A BIOISOSTERES’ APPLICATION AS INTERNAL STANDARD FOR SIMULTANEOUS DETERMINATION OF THREE TRIAZINE ANTICOCCIDIAL CHEMICALS BY UPLC

Abstract

A new chemical entity, 2-(3- methyl-4-(4-nitrophenoxy) phenyl)-1, 2, 4-triazine-3, 5 (2H, 4H)- dione (NZL), possesses remarkable anticoccidial activity. Its nonclassical bioisosterism, whereas one of its analogues, triclabendazole sulfoxide (TCB-SO), was used as the internal standard (IS) for the determination of NZL in plasma. TCB-SO's physicochemical data were proved to be more similar to NZL than the analogues, namely diclazuril (DIC) and toltrazuril (TOL), which were commonly used in previous reports. By investigating the effect of mobile phase's pH on the retention of the four substances (i.e., NZL, DIC, TOL, and TCB-SO), simultaneous separation of these analytes could be achieved, demonstrating TCB-SO's application as IS for simultaneous quantification. The 5 mM acetic acid-ammonium acetate buffer (pH 4.5) was used as aqueous phase that successfully overcame the peak-overlapping (when ammonium acetate buffer at pH 6.5 was used) of analytes and peak-tailing (when 0.1% formic acid was used) of NZL. The low limit of quantification (LLOQ) of NZL was determined to be 0.08 µg mL−1 with a good linearity range of 0.08–50 µg mL−1, fully meeting with the requirement for pharmacokinetic determination. This method has been successfully applied to study the protein binding rate in vitro and the pharmacokinetics of broilers.