Ultrasound-Assisted Extraction Combined with HPLC-UV for Fast Determination of Sulfamethazine and Its N4-Acetyl Metabolite in Plasma and Phosphate Buffer

Abstract

A simple and cost effective HPLC-UV method for fast determination of sulfamethazine (SMZ) and its N₄-acetyl metabolite, N₄-acetyl sulfamethazine (AcSMZ), in plasma and phosphate buffer was developed and validated. The sample treatment was performed by ultrasound-assisted extraction with 1 mL ethyl acetate twice, each time 5 min. Moreover, there was no need for centrifugation and further clean-up, which shortened the measurement time. The total analysis time was less than 30 min. The method proved to be a selective and accurate tool for detection of SMZ and AcSMZ in plasma and phosphate buffer with satisfactory recoveries (85.43–99.29%) and little matrix effects (0.86–0.99). The proposed approach was also applied to the evaluation of pharmacokinetic and drug-metabolite protein binding interaction studies. The results show that SMZ and AcSMZ are non-concentration-dependent, and the metabolite does not bind competitively with the drug to the plasma protein.