Use of guanidine hydrochloride and ammonium sulfate in comprehensive in-line sorption enrichment of xenobiotics in biological fluids by high-performance liquid chromatography

A novel approach has been developed for direct injection of physiological fluids on an in-line extraction pre-column followed by column switching in order to introduce the adsorbed xenobiotic onto the analytical column. The physiological fluid is pre-treated with guanidinium solution in water (200 μl of fluid plus 300 μl of a reagent containing 8.05 M guanidinium and 1.02 M ammonium sulfate) in order to denature protein binding sites and to serve as a universal solvent for a divergent range of polar to non-polar xenobiotics in a hydrophilic medium. A 0.5 M ammonium sulfate solution (500 μl) is used as a pre- and post-flush reagent for the extraction pre-column (30 mm × 2.1 mm I.D.). The pre-flush reagent prepares the sorbent environment of the C₁₈ pre-column for the hydrophobic retention of analytes. The post-flush reagent flushes non-retained sample proteins and salts to waste prior to switching the pre-column in-line with the analytical column. Universal chromatographic conditions for the analytical phase allows elution of a range of polar to non-polar xenobiotics within 20 min from an end-capped C₈ silica anaytical column (250 mm × 4.6 mm I.D.). This is effected by a linear gradient from a binary system consisting of solvent A (0.05 M KH₂PO₄) and solvent B (acetonitrile—isopropanol, 80:20, v/v).