Fig. 1. Molecular structures of orotic acid and 5-aminoorotic acid.

Fig. 2. Current–voltage curves at a gold rotating disk electrode in 0.10 M NaOH in the presence (solid line) and absence (dashed line) of orotic acid. Residual and sample voltammograms collected in the absence of dissolved O₂. Conditions: scan rate, 100 mV/s; rotation speed, 1000 rpm. Concentration of orotic acid: 1 mM. Arrows denote scan direction.

Fig. 3. Representative flow-injection peaks for increasing concentrations of OrA in 100 mM NaOH at a gold working electrode by applying the APAD waveform detailed in Table 1. Carrier flow rate: 0.3 mL/min; μM OrA concentrations: (a) 2; (b) 10; (c) 20; (d) 30; (e) 40; (f) 60; (g) 80 and (h) 150.

Fig. 4. Comparison of three pulsed potential waveforms: (A) APAD, (B) six-potential IPAD waveform [39] and (C) classical three-step PAD [40] upon anion-exchange chromatographic separation and electrochemical detection of a mixed standard solution (20 μM) containing OrA (1) and 5-aminoorotic (2). Separation accomplished by CarboPac PA1 column; eluent: 100 mM NaOH + 40 mM NaNO₃ at a flow rate of 0.25 mL/min. Volume injected: 10 μL.

Fig. 5. Separations under AEC of a partially skimmed UHT milk sample, diluted 100-fold with water before injection. Internal standard (IS) 5 μM 5-aminoorotic acid. (A) APAD and (B) six-potential IPAD waveform. Other experimental conditions as in Fig. 4.

Fig. 6. AEC–APAD separations of a whole UHT milk sample, diluted 1:40 with water. IS 15 μM 5-aminoorotic acid. The optimised APAD waveform of Table 1 was applied at a gold working electrode. Other conditions as in Fig. 4.

Fig. 7. Anion-exchange chromatographic separations of an healthy-man urine sample. Comparison of two pulsed potential waveforms: (A) APAD and (B) PAD. Eluent, 100 mM NaOH + 40 mM NaNO₃ at a flow rate of 0.20 mL/min. Volume injected, 10 μL.

Potential-time settings of the optimized APAD waveform for orotic acid determination

Peak area ratios (±SD, n = 4) of orotic and 5-amino-orotic acids evaluated in AEC with EC detection by applying three optimized potential waveforms: APAD, six-step potential IPAD and three-step potential PAD^a

Linear calibration parameters (y^a = m × x^b + b) of orotic acid evaluated by AEC–APAD^c

Orotic acid content evaluated in milk samples by AEC–APAD^a

Occurrence of orotic acid (mean value ± SD) in human urine evaluated by AEC–APAD^a