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Pipette-tip solid-phase extraction using polypyrrole as efficient
adsorbent for multidetermination of avermectins and milbemycins in
milk
Diego Hernando Angulo, Roseane Andrade Teixeira, Ricky Cássio Santos da Silva, Bruna Carneiro Pires, Flávia Viana Avelar Dutra and Keyller Bastos. Borges* Departamento de Ciências Naturais, Universidade Federal de São João del-Rei, Campus Dom Bosco, Praça Dom Helvécio 74, Fábricas, 36301-160, São João del-Rei, Minas Gerais, Brazil
- Correspondence: Prof. Keyller Bastos Borges, PhD, Departamento de Ciências Naturais, Universidade Federal de São João del-Rei, Campus Dom Bosco, Praça Dom Helvécio 74, Fábricas, 36301-160, São João del-Rei, Minas Gerais, Brazil. *e-mail: keyller@ufsj.edu.br Phone: +55 32 3379 – 2481 Fax: +55 32 3379 – 2483
Abstract In this work, we developed a method for the multidetermination of avermectins (AVM) (abamectin - ABA 1b and ABA 1a, eprinomectin - EPR and ivermectin - IVM) and milbemycins (moxidectin - MOX) in milk samples employing pipette-tip solid-phase extraction using polypyrrole (PT–PPy–SPE) as adsorbent and analyzed by HPLC-UV. PPy (adsorbent material) was characterized by scanning electron microscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy and X-ray diffraction and the data agree with the literature. The sample preparation included the clean-up of the milk by protein precipitation (PP) with acetonitrile and extraction of the analytes by PT–PPy–SPE. The chromatographic method was developed in reverse phase and isocratic mode with flow rate at 1.2 mL min–1^ and ultraviolet detection at 250 nm. The mobile phase composition was acetonitrile: methanol: water (55: 25: 20, v/v/v). The studied parameters of sample preparation and the optimized conditions were: washing solvent (300 μL water), volume and type of eluent (700 μL methanol), volume and pH of sample (1 mL and pH 10), amount of adsorbent material (50 mg PPy), and without addition of salt (NaCl). The method showed to be linear over the concentration range from 20 to 3000 ng mL−1^ with coefficients of correlation ( r ) ≥ 0.99 for all analytes and recoveries around 100%. The method developed and validated was applied in real milk samples of cow treated with Ivomec^ (IVM 3.5%), in which were found 21.51±2.94 ng mL–1^ of IVM. Finally, the results proved that PT–PPy–SPE coupled to HPLC-UV is economical, simple and easy-to-perform technique. Keywords : avermectins, milbemycyns, polypyrrole, pipette-tip solid-phase extraction, milk, HPLC.
similar biological activity, besides specific toxicological properties. AVM are characterized by the presence of a sugar molecule substituent at 13-position and of a secondary butyl or isopropyl at 25-position. AVM are used for the control of insects and mites in vegetables and fruits by action against the animal's central nervous system [6]. [6] L. Giannettia., A., Giorgia, F., Neccia, G., Ferretti, F Buiarelli., B. Neria. (2011). Validation study on avermectine residues in foodstuffs. Anal. Chem. (700): 11-15. www.elsevier.com/locate/aca. In Europe as well as in the United States, ABA, doramectin (DOR), and ivermectin (IVM) are prohibited for the treatment of lactating animals. The eprinomectin (EPR) and moxidectin (MOX) dairy cows maximum residue limits have been set at 20000 ng Kg-−1^ and 40000 ng Kg−1, respectively, as maximum permissible without affecting food safety or human health [7]. Different Codex Alimentarius Commission based on evaluations of the Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) established values of 15000 ng Kg−1^ for DOR, 5000 ng kg−1^ for ABA, 10000 ng Kg−1^ for IVM, 20000 ng Kg− for EPR and values between 10000 and 20000 ng Kg−1^ for MOX [8, 9]. [7] J. L. Shipp, K. Wang, G. Ferguson. (2000). Residual toxicity of avermectin b1 and pyridaben to eight commercially produced beneficial arthropod species used for control of greenhouse pests. Biol. Control (17): 125-131. https://doi.org/10.1006/bcon.1999.0784. [8] W.L Shoop., H.W Haines., B.H Michael., C.H. Eary. (1993). Mutual resistance to avermectins and milbemycins: oral activity of ivermectin and moxidectin against ivermectin-resistant and susceptible nematodes. Vet. Rec. (133): 445-447. http://veterinaryrecord.bmj.com/content/133/18/445. [9] R. Prichard., C. Ménez., A. Lespine. (2012). Moxidectin and the avermectins: Consanguinity but not identity. Int J Parasitol Drugs Drug Resist. (2): 134-153. https://doi.org/10.1016/j.ijpddr.2012.04.001. In Brazil, the Brazilian Residues and Contaminants Control Plan (PNCRC) monitor veterinary drugs and the MRL established for food is 10 mg L−1^ for ABA, IVM and MOX. For DOR and EPR, the MRL was set as 15 and 20 mg L−1, respectively [10].
Due to the lipophilic properties of the AVM and the long-term permanence of their residues in the bodies of animals aside from their secretions (urine, fecal matter, semen), as well as in milk is a toxic risk for the health of consumers. So, their detection and monitoring become imperative [9]. [9] R. Prichard., C. Ménez., A. Lespine. (2012). Moxidectin and the avermectins: Consanguinity but not identity. Int J Parasitol Drugs Drug Resist. (2): 134-153. https://doi.org/10.1016/j.ijpddr.2012.04.001. [10] Brasil, Ministério da Agricultura, Pecuária e Abastecimento, Secretaria de Defesa Agropecuária, Instrução Normativa No. 11/2016. www.agricultura.gov.br/assuntos/laboratorios/rede-nacional-de- laboratoriosagropecuarios/documentos-rede-nacional-de-laboratorios-agropecuarios/ modelo-p_-pag_poa_microbial_11_11_2016.pdf The route of administration affects the efficiency of the AVM against ectoparasites [9,11]. The formulation may affect the initial absorption process as well as the phase of elimination [12]. The mean elimination phase varies for each AVM and the differences are likely to be related to lipophilic and potential efflux, through ATP- binding cassette transporters (ABC transporters) [12]. The epithelium of the mammary gland, like other biological membranes, acts as a lipid barrier, and the high lipophilicity of the AVM is in favor of interaction and elimination in milk [13]. [9] R. Prichard., C. Ménez., A. Lespine. (2012). Moxidectin and the avermectins: Consanguinity but not identity. Int J Parasitol Drugs Drug Resist. (2): 134-153. https://doi.org/10.1016/j.ijpddr.2012.04.001. [10] Brasil, Ministério da Agricultura, Pecuária e Abastecimento, Secretaria de Defesa Agropecuária, Instrução Normativa No. 11/2016. www.agricultura.gov.br/assuntos/laboratorios/rede-nacional-de- laboratoriosagropecuarios/documentos-rede-nacional-de-laboratorios-agropecuarios/ modelo-p_-pag_poa_microbial_11_11_2016.pdf [11] A. Lespine., M. Alvinerie., J. Vercruysse., R.K Prichard., P. Geldhof. (2008). ABC transporter modulation: a strategy to enhance the activity of macrocyclic lactone anthelmintics. Trends Parasitol. (24): 293-298. http://hdl.handle.net/1854/LU-429782. [12] Council Directive 96/23/Ec. (2012). Measures to monitor certain substances and residues thereof in live animals and animal products and repealing. Directives
which is the most harmful in Brazil [19, 20-24]. A subcutaneous dose of IVM at a concentration of 0.2 mg kg−1^ is used for treatment and prevention of infestation. Higher doses do not accelerate action, but the efficacy may reach 100% when treated within the residual time to ensure its action for four weeks [21]. Minas Gerais (Brazil) state is considered a risk area of hemoparasite incidence [22], for this reason the use of IVM is highly recommend. Therefore, AVM and MBM monitoring is necessary, mainly so that the presence of residues of these drugs are not found in food, especially in milk. [19] Brasil, Ministério da Agricultura, Pecuária e Abastecimento, Secretaria de Defesa Agropecuária, Instrução Normativa No. 11/2016. http://www.agricultura.gov.br/assuntos/laboratorios/rede-nacional-de-laboratorios- agropecuarios/documentos-rede-nacional-de-laboratorios-agropecuarios/ Modelop.Pg_POA_Microbial_06.06.17.pdf. [20] E. Castro Janer., G.M. Klafkec., M.L. Capurrob., T.T.S. Schumaker (2015) Cross- resistance between fipronil and lindane in Rhipicephalus (Boophilus) microplus. Vet. Parasitol. (210): 77–83. http://dx.doi.org/10.1016/j.vetpar.2015.03.011. [21] L.V. Costa Gomes., W.D. Zanetti Lopes., B.C. Cruz., W.F. Teixeira., G. Felippelli., W.G. Maciel., M.D. Bichuette., M.A. Ruivo., M.H. Alcantara Colli., R. Silveira Carvalho., A. Campanha Martinez., V.E. Soares., A.J. Da Costa. (2015). Acaricidal effects of fluazuron (2.5 mg/kg) and a combination of fluazuron (1.6 mg/kg)
- ivermectin (0.63 mg/kg), administered at different routes, against Rhipicephalus (Boophilus) microplus-parasitizing cattle. Exp Parasitol. (153): 22–28. http://dx.doi.org/10.1016/j.exppara.2015.02.004. [22] B.C. Cruza., W.D. Zanetti Lopes., W.Q. Maciel., G. Felippellia., F.C. Fávero., W.F. Pires Teixeira., R. Silveira Carvalho., M. Araújo Ruivoc., M.H. Alcantara Colli., C.A. Massamitsu Sakamoto., A.J Da Costa., G. Pereira De Oliveira. (2015) Susceptibility of Rhipicephalus (Boophilus) microplus to ivermectin (200, 500 and 630 μg/kg) in field studies in Brazil. Vet. Parasitol. (207): 309–317. http://www.sciencedirect.com/science/journal/03044017/207?sdc=1. [23] L.H. De Oliveira Souza., M.V Garcia., J. Cavalcante Barros., W.R. Koller., R. Andreotti. (2016) Evaluation of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) resistance to different acaricide formulations using samples from Brazilian properties. Braz. J. Vet. Parasitol. Jaboticabal. (25): 163-171. http://dx.doi.org/10.1590/S1984-29612016026. [24] M. Nasr., El-Bahy., K. Eman., I. Bazh Hazem., M. Shaheen. (2015) Efficacy of deltamethrin, diazinon, and ivermectin on Boophilus annulatus ticks (in vitro and in vivo study) Parasitol Res. (1): 114:29–36. http://dx.doi.org/10.1007/s00436-014-4129-
For the determination of AVM in milk, an initial clean up using the protein precipitation (PP) is performed, where aliquots of acetonitrile are added to volumes greater of milk, and then this mixture is centrifuged [25, 26]. When the organic phase is separated from the aqueous, triethylamine is added and the contents are dissolved in 10 parts of water [26]. There are some works that reported the determination of AVM in milk [19-63]. Therefore, the use of miniaturized SPE techniques using PPY as adsorbent material has not been reported in the literature for the determination of AVM and MBM in milk. [25] M. Danaher., L. C. Howells., V. Cerkvenik-Flajs., M. O’keeffe. (2006). Review of methodology for the determination of macrocyclic lactone residues in biological matrices. J. Chromatogr. B (2):175–203. http://dx.doi.org/10.1016/j.jchromb.2006.07.035. [26] T. Szprengier-Juszkiewicz., P. Jedziniak., O. Małgorzata., J. Żmudzki. (2012). Control of residues of five macrocyclic lactones in cow milk by liquid chromatography with fluorescence detection. Versita. (56): 595-599. http://dx.doi.org/10213-012-0105-
Usually, AVM are extracted using conventional SPE techniques and analyzed by HPLC–FL or/and LC–MS–MS [19-63]. Most current extraction methods are long and tedious employing the liquid-liquid extraction (LLE) or SPE, in addition, a previous step to precipitate proteins (PP) is employed [27-30]. [27] N. Campillo., P. Viñas., G. Férez-Melgarejo., M. Hernández-Córdoba. (2013). Dispersive liquid–liquid microextraction for the determination of macrocyclic lactones in milk by liquid chromatography with diode array detection and atmospheric pressure chemical ionization ion-trap tandem mass spectrometry. J. Chromatogr. A. (1282): 20-
- http://doi.org/10.1016/j.chroma.2013.01.086. [28] M. M. Aguilera., P. Plaza-Bolaños., R. Romero-González., J. L. Martínez Vidal., A. Garrido Frenich. (2011). Comparison of the efficiency of different extraction methods for the simultaneous determination of mycotoxins and pesticides in milk samples by ultra-high-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem, (s.d): 2863–2875. https://doi.org/10.1007/s00216-011-4670-7.
(PPy) [39-43]. PPy possess attractive caracteristics as adsorbent material due to its properties as: hydrophobicity propitious, an acid-base character, π-π interaction, polarity, apparently porous structure, ion exchange properties, hydrogen bonds and electroactivity [39-43]. [35] K.B. Borges, E.C. Figueiredo, M.E.C. Queiroz Preparo de amostras para análise de Compostos Orgânicos, in: K.B. Borges, E.C. Figueiredo, M.E.C. Queiroz (Eds.), LTC−Livros Técnicos Científicos Editora Ltda, Rio de Janeiro, 2015. [36] J. Raich-Montiu, K. A. Krogh., M. Granados., J. Å. Jönsson., B. Halling-Sørensen. (2008). Determination of ivermectin and transformation products in environmental waters using hollow fiber-supported liquid membrane extraction and liquid chromatography–mass spectrometry/mass spectrometry. J. Chromatogr. A. (1187): 275–
- https://doi.org/10.1016/j.chroma.2008.02.063. [37] H. Bagheri. M. Saraji, (2003). Conductive polymers as new media for solid-phase extraction: isolation of chlorophenols from water sample, J. Chromatogr. A. (986): 111–119. http://dx.doi.org/ 10.1016/S0021-9673(02)01972-6. [38] B. Carneiro Pires., F.V. Avelar Dutra., T.A. Nascimento., K.B. Borges (2017). Preparation of PPy/cellulose fibre as an effective potassium diclofenac adsorbent. React Funct Polym. (113): 40–49. http://dx.doi.org/10.1016/j.reactfunctpolym.2017.02.002. [39] T.A. Nascimento., F.V Avelar Dutra., B. Carneiro Pires., C.R. Teixeira Tarley., V. Mano., K.B. Borges. (2016). Preparation and characterization of a composite based on polyaniline, polypyrrole and cigarette filters: adsorption studies and kinetics of phenylbutazone in aqueous media. RSC Adv. (6): 64450-64460. http://dx.doi.org/10.1039/c6ra14071h. [40] F.V. Avelar Dutra., B. Carneiro Pires., T.A. Nascimento., V. Mano., K.B. Borges. (2017). Polyaniline-deposited cellulose fiber composite prepared via in situ polymerization: enhancing adsorption properties for removal of meloxicam from aqueous media. RSC Adv. (7): 12639-12650. http://dx.doi.org/12639- 10.1039/c6ra27019k. [41] J Meng., J Bu., C Deng., X Zhang. (2011). Preparation of polypyrrole-coated magnetic particles for micro solid-phase extraction of phthalates in water by gas chromatography–mass spectrometry analysis. J. Chromatogr. A. (12): 1585–1591. http://dx.doi.org/10.1016/j.chroma.2011.01.057. [42] H. Bagheri., M. Saraji. (2003). Conductive polymers as new media for solid-phase extraction: isolation of chlorophenols from water sample. J. Chromatogr. A. (986): 111-119. http://dx.doi.org/10.1016/S0021-9673(02)01972-6. [43] F. Khalilian., M. Rezaee., M. K. Gorgabi. (2015). Magnetic polypyrrole/Fe3O particles as an effective sorbent for the extraction of abamectin from fruit juices using
magnetic solid-phase extraction combined with dispersive liquid–liquid microextraction. Anal. Methods. (7): 2182-2190. http://dx.doi.org/10.1039/c4ay03074e. The general limits of conventional methods for extraction and determination of AVM and MBM include the difficulty in extraction and multiple determination and do not have reported studies in literature for miniaturized. Based on the above stated difficulties, in this study we developed and validated an miniaturized and efficient method employing the PT–PPy–SPE for the extraction of AVM and MBM from milk samples prior simultaneous determination by HPLC–UV.
2. Experimental 2.1 Chemicals and reagents All analytical standards were obtained from Sigma-Aldrich®^ (Steinheim, Germany): ABA (Pestanal®, analytical standard) assay HPLC 97.6 area% (3% ABA 1b
- 92.1 % ABA1a), EPR (Pestanal®, analytical standard) assay HPLC 97.7 area% (97.34% w/w, B1a + B1b), MOX (Vetranal®, analytical standard) assay HPLC 97.7 area % and IVM (Pharmaceutical Secondary Standard, Certified Reference Material) 98.97% B1a. HPLC grade solvent methanol, toluene, acetonitrile, acetone, tetrahydrofuran was obtained from J. T. Baker®^ (Mexico City, MX, Mexico). Pyrrole reagent grade assay 98% was obtained from Sigma-Aldrich®^ (Steinheim, Germany). Water was distilled and purified using a Millipore Milli- Q Plus system (Bedford, MA, USA). All other chemicals were of analytical grade with the highest purity available. 2.2 Instrumentation and separated conditions An Agilent HPLC model 1260 system (Agilent Technologies, Palo Alto, CA, USA) compost for a quaternary pump (G1311 B), a thermostat model 1290 (G1330B), automatic injector model 1260 Hip ALS (G1367E), a column oven model 1290 TCC
dissolved in 100 mL Milli- Q water. The solution remained under constant stirring and 1.6 mL of pyrrole was added to the reaction medium by dripping for 4 h. PPy was obtained as a solid dark material and it was vacuum filtered and washed with Milli- Q water and ethanol. Finally, PPy was dried an oven at 60 °C for 24 h. 2.5. PPy characterization Scanning Electron Microscopy (SEM) performed the morphological characterization of PPy. The SEM images were obtained using Hitachi Analytical Table Top Microscope TM3000 (Hitachi, Tokyo, Japan) with the voltage acceleration at 5 kV. The structural characterization of the PPy was carried out by Fourier Transform Infrared Spectroscopy (FTIR) with a spectrometer (Bomem Hartmann & Braun, MB series, Quebec, Canada), operating between 4000 cm−1^ and 400 cm−1, at 4 cm−1^ resolution. A KBr tablet was used as an optical window taking advantage of its low cost good transmission. The thermal stability of the PPy was investigated by Thermogravimetric Analyzes (TGA) using a thermo balance 2950 Thermal Analysis Instrument (TA Instrument, New Castle, DE, USA) with a heating rate of 10 °C min-1^ under nitrogen flow (100 mL min-1) using a temperature range from room temperature up to 600 °C. The morphological structure of PPy was carried out X-ray diffraction (XRD). The X-ray diffraction equipment used was Shimadzu, model XRD-6000 (SHIMADZU CK- Chiyoda-ku, Tokyo, Japan) using CuKα radiation (λ = 1.54 Å). The analyses were performed in the 2θ range operating between 5 and 75 degrees. 2.6. Spiked milk samples To development and validation of chromatographic method for the simultaneous determination of AVMs and MBMs by HPLC, the milk samples were
obtained at a local supermarket. These blank milk samples were spiked with 50 μL of the standards solutions of ABA, IVM, EPR and MOX with concentrations that ranged from 10 to 3000 ng mL−1. The samples were taken to the refrigerator for 15 min and in the absence of light to avoid degradation [41] before protein precipitation (PP) and PT– PPy–SPE procedures. 2.7. PP procedure An aliquot of 5 mL of spiked milk was mixed with 20 mL of acetonitrile in a Falcon-type test tube of 50 mL. They were centrifuged using a Clinical Centrifuge Centribio/Daiki 80-2B (Ramos, RJ, Brazil) at 3000 rpm for 10 min. An aliquot of 15 mL of the supernatant was transferred to a 50 mL Falcon-like assay tube and 50 μL triethylamine was added. Next, it was centrifuged again in the equal conditions. The resulting extract was diluted to 50 mL using Milli- Q water and brought to the refrigerator (5 ±2 oC) for the PT–PPy–SPE procedure. 2.8. PT – PPy – SPE production and procedure In this procedure, pipette-tip of 1000 μL (polypropylene) were used to prepare the device with 50 mg PPy bundled using cotton wool on both ends to prevent loss of the adsorbent material. A pipette-tip of 200 μL was used as adapter to couple with a commercial syringe, which was used as pump manual ( Figure S1 ). Our research group has been working with this device, in which it has presented good results [37, 40-43]. Prior to the extraction, the cartridge with PPy was activated with 1 mL of ultrapure water.
expressed in terms of (relative standard deviation) (RSD %) and relative error (RE %), respectively. Intraday studies (precision and accuracy) were performed in sextuplicate to demonstrate the repeatability of the method. Intermediate precision and accuracy (Interday tests) were performed in three different days (n=3). The concentrations used were 300, 1000 and 2000 ng mL−1^ [19]. The robustness of the method was determined by analyzing the same samples by performing variations in the method conditions. Parameters, such as flow rate (± 0. units), injection volume (± 0.10 units), and mobile phase composition (30% methanol + 15% water + 55% acetonitrile; 27% methanol + 18% water + 55% acetonitrile, 20% methanol + 20% water + 60% acetonitrile). The concentrations of AVMs residues were evaluated, applying Student's t-test, with a level of significance established at a p -value ≤ 0.05. If the influence of the parameter is within a previously specified tolerance, it is said to be within the robustness range of the method [19]. The studies should evaluate the stability of the analytes during sample collection and handling after typical storage scenarios such as long-term storage (when frozen at the intended storage temperatures), short term storage (during a series of sample analyses at room temperature), and after freeze and thaw cycles. Thus, to check the stability of AVMs and MBMs the following parameters were studied: influence of freeze (−20 °C) and thaw (25 ± 2 °C) cycles, short-term room temperature (12 h at the bench top), long-term storage at −20 °C (2 and 4 days). Finally, a one-way ANOVA test was applied, with the level of significance set at a p -value ≤ 0.05 [19]. 2.6 Application of method in real samples For the application of method, it was collected three real samples from animals treated with IVM of 50 mL of milk from two producers (Sites A and B) dedicated to
milk production located in city São João del-Rei, Minas Gerais, Brazil. The samples collected have rigorous control to the internal and external parasites and were monitored by veterinarians of farms [44]. The efficacy of IVM reach 100% with a single subcutaneous administration (200 μg kg−1) on the 28th day of medication. [45, 46]. With this information, the milk samples were collected 24 h after the drug administration to monitor the IVM residues during the first cycle of treatment. [44] D. Kolberg., M. Presta., C. Wickert., M. Adaime., R. Zanella. (2009). Rapid and Accurate Simultaneous Determination of Abamectin and Ivermectin in Bovine Milk by High Performance Liquid Chromatography with Fluorescence Detection. J. Braz. Chem. Soc. (7): 120-126. http://dx.doi.org/10.1590/S0103-50532009000700004. [45]. DD Colwell. (2002). Presistant activity of moxidectin pour-on and injectable against sucking and biting louse infestations of cattle. Vet Parasitol. (140): 319–326. https://doi.org/10.1016/S0304-4017(01)00641-0. [46] D. I. S. Kolberg., M. A. Presta., C. Wickert., M. B. Adaime., R. Zanella. (2009). Rapid and Accurate Simultaneous Determination of Abamectin and Ivermectin in Bovine Milk by High Performance Liquid Chromatography with Fluorescence Detection. J. Braz. Chem. Soc. (7): 1220-1226. http://dx.doi.org/10.1590/S0103-
The samples were thawed at room temperature and with stored in Falcon tubes at -20 °C. After that, it was divided into 1 mL aliquots labeled according to producer on the date of collection and the used drug. Samples were collected from site A (samples 1, 2, and 3) and site B (samples 4, 5, and 6) in animals treated with Ivomec®^ injectable (IVM 3.5%). All drugs were obtained from Merial Saúde Animal Ltda, Fazenda São Francisco (Paulínia, SP, Brazil).
3. Results and discussion 3.1 Development of HPLC method Firstly, the isocratic and reverse mode was chosen for the separation of analytes from milk samples. It was used water, methanol, and acetonitrile for the
3.2.1 FTIR
For the multi extraction of AVMs and MBMs residues, PPy was studied as the adsorbent material. The results obtained of FTIR spectra presented typical bands of pyrrole ( Figure 3A). Bands at 1530 and 1453 cm−1^ (C=C and C-C, respectively), were observed. Bands at 1294 and 1030 cm−1^ corresponding to the vibration band for sp^2 bonds were observed. The band at 1156 cm−1^ was assigned to the N-C stretching mode. The 899 cm−1^ peaks were attributed to the sp^2 bonds indicating polymerization of the pyrrole. 3.2.2 TGA and drTGA The curves show three different thermal events. The first thermal event presents a slight loss of matter (5%) due to the evaporation of water around 90 ºC. The second thermal event indicates the beginning of the decomposition process of PPy showing a slight loss of matter around 250 ºC. The third thermal event, which corresponds to a significant detriment of the amount of polymeric matter, with the complete volatilization the total formation of a solid species, is the thermal stability. The drTGA curve of the PPy (Figure 3B) is very possible to conclude that material showed good thermal stability with degradation of the polymer matrix around 500 °C. 3.2.3 SEM The Figures S1A, S1B and S1C showed the SEM images of PPy at 500, 2000, and 4000×, respectively. In fact, the size of the particles and superficial area, could determine the extraction efficiency of PPy. The SEM showed the agglomerate polymers
exhibited homogeny size particles. This morphological profile is very common for conducting polymers synthesized by the oxidative synthesis method [38]. [38] B. Carneiro Pires., F.V. Avelar Dutra., T.A. Nascimento., K.B. Borges (2017). Preparation of PPy/cellulose fibre as an effective potassium diclofenac adsorbent. React Funct Polym. (113): 40–49. http://dx.doi.org/10.1016/j.reactfunctpolym.2017.02.002. 3.2.4 XDR The results obtained of XDR spectra ( Figure 4 ) presented only one band for PPy. It was centered around 24.6°, which can be assigned to the repeat unit of pyrrole ring, which is the principal monomer to polypirrol, implying the polymer chain is highly oriented. The broad feature of the band reveals the amorphous nature. These results are also consistent with the PPy materials obtained from conventional chemical method [41, 43]. [41] J Meng., J Bu., C Deng., X Zhang. (2011). Preparation of polypyrrole-coated magnetic particles for micro solid-phase extraction of phthalates in water by gas chromatography–mass spectrometry analysis. J. Chromatogr. A. (12): 1585–1591. http://dx.doi.org/10.1016/j.chroma.2011.01.057. [43] F. Khalilian., M. Rezaee., M. K. Gorgabi. (2015). Magnetic polypyrrole/Fe3O particles as an effective sorbent for the extraction of abamectin from fruit juices using magnetic solid-phase extraction combined with dispersive liquid–liquid microextraction. Anal. Methods. (7): 2182-2190. http://dx.doi.org/10.1039/c4ay03074e. 3.3. PT – PPy – SPE optimization To carry out the best extraction efficiency of PT–PPy–SPE coupled HPLC-UV for the multidetermination of ABA 1a, ABA 1b, EPR, MOX and IVM, some parameters were evaluated such as washing solvent, elution solvent, amount of material (PPy), effect of pH, sample volume, eluent volume effect and salting out effect (addition of NaCl).
