Amine/phenyl gradient derived base layer as a comprehensive extractive phase for headspace cooled in–tube microextraction of volatile organic compounds in saliva

•A gradient derived base layer is synthesized as comprehensive extractive phase.•The silanol thick/uniform layer facilitated the gradient surface formation.•Amine gradient coating on the base layer formed by controlled rate infusion.•Headspace cooled in–tube microextraction was also effective to analyze saliva.•The setup is capable to determine volatile organics in biological samples. A gradient derived base layer extractive phase was synthesized and applied for the determination of volatile organic compounds (VOCs) in saliva samples using the headspace cooled in–tube microextraction (HS–CITME) method. The base layers from three different sols of phenyltriethoxysilane (PTES), octyltrimethoxysilane (OTMS) and methyltrimethoxysilane (MTMS) as nonpolar precursors were individually dip coated on the stainless steel wires (SSW). Then, the hydrolyzed polar precursor aminopropyltriethoxysilane (APTES) reacted with the silanol groups already formed on the surface of SSWs via controlled rate infusion (CRI) method. The presence of polar and non–polar functional groups on the surface of substrate was evaluated by Fourier–transform infrared spectroscopy (FTIR) while the morphology and thickness of the most suitable gradient coating (amine/phenyl) were also investigated by scanning electron microscopy (SEM). Assessment of the gradient extractive phase efficiency was carried out determining a group of VOCs with different polarities coupled with gas chromatography–mass spectrometry (GCMS) and the improved performance of the synthesized base layer coatings was observed. Furthermore, a cooling device was designed and implemented to the extracting system to improve the efficiency by influencing the exothermic nature of process. The data were analyzed by principal component analysis (PCA), and hierarchical cluster analysis (HCA) and the results were interpreted by polarities of analytes. Finally, under the optimized conditions, the limits of detection (LOD) and limits of quantification (LOQ) were 0.15 and 0.50 ng L−1, respectively. The intra–day and inter–day relative standard deviations (RSDs) at 5 and 50 ng L−1 (n = 3) using a single extractive phase were 2–6 and 10–17, respectively. The data associated with RSDs% for three extractive phases were between 16 and 19 %. Eventually, the method was conveniently applied to the extraction of VOCs from saliva samples of smokers and satisfactory relative recoveries (RR%) (95–108 %) were achieved and low quantities of VOCs were dete