Investigation of Antibiotic Residues in Meat, Milk and Egg Samples

Abstract

Investigation of Antibiotic Residues in Meat, Milk, and Egg Samples Broad-spectrum antibiotics are widely administrated in poultry and cattle farms for the prevention and treatment of infectious diseases and in some cases are inappropriately used as feed additives to enhance growth in food-producing animals. The presence of antibiotic residues in animal protein is a public health concern because long-term regular consumption of foods containing antibiotic residues may pose health risks due to continuous exposure to these residual antibiotics. The extensive use of sulfa drugs (SAs), fluoroquinolones (FQs), tetracyclines (TCs), chloramphenicol (CAP), derivatives of nitrofuran metabolites (NFs) in cattle and poultry farms production has been recognized as a key contributor of antibiotic resistance. This study aimed to identify and quantify residual amounts of sulfa drugs namely sulfadimethoxine (SMX), sulfadiazine (SDZ), sulfamethazine (SMT), fluoroquinolones like ciprofloxacin (CIP), enrofloxacin (ENR), levofloxacin (LEV), tetracyclines namely tetracycline (TC) oxytetracycline (OTC), chlortetracycline (CTC), chloramphenicol, derivatives of nitrofuran metabolites of 3-Amino-2-oxazolidinone (AOZ), 3-Amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), and 1-Aminohydantion (AHD) in poultry meat, beef, egg, milk, and poultry feed samples using reported methods after modification and validation. Poultry meat, beef, egg, milk, and poultry feed samples were collected from poultry farms, and slaughter houses, different local markets and super shops around Dhaka and Gazipur cities, Bangladesh. Sample preparation was carried out using modified QuEChERS (Quick Easy Chief Effective Rugged and Safe) method and solid phase extraction using n-hexene, ethyl acetate, methanol, water, acetic acid and other solvents where needed. Clean-up was carried out using primary secondary amine (PSA) and C18 gel (particle form). Antibiotic residues were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) system consists of a Triple Quadrupole (QqQ) mass spectrometer equipped with electrospray ionization (ESI) and liquid chromatography (LC) separation system. The mass spectrometer was operated in both positive and negative ion modes with a capillary voltage of 3-4 kV. Method parameters were optimized in different optimized conditions such as desolvation and column oven temperature, pressure, gas flow. Chromatographic separation was performed on C18 column using optimized mobile phase compositions. Identifications were based on LC- vii MS/MS analysis, and quantifications were performed by comparing analyte peaks with those corresponding standards. Method linearities were measured within concentration range of 1.25-100 μg L-1, 5-200 μg L-1, 1.5-100 μg L-1, 1.5-100 μg L-1, and 1-200 μg L-1 for SAs, FQs, TCs, CAP and NFs, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were established as the minimum analyte level that produced a signal to noise ratio (S/N) of 3 and 10, respectively ensuring that the signal could be distinguished from baseline noise with confidence. Method validations were performed using spiked sample at ppb levels such as 5 (n=3) and 10 (n=3) ppb for SAs and NFs, 20 and 25 ppb for FQs, 10 and 15 ppb for TCs and CAP, respectively. Recoveries were evaluated for poultry meat, beef, egg, milk and poultry feed samples; control samples spiked with two levels of CRM (certified reference materials) for each class of antibiotics, spiking levels 5 and 10 ppb were in the range of 91-101% with RSD% from 6.12-8.22% in poultry meat, 96-103% with RSD% from 3.38-9.85% in beef, 93-98% with RSD% from 4.32-7.55% in egg, 93-97% with RSD% from 4.39-8.73% in milk, and 88-97% with RSD% from 4.98-7.85% in poultry feed for SMX, SDZ, SMT. FQs recoveries were done at two concentration levels of 20 and 25 ppb and found to be in the range of 97-101% with RSD% from 5.40-7.62% in poultry meat, 98-101% with RSD% from 3.82-7.25% in beef, 99-102% with RSD% from 4.31-9.49% in egg, 98-102% with RSD% from 4.24-9.31% in milk, and 93-98% with RSD% from 3.66-5.75% in poultry feed for CIP, ENR, LEV. TCs recoveries were done such as 10 and 15 ppb level with the range 100-103% with RSD% from 4.00-8.79% in poultry meat, 100-108% with RSD% from 4.00-10.90% in beef, 88-102% with RSD% from 6.21-9.70% in egg, 99-102% with RSD% from 5.59-9.01% in milk, and 87-97% with RSD% from 4.58-7.15% in poultry feed for TC, OTC, and CTC. CAP recoveries were done at 10 and 15 ppb level and found in the range 94-100% with RSD% from 8.38-8.52% in poultry meat, 102% with RSD% from 7.47-8.24% in beef, 97-99% RSD% from 7.88-8.17% in egg, 98-99% with RSD% from 8.59-9.97% in milk, and 99-100% with RSD% from 8.03-8.14% in poultry feed. NFs recoveries were done at 5 and 10 ppb level with the range 95-104% with RSD% from 3.26-7.17% in poultry meat, 95-102% with RSD% from 4.63-9.14% in beef, 92-101% RSD% from 5.44-8.02% in egg, 95-103% with RSD% from 4.15-7.86% in milk, and 95-103% with RSD% from 3.97-6.79% in poultry feed for NP-AOZ, NP-AMOZ, NP-AHD. In all of the experiments, recovery values remained within the accepted ranges of 70-120% viii specified by CODEX guidelines. World Health Organization (WHO), Food and Agriculture Organization (FAO) and Bangladesh Food Safety Authority (BFSA) have imposed maximum residue limits (MRLs) to protect consumers from harmful exposure to veterinary drug residues. In our present studies, among the targeted 750 analyzed samples, residues were detected in 13 samples collected from different place of Dhaka and Gazipur cities however detected residual concentrations were below MRL values.