Very few relevant methods have been described for the detection and quantitation of phenolic compounds in faecal matrix. Extraction with conventional organic
solvents such as
chloroform/
methanol (2:1, Folch
reagent),
methanol and
ethanol (72%) showed high extraction efficiency for
lipids and also gave good recovery of the major phenolic compounds present in the matrix. However, in comparison with a newly developed
phosphate buffer method, the yield of minor phenolics was negligible when detected by these conventional methods. Conventional methods also lead to contamination of the ion source of the mass spectrometer and rapid deterioration of column performance mostly due to the high concentration of
lipids. However, if the faecal matrix is initially extracted with
phosphate buffer, and the extract acidified and re-extracted with
diethyl ether, the range and yield of phenolic compounds are enhanced and the problem of
lipid contamination is substantially alleviated. Following pilot studies and optimisation of the procedure, individual phenolic compounds (n = 29) were identified by nano-electrospray ionisation mass spectrometry (nano-ESI-MS), nano-ESI-tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/EI-MS) and quantitated (n = 27) by GC/MS in subsets (n = 5) of faecal samples, collected during the European Agency for
Cancer Prevention
calcium/fibre intervention study from four European countries (Italy, Germany, Spain and Denmark). A range of phenolic compounds (mainly
acids) was detected, dominated by phenylacetic, benzoic, phenylpropionic and m-hydroxyphenylpropionic
acids, representing on average 9.91 (93%), 8.25 (92%), 9.45 (95%) and 11.05 (98%) mM in the Italian, German, Spanish and Danish samples, respectively. The new method should enable large epidemiologic, case-control and intervention studies on the relevance of phenolic
antioxidants in the aetiology of
colorectal cancer to be conducted in the future.