Measurement of Urinary Free Cortisol (UFC) using APCI-Liquid Chromatography - Tandem Mass Spectroscopy (LC TMS) and Comparison with two Different Cortisol Immunoassays

Joy Mutton, Biochemistry

J Mutton, J Heynes, C Webster, DM Kennedy

UFC measurement aids diagnosis of Cushing€™s syndrome and has traditionally been measured by immunoassay.  Due to poor specificity of immunoassays,  we have developed a LC-TMS method for UFC.

Samples were extracted with dichloromethane and deuterated 17-OHP  was used as internal standard. The method was linear up to 2000nmol/L,  with a limit of detection of 10nmol/L.  Imprecision was acceptable  (within-batch CV =9.6%  and 5.9%, between-batch CV= 7.7% and 14.7% at 122 and 355nmol/L respectively).  Accuracy was good with no significant difference between our method and other TMS users using NEQAS samples (n=12,Wilcoxon Rank Test,p=0.09).  Mean recoveries (n=3) of 94.5%, 96.2% and 102.9% were obtained when  urine was spiked  with 100, 250 and 600nmol/L of cortisol respectively.

Comparisons of UFC measurement by TMS, Elecsys and Immulite immunoassays were made using 49 urine samples.  UFC concentration by TMS correlated significantly with the immunoassays (Elecsys:Rs=0.89,p<0.0001, Immulite:Rs=0.90,p<0.0001) although TMS results were lower. TMS showed  no significant interference by cortisone, 11-deoxycortisol, prednisone and prednisolone. However, the Elecsys showed   a 565% increase and the Immulite a 235%  increase in UFC when spiked with ethanolic prednisolone compared to ethanol alone.

Overall, the TMS UFC method was more specific and sensitive than the immunoassays with acceptable precision and accuracy.