Development of a urinary free cortisol assay using solid-phase extraction-capillary electrophoresis.
In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE-CE method for the rapid analysis of UFC. UFC was concentrated using SPE C18 cartridges (3M Empore) under a vacuum and eluted with acetonitrile-SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile-SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10-15 min with a recovery of 89-94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 microg/l with SPE compared to 500 microg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.
Rao
L V
LV
Department of Pathology, University of Texas Medical Branch, Galveston, 77555-0551, USA.
Petersen
J R
JR
Bissell
M G
MG
Okorodudu
A O
AO
Mohammad
A A
AA
D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D006854:Hydrocortisone / Q000652:urine*; D012015:Reference Standards; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet
D006854:Hydrocortisone