Development of a generic method to the solid-phase extraction of acidic compounds from complex matrices

A mixed-mode solid-phase extraction procedure was developed for the isolation and purification of acidic compounds from complex biological matrices. Urine samples were spiked with several acidic drugs and diluted in ammonium acetate buffer. Isolute HAX columns (a mixed-mode phase consisting of both hydrophobic and ion-exchange ligands) were conditioned with methanol and ammonium acetate prior to sample loading. Once the samples were loaded, the cartridges were rinsed sequentially with ammonium acetate and a 50:50 ratio of methanol and deionized water. The analytes were eluted with an 80:20 ratio of methanol and acetic acid. The eluates were evaporated to dryness and reconstituted to a final volume with a 98:2:0.1 ratio of deionized water, acetonitrile, and trifluoroacetic acid. Samples were analyzed by high-performance liquid chromatography. The absolute recoveries for most of the tested acidic drugs exceeded 80% at an original concentration of 1 microg/mL. Hydrophobic and ion-exchange sorbents were also investigated separately; however, the retention of the analytes suffered during sample application as well as the purity of the eluted extract. Results on anion-exchange columns show that the correct choice of counter-ion is extremely important to the retention of acidic analytes. The highest absolute recoveries were obtained when acetate was the counter-anion on the ion-exchange sorbent.