Fluorescence polarisation immunoassays for strobilurin fungicides kresoxim-methyl, trifloxystrobin and picoxystrobin

Author’s Accepted Manuscript Fluorescence Polarisation Immunoassays for Strobilurin Fungicides Kresoxim-methyl, Trifloxystrobin and Picoxystrobin Anna Kolosova, Ksenia Maximova, Sergei A. Eremin, Anatoly V. Zherdev, Josep V. Mercader, Antonio Abad-Fuentes, Boris B. Dzantiev www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(16)30814-1 http://dx.doi.org/10.1016/j.talanta.2016.10.063 TAL16983

To appear in: Talanta Received date: 27 July 2016 Revised date: 11 October 2016 Accepted date: 15 October 2016 Cite this article as: Anna Kolosova, Ksenia Maximova, Sergei A. Eremin, Anatoly V. Zherdev, Josep V. Mercader, Antonio Abad-Fuentes and Boris B. Dzantiev, Fluorescence Polarisation Immunoassays for Strobilurin Fungicides Kresoxim-methyl, Trifloxystrobin and Picoxystrobin, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.10.063 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 galley proof before it is published in its final citable 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.

Fluorescence Polarisation Immunoassays for Strobilurin Fungicides Kresoxim-methyl, Trifloxystrobin and Picoxystrobin Anna Kolosovaa,1*, Ksenia Maximovab,1,2, Sergei A. Ereminb, Anatoly V. Zherdeva, Josep V. Mercaderc, Antonio Abad-Fuentesc, Boris B. Dzantieva a

A.N. Bach Institute of Biochemistry, Federal Research Centre ‘Fundamentals of Biotechnology’

of the Russian Academy of Sciences, Leninsky prospect 33, 119071 Moscow, Russia b

M.V. Lomonosov Moscow State University, Faculty of Chemistry, Department of Chemical

Enzymology, Leninsky Gory 1, 119991 Moscow, Russia c

Department of Biotechnology, Institute of Agrochemistry and Food Technology, Consejo

Superior de Investigaciones Científicas (IATA_CSIC), Agustín Escardino 7, 46980, Paterna, València, Spain *

Corresponding author: [email protected]

Abstract Fluorescence polarisation immunoassays (FPIAs) based on monoclonal antibodies for detection of three strobilurin fungicides - kresoxim-methyl (KM), trifloxystrobin (TF) and picoxystrobin (PC), were developed and optimised. Fluorescein-labeled derivatives of target antigens (tracers) were synthesised and purified by thin-layer chromatography. Influence of tracer structures on the assay parameters was investigated. For KM and TF, the best assay performance was achieved with the homologous pairs of reagents. For the PC assay, the heterologous tracer, i.e. fluoresceinlabeled derivative of TF, was used. The developed FPIAs were applied to the determination of KM, TF and PC in red wine. Most optimal sample preparation was achieved with cross-linked poly(vinylpyrrolidone) as a sorbent. This clean-up is simple, rapid and allows determination of all three strobilurin fungicides in one sample. Detection limits of the developed FPIAs in red wine were 28, 6 and 5 ng/mL for KM, TF and PC, respectively. Recovery in spiked samples averaged between 80 and 104%. Intra- and inter-assay coefficients of variance were less than 12%. The developed FPIA methods can be applied to screening of wine samples for KM, TF and PC residues without complicated cleanup.

1

These authors contributed equally to this work Present address: FEMTO-ST Institute, department MN2S, Bourgogne Franche-Comté University, 15B av. des Montboucons, 25030 Besançon, France 2

Keywords: strobilurin; fungicide; fluorescence polarisation immunoassay; tracer heterology; wine

Introduction

Synthetic strobilurins comprise a modern growing family of fungicides. Anke and coworkers was first who, in 1977, identified their natural analogues, strobilurins A and B, produced by the fungus Strobilurus tenacellus and Oudemansiella mucida [1, 2]. Natural strobilurins are photolabile and therefore, not suitable for plant protection. Since their discovery, various chemical modifications have been performed to produce synthetic strobilurin fungicides with desired properties, which were extensively reviewed [2-5]. All currently manufactured strobilurins are synthetic nature-derived chemicals, which comprise a common aromatic bridge providing photochemical stability to the molecule and holding both the toxophore moiety responsible for the fungicide activity (typically, an enol ether, an oxime ether ester, or an oxime) and a distinctive bulky substituent at the ortho-position [4, 6]. The first molecules to be produced, azoxystrobin and kresoxim-methyl, as well as most strobilurin-active principles of the second generation, contain the same biologically active moiety as the natural compound or with minor changes involving the acrylate, acetate or acetamide chemical group with E configuration of the double bond in the toxophore moiety. Picoxystrobin and azoxystrobin are the only two synthetic strobilurin fungicides which retain the natural toxophore group. Nevertheless, by application of quantitative structural-activity relationships, other compounds with more drastic chemical modifications without losing the antifungal activity have been synthesised [7]. Strobilurins belong to the fungicides of the Quinone outside Inhibitors (QoI) group. These agrochemicals exhibit their fungicide activity through binding to the Qo site of cytochrome b, thus blocking the electron transfer between cytochrome b and cytochrome c1 and inhibiting mitochondrial respiration [5, 8]. Strobilurin fungicides constitute a major class of modern crop and culture protection compounds. They are currently used worldwide for disease control in most cereals, fruits, and vegetables [4]. Strobilurin fungicides are relatively readily degraded and represent low risk to human health as well as to birds, mammals, and bees, although they vary in their toxicity to aquatic organisms [9]. Even though fungicides are usually applied according to the rules of Good Agricultural Practices, residues are regularly detected in agricultural commodities, foods and aquatic environments [10-13]. The presence of trace amounts of such xenobiotics can result in adverse effects to humans and other non-target organisms. With acute oral, dermal, and

inhalation effects, strobilurin fungicides are classified Category III – Caution [4]. Symptoms and signs in humans may include eye and respiratory irritation, weakness, dizziness, skin redness and chest pain [14]. Hence, these substances must be controlled to safeguard human health and to protect the environment. In addition, the presence of multiple pesticide residues, even at low concentrations, may induce higher toxicological and carcinogenic effects [13]. Therefore, the regulatory bodies of most countries have defined maximum residue levels (MRLs) for various pesticides, including strobilurins, in food and feed commodities [15, 16]. Kresoxim-methyl (KM, Figure 1a) released by BASF in 1992 belongs to the first generation of strobilurins. In 1999, it was included in Annex I of the EU Council Directive 91/414/EEC as an approved non-systemic active substance for plant protection [17]. For most fruits, vegetables and cereals, European and Russian MRLs range between 0.05 and 1 mg/kg [16, 18]. Trifloxystrobin (TF, Figure 1b) has been registered in more than 80 countries. Nowadays it is commercialised by Bayer Crop Science under different trade names (Flint, Delaro, Madison, etc.) as the only active ingredient or formulated together with other fungicides. TF is one of the best-sold strobilurins. It is widely employed to fight fungal diseases in a variety of crops such as cereals, strawberries, oranges, grapes, tomatoes, cucumbers, etc. In the EU, it was approved as low risk active substance for plant protection in 2003 [19]. European MRLs for TF in most products range from 0.02 to 15 mg/kg [18], while Russian MRLs - from 0.02 to 5 mg/kg [16]. According to 2013 EU report on pesticide residues in food, TF was one of the frequently found pesticides [20]. Picoxystrobin (PC, Figure 1c), launched as a fungicide in 2001, has been approved in the EU in 2003. This strobilurin shows preventive and curative properties. It is currently formulated and commercialised under different trademarks by DuPont and Syngenta for cereal and oilseed crop protection [4, 5, 21]. PC is one of the fungicides accepted by the British Beer and Pub Association (BBPA) and the Brewing Research International (BRI) for use on barley and wheat intended for malting and brewing [22]. According to EC regulation 396/2005, the MRL for PC in rye and wheat is 0.05 mg/kg, whereas the MRL of 0.3 mg/kg has been established for barley and oat [15, 18]. Russian MRL for most cereals and oilseeds is also 0.05 mg/kg, and for the cereals for bread making is 0.2 mg/kg [16]. Nowadays, monitoring of pesticide residues is compulsory for governments and private corporations involved in food processing. Thus, suitably validated and standardised analytical methods, as well as rapid screening tests for cost-effective food control on a large scale, are necessary for the detection of fungicides. Chromatographic methods are currently most employed techniques for pesticides’ residue determination. Mass and tandem mass spectrometry

(MS and MS-MS) are widely used as detection systems for such analysis. To determine levels of strobilurin fungicides in different matrices, a variety of methods have been proposed, mainly as multiresidue strategies applying high-performance liquid chromatography (HPLC) or gas chromatography (GC) with different detection systems and sample preparation techniques [12, 23-26]. Although sensitive and reliable, these methods are mostly expensive, laborious, time consuming, and require sophisticated equipment and skilled personnel. Among other techniques, electrochemical detection of strobilurin fungicides has been reported [21, 27]. Flow injection chemiluminescence method with off-line ultrasonic treatment for monitoring of azoxystrobin in water samples [28], as well as flow-through optosensor combined with photochemically induced fluorescence for determination of azoxystrobin in grapes, must and wine [29], are also worth mentioning. Immunochemical methods represent alternative and/or complementary techniques for pesticide monitoring [9, 30]. Such features of these methods as sensitivity, specificity, rapidity, simplicity and cost-effectiveness convert them into very powerful tools for pesticide residue analysis involved in large monitoring programs when high sample throughput and on-site screening analysis are required. Undoubtedly, the most common immunochemical method for small organic molecules is the competitive enzyme-linked immunosorbent assay (ELISA). During the last decade, ELISA methods enabling sensitive determination of some strobilurin fungicides (KM, TF, PC, azoxystrobin and pyraclostrobin) in different foodstuffs have been continuously reported [7, 9, 19, 22, 31-34]. These techniques made use of both polyclonal and monoclonal antibodies. Apart from that, a general strategy was established for the synthesis of functionalised derivatives and the introduction of spacer arms to strobilurins [6, 7, 9, 32]. Basic studies regarding the relationship between the structure of strobilurin haptens and the activity of the generated antibodies have been published [6, 35, 36]. Produced immunoreagents were also evaluated in ELISAs in terms of influence of their structure on the assay parameters. Recently, an alternative immunochemical approach has been demonstrated for the determination of a novel strobilurin fungicide benzothiostrobin by competitive and non-competitive phage immunoassays [37]. One of the most popular sample preparation procedures for pesticide residue determination in various food matrices is the QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction methodology. It has been first published by Anastassiades and co-workers [38] and since then, was broadly applied, mostly with the chromatographic methods. However, this approach was also used for some strobilurin ELISAs [31, 33]. The detection limits of ELISA techniques are comparable to or even lower than the ones obtained with the chromatographic methods [9, 31, 34, 35]. However, ELISA is a heterogeneous

method, and separation of free and antibody-bound analyte as well as a long reaction time (1-2 h) are usually needed; besides, this method involves multiple washing steps. A very promising way for simplification of immunoassays for routine applications is a shift from heterogeneous methods (with separation) to homogeneous assays (without separation). Fluorescence polarization immunoassay (FPIA) is one of the most extensively used homogeneous techniques, which meets the requirements of a simple, reliable, fast and costeffective analysis. Applications of this method to the determination of different compounds, including pesticides and mycotoxins, have been proposed in several works, such as [39-41]. However, the existing application of FPIA to determination of strobilurin fungicides is limited to detection and quantification of azoxystrobin in grape extract and river, lake, and well water samples [34]. With the use of polyclonal antibodies, the authors developed and compared three immunoassay formats, i.e. ELISA, FPIA and time-resolved fluorescence immunoassay. They concluded that while the three formats were comparable in terms of performance, the FPIA was the least labour-intensive and required the least time to perform. To our knowledge, this is the only paper reporting the FPIA method for a strobilurin fungicide so far. The present paper focuses on the development and optimization of FPIA using monoclonal antibodies (MAbs) for the determination of three strobilurin fungicides (KM, TF and PC) and application of this technique to wine samples.

Materials and methods

Materials Analytical grade KM, TF, PC and other related fungicides were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Bovine serum albumin (BSA, fatty acid free fraction V), poly(ethylene glycol) 6000 (PEG 6000), N,N-dicyclohexylcarbodiimide (DCC), Nhydroxysuccinimide (NHS), fluorescein isothiocyanate (FITC, isomer I), sodium azide, common salts and solvents were also supplied by Sigma Aldrich Chemical Co. NH2-derived silica (Bond Elute

NH2)

was

from

Varian

Inc.

(Palo

Alto,

CA,

USA),

and

cross-linked

poly(vinylpyrrolidone) (Divergan) was from BASF (Florham Park, NJ, USA). Thin-layer chromatography (TLC) plates (silica gel 60, fluorescent, 1 mm, 20 x 20 cm) were obtained from Merck Co. (Darmstadt, Germany). All chemicals and organic solvents were of reagent grade or higher. Sodium borate buffer supplemented with 0.1% sodium azide (0.5 M, pH 9.0, BB) was used in all FPIA experiments.

Standard solutions of KM, TF, PC, and other strobilurins were prepared by dilution of stock solutions of these compounds (1 mg/mL, in methanol). Fluoresceinthiocarbamyl ethylenediamine (EDF) was synthesised from FITC and ethylenediamine as described previously [42] with modifications [43]. Haptens KM6, KMgg, KMβag, KMgab, TF0, TFgg, TFβag, and TFgab (Figure 2), as well as monoclonal antibodies against KM (MAb anti-KM6), TF (MAb anti-TF0), and PC (MAb anti-PCo6), were produced and characterised at the Department of Biotechnology, Institute of Agrochemistry and Food Technology (València, Spain) as previously described [9, 22, 32].

Apparatus

Measurements of fluorescence polarisation and intensity were performed using TDx Analyzer (Abbott Laboratories, USA) in semi-automatic PhotoCheck mode. TDx glass cuvettes (up to 10 in one run) were loaded into the special carousel followed by the measurement of polarization (in mP units) and intensity (in conventional units) of fluorescence. The total time for measurement of 10 samples was about 7 min. Agilent 6410 Triple Quadrupole LC/MS tandem mass spectrometer was used to obtain mass spectra for the tracers. The spectra were recorded in electropositive mode.

Synthesis of fluorescein-labelled KM tracers

NHS (4.6 mg, 40 µmol) and DCC (8.6 mg, 40 µmol) were dissolved in 0.5 mL of absolute DMF and 15 µmol of KM derivative (KM6, KMgg, KMβag and KMgab) was added. The reaction mixture was stirred for 4 h at room temperature. Formed precipitate was separated by centrifugation of the reaction mixture at 14,000 rpm for 5 min. EDF (6.7 mg, 15 µmol) was then added to the supernatant. The reaction mixture was stirred for 2 h at room temperature and then left overnight. Small portions of the reaction mixture (20-50 µL) were separated by TLC using chloroform as eluent. The plates were dried and eluted again using chloroform/methanol (4:1, v/v). The major yellow band at Rf 0.9 was collected, eluted with 0.5 mL of methanol and stored at -20 °C in the dark. The tracer concentrations were estimated spectrophotometrically at 492 nm (extinction coefficient - 8.78 x∙104 (M-1 cm-1)).

Synthesis of fluorescein-labelled TF tracers

The synthesis procedure was similar to that for KM-EDF tracers, only 11 µmol TF derivative (TF0, TFgg, TFβag, TFgab) and 4.9 mg (11 µmol) EDF were used. After tracer purification, the major yellow band at Rf 0.9 was collected and eluted with 0.5 mL of methanol, and stored at -20 °C in the dark. The tracer concentrations were estimated similarly to KM tracers.

Fluorescence Polarisation Immunoassay

Dilution curves

To 0.5 mL tracer solution (10 nmol/L) in the TDx glass cuvettes was added 0.5 mL MAb in various dilutions, mixed, and analysed using fluorescence polarisation. Solution containing BB (0.5 mL) instead of MAb was used as a background check.

Competitive FPIA procedure

Fifty microliters of strobilurin standard solution (or sample), 0.5 mL tracer solution, and 0.5 mL MAb in optimal dilution were added sequentially to the cuvette and mixed followed by the measurement of fluorescence polarisation (mP). Standard curves were plotted as mP (or mP/mP0) versus logarithm of analyte concentration, where mP0 - fluorescence polarisation value at zero competition. To determine intra- and inter-assay coefficients of variance (CVs), three replicates of three solutions containing different concentrations of strobilurin fungicide within the dynamic range of the standard curve (low, medium and high) were tested three times a day on three different days. The mean concentration and CV were calculated on nine determinations for each solution within one day and between days. Cross-reactivity (CR) for different strobilurin fungicides was determined by performing competitive assays and comparing the analyte concentration giving half-maximal inhibition (IC50, ng/mL) of the mean blank (zero competition, mP0) and calculated as % CR = (IC50 for KM (TF, PC)/IC50 for analyte) x 100 Strobilurin concentrations in spiked samples were calculated after fitting of the standard curve using the four-parameter logistic model.

Association constants

Association constants of antigen-antibody complex were determined using Scatchard plot [44] as a slope of the curve plotted as B/F versus B, where B is the concentration of antigenantibody complex, and F is the concentration of free antigen. Based on fluorescence polarisation measurements, B/F was calculated according to the formula proposed in [45]:

(B/F)i = (mPi – mPmin)/(mPmax – mPi), where mPi – measured fluorescence polarisation, mPmax - fluorescence polarisation at the maximum antigen-antibody binding, mPmin - fluorescence polarisation at the minimum antigen-antibody binding. B values were determined as

Bi = T x (B/F)i/((B/F)i + 1), where T – initial antigen concentration in the system.

Sample preparation

Red wine (Cabernet Sauvignon) was purchased from a local retail store (Moscow, Russia). Strobilurin-free wine (previously confirmed by HPLC) was spiked with KM, TF or PC at different concentration levels and treated with the respective procedure along with the blank (non-spiked) wine sample. Three sample preparation procedures were used in our study. For the first procedure [46], 1 mL of sodium acetate buffer (0.2 M) containing 0.17 M NaCl and 0.1% BSA (pH 5.0) was added to 0.5 mL red wine and mixed for 10 min. The treated sample was centrifuged at 12,100 x g for 40 min and the supernatant was analysed in five replicates by FPIA. For the second procedure [47], 0.5 mL of the solution containing 1% PEG 6000 and 5% NaHCO3 (pH 8.5) was added to 0.5 mL red wine. Then the sample was passed through the column with different amounts of NH2-derived silica and analysed in five replicates by FPIA. The third method was direct clean-up of the red wine (1 mL) on the column with different amounts of cross-linked PVP. The purified sample was analysed in five replicates by FPIA.

Results and discussion

Synthesis and characterisation of fluorescent-labelled derivatives (tracers)

In common with other immunochemical techniques, FPIA requires production and characterisation of immunoreagents as well as optimisation and validation of an analytical system. Antibodies and fluorescent-labelled haptens (tracers) are key components in the development of FPIA. It was shown that assay specificity generally depends on the immunogenic hapten, whereas assay sensitivity is usually affected by the competitive hapten [7, 9, 32, 35, 36, 48]. Several KM and TF derivatives were used in our study (Figure 2). In these derivatives, β– iminoacetate toxophore contained different spacer arms with a terminal carboxyl group. For KM6, it was a 6-carbon hydrophobic spacer obtained with 6-aminohexanoic acid. Other KM derivatives contained dipeptide linkers, Gly-Gly, β-Ala-Gly and Gly-γ-aminobutyric acid, for KMgg, KMβag and KMgab, respectively. Thus, a polar chemical group (an amide) was introduced inside the spacer arm, while maintaining similar linker lengths. This approach made it possible to investigate the influence of a spacer heterology on the assay performance. Spacer arms prepared with dipeptides have been previously reported for the improvement of assay sensitivity or catalytic antibody production [49, 50]. Haptens TFgg, TFβag, and TFgab contained the dipeptide linkers similar to the KM haptens. To obtain the fluorescent-labelled derivatives (tracers), all KM and TF haptens were conjugated with EDF using activation of the hapten free carboxyl group and subsequent formation of the amide bond with EDF. For the tracer TF0-EDF, the fluorescent label was conjugated directly to the TF moiety. The tracers were purified by TLC and the reaction products with Rf = 0.9 were obtained. To control the tracer syntheses, structures of the tracers KM6-EDF and TF0-EDF were confirmed by mass-spectrometry (Figure S1). The peaks with m/z 845.2 (Figure S1a) and 826.6 (Figure S1b) corresponded to the molecular weights of KM6-EDF and TF0-EDF, respectively.

Antibody dilution curves

Tracer working dilutions were optimised for the assay. The FPIA is a homogeneous assay technique based on differences in polarisation of the fluorescence labelled species in the free and bound fractions. It involves competition between a free analyte and a tracer for binding to a

specific antibody. Hence, the tracer concentration is one of the key parameters for development and optimisation of the FPIA procedure, which markedly influences the assay sensitivity. Tracer determines intensity of the emitted polarised light and contributes to the competition for antibody binding. Thus, the lowest possible tracer concentration, which allows reliable detection of a label and produces the minimum effect on the competition, should be used to develop a sensitive assay. Commonly for FPIA, total fluorescence intensity of the solution with the optimum tracer concentration is 10-15 times higher than the background signal (for the buffer). For strobilurin tracers, the lowest concentration was approximately 10 nM in the final reaction, corresponding to the working dilutions of 1:30,000; 1:5100; 1:6250 and 1:4900, for the tracers KM6-, KMβag-, KMgab- and KMgg-EDF, respectively. For TF0-, TFβag-, TFgab- and TFgg-EDF, the working dilutions were 1:40,000; 1:5500; 1:5500 and 1:17,500, respectively. All synthesised KM and TF tracers were tested for binding with antibodies using the FPIA dilution curves obtained for KM and TF MAbs against immunogens KM6-BSA and TF0BSA, respectively (Figure 3). The MAb working concentrations were determined as the antibody concentration giving 80% binding with a tracer. For the quantitative assessment of tracers’ binding with MAbs, association constants have been estimated using Scatchard plot (Table 1). They averaged from 4 x 107 to 4 x 109 M-1. It is easy to notice that structures of tracers KM6-EDF and TF0-EDF are homologous to the respective immunogens. All KM tracers demonstrated good binding with the antibodies (Figure 3a). Interestingly, the highest antibody affinity was observed for tracer KMgab-EDF with the bridge heterology and longest spacer arm. For immunoassays, strongest binding is commonly observed for a homologous pair of reagents. Bearing in mind the fact that FPIA is a homogeneous technique and all the interactions take place in the aqueous medium, we could assume that in case of KMgab-EDF, longer and more hydrophilic linker allowed a favourable molecular conformation and better exposure of the KM moiety resulting in stronger antibody binding compared to the homologous tracer. For the TF tracers, homologous to the immunogen TF0-EDF exhibited strongest antibody binding (Figure 3b). TFgg- and TFgab-EDF gave much weaker binding, most probably due to the linkers incorporated in the structure of these tracers. On the contrary, structure of the immunogen TF0-BSA does not involve any spacer between the TF moiety and the protein carrier. Probably in this case, it was the main factor to consider. Virtually no binding with MAbs was observed for TFβag-EDF. It is worth mentioning that for the KM tracers, antibody affinity for the tracer with β-Ala-Gly spacer arm was also the lowest. Perhaps this bridge structure gave the least favourable conformation of the tracer molecule in the solution and steric hindrance probably occurred.

Obtained results suggest different tendencies in MAb binding with KM and TF tracers. For KM FPIA, all the tracers and the immunogen had the spacer arms, and antibody binding was mostly determined by the tracer molecular conformation in the solution, which largely depended on the linker structure and length. Whereas for TF, the presence or absence of the bridge in the immunoreagent molecule was crucial. For the development of FPIA for PC, structure similarity between PC and TF (Figure 1) was taken into consideration. Therefore, tracer TF0-EDF was tested for binding with MAb against PC. Structure of the PC immunogen is shown in the Figure 4. Dilution curve demonstrated some binding between the tracer and MAb (data not shown). Association constant for this pair of reagents was 4.6 x 106 М-1 (Table 1). Therefore, tracer TF0-EDF could be used for the development of FPIA method for PC determination.

Competitive FPIA procedures

Competition between free and fluorescein-labelled strobilurins for binding with antibodies was investigated using different tracers and assay conditions were optimised. For KM assay, all the tracers gave similar assay sensitivity (Table 2) and dose-response curves had nearly identical slopes (Figure 5a). Homologous tracer KM6-EDF was chosen for further assay development. FPIA with this tracer was most sensitive and characterised by the lowest detection limit (21 ng/mL), broad dynamic range (59-22,200 ng/mL) and low variability (intra- and interassay CVs were less than 3%). Commonly, immunoassay methods can benefit from utilizing competitors for which the antibody has slightly lower affinity than to the analyte. In case of FPIA, the highest antibody affinity was observed for the heterologous tracer KMgab-EDF. Thus, FPIA with homologous tracer KM6-EDF with lower MAb affinity appeared to have the best performance. The same MAb and haptens were investigated by Mercader et al. [9] for the development of indirect competitive ELISA. In their work, heterologous coating conjugates resulted in slightly lower IC50 values compared to the homologous conjugate. This tendency was in agreement with the commonly found variations when immunoreagents’ heterology is used in MAb-based assays [9, 51]. The best assay sensitivity was achieved with the heterologous conjugate obtained from KMgab hapten. Homogeneous immunoassay techniques, such as FPIA, are generally known to be less sensitive than heterogeneous ones. The detection limit of the current method was higher than that for ELISA developed with the same MAb (0.3 ng/mL). As a rapid and simple method for

screening of a large number of samples, FPIA, however, has substantial advantages, such as short assay time (7–10 min compared to hours) and absence of separation or washing as everything is performed in a single step in comparison with excessive handling required in ELISA. For TF FPIA, homologous tracer TF0-EDF with the highest antibody affinity resulted in the best assay performance (Figure 5b). Detection limit was 5 ng/mL and with appropriate dilutions, TF could be determined from 8 to 135 ng/mL. Within- and between-assay CVs were less than 5%. Heterologous to the immunogen tracers TFgg- and TFgab-EDF resulted in slightly higher slopes than the homologous tracer did (Table 2). However, the dose-response curves with these tracers were shifted in the higher concentration region and therefore, the detection limits were also much higher than for the homologous assay. The same MAb and haptens were used by Mercader et al. [32] for the development of indirect competitive ELISA. For the coating conjugates of ovalbumin with TF haptens, they observed the same tendency as for KM ELISA, i.e. modest reductions of the IC50 values with the conjugates using dipeptides compared to the homologous conjugate. Similar to KM ELISA, the assay with the lowest IC50 value was achieved with the heterologous conjugate OVA-TFgab. As described before, tracer TF0-EDF also showed an appropriate binding with MAb against PC, which was used for the development of FPIA for this strobilurin fungicide (Figure 6). Detection limit for this method was 3 ng/mL and assay dynamic concentrations ranged from 5 to 950 ng/mL. The FPIA was characterised by good reproducibility: intra- and inter-assay CVs were less than 4%.

Cross-reactivity

Cross-reactivity (CR) of different strobilurin fungicides was investigated using the developed assays (Table 3). Calibration curves were prepared up to 0.1 mg/L and measured by FPIA. FPIA for TF was highly specific: for azoxystrobin and PC calculated CRs were 1.9 and 2.5%, respectively. For other compounds, CRs were negligible (<0.1%). Similar results were obtained for the above-mentioned TF ELISA [32]. Noticeable CR was observed only in case of azoxystrobin, PC and dimoxystrobin with the values of 3.1, 2.3, and 1.3%, respectively. For PC FPIA, the highest CR of 9.7% was observed for dimoxystrobin. This compound was also recognised by MAb against KM with CR value of 55% in FPIA. Cross-reactivity for dimoxystrobin in the indirect ELISA was 409% with respect to KM [9]. This result is not surprising due to close structural similarity between KM and dimoxystrobin. Moreover, the

toxophore moiety of dimoxystrobin contains the amide group likewise the amide incorporated into KM immunogen structure during derivatisation of the KM molecule. Those results suggest that the toxophore moiety is one of the epitopes recognised by the MAb.

FPIA of KM, TF and PC in red wine

Maximum residue limits (MRLs) for the presence of pesticides in processed food products are not commonly regulated, so tolerances established for raw commodities like grapes generally apply to wine [52]. Despite the fact that strobilurin fungicides are relatively readily degraded and their level in wine is usually lower than in grapes, their residues have been repeatedly reported in recent studies as often encountered in wines [10, 11]. Recently, ELISAs have been applied to monitoring of multiple fungicide residues in international wines [52]. Commercial wine samples (n = 280) of different geographical areas and characteristics were included in the survey. More than 40% of the samples contained at least one of the targets (>10 µg/L), while residue contents higher than 100 µg/L were found in 8.4% of the samples. This study shows that contamination of commercial wines with fungicides is an issue of worldwide relevance with potential implications for consumer health and international trade. Red wine is generally more complex matrix than white wine and is rich in flavonoids and tannins. Some flavonoid compounds mainly determine the colour of red wine. Tannins, which contain some flavonoid polyphenols, are most commonly found in red wine [53]. Various sample clean up procedures have been developed for the analyses of wine. For fungicide determination in wine by chromatographic methods, liquid as well as solid-phase extraction using different organic solvents is applied [12, 23-26]. However, such clean-up procedures are often laborious and time-consuming. Hence, for the FPIA suggested as a rapid alternative method, sample preparation should be kept as simple as possible. Apart from that, organic solvents have strongly negative effect on immunoglobulins. FPIA is susceptible to interference with different components existing in some matrices. Thus, polyphenols from wine can interfere with an assay due to their non-specific binding with antibodies. To correct for matrix interferences in wine, sample dilution in water or buffer is commonly used in ELISA [52, 54]. Dilutions of up to 250 times were possible due to low detection limits of some ELISAs. Precipitation of tannins in the acetate buffer as well as solidphase extraction using C-18 column were also employed in ELISA for fungicide determination in wine [46]. For the developed FPIA, detection limit did not allow high sample dilution. Therefore, different approaches were used in our study.

The first clean up procedure was based on the precipitation of tannins in the acetate buffer [46]. Samples of red wine were mixed with the buffer (1:2) and after centrifugation, the supernatants were analysed by FPIA. To study the matrix effect, the standard curves for three strobilurins were prepared in the supernatant and compared with the respective standard curve in deionised water. Effects observed for KM and PC were similar to those for TF (Figure 7a). It was clear that, although simple and rapid, this sample preparation did not allow sufficient reduction of matrix effects and therefore, was not suitable. Another approach used in our investigation was similar to that described in [47]. Application of some polymers, such as PEG or PVP, to the preparation of wine samples has been reported for mycotoxin immunoassays [55, 56]. In our study, wine sample was diluted (1:1) with a solution containing 1% PEG and 5% NaHCO3. Then the sample was passed through a column with NH2derived silica. Different sorbent amounts were investigated and standard curves in the treated red wine were compared to that in deionised water (Figure 7b). Near-optimal standard curves could be obtained with 0.3 g sorbent. However, all the recoveries determined for KM, TF and PC using this sample preparation procedure were lower than 36% (Table S1), probably due to the partial adsorption of strobilurins on the column. Therefore, this sample clean-up could not be used for the determination of strobilurin fungicides in wine by FPIA. The third approach to the sample preparation was wine clean up with the use of cross-linked PVP (Divergan) as a sorbent. This sorbent is known to adsorb polyphenols. Small amounts of Divergan are used in winemaking for clarification of wine. In our work, 1 mL wine was passed through the column with PVP. To optimise the procedure, different sorbent amounts were investigated. Figure 8 shows standard curves for TF in the treated red wine and in deionised water. Similar results were obtained for KM (Figure S2) and PC (Figure S3). Thus, using 0.5 g of the Divergan sorbent for 1 mL wine allowed elimination of matrix interferences. It is worth mentioning that with this procedure, there was no need for additional sample dilution. Apart from that, this sample cleanup is simple, rapid and allows determination of all three strobilurin fungicides in one sample. Furthermore, recoveries were determined using spiked wine samples. Red wine was spiked with KM, TF and PC at different concentration levels, subsequently passed through the Divergan column and analysed in five replicates by FPIA (Table 4). Obtained recoveries averaged between 80 and 104%. No false positive results for blank (non-spiked) samples were observed. Detection limits of the developed FPIAs in red wine were 28, 6 and 5 ng/mL for KM, TF and PC, respectively. Intra- and inter-assay CVs were less than 12%. The developed FPIA

techniques allowed accurate determination of KM, TF and PC in wine at concentrations well covering the respective MRLs. Obtained recoveries for each strobilurin demonstrated efficiency of the developed assays and their applicability to the analysis of wine.

4. Conclusion

In conclusion, FPIA methods based on monoclonal antibodies for determination of three strobilurin fungicides, KM, TF and PC, were developed and optimised. Tracers with different structures (four KM and four TF ones) were synthesised, purified and examined for antibody binding. Influence of the tracer structure on the parameters of KM and TF FPIAs was investigated. The best assay performances were achieved with the homologous pairs of reagents. For PC FPIA, heterologous to the immunogen tracer TF0-EDF was used. The developed assays were applied to the analysis of red wine. Three sample preparation procedures were examined. The best results were obtained with the cross-linked PVP (Divergan) as a sorbent. Detection limits of the developed FPIAs in red wine were 28, 6 and 5 ng/mL for KM, TF and PC, respectively. Strobilurin spikes in red wine were determinable by the FPIAs with good recovery. Based on these results, the developed assays appear to meet the performance criteria for monitoring of KM, TF and PC residues in wine samples without complicated cleanup.

Acknowledgement

The work was supported by the Russian Science Foundation (project number 14-1600149). We would like to acknowledge Ms. Anna Boroduleva from the Faculty of Chemistry, Lomonosov Moscow State University, for recording MS spectra of the tracers.

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Figure 1. Structures of kresoxim-methyl (a), trifloxystrobin (b) and picoxystrobin (c) Figure 2. Structures of kresoxim-methyl (a) and trifloxystrobin (b) haptens Figure 3. Dilution curves for KM MAb with different KM tracers (a) and TF MAb with different TF tracers (b) Figure 4. Structure of picoxystrobin immunogen Figure 5. FPIA standard curves for KM (a) and TF (b). For KM FPIA, MAb KM6-BSA and tracers KM6-, KMgg-, KMβag- and KMgab-EDF were used; for TF FPIA, MAb TF0-BSA and tracers TF0-, TFgg-, and TFgab-EDF were used.

Figure 6. FPIA standard curve for PC. Anti-PC MAb and tracer TF0-EDF were used. Figure 7. FPIA standard curves for TF in red wine and deionised water using different sample preparations: (a) precipitation of tannins in the acetate buffer and (b) with the solution containing 1% PEG and 5% NaHCO3 followed by the purification with different amounts of NH2–derived silica. Sample volume – 1 mL. Homologous pair of reagents MAb TF0-BSA and tracer TF0EDF was used. Figure 8. FPIA standard curves for TF in red wine and deionised water using sample preparation with different amounts of cross-linked PVP. Sample volume – 1 mL. MAb TF0-BSA and tracer TF0-EDF were used.

Table 1. MAb working concentrations and association constants for different KM and TF tracers MAb working concentration, µg/mL 4.4

Tracer

Association constant, М-1

KMβag-EDF

(4.0 ± 1) x 107

KMgg-EDF

(1.9 ± 0.2) x 108

2.6

KMgab-EDF

(1.5 ± 0.3) x 10

9

1.3

KM6-EDF

(1.7 ± 0.4) x 108

2.9

TFβag-EDFa

No binding

-

TFgg-EDFa

(2.3 ± 0.4) x 108

1.6

TFgab-EDFa

(2.6 ± 0.2) x 108

1.6

TF0-EDFa

(4.2 ± 0.3) x 109

0.4

b

TF0-EDF a b

(4.6±0.3) x 10

6

5.0

binding with anti-TF MAb binding with anti-PC MAb

Table 2. Assay parameters for KM and TF using different tracers Dynamic concentration rangeb, ng/mL 59 – 22,200

Slopec

KM6-EDF

Detection limita, ng/mL 21

KMβag-EDF

49

55 – 9250

0.82

KMgg-EDF

129

180 – 10,000

0.70

KMgab-EDF

67

75 – 8900

0.80

TF0-EDF

5

8 – 135

1.53

Tracer

0.89

KMgg-EDF

135

240 – 2960

1.66

TFgab-EDF

115

153 – 1280

1.98

a

determined as the concentration of strobilurin that gives an observed signal three times the standard deviation of the mean blank (zero competition, mP0) b determined as the strobilurin concentration required for 10-90% inhibition of mP0 c absolute value, determined at 50% inhibition (IC50) of mP0

Table 3. Cross-reactivity of the MAbs against KM, TF and PC in FPIA Cross-reactivity in FPIA, %

Compound, structure

KM

TF

PC

100

<0.1

1.0

<0.1

100

<0.1

<0.1

2.5

100

<0.1

1.9

<0.1

<0.1

<0.1

<0.1

KM CH3 O

H3C

O

O

N

CH3

O

TF F3C

N

O

H3C

O

C

O

N

CH3

O

PC F3C

N

O O

H3C

O

C

CH3

O

Azoxystrobin N

N

O CN

O H3C

O

O

C

CH3

O

Pyraclostrobin

N Cl

O

N H3C

O

C O

N

O

CH3

Dimoxystrobin CH3 O

H3C CH3

55 H N

C

N

O

<0.1

9.7

CH3

O

Table 4. Recovery of strobilurins in red wine for the sample preparation using cross-linked PVP as a sorbent Amount added, ng/mL Amount determineda, ng/mL Recovery, % Kresoxim-methyl 60

61 ± 3

102 ± 5

80 100 150

78 ± 3 95 ± 4 137 ± 5

98 ± 4 95 ± 4 91 ± 4

200

192 ± 5

96.0 ± 2.5

Trifloxystrobin 10 20 30 50 100

9±1 19 ± 1 25 ± 2 45 ± 3 93 ± 4

90 ± 10 95 ± 5 83 ± 7 90 ± 6 93 ± 4

Picoxystrobin 10 20 50 100 150 a

8±1 18 ± 1 44 ± 2 94 ± 3 156 ± 4

80 ± 10 90 ± 5 88 ± 4 94 ± 3 104.0 ± 2.7

data are the mean of five assays

Highlights x

Fluorescence polarisation immunoassays for three strobilurins were developed

x

Tracers with different structures were synthesised

x

Influence of tracer structure on the assay performance was investigated

x

Rapid and simple determination of strobilurins in red wine with good recovery

x

Optimal sample clean-up with cross-linked poly(vinylpyrrolidone)

CH3 O

H3C

O

O

N

CH3

O

(a)

F3C

O

N

H3C

O

C

N

O

CH3

O

(b)

F3C

N

O

H3C

O

C O

(c)

Figure 1

O

CH3

CH3 O R

N O

O

H3 C

(a)

CH3 F3C

N

O R N H3C

O

O

(b)

KM(TF)gg: KM(TF)βag: KM(TF)gab: KM6: TF0:

Figure 2

R = NHCH2CONHCH2CO2H R = NHCH2CH2CONHCH2CO2H R = NHCH2CONHCH2CH2CH2CO2H R = NHCH2CH2CH2CH2CH2CO2H R = OH

KM6-EDF KMβag-EDF KMgg-EDF KMgab-EDF

1,0

mP/mP0

0,8

0,6

0,4

0,2

0,0

0,1

1,0

10,0

Concentration of MAb (Pg/mL)

(a)

TFgg-EDF TFβag-EDF TFgab-EDF TF0-EDF

250

mP

200

150

100

50

0,0

0,1

1,0

Concentration of MAb (Pg/mL) (b)

Figure 3

10,0

Figure 4

KM6-EDF KMβag-EDF KMgab-EDF KMgg-EDF

100 90

mP/mP0, %

80 70 60 50 40 30 0

10

100

1 000

10 000

100 000

Concentration of KM (ng/mL)

(a)

TF0-EDF TFgg-EDF TFgab-EDF

100

mP/mP0, %

90 80 70 60 50 40 30 0

1

10

100

1 000

Concentration of TF (ng/mL)

(b)

Figure 5

10 000

100 000

110

100

mP

90

80

70

60 0

1

10

100

1 000

Concentration of PC (ng/mL)

Figure 6

10 000

110

water red wine

100 90

mP/mP0, %

80 70 60 50 40 30 0

1

10

100

1 000

10 000

100 000

Concentration of TF (ng/mL)

(a)

110

water wine, 0.1 g sorbent wine, 0.2 g sorbent wine, 0.3 g sorbent

100 90

mP/mP0, %

80 70 60 50 40 30 0

1

10

100

Concentration of TF (ng/mL)

(b)

Figure 7

110

water wine, 0.5 g sorbent wine, 0.2 g sorbent

100

mP/mP0, %

90 80 70 60 50 40 30 0

1

10

100

Concentration of TF (ng/mL)

Figure 8

F3C

CH3 N O OCH3

N

OCH3

Trifloxystrobin

O

O O OCH3

N

OCH3

Kresoximmethyl

CH3 F3C

N

O

OCH3

OCH3

Picoxystrobin

O