382 Ana Mar´ıa Bueno Ana Mar´ıa Contento ´ Angel R´ıos Department of Analytical Chemistry and Food Technology, University of Castilla–La Mancha, Campus de Ciudad Real, Ciudad Real, Spain Received September 14, 2013 Revised November 29, 2013 Accepted November 29, 2013
J. Sep. Sci. 2014, 37, 382–389
Research Article
Determination of sulfonamides in milk samples by HPLC with amperometric detection using a glassy carbon electrode modified with multiwalled carbon nanotubes A sensitive and accurate method for determining five sulfonamides based on HPLC with amperometric detection and using a glassy carbon electrode modified with multiwalled carbon nanotubes is proposed. Optimal conditions for the quantitative separation of selected sulfonamides were studied, and glassy carbon electrodes with and without modification with carbon nanotubes were systematically investigated as electrodic materials. Statistical analysis of the obtained results demonstrated that these modified electrodes achieved considerably better stability and sensitivity than the conventional unmodified ones. Detection limits were in the 1.2–6.0 ng/mL range. The usefulness of the method was demonstrated by the analysis of milk samples, taking into account the European legislation on residues in food products, following both a screening method to classify the samples and a confirmation method to provide more detailed information in the case of positive samples. Keywords: Amperometric detection / Carbon nanotubes / Liquid chromatography / Modified electrodes / Sulfonamides DOI 10.1002/jssc.201301011
1 Introduction Sulfonamides (SAAs) are a class of antibacterial drugs used in farm animals for the treatment of a variety of bacterial infections. In food-producing animals, sulfonamides are used not only for therapeutic but also for prophylactic purposes [1, http://fri.wisc.edu/docs/pdf/FRIBrief_VetDrgRes.pdf]. The quantification of these antibacterial drugs in milk represents a challenge, since the antibacterial drugs and their metabolites are secreted in milk. Although the use of veterinary drugs has helped to increase the food supply, negative consequences, such as presence of drug residues in food, cannot be ignored. The presence of drug residues in foods can be a health hazard to consumers. First, the carcinogenicity of some drugs may be a serious concern [2–5]. Second, continuous exposure of certain micro-organisms to these drugs may result in the development of drug-resistant strains. Because of the toxicology of the residues of these compounds for the human health, the European Union (EU) has set the maximum combined residues for all substances in the sulfonamide group at 100 g/kg [6]. ´ Correspondence: Professor Angel R´ıos, Department of Analytical Chemistry and Food Technology, University of Castilla–La Mancha, Campus de Ciudad Real, E-13004 Ciudad Real, Spain E-mail: [email protected] Fax: +34 926295232
Abbreviations: CNT, carbon nanotube; ED, electrochemical detection; GCE, glassy carbon electrode; MWCNT, multiwalled carbon nanotube C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Several analytical methods have been developed to separate and detect these compounds in different samples. GC– MS methods were developed for detecting sulfonamides after derivatization [7–9]. Several methods using CE with UV [10], fluorescence [11], electrochemical (ED) [12], and MS [13] detection have been used for the determination of these drugs in meat, tissues, tablets, and human urine samples, respectively. But the most commonly used methods include LC using UV diode array [14–19], fluorescence [20–22], MS [22–27], or MS/MS detection [28–30]. These methods require significant amounts of time associated with clean-up steps, sophisticated instrumentation, and skilled operators. There are only a few references using ED, probably because of problems related to electrode fouling and deactivation [31–33]. Thus, in this way, the voltammetric behavior of eight sulfonamides at the glassy carbon electrode (GCE) was studied and low detection limits were obtained [31]. The same electrode was used in the oxidation [34] and reduction [35] of sulfadiazine in pharmaceuticals with good results. A similar study for the electroanalytical oxidation of sulfamerazine was carried out by Pingarr´on et al. [33]. Some previous studies used borondoped conductive diamond electrodes for the ED determination of several sulfonamides [36, 37], characterized by an excellent performance with high sensitivity and reproducibility. Thus, this type of electrode was used by Souza et al. in pharmaceuticals, in order to determine sulfadiazine and sulfamethoxazole by square-wave voltammetry [37]. The results obtained show very low detection limits and good recoveries. Colour Online: See the article online to view Fig. 4 in colour. www.jss-journal.com
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Figure 1. Chemical structures of the SAAs.
Recently, many studies have revealed that modification of electrode surface with nanomaterials is a promising avenue. Carbon nanotubes (CNTs), consisting of cylindrical graphene sheets with nanometer diameter, have attracted much attention due to their unique mechanical, chemical, and electronic properties [38, 39] since their discovery by Iijima [40] in 1991. All these properties make them suitable for the modification of electrodes [41–43] and suggest great promise for amperometric detection [44–46]. The performance of electrodes modified with multiwalled CNTs (MWCNTs) is superior to the performance of other conventional carbon electrodes in terms of electron transfer rate, reversibility, and conductivity [47, 48]. CNTs were incorporated into a paste electrode in the determination of sulfamethoxazole for the first time by Arvand et al. [48]. Results showed a marked decrease on overvoltage for sulfamethoxazole oxidation and enhanced the S/N characteristics compared to those observed at the carbon paste electrode. In this work, we report the development of an accurate and precise method for the determination of five representative sulfonamides (sulfisoxazole, SXZ; sulfamerazine, SMZ; sulfathiazole, STZ; sulfanilamide, SAA; and sulfadiazine, SDZ) based on the amperometric response at MWCNTsmodified GCEs after separation by HPLC. The structures of these five compounds are shown in Fig. 1. A high sensitivity, together with a good repeatability and reproducibility of the response obtained with these electrodes were achieved. The proposed method was applied to the analysis of these compounds in different types of milk samples by using a screening method or a confirmatory method, according to the experimental chromatographic conditions used. The screening/confirmation strategy avoids the detailed analysis of the samples, especially when samples are negative (most samples will be negative samples). In addition, the screening method is closer to the needs of the consumer and to the legislation requirements. From a technical viewpoint, or for decisionmaking purposes, the information about positive samples must be completed with the particular concentrations. Thus, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the screening/confirmation strategy meets this double important role.
2 Materials and methods 2.1 Apparatus and electrodes Liquid chromatographic experiments were carried out with an HP 1090 Liquid Chromatograph (Agilent, USA) and amperometric detector (Metrohm 791 VA, Gomensoro S.A., Spain) using Labview software. The wall-jet flow-cell consisted of an Ag/AgCl/3M KCl reference electrode (Metrohm Model 60727000, Gomensoro, Spain), a platinum auxiliary electrode, and glassy carbon GCE (Metrohm Model 60805010, Gomensoro, shaft diameter bottom 7 mm) or GCE-CNTs as working electrode. A Zorbax SB-C18 column (Agilent, 150 × 4.6 mm id; particle size, 3.5 m) was used for the separation of the drugs. A solution of 0.05 M K2 HPO4 pH 3/ACN/MeOH (80:15:5) at room temperature (20 ± 1⬚C) was used as the mobile phase in the LC experiments. The potential detection applied was 1200 mV for all measurements. The length of the tubing connecting the HPLC and the detector was 10 cm. The wall-jet flow-cell consisted of an Ag/AgCl/3 M KCl reference electrode (Metrohm Model 60727000), a platinum auxiliary electrode, and glassy carbon GCE (Metrohm Model 60805010, shaft diameter bottom 7 mm) or GCE-CNTs as working electrode. Samples were extracted using an SPE-Vacuum manifold from Supelco (Madrid, Spain). An ultrasonic bath (Ultrasons J.P. Selecta, Barcelona, Spain) was used to clean the surface of the electrodes and homogenize the solutions. 2.2 Materials and standards SAA, SXZ, STZ, SMZ, SDZ, trichloroacetic acid, potassium monobasic phosphate and dibasic, and sodium hydroxide www.jss-journal.com
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were purchased from Sigma Chemical (St. Louis, MO, USA). MWCNTs with 95% purity were obtained from NanoLab (Brighton, MA). Methanol and acetonitrile were of HPLC grade and acquired from Panreac (Barcelona, Spain). Nafion was supplied from Fluka (USA). In all cases, water was of high quality, purified in a Milli-Q system (Millipore, Bedford, MA, USA). A stock standard solution (1 mM) of each sulfonamide was prepared in MeOH and stored in the dark at 5⬚C. Working standard solutions were prepared daily by diluting the stock solutions with carrying medium (0.05 M K2 HPO4 pH 3/ACN/MeOH 80:15:5). 2.3 Preparation of the CNTs dispersion and GCE-CNTs The CNTs dispersion was obtained by dispersing 1.0 mg of MWCNTs in 1.0 mL of 0.1% v/v Nafion solution followed by sonication for 15 min. The GCE surface was polished with alumina, rinsed with distilled water, sonicated in water and acetone with an ultrasonic bath, and dried in air. An aliquot (10 L) of the dispersion was dropped on the electrode surface and the solvent was evaporated under an infrared heat lamp [32]. The CNTs dispersion was prepared each week, but it did not lose its ED properties for longer periods.
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To modify the surface of the electrode, dispersions of CNTs in Nafion at concentrations between 0.5 and 5 mg/mL were prepared. The amperometric response for 3 g/mL sulfanilamide was checked, and the maximal current was obtained for 1 mg/mL of CNTs dispersion. Larger concentrations of CNTs considerably increased the baseline noise. Moreover, different volumes (between 5and 20 L) of 1 mg/mL CNTs dispersion in Nafion were checked by measuring the current response for 3 g/mL SAA. The SAA peak current increased with the volume of CNTs dispersion up to 10 L, followed by a decrease in the current signal for longer volumes. Therefore, 10 L of 1 mg/mL of CNTs dispersion in Nafion was used to modify the GC electrode. Finally, to assess the correct adsorption of CNTs dispersion over the surface electrode and evaporate the solvent, the modified electrode was exposed under an IR lamp for 10 min. These optimized conditions were used to prepare the GCE-CNTs in all cases. The generation of a new modified electrode was required every working day.
3.2 Optimization of chromatographic conditions First, the length of the tubing connecting the HPLC system and the amperometric detector was reduced as much
2.4 Samples preparation Whole, semi-skim and skim milk samples (Hacendado, Mercadona) were acquired in a local market. These samples were doped with several concentrations of studied SAAs and 2.5 mL of the trichloroacetic acid 20% w/v was added to 5.0 mL of each milk sample in order to precipitate the proteins. After 15 min, samples were filtered with pleated paper filter, adjusted to pH 5 with NaOH 0.5 M and properly diluted with mobile phase (0.05 M K2 HPO4 pH 3/ACN/MeOH, 80:15:5) to 10 mL. Then, samples were filtered and the filtrate was adjusted to pH 4.5–5 with NaOH 0.5 M and applied to a Strata-X cartridge preconditioned with 5 mL of methanol and 10 mL of water. The cartridge was washed with 5 mL of methanol/water (5:95) and eluted with 5 mL of methanol/acetonitrile (50:50). The solution was evaporated to dryness under a stream of nitrogen and the extract was re-dissolved with 200 L of mobile phase. Then, it was injected into the HPLC–ED system. Infant milk powder (Nestl´e, Spain) was acquired in a local pharmacy. A 50 g portion of the powder was dissolved in 100 mL of milli-Q water. Then, 5.0 mL of this solution was doped with different concentrations of SAAs and treated following the same procedure described above for milk samples.
3 Results and discussion 3.1 Optimization of GCE-CNTs preparation Several studies on the preparation of the GCE-CNTs were carried out in order to obtain the best analytical response. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Chromatograms for (A) conventional GCE and (B) GCECNTs versus Ag/AgCl of a standard mixture containing 3 g/mL concentrations of each compound. Conditions: mobile phase: 0.05 M KH2 PO4 pH 3/ACN/MeOH (80:15:5), injection volume: 20 L, potential detection: 1.2 V, and flow rate: 1.0 mL/min.
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Figure 3. Background current versus time profiles for (A) conventional GCE and (B) GCE-CNTs at 1.2 V under flow conditions. Mobile phase: 0.05 M KH2 PO4 pH 3/ ACN/MeOH (80:15:5).
as possible in order to minimize the dispersion of the analytes (ca. 10 cm). In preliminary studies, in order to address the chromatographic separation of SAAs, several types of C18 columns with different lengths and different diameters of particle were used. The best separation of the five studied SAAs
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was obtained when a diameter of particle of 3.5 m was used. Moreover, different binary mobile phases, prepared by different ratios of acetonitrile or methanol and buffer solution, at several pH values, were tested. Ternary mobile phases containing acetonitrile/methanol/buffer solution, at several pH values and concentrations, were also assayed. As a compromise between adequate retention times and good sensitivity, an acetonitrile/methanol/0.05 M phosphate buffer (pH 3, 15:5:80) mobile phase was selected. The effect of the mobile-phase flow rate was tested between 0.5 and 1.5 mL/min. As expected, both retention time and peak width decreased as the flow rate increased for all the studied compounds. However, a lower resolution between peaks was observed for higher flow rates. The best resolution between all the peaks was obtained when 1 mL/min was used. Therefore, this value was selected as the optimum value. Under these conditions, the retention times (min ± SD, n = 3) were as follows: 2.66 ± 0.01 (SAA), 6.04 ± 0.01 (SDZ), 8.04 ± 0.01 (STZ), 9.52 ± 0.01 (SMZ), 26.63 ± 0.87 (SXZ). Under the selected chromatographic conditions, the choice of the potential to be applied to the CNT-GC electrode for its use as an amperometric detector was carried out by plotting the current values measured at different applied potentials. For this purpose, 20 L aliquots of 3 g/mL of SAAs solutions into a mobile-phase solution consisting of 0.05 M K2 HPO4 pH 3/ACN/MeOH (80:15:5) and a flow rate of 1 mL/min was used. The maximum current for all the studied compounds was observed at 1.2 V. Therefore, this value of potential was selected for the determination of the studied SAAs. Under these experimental conditions, no cleaning or pretreatment of the electrode after each injection was required. It was enough to condition the modified electrode at the beginning of the work, in order to obtain a fresh electrode surface. No appreciable fouling signals were observed after successive scans. The modified electrode was regenerated after 20 consecutive injections.
Table 1. Analytical parameters using the external calibration method with GCE
R2
y = (a ± tsa ) + (b ± tsb )x
RSD (%) intra day
RSD (%) inter day
LOD (g/mL)a)
LOQ (g/mL)b)
(A) By using the GCE STZ 0.09–9.2 SXZ 0.08–10.1 SAA 0.10–10.0 SDZ 0.10–6.4 SMZ 0.09–8.7
0.994 0.994 0.994 0.994 0.998
y = (−1.8 ± 1.7) + (3.1 ± 0.3)x y = (0.1 ± 0.2) + (1.1 ± 0.1)x y = (1.7 ± 0.8) + (1.6 ± 0.1)x y = (0.7 ± 0.8) + (0.8 ± 0.1)x y = (−1.2 ± 0.6) + (1.5 ± 0.1)x
4.8 5.5 6.1 4.3 5.4
6.6 7.3 7.8 6.4 7.4
0.005 0.015 0.010 0.020 0.011
0.018 0.050 0.034 0.068 0.036
(B) By using the CNTs-GCE STZ 0.07–8.5 SXZ 0.06–7.3 SAA 0.08–9.8 SDZ 0.08–8.9 SMZ 0.09–10.5
0.994 0.994 0.994 0.994 0.998
y = (−4.4 ± 0.9) + (6.5 ± 0.1)x y = (−1.6 ± 1.6) + (2.2 ± 0.4)x y = (−2.7 ± 2.9) + (5.3 ± 0.5)x y = (−1.4 ± 0.7) + (1.3 ± 0.1)x y = (−2.0 ± 0.2) + (2.6 ± 0.1)x
3.5 4.7 3.8 4.3 4.1
5.2 5.9 5.2 6.1 5.5
0.001 0.004 0.002 0.006 0.003
0.004 0.012 0.005 0.020 0.010
Compound
Lineal range (g/mL)
a) LOD expressed as 3sa /b. b) LOQ expressed as 10sa /b.
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Figure 4. Chromatograms for (A) screening test and (B) confirmation procedure by HPLC of four milk samples obtained with GCE-CNTs versus Ag/AgCl. Conditions: (A) mobile phase: 0.05 M KH2 PO4 pH 3/ACN/MeOH (40:20:40), injection volume: 20 L, potential detection: 1.2 V, and flow rate: 0.7 mL/min; (B) mobile phase: 0.05 M KH2 PO4 pH 3/ACN/MeOH (80:15:5), injection volume: 20 L, potential detection: 1.2 V, and flow rate: 1.0 mL/min.
3.3 Analytical characteristics of GCE-CNTs and conventional GCE Both electrodes, as amperometric detectors for HPLC, were compared in terms of sensitivity and background current stabilization. Figure 2 shows the comparison of LC chromatograms obtained for GCEs (Fig. 2A) and for GCE-CNTs (Fig. 2B) using 20 L of 3 /mL solutions of studied compounds, injected into the mobile phase at a flow rate of 1 mL/min. As it can be seen, the ED response is considerably higher for all studied SAAs using GCE-CNTs than those using conventional GCEs. The rapid stabilization of the background current was very advantageous in order to reduce the analysis time, which in turn allows the analysis of large numbers of samples for a short time. In this way, Fig. 3 shows the comparison of the background current stabilization time between both electrodes. It is important to highlight the differences between the background current stabilization time for both electrodes. Although the GCE appeared to have stabilized after 30 min, a continuous decrease in the background current is evident for longer times. Moreover, the GCE-CNTs electrode shows stability after 15 min, and hence it can be concluded that this modified electrode exhibits some resistance to oxidative processes. The long stabilization time for the GCE is believed to be due to the oxidation of the electrode itself, together with oxygen evolution [24]. External calibration for the analytes using both electrodes was carried out. Chromatograms for the studied SAAs were obtained for series of sulfonamides solutions at various concentrations ranged between 0.1 and 10 g/mL. The calibration graph of the peak current versus concentration was constructed using data from these measurements and the least squares were evaluated using the linear regression
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method. The analytical parameters for the calibration are summarized in Table 1. LODs and LOQs were calculated using the expressions (yB + 3sB−a )/b and (yB + 10sB−a )/b, respectively, where sB is the SD of ten measures of the experimental blank signal (yB ), and a and b are the
Figure 5. Chromatograms for whole (A) and infant milk (B) samples obtained with GCE-CNTs versus Ag/AgCl. Conditions: mobile phase: 0.05 M KH2 PO4 pH 3/ACN/MeOH (80:15:5), injection volume: 20 L, potential detection: 1.2 V, and flow rate: 1.0 mL/min.
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tration of 3 g/mL for each compound. The RSDs obtained were lower than 10%.
intercept and the slope of the calibration equation. A very good linear dependence with the concentration and excellent values for the coefficient of determination were obtained for all analytes. The main different between the two electrodes was the sensitivity, in terms of calibration slope, which was clearly greater for the modified GCE-CNTs in all cases. The repeatability for five measurements of the current peak for solutions containing 3 g/mL of each compound, under optimized conditions, was satisfactory, with RSDs lower than 5%. The reproducibility of the current peak was tested over two days using solutions prepared at a concen-
3.4 Analytical applications 3.4.1 Screening of samples In preliminary steps in the analysis of samples, a screening test based on the HPLC GCE-CNTs method is proposed. For this purpose, according to the European Union that has set the maximum combined residues of all substances in the
Table 2. Recovery studies carried out in milk samples
Sample
SAA (g/kg)
SDZ (g/kg)
STZ (g/kg)
SMZ (g/kg)
SXZ (g/kg)
Added
Found
%R
Found
%R
Found
%R
Found
%R
Found
%R
Whole milk
50 60 70 80 90 100 110 120 130 140
49.7 58.4 70.4 77.4 91.4 97.1 107.8 123.4 129.0 136.8
99.4 97.3 100.6 96.7 101.5 97.1 98.0 102.8 99.2 97.7
48.4 61.1 72.6 77.4 87.6 101.3 114.4 119.3 126.8 137.5
96.9 101.8 103.7 96.8 97.3 101.3 104.0 99.4 97.5 98.2
48.6 60.5 69.2 77.8 87.1 103.4 108.0 116.5 129.2 145.7
97.3 100.9 98.9 97.2 96.8 103.4 98.2 97.1 99.4 104.1
48.2 59.5 73.4 79.9 91.4 97.1 107.8 119.3 127.0 135.7
96.5 99.1 104.8 99.9 101.5 97.1 98.0 99.4 97.7 96.9
49.2 56.9 71.7 76.6 87.5 98.9 114.5 123.1 125.4 137.8
98.5 94.8 102.4 95.8 97.2 98.9 104.1 102.6 96.5 98.4
Semi-skim milk
50 60 70 80 90 100 110 120 130 140
48.4 58.5 71.2 82.8 88.4 96.8 107.0 122.2 134.4 141.3
96.9 97.5 101.7 103.5 98.2 96.8 97.3 101.8 103.4 100.9
50.1 62.0 71.3 77.8 87.1 97.7 109.3 122.9 127.4 136.5
100.2 103.4 101.8 97.3 96.8 97.7 99.4 102.4 98.0 97.5
47.9 57.7 69.6 82.2 86.7 98.9 114.3 116.8 125.4 134.0
95.8 96.1 99.4 102.8 96.3 98.9 103.9 97.3 96.5 95.7
48.6 57.1 72.2 78.7 90.6 93.9 112.6 118.1 127.1 141.5
97.3 95.2 103.2 98.4 100.7 93.9 102.4 98.4 97.8 101.1
52.2 61.4 68.4 79.4 92.5 96.1 109.1 124.7 135.3 134.8
104.3 102.3 97.7 99.3 102.8 96.1 99.2 103.9 104.1 96.3
Skim milk
50 60 70 80 90 100 110 120 130 140
47.6 58.6 69.4 81.9 88.6 103.2 105.9 122.3 135.6 136.2
95.2 97.6 99.1 102.4 98.5 103.2 96.3 101.9 104.3 97.3
47.8 58.9 67.7 79.9 87.6 96.6 114.2 117.2 129.2 145.3
95.6 98.1 96.7 99.9 97.3 96.6 103.8 97.7 99.4 103.8
48.6 62.6 71.3 77.0 92.9 98.5 112.6 118.9 126.9 133.3
97.3 104.3 101.9 96.3 103.2 98.5 102.4 99.1 97.6 95.2
52.2 57.7 72.0 77.9 91.4 105.3 111.0 117.8 129.0 134.4
104.5 96.2 102.8 97.4 101.5 105.3 100.9 98.2 99.2 96.0
48.8 59.6 67.1 76.9 93.4 98.1 113.7 116.5 132.5 134.0
97.5 99.3 95.9 96.1 103.8 98.1 103.4 97.1 101.9 95.7
Infant milk powder
50 60 70 80 90 100 110 120 130 140
50.8 58.0 70.3 77.8 89.8 98.4 112.6 117.0 134.4 134.8
101.5 96.7 100.4 97.3 99.8 98.4 102.4 97.5 103.4 96.3
52.6 58.3 72.0 78.8 87.9 100.9 105.7 117.8 129.2 145.5
105.3 97.2 102.8 98.5 97.7 100.9 96.1 98.2 99.4 103.9
48.2 62.5 72.7 79.4 86.5 102.8 109.2 125.0 125.6 136.5
96.3 104.1 103.9 99.2 96.1 102.8 99.3 104.2 96.6 97.5
50.1 58.8 70.3 78.2 85.9 98.4 112.0 120.2 132.0 137.5
100.2 98 100.4 97.7 95.4 98.4 101.8 100.2 101.5 98.2
49.6 58.0 72.9 82.3 88.8 100.1 111.0 117.0 133.9 147.1
99.3 96.6 104.2 102.9 98.7 100.1 100.9 97.5 103.0 105.1
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sulfonamide group at 100 g/kg (threshold) [6], a unique peak containing the overall response for the total amount of the SAAs was obtained. The conditions to achieve a single peak as the sum of the oxidation of all SAAs studied were optimized. Different mobile ternary phases containing acetonitrile/methanol/buffer solution at several ratios were tested. The optimum peak was obtained with acetonitrile/methanol/0.05 M phosphate buffer (pH 3; 20:40:40) solution as mobile phase. Moreover, mobile-phase flow rate was tested between 0.5 and 1.5 mL/min, and a flow rate of 0.7 mL/min was the optimum to obtain a well-defined peak. Under these optimized conditions, several spiked milk samples, with different amounts of SAAs were analyzed. Figure 4 shows (A) the screening protocol and (B) confirmative procedure by HPLC–ED methodology proposed using GCE-CNTs, and the results obtained for four representative samples: (i) sample into reliability region with negative response according to the threshold; (ii) and (iii) samples into unreliability region (inconclusive samples according to the threshold); and (iv) a sample into reliability region with positive response according to the threshold. This proposed methodology allows the rapid and simple classification of milk samples according to the legislation (threshold) and the confirmation of the inconclusive samples.
3.4.2 Quantitative analysis in milk samples The developed HPLC GCE-CNTs method was used to confirm and determine the studied SAAs in different types of milk samples, such as whole, semi-skim, skim, and infant milks. First, milk samples were spiked with SAA, SDZ, SMZ, STZ, and SXZ at variable concentrations (50–140 g/kg) in order to study the presence of potential matrix effects. It is clear that in natural collected milk samples, it is not possible to assure that the antibacterial drugs and their metabolites could present some potential matrix effect with respect to spiked samples. This possibility, which is not a trivial matter, will require a specific study with the assistance of responsible veterinarians for administering antibacterial drugs. Although this is beyond the scope of this work in general analytical terms, this potential problem (if appearing in specific cases) could be solved by the appropriate modification of the sample preparation procedure, the modification of the chromatographic conditions, and/or the use of the standard addition method for the calibration. For the purpose of this work, the preparation of the spiked samples is described in Section 2.4. In all the analyzed samples, no chromatographic interferences were observed for the studied SAAs. Figure 5 shows the chromatograms obtained for a whole (A) and infant (B) milk samples. This method can be used as an HPLC confirmation procedure after the previous screening method described before in order to identify the peaks obtained by comparing the retention times of samples with those of standard compounds. Efficient, reproducible, and sensitive separation and the detection of the SAAs of interest were obtained C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in less than 30 min. Table 2 shows the results obtained for the determination of the five SAAs. As can be seen in this table, the concentrations added and found were generally in good agreement with high recoveries (96–104%).
4 Conclusions The novelty of this work is the use of GCE-CNTs for amperometric detection after the HPLC separation of several SAAs (sulfanilamide, sulfamerazine, sulfadiazine, sulfathiazole, and sulfisoxazole). This work has demonstrated that this electrode exhibits a good electrocatalytic behavior for these compounds, increasing the sensitivity compared with a conventional GCE. Good linearity, precision, and detection and quantification limits were obtained. The proposed method was used for the determination of SAAs in milk samples to obtain very satisfactory percentage recoveries. It is also important to remark on the capacity of the methodology to be adapted as a simple and rapid screening method to give information about the potential contamination of milk samples, and moreover to be used as a common confirmatory method, providing individual information of every sulfonamide compound. Financial support from the Spanish Ministry of Science and Innovation (Project CTQ2010–15027) is gratefully acknowledged. The authors have declared no conflict of interest.
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Research Article
Determination of sulfonamides in milk samples by HPLC with amperometric detection using a glassy carbon electrode modified with multiwalled carbon nanotubes A sensitive and accurate method for determining five sulfonamides based on HPLC with amperometric detection and using a glassy carbon electrode modified with multiwalled carbon nanotubes is proposed. Optimal conditions for the quantitative separation of selected sulfonamides were studied, and glassy carbon electrodes with and without modification with carbon nanotubes were systematically investigated as electrodic materials. Statistical analysis of the obtained results demonstrated that these modified electrodes achieved considerably better stability and sensitivity than the conventional unmodified ones. Detection limits were in the 1.2–6.0 ng/mL range. The usefulness of the method was demonstrated by the analysis of milk samples, taking into account the European legislation on residues in food products, following both a screening method to classify the samples and a confirmation method to provide more detailed information in the case of positive samples. Keywords: Amperometric detection / Carbon nanotubes / Liquid chromatography / Modified electrodes / Sulfonamides DOI 10.1002/jssc.201301011
1 Introduction Sulfonamides (SAAs) are a class of antibacterial drugs used in farm animals for the treatment of a variety of bacterial infections. In food-producing animals, sulfonamides are used not only for therapeutic but also for prophylactic purposes [1, http://fri.wisc.edu/docs/pdf/FRIBrief_VetDrgRes.pdf]. The quantification of these antibacterial drugs in milk represents a challenge, since the antibacterial drugs and their metabolites are secreted in milk. Although the use of veterinary drugs has helped to increase the food supply, negative consequences, such as presence of drug residues in food, cannot be ignored. The presence of drug residues in foods can be a health hazard to consumers. First, the carcinogenicity of some drugs may be a serious concern [2–5]. Second, continuous exposure of certain micro-organisms to these drugs may result in the development of drug-resistant strains. Because of the toxicology of the residues of these compounds for the human health, the European Union (EU) has set the maximum combined residues for all substances in the sulfonamide group at 100 g/kg [6]. ´ Correspondence: Professor Angel R´ıos, Department of Analytical Chemistry and Food Technology, University of Castilla–La Mancha, Campus de Ciudad Real, E-13004 Ciudad Real, Spain E-mail: [email protected] Fax: +34 926295232
Abbreviations: CNT, carbon nanotube; ED, electrochemical detection; GCE, glassy carbon electrode; MWCNT, multiwalled carbon nanotube C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Several analytical methods have been developed to separate and detect these compounds in different samples. GC– MS methods were developed for detecting sulfonamides after derivatization [7–9]. Several methods using CE with UV [10], fluorescence [11], electrochemical (ED) [12], and MS [13] detection have been used for the determination of these drugs in meat, tissues, tablets, and human urine samples, respectively. But the most commonly used methods include LC using UV diode array [14–19], fluorescence [20–22], MS [22–27], or MS/MS detection [28–30]. These methods require significant amounts of time associated with clean-up steps, sophisticated instrumentation, and skilled operators. There are only a few references using ED, probably because of problems related to electrode fouling and deactivation [31–33]. Thus, in this way, the voltammetric behavior of eight sulfonamides at the glassy carbon electrode (GCE) was studied and low detection limits were obtained [31]. The same electrode was used in the oxidation [34] and reduction [35] of sulfadiazine in pharmaceuticals with good results. A similar study for the electroanalytical oxidation of sulfamerazine was carried out by Pingarr´on et al. [33]. Some previous studies used borondoped conductive diamond electrodes for the ED determination of several sulfonamides [36, 37], characterized by an excellent performance with high sensitivity and reproducibility. Thus, this type of electrode was used by Souza et al. in pharmaceuticals, in order to determine sulfadiazine and sulfamethoxazole by square-wave voltammetry [37]. The results obtained show very low detection limits and good recoveries. Colour Online: See the article online to view Fig. 4 in colour. www.jss-journal.com
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Figure 1. Chemical structures of the SAAs.
Recently, many studies have revealed that modification of electrode surface with nanomaterials is a promising avenue. Carbon nanotubes (CNTs), consisting of cylindrical graphene sheets with nanometer diameter, have attracted much attention due to their unique mechanical, chemical, and electronic properties [38, 39] since their discovery by Iijima [40] in 1991. All these properties make them suitable for the modification of electrodes [41–43] and suggest great promise for amperometric detection [44–46]. The performance of electrodes modified with multiwalled CNTs (MWCNTs) is superior to the performance of other conventional carbon electrodes in terms of electron transfer rate, reversibility, and conductivity [47, 48]. CNTs were incorporated into a paste electrode in the determination of sulfamethoxazole for the first time by Arvand et al. [48]. Results showed a marked decrease on overvoltage for sulfamethoxazole oxidation and enhanced the S/N characteristics compared to those observed at the carbon paste electrode. In this work, we report the development of an accurate and precise method for the determination of five representative sulfonamides (sulfisoxazole, SXZ; sulfamerazine, SMZ; sulfathiazole, STZ; sulfanilamide, SAA; and sulfadiazine, SDZ) based on the amperometric response at MWCNTsmodified GCEs after separation by HPLC. The structures of these five compounds are shown in Fig. 1. A high sensitivity, together with a good repeatability and reproducibility of the response obtained with these electrodes were achieved. The proposed method was applied to the analysis of these compounds in different types of milk samples by using a screening method or a confirmatory method, according to the experimental chromatographic conditions used. The screening/confirmation strategy avoids the detailed analysis of the samples, especially when samples are negative (most samples will be negative samples). In addition, the screening method is closer to the needs of the consumer and to the legislation requirements. From a technical viewpoint, or for decisionmaking purposes, the information about positive samples must be completed with the particular concentrations. Thus, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the screening/confirmation strategy meets this double important role.
2 Materials and methods 2.1 Apparatus and electrodes Liquid chromatographic experiments were carried out with an HP 1090 Liquid Chromatograph (Agilent, USA) and amperometric detector (Metrohm 791 VA, Gomensoro S.A., Spain) using Labview software. The wall-jet flow-cell consisted of an Ag/AgCl/3M KCl reference electrode (Metrohm Model 60727000, Gomensoro, Spain), a platinum auxiliary electrode, and glassy carbon GCE (Metrohm Model 60805010, Gomensoro, shaft diameter bottom 7 mm) or GCE-CNTs as working electrode. A Zorbax SB-C18 column (Agilent, 150 × 4.6 mm id; particle size, 3.5 m) was used for the separation of the drugs. A solution of 0.05 M K2 HPO4 pH 3/ACN/MeOH (80:15:5) at room temperature (20 ± 1⬚C) was used as the mobile phase in the LC experiments. The potential detection applied was 1200 mV for all measurements. The length of the tubing connecting the HPLC and the detector was 10 cm. The wall-jet flow-cell consisted of an Ag/AgCl/3 M KCl reference electrode (Metrohm Model 60727000), a platinum auxiliary electrode, and glassy carbon GCE (Metrohm Model 60805010, shaft diameter bottom 7 mm) or GCE-CNTs as working electrode. Samples were extracted using an SPE-Vacuum manifold from Supelco (Madrid, Spain). An ultrasonic bath (Ultrasons J.P. Selecta, Barcelona, Spain) was used to clean the surface of the electrodes and homogenize the solutions. 2.2 Materials and standards SAA, SXZ, STZ, SMZ, SDZ, trichloroacetic acid, potassium monobasic phosphate and dibasic, and sodium hydroxide www.jss-journal.com
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were purchased from Sigma Chemical (St. Louis, MO, USA). MWCNTs with 95% purity were obtained from NanoLab (Brighton, MA). Methanol and acetonitrile were of HPLC grade and acquired from Panreac (Barcelona, Spain). Nafion was supplied from Fluka (USA). In all cases, water was of high quality, purified in a Milli-Q system (Millipore, Bedford, MA, USA). A stock standard solution (1 mM) of each sulfonamide was prepared in MeOH and stored in the dark at 5⬚C. Working standard solutions were prepared daily by diluting the stock solutions with carrying medium (0.05 M K2 HPO4 pH 3/ACN/MeOH 80:15:5). 2.3 Preparation of the CNTs dispersion and GCE-CNTs The CNTs dispersion was obtained by dispersing 1.0 mg of MWCNTs in 1.0 mL of 0.1% v/v Nafion solution followed by sonication for 15 min. The GCE surface was polished with alumina, rinsed with distilled water, sonicated in water and acetone with an ultrasonic bath, and dried in air. An aliquot (10 L) of the dispersion was dropped on the electrode surface and the solvent was evaporated under an infrared heat lamp [32]. The CNTs dispersion was prepared each week, but it did not lose its ED properties for longer periods.
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To modify the surface of the electrode, dispersions of CNTs in Nafion at concentrations between 0.5 and 5 mg/mL were prepared. The amperometric response for 3 g/mL sulfanilamide was checked, and the maximal current was obtained for 1 mg/mL of CNTs dispersion. Larger concentrations of CNTs considerably increased the baseline noise. Moreover, different volumes (between 5and 20 L) of 1 mg/mL CNTs dispersion in Nafion were checked by measuring the current response for 3 g/mL SAA. The SAA peak current increased with the volume of CNTs dispersion up to 10 L, followed by a decrease in the current signal for longer volumes. Therefore, 10 L of 1 mg/mL of CNTs dispersion in Nafion was used to modify the GC electrode. Finally, to assess the correct adsorption of CNTs dispersion over the surface electrode and evaporate the solvent, the modified electrode was exposed under an IR lamp for 10 min. These optimized conditions were used to prepare the GCE-CNTs in all cases. The generation of a new modified electrode was required every working day.
3.2 Optimization of chromatographic conditions First, the length of the tubing connecting the HPLC system and the amperometric detector was reduced as much
2.4 Samples preparation Whole, semi-skim and skim milk samples (Hacendado, Mercadona) were acquired in a local market. These samples were doped with several concentrations of studied SAAs and 2.5 mL of the trichloroacetic acid 20% w/v was added to 5.0 mL of each milk sample in order to precipitate the proteins. After 15 min, samples were filtered with pleated paper filter, adjusted to pH 5 with NaOH 0.5 M and properly diluted with mobile phase (0.05 M K2 HPO4 pH 3/ACN/MeOH, 80:15:5) to 10 mL. Then, samples were filtered and the filtrate was adjusted to pH 4.5–5 with NaOH 0.5 M and applied to a Strata-X cartridge preconditioned with 5 mL of methanol and 10 mL of water. The cartridge was washed with 5 mL of methanol/water (5:95) and eluted with 5 mL of methanol/acetonitrile (50:50). The solution was evaporated to dryness under a stream of nitrogen and the extract was re-dissolved with 200 L of mobile phase. Then, it was injected into the HPLC–ED system. Infant milk powder (Nestl´e, Spain) was acquired in a local pharmacy. A 50 g portion of the powder was dissolved in 100 mL of milli-Q water. Then, 5.0 mL of this solution was doped with different concentrations of SAAs and treated following the same procedure described above for milk samples.
3 Results and discussion 3.1 Optimization of GCE-CNTs preparation Several studies on the preparation of the GCE-CNTs were carried out in order to obtain the best analytical response. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Chromatograms for (A) conventional GCE and (B) GCECNTs versus Ag/AgCl of a standard mixture containing 3 g/mL concentrations of each compound. Conditions: mobile phase: 0.05 M KH2 PO4 pH 3/ACN/MeOH (80:15:5), injection volume: 20 L, potential detection: 1.2 V, and flow rate: 1.0 mL/min.
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Figure 3. Background current versus time profiles for (A) conventional GCE and (B) GCE-CNTs at 1.2 V under flow conditions. Mobile phase: 0.05 M KH2 PO4 pH 3/ ACN/MeOH (80:15:5).
as possible in order to minimize the dispersion of the analytes (ca. 10 cm). In preliminary studies, in order to address the chromatographic separation of SAAs, several types of C18 columns with different lengths and different diameters of particle were used. The best separation of the five studied SAAs
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was obtained when a diameter of particle of 3.5 m was used. Moreover, different binary mobile phases, prepared by different ratios of acetonitrile or methanol and buffer solution, at several pH values, were tested. Ternary mobile phases containing acetonitrile/methanol/buffer solution, at several pH values and concentrations, were also assayed. As a compromise between adequate retention times and good sensitivity, an acetonitrile/methanol/0.05 M phosphate buffer (pH 3, 15:5:80) mobile phase was selected. The effect of the mobile-phase flow rate was tested between 0.5 and 1.5 mL/min. As expected, both retention time and peak width decreased as the flow rate increased for all the studied compounds. However, a lower resolution between peaks was observed for higher flow rates. The best resolution between all the peaks was obtained when 1 mL/min was used. Therefore, this value was selected as the optimum value. Under these conditions, the retention times (min ± SD, n = 3) were as follows: 2.66 ± 0.01 (SAA), 6.04 ± 0.01 (SDZ), 8.04 ± 0.01 (STZ), 9.52 ± 0.01 (SMZ), 26.63 ± 0.87 (SXZ). Under the selected chromatographic conditions, the choice of the potential to be applied to the CNT-GC electrode for its use as an amperometric detector was carried out by plotting the current values measured at different applied potentials. For this purpose, 20 L aliquots of 3 g/mL of SAAs solutions into a mobile-phase solution consisting of 0.05 M K2 HPO4 pH 3/ACN/MeOH (80:15:5) and a flow rate of 1 mL/min was used. The maximum current for all the studied compounds was observed at 1.2 V. Therefore, this value of potential was selected for the determination of the studied SAAs. Under these experimental conditions, no cleaning or pretreatment of the electrode after each injection was required. It was enough to condition the modified electrode at the beginning of the work, in order to obtain a fresh electrode surface. No appreciable fouling signals were observed after successive scans. The modified electrode was regenerated after 20 consecutive injections.
Table 1. Analytical parameters using the external calibration method with GCE
R2
y = (a ± tsa ) + (b ± tsb )x
RSD (%) intra day
RSD (%) inter day
LOD (g/mL)a)
LOQ (g/mL)b)
(A) By using the GCE STZ 0.09–9.2 SXZ 0.08–10.1 SAA 0.10–10.0 SDZ 0.10–6.4 SMZ 0.09–8.7
0.994 0.994 0.994 0.994 0.998
y = (−1.8 ± 1.7) + (3.1 ± 0.3)x y = (0.1 ± 0.2) + (1.1 ± 0.1)x y = (1.7 ± 0.8) + (1.6 ± 0.1)x y = (0.7 ± 0.8) + (0.8 ± 0.1)x y = (−1.2 ± 0.6) + (1.5 ± 0.1)x
4.8 5.5 6.1 4.3 5.4
6.6 7.3 7.8 6.4 7.4
0.005 0.015 0.010 0.020 0.011
0.018 0.050 0.034 0.068 0.036
(B) By using the CNTs-GCE STZ 0.07–8.5 SXZ 0.06–7.3 SAA 0.08–9.8 SDZ 0.08–8.9 SMZ 0.09–10.5
0.994 0.994 0.994 0.994 0.998
y = (−4.4 ± 0.9) + (6.5 ± 0.1)x y = (−1.6 ± 1.6) + (2.2 ± 0.4)x y = (−2.7 ± 2.9) + (5.3 ± 0.5)x y = (−1.4 ± 0.7) + (1.3 ± 0.1)x y = (−2.0 ± 0.2) + (2.6 ± 0.1)x
3.5 4.7 3.8 4.3 4.1
5.2 5.9 5.2 6.1 5.5
0.001 0.004 0.002 0.006 0.003
0.004 0.012 0.005 0.020 0.010
Compound
Lineal range (g/mL)
a) LOD expressed as 3sa /b. b) LOQ expressed as 10sa /b.
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Figure 4. Chromatograms for (A) screening test and (B) confirmation procedure by HPLC of four milk samples obtained with GCE-CNTs versus Ag/AgCl. Conditions: (A) mobile phase: 0.05 M KH2 PO4 pH 3/ACN/MeOH (40:20:40), injection volume: 20 L, potential detection: 1.2 V, and flow rate: 0.7 mL/min; (B) mobile phase: 0.05 M KH2 PO4 pH 3/ACN/MeOH (80:15:5), injection volume: 20 L, potential detection: 1.2 V, and flow rate: 1.0 mL/min.
3.3 Analytical characteristics of GCE-CNTs and conventional GCE Both electrodes, as amperometric detectors for HPLC, were compared in terms of sensitivity and background current stabilization. Figure 2 shows the comparison of LC chromatograms obtained for GCEs (Fig. 2A) and for GCE-CNTs (Fig. 2B) using 20 L of 3 /mL solutions of studied compounds, injected into the mobile phase at a flow rate of 1 mL/min. As it can be seen, the ED response is considerably higher for all studied SAAs using GCE-CNTs than those using conventional GCEs. The rapid stabilization of the background current was very advantageous in order to reduce the analysis time, which in turn allows the analysis of large numbers of samples for a short time. In this way, Fig. 3 shows the comparison of the background current stabilization time between both electrodes. It is important to highlight the differences between the background current stabilization time for both electrodes. Although the GCE appeared to have stabilized after 30 min, a continuous decrease in the background current is evident for longer times. Moreover, the GCE-CNTs electrode shows stability after 15 min, and hence it can be concluded that this modified electrode exhibits some resistance to oxidative processes. The long stabilization time for the GCE is believed to be due to the oxidation of the electrode itself, together with oxygen evolution [24]. External calibration for the analytes using both electrodes was carried out. Chromatograms for the studied SAAs were obtained for series of sulfonamides solutions at various concentrations ranged between 0.1 and 10 g/mL. The calibration graph of the peak current versus concentration was constructed using data from these measurements and the least squares were evaluated using the linear regression
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method. The analytical parameters for the calibration are summarized in Table 1. LODs and LOQs were calculated using the expressions (yB + 3sB−a )/b and (yB + 10sB−a )/b, respectively, where sB is the SD of ten measures of the experimental blank signal (yB ), and a and b are the
Figure 5. Chromatograms for whole (A) and infant milk (B) samples obtained with GCE-CNTs versus Ag/AgCl. Conditions: mobile phase: 0.05 M KH2 PO4 pH 3/ACN/MeOH (80:15:5), injection volume: 20 L, potential detection: 1.2 V, and flow rate: 1.0 mL/min.
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tration of 3 g/mL for each compound. The RSDs obtained were lower than 10%.
intercept and the slope of the calibration equation. A very good linear dependence with the concentration and excellent values for the coefficient of determination were obtained for all analytes. The main different between the two electrodes was the sensitivity, in terms of calibration slope, which was clearly greater for the modified GCE-CNTs in all cases. The repeatability for five measurements of the current peak for solutions containing 3 g/mL of each compound, under optimized conditions, was satisfactory, with RSDs lower than 5%. The reproducibility of the current peak was tested over two days using solutions prepared at a concen-
3.4 Analytical applications 3.4.1 Screening of samples In preliminary steps in the analysis of samples, a screening test based on the HPLC GCE-CNTs method is proposed. For this purpose, according to the European Union that has set the maximum combined residues of all substances in the
Table 2. Recovery studies carried out in milk samples
Sample
SAA (g/kg)
SDZ (g/kg)
STZ (g/kg)
SMZ (g/kg)
SXZ (g/kg)
Added
Found
%R
Found
%R
Found
%R
Found
%R
Found
%R
Whole milk
50 60 70 80 90 100 110 120 130 140
49.7 58.4 70.4 77.4 91.4 97.1 107.8 123.4 129.0 136.8
99.4 97.3 100.6 96.7 101.5 97.1 98.0 102.8 99.2 97.7
48.4 61.1 72.6 77.4 87.6 101.3 114.4 119.3 126.8 137.5
96.9 101.8 103.7 96.8 97.3 101.3 104.0 99.4 97.5 98.2
48.6 60.5 69.2 77.8 87.1 103.4 108.0 116.5 129.2 145.7
97.3 100.9 98.9 97.2 96.8 103.4 98.2 97.1 99.4 104.1
48.2 59.5 73.4 79.9 91.4 97.1 107.8 119.3 127.0 135.7
96.5 99.1 104.8 99.9 101.5 97.1 98.0 99.4 97.7 96.9
49.2 56.9 71.7 76.6 87.5 98.9 114.5 123.1 125.4 137.8
98.5 94.8 102.4 95.8 97.2 98.9 104.1 102.6 96.5 98.4
Semi-skim milk
50 60 70 80 90 100 110 120 130 140
48.4 58.5 71.2 82.8 88.4 96.8 107.0 122.2 134.4 141.3
96.9 97.5 101.7 103.5 98.2 96.8 97.3 101.8 103.4 100.9
50.1 62.0 71.3 77.8 87.1 97.7 109.3 122.9 127.4 136.5
100.2 103.4 101.8 97.3 96.8 97.7 99.4 102.4 98.0 97.5
47.9 57.7 69.6 82.2 86.7 98.9 114.3 116.8 125.4 134.0
95.8 96.1 99.4 102.8 96.3 98.9 103.9 97.3 96.5 95.7
48.6 57.1 72.2 78.7 90.6 93.9 112.6 118.1 127.1 141.5
97.3 95.2 103.2 98.4 100.7 93.9 102.4 98.4 97.8 101.1
52.2 61.4 68.4 79.4 92.5 96.1 109.1 124.7 135.3 134.8
104.3 102.3 97.7 99.3 102.8 96.1 99.2 103.9 104.1 96.3
Skim milk
50 60 70 80 90 100 110 120 130 140
47.6 58.6 69.4 81.9 88.6 103.2 105.9 122.3 135.6 136.2
95.2 97.6 99.1 102.4 98.5 103.2 96.3 101.9 104.3 97.3
47.8 58.9 67.7 79.9 87.6 96.6 114.2 117.2 129.2 145.3
95.6 98.1 96.7 99.9 97.3 96.6 103.8 97.7 99.4 103.8
48.6 62.6 71.3 77.0 92.9 98.5 112.6 118.9 126.9 133.3
97.3 104.3 101.9 96.3 103.2 98.5 102.4 99.1 97.6 95.2
52.2 57.7 72.0 77.9 91.4 105.3 111.0 117.8 129.0 134.4
104.5 96.2 102.8 97.4 101.5 105.3 100.9 98.2 99.2 96.0
48.8 59.6 67.1 76.9 93.4 98.1 113.7 116.5 132.5 134.0
97.5 99.3 95.9 96.1 103.8 98.1 103.4 97.1 101.9 95.7
Infant milk powder
50 60 70 80 90 100 110 120 130 140
50.8 58.0 70.3 77.8 89.8 98.4 112.6 117.0 134.4 134.8
101.5 96.7 100.4 97.3 99.8 98.4 102.4 97.5 103.4 96.3
52.6 58.3 72.0 78.8 87.9 100.9 105.7 117.8 129.2 145.5
105.3 97.2 102.8 98.5 97.7 100.9 96.1 98.2 99.4 103.9
48.2 62.5 72.7 79.4 86.5 102.8 109.2 125.0 125.6 136.5
96.3 104.1 103.9 99.2 96.1 102.8 99.3 104.2 96.6 97.5
50.1 58.8 70.3 78.2 85.9 98.4 112.0 120.2 132.0 137.5
100.2 98 100.4 97.7 95.4 98.4 101.8 100.2 101.5 98.2
49.6 58.0 72.9 82.3 88.8 100.1 111.0 117.0 133.9 147.1
99.3 96.6 104.2 102.9 98.7 100.1 100.9 97.5 103.0 105.1
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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sulfonamide group at 100 g/kg (threshold) [6], a unique peak containing the overall response for the total amount of the SAAs was obtained. The conditions to achieve a single peak as the sum of the oxidation of all SAAs studied were optimized. Different mobile ternary phases containing acetonitrile/methanol/buffer solution at several ratios were tested. The optimum peak was obtained with acetonitrile/methanol/0.05 M phosphate buffer (pH 3; 20:40:40) solution as mobile phase. Moreover, mobile-phase flow rate was tested between 0.5 and 1.5 mL/min, and a flow rate of 0.7 mL/min was the optimum to obtain a well-defined peak. Under these optimized conditions, several spiked milk samples, with different amounts of SAAs were analyzed. Figure 4 shows (A) the screening protocol and (B) confirmative procedure by HPLC–ED methodology proposed using GCE-CNTs, and the results obtained for four representative samples: (i) sample into reliability region with negative response according to the threshold; (ii) and (iii) samples into unreliability region (inconclusive samples according to the threshold); and (iv) a sample into reliability region with positive response according to the threshold. This proposed methodology allows the rapid and simple classification of milk samples according to the legislation (threshold) and the confirmation of the inconclusive samples.
3.4.2 Quantitative analysis in milk samples The developed HPLC GCE-CNTs method was used to confirm and determine the studied SAAs in different types of milk samples, such as whole, semi-skim, skim, and infant milks. First, milk samples were spiked with SAA, SDZ, SMZ, STZ, and SXZ at variable concentrations (50–140 g/kg) in order to study the presence of potential matrix effects. It is clear that in natural collected milk samples, it is not possible to assure that the antibacterial drugs and their metabolites could present some potential matrix effect with respect to spiked samples. This possibility, which is not a trivial matter, will require a specific study with the assistance of responsible veterinarians for administering antibacterial drugs. Although this is beyond the scope of this work in general analytical terms, this potential problem (if appearing in specific cases) could be solved by the appropriate modification of the sample preparation procedure, the modification of the chromatographic conditions, and/or the use of the standard addition method for the calibration. For the purpose of this work, the preparation of the spiked samples is described in Section 2.4. In all the analyzed samples, no chromatographic interferences were observed for the studied SAAs. Figure 5 shows the chromatograms obtained for a whole (A) and infant (B) milk samples. This method can be used as an HPLC confirmation procedure after the previous screening method described before in order to identify the peaks obtained by comparing the retention times of samples with those of standard compounds. Efficient, reproducible, and sensitive separation and the detection of the SAAs of interest were obtained C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in less than 30 min. Table 2 shows the results obtained for the determination of the five SAAs. As can be seen in this table, the concentrations added and found were generally in good agreement with high recoveries (96–104%).
4 Conclusions The novelty of this work is the use of GCE-CNTs for amperometric detection after the HPLC separation of several SAAs (sulfanilamide, sulfamerazine, sulfadiazine, sulfathiazole, and sulfisoxazole). This work has demonstrated that this electrode exhibits a good electrocatalytic behavior for these compounds, increasing the sensitivity compared with a conventional GCE. Good linearity, precision, and detection and quantification limits were obtained. The proposed method was used for the determination of SAAs in milk samples to obtain very satisfactory percentage recoveries. It is also important to remark on the capacity of the methodology to be adapted as a simple and rapid screening method to give information about the potential contamination of milk samples, and moreover to be used as a common confirmatory method, providing individual information of every sulfonamide compound. Financial support from the Spanish Ministry of Science and Innovation (Project CTQ2010–15027) is gratefully acknowledged. The authors have declared no conflict of interest.
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