MSACL 2025 Abstract

Cortisol and Cortisone in Scales of Atlantic Cod by LC-MS/MS - A Possible Extrapolation to Clinical Study of Chronic Stress Biomarkers

Daniela Dulgheriu (1), Martine Maria Storvestre (2), Thrond Oddvar Haugen (2), Jan Ludvig Lyche (1)
(1) Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Department of Paraclinical sciences, Pharmacology/Toxicology Unit, Oluf Thesens vei 22,1433 Aas, Norway (2) Norwegian University of Life Sciences, Faculty of Environmental Sciences and Natural Resource Management, Ecology and Natural Resource Management Section, Postbox 5003 NMBU 1432 Aas, Norway

Daniela Dulgheriu, MD (Presenter) Norwegian University of Life Sciences

Presenter Bio: My background is masters degree in organic chemistry.
I have over 15 years of work experience with liquid chromatography-tandem mass spectrometry technique. My main area of work is LC-MS/MS method development of small molecules such as veterinary drugs, steroid hormones, catecholamines ,nucleotides ,opioids and pesticides , with applications in clinical-TDM studies, environmental, toxicology and food safety.

Relevant Financial Disclosures (within past 24 months, reported on Aug 20, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Cortisol analysis is used in research to study the relationship between stress, health and disease. High cortisol levels in blood are one of the major endocrine primary stress responses in vertebrates and are widely used as stress indicators in clinical studies - mainly for acute stress measurement. However, it does not reflect long-time stress exposure, and blood sampling is an invasive and stressful procedure, which may influence the results. Thus, alternative matrices which may reflect long-term stress exposure and cortisol accumulation over time need to be investigated. Hair of mammals, feathers of birds and fish scales have been previously used for chronic stress assessment. These analyses can be used to identify potential stress-related disorders, monitor glucocorticoid treatment, and assess the impact of stress on various health conditions. Scales are a target for endocrine stimuli and can provide insights into chronic stress caused by environmental stressors and anthropogenic disturbances. Prolonged and persistent situations such as pollution or boat noise may cause stress in wild fish.

OBJECTIVES:
The aim of this study was to measure concentrations of cortisol and cortisone in scales of Atlantic cod from different regions of Oslofjord - as chronic stress assessment, to understand any potential stress effects on wild fish populations.

METHODS:
After removal of the mucus, fish scales were washed thrice by using isopropanol, then dried on paper and stored at -80°C until subsequent analysis. A sensitive LC-MS/MS (liquid chromatography-tandem mass spectrometry) analytical method for quantification of cortisol and cortisone without derivatization, in fish scales was developed and validated, with detection levels of both cortisol and cortisone in 20 mg dry fish scale sample of 0.05 ng/g and good extraction recoveries (80% for cortisol and 90% for cortisone). Stable isotope labelled internal standards of cortisol and cortisone were used to spike each sample, which ensured a reliable quantification of steroid hormones in fish scale matrix. Due to the endogenous nature of steroid hormones, assay performance was evaluated by spiking cleaned and dried fish scales with the corresponding isotope-labelled standards analogues of each compound as a surrogate standard.

RESULTS:
This new methodology using solid phase extraction (SPE), allows simultaneous quantification of cortisol and cortisone in just 10-20mg of dried fish scales. Additionally the sample preparation time is much shorter and effective compared to other methods found in literature.

CONCLUSIONS:
This analytical method allows chronic stress quantification by measuring cortisol and cortisone levels from fish scales-as welfare indicator and could be extrapolated to other similar matrices, such as hair to investigate chronic stress in humans.