Stacking and separation of urinary porphyrins in capillary electrophoresis: optimization of concentration efficiency...

We demonstrated that anionic porphyrins could be stacked and separated in micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) by applying acetonitrile and high salt content in human urine sample matrix. The introduction of sample containing acetonitrile and sodium chloride into the CE capillary at more than 10% of the total capillary volume resulted in the improvement of peak resolution and the enhancement of detection sensitivity. The achieved acetonitrile stacking enrichment factors of six porphyrins ranged from 12 to 32 in MEKC and from 28 to 33 in MEEKC, respectively. The stacking technique was successfully applied for analyzing porphyrins present in urine samples that were deproteinized with acetonitrile. For the analysis of coproporphyrin isomers, addition of the sodium cholate (SC) into micelle and microemulsion solutions provided adequate resolution. Calibration curves obtained for the determination of coproporphyrin isomers were found linear between 30 and 400 nmol L(-1), and the limit of detection (LOD) was 20 nmol L(-1) in MEEKC. Intra- and interday precisions (n=11) in the microemulsion separation system for the isomers at spiked concentrations of 40-400 nmol L(-1) in urine were in the range of 0.1-0.4% and 0.7-7.6% for migration time and peak area, respectively. Coproporphyrin III, coproporphyrin I and uroporphyrin were detected at levels of 80.7 nmol L(-1), 32.3 nmol L(-1) and 19.8 nmol L(-1), respectively, in the urine samples collected from healthy individuals. Different porphyrin profiles, however, were observed in urine samples from porphyria cutanea tarda (PCT) patients.