Measurement of cortisol and cortisone in children's hair using ultra performance liquid chromatography and tandem mass spectrometry

Objectives: Children's stress research may benefit from the establishment of a standardized and validated methodology to measure cortisol and cortisone in hair, as this biomatrix is non-invasive, easily obtainable and more practicable compared to other commonly used matrices in stress research. Methods: A new analytical method using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is developed and validated to simultaneously detect cortisol and cortisone in children's hair. Applicability of the UPLC-MS/MS method is tested in 6 cm hair samples of 223 elementary school girls, and cortisol-to-cortisone ratios are calculated. Results: The LOD and LOQ were 2 pg mg−1 and 5 pg mg−1 respectively, for both cortisol and cortisone. Good linear responses in the ranges 5–1000 pg mg−1 (R2 > 0.99) and extraction recoveries of >84% and >87% were observed for cortisol and cortisone respectively. Relative standard deviation percentages were <13.4% (cortisol) and <14.1% (cortisone) for intra-assay precision, and <18.8% for inter-assay precision for both analytes at LOQ. %Bias was <18.9% for intra- and inter-day accuracy at LOQ, and <12.3% at the other concentrations. The median hormone concentrations (pg mg−1, range) were 8.80 (5–1330.48), 8.62 (5–69.6) and 0.80 (0.11–54.4) for cortisol (N = 39), cortisone (N = 168) and the cortisol-to-cortisone ratio (N = 31) respectively. Conclusions: We presented a UPLC-MS/MS method with good precision and accuracy to simultaneously measure cortisol and cortisone in hair samples 6 cm long. Our observations suggest that it is advisable for future research not to analyse full hair segments up to 6 cm long because of inter-segment loss, but to restrict to more proximal fragments which may more accurately represent HPA activity.