Determination of Cotinine in Urine and Saliva Using Hydrophilic Interaction Liquid Chromatography and Tandem Mass Spectrometry

Abstract

A new simplified method for monitoring of cotinine in urine and saliva, based on hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS) is presented. Work-up techniques based on filtration, using 0.45 μm pore sizepolypropylene filter, of crude urine and saliva and alkaline extraction of cotinine with methylene dichloride were studied. Multiple reaction monitoring (MRM) of the protonated molecular ions of cotinine and tri-deuterium labelled cotinine resulted in selective determinations with linear calibration graphs (correlation coefficients >0.999) The precision for 10 samples, containing 100 ng cotinine /ml, prepared from urine and saliva by methylene dichloride extraction or filtration were between 2 and 5 %. The detection limit for extracted and filtrated saliva was 2 fmol and for extracted and filtrated urine it was 5 and 9 fmol, respectively. Agreement was obtained for the two different work-up techniques. Extraction of urine/saliva enabled detection at lower levels (down to 5 pg cotinine/ml of urine) compared to filtration, since the possibility of enrichment of the sample but also due to reduction of interfering compounds present in the matrix. However, when performing large exposure studies with many samples to be analysed, the simplified and time saving work-up procedure obtained with filtration is valuable. The higher organic content of the effluent in the HILIC mode and a more efficient separation of polar compounds present in the matrix, favoured the electrospray ionisation compared to reversed phase (RP) LC, thus enabling lower detection limits.