Sterols, and specifically oxysterols, play important roles in the biosynthesis of bile acids and steroid hormones as well as possessing biological activities in their own right. Analysis of oxysterols is complicated due to their low abundance in biological systems and poor ionisation characteristics in mass spectrometry. Over the past decade, we have developed a liquid chromatography-mass spectrometry method termed enzyme-assisted derivatisation for sterol analysis (EADSA). Our derivatisation procedure relies on two solid-phase extraction steps to (i) separate cholesterol from oxysterols and (ii) remove excess derivatisation reagents. Recent inter-batch variation in C18 reversed-phase cartridges has led us to experiment with alternative columns. Here, we present our findings and report an improved sample preparation procedure using polymeric hydrophilic-lipophilic balanced reversed-phase cartridges.