Free urinary
cortisol (UFF) measurement is one of the most useful screening tests for
Cushing's syndrome. Immunoassays employed today by most clinical laboratories present limitations, specially concerning specificity. These limitations restrain a widespread application of the method, as well as the comparison of results obtained by the use of different methods. We present the development and characterization of a UFF and
cortisone method based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). A 200 microL aliquot from a 24 h urine sample is mixed with a
solution containing a known quantity of deuterated
cortisol and on-line extracted in solid phase (C18). The eluate is transferred to a second C18 column (Phenomenex Luna, 3 micro, 50 x 2 mm) and the isocratic mode elution profile is directly applied to a tandem mass spectrometer model Quattro Micro operating in positive mode atmospheric pressure chemical ionization (APCI). All process is automated and the quantification is performed by isotopic dilution, based on the analyte and the deuterated internal standard peak area ratios. The specificity study showed that all the
steroids tested presented cross reactivity of <1% for
cortisol and
cortisone. Functional sensitivity is <1 microg/L for both
steroids, and the interassay CV <8%. Recovery and linearity studies were satisfactory and comparison of results obtained using a RIA for UFF and the present method in 98 routine samples showed a correlation of r= 0.838, with the results obtained with LC-MS/MS significantly lower (medians of 22.0 vs. 49.4 microg/24 h for RIA) (P<0.0001). Reference values for
cortisol were defined as values between 11 and 43 microg/24 h, compatible to those recently described for similar methods. The concomitant measurement of UF
cortisone allows the study of the activity of the
enzyme 11beta-HSD2 and the diagnosis of the
apparent mineralocorticoid excess syndrome. The method represents the first
steroid assay of a new generation, based on automated preparative methods and tandem mass spectrometry, described in our country.