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

Lactulose and mannitol have been used to assess intestinal permeability and several methodologies have been used. ObjectivesThis study aimed to validate the high-performance liquid chromatography method coupled with tandem mass spectrometry to measure mannitol and lactulose sugars. Material and MethodsWe 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(R) 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. ResultsThe 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). ConclusionsThe 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.