Separation of 9 Sulfonamide Drugs in aparts per thousand4 Min by Ultra-High Performance Supercritical Fluid Chromatography (UHPSFC): with a Feasibility Study for Detection in Milk

Nine widely used veterinary sulfonamide drugs were baseline separated (R (s) a parts per thousand yen1.5) in just over 4 min using a 3 x 100 mm, 1.8 mu m RX-Sil column, with 9.2 % methanol in carbon dioxide, at 110 bar and 30 A degrees C, with direct UV detection at 260 nm using a 3 mm, 2 mu L tapered flow cell. Pressure drop was only 172 bar. Optimization was difficult due to the similarity in structures. Small changes in modifier concentration, temperature and pressure, each tended to improve the resolution of some peak pairs but degraded the resolution of others. There were four critical pairs, each responding differently to changes in conditions. Optimization was performed by plotting resolution between pairs as a function of modifier concentration first, temperature second, and outlet pressure third. Retention time was then minimized by changing flow rate. The estimated limit of quantitation (LOQ, S/N > 10), for direct injections, was a parts per thousand 200-400 ng/g of each, inadequate for regulatory requirements. Solid phase extraction (SPE) attempted to pre-concentrate samples spiked with sulfamethazine by a parts per thousand 20:1. From water, the limit of detection (LOD) was a parts per thousand 2.7 ng/mL with LOQ a parts per thousand 9 ng/mL using UV at 260 nm. The LOD for milk was 6.2 ng/mL, and LOQ was 20.1 ng/mL. A better pre-concentration step or a more sensitive detector such as MS-MS is required. Even with these inadequacies, SFC was shown to be a feasible, faster, "greener" alternative to HPLC for the separation of these drugs.