V.J. Piccirillo, D. Sargent, J.T. Stevens, M.L. Weiner,;1; Mechanisms of pyrethroid neurotoxicity: implications for cumulative risk assessment. Toxicol. 171 (2002) 3-59.

PT

ED

[3] K. Palmquist, A. Fairbrother, J, Salatas, Pyrethroid Insecticides: Use,;1; Environmental Fate, and Ecotoxicology. In: F. Perveen (Ed.), Insecticides Advances in Integrated Pest Management, InTech, (2012) 251-278. [4] C. Bouwknegt, P.A. van Rijn, J.J.M. Schipper, D. Hölzel, J. Boonstra, A.M. Nijhof, W.M.A van Rooij, F. Jongejan, Potential role of ticks as vectors of bluetongue virus. Exp. Appl. Acarol. 52(2) (2010) 183-192.

A

CC E

[5] European Parliament and Council, Regulation;1; (EC) No 470/2009 of the European Parliament and Council and of the Council of 6 May 2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/90 and amending Directive 2001/82/EC of the European Parliament and of the Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council. Off. J. Eur. Union. L152 (2009):11–22.

[6];1; European Union Parliament and Council, Regulation (EC) No 396/2005 of the European Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC. Off. J. Eur. Union. L70 (2005):1-16.

[7];1; The European Union, Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin, Off. J. Eur. Comm. L15 (2010) 1–72.

IP T

[8];1; The European Communities, Commission Decision 212/2013 of 11 March 2013, replacing Annex I to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards additions and modifications with respect to the products covered by that Annex, Off. J. Eur. Commun. L221 (2013) 30-52.

SC R

[9] P. Stefanelli, A. Santilio, L. Cataldi, R. Dommarco,;1; Multiresidue analysis of organochlorine and pyrethroid pesticides in ground beef meat by gas chromatography-mass spectrometry, J. Environ. Sci. Health B 44 (2009) 350-356.

N

U

[10] EU;1; – Pesticides database (available at http://eceuropaeu/food/plant/pesticides/eupesticides- database /public/?event=homepage&language=EN; accessed 07/04/16).

ED

M

A

[11] L. Cherta, T. Portolés, J. Beltran, E. Pitarch, J.G. Mol, F. Hernández,;1; Application of gas chromatography–(triple quadrupole) mass spectrometry with atmospheric pressure chemical ionization for the determination of multiclass pesticides in fruits and vegetables, J. Chromatogr A 1314 (2013) 224-240.

CC E

PT

[12] A. Di Muccio, P. Pelosi, D.A. Barbini, T. Generali, A. Ausili, F. Vergori,;1; Selective extraction of pyrethroid pesticide residues from milk by solid-matrix dispersion, J. Chromatogr. A 756 (1997) 5160.

A

[13] M. Shamsipur, N. Yazdanfar, M. Ghambarian,;1; Combination of solid-phase extraction with dispersive liquid–liquid microextraction followed by GC–MS for determination of pesticide residues from water, milk, honey and fruit juice, Food Chem. 204 (2016) 289-297.

[14] C. Anagnostopoulos, K. Liapis, S.A. Haroutounian, G.E. Miliadis,;1; Development of an Easy Multiresidue Method for Fat-Soluble Pesticides in Animal Products Using Gas Chromatography– Tandem Mass Spectrometry, Food Anal. Methods 7 (2014) 205-216.

[15] G.F. Pang, Y.Z. Cao, J.J. Zhang, C.L. Fan, Y.M. Liu, X.M. Li, G.Q. Jia, Z.Y. Li, Y.Q. Shi, Y.P. Wu, T.T. Guo,;1; Validation study on 660 pesticide residues in animal tissues by gel permeation chromatography cleanup/gas chromatography–mass spectrometry and liquid chromatography– tandem mass spectrometry, J. Chromatogr. A 1125 (2006) 1-30.

IP T

[16] F. Jia, W. Wang, J. Wang, J. Yin, Y. Liu, Z. Liu,;1; New strategy to enhance the extraction efficiency of pyrethroid pesticides in fish samples using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method, Anal. Methods 4 (2012) 449-453.

SC R

[17];1; United States Department of Agriculture Food Safety and Inspection Service, Determination of Chlorinated Hydrocarbons (CHCs) and Chlorinated Organophosphate Hydrocarbons (COPs) with Gel Permeation Chromatography (GPC) (2010) 1-29.

N

U

[18] European Union,;1; Official Method of Analysis EN 1528/1-4, European Committee of Standardization, Brussels, Belgium, (1996).

M

A

[19] C.J. Akre, J.D. MacNeil,;1; Determination of eight synthetic pyrethroids in bovine fat by gas chromatography with electron capture detection, J. AOAC. Int. 89 (2006) 1425-1431.

PT

ED

[20] Z.C. Kodba, D.B. Vončina,;1; A Rapid Method for the Determination of Organochlorine, Pyrethroid Pesticides and Polychlorobiphenyls in Fatty Foods Using GC with Electron Capture Detection, Chromatographia 66 (2007) 619-624.

CC E

[21] J.W. Park, A.M. Abd El-Aty, M.H. Lee, S.O. Song, J.H. Shim,;1; Residue analysis of organophosphorus and organochlorine pesticides in fatty matrices by gas chromatography coupled with electron-capture detection, J. Biosci. 61 (2006) 341-346.

A

[22] F. Sun, F. Lin, S.S. Wong, G.C. Li,;1; The Screening of Organophosphorus, Organochlorine and Synthetic Pyrethroid Pesticides Residues in Beef Fat by Tandem Solid-Phase Extraction Technique, J. Food Drug Anal. 11 (2003) 258-265.

[23] Z.M. Chen, Y.H. Wang,;1; Chromatographic methods for the determination of pyrethrin and pyrethroid pesticide residues in crops, foods and environmental samples, J. Chromatogr. A 754 (1996); 367-395.

[24] R. Juhler,;1; Optimized method for the determination of organophosphorus pesticides in meat and fatty matrices, J. Chromatogr. A 786 (1997) 145-153.

IP T

[25];1; European Reference Laboratory, EUPT AO 11, 11th European Proficiency Test on Pesticides in Food of Animal Origin and Commodities with High Fat Content (2016) 1-88.

SC R

[26] C. Corcellas, E. Eljarrat, D. Barceló,;1; Chapter 9 - Determination of Pyrethroid Insecticides in Environmental Samples by GC–MS and GC–MS–MS. In: F. Imma, E.M. Thurman, eds. Comprehensive Analytical Chemistry, Elsevier, 61 (2013) 203-230.

N

U

[27] T. Portolés, J.G.J. Mol, J.V. Sancho, F. Hernández,;1; Advantages of Atmospheric Pressure Chemical Ionization in Gas Chromatography Tandem Mass Spectrometry: Pyrethroid Insecticides as a Case Study, Anal. Chem. 84 (2012) 9802-9810.

ED

M

A

[28] R.P. Carneiro, F.A.S. Oliveira, F.D. Madureira, G. Silva, W.R. de Souza, R.P. Lopes,;1; Development and method validation for determination of 128 pesticides in bananas by modified QuEChERS and UHPLC–MS/MS analysis, Food Control 33 (2013) 413-423.

PT

[29] S.W.C. Chung, C.H. Lam,;1; Development and validation of a method for determination of residues of 15 pyrethroids and two metabolites of dithiocarbamates in foods by ultra-performance liquid chromatography–tandem mass spectrometry, Anal. Bioanal. Chem. 403 (2012) 885-896.

CC E

[30] W. Li, M.K. Morgan, S.E. Graham, J.M. Starr,;1; Measurement of pyrethroids and their environmental degradation products in fresh fruits and vegetables using a modification of the quick easy cheap effective rugged safe (QuEChERS) method, Talanta 151 (2016) 42-50.

A

[31] D.B. Martínez, P.P. Vázquez, M.M. Galera, M.D.G. García,;1; Determination of Pyrethroid Insecticides in Vegetables with Liquid Chromatography Using Detection by Electrospray Mass Spectrometry, Chromatographia 63 (2006) 487-491.

[32] C. Soler, J. Mañes, Y. Picó,;1; Liquid chromatography–electrospray quadrupole ion-trap mass spectrometry of nine pesticides in fruits, J. Chromatogr. A 1048 (2004) 41-49.

[33] A. Valverde, A. Aguilera, M. Rodrı ́guez, M. Boulaid,;1; What are we determining using gas chromatographic multiresidue methods: tralomethrin or deltamethrin?, J. Chromatogr. A 943 (2001) 101-111.

IP T

[34];1; Commission Decision 2002/657/EC of 12 August 2002, implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, Off. J. Eur. Commun. L221 (2002) 8–36.

SC R

[35] M. LeDoux,;1; Analytical methods applied to the determination of pesticide residues in foods of animal origin. A review of the past two decades, J. Chromatogr. A 1218 (2011) 1021-1036.

A

N

U

[36] F. Bordet, D. Inthavong, J.M. Fremy,;1; Interlaboratory Study of a Multiresidue Gas Chromatographic Method for Determination of Organochlorine and Pyrethroid Pesticides and Polychlorobiphenyls in Milk, Fish, Eggs, and Beef Fat, J. AOAC Int. 85 (2002) 1398-1409.

ED

M

[37] E. Dickinson, D.J. McClements,;1; Fat Crystallization in Oil-in-Water Emulsions. In: E. Dickinson, D.J. McClements, eds. Advances in Food Colloids. Glasgow, UK. Chapman and Hall (1995) 211–246.

CC E

PT

[38] J. Zhan, D.M. Xu, S.J.Wang, J. Sun, Y.J. Wu, M.L. Ni, J.Y. Yin, J. Chen, X.J. Yu, Z.Q. Huang,;1; Comprehensive screening for multi-class veterinary drug residues and other contaminants in muscle using column-switching UPLC-MS/MS, Food Addit. Contam. A (2013) 1888-1899.

[39] S.J. Lehotay, K. Mastovska, S.J. Yun,;1; Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrixes, J. AOAC Int. 88 (2005) 630-638.

A

[40] G. Wu, X. Bao, S. Zhao, J. Wu, A. Han, Q. Ye,;1; Analysis of multi-pesticide residues in the foods of animal origin by GC–MS coupled with accelerated solvent extraction and gel permeation chromatography cleanup, Food Chem. 126 (2011) 646-654.

[41] S.R. Rissato, M.S. Galhiane, F.R.N. Knoll, B.M. Apon.;1; Supercritical fluid extraction for pesticide multiresidue analysis in honey: determination by gas chromatography with electron-capture and mass spectrometry detection, J. Chromatogr. A 1048 (2004) 153-159.

[42] M. Weichbrodt, W. Vetter, B. Luckas,;1; Microwave-assisted extraction and accelerated solvent extraction with ethyl acetate-cyclohexane before determination of organochlorines in fish tissue by gas chromatography with electron-capture detection, J. AOAC Int. 83 (2000) 1334-1343.

IP T

[43] European Union.;1; Official Method of Analysis EN 1528/1-4, European Committee of Standardization, Brussels, Belgium, 1996.

SC R

[44] B. Kinsella, J. O’Mahony, E. Malone, M. Maloney, H. Cantwell, A. Furey, M. Danaher,;1; Current trends in sample preparation for growth promoter and veterinary drug residue analysis, J. Chromatogr. A 1216 (2009) 7977-8015.

A

N

U

[45] R.P. Lopes, D.V. Augusti, A.G.M. Oliveira, F.A.S. Oliveira, E.A. Vargas, R. Augusti,;1; Development and validation of a methodology to qualitatively screening veterinary drugs in porcine muscle via an innovative extraction/clean-up procedure and LC-MS/MS analysis, Food Addit. Contam. A 28 (2011) 1667-1676.

ED

M

[46] R.P. Lopes, D.V. Augusti, L. Francisco de Souza, S.A. Santos, J.A. Lima, E.A. Vargas, E. Augusti, Development and validation;1; (according to the 2002/657/EC regulation) of a method to quantify sulfonamides in porcine liver by fast partition at very low temperature and LC-MS/MS, Anal. Methods 3 (2011) 606-613.

CC E

PT

[47] G. Rübensam, F. Barreto, R.B. Hoff, T.M. Pizzolato,;1; Determination of avermectin and milbemycin residues in bovine muscle by liquid chromatography-tandem mass spectrometry and fluorescence detection using solvent extraction and low temperature cleanup, Food Control 29 (2013) 55-60.

A

[48] J. Xie, T. Peng, A. Zhu, J. He, Q. Chang, X. Hu, H. Chen, C. Fan, W. Jiang, M. Chen, J. Li, S. Ding, H. Jian,;1; Multi-residue analysis of veterinary drugs, pesticides and mycotoxins in dairy products by liquid chromatography–tandem mass spectrometry using low-temperature cleanup and solid phase extraction, J. Chromatogr B 1002 (2015) 19-29.

[49] S.W.C. Chung, C.H. Lam,;1; Development of a 15-class multiresidue method for analyzing 78 hydrophilic and hydrophobic veterinary drugs in milk, egg and meat by liquid chromatographytandem mass spectrometry, Anal. Methods 7 (2015) 6764-6776.

[50] S.M. Goulart, M.E.L.R. de Queiroz, A.A. Neves, J.H. de Queiroz,;1; Low-temperature clean-up method for the determination of pyrethroids in milk using gas chromatography with electron capture detection, Talanta 75 (2008) 1320-1323.

IP T

[51] J. Zhan, Y.Y. Zhong, X.J. Yu, J.F. Peng, S. Chen, J.Y. Yin, J.J. Zhang, Y. Zhu,;1; Multi-class method for determination of veterinary drug residues and other contaminants in infant formula by ultra performance liquid chromatography–tandem mass spectrometry, Food Chem. 138 (2013) 827-834.

SC R

[52] G.P. de Pinho, A.A. Neves, M.E.L.R. de Queiroz, F.O. Silvério,;1; Optimization of the liquid–liquid extraction method and low temperature purification (LLE–LTP) for pesticide residue analysis in honey samples by gas chromatography, Food Control 121 (2010) 1307-1311.

A

N

U

[53] S. Chen, X. Yu, X. He, D. Xie, Y. Fan, J. Peng,;1; Simplified pesticide multiresidues analysis in fish by low-temperature cleanup and solid-phase extraction coupled with gas chromatography/mass spectrometry, Food Chem. 113 (2009) 1297-1300.

ED

M

[54] C. Lentza-Rizos, E.J. Avramides, F. Cherasco,;1; Low-temperature clean-up method for the determination of organophosphorus insecticides in olive oil, J. Chromatogr. A 912 (2001) 135-142.

A

CC E

PT

[55] R.A. Henry,;1; A Novel Chemical Route to Stable, Regenerable Zirconia-Based Chiral Stationary Phases for HPLC, Am. Lab 37 (2005) 22-24.

N

U

SC R

IP T

Figure:

A

CC E

PT

ED

M

A

Figure 1. LC-MS/MS trace of fat sample fortified at a concentration of 100 µg/kg of each analyte.

I 2h/60C

140

2h/70C

4h/70C

100 80

Recovery (%)

A

4h/60C

M

Recovery (%)

120

N U SC R

Avian

160

60 40

ED

CC E

160

PT

0

2h/60C

4h/60C

2h/70C

4h/70C

2h/60C

4h/60C

2h/70C

4h/70C

140 120 100 80 60

Bovine

20 0

2h/60C

4h/60C

2h/70C

160

4h/70C

140

140

120

120

100

100

80 60

Recovery (%)

Recovery (%)

Porcine

40

20

A

160

80 60

40

40

20

20

0

0

Ovine

Figure 2. Assessment of the impact different temperature (60°C and 70°C) and times (2 h and 4 h) on the stability of pyrethrin and pyrethroid during the adipose tissue melting process. Results are expressed as mean recovery (%) ± S.D (n = 3 different samples for each species) relative to fat samples spike post melting. The sample fortification and spiking level was 250 µg kg -1.

Tables:

1

NL = Not listed.

CC E A

Porcine

U

SC R

50 NL1 50 50 20 NL1 100 100 NL1 20 10 NL1 50 50 50 20 10 NL1 NL1

The MRLs are according to Pesticide Database. The MRLs in parenthesis are taken from 37/2010 EC.

PT

2

50 NL1 3000 200 500 NL1 2000 (200)2 500 (50)2 NL1 200 10 150 50 50 50 20 300 NL1 NL1

A

50 NL1 3000 200 (50)2 500 NL1 2000 (200)2 500 (50)2 NL1 250 10 150 50 (500)2 50 50 20 300 NL1 NL1

ED

Acrinathrin Allethrin Bifenthrin Cyfluthrin Cyhalothrin Cyphenothrin Cypermethrin Deltamethrin Fenpropathrin Fenvalerate Flucythrinate Flumethrin Permethrin Phenothrin Pyrethrins Resmethrin Tau-Fluvalinate Tetramethrin Tralomethrin

N

Bovine

M

Compound

Maximum Residue Limits (µg kg-1) Ovine Avian

IP T

Table 1. Maximum residues limits for pyrethrin and pyrethroid pesticide residues in avian, bovine, ovine and porcine fat.

50 NL1 3000 200 500 NL1 2000 500 NL1 30 10 NL1 50 50 50 20 300 NL1 NL1

Table 2. UHPLC-MS/MS conditions for pyrethrin and pyrethroid pesticides.

14.759 β-Cyfluthrin

8.811 & 8.987

λ-Cyhalothrin 9.782 Cypermethrin

9.046 & 9.316

Cyphenothrin

9.283 & 9.954

Deltamethrin

9.437

Fenpropathrin

8.150

Fenvalerate 9.759 Flucythrinate

8.322

Flumethrin

14.386 & 14.662

Permethrin

ED

10.155 & 10.990 Phenothrin

Pyrethrin I, II

PT

10.357 & 11.043

8.312

CC E

Resmethrin

9.536 & 10.057

τ-Fluvalinate Tetramethrin

A

Tralomethrin

Deltamethrin-D5 Fenvalerate-D5

Rt, retention time CE, collision energy c CA, cell accelerator b

6.376

10.268 & 11.616

Bifenthrin-D5 Cypermethrin-D6

a

12.24

14.572 9.012 9.369 9.691

79

9 41

4

Bifenthrin-D5

67

5 99

4

None

94

9 9 45

4

Bifenthrin-D5

67

9 9

4

99

9 5 9

4

9 9

4

9 21

4

13

4

79 73 117

4

9 13 45

4

76

9 50

4

114

9 21

4

73

17 5 13

4

81

13 13 49

4

67

5 13 33

4

111

9 21 45

4

67

9 25

4

67

21 13

4

79

13 13

4

108 114 108 99

9 9 13 13

7 7 7 7

79

Cypermethrin-D6 Cypermethrin-D6

9 33

67

IP T

Bifenthrin

Internal Standard

SC R

6.797 & 7.064

CA (V)c

U

Allethrin I

559.2 → 208.0 559.2 → 181.0 303.2 → 169.1 303.2 → 135.1 303.2 → 93.1 442.2 → 181.0 440.2 → 181.1 440.2 → 166.1 453.1 → 192.9 451.1 → 190.9 469.1 → 227.0 467.1 → 450.0 467.1 → 225.0 435.1 → 193.0 433.1 → 191.0 393.2 → 151.1 393.2 → 123.1 525 → 282.9 523 → 280.8 350.2 → 125.1 350.2 → 97.2 439.1 → 168.9 437.1 → 167.1 437.1 → 124.9 469.2 → 412.1 469.2 → 157.1 527.1 → 267.0 527.1 → 239.0 410.1 → 183.0 408.1 → 355.1 408.1 → 183.0 351.2 → 249.1 351.2 → 183 351.2 → 153.1 329.2 → 161.1 329.2 → 143.1 329.2 → 105.1 339.2 → 171.0 339.2 → 143.1 339.2 → 128.0 503.1 → 208.0 503.1 → 181.0 332.2 → 164.0 332.2 → 135.1 684.9 → 442.6 682.9 → 440.6 445.3 → 186 439.2 → 197 528.1 → 280.8 442.3 → 167.1

CE (eV)b

N

13.401

Fragmentor (V)

A

Acrinathrin

Transiiton (m/z)

M

Rt (mins)a

Analyte

Cypermethrin-D6 Cypermethrin-D6

Deltamethrin-D5

Cypermethrin-D6

Fenvalerate-D5 Cypermethrin-D6

Bifenthrin-D5 Cypermethrin-D6

Cypermethrin-D6

Cypermethrin-D6

Cypermethrin-D6

Cypermethrin-D6

None Cypermethrin-D6 IS IS IS IS

A ED

PT

CC E

IP T

SC R

U

N

A

M

Table 3. Matrix effects study results for pyrethrin and pyrethroid analytes in the fat tissue of different animal species. Bovine (n = 10)

Ovine (n = 9)

Porcine (n = 10)

MEa

RSD b

ME

ME

ME

(%)

(%)

(%)

RSD (%)

(%)

RSD (%)

(%)

RSD (%)

Acrinathrin

-164.3

14.0

-209.5

10.6

-153.4

19.1

-193.8

9.3

Allethrin I

-21.6

12.7

-32.8

8.8

-15.0

12.4

-36.6

10.9

Bifenthrin

-22.8

14.2

-36.9

9.0

-13.6

17.2

-36.9

11.3

Cyfluthrin

-69.5

14.3

-44.7

12.9

-46.2

15.1

-69.1

14.4

Cyhalothrin

-91.8

13.4

-114.9

11.1

-80.5

16.3

-122.9

10.6

Cypermethrin

-58.0

12.2

-67.2

9.0

-47.1

14.0

-72.9

9.8

Cyphenothrin

-21.1

12.4

-29.0

10.1

-11.2

15.4

-33.0

12.7

Deltamethrin

-51.5

12.3

-70.3

8.9

-46.8

14.4

-74.8

11.0

Fenpropathrin -31.5

12.3

-43.7

-22.5

14.9

-49.8

10.5

Fenvalerate

-33.7

13.5

-42.7

10.3

-23.5

14.5

-54.9

12.2

Flucythrinate

-33.2

13.0

-48.4

9.1

-27.7

13.0

-54.0

11.7

Flumethrin

-75.9

12.1

-99.4

10.7

-68.6

16.4

-105.7

12.9

Permethrin

-11.3

13.1

-17.8

12.3

-2.4

15.1

-20.9

12.4

Phenothrin

-25.2

13.0

-39.1

9.9

-15.2

14.1

-41.6

11.8

SC R

U

N

A

M

ED

PT

8.7

IP T

Avian (n = 10)

CC E

Analyte

-23.7

13.3

-39.8

10.3

-19.7

13.0

-41.2

12.5

Resmethrin

-12.6

11.5

-25.1

9.8

-5.1

15.5

-25.3

11.1

Fluvalinate

-379.7

11.3

-511.3

12.7

-388.0

39.5

-499.0

12.3

Tetramethrin

-31.1

12.1

-43.0

8.6

-22.0

35.5

-48.7

9.6

Tralomethrin

-37.7

14.3

-55.5

9.6

-32.4

36.3

-58.9

9.4

A

Pyrethrin I, II

a b

ME, matrix effects RSD, relative standard deviation

I N U SC R

Table 4. Within-laboratory repeatability and reproducibility validation results for bovine fat samples at MRL. Analyte

Validation Levels (µg kg-1)

Within-laboratory Repeatability (WLr) (n=8)

Within-laboratory Reproducibility (WLR) (n=8)

A

CC E

PT

ED

M

A

Trueness (%) CV (%) Trueness (%) CV (%) 0.5VL 1.0VL 1.5VL 0.5VL 1.0VL 1.5VL 0.5VL 1.0VL 1.5VL 0.5VL 1.0VL 1.5VL Acrinathrin 50 103 93 107 5.5 14.3 13.2 104 95 96 13.0 19.2 24.9 Allethrin 10 108 111 115 21.5 17.9 9.1 119 109 118 26.9 17.0 12.7 Bifenthrin* 10 118 109 106 20.8 4.7 4.8 137 112 109 19.9 8.7 11.8 Cypermethrin 200 110 105 104 12.5 11.6 11.4 117 113 115 8.9 14.4 9.6 Cyphenothrin 10 120 104 104 18.6 7.1 12.2 143 108 106 14.8 9.3 18.9 Deltamethrin 50 103 104 97 9.1 11.2 6.0 106 107 111 11.0 14.6 11.1 Fenpropathrin 10 116 104 108 16.0 7.5 12.3 135 102 104 16.7 10.7 17.0 Fenvalerate 250 108 103 101 16.3 14.1 9.7 115 111 123 14.5 26.5 23.3 Flucythrinate 50 110 109 114 5.8 6.4 12.5 110 105 107 10.1 12.0 18.7 Flumethrin 150 87 90 92 8.3 7.5 7.7 89 93 94 12.2 7.3 13.1 Permethrin 500 87 95 100 7.9 7.6 12.1 94 100 98 8.3 15.5 14.3 Phenothrin 50 101 104 110 6.3 7.3 11.2 110 109 107 8.0 14.7 17.5 Pyrethrin I, II 50 104 103 107 4.8 6.3 7.0 105 101 99 7.6 9.8 12.4 Resmethrin 100 89 91 97 5.7 5.4 11.7 95 93 96 7.5 11.2 16.5 Tetramethrin 10 96 97 99 17.6 8.8 8.3 111 95 94 18.3 11.1 9.1 Tralomethrin 50 102 90 101 15.2 26.8 19.5 111 96 104 13.3 28.4 17.5 β-Cyfluthrin 50 99 97 101 15.0 16.5 12.0 94 99 94 14.3 20.3 17.3 λ-Cyhalothrin 500 94 95 101 5.2 7.8 12.6 97 95 100 7.2 12.3 15.2 τ-Fluvalinate 300 91 90 100 5.7 6.4 13.0 94 95 99 8.5 14.7 18.9 Bovine Validation; WLR = 3 runs on 3 different days by three different analysts Key: VL = validation level, MRL = maximum residue limits, * Bifenthrin has an MRL of 3000 µg/kg, Bovine Validation; WLr = 3 runs on 3 different days by one analyst. Key: VL = validation level, MRL = maximum residue limits, * Bifenthrin has an MRL of 3000 µg/kg,

I N U SC R

Table 5. Within-laboratory reproducibility validation results for ovine and avian fat samples at MRL.

Trueness (%) 0.5VL 1.0VL 1.5VL 107 116 95 107 108 102 108 110 108 113 116 115 122 112 108 90 92 92 97 98 91 102 102 102 107 111 106 88 87 88 97 99 91 107 111 103 97 103 98 95 101 92 102 105 97 115 111 100 108 110 110

0.5VL 11.1 11.1 5.5 7.5 10.7 5.9 9.3 8.3 5.5 14 8.7 7.3 5.7 5.8 5.2 15.5 15.1

CV (%) 1.0VL 1.5VL 11.6 15.9 8.4 9.7 4.7 4.3 7 9.7 8.4 13.4 4.3 5.4 4.9 10.6 8.9 8.4 6.4 14 19.8 19.1 9.3 16.6 7.6 13.7 7.1 15.1 5.0 12.6 5.5 12.0 14.7 17.6 14.8 17.9

Validation Levels (µg kg-1)

101

106

97

12.5

12

20.1

300

106

111

110

11.8

9.1

18.6

10

M

A

500

50 10 50 100 10 100 10 250 50 10 50 50 50 100 10 10 50 20

Avian Fat Within-laboratory Reproducibility (WLR) (n=8) Trueness (%) CV (%) 0.5VL 1.0VL 1.5VL 0.5VL 1.0VL 1.5VL 95 107 109 32.3 23.1 30.8 102 102 103 12.5 7.4 13.3 101 104 104 7.7 5.1 4.4 101 103 102 9.1 7.4 5.8 121 114 116 18.4 13.2 20.9 99 97 96 7.1 7.9 4.9 104 107 111 12.3 9.9 14.5 101 99 101 12.8 6.2 5.6 97 108 120 19.2 12.6 16.0 110 107 107 16.0 11.9 14.5 94 104 117 30.7 17.6 21.1 99 109 115 25.5 14.7 17.9 87 95 106 20.4 13.2 17.2 84 95 101 24.9 13.7 16.7 94 101 109 12.5 10.4 10.9 94 101 110 27.8 14.7 14.1 106 107 105 14.8 8.6 7.4 94 107 118 23 18.5 22.6 125

123

118

23.7

16.2

A

τ-Fluvalinate

Ovine Fat Within-laboratory Repeability (WLr) (n=8)

ED

50 10 10 200 10 50 10 250 50 150 50 50 50 100 10 50 50

CC E

Acrinathrin Allethrin Bifenthrin* Cypermethrin Cyphenothrin Deltamethrin Fenpropathrin Fenvalerate Flucythrinate Flumethrin Permethrin Phenothrin Pyrethrin I, II Resmethrin Tetramethrin Tralomethrin β-Cyfluthrin λ-Cyhalothrin

Validation Levels (µg kg-1)

PT

Analyte

Ovine Validation; WLr = 3 runs on 3 different days by one analyst. WLR = 3 runs on 3 different days by three different analysts. Key: VL = validation level, MRL = maximum residue limits, * Bifenthrin has an MRL of 3000 µg/kg. Avian Validation; WLr = 3 runs on 3 different days by one analyst. WLR = 3 runs on 3 different days by three different analysts. Key: VL = validation level, MRL = maximum residue limits, * Bifenthrin has an MRL of 50 µg/kg.

24.5