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.

Materials 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). 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 ng/mL, 500 ng/mL and 1000 ng/mL) and in triplicate, which showed recovery values above the recommended (>120%). Intermediate precision was determined at 24 hours 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).

Conclusion: 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.

Keywords: Liquid chromatography coupled with tandem mass spectrum; Lactulose; Mannitol test validation; Intestinal epithelium barrier function; Children malnutrition; Molecular biomarkers