Assay of neopterin in serum by means of two-dimensional high-performance liquid chromatography with automated column switching using three retention mechanisms

An automated two-dimensional HPLC method for the determination of neopterin in serum is described. Neopterin is separated from proteins on a short octadecylsilica column by size exllusion and from the majority of the other serum components by adsorption. The fraction containing neopterin is transferred by column switching to a solvent-generated cation-exchange column using dodecylsulfonic acid as surface activator. Parameters influencing the separation performance and senstivity of the fluorescence detection are discussed. The efficiency of the cleaning of the first column was optimized. The method was validated. It achieves a precision of 1% (R.S.D.) and a detection limit of about 0.3 nmol/1. The accuracy is nearly 100%. The method allow a high sample throughput, requiring 15 min per sample.