Validation of an LC-MS/MS Method for Urinary Lactulose and Mannitol Quantification: Results in Patients with Irritable Bowel Syndrome

Abstract

<i>Aim</i>. Lactulose/mannitol ratio is used to assess intestinal barrier function. Aim of this work was to develop a robust and rapid method for the analysis of lactulose and mannitol in urine by liquid chromatography coupled to tandem mass spectrometry. Lactulose/mannitol ratio has been measured in pediatric patients suffering from irritable bowel syndrome. <i>Methods</i>. Calibration curves and raffinose, used as internal standard, were prepared in water : acetonitrile 20 : 80. Fifty <i>μ</i>L of urine sample was added to 450 <i>μ</i>L of internal standard solution. The chromatographic separation was performed using a Luna NH₂ column operating at a flow rate of 200 <i>μ</i>L/min and eluted with a linear gradient from 20% to 80% water in acetonitrile. Total run time is 9 minutes. The mass spectrometry operates in electrospray negative mode. Method was fully validated according to European Medicine Agency guidelines. <i>Results and Conclusions</i>. Linearity ranged from 10 to 1000 mg/L for mannitol and 2.5 to 1000 mg/L for lactulose. Imprecision in intra- and interassay was lower than 15% for both analytes. Accuracy was higher than 85%. Lactulose/mannitol ratio in pediatric patients is significantly higher than that measured in controls. The presented method, rapid and sensitive, is suitable in a clinical laboratory.