Simultaneous high-performace liquid chromatographic determination of sulfamonomethoxine and its hydroxy/N4-acetyl metabolites following centrifugal ultra-filtration in animal blood plasma

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of sulfamonomethoxine (N-1-(6-methoxy-4-pyrimidinyl) sulfanilamide, SMM) and its hydroxy metabolites, i.e., N-1-(6-methoxy-2-hydroxy-4-pyrimidinyl) sulfanilamide (2-OH-SMM), N-1-(6-hydroxy-4-pyrimidinyl) sulfanilamide (6-OH-SMM), N-1-(2,6-dihydroxy-4-pyrimidinyl) sulfanilamide (2,6-diOH-SMM), acid N-4-acetyl SMM (N-4-AcSMM) in the blood plasma of food-producing animals. Sample preparation was performed solely with an Ultrafree(TM) C3 as a centrifugal ultra-filtration unit. A Mightysil RP-4 GP column and an isocratic mobile phase of 4% (v/v) acetic acid solution - acetonitrile - N,N-dimethylformamide (86:10:4, v/v/v) were used with a photo-diode array detector Average recoveries of spiked SMM, three OH-SMMs and N-4-AcSMM (0.1, 0.5 and 2.0 mug mL(-1), respectively), were >93.2%, with coefficients of variation between 0.4 and 4.4%. The limit of detection (LOD) and limit of quantitation (LOQ) were less than or equal to 0.043 mug mL(-1) and less than or equal to 0.080 mug mL(-1), respectively The total time required for the analysis of one sample was less than 20 min.