The Bioequivalence And Residue Detection Method Study Of Compound Sulfadiazine Suspension In Broiler Chicken

Sulfadiazine(SD) is a kind of synthetic benzene sulfonamide drugs, sulfa drugs are widely used in veterinary clinical because of its broad-spectrum antimicrobial drugs. It has significant inhibitory effect for a variety of Gram-positive bacteria and some Gram-negative bacteria. Trimethoprim, a synthetic diaminopyrimidines, is mainly combined with sulfa drugs into compound preparation for the expansion of the antibacterial spectrum, to enhanced antibacterial activity, and it was used widely in clinical practice. Sulfadiazine compound suspension is white or pale yellow compound, the main component were sulfadiazine(SD) and trimethoprim(TMP); it belongs to broad-spectrum antibiotics and was mainly used for bacterial diseases and protozoa infections caused by sensitive bacteria for chicken and ducks.Long-term irrational use of sulfa drugs can cause drug residues in animal products, and for the animals themselves, sulfa drugs overdose in feed, not only damage the body but also affect performance, the consumption of the residual food of animal origin will destroy the human’s body’s normal function, it also cause damage to the environment. At present, the European Union and other international countries had made specific provisions on MRLs for sulfa drugs residue. Therefore, detecting sulfonamides residues in animal foods has important implications for human health.This paper aims to establish a simple, rapid and sensitive detection method for simultaneously determination of sulfadiazine and trimethoprim in broiler plasma by rapid ultra-performance liquid chromatography mass spectrometry, And the method was applied to the bioequivalence study. And this paper aims to develop a residue method for simultaneously determination of sulfadiazine and trimethoprim in broiler muscle and liver by liquid chromatography tandem mass spectrometry. The main conclusions are as follows:1. To ensure the smooth progress of the bioequivalence experiment, this paper first carried out content determination for the two compound preparation. The content determination was in accordance with quality standards of the import veterinary drugs. According to the external standard method with peak area to calculation content, As a result, The content of sulfadiazine in the reference and test preparation were 99.78%, 99.82% of labeled content, trimethoprim content in the reference and test preparations were 99.15%, 97.66% of labeled content, The result showed that the two preparations are in line with the quality standards and showed no significant differences, laying the foundation for bioequivalence experiments.2. Establish a method for simultaneous detection sulfadiazine and trimethoprim in broiler plasma by ultra-performance liquid chromatography mass spectrometry. In this method, the sample is extracted with methanol, detected by ultra-high performance liquid chromatography mass spectrometry, quantified by internal standard. The results showed that the two analytes in plasma showed good linearity and the correlation coefficients were greater than 0.99, Accuracy and precision were measured using the four concentrations of the standard addition to matrix. The intra and inter-day relative standard deviations were in range of 1.76-13.41%, 2.3-10.6%, the mean average recovery were in the range of 80-110%, meanwhile the stability were good. The limits of detection of sulfadiazine and trimethoprim in plasma were 0.05μg/m L and 0.01μg/m L.3. Bioequivalence study : this study adopted a parallel design, 40 chickens for test and reference preparation respectively, a single oral lavage, blood samples were collected from the brachial vein immediately before dosing(pre-dose) and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 8, 12, 24, 36 and 48 h after administrating a total dose of 38.4mg/kg. Plasma samples were analysised by the method for simultaneous detection sulfadiazine and trimethoprim by ultra-performance liquid chromatography mass spectrometry. The result was analysised by software DAS3.0. The AUC and Cmax for test preparation were within the equivalent range 80-125%, 70-143%(done logarithmic conversion) of the reference formulation. Results showed that: the compound sulfadiazine suspension test preparation and reference preparation is biological bioequivalent.4. Establish a method for simultaneous detection of sulfadiazine and trimethoprim in chicken muscle and liver by liquid chromatography tandem mass spectrometry. In this method, the sample is extracted with acetonitrile, removed fat by hexane, after concentrated it was reconstitution in acetonitrile, detected by liquid chromatography tandem mass spectrometry, quantified by internal standard. The results showed that the two analytes showed good linearity and the correlation coefficients R2 were greater than 0.99, The intra and inter-day relative standard deviations were in the range of 2.2-10.2% and the mean average recovery were in the range of 88-109%, The stability were good in chicken muscle and liver. And the limits of detection of sulfadiazine and trimethoprim were 4μg/kg, 0.8μg/kg in muscle,and 20μg/kg,4μg/kg in liver. Detecting the sample tissue of chicken with the established residue method proves the feasibility of this method.In summary, This method of this paper established for simultaneous detection of sulfadiazine and trimethoprim in plasma is simple and feasible by ultra-high performance liquid chromatography mass spectrometry, and make judgment for bioequivalence; The residue method established in this paper was also feasible.