Development and Validation of a Quantification Method of Cotinine in Urine Using Two Innovative Technologies: Supported Liquid Extraction and QDa Detection

Objective: Cotinine is the best biomarker of tobacco smoke exposure because of its long half-life. Several methods are developed to quantify cotinine in biological fluids, including high performance liquid chromatography. This method requires an extraction step in order to clean-up the sample and concentrate the analyte of interest. The aim of this study was to optimise the extraction step and to develop an easy, reproducible and specific method to measure cotinine in urine. Methods: An extraction of neutrals was chosen and performed by Supported Liquid Extraction (SLE). SLE consists in liquidliquid extraction in the presence of a sorbent enabling efficient extraction with less organic solvent and without any emulsion formation. Urine was basified by treatment with NH4OH in order to neutralise cotinine before loading on SLE plate. After drying, neutrals were eluted with a mix containing dichloromethane and isopropyl alcohol. Solvent was then evaporated and samples were reconstituted with water. Detection of cotinine was performed by mass detection using a QDa detector after UHPLC separation with a C18 column at a flow rate of 0.4 ml/min. A gradient elution of H2O +0.1% NH4OH and CH3CN was used. The method was validated based on linearity, precision, recovery and limits of detection and quantification. Results: The range of linearity was 0.001 µg/ml -5 µg/ml with a determination coefficient of 0.997. The precision was evaluated by the Relative Standard Deviation (RSD) for intra- and interassay and was below 5% and 10% respectively. 3 levels of concentration were tested to assess the recovery rate which was consistent and higher than 96%. Conclusion: Cotinine concentration can be measured in urine by SLE extraction and UHPLC-QDa detection. This method is easy, reproducible and allows quantification of low concentrations. It is a good solution to assess patients’ tobacco smoke exposure in medical laboratory.