Photophysics of faecal pigments stercobilin and urobilin in aliphatic alcohols: introduction of a sensitive method for their detection using solvent phase extraction and fluorometry

Faecal pigments (FPs) are ubiquitous in the environment and are a primary contaminant in groundwater and surface water. This article presents a new analytical paradigm by a fluorescence coupled extraction-based method involving FP fluorescence enhancement and minimization of background fluorescence for high sensitivity detection. FPs show higher fluorescence intensity in aliphatic alcohols due to the breaking down of higher-order H-aggregates into lower-order H-aggregates (dimers). DFT studies using the B3LYP functional and LANL2DZ basis set show pi-pi stacking and hydrogen-bonding contributions towards forming H-aggregated dimers of FPs in the implicit and explicit solvent environments of 1-hexanol. This study is the first report on the extractability of FPs using 1-hexanol as an efficient extraction medium in comparison to higher-order aliphatic alcohols (1-butanol, 1-hexanol and 1-octanol). Furthermore, FP-Zn(ii) complexes in 1-hexanol medium significantly enhance the fluorescence emission intensity (similar to 14-17 times), and the emission intensity remains stable over time. This further helps to increase the detection limit of FPs in the picomolar to sub-picomolar concentration range. This study proposes a protocol involving extraction of FPs by 1-hexanol followed by the complexation of FPs with Zn(ii) in the alcohol media and subsequent fluorimetric detection of the FP-Zn(ii) complex with a high level of sensitivity, enabled by reduced interference from the background fluorescence of humic acid. The complexation behaviour of FPs with various metal salts was also examined, which provided an understanding of the fluorescence behaviour of FPs with various other metal ions commonly present in natural environmental water. The proposed analytical method has been further validated using real water samples.

Automated summary

This study introduces a new analytical paradigm for high sensitivity detection of fecal pigments (FPs) by fluorescence enhancement and background fluorescence minimization. The use of 1-hexanol as an efficient extraction medium for FPs is proposed, along with the complexation of FP-Zn(ii) in alcohol media to enhance fluorescence emission intensity and reduce interference, enabling the detection of FPs at picomolar to sub-picomolar concentrations. The study also examines the complexation behavior of FPs with various metal salts and validates the proposed analytical method using real water samples.