Large-volume sample stacking-capillary electrophoresis used for the determination of 3-nitrotyrosine in rat urine

Large-volume sample stacking using the electroosmotic flow (EOF) pump technique has been investigated for the quantification of 3-nitrotyrosine in urine of diabetic rats. The best separation conditions for these highly complex samples were obtained using capillary electrophoresis (CE) in the reversed polarity mode (i.e., injecting at the cathode and detecting at the anode) using cetyltrimethylammonium bromide (CTAB) in the running buffer. The optimum CE separation conditions were achieved using a phosphate buffer prepared with 0.15 M phosphoric acid and 0.5 mM CTAB adjusted to pH 6.4 with sodium hydroxide. In such CE conditions, the limit of detection (LOD) was 1.77 μM for 3-nitrotyrosine with normal injection mode, meanwhile with the large-volume sample stacking technique a more than 20-fold improvement was observed (i.e., LOD = 0.08 μM was obtained) without noticeable loss of resolution. This value allowed the detection of 3-nitrotyrosine in urine from diabetic rats. To our knowledge, this work is one of the few applications showing the great possibilities of these stacking procedures to analyse biological samples by CE.