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. 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The research ethics committee of the Federal University of Ceara, in accordance with resolutions 19/96 and complementary ones, approved at the meeting of April 4, 2013, amendment and protocol summer 1 of 03/08/201, and free and informed consent form 2 of 03/08/2013 referring to study 238/05 entitled "Intervention with micronutrients and long-term impact in Brazil". I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript