Multiclass analysis of antibacterial residues in milk using RP-liquid chromatography with photodiode array and fluorescence detection and tandem mass spectrometer confirmation

Abstract

A simplified procedure for simultaneous quantification of ceftiofur (CEF), fluoroquinolone (FQ) and sulfonamide (SA) antibacterials in bovine milk was developed. The reverse-phase liquid chromatography (RP-LC) multiclass method for analysis of eleven distinct compounds, from three antibacterial classes, was validated in line with Commission Decision 2002/657/EC. Confirmation of the analytes identities was performed by electrospray mass spectrometry detection. The analytes were extracted from milk matrix by liquid-liquid extraction with acidified ultrapure water and directly analyzed in the chromatograph. The SA compounds were pre-column derivatized with fluorescamine for fluorescence detection. The method provided good results regarding the analytical parameters of linearity, selectivity, sensitivity, precision, recovery, decision limit (CCα), detection capability (CCβ), limit of detection (LOD), limit of quantification (LOQ), stability and robustness. Analytes were extracted by liquid-liquid extraction in the fortified matrix and the compounds identity was confirmed by their precursor ion and fragments through tandem mass spectrometry analysis. Additionally, milk samples from two state capitals in the South Region of Brazil were analyzed by both the quantitative and confirmatory methods. The validation process showed correlation coefficients (r(2)) greater than 0.98 for all the analytes, with recovery rates up to 98% for all the studied drugs. LOD and LOQ limits ranged from 8.0 to 20.0 ng mL(-1) and 10.0 to 32.0 ng mL(-1), demonstrating good specificity of the method. The intra-day and inter-day precisions for all the analytes were below or equal to 7.40 and 10.13, respectively. The studied antibacterials were not detected in milk samples. The developed method represents an efficient alternative for multi-residue analysis in milk, being suitable and especially viable for monitoring in developing countries.

Survey of pyrethroid, macrocyclic lactone and antibacterial residues in bulk milk tank from Minas Gerais State, Brazil

“…Thus, in attempt to protect human health and ensure a high-quality product, several governmental authorities and international committees (Brasil 1999, European Commission 2010, FAO/WHO 2011 have set MRLs for various veterinary drug residues. Antimicrobials, macrocyclic lactones and pyrethroids are the most widely used veterinary drugs in dairy cattle management for disease therapy and control of bacterial and parasitic infections (Bilandžić et al 2011, Toaldo et al 2012. Therefore, most of these studies that have assessed the presence of veterinary drug residues in bulk tank milk in Brazil have used rapid screening methods (Cerqueira et al 2014, Trombete et al 2014 or analyzed a single or limited class of active compounds (Ciscato et al 2002, Hernandes et al 2009, Pacheco-Silva et al 2014.…”

“…Although these screening methods (i.e. immunological or microbial inhibition assays) are widely used to detect the presence of veterinary drugs in foods, more accurate chromatographic methods coupled with highly specific and sensible detection systems, such as tandem mass spectrometry, are required to identify, confirm the presence of and quantify these compounds (Molina et al 2003, Samanidou & Nisyriou 2008, Toaldo et al 2012) because they provide full or complementary information enabling the unequivocal identification and quantification of the analyte at levels of interest (Samanidou & Nisyriou 2008).…”

Climate conditions associated with the occurrence of antimicrobial and macrocyclic lactone residues in bulk tank milk

“…Antimicrobials and macrocyclic lactones are the most widely used veterinary drugs in dairy cattle management for treatment of disease and the control of bacterial and parasitic infections (Bilandžić et al, 2011;Toaldo et al, 2012). Extensive and improper use of these active compounds can lead to the presence of their residues in milk, causing human health risks, the development of microbial drug resistance, the spread of resistant pathogens, loss of industrial output and technological problems in dairy products (Ruegg and Tabone, 2000;Toaldo et al, 2012).…”

“…Extensive and improper use of these active compounds can lead to the presence of their residues in milk, causing human health risks, the development of microbial drug resistance, the spread of resistant pathogens, loss of industrial output and technological problems in dairy products (Ruegg and Tabone, 2000;Toaldo et al, 2012).…”

“…One possible cause of false-positive results in these screening tests is the presence of indigenous antimicrobial agents and free fatty acids (Molina et al, 2003). Although these screening methods (i.e., immunological or microbial inhibition assays) are widely used to detect the presence of veterinary drugs in foods, more accurate chromatographic methods coupled with highly specific and sensible detection systems, such as tandem mass spectrometry, are required to identify, confirm the presence of and quantify these compounds (Molina et al, 2003;Samanidou and Nisyriou, 2008;Toaldo et al, 2012) because they provide full or complementary information enabling the unequivocal identification and quantification of the analyte at levels of interest (Samanidou and Nisyriou, 2008).…”