Determination of free cortisol and free cortisone in human urine by on-line turbulent flow chromatography coupled to fused-core chromatography-tandem mass spectrometry (TFC-HPLC-MS/MS).

[cushing syndrome]

Urinary free cortisol and urinary free cortisone are decisive markers for the diagnosis of syndromes related to the dysfunction of the adrenal gland or to evaluate certain enzymatic disorders . Here , we present a new method , designed for routine laboratory use , which enables quick determination of these analytes with minor sample workup . Turbulent flow chromatography shortens sample preparation , and connection to a fused-core particle-packed column (rugged amide-embedded C 18 phase ) permits a rapid and effective separation of the analytes , as well as additional separation from other related and isobaric compounds present in urine . Urinary isobaric compounds were successfully identified . The method requires only 100 μl of urine supernatant per sample . The total time between injections is 9 .5 min . The solvents used for both turbulent and analytical chromatography are water and methanol , and the relatively low flows needed during the method resulted in an extended life of the columns . Linearity showed a R (2 ) > 0 .994 . Limit of detection and limit of quantification are 0 .5 and 1 .0 ng /ml for cortisone and 1 .0 and 2 .0 ng /ml for cortisol . Recoveries ranged from 99 .7 to 109 .1 % for cortisone and from 98 .7 to 102 .9 % for cortisol . Accuracy values (relative errors ) for intra- and inter-assay experiments were always below 8 %, whereas precision (percent CV ) ranged from 3 .7 to 10 .7 %. No matrix effects were detected during the validation process . The reproducibility for each analyte 's retention time was excellent , with a coefficient of variation always below 0 .2 %. The final validation step included the study of urine samples from healthy children and from children previously diagnosed with corticoidal disorders . The high selectivity achieved enables quick data handling .