Ionic liquid-based aqueous biphasic systems as one-step clean-up, microextraction and preconcentration platforms for the improved determination of salivary biomarkers

Abstract

Although saliva is a convenient human fluid for biomonitoring, current analytical set-ups for its analysis exhibit technical, sensitivity and instrumental compatibility constraints. To overcome these drawbacks, while following the Green Analytical Chemistry trends, this study is the first to propose the use of aqueous biphasic systems (ABSs) comprising the low cytotoxic butylguanidinium chloride ionic liquid (IL) and different salts as a saliva clean-up, microextraction and preconcentration tool. Among the developed and characterized ABSs, the one composed of the IL and K₂HPO₄ was selected to develop an integrated analytical procedure in saliva. Sample clean-up is achieved by removing 70% of the salivary proteins through precipitation as a solid interphase of the ABS – creating a three-phase partitioning (ABS/TPP) system – while a miniaturized extraction and preconcentration approach is simultaneously performed. The ABS/TPP, together with high-performance liquid chromatography and fluorescence detection (HPLC-FD), was optimized for bisphenols as representative biomarkers in saliva. Optimum conditions included 0.35 g of IL, 0.60 g of salt, 1.1 g of saliva, 1 min of stirring, and centrifugation. The ABS/TPP-HPLC-FD method exhibited enrichment factors up to ca. 3, extraction efficiencies higher than 80.5% despite using a miniaturized technique, and limits of detection down to 0.40 ng mL⁻¹. The inter-day precision, expressed as relative standard deviation, was lower than 9.1%, achieving average relative recoveries of 106%. The method was successfully performed when analyzing male and female saliva. The green nature of the method as compared to other state-of-the-art techniques was demonstrated using several green metrics, scoring 0.63 in AGREEprep.