A simple liquid chromatography-tandem mass spectrometry method for urinary free cortisol analysis: suitable for routine purpose

Article in press - uncorrected proof
Clin Chem Lab Med 2010;48(10):1433–1437 2010 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2010.282

A simple liquid chromatography-tandem mass spectrometry method for urinary free cortisol analysis: suitable for routine purpose

Silvia Persichilli*, Jacopo Gervasoni, Federica Iavarone and Cecilia Zuppi
Istituto di Biochimica e BiochimicaClinica, Universita ` Cattolica del Sacro Cuore, Rome, Italy

Keywords: cortisol; immunoassay; liquid chromatographytandem mass spectrometry.

Introduction Abstract
Background: The best index of adrenal dysfunction is urinary free cortisol (UFC) measurements performed using a 24-h urine collection. This measurement is also useful in the investigation of Cushing’s syndrome. In this paper, wereport a simple and selective method for the analysis of UFC by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) suitable for use in a high-volume clinical laboratory routine. The results were compared to those obtained using a commercial immunoassay method used in our laboratory. Methods: Urine samples containing 50 ng of internal standard (Cortisol-9,11,12,12-d4) were deproteinized usingcentrifugal filters with a molecular weight 10,000 Da cut-off and injected on a reversed phase column. Cortisol was analyzed in highly selective reaction monitoring in positive atmospheric pressure chemical ionization mode, at a resolution of 0.4 amu full width half maximum, and following the transitions related to the precursor 363.2 for cortisol and 367.2 for deuterated cortisol. The methodvalidation included analysis of precision, linearity, sensitivity, recovery and interference from structurally similar steroids. UFC from 230 subjects was measured using LC-MS/MS and electrochemiluminescence immunoassay (ECLIA) methods. Results: The calibration curves exhibited linearity and reproducibility in the range 7–10,000 nmol/L. Total imprecision was lower than 10%. The limit of detection andlimit of quantification were 2 and 7 nmol/L, respectively. Mean recovery was higher than 90%. Structurally similar steroids do not interfere in the proposed method, but cause a significant change in the ECLIA results. Cortisol values obtained using the ECLIA method were always higher than those obtained using the LC-MS/MS method, with the bias directly proportional to cortisol concentrations. Thereference values calculated using 180 normal subjects were 11–70 mg/day. Conclusions: The proposed method is sensitive, simple, free from interferences and reliable for routine use. Clin Chem Lab Med 2010;48:1433–7.
*Corresponding author: Dr. Silvia Persichilli, Istituto di Biochimica e Biochimica Clinica, Universita Cattolica del Sacro ` Cuore, Largo A. Gemelli 8, 00168 Rome, Italy Phone: q39 063015 4222, Fax: q39 06 3015 6783, E-mail: spersichilli@rm.unicatt.it Received February 9, 2010; accepted April 29, 2010; previously published online July 7, 2010

Cortisol is the main glucocorticoid hormone in humans. Plasma concentrations have a circadian rhythm. Therefore, urinary free cortisol (UFC) measured in a 24-h collection is the best index of adrenal function, and the test of choicefor the diagnosis of Cushing’s syndrome (1, 2). Several methods have been developed for UFC analysis in clinical laboratories ranging from radioimmunoassay (RIA) to HPLC. However, these methods have now been almost completely replaced by high throughput immunoassays (3). Immunoassay methods use antibodies directed against the C-3 position of cortisol wi.e., via o-(carboxymethyl oxyme) bridgex, andare strongly affected by interfering compounds, such as exogenous or endogenous steroids containing the same 3-keto group (4, 5). For these reasons, most immunoassay manufacturers suggest using a liquid-liquid extraction procedure before analysis to eliminate interfering compounds. This pre-assay treatment is time consuming and only partially eliminates interferences; removing only conjugate...