Interference of 6 beta-hydroxycortisol in the quantitation of urinary free cortisol by immunoassay and its elimination by solid phase extraction.

OBJECTIVE

To study the cross-reactivity of 6 beta-hydroxycortisol (6 beta-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 6 beta-OHF from the specimen.

METHODS

Interference by 6 beta-OHF was studied in three enzyme immunoassay and three radioimmunoassay kits. Interference was assessed by the multiple regression equation derived from a 4 x 4 matrix composed of four levels of cortisol and four levels of 6 beta-OHF. Sep-Pak Plus C18 cartridges were used to remove 6 beta-OHF from the specimens, and optimum conditions for fractionating 6 beta-OHF from cortisol were determined by eluting the cartridge with a stepwise increment in methanol concentration. HPLC was used to monitor cortisol and 6 beta-OHF in the sample and cartridge eluates.

RESULTS

Cross-reactivity of 6 beta-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 6 beta-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 6 beta-OHF. SPE of samples completely removed 6 beta-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 mumol/L of 6 beta-OHF.

CONCLUSIONS

Elevated urinary 6 beta-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 6 beta-OHF from urine specimens, therefore, increasing accuracy and precision in UFC measurements by immunoassay.