Application of nanofiber‐packed SPE for determination of salivary‐free cortisol using fluorescence precolumn derivatization and HPLC detection

Salivary cortisol has emerged as an easy-to-collect biologic marker of stress in many researches. In this study, we present a method for the determination of salivary-free cortisol using HPLC method with fluorescence precolumn derivatization, which is based on a novel extraction from the strongly acidic medium (fluorescent derivatives of cortisol in sulfuric acid medium) by electrospun polystyrene nanofibers packed SPE. For high-throughput sample extraction, an array pretreatment device based on nanofibers packed SPE micro-column was designed. The LOD of cortisol was 0.01 microg/L (S/N=3). The RSDs (n=6) for all analytes were below 8.0%, and the recoveries were 110, 102.4, and 99.4% (n=3) for saliva spiked with 0.1, 10, and 20 microg/L of cortisol, respectively. The proposed method was then successfully applied in the determination of free cortisol in human saliva. The salivary cortisol concentrations in the real samples ranged from 0.22 to 7.45 microg/L. The nanofiber-packed SPE overcame the low extraction recovery and bad clean-up effect of the conventional methods, and increased the sensitivity and selectivity of the method.