P18 - A new LC-MS/MS method for the determination of serum cortisol, cortisone, and dexamethasone: utility in the interpretation of the low-dose dexamethasone suppression test

Introduction The low-dose dexamethasone suppression test (DST) is used for diagnosis of hypercortisolism and it can be influenced by dexamethasone (DEX) bioavailability. Purpose To apply a new LC-MS/MS method for the quantification of cortisol (F), cortisone (E), and DEX to assess DST adequacy and to derive the cut-off values of F and E after DST for the diagnosis of hypercortisolism. Methods Clinical data and patient samples were collected during the execution of the DST at the Division of Endocrinology of the A.O.U. Città della Salute e della Scienza di Torino from 2022 to 2024. The concentration of F after DST was measured using the chemiluminescent immunoassay (CLIA) method; F, E, and DEX were determined on the same sample using LC-MS/MS. The diagnosis of hypercortisolism was established through the DST performed with the CLIA method. Results A total of 416 patients was included (M/F: 146/270, age 52.9±18.5 years). 29 had overt hypercortisolism, 107 mild autonomous cortisol secretion, 137 non- functioning adrenal incidentaloma and 143 had no pituitary or adrenal pathology. The metabolic-hypertensive profile was worse in patients with hypercortisolism. DEX levels did not differ among the diagnostic groups (p<0.914). The 2.50 percentile of DEX concentration across all DSTs was 1.25 ng/mL, while for negative DSTs it was 1.31 ng/mL. Among the ten patients below this cut-off, three had hypercortisolism, one was taking combined oral contraceptive pill and six suppressed cortisol correctly after DST. F and E measured by LC-MS/MS after DST showed comparable accuracy to the CLIA method in defining test positivity (F-MS cut-off: 20.32 ng/mL, AUC 0.970; E-MS cut-off: 4.1 ng/mL, AUC 0.945) and in predicting the final diagnosis of hypercortisolism (F-CLIA AUC: 0.933; F-MS cut-off: 23.49 ng/mL, AUC 0.928; E- MS cut-off: 4.53 ng/mL, AUC 0.923). Conclusions The LC-MS/MS method for the determination of F, E, and DEX is reliable even at minimally detectable DEX levels and has high accuracy. The cut-off values of F after DST for defining test positivity and diagnosing hypercortisolism are slightly higher than the F-CLIA cut-off (20.32/23.49 vs. 18.10 ng/mL), while the corresponding cut- off values for E after DST (4.1/4.53 ng/mL) were defined de novo.