Hubert P, Chiap P, Evrard B, Delattre L, Crommen J
Laboratory of Drug Analysis, University of Liège, Belgium.
J Chromatogr. 1993 Dec 8;622(1):53-60. doi: 10.1016/0378-4347(93)80249-4.
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 N4-acetyl metabolite in ovine plasma. The sample clean-up was performed by solid-phase extraction (SPE) on C18 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-microliters 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 N4-acetylsulfamethazine were determined photometrically at 262 nm. Under these conditions, linear calibration curves ranging from 2 to 250 micrograms 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 micrograms ml-1.
已开发出一种自动样品制备程序，随后将样品提取物在线注入高效液相色谱（HPLC）系统，用于定量分析绵羊血浆中的磺胺二甲嘧啶及其N4-乙酰代谢物。样品净化通过在C18一次性萃取柱（DEC）上进行固相萃取（SPE）来完成。所有样品处理操作均由机器人自动进样器完成。首先用甲醇和pH 7.4的磷酸盐缓冲液对DEC进行预处理。将1.0 ml血浆样品加载到DEC上后，用相同缓冲液冲洗DEC。用甲醇（0.25 ml）进行洗脱步骤，然后通过加入0.75 ml体积的pH 6.4磷酸盐缓冲液稀释洗脱液。将20微升所得溶液注入到一根短保护柱后的十八烷基硅胶柱上。HPLC流动相为甲醇-pH 6.4的磷酸盐缓冲液（25:75，v/v）。磺胺二甲嘧啶和N4-乙酰磺胺二甲嘧啶在262 nm处进行光度测定。在这些条件下，两种化合物均获得了2至250微克/毫升的线性校准曲线。在血浆浓度为50微克/毫升时，药物回收率高于90%，典型的相对标准偏差值为日内0.7%和日间2.0%。
