The electroanalysis of mannitol, xylose and lactulose at copper electrodes: voltammetric studies and bioanalysis in human urine by means of HPLC with electrochemical detection.

The electrochemical behavior of mannitol, xylose and lactulose has been investigated at a copper working electrode. A sensitive, accurate and precise method employing HPLC with electrochemical detection in the d.c. amperometric mode, has been developed and validated for the determination of mannitol and lactulose in human urine. The ratio of these probe carbohydrates is altered in conditions that cause damage to the intestinal mucosal barrier. Systematic studies employing cyclic voltammetry indicate that the electrode reaction involves an electrocatalytic oxidation of each carbohydrate in a process yielding a single irreversible anodic wave that is dependent on the ionic strength of the sodium hydroxide supporting electrolyte solution. High performance liquid chromatography with electrochemical detection was performed using a thin-layer cell housing a custom manufactured copper working electrode. The optimized HPLC method can detect 72, 57 and 419 pg of mannitol, xylose and lactulose injected on column, respectively. The corresponding linear calibration ranges are 359 pg-2.24 microgram, 57.4 pg-896 ng and 419 pg-262 ng, respectively. Solid-phase extraction of human urine on polar sorbents, and direct injection after simple 1 + 99 dilution in 0.025 M NaOH were compared for bioanalysis. Direct injection was selected for further method developed as the technique proved robust and simple. The optimized method was validated for the determination of mannitol and lactulose in human urine over the concentration ranges predicted when assessing intestinal permeability (0.25-2.5 mg ml-1 mannitol and 0.05-1.0 mg ml-1 lactulose). Over these ranges intra- and inter-assay bias is < +/- 6.5%, and imprecision (coefficient of variation) is < 9% for each carbohydrate. The validated method provides a useful alternative to HPLC with pulsed-amperometric detection at gold electrodes.