Efficient development of a magnetic molecularly imprinted polymer for selective determination of trimethoprim and sulfamethoxazole in milk

Microchemical Journal 154 (2020) 104648

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Efficient development of a magnetic molecularly imprinted polymer for selective determination of trimethoprim and sulfamethoxazole in milk

T

Laíse Aparecida Fonseca Dinalia, Hanna Leijoto de Oliveiraa, Leila Suleimara Teixeiraa, ⁎ Anny Talita Maria da Silvaa, Kaíque A D'Oliveirab, Alexandre Cuinb, Keyller Bastos Borgesa, a

Departamento de Ciências Naturais, Universidade Federal de São João del-Rei (UFSJ), Campus Dom Bosco, Praça Dom Helvécio 74, Fábricas, 36301-160, São João delRei, Minas Gerais, Brazil b Departamento de Química, Universidade Federal de Juiz de Fora (UFJF), Campus Universitário, Martelos, 36036-330, Juiz de Fora, Minas Gerais, Brazil

ARTICLE INFO

ABSTRACT

Keywords: Antibiotics Sample preparation Solid phase extraction Magnetic polymers HPLC

A molecularly imprinted polymer was synthesized on the surface of magnetic nanoparticles for subsequent application in the extraction of trimethoprim (TMT) and sulfamethoxazole (SMX) residues in bovine milk. The magnetic polymer was suitably characterized and demonstrated high stability and optimum adsorption capacity under the conditions of analysis. MMIP was used in the preparation of milk samples by the magnetic solid phase extraction technique whose optimized parameters were: sample volume, elution solvent and its volume, amount of adsorbent, stirring time, and pH of the sample. The relative recoveries of analytes were 98.66 ± 5.59% and 75.57 ± 0.72% for TMT and SMX, respectively. The method developed was linear in the range of concentration of 0.05–20 µg mL−1 with correlation coefficients of 0.997 and 0.994 for TMT and SMX, respectively. It was also demonstrated satisfactory accuracy and precision, as well as adequate robustness and good drugs stability in the matrix. The method was applied satisfactorily in real milk samples contaminated with residues of these antibiotics.

1. Introdution The association of trimethoprim (TMT) with sulfamethoxazole (SMX) is commonly used in the treatment of infections in dairy cows because of the synergistic effect, which provides a broad spectrum of action, lower bacterial resistance, and bactericidal effect [1–4]. The association inhibits the synthesis of folic acid in microorganisms that need to synthesize it by sequentially blocking the biosynthesis of tetrahydrofolate, the active form of folinic acid, which acts as a coenzyme for the formation of nucleic acids [5–7]. Due to large-scale dairy production and to indiscriminate use of these antimicrobials in livestock, unwanted residues can be found in milk for human consumption [8,9]. Thus, it is necessary to develop increasingly fast, selective, and accurate analytical methodologies for the detection, control, and monitoring of these wastes. In this context, sample preparation is an important step in the development of an analytical method, because it has the purpose of extracting one or more analytes from complex matrices and eliminating a large part of the interferences, guaranteeing better selectivity of the method [10]. Solid phase extraction (SPE) in conventional form utilizes extraction cartridges that are packaged with an adsorbent material, ⁎

generally C18 [11]. Selective adsorbent materials such as molecularly imprinted polymers (MIPs) are also widely used in SPE; this technique is better known as molecularly imprinted solid phase extraction (MISPE) [12,13]. MIPs are synthetic polymers that have selective cavities for a given molecule with a mechanism based on natural recognition systems, such as antigen-antibody, drug-receptor, and enzyme-substrate interactions [14,15]. The search for simplification of the preparation stages has led to the development of new techniques in SPE, such as magnetic solid phase extraction (MSPE). This term was used for the first time in 1999 by Saríková and Safarík [16]. However, Towler et al. (1996) had already reported the use of magnetic nanoparticles as an adsorbent in extraction processes [17]. The direct contact between the solid and liquid phases in the MSPE contributes to a better efficiency of the extraction process, less sample preparation time and eases phase separation [18,19]. In recent years, there has been a growing number of works that combine molecular printing technology with materials that have magnetic properties, resulting in the so-called magnetic molecularly imprinted polymers (MMIP) first described by Ansell and Mosbach in 1998 [20,21]. MMIPs are materials consisting of a magnetic base, coated with a

Corresponding author. E-mail address: [email protected] (K.B. Borges).

https://doi.org/10.1016/j.microc.2020.104648 Received 21 November 2019; Received in revised form 15 January 2020; Accepted 15 January 2020 Available online 16 January 2020 0026-265X/ © 2020 Elsevier B.V. All rights reserved.

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non-magnetic organic polymer and present high adsorption capacity and selectivity to the analyte. They also have some benefits in relation to conventional MISPE, such as less solvent quantity, no need for cartridge packing, as well as the facility in separation of the phases, less steps in the extraction process, reduction of analysis time, and improvements in kinetics and link capacity, because of direct contact between the solid phase and the sample [22,23]. Noting that in the literature there have been no reports to date of the use of MMIP for simultaneous extraction of TMT and SMX in any type of matrix, the main objectives of this work were to synthesize and characterize the materials Fe3O4, Fe3O4@SiO2, MMIP, and non-imprinted magnetic polymer (MNIP) and develop a fast and simple analytical method for separation of TMT and SMX by HPLC-UV. In addition, to optimize sample preparation using MMIP as adsorbent in MSPE and to apply the method in real samples of bovine milk.

thermostat model 1290 (G1330B), automatic injector model 1260 Hip ALS (G1367E), column oven model 1290 TCC (G1316C), UV/VIS detector model 1260 VWD (G1314F), and an Agilent Open LAB chromatography Data System®. Chromatographic separation of the antibiotics was performed in isocratic mode on a Phenomenex® Luna C8 (250 mm × 4.6 mm, 5 µm) column, with mobile phase consisting of solution A and methanol (95: 5, v/v). Solution A was constituted by 0.1% triethylamine pH 6.5: ACN (80: 20, v/v). The other chromatographic conditions were: flow rate at 1.5 mL min−1, injection volume of 5 µL, temperature at 25 °C, and UV detection at 280 nm. 2.4. Synthesis and coating of Fe3O4 nanoparticles In 80 mL of ultrapure water, preheated to 80 °C, 15 mmol of FeCl3•6H2O and 10 mmol of FeSO4•7H2O were dissolved under vigorous stirring. Subsequently, 50 mL of 28% (v/v) ammonium hydroxide (NH4OH) were added, slowly changing the coloration of the solution from yellow to black. The mixture was maintained at 80 °C for 30 min to precipitate the Fe3O4 nanoparticles. The precipitate was collected magnetically and washed with deionized water until the pH was between 6.5 and 7.5. The particles were dried in an oven at 60 °C for 24 h. Subsequently, 600 mg of Fe3O4 were mixed with 60 mL of a solution of ethanol: ultrapure water (5:1, v/v). The suspension was kept in an ultrasonic bath for 20 min. A volume of 10 mL of 28% (v/v) NH4OH and 4 mL of TEOS was then added and the reaction was maintained at room temperature under mechanical stirring for 12 h. The modified magnetic nanoparticles (Fe3O4@SiO2) were then magnetically separated and washed with ultrapure water until the wash pH had a value between 6.5 and 7.5. The particles were dried in an oven at 60 °C for 24 h.

2. Experimental 2.1. Reagents and solvents The reagents employed in the synthesis of the materials were: iron chloride III and iron sulfate II purchased from Neon® (Suzano, SP, Brazil), ammonium hydroxide from Qhemis® (Indaiatuba, SP, Brazil), ethanol from PanReac AppliChem® (Castellar del Vallès, Barcelona, Spain), TEOS from Merck® (Darmstádio, Germany), methacrylic acid (MA) and ethyleneglycol dimethacrylate (EGDMA) from Sigma Aldrich® (St. Louis, MO, USA), 4,4′-azobis(ácido 4-cianovalérico) from Santa Cruz Biotechnology® (Dallas, TX, USA) and chloroform from Dinâmica® (Diadema, SP, Brazil). The solvents used in mobile phase were: acetonitrile (ACN) from J. T. Baker® (Mexico City, MX, Mexico) and methanol from Dinâmica® (Diadema, SP, Brazil). Other reagents and solvents, namely triethylamine, sodium hydroxide, acetic acid, and acetone were purchased from Vetec® (Rio de Janeiro, RJ, Brazil), Synth® (Diadema, SP, Brazil), Cromato® (Diadema, SP, Brazil), and Macronк Chemicals® (Phillipsburg, NJ, USA) respectively, and distilled/purified water from a Millipore system Milli-Q Plus® (Bedford, MA, USA). All reagents and solvents used were analytical grade and HPLC grade, respectively.

2.5. Synthesis of the mmip and mnip In a flask containing 500 mg of Fe3O4@SiO2, 20 mL of chloroform were added. In another flask, 0.4 mmol of TMT (template) and 2.0 mmol of MA (functional monomer) were dissolved in 20 mL of chloroform. The flasks were simultaneously submitted to the ultrasonic bath for 1 h to form the monomer-template complex and dispersion of magnetic particles, respectively. The contents of both flasks were subsequently poured into an amber flask containing 12 mmol of EGDMA and 80 mg of 4,4′-azobis (4-cyanovaleric acid). The flask was sealed and maintained at 75 °C under mechanical stirring for 24 h. After drying, the MMIP was washed with a solution of methanol: acetic acid (7: 3, v/ v) until complete removal of the template, which was monitored by the injection of the wash solution under HPLC-UV. The MNIP was synthesized under the same conditions, but without the presence of the template. The schematic representation of MMIP preparation is shown in Fig. 1.

2.2. Standard solution TMT and SMX standards were purchased from the United States Pharmacopeia Reference Standard. The secondary patterns used were: TMT (98.85%, w/w) and SMX (98.95%, w/w). TMT and SMX standard solutions were prepared in methanol at the concentration of 1 mg mL−1, maintained under photoprotection, and refrigerated at −20 °C. From these standards, solutions at the concentration of 40 µg mL−1 for the procedure of optimization of sample preparation and construction of the analytical calibration curve, from 0.05-20 µg mL−1 were prepared.

2.6. Characterizations The thermal behavior of the synthesized materials was determined by thermogravimetry (TGA) in a thermobalance (2950 TA Instrument, New Castle, USA) heated from 25 to 1000 °C, with a heating rate of 10 °C min−1 and under nitrogen flow (50 mL min−1). The morphology of the materials was determined by Scanning Electron Microscopy

2.3. Instrumentation The analyzes were performed on an HPLC Agilent® (Palo Alto, CA, EUA) equipped with a quaternary pump, model 1260 (G1311B),

Fig. 1. Schematic representation of MMIP preparation. 2

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equipped with an Energy Dispersive Spectrometer (SEM/EDS) under a microscope (TM3000 Hitachi Analytical Table Top, Tokyo, Japan) with voltage acceleration at 5 kV. The crystal structure of the materials was evaluated by X-Ray Diffraction (XRD) on a Bruker X-ray diffractometer (AXS D8 da Vinci Advance, Germany) with copper radiation kα1 = 1,54,059 Å and kα2 = 1,54,443 Å. The mean crystallite size was evaluated from the obtained diffractograms and calculated using the Scherrer equation (Eq. (1)) [24]. Fourier Transform Infrared (FTIR) analyzes were performed on a Fourier Transform Spectrometer (Bruker Optik GmbH Vertex 70, Germany) operating between 4000 and 400 cm−1 at 4 cm−1 resolution by the conventional method (KBr tablet). The characterization of specific surface area, pore volume and pore size distribution were determined from the N2 adsorption-desorption isotherms together with the Brunauer – Emmett – Teller equation (BET) and the Barrett – Joyner – Halenda (BJH) method in Quantachrome Autosorb-iQ2 analyzer.

D=

K cos

methanol for HPLC-UV analysis. Drying the samples prior to injection is a standard procedure for eliminating potential interferents. 2.10. Selective experiment The print effect test was performed to verify the MMIP selectivity for the TMT (used as a template in the synthesis of the material) in milk samples enriched with other drugs used in dairy cattle. The objective was to evaluate whether the material can accurately recognize TMT in milk samples even in the presence of other drugs. Thus marbofloxacin, norfloxacin, ciprofloxacin, enrofloxacin, ivermectin, eprinomectin, abamectin, moxidectin, tetramisol and lidocaine were used as contaminants. The tests were performed in test tubes containing 500 µL of sample enriched with TMT and the other drugs, along with 30 mg of MMIP. After adsorption performed under vortexing at 2000 rpm for 60 s, the samples were separated from the adsorbent material, oven dried at 60 °C, re-suspended, and analyzed by HPLC/UV. From this assay, the parameters pertinent to the selective performance of the MMIP were determined, such as the distribution coefficient (Kd), calculated from Eq. (2), and the selectivity coefficient (K) obtained through Eq. (3) [30].

(1)

Where D is the size of the crystallite; K is a dimensionless constant used according to the shape of the particle, in this case a value of 0.9 was used considering the shape of the spherical crystallite [25]; λ is the wavelength of the radiation used; β (in radian) was determined to be the full width at half-maximum and θ corresponds to the Bragg angle.

Kd =

2.7. Wettability evaluation

(Ci

Cf ) Cf

×

V(mL) mA(g )

(2)

Where Ci and Cf represent the initial and final concentration of the solution; V volume of the solution, and mA the mass of the adsorbent material.

The interaction between a drop of water and the surface of the material forms a contact angle (θc) that can provide information on the degree of affinity of the material by water. The θc can be obtained through the intersection between the liquid and the solid surface [26]. The formation of angles smaller than 90° indicate that the adhesion forces are greater than the cohesive forces and therefore represent the hydrophilicity of the material. Angles greater than 90° indicate that the adhesive forces are less intense, demonstrating the hydrophobic characteristic of the material. The surfaces may also be superhydrophobic, with angles greater than 150° or superhydrophilic, with angles smaller than 10° [27].

K=

Kd (TMT ) Kd (Interferences)

(3)

2.11. Method validation In this study, the selectivity, linearity, intraday and interday precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), robustness, and stability were evaluated. [31,32]. Selectivity was evaluated through the interposition of the chromatogram of the matrix (blank) with the chromatogram of the matrix spiked with the TMT and SMX standards. The linearity of the method was determined through the calibration curve in the range of 0.05–20.0 µg mL−1. The relationship between the analytical responses and each level of concentration was evaluated from the linear regression equation determined by the ordinary least squares method together with the correlation coefficient (r) [31]. The analysis of variance (ANOVA) was used to prove the linearity of the method. The precision of the method was expressed through intraday precision (n = 6) that demonstrates the repeatability in a short period of time, and through interday accuracy (n = 3) that determines the agreement between the results obtained in the same laboratory, but on different days [33]. The relative standard deviation (RSD) was used to evaluate the proximity of the instrumental responses for each concentration level evaluated. Accuracy was evaluated from solutions at the same concentrations as those used for precision (n = 6) and was measured from the relative error (RE) for each concentration studied, relating the real and nominal concentration. The LOD was defined experimentally (n = 6) by the lowest concentration of the analyte that can be detected by the instrumental technique [34]. The LOQ was given by the lowest analyte concentration determined with acceptable precision and accuracy under the experimental conditions (n = 6) [33]. For determination of the LOQ and LOD, after sample preparation the analytes were re-suspended in 80 mL of methanol and 50 μL were injected into the HPLC / UV. The robustness of a method establishes its ability to produce reliable

2.8. Bovine milk samples For the development of this work were used samples of whole milk sold in local supermarkets. It was necessary to perform the decantation procedure of whole milk proteins in order to obtain the bovine whey. In this way, 100 mL of contaminant free milk and 100 mL of deionized water were heated in a water bath at a temperature of 42 °C, then 2 mol L − 1 acetic acid was then added dropwise until decantation of the proteins. The serum was filtered on filter paper and mixed with 50 µL of triethylamine [28,29]. Whey from bovine milk spiked with TMT and SMX standards, at the concentration of 40 µg mL−1, was used for analytical method optimization. 2.9. Optimization of sample preparation MSPE was applied as sample preparation procedure. For a better performance of analytes extraction using this technique some parameters were optimized: elution solvent, material quantity (MMIP), stirring time for adsorption/desorption, sample pH, and the eluent and sample volumes. Thus, some initial sample preparation conditions were adopted: milk sample spiked with TMT and SMX standards at concentration of 40 µg mL−1 at pH 8.0, sample volume of 750 µL, 20 mg of adsorbent material, 750 µL of ACN as the elution solvent and 60 s stirring time for adsorption/desorption. The adsorption and desorption processes were performed in vortex (IKA® MS 3 Basic) and the separation was performed magnetically. After each extraction, the eluted aliquots were dried in an oven at 60 °C and re-suspended in 750 µL of 3

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results in the face of small variations of the analytical parameters [35]. Thus, the robustness was evaluated in the concentration of 20 µg mL−1 (n = 3) varying the parameters of flow rate, pH, and mobile phase ratio. The impact of each variation was evaluated using the precision and accuracy criteria, RSD% and RE%, respectively. The variables were also submitted to the one-way ANOVA statistical test, which evaluates the difference, within a single variable, composed of two, three, or more independent groups. The level of significance was 5% (p < 0.05). Stability was determined from the TMT and SMX solutions at the concentrations of 3.5 and 17.0 µg mL−1 in bovine whey at pH 8.0, with 12 h intervals at room temperature, 48 and 96 h freezing, and three freezing and thawing cycles. For all stability parameters comparison was performed with freshly prepared solutions in the same concentrations. Stability was evaluated through the RSD values obtained for each evaluated level. The results between the fresh solutions and the solutions submitted to the stability conditions were treated statistically through the t-test (Student's test) with a significance level of 5% (p < 0.05).

because of coating with silica and later with the polymer. The coating gives the material an amorphous characteristic that was proved by the presence of a wide peak at 2θ = 20° for the coated materials, being more intense for the MMIP and MNIP. It was also noted that the diffraction angles obtained experimentally from the silica coated particles and polymer were the same as Fe3O4, showing good adhesion to the magnetic core, and the crystalline structure of the magnetite was preserved. It is important to mention that the material does not lose its magnetic properties even after coating with MIP (Figure S2). From the peak of diffraction with intensity of 100% (311), it was possible to qualitatively estimate the mean crystallite size for the Fe3O4 sample, which was 10.11 nm. For a better crystallite size analysis, a measurement with a larger range and longer data accumulation time should be made. This result indicates that the iron oxide nanoparticles were synthesized successfully in this work. Even from these data the possibility of the presence of maghemite (γ-Fe2O3) traces, the oxidized form of the magnetite that may have formed during the coprecipitation and silanization process cannot be ruled out. However for this work, the identification of these traces was not important, since γ-Fe2O3 also exhibits magnetic properties similar to magnetite [42].

2.12. Method application The concentrations of TMT and SMX in milk vary according to the age of the animal and the form of administration of the drugs. SMX is absorbed rapidly and is bio-transformed in the liver, mainly by acetylation and oxidation. It is mainly eliminated by the kidneys and can be eliminated by secretions, sweat, saliva and milk. In milk the concentration of SMX is about 1 to 3.5 times lower than the concentration in plasma, because there exists a high percentage of plasma protein binding, being about 70% [5,36]. TMT is easily absorbed, and has maximum plasma levels around 4 h. It has a concentration in milk of about 1 to 3 times higher than the concentration in plasma, due to the low plasma protein binding of about 44% [37]. The real milk samples were supplied by a rural producer in São João del-Rei, MG, Brazil and kept in a freezer at −20 °C. The milk proteins were then precipitated and the sample preparation and the analytical methodology developed in this work were applied.

4.1.1. TGA TGA reports on the thermal stability of the synthesized materials, through the variation of the mass as a function of the temperature. As can be seen from Fig. 2B, a first mass loss event was identified in all samples at temperatures below 100 °C because of moisture and unreacted compounds in the synthesis of materials. For the iron nanoparticles and modified nanoparticles, little weight loss was observed, being a total of 2.5% and 11.2%, respectively. This occurred because of the high thermal resistance of the magnetite. For the MMIP and MNIP, the rapid mass loss, 75% on average, was observed at approximately 350 °C and occurred due to the thermal decomposition. After this event, at approximately 500 °C, there was a slight weight loss due to possible products formed during the decomposition of the materials. It is further observed that after decomposition, about 13 % by mass remained for MMIP and MNIP due to the magnetic particles that are thermally stable in the temperature range analyzed.

3. Results and discussion 3.1. Development of HPLC-UV method After the literature review and adjustment of the chromatographic parameters, it was possible to obtain the ideal separation (Fig. S1). As can be seen in Table S1, it was verified that the RSD values of the chromatographic run for the same concentration level were lower than 2.0% and, through the Fa calculations, values below the maximum allowed limit (approximately 1) were obtained, demonstrating that the peaks were symmetrical and valid. Well-resolved peaks (Rs> 1.5) were obtained indicating complete separation of constituents. The efficiency of the column was measured by the number of theoretical plates, which presented values higher than 2000, demonstrating good efficiency of the analytical column [38,39].

4.1.2. FTIR Fig. 2C shows two characteristic absorption bands of Fe-O (Fe3O4) vibration at approximately 580 cm−1 and 445 cm−1. For Fe3O4@SiO2, in addition to Fe-O bands, an intense absorption peak was observed at 1096 cm−1. This peak is attributed to the asymmetrical elongation of the Si-O-Si bond. It was also observed the presence of two absorption bands at 800 cm−1 and 471 cm−1 corresponding to the Si-O bond flexural vibration. Finally, a characteristic band at 954 cm−1, due to the Si-OH elongation, was recorded. In Fig. 2D, in addition to the absorption bands already described, there are other characteristic bands of the reagents used in the synthesis of the MMIP and MNIP. In this case, the FTIR presents an intense band, due to the elongation vibration of the C = O carboxylic bond of MA, at 1727 cm −1. The bands at 1257 cm−1 and 1160 cm−1 refer to the symmetrical and asymmetric CeO bond elongations for the ester functional group, from EGDMA. In addition it is possible to observe a absorption band at 1451 cm−1, due to angular deformation of the CH2eCH2 bond; a band at 1637 cm−1 referring to the elongation C = C; a band at 1390 cm−1 due to the symmetrical flexural vibration of the methyl groups; and two CeH bond elongation bands of the CH3 and CH2 groups at 2997 cm−1 and 2939 cm−1. The broad band at 3442 cm−1 corresponds of the stretching of the OeH bonds of the carboxylic acid from MA, and this was confirmed by the intense band of the elongation of the C = O bond at 1727 cm−1 [43]. These results indicate the efficiency of magnetic particle coating steps and indicate that the polymerization process was satisfactory.

4. Characterizations 4.1. XRD XRD analysis was a fundamental technique for the knowledge of the crystalline structure and the microstructure of the materials [40]. It was possible to observe from Fig. 2A six characteristic magnetite peaks (Fe3O4) referring to Miller indices (220), (311), (400), (422), (511), and (440) and their respective positions in the range 2θ = 20 to 70°, which is in agreement with the crystalline Fe3O4 diffraction pattern, available in the American Mineralogist Crystal Structure Database [41]. It was observed that the intensity of the Fe3O4@SiO2, MMIP, and MNIP peaks, significantly decay in comparison to the intensity of the magnetite, 4

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Fig 2. (A) XRD (B) TGA (C) and (D) FTIR of the synthesized materials: Fe3O4, Fe3O4@SiO2, MMIP and MNIP.

Fig. 3. SEM at magnification of 1500×of (A) Fe3O4; (C) Fe3O4@SiO2; (E) MMIP and (G) MNIP and wettability (θ) of (B) Fe3O4; (D) Fe3O4@SiO2; (F) MMIP and (H) MNIP. 5

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adsorbent capacity for other analytes with some similarity in molecular structure, thus ACN was chosen for the next optimization steps, as it also favored SMX recovery.

Table 1 Specific surface area and pore size of materials determined by BJH and BET equation. Parameter Specific surface area (m2 g Pore size (nm) Pore volume (cm3 g − 1)

− 1

)

MMIP

MNIP

13.5 8.4 0.048

16.2 10.1 0.086

4.2.2. Effect of volume of the elution solvent The effect of volume of elution solvent was evaluated by varying the volumes of 200, 500 and 750 µL of ACN. As can be seen in Fig. 4B, the volume of 750 µL was the most efficient in eluting the analytes. According to the results obtained, 500 µL was insufficient to extract a good amount of both analytes.

4.1.3. SEM/EDS SEM allowed observing of the morphology of the synthesized materials at magnification 1500 × . As can be observed in Fig. 3A,C,E and G, for all materials, it was possible to verify irregular, heterogeneous and non-defined surfaces, which favors the existence of porosity and consequently the adsorption efficiency. From the analysis by EDS, it was possible to semi-quantitatively verify the presence and variation of the chemical elements in the synthesized materials (Table S2). For Fe3O4, in addition to the large amounts of Fe and O, traces of the Si and C contaminants are noted. When the iron nanoparticles were coated with TEOS (Fe3O4@SiO2) the amount of Fe reduced and the amounts of O and Si increased, as expected. For MMIP and MNIP, there is great proximity in the elemental quantities, except for the presence of F in the MMIP, due to some impurity. This similarity proves the efficiency of the MMIP washes for template removal. Moreover, the significant increase of C and the reduction of Fe and Si indicate that all coating steps were satisfactorily performed and the magnetic properties of Fe3O4 were maintained.

4.2.3. Effect of MMIP amount Adsorption occurs by the retention of the analyte on the surface of the adsorbent material. Fig. 4C shows a greater recovery, especially of TMT, when 30 mg of MMIP was used, possibly because of the greater surface area that favored better adhesion of the analyte to the binding sites. Thus, 30 mg of MMIP was the amount defined for continuity of the optimization. 4.2.4. Effect of sample volume In MSPE, the sample volume tends to be smaller than that used in conventional SPE, and direct contact between the adsorbent and the sample favors a more effective extraction. As seen in Fig. 4D, 500 µL of sample presented better recovery for both analytes, obtaining the optimum ratio between the adsorbate and the adsorbent. Larger volumes did not favor recovery due to the saturation of the MMIP binding sites by the greater amount, in mass, of the analytes.

4.1.4. BET From the N2 adsorption-desorption assay and the mathematical treatments using the BET equation and the BJH method, it was possible to determine the specific surface areas, pore volume and pore size distribution of MMIP and MNIP, as can be seen in Table 1. It can be observed that the surface area of MMIP is close to that of MNIP and the values obtained correspond to the use of chloroform as porogenic solvent in the synthesis of these materials [44]. However, the pore size indicates that both materials are mesoporous, which contributes to the great recovery of the analytes studied in this work.

4.2.5. Effect of stirring time The adsorption of the analyte was given through direct contact between the sample solution and the MMIP, thus contact time was optimized, as observed in Fig. 4E. The agitation time of 60 s was the most efficient for TMT and SMX recovery, because it was the ideal time of interaction between the analytes and MMIP in the adsorption process, and also to disrupt interactions in the desorption process. 4.2.6. Effect of sample pH The pH of the sample was a fundamental parameter for the adsorption and consequently the recovery of the analytes. The pH should favor the neutrality of the chemical species to be adsorbed, because the presence of charges on the surface of the material can affect adsorption efficiency. The pH 8.0 showed the best performance, with recovery of 98.66 ± 5.59% for TMT, and 75.57 ± 0.72% for SMX, as seen in Fig. 4F. At this pH, TMT exhibits the majority of electrically neutral microspecies, which favored recovery around 100% of this analyte [45].

4.1.5. Wettability of materials Fig. 3B and D show the contact angles of 90° and 115° for Fe3O4 and Fe3O4@SiO2, respectively. Fe3O4 presented a hydrophilic character, but the TEOS coating provided a hydrophobic characteristic to the material. The wettability of the MMIP and MNIP (Fig. 3F and H) presented 120° intersection angles for both materials, indicating that the coating with the polymer maintained the hydrophobic characteristic since the synthesis occurred on the surface of the modified magnetic particles with TEOS. However, it can be said that MMIP is a hydrophobic adsorbent, or a non-polar adsorbent, which matches most polymeric adsorbents. This contributes to adsorption of analytes with low polarity of aqueous matrices, such as whey.

4.2.7. Comparison with other adsorbent materials After defining, the parameters discussed so far, a comparison was performed between the relative recoveries of the MMIP against other materials, among them MNIP, the iron particles, and the magnetic support used in the synthesis of MMIP and MNIP. As seen in Fig. S3A, the MMIP presented higher recovery of the analytes, especially of TMT, due to the selective sites for this analyte. For SMX, MMIP also showed a greater recovery because of a slight resemblance to TMT in molecular structure.

4.2. MSPE optimization 4.2.1. Elution solvent The choice of the elution solvent should be based on the properties of the analyte and the adsorbent material. This was a factor of extreme importance, because after the adsorption process, the eluent must be able to crack the interactions between the adsorbate and the adsorbent, favoring the recovery of the analyte and not elution of the interferences. Among the solvents evaluated, ACN has the highest value of relative polarity, with an eluotropic value (Ɛ°) of 0.5 [11]. Therefore, this solvent was the best to break the interactions between TMT and MMIP, which was evidenced by the higher recovery of this analyte, as shown in Fig. 4A. Although MMIP has selective sites for TMT, it also has

4.2.8. Reuse of the MMIP The reuse capacity of the MMIP was evaluated. The same material showed good recovery for TMT until when reused for the fourth time, as evidenced in Fig. S3B. This ability to reuse was because of the non-use of acid in the elution solvent, which contributed to the preservation of the binding sites. The SMX gradually lost its recovery with reuse, a justifiable fact, since the material does not present a selective site for this analyte. 6

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Fig 4. Optimization of sample preparation: (A) Elution solvent; (B) Eluent solvent volume; (C) Amount of MMIP; (D) Sample volume; (E) Stirring time and (F) pH of the sample.

4.3. Selective experiment

in question. According to the values obtained, it was possible to confirm that the material has a higher affinity for TMT than for the other analytes. These values also confirm the selectivity of MMIP for TMT and demonstrate that it is possible to recognize it among the drugs tested.

Fig. S4 demonstrates TMT and SMX relative recoveries in presence some drugs that can to be interferents in analysis. It was observed that the highest recoveries were related to TMT, which was used as template, followed by SMX. It was possible to observe that all the other drugs presented recoveries inferior to 45%. Table S3 shows the values of the distribution coefficient (Kd) and the selectivity coefficient (K) obtained for TMT and for the other drugs. The Kd value demonstrates the affinity between the analyte and the adsorbent material, thus representing the trend of migration of the molecule into the material. The highest value was obtained for TMT, indicating the high template affinity by the MMIP. This suggests that there was efficiency of the polymerization process. In addition, the values of K represent the ratio of the affinity of the MMIP by the template and for the other drugs, so the higher K values indicate low affinity for the material by the analyte

4.4. Method validation After optimization of the separation of analytes, the chromatographic system compliance tests, and the optimization of the sample preparation, the method was validated, considering the validation parameters and their respective acceptance criteria [31]. The selectivity of the method can be assessed from Fig. S5A. Although there is some interfering from extracted samples, they did not impede the clear identification of the TMT and SMX analytical signals. This method was considered linear in the range 0.05–20.0 µg mL−1 through the linear regression equations for each analyte, and their 7

Microchemical Journal 154 (2020) 104648

L.A.F. Dinali, et al.

4.5. Comparison with previously reported methods

Table 2 Linearity, LOQ, LOD for the method of separation of TMT and SMX in milk serum.

Linear equationa rb Range (µg mL−1) RSD (%)c F value LOD (µg mL−1) LOQ (µg mL−1) RSD (%)c RE (%)d

TMT

SMX

y = 584,332 x + 1,718,588 0.998 0.05–20 3.070 7570.69 0.01 0.05 10.57 13.21

y = 1,097,738 x + 1,485,026

Several authors have developed methods for separating TMT and SMX in different types of samples, and in the most part of these works the sample preparation was through SPE. Table 4 shows some of these works, as well as the analytical methods used, LOQ, LOD, linear range, in addition to the analysis conditions. It is important to note that no works were found that used the MMIP as adsorbent in MSPE for extraction of TMT and SMX in any type of matrix. In addition, LOD and LOQ values are close to, and even better than, the other methods for quantifying the same drugs in milk.

0.997 0.05–20 0.281 3757.34 0.02 0.05 7.29 14.44

4.6. Application of the method to real milk samples

a

Calibration curves determined in triplicate (n = 3) at concentrations of 0.05; 3.5; 6.5; 10.5; 13.0; 17.0; 20.0 µg mL−1; y = ax + b, where y is the peak area of the analyte, a is the angular coefficient, b is the linear coefficient and x is the solution concentration measured in µg mL−1. b r = linear correlation coefficient. c RSD% = relative standard deviation of slope of the calibration curve; c RSD% = relative standard deviation for LOQ. d RE% = Relative error for LOQ.

Pereira and Cass [9] and Nuñez et al. [46] determined that after 12 h of application of TMT and SMX in cattle it is possible to detect them in milk samples. Fig. S5B presents a chromatogram referring to analysis of a real sample of bovine milk acquired from a rural producer. In this milk sample, the concentrations of 7.07 and 1.58 µg mL−1 of TMT and SMX, respectively, were obtained. The higher concentration of TMT and lower of SMX were consistent with the fact of 40% of the TMT and 70% of SMX bind to plasma proteins, thus TMT was excreted in milk in a higher concentration than SMX. These results show that cattle were probably being treated for some disease, among them mastitis. It also demonstrates the importance of monitoring food samples, thus the milk can be discarded when the cattle are undergoing some treatment, respecting the withdrawal time of each drug and to ensure food safety for the consumer. Following these procedures, milk consumers will have the product free of drug residues.

respective correlation coefficients, both above 0.99. The RSD values for the slope of each curve were below 15% and the F (ANOVA) values are above the tabulated F (4.20), indicating that the analytical response effectively varies according to the concentration, confirming the linearity of the method [31]. Table 2 provides the linearity data, as well as the LOD and LOQ of the method. The LOQ features the RSD and RE values below 20%, demonstrating that this level of concentration exhibits acceptable precision and accuracy. In Table 3 it is possible to observe the data for precision and accuracy, in which RSD and RE were lower than 15%. The robustness evaluation data can be observed in Table S4. Note that the obtained values of RSD and RE, for both analytes, were lower than 15% and Pvalues were higher than 0.05, exhibiting that the method is not susceptible to any of the variations studied. The results obtained for stability are available in Table S5. RSD values for the two analytes were less than 15%. The comparisons between the solutions studied and the freshly prepared samples had p-values higher than 0.05, indicating that the difference was not significant. The solutions of the analytes in bovine milk were stable under the conditions evaluated.

5. Conclusions A new method for TMT and SMX determination was developed by a combination of MSPE technique and HPLC-UV. The characterization of the materials allowed to observe some properties such as good adhesion of the polymer to the magnetic core, which justifies the excellent magnetic properties, as well as good thermal stability, characteristic bands of the substances used in the synthesis, and a heterogeneous and irregular surface. Sample preparation using MMIP as adsorbent material in MSPE was optimized and presented relative recovery of around 100% for TMT, because of the selective cavities of the material for this analyte and close to 80% for SMX. This method presented adequate sensitivity and reliability, providing an excellent baseline resolution of the analytes. MSPE also showed fast and easy execution, low solvent consumption and excellent recovery. Validation of the method demonstrated linearity, selectivity, precision and accuracy, within the limits of acceptance, and acceptable LOD and LOQ for both drugs. The method proved to be useful for determination of TMT and SMX in bovine milk samples, which can also be extended to other matrices and applications.

Table 3 Precision and accuracy of the analytical method for determination of TMT and SMX in bovine milk. TMT Nominal concentration (µg mL−1) Intraday (na = 6) Real concentration (µg mL−1) Precision (RSD,%)c Accuracy (RE,%)d Interday (nb = 2) Real concentration (µg mL−1) Precision (RSD,%)c Accuracy (RE,%)d a b c d

SMX

3.5

10.5

17.0

3.5

10.5

17.0

3.397

10.560

16.416

3.084

10.681

17.015

5.489 −2.941

1.923 0.569

2.444 −3.436

13.281 −11.892

2.761 1.721

1.492 0.088

3.689

10.496

16.298

3.084

10.681

17.015

9.070 5.398

1.634 −0.041

2.383 −4.132

1.756 −11.892

0.300 1.721

0.486 0.088

Author statement The authors state that the article being submitted is original, does not infringe copyright laws or any other third-party property rights, has not been previously published, and is not being considered for publication elsewhere. The authors confirm that the final version of the manuscript has been reviewed by native English speakers (English Proof Service) and approved by all authors. Declaration on Conflict of Interest

n = number of replicates of the analyzes. n = number of days. RSD = relative standard deviation between replicates. RE = relative error between actual and nominal concentration.

The authors declare no conflict of interest, particularly no financial and personal relationships with other people or organizations that could inappropriately influence this work. 8

Residual water Water

HPLC-UV

9 SPE-Column RAM/on line

Egg Human plasma Buffalo meat Bovine milk Bovine milk

LC-UV LC-MS/MS HPLC-PDA

HPLC-UV

HPLC-UV

MSPE

SPE-column RAM/on line

SPE

SPE/on line

SPE-Column RAM/online

HPLC/ Electrochemical HPLC-UV

SPE

Bar adsorptive microextraction (BAµE)

SPE

SPE

Sample preparation

Human plasma Bovine milk

LC-MS/MS

HPLC-DAD

Fish plasma

LC-MS/MS

TMT,SMX, ibuprofen, diclofenac, naproxen, salbutamol TMT, SMX TMT, SMX, sulfadiazol, sulfadimethoxine TMT, SMX TMT, SMX TMT, SMX TMT, SMX TMT, SMX, sulfadimidine, sulfadoxine TMT, SMX TMT, SMX

Sample

Instrumental Technique

Analytes

0.031 µg g (all)

25 ng mL−1 50 ng mL−1 0.01 µg mL−1 0.02 µg mL−1

25 ng mL−1 50 ng mL−1 0.05 µg mL−1 (all)

− 1

25 µg mL−1 50 µg mL−1 80 ng mL−1 (all) 0.05 µg mL−1 0.50 µg mL−1 0.062 µg g − 1 (all)

0.53 µg mL−1 0.26 µg mL-1 (Sulfas) –

50–4000 ng mL−1 25–800 ng mL−1 0.05–20 µg mL−1

0.03–30 µg mL−1 0.88–80 µg mL−1 25–400 µg mL−1 50–800 µg mL−1 0.08–1 µg mL−1 0.08–2 µg mL−1 0.05–5 µg mL−1 0.50–60 µg mL−1 0.031–2 µg g − 1 (all)

62.09–88.66 ng L − 24.71–27.52 ng L − 0.16 a 8 µg mL−1 (all)

0.02 ng L − 1 (all) 0.16 µg mL−1 0.08 µg mL−1 (Sulfas), 15 pg 44 pg 15 µg mL−1 25 µg mL−1 40 ng mL−1 25 ng mL−1 –

–

1–100 ng mL−1 (all) 2–200 ng mL−1 (Ibuprofen)

–

1 ng mL−1 (all) 2 ng mL−1 (ibuprofen)

Linear range

LOD

LOQ

Table 4 Review of literature on analytical methods for determination of TMT and SMX in different matrices.

1

1

C8 RAM-BSA (100 mm × 4.6 mm, 10 µm). Luna® C8 (150 mm × 4.6 mm, 10 µm) Luna C8 (250 mm × 4,6 mm, 5 µm)

C8 RAM-BSA (100 mm × 4.6 mm, 10 µm) Luna® C8 (150 mm × 4.6 mm, 10 µm) C18 RAM-BSA (100 mm × 4.6 mm, 10 µm) Luna® C18 (150 mm × 4.6 mm, 10 µm) HPLC-UV: Purospher® C18 (125 mm × 4.0 mm, 5 µm) LC-MS/MS: Gemini® C18 (150 mm × 4.6 mm, 5 µm) RP-C18 (250 mm × 4.6 mm, 5 µm)

Thermo Hypersil Gold C18 (50 mm × 4.6 mm, 5 µm)

Phenomenex Kinetex C18 (150 mm × 4.6 mm, 2.6 µm)

C18 reverse phase

Waters® CORTECS C18 (150 mm × 2.1 mm, 2.7 µm)

Column

This work

[9]

[54]

[53]

[52]

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Reference

L.A.F. Dinali, et al.

Microchemical Journal 154 (2020) 104648

Microchemical Journal 154 (2020) 104648

L.A.F. Dinali, et al.

Acknowledgements [19]

The authors would like to thank the Brazilian agencies CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) for financial support. L. A. F. Dinali thanks to Universidade Federal de Juiz de Fora (UFJF) for technical support in XRD analysis. Also, this study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, and part of the project involving the Rede Mineira de Química (RQ-MG) supported by FAPEMIG (Project: REDE-113/10; Project: CEX-RED-001014).

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Supplementary materials

[24]

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.microc.2020.104648.

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11