Cortisol Stability at 16 and 30 Years in Urine Specimens Stored at −20°C

Abstract

Objective

This study aims to investigate whether urinary cortisol can be reliably measured after long-term storage at −20°C, using urine samples collected in 1993 from rural Bangladeshi women, and assayed after 16 and 30 years (2009 and 2023).

Methods

Specimens were collected by participants, placed in coolers with ice packs, transported to a field lab, and then refrigerated for up to 1 week before processing. Specific gravity was measured, and a 6.5 mL sample was archived at −20°C. Urinary cortisol was assayed in 2009 as part of a prior study using enzyme-linked immunosorbent assay (ELISA). In 2023, cortisol was measured in a subsample of 200 specimens using the same assay.

Results

Of the 200 specimens, 7 (3.5%) were measured with low precision. Cortisol could not be detected in nine specimens (4.5%). In the remaining 184 specimens, urinary log cortisol levels measured in 2009 and 2023 were positively correlated (r = 0.973, 95% CI: 0.964–0.980; p < 0.0001 and nonparametric r_s = 0.795; p < 0.0001). A paired t-test revealed a bias toward slightly greater values in the 2023 measurements (p < 0.0001).

Conclusion

These results suggest that urinary cortisol remains largely intact and measurable in specimens stored at −20°C for three decades, and there is a strong correlation between cortisol measured after 16 and 30 years in storage.