New method of aqueous two phase with solid phase extraction (ATP-SPE) for detection of sulfonamides

For aqueous two-phase extraction (ATPE), adsorbents such as primary secondary amine (PSA) are usually used to remove fat and protein in meat samples. However, it was determined that PSA adsorbed approximately 5% of measured sulfonamides (SAs), which is the reason for the low recovery rate. To overcome this adverse PSA effect, a solid phase extraction (SPE) method was used for purification and enrichment after the formation of aqueous two-phase. Based on these, A new aqueous two-phase sample pretreatment method with solid phase extraction (ATP-SPE) to detect sulfonamides in fish meat is proposed. Fluorescence quenching showed that for 6 SAs, hydrogen bonding and van der Waals force played the major role in the binding reaction with fish serum albumin protein. In addition, in the 80% ACN aqueous solution, a slow protein denaturation took place and the drugs bound with the protein were released, which is more conducive to the aqueous two-phase extraction. In order to obtain better separation effect, the factors for synthesis of hydrophobic-lyophobic balance (HLB) packing for SPE were explored. Stirring speed is the most important factor affecting the particle size of microspheres. After purification and enrichment by SPE, samples were directly analyzed by HPLC. The analytical process was validated in the fish sample matrix by the analysis of spiked blank samples. The mean recoveries of this method were 80.1-95.1% with RSD values of 1.37-3.95%. The detection limits were 5.8-11.7 ng/g. The new ATP-SPE pretreatment method provides a new way for better recovery of veterinary drugs in meat products.