Determination of salivary cotinine through solid phase extraction using a bead-injection lab-on-valve approach hyphenated to hydrophilic interaction liquid chromatography

Cotinine, the first metabolite of nicotine, is often used as a biomarker in the monitoring of environmental tobacco smoke (ETS) exposure due to its long half-life. This paper reports on the development of an at-line automatic micro-solid phase extraction (μSPE) method for the determination of salivary cotinine followed by its analysis via hydrophilic interaction liquid chromatography (HILIC). The SPE methodology is based on the bead injection (BI) concept in a mesofluidic lab-on-valve (LOV) flow system to automatically perform all SPE steps. Three commercially available reversed-phase sorbents were tested, namely, Oasis HLB, Lichrolut EN and Focus, and the spherically shaped sorbents (i.e., Oasis HLB and Focus) provided better packing within the SPE column and hence higher column efficiency. An HILIC column was chosen based on its potential for achieving higher sensitivity and better retention of polar compounds such as cotinine. The method uses an isocratic program with acetonitrile:100 mM ammonium acetate buffer, pH 5.8 in 95:5 v/v ratio as the mobile phase at a flow rate of 1.0 mL min−1. Using this approach, the linear calibration range was from 10 to 1000 ng which corresponded to 5-500 μg L−1. The corresponding μSPE-BI-LOV system was proven to be reliable in the handing and analysis of viscous biological samples such as saliva, achieving a sampling rate of 6 h−1 and a limit of detection and quantification of 1.5 and 3 μg L−1, respectively.