Synthesis and Application of a Nitrobenzeneboronic Acid-substituated Silica for Affinity Chromatography

A boronic acid-substituted silica was synthesized using γ-glycidoxypropyltrimethoxysilane as the spacer arm and 3-amino-4-nitrobenzeneboronic acid as the ligand. The 3-amino-4-nitrobenzeneboronic acid was modified from m-aminobenzeneboronic acid. Then, boronic acid-substituted silica was packed in a stainless-steel column (35 mm × 4.6 mm I.D.) under a pressure of 20.7 MPa as an affinity pre-column. A coupled-column liquid chromatographic method for the direct analysis of 11 urinary nucleosides is described. Efficient on-line clean-up and concentration of 11 nucleosides from urine samples were obtained using the affinity pre-column as the first column (extraction column) and an ODS column (250 mm × 4.6 mm I.D.) as the second column (analytical column). The mobile phases applied consisted of 0.25 M ammonium acetate (pH 7.95) on the extraction column, and 25 mM potassium dihydrogen phosphate (pH 4.5) on the analytical column, respectively. Determination of urinary nucleosides was performed on the analytical column using a linear gradient elution comprising 25 mM potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Calibration plots of 11 standard nucleosides showed good RSD, good recovery, and excellent linearity (r > 0.995).