Simultaneous determination of hydroxycinnamates and catechins in human urine samples by column switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry

A quantitative liquid chromatography mass spectrometry (LC-MS) methodology with online sample clean up by column switching is described for the simultaneous determination of the hydroxycinnamates, caffeic acid and chlorogenic acid, and of the catechins, epicatechin and catechin in human urine samples. Enzymatically treated urine samples were directly injected onto the LC-MS system, where sample clean up was performed by a reversed-phase Zorbax 300SB C(3) column and selective elution of the target compounds onto a Zorbax SB C(18) column resulted in final separation prior to detection by atmospheric pressure chemical ionization (APCI) MS using single ion monitoring (SIM) in negative mode. Linear calibration graphs were achieved in the dynamic range of 10-1000 ng/ml urine. The inter- and intraassay coefficients of variation (C.V.%) for the analysis of the four compounds in quality control urine samples were between 7.8 and 10.9, n=17 (reproducibility), and the repeatability of the assay was between 2.5 and 5.0% (n=12). Analyses of urine samples from a human dietary intervention study with intake of 600 g of fruits and vegetables were demonstrated. To our knowledge, this is the first method described that allows simultaneous determination of both hydroxycinnamates and catechins in biological samples.