Analytical MethodsRapid immunochemical analysis of the sulfonamide-sugar conjugated fraction of antibiotic contaminated honey samples
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
References (33)
- et al.
Magnetic solids in analytical chemistry: A review
Analytica Chimica Acta
(2010) - et al.
Magnetic gold nanoparticles in SERS-based sandwich immunoassay for antigen detection by well oriented antibodies
Biosensors & Bioelectronics
(2013) - et al.
A new and simple method to determine trace levels of sulfonamides in honey by high performance liquid chromatography with fluorescence detection
Journal of Chromatography A
(2009) A rapid and sensitive method for the quantifation of microgram quantities of protein utilizing the principle of protein-dye binding
Analytical Biochemistry
(1976)- et al.
Immunoassay for folic acid detection in vitamin-fortified milk based on electrochemical magneto sensors
Biosensors and Bioelectronics
(2009) - et al.
Quantitative analysis of twelve sulfonamides in honey after acidic hydrolysis by high-performance liquid chromatography with post-column derivatization and fluorescence detection
Journal of Chromatography A
(2004) - et al.
Development of a group-specific immunoassay for sulfonamides: Application to bee honey analysis
Talanta
(2007) - et al.
Antimicrobials in beekeeping
Veterinary Microbiology
(2012) - et al.
Determination of tetracyclines residues in honey using high-performance liquid chromatography with potassium permanganate–sodium sulfite–β-cyclodextrin chemiluminescence detection
Journal of Chromatography B
(2005) - et al.
Establishment of magnetic beads-based enzyme immunoassay for detection of chloramphenicol in milk
Food Chemistry
(2012)
Electrochemical magneto immunosensing of antibiotics residues in milk
Biosensors & Bioelectronics
Generation of broad specificity antibodies for sulfonamide antibiotics and development of an enzyme-linked immunosorbent assay (ELISA) for the analysis of milk samples
Journal of Agricultural and Food Chemistry
Traceability of sulfonamide antibiotic treatment by immunochemical analysis of farm animal hair samples
Analytical and Bioanalytical Chemistry
Contaminants of bee products
Apidologie
Official Journal of the European Communications
Immunochemical assays for direct sulfonamide antibiotic detection in milk and hair samples using antibody derivatized magnetic nanoparticles
Journal of Agricultural and Food Chemistry
Cited by (12)
Ni(OH)<inf>2</inf> nanoarrays based molecularly imprinted polymer electrochemical sensor for sensitive detection of sulfapyridine
2019, Sensors and Actuators, B: ChemicalCitation Excerpt :Hence, many countries set strict standards of sulfonamide residues in freshwater fish. For example, sulfonamide residues cannot be exceeded 100 μg kg−1 in food in China, America and many European countries [7,8]. Thus, developing a fast, highly selective, and sensitive method to detect sulfonamide is very important for public health.
Upconversion luminescence resonance energy transfer-based aptasensor for the sensitive detection of oxytetracycline
2015, Analytical BiochemistryEnvironmentally friendly analysis of sulphonamides in Brazilian honey through automated and miniaturised sample preparation coupled with LC-MS/MS
2022, Food Additives and Contaminants - Part AA Magnetic, Core–Shell Structured, pH-Responsive Molecularly Imprinted Polymers for the Selective Detection of Sulfamethoxazole
2021, Journal of Inorganic and Organometallic Polymers and MaterialsPhages in Therapy and Prophylaxis of American Foulbrood – Recent Implications From Practical Applications
2020, Frontiers in Microbiology