| What is fed to animals produced for human consumption can have important implications for the health of the public. Animal feed/feed ingredients regulated as drugs are regulated more stringently than foods and require premarket approval based on safety and efficacy testing. These concerns arise not only from what is fed to animals but also from gaps in regulations and systems intended to ensure the safety of feed and the food supply. While knowing what is in animal feed is important in order to protect the public?s health, whether a feed or feed ingredient is regulated as a drug depends on its intended use. The aim of this research provides the development of analytical methods capable of simultaneous screening, confirmation and quantification of maximum number veterinary drugs covering a wide diversity of polarities and p Ka values. The method employs ultrasonic–assisted extraction combined with D-SPE including variety of target matrices by Liquid Chromatography Tandem Mass Spectrometry(LC-MS/MS). It focuses on food producing animal species and authorized and prohibited medicinal additives in animal feed. The analytical technique used in all method was the highly selective and sensitive LCMS/MS. This research resulted in the development and validation of methods for analysis in feed, urine and serum and plasma. Mostly veterinary drugs and antibiotics are prohibited for use as feed additives and quantification of 200 veterinary drugs from 25 different classes, beta-lactam, beta agonist, beta-blockers, sulfonamides, NSAID, steroids, quinolones, floroquinolones, macrolides, coccidiostats; piperidines, amines, penincillin, glycosides, aminoglycosides, piperazines, purines, amides, tetracyclines, coccidiostats, Imidazoles and imadazolines, etc. The validation of the developed methods is also the part of this research. All validation protocol was designed in accordance with EU legislation; Commission Decision 2002/657/EC technical criteria that must be applied in the screening and confirmation of veterinary drug residues. This legislation is concerned with the performance of analytical methods and the interpretation of results. Validation criteria were examined using protocols set out in this legislation and in-house validation included specificity, linearity, accuracy, precision, repeatability, reproducibility, decision limits(CCα), detection capabilities(CCβ), to ensure that the method was fit for monitoring purpose a wide variety of veterinary drugs. We prepared Single laboratory validation plan consecutively 4 days for matrices, recovery, repeatability(intra-day) and reproducibility(inter-day) were evaluated by analyzing of 20 blank matrix spiked at the same concentration level 1,2,5,10,50 and 100 μg/kg and their five replicates were measured including matrix blank and calculated their mean recovery, relative standard deviation, CCα, CCβ, LOD and LOQ. The different physicochemical properties of veterinary drugs make the development of multi-class analytical method very challenging. Initially optimization of extraction method and instrumental parameters started for setting the reliable screening method, Extraction and clean-up procedures were optimized with fortified Blank feed, samples were extracted by ultrasonic assisted extraction with mixture of methanol/acetonitrile/Mc Ilvaine buffer at p H 4.6(37.5/37.5/25, v/v/v) containing 0.3% of EDTA-Na2, followed by clean up using dispersive solid-phase extraction(D-SPE) with primary secondary amine(PSA), PSA was inspired by the QUECh ERS?s method. Feed average recoveries ranged from 52%(Promethazine Hcl) to 109 %(Valnemulin Hcl) at three spiked levels with associated relative standard deviations(RSD%) of 2% and 20%, overall 77% analytes showed recoveries 65-109% and furthermore the Decision limits(CCα) ranging from 0.2-1.3 μg/kg and detection capability(CCβ) 0.3-1.5 μg/kg. Blank urine Samples fortified and beta glucuronidase added for enzymatic activity and incubated for 3 hours then extracted with mixture of methanol/acetonitrile/sodium succinate buffer at p H 4.6(37.5/37.5/25, v/v/v) containing 0.3% of EDTA-Na2, followed by clean up using dispersive solid-phase extraction(D-SPE) with primary secondary amine(PSA). Recoveries based on the matrix matched standard calibration for urine ranged from 50%-133% with RSD 2% to 23% and experimental ranged LOD 0.02 to 33.3μg/L and LOQ 0.07 to 111μg/L. fortified blood Samples have the same procedure as feed and Recoveries based on the matrix matched standard calibration for blood and ranged from 48 to122 % with limit of detection is 0.02 to 30μg/L, limit of quantification ranged 0.07 to100μg/L.The intent is to provide the relatively simple, reproducible and sensitive method for feeding stuffs currently wide variety of raw materials is allowed in the manufacture of animal feed and given to major food-producing animals(including cattle, poultry, pigs and major animal species produced in aquaculture. the results proved the suitability and potential of the method for the detection and identification of all tested veterinary drugs and its applicability to other complex matrices. The feed Samples collected by local authorities by conducting survey for animal feed safety, samples tested and found positive for many veterinary drugs and results were confirmed using liquid chromatography tandem mass. The inspection of 57 feed samples collected from various feed mills of five provinces of China, and result revealed that feed samples contain the veterinary drug concentration in feed samples was, respectively, in the range of 0.18 μg/kg-111 μg/kg, the most frequently veterinary drug found was sulfadiazine and for urine 22 urine samples collected from farmers and slaughter house the veterinary drug concentration was, respectively, in the range of 0.2-1.9μg/L.At present many legislation and Guidance on veterinary drugs in China and others countries providing List of approved and unauthorized veterinary drugs and their MRLs used as medicinal additives in animal feed in animal production for therapeutics and prophylactic purpose but banned for use as growth promoter. This present research results are reliable and for controlling the contamination within the food processing chain, potentially resulting in deleterious health effects in animals and consumers. The main purpose of this study was to quantify the veterinary drugs in commercially available animal feeds by various ways directly analysis of feed or checked their presence in urine and blood samples of food producing animals... |
