Determination of sulfonamides in milk after precolumn derivatisation by micellar liquid chromatography

Analytica Chimica Acta 593 (2007) 152–156

Determination of sulfonamides in milk after precolumn derivatisation by micellar liquid chromatography M´onica Ana Raviolo b , Maria Rambla-Alegre a , Jenifer Clausell-Tormos c , Maria-Elisa Capella-Peir´o a , Samuel Carda-Broch a , Josep Esteve-Romero a,∗ ` Area de Qu´ımica Anal´ıtica, Q.F.A., Universitat Jaume I, Campus Riu Sec, 12080 Castell´o, Spain Dpto. de Farmacia, Fac. de Ciencias Qu´ımicas, Universidad Nacional de C´ordoba, C´ordoba, Argentina c Institut de Science et d’Ing´ enierie Supramol´eculaires (ISIS), Universit´e Louis Pasteur, Strasbourg, France a

b

Received 27 February 2007; received in revised form 30 April 2007; accepted 2 May 2007 Available online 6 May 2007

Abstract A simple method to identify and determine six sulfonamides (sodium sulfacetamide, sulfamethizole, sulfaguanidine, sulfamerazine, sulfathiazole and sulfamethoxazole) in milk by micellar liquid chromatography (MLC) is reported. The assay makes use of a precolumn diazotisation-coupling derivatisation including the formation of an azo dye that can be detected at 490 nm. Furthermore, the use of MLC as an analytical tool allows the direct injection of non-purified samples. The separation was performed with an 80 mM SDS-8.5% propanol eluent at pH 7. Analysis times are below 16 min with a complete resolution. Linearities (r > 0.9999), as well as intra- and inter-day precision (below 2.7%), were studied in the validation of the method. The limits of detection and quantification ranged from approximately 0.72 to 0.94 and 2.4 to 3.1 ng mL−1 , respectively. The detection limit was below the maximum residue limit established by the European Community. Finally, recoveries in spiked milk samples were in the 83–103% range. © 2007 Elsevier B.V. All rights reserved. Keywords: Sulfonamides; Azo dye precolumn derivatisation; Micellar liquid chromatography; Milk

1. Introduction Sulfonamides are widely used to prevent and control a number of veterinary diseases, such as gastrointestinal and respiratory infections, as well as growth-promoting purposes and as a prophylactic [1–4]. When inappropriate abusive antibiotic-based treatments are applied to treat livestock diseases, undesirable residues can remain in both the animal tissues and biofluids, including milk. These residues are a great public health concern due to the risk of developing drug resistance, which leads to an inefficiency of this medicine for therapeutic use [5,6]. Consequently, in order to prevent health problems, the European Community has adopted a maximum sulfonamide residue level of 100 ␮g of total sulfonamides kg−1 in edible animal tissue, including milk [5]. Therefore, the determination of such residues in meat and other animal by-products, such as

∗

Corresponding author. Tel.: +34 964 728092; fax: +34 964 728066. E-mail address: [email protected] (J. Esteve-Romero).

0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.05.001

milk used for human consumption, has become an important task. Several analytical methods have been developed to determine sulfonamides in animal tissue and biofluids using high-performance liquid chromatography (HPLC) with UV, fluorescence, or mass spectrometry [2,4,7,8–11]. Other methods include gas chromatography [12,13], capillary zone electrophoresis [3,14,15] and micellar electrokinetic capillary chromatography [16]. The difficulties involved in the analysis of complex biological matrices are the presence of interferences that are co-extracted. For this reason, the methods mentioned above need previous treatment of the sample, which includes several steps, such as extraction with organic solvent [4,13], solid-phase extraction [1,7] or centrifugal ultrafiltration [9,17]. The sample treatment can generate variable and low recoveries and, moreover, this process can be toxic due to the use of methylene chloride, acetonitrile, and methanol for the extraction and/or mobile phase of the HPLC. Micellar liquid chromatography (MLC) is an alternative method to HPLC [18–20]. In MLC, the mobile phase is

M.A. Raviolo et al. / Analytica Chimica Acta 593 (2007) 152–156

composed of a surfactant at a higher concentration than the critical micellar concentration and an organic modifier, such as propanol, butanol or pentanol, which decrease the retention factor and increase efficiency. The exact prediction of any solute retention is possible through micellar mobile phases by utilising models that could also be used to optimise the separation of solute mixtures [21,22]. One of the main advantages that MLC offers is the possibility of determining drugs in complex matrices without previously having to extract the proteins present in the different samples. In recent years, MLC has proved to be a useful technique in the analysis of diverse groups of substances in different matrices [23–26]. MLC uses mobile phases that are non-toxic, non-flammable, biodegradable and relatively inexpensive in comparison to other methods. On the other hand, the sulfonamides are arylamines and contain a primary aromatic amine (Fig. 1). For this reason they can be derivatised and quantified in the visible band, by the formation of azo dyes using the diazotisation-coupling reaction. Derivatisation [27,28] includes a first reaction of diazotisation with sodium nitrite, a second step for elimination of excess sodium nitrite by reaction with sulfamic acid, and finally coupling with the Bratton–Marshall reagent [N-1(naphythyl)ethylene-diamine dihydrochloride or NED]. The use of a micellar medium of sodium dodecyl sulfate (SDS) offers some advantages such as the fact that it requires no changes in pH, decreases reaction times and enhances the molar absorption of the azo dyes [29–31]. The present paper describes a procedure for simplified sample preparation for simultaneous determination of six sulfonamides in milk. The strategy of forming chromogen sulfonamides together with the use of micellar liquid chromatography increases selectiveness in biological matrices. Moreover, MLC is used as an analytical method allowing direct injection of

153

non-purified samples. It is fast, selective, economical and does not cause any environmental pollution. In consequence, it offers important savings in costs and labour. 2. Experimental 2.1. Chemicals and reagents The sulfonamides studied were: sodium sulfacetamide, sulfamethizole, sulfaguanidine, sulfamerazine, sulfathiazole and sulfamethoxazole, all from Sigma (St. Louis, USA). The sodium nitrite, sulfamic acid, N-(1-naphthyl)ethylene-diamine dihydrochloride, sodium dodecyl sulfate, 1-propanol, 1-butanol, 1-pentanol, disodium hydrogenphosphate, sodium dihydrogenphosphate, hydrochloric acid and sodium hydroxide used were from Merck (Darmstadt, Germany). Stock solutions and mobile phases were prepared in ultrapure water (Millipore S.A.S., Molsheim, France). The solutions and the mobile phases were filtered through 0.45 ␮m nylon membranes (Micron Separations, Westboro, MA, USA). 2.2. Instrumentation The chromatographic system used for the optimisation procedure and for validation of the method was an Agilent Technologies model 1100 (Palo Alto, CA, USA). It was equipped with a quaternary pump, an autosampler with 2 mL vials fitted with a Rheodyne valve (Cotati, CA, USA), and a diode array detector (range 190–700 nm). A Kromasil 100 C18 column (5 ␮m particle size, 250 mm × 4.6 mm i.d., from Scharlab (Barcelona, Spain) thermostated at 25 ◦ C was used in the separations. The flow rate, injection volume and wavelength detection were 1 mL min−1 , 20 ␮L and 490 nm, respectively. The signal

Fig. 1. Structure of the sulfonamides.

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was acquired by a personal computer connected to the chromatograph by means of a Hewlett Packard Chemstation. Michrom software [21,22] was used for chromatographic data treatment. A GLP 22 potentiometer (Crison, Barcelona) equipped with a combined Ag/AgCl/glass electrode was used to perform pH measurements. The analytical balance used was a MettlerToledo AX105 Delta-Range (Greifensee, Switzerland). 2.3. Preparation of solutions, samples and mobile phases For optimisation studies, stock solutions (100 ␮g mL−1 ) of each sulfonamide were prepared in water. For application studies, commercial milk was purchased in the local market and fortified with the sulfonamides; it was then injected into the chromatograph without any pretreatment other than derivatisation and filtration. Stock and milk solutions were stored at 4 ◦ C. The micellar mobile phase was prepared using SDS, which was buffered with disodium hydrogenphosphate 10 mM at pH 7 using 100 mM of sodium hydroxide, and 1-propanol, 1-butanol or 1-pentanol was added to achieve the desired concentration. 2.4. Derivatisation procedure Different volumes from the solutions of sulfonamides (water or milk) were introduced into a 25 mL volumetric flask, together with 10 mL of a 50 mM SDS-150 mM HCl solution and 1 mL of 100 mM sodium nitrite. After 5 min, 1 mL of 300 mM sulfamic acid was added and the mixture was left to react for an additional 10 min. Finally, 0.5 mL of 30 mM NED was added, and the volume was completed with water. The azo dyes were formed immediately and were stable for at least 1 month, with the exception of sodium sulfacetamide. The degradation of this compound was observed by the attenuation of the chromatographic peak and the appearance of a second peak. For this reason, the sulfacetamide azo dye was always injected within 1 h of its preparation, and under these conditions only one peak was observed. 3. Results and discussion 3.1. Optimisation strategy and mobile phase selection In order to find the best composition of the mobile phase that allows the simultaneous analysis of the six sulfonamides considered in this study, each of them was injected in mobile phases at pH 7 containing SDS (mM)/modifier (%, v/v): SDS/pentanol (50/1, 50/5, 100/3, 100/5, 125/3, 150/1, 150/3, 150/5), SDS/butanol (50/1, 50/4, 50/7, 75/5.5, 100/4, 100/7, 150/1, 150/7), SDS/propanol (50/2.5, 50/12.5, 100/2.5, 100/7.5, 125/5, 150/2.5, 150/7.5, 150/12.5). From the peaks that were obtained in these mobile phases, measurements of different parameters for each of the sulfonamides were determined, namely, capacity factor (k), efficiency (N) and asymmetry factor (B/A). These data were used together with a mathematical model (Eq. (1)) and an interpretative optimization procedure to predict

the chromatogram resolution of the six sulfonamides [19]. k=

KAS (1/1 + KAD ϕ) 1 + KAM (1 + KMD ϕ/1 + KAD ϕ)[M]

(1)

When pentanol and butanol were used, overlapping of some of the compounds took place, whereas propanol allows the complete resolution of the six sulfonamides. Using SDS–propanol mobile phases and under the criteria of maximum resolution–minimum analysis time, the mobile phase that was selected as being optimal was 80 mM SDS–8.5% (v/v) propanol-pH 7. In this mobile phase, the parameter values for each sulfonamide (tret , k, N and B/A) were 4.5, 1.7, 2300, 1.2 for sodium sulfacetamide; 5.0, 2.1, 1800, 1.1 for sulfamethizole; 7.0, 3.4, 1600, 1.1 for sulfaguanidine; 9.9, 5.2, 1700, 1.1 for sulfamerazine; 11.6, 6.5, 1000, 1.0 for sulfathiazole and 13.2, 7.3, 1700, 1.3 for sulfamethoxazole, respectively. 3.2. Milk blank behaviour When milk blanks are injected directly into a micellar chromatographic system consisting of 80 mM SDS–8.5% (v/v) propanol-pH 7, and the wavelength used for the detection is 260 nm, (maximum absorption of sulfonamides without derivatisation), the chromatogram (Fig. 2a) shows bigger peaks at short times (below 4 min) for the main components of milk, including proteins [32]. On the other hand, the chromatogram of the same milk matrix treated with the diazotisation and coupling derivatisation, detected at 490 nm in this case, corresponding to the maximum absorption of derivatised sulfonamides buffered at pH 7, shows three small peaks at 1.5, 2.0, and 3.6 (Fig. 2b). Note the change of scale because in the scale in Fig. 2a, the three peaks from Fig. 2b are minimised. These three peaks must be assigned to some endogenous compounds that undergo derivatisation or display a yellow colour but, in any case, the important thing is that these peaks did not interfere with the determination of the sulfonamides. Fig. 2c shows a spiked milk sample containing 25 ng mL−1 of each sulfonamide that was derivatised. 3.3. Method validation Calibration curves were constructed using the areas of the chromatographic peaks (triplicate injections) obtained at nine different concentrations, in the range 0.2–20 ␮g mL−1 for sodium sulfacetamide, sulfamethizole, sulfamerazine and sulfamethoxazole and eight concentrations, in the range 0.2–10 ␮g mL−1 for sulfaguanidine and sulfathiazole. The limits of detection (LODs) and quantification (LOQs) for sulfonamides were determined with the 3 s and 10 s criteria, respectively, using 10 injections of a milk blank. Table 1 shows the slopes, intercepts, and regression coefficients of the calibration curves, LOD and LOQ. As shown in this table, satisfactory regression coefficients (r > 0.9999) for the calibration curves were obtained. Moreover, the LOD of each sulfonamide ranged from approximately 0.72 to 0.94 ng mL−1 , values that were below the maximum residue limit established by the European Community [5] and were comparable to or even better than reported methods [4,17,32].

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Fig. 2. Chromatograms obtained from milk samples: (a) blank milk at 260 nm; (b) blank milk treated with the diazotization and coupling reagents at 490 nm; (c) milk spiked with 25 ng mL−1 of each sulfonamide. Sulfonamides and retention times (min) were: (1) sodium sulfacetamide, 4.5, (2) sulfamethiazole, 5.0, (3) sulfaguanidine, 7.0, (4) sulfamerazine, 9.9, (5) sulfathiazole, 11.6, and (6) sulfamethoxazole, 13.2.

Precision, defined as the relative standard deviation or coefficient of variation, was determined by intra and inter assays. These were determined at low, medium and high concentrations, according to the calibration curve ranges (Table 2). Intra-day precision was calculated by measuring the areas of the peaks obtained from 10 injections of three test solutions

on the same day, and intra-assay values were taken on 10 days over a 3-month period, at three different drug concentrations. As shown in Table 2, the overall mean precision, defined as the percentage of the relative standard deviation (% C.V.), was 0.41–2.65% and 0.27–3.72% for intra- and inter-day values, respectively.

Table 1 Parameters of the calibration curves: slope, intercept, regression coefficient (r), limit of detection (LOD, ng mL−1 ) and quantification (LOQ, ng mL−1 ) for the sulfonamides studied Compound

Slopea

Sulfacetamide Sulfamethizole Sulfaguanidine Sulfamerazine Sulfathiazole Sulfamethoxazole

109.846 109.934 124.537 96.095 121.833 113.034

a

Intercepta ± ± ± ± ± ±

0.286 0.071 0.112 0.182 0.115 0.149

Average ± standard deviation of n measurements.

−3.274 −4.942 −5.333 1.204 −4.117 0.772

± ± ± ± ± ±

2.422 0.571 0.600 1.531 0.620 1.190

n

r

LOD

LOQ

9 9 8 9 8 9

0.9999 0.9999 0.9999 0.9999 0.9999 0.9999

0.82 0.82 0.72 0.94 0.74 0.79

2.7 2.7 2.4 3.1 2.5 2.7

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Table 2 Intra- and inter-day precision (%C.V.) Compound

Sulfacetamide Sulfamethizole Sulfaguanidine Sulfamerazine Sulfathiazole Sulfamethoxazole

ple, has a low degree of toxicity and the risk of polluting the environment by using organic solvents is reduced.

Intra

Inter

c1

c2

c3

c1

c2

c3

2.26 1.53 1.91 1.94 2.65 1.72

1.61 0.71 0.85 0.41 0.92 0.67

1.08 0.83 0.83 0.98 0.52 0.49

1.64 3.72 2.53 3.14 2.66 2.06

0.52 0.63 1.05 0.98 0.82 0.77

0.27 0.29 0.41 0.33 1.45 0.65

c1 , c2 and c3 were 0.4, 6 and 17.5 ␮g mL−1 for sulfacetamide, sulfamethizole, sulfamerazine and sulfamethoxazole and 0.4, 6 and 10 ␮g mL−1 for sulfaguanidine and sulfathiazole. Table 3 Recovery of sulfonamides spiked samples (n = 10), by triplicate measurements at three different spiking levels in milk samples Compound

Recovery (%)a c1

Sulfacetamide Sulfamethizole Sulfaguanidine Sulfamerazine Sulfathiazole Sulfamethoxazole

99.60 101.43 95.03 99.47 95.14 92.97

c2 ± ± ± ± ± ±

1.94 0.70 5.93 0.75 3.68 1.68

97.30 91.73 90.47 98.53 93.76 95.53

c3 ± ± ± ± ± ±

0.73 1.51 3.25 1.37 0.18 1.44

94.93 97.67 83.68 103.00 100.56 95.07

± ± ± ± ± ±

0.97 1.20 1.63 0.65 1.55 3.57

a Average ± standard deviation of triplicate measurements; c , c and c were 1 2 3 0.4, 6 and 17.5 ␮g mL−1 for sulfacetamide, sulfamethizole, sulfamerazine and sulfamethoxazole and 0.4, 6 and 10 ␮g mL−1 for sulfaguanidine and sulfathiazole.

3.4. Analysis of spiked milk samples The sample recovery of this newly developed procedure was examined by adding sulfonamides to sulfonamide-free milk samples and then evaluating them. Table 3 summarises the recoveries from milk samples (n = 10), by triplicate measurements at three different spiking levels: 0.4, 6, and 17.5 ␮g mL−1 for sodium sulfacetamide, sulfamethizole, sulfamerazine and sulfamethoxazole and 0.4, 6 and 10 ␮g mL−1 for sulfaguanidine and sulfathiazole. Good recoveries ranging from 83.68 to 103.00% were determined, which means that this sample preparation method was suitable for the analysis of sulfonamide in milk samples. 4. Conclusions The method proposed in this work offers several advantages. Derivatisation of sulfonamides with NED is simple, fast and reproducible, with absorptivities at visible wavelengths that improve the signal-to-noise ratio when dealing with complex samples (e.g. milk). This method does not require complex procedures such as sample extraction and/or sample cleaning, and there is no need for large volumes of samples and solvents. Results indicate that the procedure described here is useful for the screening and quantification of the six sulfonamides in milk. Analysis times are below 16 min, with LODs and LOQs that are smaller than or similar to the values reported in the literature. Finally, it can be concluded that our method of MLC is sim-

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