The Study Of Pharmacokinetics And Plasma Protein Binding Properties In Vitro Of Sulfamethazine And Its Acetyl Metabolite In Rabbits

As an effective agent against a broad spectrum of microorganisms, sulfamethazine (SMZ) has been widely used in livestock farming as a preventive and therapeutic agent, or added into the feeds to improve their anti-bacterial ability. However, such treatment has led to more and more serious sulfamethazine residue in animal-source food these years. In this paper, we have established and validated a HPLC-UV method for fast determination of SMZ and its N4-acetyl metabolite:N4-acetyl sulfamethazine (AcSMZ) in plasma and phosphate buffer. With the proposed approach, the evaluation of pharmacokinetic of SMZ and AcSMZ and drug-metabolite protein binding interaction studies had also been carried out. The main research contents and results are as follows:Acetyl chloride reacted with SMZ in the presence of triethylamine as acid scavenger, and heavy crystallization with ethyl acetate, the main metabolite of SMZ was finally synthesized.A simple and cost effective HPLC-UV method for fast determination of (SMZ) and AcSMZ in plasma and phosphate buffer was developed. The sample treatment was performed by ultra-sound assisted extraction with lmL ethyl acetate twice, each time for5min, and the ethyl acetate phase was reduced to incipient dryness by gentle nitrogen gas. Subsequently, the residue was reconstituted in500μL acetonitrile. The determination of SMZ and AcSMZ was carried out using HPLC-PDA detector with reversed C18Phenomsil column (250mm×4.6mm,5μm) and isocratic elution with acetonitrile: water (30/70, v/v) as the mobile phase, flow speed at1mL/min, detective wavelength set at265nm, and column temperature set at℃30. The total analyzing time was less than30min. The method was validated and found to be higly selective without any other potential interference. Good linear relationship was observed within the concentration range of0.1-50μg/mL. The instrumental limits of detection (LOD) were15and20ng/mL for AcSMZ and SMZ, respectively. The instrumental limits of quantification (LOQ) were50and67ng/mL for AcSMZ and SMZ, respectively. Satisfactory recoveries of the targets (85.43-99.29%) were demonstrated at three different standard-spiked levels. The instrumental intra-day precisions were0.66-4.40%, and the instrumental inter-day precisions were1.19-9.05%. The detection of the targets was free from the matrix effects. One HPLC C18column had been used in over2000analytical cycles with matrix, and still possesses satisfactory chromatographic performance. The pharmacokinetics of SMZ and AcSMZ in the rabbit plasma were investigated. The plasma concentration of SMZ and AcSMZ was detected after the administration of SMZ to9rabbits at the dose of100mg/kg, and processed by non-compartmental statistical analysis with Drug and Statistic (DAS)3.1.4pharmacokinetic software.6out of9rabbits were fast acetytors, and the rest were slow acetylators. The mean elimination half-lives (t1/2) of AcSMZ in fast and slow acetylators were7.66±3.28h and9.68±3.96h, respectively. The mean elimination half-lives (t1/2) of SMZ in fast and slow acetylators were15.96±7.78h and16.08±7.47h, respectively. The total body clearance (CL) of AcSMZ in fast and slow acetylators were3.50±0.75mL/(min kg) and4.67±2.08mL/(min kg), respectively. The total body clearance (CL) of SMZ in fast and slow acetylators were13.25±2.43mL/(min kg) and5.00±1.00mL/(min kg), respectively. The area under the plasma contration-time curve (AUC(0→t)) of AcSMZ in fast and slow acetylators were455.66±66.28mg h L-1and388.65±137.52mg h L-1, respectively. The area under the plasma contration-time curve (AUC(0→t)) of SMZ in fast and slow acetylators were126.72±21.96mg h L-1and339.50±69.44mg h L-1, respectively. The diversity of half-lives in different animals may be attributed to species variations, age, breed of the animals and the environmental conditions in which these studies were carried out.The binding of drug with plasma protein greatly influences the distribution、 metabolism and clearance of the drug. In this article, we studied the binding properties of SMZ and AcSMZ with the plasma protein. After the equilibrium dialysis, the concentrations of plasma and dialysis samples were determined with the established method. The plasma protein binding rates did not differ significantly between fast and slow acetylators, the mean rates were88.74-92.96%for AcSMZ and64.57-68.91%for SMZ. The binding of SMZ and AcSMZ were non-concentration-dependent. AcSMZ did not compete with SMZ to bind to plasma protein. Increasing the equilibrating temperature from4℃to37℃resulted in the decrease of affinity of target compounds and plasma protein, so as to lower the plasma protein binding rates. The above research will provide reference statistics for the metabolic rule studies of SMZ.