LAUSR

&NA; Cortisol (F) and cortisone (E) are metabolized to A‐ring reduced metabolites in the reactions catalyzed by 5&agr;‐ and 5&bgr;‐reductase. 5&agr;‐tetrahydrocortisol (alloTHF) and 5&bgr;‐tetrahydrocortisol (THF) are produced from F. The metabolism of E takes place in analogy to form alloTHE and THE. Up to now, the analysis of endogenous glucocorticoids did not consider alloTHE, limiting the metabolism of E to THE only. Nevertheless, such simplification can generate inaccuracy in the assessment of the function of enzymes crucial for glucocorticoids metabolism: 11&bgr;‐hydroxysteroid dehydrogenase type 1 and type 2 (11&bgr;‐HSD1 and 11&bgr;‐HSD2), as well as 5&agr;‐ and 5&bgr;‐reductase. The paper presents the new LC–MS/MS method for the simultaneous analysis of F and E with their tetrahydro‐ (THF and THE) and allo‐tetrahydrometabolites (alloTHF and alloTHE) in urine. The method was fully validated and allows determining both the unconjugated and total concentrations of urinary glucocorticoids. The method meets the EMA's recommendations and was proved to be useful in the analysis of clinical samples. The LLOQ of 1 ng/mL allows the determination of free urinary F, E, THF and THE, but not alloTHF and alloTHE, in samples obtained from pregnant women. The range of concentrations is wide enough for the analysis of total levels of F, E, THF, alloTHF, THE and alloTHE. The undisputed advantage of the method, distinguishing it among others, is the ability to determine F and E and their both 5&agr;‐ and 5&bgr;‐metabolites. Taking alloTHE into consideration enables the thorough analysis of the glucocorticoid equilibrium in human. HighlightsThorough analysis of glucocorticoid balance.Versatility: determination of free and total amounts of steroids.Metabolism of cortisone without simplification.Novelty: analysis of allo‐tetrahydrocortisone.Full validation and reliable analysis of clinical samples.