Methods:
The authors compared standard (finely cutting hair) vs. 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 (LCMS) vs. ELISA assays for measuring hair cortisol (n=28). The extraction process included sequential incubations in methanol and acetone, repeated twice. Hair protein was measured via 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 LCMS.
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