Ternary gradient elution markedly improves silver-ion high performance liquid chromatography of unsaturated sterols

A wide variety of unsaturated sterols can accumulate in eukaryotic cells as a consequence of normal metabolism, genetic disorders, and actions of enzyme inhibitors. Resolving these sterol mixtures into individual components by conventional chromatographic methods is inefficient because unsaturated sterols differ little in polarity, hydrophobicity, and volatility. Although sterol mixtures are well-resolved by silver-ion high performance liquid chromatography (Ag(+)-HPLC), existing methods require derivatization to acetates for best results, and the isocratic mobile phases lead to long analysis times and low sensitivity for late-eluting sterols. We show that these problems can be overcome with ternary gradient elution using acetone, hexanes, and acetonitrile. Separation of a mixture of 20 underivatized sterols gave dramatically shortened analysis times, with good peak shapes for both early- and late-eluting components. In a similar separation of blood sterols from a patient with Smith-Lemli-Opitz syndrome, the band for 7-dehydrocholesterol was much narrower than with isocratic elution. Column re-equilibration was rapid, and the separations could be monitored with ultraviolet spectroscopy at 210 nm, which affords universal, non-destructive detection of unsaturated sterols. Also discussed are retention mechanisms and reproducibility of Ag(+)-HPLC separations. The overall results represent a major advance in chromatographic methods for resolving mixtures of unsaturated sterols differing in the number and position of olefinic bonds.