Magnetic solid phase extraction followed by high-performance liquid chromatography for the determination of sulphonamides in milk samples

Food Chemistry 157 (2014) 511–517

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Analytical Methods

Magnetic solid phase extraction followed by high-performance liquid chromatography for the determination of sulphonamides in milk samples Israel S. Ibarra a, Jose M. Miranda b, Jose A. Rodriguez a, Carolina Nebot b, Alberto Cepeda b,⇑ a

Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Hidalgo, Carr. Pachuca-Tulancingo Km. 4.5, 42076 Pachuca, Hgo, Mexico Dpto. Química Analítica, Nutrición y Bromatología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Pabellón 4 planta baja, Campus Universitario s/n, 27002 Lugo, Spain b

a r t i c l e

i n f o

Article history: Received 30 March 2013 Received in revised form 17 January 2014 Accepted 17 February 2014 Available online 26 February 2014 Keywords: Magnetic solid phase extraction High performance liquid chromatography Phenyl silica adsorbent Sulphonamides Milk

a b s t r a c t A simple and effective method based on magnetic solid-phase extraction combined with high performance liquid chromatography was used for the determination of nine sulphonamides in milk samples. The extraction and cleanup via silica-based magnetic adsorbent dispersion in milk samples followed by the magnetic isolation and desorption of the analytes using NaOH–methanol. Three different magnetic phenyl silica adsorbents were synthesized by varying the molar ratio of phenyltrimethylsilane and tetramethylorthosilicate; these adsorbents were evaluated for sulphonamides retention in terms of their pH and degree of hydrophobicity. The optimal conditions were a pH of 6.0 and a magnetic:sorbent ratio of 2:1. Under optimal conditions, limits of detection ranging from 7 to 14 lg L1 were obtained. The method was validated according with the European Commision Decision 2002/657/EC. The proposed method was applied to analyse sulphonamides in 27 milk samples of different brands. Thirteen samples tested were positive for the presence of sulphonamides. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Sulphonamides (SAs) are a group of synthetic antimicrobials that are frequently employed for clinical and veterinary purposes to control bacterial infection. These antimicrobials are used for their broad antibacterial spectrum, high efficacy, and low cost (Wang et al., 2012). A variety of SAs are available. However, the SAs most frequently employed in veterinary medicine are sulfadiazine (SDZ), sulfathiazole (STZ), sulfamethazine (SMZ), sulfamethoxypyridazine (SMPZ), sulfachloropyridazine (SCP), sulfamethoxazole (SMX), sulfisoxazole (SFX), sulfadimethoxine (SDM) and sulfaquinoxaline (SQX) (Korpimaki et al., 2004; Wang et al., 2007, 2012). In animal husbandry, SAs are directly administered or added to the feed of poultry, pigs, and cattle to prevent and treat gastrointestinal and respiratory diseases. Additionally, SAs are used in some countries as growth promoters for food-producing animals (Pereira et al., 2012; Wang et al., 2012). The uncontrolled use of sulphona-

⇑ Corresponding author. Address: Laboratorio de Higiene Inspección y Control de Alimentos, Facultad de Veterinaria, Pabellón 4 p.b., Campus Universitario, 27002 Lugo, Spain. Tel.: +34 982285900; fax: +34 982254592. E-mail addresses: [email protected] (J.M. Miranda), alberto.cepeda@ usc.es (A. Cepeda). http://dx.doi.org/10.1016/j.foodchem.2014.02.069 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

mides can lead to the accumulation of these drugs in animal tissues. The presence of these residues, despite their minimal amounts, can induce adverse effects in humans, such as allergic reactions in hypersensitive individuals. Another long-term effect may be carcinogenicity, and prolonged exposure can result in the selection of resistant bacteria in the human body (Di Corcia & Nazzari, 2002; Koesukwiwat, Jayanta, & Leepipatpiboon, 2007; Zayas-Blanco, García-Falcón, & Simal-Gándara, 2004). In dairy cows, SAs are applied in significant amounts and are excreted in the milk. To ensure safety for consumers, the European Union (EU) commission and Drug Administration (FDA) has established a maximum reside limit (MRL) of 100 lg L1 and 100 lg kg1, respectively for SAs in food of animal origin, such as milk (Commission Regulation (EC) No. 281/96, 1999; Commission Regulation (EEC) No. 2377/90, 1990; Koesukwiwat et al., 2007; Wenjun, Chunming, & Minglin, 2011). A variety of analytical methodologies have been developed and reported for the determination of SA residues in foodstuffs at the lg kg1 or lg L1 levels. These methods, used individually or sequentially according to the complexity of the analytical matrix, include immunoassay (Keizer, Bienenmann-Ploum, Bergwerff, & Haasnoot, 2008), capillary electrophoresis (Ming-Ren & Su-Yi, 2003; Soto-Chinchilla, García-Campaña, Gámiz-Gracia, & CrucesBlanco, 2004), gas chromatography (Reeves, 1999), high

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performance liquid chromatography/mass spectrometry (HPLC/MS) (Bogialli, Ascenzo, Di Corcia, Laganà, & Nicolardi, 2008; Di Corcia & Nazzari, 2002), and high performance liquid chromatography (HPLC) (Hyun-Hee, Jung-Bin, Yun-Hee, & Kwang-Geun, 2009; Zayas-Blanco et al., 2004). However, some techniques can be expensive, and many laboratories do not have access to this instrumentation, especially in developing countries. Consequently, the lack of residue control in animal-origin foods for developing nations is a serious handicap that impedes the exportation of these products to countries in which MRLs are established. In this way, HPLC has become a useful technique for analysing antibiotics because it offers high separation efficiency and short analysis times. However, this technique is sometimes insufficient for the direct determination of such antibiotic residues. Trace SA residues must be preconcentrated in food samples such as milk, so they can be detected by HPLC. One of the most difficult stages in the analysis of antibiotics is the extraction, cleanup, and preconcentration of the samples. Solid phase extraction (SPE) is currently the most commonly method employed for simultaneous extraction and cleanup. However, the SPE procedure is expensive and sometimes labour-intensive (Zayas-Blanco et al., 2004). For SAs extraction, the sorbent selection depends on several factors. Common adsorbents are silica-based (C8, C18) (Heller et al., 2002; Reeves, 1999). Recently, adsorbents combined with different types of polymers, such as hydrophilic–lipophilic balanced (HLB) polymers, have been used as an alternative to chromatographic separations, and, in some cases, these combined adsorbents were found to be more selective during the separation of different compounds (Ming-Ren & Su-Yi, 2003). Magnetic solid phase extraction (MSPE) has received considerable attention in recent years due to its potential applications to cell isolation, enzyme immobilisation, protein separation and the preconcentration of organic compounds (Meng et al., 2011; Safarik & Safarikova, 1999). This technique is based on the dispersion of a magnetic adsorbent in the liquid sample. The magnetic adsorbents with analytes adsorbed on the surface can be isolated and eluted with an appropriate solvent. The most attractive property of MSPE is the easy isolation of the magnetic adsorbents from sample solutions by applying an external magnetic field, which minimises the sample treatment time. MSPE has allowed the selective separation of organic compounds, including antibiotics, anti-inflammatory drugs, pesticides and phenolic compounds that are present in different matrices (Aguilar-Arteaga, Rodriguez, Miranda, Medina, & Barrado, 2010; Ibarra, Rodriguez, Paez-Hernandez, Santos, & Miranda, 2012). In this work, a new method for analysing the SAs in milk samples was developed to analyse whole milk samples according to the EU MRLs. This method involves sample pretreatment by MSPE using a paramagnetic adsorbent. The solids were magnetite-embedded with silica functionalized with phenyl chains. The magnetic solids were synthesized, characterised and employed in the cleanup and preconcentration during SAs determination. The magnetic solids are selective and easier to use than classic methodologies, such as SPE.

of acetonitrile with 1% formic acid, and the second a solution of water with 1% formic acid. Both solutions were filtered through a 0.45-lm filter prior to use. Sulfachloropyridazine (98%), sulfadiazine (99%), sulfadimethoxine (99%), sulfamethazine (99%), sulfamethoxazole (98%), sulfamethoxypyridazine (98%), sulfaquinoxaline (99%), sulfathiazole (98%), and sulfisoxazole (98%) were obtained from Sigma (St. Louis, MO, USA). The different standard solutions, prepared daily by dilution of a stock solution of each sulphonamide at a concentration of 0.6 g L1, were prepared by dissolving the pure substances in methanol. These solutions were stored in the dark and refrigerated at 4 °C. Sulfamethoxydiazine (SMTD), at a concentration of 5 lg L1, was used as the internal standard (IS). Magnetite embedded in phenyl silica adsorbents was synthesized by the sol–gel method using magnetite, phenyltrimethoxysilane (97%, PTMS) and tetramethylorthosilicate (98%, TMOS) from Sigma. Emulsion polymerisation of the monomers was performed using Triton X-100 and cetyltrimethyl ammonium bromide (CTAB) from Sigma. 2.2. Apparatus The structures of the products obtained were determined by Xray diffraction (XRD) using a Philips type powder diffractometer fitted with a Philips PW 1710 control unit, Vertical Philips PW 1820/00 goniometer and FR 590 Enraf Nonius generator (Philips, Almelo, Netherlands). The instrument was equipped with a graphite diffracted beam monochromator and copper radiation source (k(Ka1) = 1.5406 Å), operating at 40 kV and 30 mA. The X-ray powder diffraction pattern was collected by measuring the scintillation response to Cu Ka radiation versus the 2h value over a 2h range of 5–70, with a step size of 0.02° and a counting time of 2 s per step. The solid products were characterised using a Fourier transform infrared (FTIR) spectrophotometer (PerkinElmer, model IRDM). Dry samples were prepared in the form of KBr tablets (1:100, w/w).The morphological analysis of the magnetic adsorbents was performed using a JEOL JEM-1011 Transmission Electron Microscope (TEM) and JEOL JSM-6360LV scanning electron microscope (SEM; JEOL (Europe) B.V. Belgium). The HPLC–DAD analyses were carried out on a Waters 2695 HPLC system, which is a liquid chromatography connected to a Waters 996 diode array detector (DAD) (Milford, MA, USA), and the dates were collected using Waters Empower Pro software. The separation was performed using a Gemini 3 l C18 110 Å column (50  4.60 mm, 3 lm) supplied by Phenomenex (Macclesfield, UK). The samples were injected automatically (10.0 lL). Their separation was performed using two different mobile phases: an aqueous solution of 0.1% formic acid (Phase A) and a solution of 0.1% formic acid in acetonitrile (Phase B). The mobile phase gradient used was as follows: step 1: 0–4 min, 2% B, step 2: 4–5 min, 2–10% B, step 3: 5–27 min, 10–30% B, step 4: 27–30 min, 30–55% B, step 5: 30– 30.5 min, 55% B, step 6: 30.5–31 min, 55–2% B, step 7: 31–35 min, 2% B. The total flow rate was kept constant at 0.5 mL/min during the separation. The chromatogram was monitored at 260 nm, and UV spectra of individual peaks were recorded in the range of 200– 400 nm.

2. Experimental 2.3. Synthesis of the adsorbent 2.1. Reagents and chemicals All solutions were prepared by dissolving the respective analytical grade reagent in deionized water with a resistivity not less than 18.0 MX cm, provided by a Milli-Q system (Millipore, Bedford, MA, USA). Sodium hydroxide and acetic acid were obtained from J.T. Baker (Phillipsburg, NJ, USA). Methanol was supplied from Sigma–Aldrich (Taufkirchen, Germany). The mobile phases used for HPLC experiments consisted of two solutions, one a solution

The synthesis of the adsorbents was carried out in two steps. First, magnetite was obtained through the partial oxidation and precipitation of Fe(II) in the presence of oxygen in basic media (Barrado, Prieto, Vega, & Fernández-Polanco, 1998). Fifty millilitres of 1.25 mM FeSO47H2O solution were stirred at 60 ± 5 °C, and the solution was adjusted to a pH of 10 ± 0.2 with 6 M NaOH solution. After 1 h, the magnetic precipitates were isolated and washed with deionized water. The magnetic adsorbents were obtained by

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emulsion polymerisation by mixing the previously synthesized magnetite. The synthesized magnetite was added to a flask containing PTMS and TMOS at different molar ratios. The functional monomers were added at a total concentration of 3 mM. The silica precursors were previously solubilised in 24 mL of a solution containing 2.0% (w/v) Triton X-100, 0.02% (w/v) CTAB, 12.5% (v/v) methanol and 200 lL, with 28% (w/v) NH3 as the catalyst. The mixture was heated and refluxed at 120 ± 5 °C for 16 h with stirring. 2.4. Sample treatment The proposed MSPE methodology was performed as follows: Initially, 0.1 g of the magnetic adsorbent was added to an Erlenmeyer flask. The particles were conditioned with 5.0 mL of methanol in an ultrasonic bath for 5 min. The adsorbent was then isolated by applying an external magnetic field and washed twice with 10.0 mL of deionized water for 3 min, and the supernatant was discarded. Subsequently, an adequate volume (10.00 ± 0.08 mL) of milk sample was mixed with the preactivated magnetic adsorbents. After sonication for 15 min, an external magnetic field was applied to isolate the adsorbent with the adsorbed analytes. The liquid phase was decanted, while the solid phase was washed three times with 5.0 mL of acetate buffer (pH 4.0). The SAs were eluted from the magnetic adsorbent by dispersion of the solid with 3.0 mL of methanol solution containing 1  103 M NaOH for 5 min. The resulting solution was evaporated to dryness under an air stream, and the residue was reconstituted to 500 lL in 1% formic acid/ 5 lg L1 of IS. Finally, the solution was filtered through a 0.2 lm nylon filter and analysed by HPLC–PAD. 3. Results and discussion 3.1. Chromatographic separation The mobile phase composition was studied to achieve the optimum conditions of separation for the nine sulphonamides and the internal standard using the stationary phase described above. In this study, two different solutions were evaluated for the mobile phase gradient: a 0.1% formic acid aqueous solution (Phase A) and a 0.1% formic acid in acetonitrile solution (Phase B). The percentage of acetonitrile in the mobile phase significantly affects the resolution of the SAs of interest.

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To evaluate this effect, three different levels (30%, 45% and 50%) in four time intervals were tested. In the case of 50%, three interval times were evaluated (5–11, 5–15 and 5–22 min). For 45% and 30%, they were tested at 5–27 min. According to the experiments performed, when the percentage of acetonitrile achieved 50% a signal overlap among four analytes of interest (sulfachloropyridazine, sulfamethoxazole, sulfadimethoxine and sulfisoxazole) was observed. A better resolution of 8 SAs was obtained using the gradient proposed using a 45%, but the resolution of the last 2 signals was not adequate with an increase of the percentage of acetonitrile to 70%. A slower mixture of the mobile phase solutions over 27 min, followed by an increase of the percentage of acetonitrile to 55%, provided an adequate resolution of the SAs analysed. During the initial three steps in the gradient, the separation of eight signals (i.e., sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfamethoxazole, sulfisoxazole, and the internal standard) is obtained, while the last steps are required for the separation and identification of sulfadimethoxine and sulfaquinoxaline. The last gradient was used for the SA analysis with MSPE–HPLC. 3.2. Characterisation of the optimum magnetic adsorbent To evaluate the retention of SAs, one of the most important characteristics of the magnetic adsorbents is their hydrophobicity. For this reason, three different magnetic adsorbents were synthesized and characterised. The magnetic adsorbents were obtained by varying the molar ratio of functionalized monomer (PTMS) and crosslinking monomer (TMOS) in the following ratios: 1.0:1.0, 2.0:1.0 and 4.0:1.0 (PTMS:TMOS). Solids with high surface areas were obtained in the four experiments (Aguilar-Arteaga et al., 2010). The magnetic adsorbents synthesized were evaluated for SA retention by MSPE. The overall morphology of the magnetic adsorbent was studied using SEM–TEM, showing that the adsorbent presented a spherical morphology (Fig. 1). The X-ray diffraction (XRD) study of the magnetic adsorbents showed, in all cases, the reflections corresponding to magnetite (m). Fig. 1 shows the typical XRD patterns of the adsorbents. The presence of a wide band at 2h angles from 5° to 9° and 15° to 25° is consistent with the amorphous phase of the silica gel. The FTIR spectrum for the magnetic adsorbent presents characteristic vibrations from 600 to 550 cm1, corresponding to the vibrations of the metal occupying tetrahedral and octahedral

Fig. 1. Diffraction patterns for the magnetic adsorbents (m: diffraction lines for magnetite). Solids synthesized from: (A) 1.0:1.0, (B) 2.0:1.0, and (C) 4.0:1.0.

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positions, respectively. The spectra show an intense stretching band at 3600–3250 cm1, which is attributed to the vibration of the silanol group (SiOH). The aromatic character of phenyl adsorbents was corroborated by a group of weak signals above 3000 cm1, corresponding to the C–H vibration of the phenyl group. A series of weak bands from 1700 to 1550 cm1 is also characteristic of C–H vibrations from aromatic groups. The bending band at 1450 cm1 is attributed to the H2O contained in the magnetic adsorbent. A stretching band at 1300–900 cm1 is assigned to the siloxane group (Si–O–Si), and the deformation band at approximately 850–750 cm1 belongs to the Si–OH group. According to the FTIR spectra, the stretching band of SiOH decreases as the PTMS:TMOS ratio increases, and the hydrophobicity of the magnetic adsorbent thus increases in the same way (Aguilar-Arteaga et al., 2010). The SEM and TEM analysis of the morphology of the magnetic adsorbents shows a homogenous solid with spherical morphology, and the particle size was determined to be in the range of 63– 100 nm. The magnetite particles partly cover the surface with silica gel; the magnetic moments are then randomly oriented such that when an external magnetic field is applied, these moments become aligned parallel to the field because the magnetic adsorbent properties are similar to those of pure magnetite. This property makes the solids suitable for use as adsorbents in magnetic-MSPE preconcentration. 3.3. Retention and elution of SAs in milk samples The pH dependence of sample extraction and isolation is frequently studied during the isolation of SAs from complex matrices. The existence of various degrees of ionisation of the SA structures is related to their acid dissociation constants (pKa). The pKa values of SAs in aqueous solution range from 1.49 to 7.12, indicating that, at pH values below 1.49, SAs are in cationic form. SAs are in a neutral state as zwitterions between pH 1.49 and 7.12. At pH values higher than 7.12, the SAs are negatively charged (Sanli, Altun, Sanli,

Alsancak, & Beltran, 2009). To evaluate the effect of pH and hydrophobicity, the retention experiments were carried out in the pH interval from 4.0 to 10.0. All of the experiments were performed using 10 ml of a 10 mg L1 standard solution of each SA and 0.1 g of magnetic adsorbent. Once the extraction was completed, the SAs remaining in the solution were determined by HPLC. The percentages of retention and recovery were calculated as a function of the concentration added and concentration found after extraction. Table 1 shows the % retention obtained using different magnetic adsorbents. The percentage of SA retention increases according to the decrease in the hydrophobicity of the magnetic adsorbent. For the whole pH range investigated, the maximum retention was obtained using the magnetic adsorbent with a 1:1 PTMS:TMOS ratio. However, the use of this adsorbent was ineffective in the preconcentration of SAs due to the adsorbent present with a high polarity, thus resulting in a low re-extraction of the analytes. For this reason, the adsorbent employed in the extraction of SAs was the magnetic adsorbent with a 2:1 PTMS:TMOS ratio. The pH effect on the SAs retention demonstrated a higher affinity of the adsorbent for their neutral state than their protonated and deprotonated forms. Therefore, the optimal conditions selected for SAs retention were a pH of 6.0 and a magnetic adsorbent ratio of 2:1 because, under these conditions, the percentage of retention ranged from 72.30% to 98.52% for all SAs determined. At pH values below 4.0, the degradation of the magnetite present in the magnetic adsorbent occurred, allowing the formation of chelates between the Fe(III) and the SAs and resulting in the degradation of the SAs (Hassan, M.K., Hassan, R.M., & Abdalla, M.A, 1991). At pH values above 9.0, the magnetite particles acquired a negative charge due to the binding of hydroxide groups, thus causing electrostatic repulsion between the adsorbent and the anionic SAs (Jordan, Marmier, Lomenech, Giffaut, & Ehrhardt, 2007). This effect explains the positive effect during elution when a 1  103 M NaOH in methanolic solution is used for the preconcentration of the SAs by MSPE. To determine the adsorption capacity and the affinity of the magnetic adsorbent for the analytes of interest, SA concentrations

Table 1 Percentages of retention (mean and % RSD, n = 3) at different pH values and magnetic adsorbent proportions (PTMS:TMOS). PTMS:TMOS

SAs

pH 4.0 (RSD)

6.0 (RSD)

8.0 (RSD)

10.0 (RSD)

1.0:1.0

SDZ STZ SMZ SMPZ SCP SMX SFX SDM SQX

77.74 81.95 81.02 79.45 91.74 85.54 99.20 96.07 97.61

(1.62) (2.34) (2.64) (1.24) (1.20) (3.05) (2.56) (2.31) (2.01)

79.10 (3.50) 78.70 (1.69) 85.94 (2.67) 84.75 (2.01) 93.92 (1.95) 87.16 (4.63) 96.17 (3.62) 99.57 (0.63) 101.24 (3.59)

52.56 54.78 68.76 68.94 70.90 69.50 56.48 73.54 92.14

(2.79) (2.47) (2.31) (2.80) (2.14) (3.87) (3.07) (2.83) (3.24)

47.66 57.08 68.66 64.39 69.25 68.69 52.26 74.76 62.41

(2.38) (3.09) (2.49) (2.55) (2.37) (2.55) (3.14) (1.55) (3.02)

2.0:1.0

SDZ STZ SMZ SMPZ SCP SMX SFX SDM SQX

71.19 71.85 73.49 69.47 87.95 66.27 96.91 95.99 99.67

(1.38) (1.56) (2.37) (3.09) (1.06) (2.24) (0.95) (1.11) (3.98)

73.36 72.30 77.25 74.73 85.10 75.48 97.41 97.10 98.52

(2.04) (3.22) (1.63) (0.71) (1.62) (1.71) (1.58) (2.07) (1.52)

51.05 46.31 64.51 60.49 70.44 68.49 87.97 77.09 95.35

(2.19) (2.53) (3.51) (4.47) (3.15) (4.47) (2.57) (1.62) (1.18)

26.92 31.61 45.73 47.33 39.42 35.35 86.56 66.20 71.26

(2.10) (2.03) (2.24) (2.20) (1.32) (1.29) (2.79) (2.65) (2.52)

4.0:1.0

SDZ STZ SMZ SMPZ SCP SMX SFX SDM SQX

23.71 16.36 48.74 48.73 54.59 57.12 64.29 86.78 97.04

(2.58) (1.92) (2.40) (1.43) (3.66) (3.98) (2.60) (2.01) (2.44)

23.80 22.45 54.28 48.68 47.75 51.77 47.61 86.14 74.44

(3.60) (2.97) (4.02) (3.62) (1.67) (3.57) (3.73) (3.96) (3.70)

19.12 24.57 48.57 44.64 34.87 44.26 40.87 68.31 70.45

(4.04) (2.84) (3.69) (2.48) (3.47) (2.72) (2.89) (3.05) (1.49)

7.86 (2.26) 10.53 (2.77) 32.14 (4.05) 27.65 (4.01) 28.12 (3.01) 28.53 (4.38) 13.41 (2.94) 31.11 (2.61) 34.48 (3.95)

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I.S. Ibarra et al. / Food Chemistry 157 (2014) 511–517 Table 2 Results of the Scatchard analysis. SA

Linear equation

Kd (lM)

Qmax (lmol g1)

SDZ STZ SMZ SMPZ SCP SMX SFX SDM SQX

3.84 ± 0.45–17.32 ± 0.05 Q 3.66 ± 0.18–17.27 ± 2.38 Q 3.51 ± 0.10–14.30 ± 1.41 Q 4.23 ± 0.19–32.03 ± 2.82 Q 3.64 ± 0.17–16.32 ± 3.11 Q 3.10 ± 0.14–15.36 ± 2.22 Q 3.38 ± 0.08–12.05 ± 1.16 Q 3.00 ± 0.14–7.91 ± 1.92 Q 3.14 ± 0.08–8.11 ± 0.99 Q

57.74 ± 0.17 57.90 ± 7.98 69.93 ± 6.89 31.22 ± 2.75 61.27 ± 11.67 65.10 ± 9.41 82.99 ± 7.99 126.42 ± 30.69 122.10 ± 14.90

221.71 ± 25.98 211.92 ± 31.01 245.45 ± 25.19 132.06 ± 13.05 223.04 ± 43.76 201.82 ± 30.56 280.49 ± 27.80 379.26 ± 50.26 384.61 ± 47.96

Table 4 CCa, CCb, inter-assay RSD (%) and Accuracy (%) values for each of the analytes investigated.

were determined using the adsorption isotherm and the corresponding Scatchard analysis. Adsorption isotherms were constructed to determine the concentration (mM) of SAs remaining after the dispersion of 0.1 g of magnetic adsorbent in 25 mL of a SA solution in the concentration interval from 2 to 70 lM. The amount of SA bound to the magnetic adsorbent at the binding equilibrium increased to a concentration of 50 lM. In the Scatchard analysis, the amount of SA bound to the magnetic adsorbent was obtained by subtracting the free concentration from the initial concentration of each SA. When the SA concentration was varied, the Scatchard plot was obtained according the following equation:

Q Q 1 ¼ max  Q ½SAs Kd Kd where Q (mmol g1) is the amount of each SA bound to the magnetic adsorbent at equilibrium, [SAs] (mM) is the free SA concentration at equilibrium, Kd is the dissociation constant (the affinity of the magnetic adsorbent for the SAs), and Qmax (mmol g1) is the maximum binding amount. The Kd and Qmax are estimated from the slope and intercept of the linear plot of Q vs. Q/[SA]. The linear regression equations for each analyte are shown in Table 2. The Kd and Qmax values correspond to a medium affinity between the adsorbent and SA, which is adequate in the elution process (BaniYaseen, 2011; Shi et al., 2011). The linearity observed in the Scatchard plot indicates the presence of homogeneous active sites on the magnetic adsorbent. Retention, elution and affinity experiments demonstrate the usefulness of the developed magnetic adsorbent

Analyte

CCa (lg L1)

CCb (lg L1)

% RSD

% Accuracy

SDZ STZ SMZ SMPZ SCP SMX SFX SDM SQX

112.99 117.16 109.62 113.16 111.57 114.58 115.65 117.04 108.86

125.99 134.32 119.24 126.32 123.14 129.16 131.31 134.08 117.73

3.31–7.36 6.82–9.74 1.59–4.88 5.42–9.64 2.91–9.60 3.37–7.34 5.68–9.32 2.89–7.00 3.26–5.01

82.04–108.49 81.88–111.80 91.74–112.82 83.13–111.42 85.55–106.25 83.24–110.27 84.13–113.15 84.90–113.45 96.67–114.98

for the SAs determination in milk samples by magnetic solid phase extraction. 3.4. Method validation Under the optimal conditions, the analytical parameters of the method combining MSPE and HPLC (MSPE–HPLC) were evaluated using a milk sample with a volume of 10.0 mL that was spiked with SAs in the concentration range of 0–500 lg L1. Each standard was prepared and analysed in triplicate. The resulting standards were thoroughly homogenised and preconcentrated using the proposed methodology described in the experimental section. The peak areas obtained were measured, and a calibration curve was constructed from the average peak areas. The calibration curve showed a linear dependence of the peak area on the SA concentration in the spiked milk sample. The regression parameters for the calibration lines are shown in Table 3. The LODs were calculated for a signal-to-noise ratio equal to 3.29 according to the IUPAC recommendations (Currie, 1995). The accuracy of the method was investigated using a quantitative confirmatory method according to the Commision Decision 2002/657/EC. The decision limit (CCa) and detection capability (CCb) were obtained employing different sulphonamide standard solution to perform a regression study between the area points observed and the concentrations injected. Five standard solutions (0.5, 1.0, 1.5, 2.0 and 5.0 MRL) and a total of three sets of 21

Table 3 Regression parameters of the calibration lines for absorbance (mAU) vs. concentration of SAs (lg L1) in 10.0 mL of milk. Analyte

Regression parameters

SDZ STZ SMZ SMPZ SCP SMX SFX SDM SQX

Intercept bo ± ts (bo)

Slope b1 ± ts (b1)

Correlation coefficient r2

Limit of detection (lg L1)

Linear range (lg L1)

0.0021 ± 0.0009 0.0014 ± 0.0014 0.0013 ± 0.0016 0.0079 ± 0.0056 0.0001 ± 0.0016 0.0036 ± 0.0018 0.0102 ± 0.0021 0.0079 ± 0.0049 0.0199 ± 0.0081

0.9501 ± 0.0125 1.6428 ± 0.0197 3.6945 ± 0.0233 5.7454 ± 0.0799 1.7434 ± 0.0224 2.0606 ± 0.0251 0.8620 ± 0.0298 5.2647 ± 0.0705 7.3368 ± 0.1160

0.9991 0.9993 0.9998 0.9990 0.9992 0.9993 0.9994 0.9991 0.9988

12 11 7 14 13 12 10 13 14

36–800 33–800 21–800 42–800 39–800 36–800 30–800 39–800 42–800

Repeatability (n = 3) 50 lg L1

lg L SDZ STZ SMZ SMPZ SCP SMX SFX SDM SQX

1

50.29 51.29 55.39 45.57 46.22 44.14 49.44 42.89 55.12

Reproducibility (n = 3) 100 lg L1

150 lg L1

1

1

% RSD

lg L

6.20 9.81 3.93 5.06 11.09 3.98 7.58 2.62 3.31

90.12 92.21 109.64 99.45 94.57 95.04 99.13 107.38 105.91

% RSD

lg L

5.54 9.32 1.79 8.02 8.23 8.57 6.14 5.90 4.72

128.95 131.41 158.68 135.22 134.81 159.89 128.37 153.88 162.64

50 lg L1 1

% RSD

lg L

5.31 8.02 5.14 3.07 2.15 2.12 1.02 7.10 4.85

44.15 51.85 54.86 51.07 46.68 47.47 48.72 51.77 47.66

100 lg L1 1

% RSD

lg L

3.64 7.68 1.20 8.40 7.42 6.41 6.75 3.40 4.39

92.44 98.39 115.36 92.67 105.49 96.55 90.18 104.88 100.22

150 lg L1 % RSD

lg L1

% RSD

7.58 4.27 2.05 4.14 4.70 2.12 1.88 8.71 5.38

147.93 154.07 151.77 142.43 135.91 156.10 142.57 161.01 151.59

9.99 9.67 5.67 7.18 5.22 9.14 8.90 5.92 3.87

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I.S. Ibarra et al. / Food Chemistry 157 (2014) 511–517

Fig. 2. Chromatograms obtained from the analysis of sulfonamides by MSPE: (A) blank sample, (B) real milk sample and (C) spiked milk sample (200 lg L-1); sulfadiazine (SDZ), sulfathiazole (STZ), sulfamethazine (SMZ), sulfamethoxypyridazine (SMPZ), sulfachloropyridazine (SCP), sulfamethoxazole (SMX), sulfisoxazole (SFX), sulfadimethoxine (SDM), sulfaquinoxaline (SQX) and internal standard (IS).

samples with six replicates for the concentration levels of 0.5, 1.0 and 1.5 were analysed on three different occasions, and the relative standard deviations (% RSDs) were determined. The CCa is the lowest concentration at which a method can discriminate with a statistical certainty of 1-a that the analyte is present above the MRL. In the case of sulphonamides with established MRL, CCa was established analysing blank samples spiked at approximately the MRL level. The concentration at the MRL plus 1.64 times the corresponding standard deviation (SD) is defined as CCa (a = 5%). The CCb is the concentration at which the method is able to detect truly contaminated samples with a statistical certainty of 1-b. CCb was calculated using the signal at CCa plus 1.64 times the standard deviation of the within laboratory reproducibility of spiked samples at the MRL level (b = 5%). The CCa and CCb values, in analytical solutions and milk samples, are shown in Table 4. The mean absolute recoveries obtained for the nine SAs in spiked milk samples were found to be in the range of 81.88–114.98%, with a RSD less than 10% in all cases using the MSPE methodology proposed. These values are higher than the values reported using SPE–CE with Oasis HLB cartridges (72–97%) (Decision, 2002). The SPE methodologies reported involve a cleanup step using liquid–liquid extraction before extraction by SPE. The main advantage of the MSPE procedure is the reduction in sample treatment time. The precision of the MSPE method proposed was measured in terms of repeatability and reproducibility for migration times and peak areas (Currie, 1995). The results were determined as the relative standard deviation (% RSD) obtained using three individual magnetic adsorbents synthesized under optimal conditions and applied to the analysis of a spiked blank sample (50, 100, and 150 lg L1). Based on these results and considering the most restrictive MRLs established by the EU regulations, the synthesis and analysis methodologies are adequate for analysing the SAs in milk samples. The proposed methodology was applied to the determination of SAs in 27 commercial milk samples from different brands. Three replicate determinations of each analyte in the selected samples were performed. According to the results obtained from the analysis of the SAs in milk, 13 samples tested were positive for the presence of different SAs, which were identified by their migration times. To confirm the presence of each analyte, a standard addition was made to the sample extract. An increase in the peak area confirmed the presence of the antibiotic residue. Twelve samples were positive for SDZ, 11 for SMPZ, 2 for SDM and one sample for SQX, with

concentrations of 42.18–119.80 lg L1, 36.64–107.53 lg L1, 82.35–120.64 lg L1, and 96.59 lg L1, respectively. The RSD was less than 10% in all analyses. The chromatograms obtained for the milk samples after extraction are shown in Fig. 2. No interference from the matrix was observed after the MSPE–HPLC analysis of a blank milk sample (Fig. 2A), a real milk sample (Fig. 2B) and a spiked milk sample (Fig. 2C), demonstrating the selectivity of the MSPEHPLC methodology for SA analysis. 4. Conclusions The proposed MSPE technique based on the synthesized magnetic adsorbent (Fe3O4–SiO2–phenyl modified) was demonstrated to be an efficient strategy for the rapid preconcentration of SAs residues in complex matrices such as milk. The methodology described is faster than classical preparation procedures, such as SPE, with a minimum sample manipulation, lower solvent consumption, and consequently lower cost. Additionally, this technique provides good results in terms of sensitivity and accuracy. When coupled to HPLC, the MSPE method yielded LODs of 7–14 lg L1, according to the more restrictive MRLs established, for the nine target SAs determined. Additionally, the MSPE preconcentration technique is also a good alternative for coupling to other analytical methodologies, such as CE. Acknowledgements The authors wish to thank CONACyT (mixed scholarship 290618) and Consellería de Cultura, Educación e Ordenacion Universitaria, Xunta de Galicia (project EM 2012/153) for financial support. References Aguilar-Arteaga, K., Rodriguez, J. A., Miranda, J. M., Medina, J., & Barrado, E. (2010). Determination of non-steroidal anti-inflammatory drugs in wastewaters by magnetic matrix solid phase dispersion–HPLC. Talanta, 80, 1152–1157. Bani-Yaseen, A. D. (2011). Spectrofluorimetric study on the interaction between antimicrobial drug sulfamethazine and bovine serum albumin. Journal of Luminescence, 131, 1042–1047. Barrado, E., Prieto, F., Vega, M., & Fernández-Polanco, F. (1998). Optimization of the operational variables of a medium-scale reactor for metal-containing wastewater purification by ferrite formation. Water Research, 32, 3055–3061.

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