The Joint Annual Scientific Meetings of the Endocrine Society of Australia and the Society for Reproductive Biology 2018

Comparison of LCMSMS, HPLC and two automated immunoassay methods in a cohort of patients with clinically confirmed Cushing’s syndrome (#240)

Lydia S Lamb 1 2 , David Henley 1 2 , Rui Zhang 3 , Conchita Boyder 3 , Brian Perriam 3 , Ee Mun Lim 1 3
  1. Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia
  2. School of Medicine and Pharmacology, University of Western Australia, Perth, Western Australia, Australia
  3. Department of Clinical Biochemistry, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Perth, Western Australia, Australia


Measurement of 24 hour urine free cortisol (UFC) is one of the initial investigations for the diagnosis of Cushing’s syndrome (CS). Automated immunoassay (AIA) methods are prone to interferences from exogenous glucocorticoids and cortisol metabolites leading to overestimation of urinary cortisol and reduced specificity. Other methods with greater specificity including high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LCMSMS) are not subject to this interference and are being used with increasing frequency.


To compare different methods of UFC measurement in patients investigated for CS.


PathWest QEII routinely measures UFC on automated immunoassay platform Siemens Immulite 2000 XPi with reference interval (RI) <900 nmol/day. Since June 2016, aliquots of UFC were collected and stored at -20°C for LCMSMS method evaluation. The LCMSMS uses a Xevo TQ-S (Waters Corporation, Milford, MA) with prior solid phase extraction and measures cortisol, cortisone, deoxycorticosterone, 11-deoxycortisol, prednisolone and dexamethasone (provisional RI <170 nmol/day). Analysis of urine cortisol was also performed on another immunoassay platform, Abbott Architect (RI <330 nmol/day), as well as our in-house HPLC method (RI <170 nmol/day).


9 patients had clinically confirmed CS: Cushing’s disease (5), ectopic ACTH secretion (2), metastatic adrenocortical carcinoma (ACC) (1) and adrenal CS (1). 7 of 9 patients had concordant increase in UFC by all methods. Two patients failed overnight dexamethasone suppression test but had normal UFC by all methods. Cortisol/cortisone ratio was elevated in 3 cases, including ectopic CS (2) and ACC (1). One additional patient had 11-deoxycorticosterone and 11-deoxycortisol secreting metastatic ACC.


Results are concordant on the specific LCMSMS and HPLC methods compared to the unextracted automated immunoassay platforms as long as appropriate diagnostic cut-offs are applied. LCMSMS has the additional benefit of looking at other corticosteroid metabolites.