MSACL 2019 EU Abstract

Validated High-Sensitive Steroid Profiling in Human Serum Using LC-MS/MS

Aurore JAFFUEL (1), Alban HUTEAU (1), Stéphane MOREAU (2).
(1) SHIMADZU France, Noisiel, FRANCE. (2) Shimadzu Europa GmbH, Duisbourg, Germany.

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Stephane Moreau (Presenter) SFP

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: Steroid profiling is of clinical significance for the diagnosis of a wide variety of diseases as steroids play a major role in the regulation of several physiological functions. Historically, these analytes have been measured using immunoassays. However, it is now established that these methods suffer from lack of specificity, due to cross-reactivity. Mass spectrometry has become a standard for accurate steroid profiling, taking advantage of its specificity, high sensitivity and the ease of sample preparation. Nevertheless, due to their small size and their large range of polarity, the analysis by LC-MS/MS is still a challenge.

METHOD: Here we present a method for high-sensitive steroid profiling in human serum using LC-MS/MS. This method was validated and is now used in routine in a French hospital. The analysis was performed using Nexera X2 high performance liquid chromatography (Shimadzu Corporation, Kyoto) and LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto). MRM parameters were optimized using LabSolutions software (Shimadzu Corporation, Kyoto). Sample preparation consists of a supported liquid extraction (SLE), followed by evaporation. The analytical separation was performed on a Raptor Biphenyl 2.7μm 50x3mm (Restek Corporation, State College, PA), using water and methanol as mobile phases, and ammonium fluoride as additive.

RESULTS: The method was validated for the analysis of 10 steroids in serum: aldosterone, 11 deoxycortisol, corticosterone, 17OHP, testosterone, androstenedione, progesterone, DHEA, DHEA sulfate, estradiol. Validated low limits of quantification (LLOQ) confirmed the high sensitivity of the method: 0.025 ng/mL for aldosterone, 0.09 ng/mL for 11-deoxycortisol, 0.5 ng/mL for corticosterone, 0.1 ng/mL for 17OHP, 0.06 ng/mL for testosterone, 0.2 ng/mL for androstenedione, 0.1 ng/mL for progesterone, 1 ng/mL for DHEA, 100 ng/mL for DHEA sulfate, 0.04 ng/mL for estradiol. For all analytes, r² of linearity models were above 0.99, with S/N > 10 for LLOQ levels. Accuracies of calibration and QC samples were comprised in between 85 and 115% for all analytes. Additionality results were correlated with external validated reference methods, presenting a good correlation.

CONCLUSION: The method proved it fits for purpose and is now used for routine analysis in this hospital.