Elsevier

Food Chemistry

Volume 178, 1 July 2015, Pages 156-163
Food Chemistry

Analytical Methods
Rapid immunochemical analysis of the sulfonamide-sugar conjugated fraction of antibiotic contaminated honey samples

Dedicated to the memories of Francisco Sanchez-Baeza and Alejandro Muriano.
https://doi.org/10.1016/j.foodchem.2015.01.037Get rights and content

Highlights

  • Rapid immunochemical screening for sulfonamide contaminated honey analysis.

  • Sulfonamides conjugated to sugars have a very low SA immunoreactivity (<5%).

  • Sulfonamide antibiotics must be first released before the analysis.

  • The whole analytical procedure is performed in less than 2 h for more than 100.

  • Detectability below action limits established in EU countries for nine sulfonamides.

Abstract

A rapid high-throughput immunochemical screening (HtiS) procedure for the analysis of the sulfonamide (SA)-sugar conjugated fraction of antibiotic contaminated honey samples has been developed. Studies performed with this matrix have indicated that sulfonamide antibiotics are conjugated to sugars rapidly and quantitatively, providing samples with very low SA immunoreactivity. Therefore, sulfonamides must be first released before the analysis, and for this purpose, a simple and fast sample preparation procedure has been established consisting of hydrolyzing the sample for 5 min, adjusting the pH and buffering the sample prior to the immunochemical analysis. Under these conditions, honey samples could be directly analyzed without any additional sample treatment, other than dilution. Recovery values of the whole analytical procedure were greater than 85%. The analysis of the same samples without the hydrolysis provided recovery values below 5%. Selectivity studies performed in hydrolyzed honey samples revealed that nine relevant sulfonamide antibiotics can be detected with limit of detection (LOD) values below the action limits established by some EU countries (Belgium, 20 μg kg−1, United Kingdom or Switzerland, 50 μg kg−1).

Introduction

Honey has been traditionally considered a natural and healthy product. However, recently, the presence of antibiotic residues in this nutrient has been reported (Bogdanov, 2005). Contamination of natural honey with antibiotics may occur after the direct treatment of bees against bacterial brood diseases, such as American foulbrood (AFB) or European Foul Brood (EFB) (Serra Bonvehí & Gutiérrez, 2008). Residues could also originate from the increasing use of antibiotics to treat bacterial infections of orchard plants and trees (McManus & Jones, 1994). Thus, important fruit-tree diseases such as Pseudomonas blossom blast are treated with antibiotics, mainly during blossom (Spotts & Cervantes, 1995). Contamination of the blossom with high concentrations of antimicrobials implies the risk of a carry-over of the residues into the honey (Heering et al., 1998, Wan et al., 2005)

Sulfonamides are among the antibiotics most frequently found in honey (Reybroeck et al., 2010, Wang et al., 2006). The majority of the sulfonamides currently used show a relatively long half-life, which may result in serious health problems in humans, such as allergic or toxic reactions (Sensderson, Naisbitt, & Park, 2006). Moreover, it has been reported that, in honey, sulfonamides tend to bind sugars via the formation of N-glycosidic bonds through their aniline group (Sheth, Yaylayan, Low, Stiles, & Sporns, 1990). Although governmental and regulatory agencies have established maximum residue limits (MRLs) for sulfonamides residues in different food commodities to safeguard public health (Commision Regulation, 1990, Food and Durg Regulations, 1991), no MRLs have been established for honey in Europe, since the use of antimicrobials to treat honeybees is not authorized. Nevertheless, since in many other countries, these practices are legal, problems arise regarding imports of honey into the EU, which calls for reliable, rapid and high-throughput screening (HTS) analytical methods to ensure that imported honey samples placed in the EU market will comply with the EU rules. As stipulated in Annex II of Council Directive, 2001/110/EC, honey must be free from organic or inorganic foreign matter to its composition. In the absence of either EU MRLs, some countries within the European Union have established their particular action limits (recommended target concentrations, non-conformity or tolerance levels) for these antibiotics (Bernal et al., 2009, Reybroeck et al., 2012). As an example, Belgium and the United Kingdom have set up tolerance levels of 20 and 50 μg kg−1, respectively, for total sulfonamides in honey, and Switzerland has set a tolerance level of 50 μg kg−1, referring to the sum of sulfonamides and their metabolites.

High-performance liquid chromatography followed by tandem mass spectrometry or fluorescence detection are the most commonly used techniques for the analysis of sulfonamides in honey (Maudens et al., 2004, Sheridan et al., 2008). Alternatively, immunochemical methods could complement the screening of antibiotic residues, based on their simplicity, low cost and high throughput capabilities (Heering et al., 1998, Pastor-Navarro et al., 2007, Serra Bonvehí and Gutiérrez, 2008, Tafintseva et al., 2009). Despite these advantages, current immunochemical analytical procedures reported for the detection of sulfonamide residues in honey samples still involve complex extraction and clean up processes using organic solvents (Heering et al., 1998, Pastor-Navarro et al., 2007, Tafintseva et al., 2009), which limit their use as first action screening methods. Moreover the necessity to release the sulfonamides from the sugar conjugates is rarely discussed. Thus, blocking of the aniline group by the sugar, could result in a decrease of the recognition of the sulfonamides by the antibody and in an underestimation of the concentration of these residues in the sample. Eventually, chemical methods can be used to release the antibiotic prior the analysis. Hence, strong acids may be employed to break the imine bond formed between the sugar and the sulfonamide (Schawaiger & Schuch, 2000), but usually these procedures yield complex samples that have to be purified prior the analysis. Sheridan et al. (2008) used a multi-screening approach to monitor 14 sulfonamide compounds and chloramphenicol applying acidic hydrolysis (1 h, room temperature, RT) to liberate the sugar-bonded sulfonamide, but the sample had to subsequently be purified by solid-phase extraction (SPE) to remove potential interferences with an absolute recovery of around 60%. Similarly, Wang et al. (2012) describe an acidic hydrolysis step (1 h, RT) followed by liquid–liquid extraction (LLE) prior LC–ESI-MS/MS analysis.

The advantages of using antibody-derivatized magnetic particles to simplify sample treatment procedures are well recognized. A significant number of immunoassays and immunosensors have exploited the benefits of using magnetic particles biofunctionalized with either antigens or antibodies, as it can be seen in recently published papers (Baniukevic et al., 2013, Font et al., 2008, Lermo et al., 2009, Orlov et al., 2013, Xu et al., 2012), and reviews (Aguilar-Arteaga et al., 2010, Kuramitz, 2009, Pedrero et al., 2012, Zhang and Zhou, 2012). Few years ago, we also reported their use on a direct ELISA (Font et al., 2008) and an electrochemical immunosensor (Zacco et al., 2007) to directly detect sulfonamides residues in milk samples without any sample treatment, although the sulfonamide selectivity profile of such immunochemical approaches was very narrow. Later on, we reported a broad-selectivity microplate-based indirect ELISA able to detect up to 10 different sulfonamide antibiotic congeners in different biological samples (Adrian, Font et al., 2009, Adrian, Gratacós-Cubarsí et al., 2009). The antibodies used were raised against an immunizing hapten maximizing recognition of the common epitope of this antibiotic family, which is the aniline group. Based on this previous knowledge, we report here the development of a rapid and efficient broad-selectivity immunochemical procedure to quantify the sulfonamide-sugar conjugate fraction of honey samples, involving a quick hydrolysis step prior the immunochemical analysis.

Section snippets

Chemicals and immunochemicals

All the sulfonamides used in this work were supplied by Riedel-de Haën (Seelze, Germany). Hapten SA1 (5-[6-(4-amino-benzenesulfonylamino)-pyridin-3-yl]-2-methyl-pentanoic acid) and hapten SA2 (5-[4-(amino) phenylsulfonamide]-5-oxopentanoic acid) were prepared as previously described (Adrian, Font et al., 2009, Font et al., 2008). Ovalbumin (OVA) and the secondary antibody peroxidase conjugate (antiIgG-HRP) were purchased from Sigma Chemicals Co. (St. Louis, Missouri). Tosyl-activated

Immunochemical assay performance in honey samples

Few years ago, we reported the development of sulfonamide immunoreagents addressed to provide wide selectivity to the immunochemical analytical procedures developed. This was accomplished by designing a hapten that maximized recognition of the common aniline group of this antibiotic family (Adrian, Font et al., 2009). In this work, we address the implementation of a high-throughput immunochemical screening method to the analysis of these residues in honey samples. However, since it has been

Conclusions

It has been demonstrated that sulfonamide antibiotics rapidly react with components of the honey matrix, probably sugars, as stated in the introduction. This fact indicates the mandatory need to perform hydrolysis prior the analysis. In that respect, an efficient and reliable immunochemical analytical procedure for the analysis of sugar-conjugated sulfonamide antibiotic residues in honey samples has been developed. The method involves hydrolysis of the sugar conjugates in just 5 min (Method B),

Acknowledgments

This work has been supported by the European Community (Conffidence project, KBBE2008-211326). CIBER-BBN is an initiative funded by the he Spanish National Plan for Scientific and Technical Research and Innovation 2013–2016, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. The Nanobiotechnology for Diagnostics group (Nb4D) group (previously named Applied Molecular Receptors

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