Hair cortisol and other analytes continue to decline and show reduced stability after two years of storage: Follow-up data from a systematic within-subject investigation.

Background: We recently highlighted pre-analytical storage duration over a one-year period as an important methodological influence on hair endocrine data. Here, we extend these findings using a 24-month follow-up of our systematic intra-individual investigation, examining mean-level changes and relative stability of hair analytes across storage durations. Methods: Hair samples were collected from 81 healthy participants from the posterior vertex. Samples were subdivided into five aliquots and analyzed either immediately or stored under standardized conditions (dark, dry, room temperature) for 3, 6, 12 or 24 months. Analyses focus on changes in the 24-month follow-up data. Concentrations of cortisol, cortisone, progesterone, dehydroepiandrosterone and endocannabinoids (AEA, 1/2-AG, OEA, SEA, PEA) were quantified using LC-MS/MS. Results: Concentrations of hair cortisol and cortisone declined linearly across the two-year storage period. Between 12 and 24 months of storage, additional numerical decreases were observed (hair cortisol: -5.6%, hair cortisone: -8.9%), which however were not significant for hair cortisol. For hair cortisol, relative stability decreased with prolonged storage, reflected in weaker correlations between immediately analyzed and stored samples. Across other analytes, storage-related effects varied but generally suggested poorer data quality with increasing storage duration. Conclusions: We provide further support for the importance of storage duration as a methodological influence on endocrine hair analysis. For hair cortisol, longer storage was associated not only with declining concentrations but also with weaker correspondence with immediately analyzed samples. Our findings reinforce the recommendation to conduct hair analyses as early as possible, ideally within three-to-six months of sampling.