Simultaneous HPLC determination with light-scattering detection of lactulose and mannitol in studies of intestinal permeability in pediatrics

Abstract

We describe a new HPLC method for the simultaneous determination of lactulose and mannitol in urine, in which cation-exchange chromatography and evaporative light-scattering detection are used. The two sugars are orally administered for the estimation of intestinal permeability in children. Samples were purified by solid phase extraction on a C18 cartridge and subsequent addition of anion-exchange resin. Cellobiose may be used as an internal standard. The chromatographic separation was carried out in 16 min at a flow rate of 0.5 mL/min, using deionized water as the mobile phase. Within-run precision (CV) measured at three concentrations was 1.6-2.3% for lactulose and 1.0-1.9% for mannitol. Between-run CVs were 2.1-4.1% and 1.3-2.7% for lactulose and mannitol, respectively. Analytical recovery of both sugar probes was 97-101%. The detection limits (signal-to-noise ratio = 3) were 0.82 mg/L for lactulose and 0.65 mg/L for mannitol. The lactulose/ mannitol ratio in control subjects was 0.024 +/- 0.006; in patients with Crohn's and coeliac diseases in active phase, the ratios were 0.200 +/- 0.082 and 0.072 +/- 0.025, respectively. The method is rapid, simple, and sensitive, and suitable for determination of intestinal permeability in children.