Fully automated determination of sulfamethazine in ovine plasma using solid-phase extraction on disposable cartridges and liquid chromatography

Abstract

An automatic sample preparation procedure followed by on-line injection of the sample extract into a HPLC system has been developed for the quantitative analysis of sulfamethazine and its N 4-acetyl metabolite in ovine plasma. The sample clean-up was performed by solid-phase extraction (SPE) on C 18 disposable extraction cartridges (DECs). All the sample handling operations were effected by a robotic auto-sampler. The DEC was first conditioned with methanol and phosphate buffer pH 7.4. After loading 1.0 ml of plasma sample onto the DEC, the latter was washed with the same buffer. The elution step was performed with methanol (0.25 ml) and the eluate was then diluted by adding 0.75 ml volume of phosphate buffer pH 6.4. A 20-μl volume of the resultant solution was injected onto an octadecyl silica column preceded by a short guard column. The HPLC mobile phase was methanol—phosphate buffer pH 6.4 (25:75, v/v). Sulfamethazine and N 4-acetylsulfamethazine were determined photometrically at 262 nm. Under these conditions, linear calibration curves ranging from 2 to 250 μg ml −1 have been obtained for both compounds. Drug recoveries were higher than 90% and typical relative standard deviation values were 0.7% (within-day) and 2.0% (between-day) at a plasma concentration of 50 μg ml −1.