Highly sensitive rapid determination of orotic acid in urine samples using a field-amplified sample stacking approach in capillary electrophoresis coupled with contactless conductivity detection

Orotic aciduria is a severe, hereditary, life-threatening condition, particularly in newborns. An increased orotic acid (OA) content in urine may be a strong indicator of this condition. In this study, we developed a rapid, simple, highly sensitive diagnostic method for use in monitoring the OA levels in urine samples, which were successfully determined using capillary electrophoresis combined with capacitively coupled contactless conductivity detection (CE-C⁴D). A straightforward analysis with an increased sensitivity towards OA and an analysis time of approximately 5 min were realized, and the limit of detection of the developed method was 0.014 mg/L in aqueous solution. The optimized composition of the separation electrolyte was 20 mM 2-(N-morpholino)ethanesulfonic acid/histidine with 0.1 mM cetyltrimethylammonium bromide at a pH of 6.5. The sensitivity of the developed method was significantly increased using a sample stacking approach with a 10% acetonitrile (v/v) plug solution. The method was validated, and satisfactory recoveries of 80.0–92.3% were obtained. The amounts of OA in five urine samples were successfully determined.