Measuring lactulose and mannitol levels using liquid chromatography coupled with tandem mass spectrum: application to clinical study of intestinal epithelium barrier function

Abstract

Lactulose and mannitol have been used to assess intestinal permeability and several methodologies have been used. Objectives: This study aimed to validate the high-performance liquid chromatography method coupled with tandem mass spectrometry to measure mannitol and lactulose sugars. Material and Methods: We used a high-performance liquid chromatography (HPLC) system coupled to an ABsciex Q-TRAP 5500 triple quadrupole mass spectrometer (MS/MS) with an ABSciex Electro Nebulization Interface (ESI) (Framingham, MA, USA). For the separation of lactulose and mannitol compounds in the HLPC, the analytical column HILIC-ZIC from ES Industries (West Berlin, USA) was used. The parameters analyzed for analytical validation were specificity/selectivity, linearity, LD, LQ accuracy, precision (repeatability and intermediate precision) and matrix effect. Results: The accuracy was demonstrated from the recovery at three concentration levels (100, 500 and 1000 ng/mL) and in triplicate, which showed recovery values above the recommended (>120%). Intermediate precision was determined at 24-hour intervals and the coefficients of variation found were less than 8.7%. The matrix effect was measured through the retention times in the standard samples and in the samples of the spiked standards in dilutions with urine samples, which varied between 99.3% and 100.3%. Urine samples from malnourished and healthy children were analyzed. The L:M ratio was considerably lower in the control group compared to the MN group (p<0.0001) and the mannitol excretion rate was higher (p<0.0001). Conclusions: The results showed that the HPLC-MS/MS method was sensitive, specific, and accurate for the determination of molecular biomarkers of lactulose and mannitol. In addition, the L:M test is a functional test capable of determining with high sensitivity the barrier function damage of the intestinal epithelium in children with malnutrition compared to health control children.