The application of activated pulsed amperometric detection (APAD) for the determination of orotic acid (OrA) in real samples at a gold working electrode in alkaline solutions, in combination with anion-exchange chromatography, is reported. Such an activated potential waveform was designed with an initial step that involves the formation of redox active species (e.g., adsorbed AuOH/AuO), which in turn is halted upon lowering the applied potential at the detection value while the adsorbed gold hydroxide/oxide species are still catalytically active. A direct comparison between the activated potential waveform and the more commonly used pulsed amperometric detection showed roughly a 20-fold increase in sensitivity. The chromatographic separation of OrA was accomplished by using a microbore anion-exchange column eluted with an isocratic mobile phase composed of 100mM NaOH + 40mM NaNO3. Orotic acid was determined at the concentration ranges of 0.2–30 uM (r = 0.9997) with an absolute detection limit of 80 pg (10L injected). The levels of OrA in cows’ milk samples evaluated by standard additions, using 5-aminoorotic acid as an internal standard, ranged from 56 to 126 mg/L. Lower levels were found in raw sheeps’ milk (<20 mg/L). The assay is shown to be very useful in clinical investigations where relatively high levels of OrA in human urine are correlated to metabolic diseases.
Pulsed Electrochemical Detection of Orotic Acid by an Activated Potential Waveform at a Gold Working Electrode Following Anion-Exchange Chromatography
NARDIELLO, DONATELLA;
2006-01-01
Abstract
The application of activated pulsed amperometric detection (APAD) for the determination of orotic acid (OrA) in real samples at a gold working electrode in alkaline solutions, in combination with anion-exchange chromatography, is reported. Such an activated potential waveform was designed with an initial step that involves the formation of redox active species (e.g., adsorbed AuOH/AuO), which in turn is halted upon lowering the applied potential at the detection value while the adsorbed gold hydroxide/oxide species are still catalytically active. A direct comparison between the activated potential waveform and the more commonly used pulsed amperometric detection showed roughly a 20-fold increase in sensitivity. The chromatographic separation of OrA was accomplished by using a microbore anion-exchange column eluted with an isocratic mobile phase composed of 100mM NaOH + 40mM NaNO3. Orotic acid was determined at the concentration ranges of 0.2–30 uM (r = 0.9997) with an absolute detection limit of 80 pg (10L injected). The levels of OrA in cows’ milk samples evaluated by standard additions, using 5-aminoorotic acid as an internal standard, ranged from 56 to 126 mg/L. Lower levels were found in raw sheeps’ milk (<20 mg/L). The assay is shown to be very useful in clinical investigations where relatively high levels of OrA in human urine are correlated to metabolic diseases.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
