Attenuated total reflection infrared microspectroscopy combined with multivariate analysis: a novel tool to study the presence of cocoa polyphenol metabolites in urine samples

The detection and quantification of polyphenols in biological samples is mainly performed by liquid chromatography in tandem with mass spectrometry (HPLC-MS/MS). This technique requires the use of organic solvents and needs control and maintenance of several MS/MS parameters, which makes the method expensive and time consuming. The main objective of this study was to evaluate, for the first time, the potential of using attenuated total reflection infrared microspectroscopy (ATR-IRMS) coupled with multivariate analysis to detect and quantify phenolic compounds excreted in human urine. Samples were collected from 5 healthy volunteers before and 6, 12 and 24 h after ingestion of 40 g cocoa powder with 250 mL of water or whole milk, and stored at -80 °C. Each sample was centrifuged at 5000 rpm for 10 min and at 4 °C and applied onto grids of a hydrophobic membrane. Spectra were collected in the attenuated total reflection (ATR) mode in the mid-infrared region (4000-800 cm(-1)) and were analyzed by a multivariate analysis technique, soft independent modeling of class analogy (SIMCA). Spectral models showed that IR bands responsible for chemical differences among samples were related to aromatic rings. Therefore, ATR-IRMS could be an interesting and straightforward technique for the detection of phenolic compounds excreted in urine. Moreover, it could be a valuable tool in studies aimed to identify biomarkers of consumption of polyphenol-rich diets.