Direct screening of lyophilised biological fluids for bile acids using an evaporative light scattering detector

Abstract

The usefulness of lyophilisation for the direct screening of biological fluids for bile acids was investigated. Human serum and urine were lyophilised without losses of the target compounds and further extracted with n-hexane in acidic medium under magnetic stirring. An integrated flow injection-liquid chromatographic system coupled to an evaporative light scattering detector (ELSD) was used for automated screening/confirmation. The continuous module allows sequential filtration of the organic phase, solvent changeover and solid-phase extraction for clean-up and preconcentration purposes. Retained bile acids were eluted with an acetonitrile–methanol (65:35, v/v) stream. For screening purposes, the effluent was directly introduced in the ELSD detector and the total bile acid content of the sample determined. For confirmatory analysis, another aliquot of the sample was processed in the screening module but the effluent was directed to the chromatographic columns, which provided the free bile acid profile of the sample. Fasting serum and urine samples obtained from healthy individuals were lyophilised and processed. Good agreement was obtained in the analysis of the two matrices assayed following the screening and confirmatory methods.