Development of a liquid chromatography-tandem mass spectometry (LC-MS/MS) method to evaluate intestinal permeability in clinical practice

The intestinal permeability test is a non-invasive method for assessing gastrointestinal mucosal integrity. The lactulose/mannitol ratio is used to assess the intestinal barrier function. Aim of this work was to develop a rapid and robust method for the simultaneous analysis of lactulose and mannitol in urine by LC-MS/MS. Calibration curves were prepared in water: acetonitrile 20:80. Raffinose was used as internal standard at 25 mg/L in water: acetonitrile 20: 80. 50 mu L of urine sample was added to 450 mu L of internal standard solution, and then vortexed and centrifuged. A 200 mu L aliquot was transferred into a vial and placed in the autosampler for LC-MS/MS analysis. The chromatographic separation was performed using a Phenomenex Luna NH2 column operating at a flow rate of 200 mu L/min and eluted with a linear gradient from 20 to 80 water in acetonitrile. The total run time was 16 min. The MS operates in electrospray negative mode. The method was validated according to European Medicine Agency guidelines. Linearity ranged from 10 to 800 mg/L for mannitol and from 2.5 to 400 mg/L for lactulose, respectively. Inter-assay imprecision (expressed as CV) was <13% for both analytes. Recovery was >85%.