Supported liquid extraction in the quantitation of plasma enterolignans using isotope dilution GC/MS with application to flaxseed consumption in healthy adults

Abstract

Dietary interventions involving foods that are enriched in lignans, such as flaxseed, are drawing attention due to their beneficial protective effects in various diseases and human conditions. Accurate quantitation of key lignan metabolites such as enterodiol (END) and enterolactone (ENL) is necessary in order to identify factors that may influence overall bioavailability. Here we describe the validation of a novel supported liquid extraction (SLE) method for isolation of plasma enterolignans, END and ENL, using 2H6-labeled isotopes with gas chromatography–mass spectrometry in micro selected ion storage (GC/MS-μSIS) mode. Following enzymatic hydrolysis and SLE extraction with 70:30 diethyl ether:ethyl acetate, enterolignans were rapidly separated within 8min. SLE in combination with GC/MS-μSIS gave high recoveries of 96.4% and 96.0% for END and ENL. Intra-assay precision ranged from 2.5 to 5.9% for both compounds whereas the inter-assay precision was 2.6–6.9%. SLE was also directly compared to liquid liquid extraction (LLE). Both techniques offered high precision and accuracy, however, SLE consistently enabled successful analyte extractions and derivatizations, unlike LLE, which had an ∼4% failure rate. SLE was also tested in a study where dietary milled flaxseed supplementation (30g/day for 1month) and enterolignan bioavailability was examined in a healthy, human population (n=10). Plasma total enterolignan levels significantly increased (P=0.002) at 4weeks relative to baseline. Average concentrations for END and ENL were 209nM and 304nM, respectively.