Methodological Considerations for Hair Cortisol Measurements in Children.

BACKGROUND: Hair cortisol levels are used increasingly as a measure for chronic stress in young children. We propose modifications to the current methods used for hair cortisol analysis to more accurately determine reference ranges for hair cortisol across different populations and age groups. METHODS: The authors compared standard (finely cutting hair) versus milled methods for hair processing (n = 16), developed a 4-step extraction process for hair protein and cortisol (n = 16), and compared liquid chromatography-mass spectrometry (LC-MS) versus enzyme-linked immunosorbent assays (ELISAs) for measuring hair cortisol (n = 28). The extraction process included sequential incubations in methanol and acetone, repeated twice. Hair protein was measured through spectrophotometric ratios at 260/280 nm to indicate the hair dissolution state using a BioTek plate reader and dedicated software. Hair cortisol was measured using an ELISA assay kit. Individual (n = 13), pooled hair samples (n = 12) with high, intermediate, and low cortisol values, and the ELISA assay internal standards (n = 3) were also evaluated by LC-MS. RESULTS: Milled and standard methods showed highly correlated hair cortisol (rs = 0.951, P < 0.0001) and protein values (rs = 0.902, P = 0.0002), although higher yields of cortisol and protein were obtained from the standard method in 13 of 16 and 14 of 16 samples, respectively (P < 0.05). Four sequential extractions yielded additional amounts of protein (36.5%, 27.5%, 30.5%, 3.1%) and cortisol (45.4%, 31.1%, 15.1%, 0.04%) from hair samples. Cortisol values measured by LC-MS and ELISA were correlated (rs = 0.737; P < 0.0001), although cortisol levels [median (interquartile range)] detected in the same samples by LC-MS [38.7 (14.4-136) ng/mL] were lower than that by ELISA [172.2 (67.9-1051) ng/mL]. LC-MS also detected cortisone, which comprised of 13.4% (3.7%-25.9%) of the steroids detected. CONCLUSIONS: Methodological studies suggest that finely cutting hair with sequential incubations in methanol and acetone, repeated twice, extracts greater yields of cortisol than does milled hair. Based on these findings, at least 3 incubations may be required to extract most of the cortisol in human hair samples. In addition, ELISA-based assays showed greater sensitivity for measuring hair cortisol levels than LC-MS-based assays.