Validation of UPLC-MS/MS Method for Determination of Urinary Lactulose/Mannitol

Abstract

Determination of urinary lactulose/mannitol is one of the most used tests to evaluate intestinal barrier function. High-performance liquid chromatography (HPLC) separation with electrospray ionization tandem mass spectrometry guarantees high levels of selectivity and reproducibility. In this paper we report an upgrade of the previous published liquid chromatography tandem mass spectrometry method, introducing more reliable internal standards and ultra-performance liquid chromatography with ethylene bridged hybrid amide columns. The ultra-performance liquid chromatography provided an efficient chromatographic separation of the two sugars in 5 min, compared to 15 min using the previous method. The limit of quantification was 10 µg/mL for mannitol and 2.5 µg/mL for lactulose, and the assay was linear up to 1000 µg/mL for mannitol and 1000 µg/mL for lactulose. The within-run precision and accuracy ranged from 0.7 to 2.9% and 97.2 to 101.2%, respectively. The between-run precision and accuracy ranged from 1.9 to 4.7% and 94.8 to 97.5%, respectively. Recovery was higher than 90.2% for both lactulose and mannitol, and the matrix effect for both lactulose and mannitol was lower than 15%. With this new method we have a real improvement in terms of accuracy and reproducibility, ensuring results in shorter time. The changes to the previous protocol make this method excellent for routine purposes.

Keywords: L/M; UPLC-MS/MS; intestinal permeability.

Figure 1

Selected reaction monitoring (SRM) chromatograms for lactulose, mannitol and their internal standards lactulose ¹³C₁₂ and d-mannitol-1 ¹³C,1-1-d_2.