Quantitative analysis of selected antibiotic drug residues in honey and manure samples using modern analytical techniques

Abstract

In this study, modern clean-up and pre-concentration techniques, namely hollow fibre liquid phase micro-extraction (HFLPME) and miniaturized pipette-tip extraction based on molecularly imprinted polymers (m-MIP-PTE), were used for the extraction of antibiotic residues from honey and manure samples, respectively. The work is divided into five papers. In paper I, modern extraction and clean-up methods of antibiotic residues from various food samples were reviewed. One of the challenges in veterinary drug residue analysis is their occurrence in trace amounts that are normally below limits of detection of most analytical instruments. Consequently, various efficient, economical, miniaturized and environmentally friendly extraction methods have been developed in recent years to pre-concentrate these drugs in an attempt to overcome the limitations of conventional extraction methods such as liquid-liquid extraction and solid phase extraction. In this work, the application of techniques including quick, easy, cheap, effective, rugged and safe (QuEChERS), molecularly imprinted polymers, dispersive liquid-liquid microextraction and hollow fiber liquid-phase micro-extraction in the pre-concentration of various antibiotic residues in animal derived food samples was discussed. The recent applications of sorptive micro-extraction techniques in the pre-concentration of antibiotic residues from food samples were reviewed in Paper II. The current trends of analytical sample preparation methods are favouring extraction and pre-concentration techniques that are compliant to the principles of green analytical chemistry. In this paper, the origin and principles of solvent-free sample preparation techniques, known as green techniques, including solid phase micro-extraction, stir bar sorptive extraction, stir cake sorptive extraction and fabric phase sorptive extraction were briefly discussed. Their application in the extraction of antibiotic residues from food samples in the years 2015 to 2020 was critically reviewed. Furthermore, possible sorption mechanisms facilitating the adsorption of these drugs on sorbents were discussed. In paper III, recent applications of solid phase extraction in the pre-concentration of antibiotic residues from livestock and poultry manure were collated and critically discussed. Application of livestock manure contaminated with antibiotics and their metabolites to farming land may pose a threat to agro-ecosystems, plant growth, aquatic life and soil life, with antibiotic resistance increasingly becoming ubiquitous. Therefore, monitoring of manure for antibiotic residues is of vital importance in order to assess the risks of environmental pollution by these drugs. Several sample pre-treatment techniques have been developed for the extraction of antibiotic residues from complex matrices

including manure over the years. However, SPE is the still the commonly used clean-up technique for this matrix. Therefore, a critical overview of studies that have been conducted in the past 6 years on the extraction of antibiotic residues from manure employing SPE were given in this review. The presence of tetracycline residues in honey samples was investigated in paper IV. Carrier mediated HFLPME was employed for the extraction and pre-concentration of these drugs prior to analysis on the LC-Q-TOF/MS. An acceptor phase solution employed in this study was 0.1 M H3PO4 containing 1.0 M NaCl (pH = 1.0), whereas 0.05 M Na2HPO4 (pH = 9.5) was used as the donor phase. Parameters that could affect the extraction efficiency of this method, such as the pH of the acceptor and donor solution, salt addition, agitation speed and extraction time were optimized before the application of the developed method to honey samples. Calibration curves showed good linearity, in the range of 20-500 μg L-1 with correlation coefficients ranging between 0.9918 and 0.9998, under the optimized conditions. The recoveries of blank honey samples at three spiking levels (75, 200 and 300 μg L-1) ranged from 56.1% to 120.8% with RSDs between 0.37% and 11.21%. LODs and LOQs were in the ranges of 0.042 to 0.661 μg kg-1 and 0.127 to 2.002 μg kg-1, respectively. Doxycycline residues were detected in a commercial honey sample at a concentration of 0.20 μg kg-1. The pre-concentration of sulfonamides from manure samples using miniaturized pipette-tip extraction based on molecularly imprinted polymers (MIPs) was studied in paper V. MIPs were synthesized by precipitation polymerization using sulfamethoxazole as a template. Characterization of the MIPs was then done using FTIR. Parameters that could influence the extraction efficiency of this technique including loading cycles, desorption cycles, type of elution solvent, sample pH, salt addition and sorbent amount were optimized. The optimized method was applied to manure samples prior to analysis on the LC-Q-TOF/MS where sulfachlorpyridazine, sulfadiazine, sulfamethoxypyridazine, sulfamethoxazole and sulfaquionoxaline were investigated. The developed method was validated and linearity in the range of 20-400 μg L-1 was obtained with regression coefficients ranging between 0.9881 and 0.9990, under the optimized conditions. The recoveries of blank manure samples at three spiking levels (100, 200 and 300 μg L-1) ranged from 67.73% to 120.48% with RSDs between 0.08% and 15.3%. LODs and LOQs were in the ranges of 0.111 to 0.319 μg kg-1 and 0.336 to 0.966 μg kg-1, respectively. Finally, the proposed method was successfully applied for the extraction of the five sulfonamides from cattle manure samples. Sulfamethoxazole and sulfamethoxypyridazine were detected at concentration levels of 0.1083 and 0.065 μg kg-1, respectively.