Development and Validation of an LC-MS/MS Method for the Determination of Glucocorticoids and Endocannabinoids in Hair: Application to Longitudinal Pregnancy Samples

Chronic stress has been associated with adverse health outcomes, including anxiety, depression, and cardiovascular disease. During pregnancy, stress-related biological changes may affect maternal and fetal health; however, assessing cumulative stress-related biomarkers remains challenging. Glucocorticoids (cortisol/cortisone) and endocannabinoids (lipid-based neurotransmitters) have been proposed as biomarkers related to stress physiology. Hair is a useful matrix for evaluating cumulative analyte exposure over extended periods because it is less affected by short-term fluctuations and circadian variation than blood, saliva, or urine. The aims of this study were to: 1) develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of glucocorticoids and endocannabinoids in hair, and 2) apply the method to hair samples collected at delivery to describe glucocorticoid and endocannabinoid concentrations across pregnancy. The method consisted of 20 mg of pulverized hair incubated in 1 mL of methanol:acetonitrile (75:25) under ultrasound at 55 °C for 2 h, and analyzed by LC-MS/MS. A total of 66 3-cm hair segments from 35 pregnant women across the three trimesters were analyzed. Glucocorticoids were detected in 62 segments (cortisol in 62, cortisone in 54), and endocannabinoids in all of them (OEA and PEA in all, 2-AG in 53, but none for AEA). In the 13 cases with segments from all three trimesters, no significant changes in endocannabinoids were observed, whereas glucocorticoids increased throughout pregnancy. These findings demonstrate that hair analysis enables reliable, longitudinal assessment of glucocorticoids and endocannabinoids during pregnancy, supporting its utility as a non-invasive monitoring tool.