Interference of 6β-Hydroxycortisol in the Quantitation of Urinary Free Cortisol by Immunoassay and Its Elimination by Solid Phase Extraction

Objectives: To study the cross-reactivity of 6β-hydroxycortisol (6β-OHF) with anticortisol antibodies and subsequent interference with urinary free cortisol (UFC) quantitation in commercial immunoassay kits. A solid-phase-extraction (SPE) technique was evaluated for removal of 6β-OHF from the specimen. Methods: Interference by 6β-OHF was studied in three enzyme immunoassay and three radioimmunoassay kits. Interference was assessed by the multiple regression equation derived from a 4 × 4 matrix composed of four levels of cortisol and four levels of 6β-OHF. Sep-Pak Plus C18 cartridges were used to remove 6β-OHF from the specimens, and optimum conditions for fractionating 6β-OHF from cortisol were determined by eluting the cartridge with a stepwise increment in methanol concentration. HPLC was used to monitor cortisol and 6β-OHF in the sample and cartridge eluates. Results: Cross-reactivity of 6β-OHF with anticortisol antibodies was variable and depended on the cortisol kit used with a range from 0.1 to 10%. Multiple regression analysis indicated that 6β-OHF interfered positively with the cortisol assay regardless of cortisol concentrations. Extraction of UFC by methylene chloride as recommended by some immunoassay kits yielded a significant constant error to the UFC results which was nearly proportional to the added levels of 6β-OHF. SPE of samples completely removed 6β-OHF from the specimen when a 40% methanol elution step was included. Consequently, specimens processed in this manner did not show interference in UFC measurements even in samples spiked with 2.38 μmol/L of 6β-OHF. Conclusions: Elevated urinary 6β-OHF may be a significant source of interference in UFC immunoassays. SPE of samples prior to analysis would be a simple and inexpensive means for removing 6β-OHF from urine specimens, therefore, increasing accuracy and precision in UFC measurements by immunoassay.