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Evaluation of a Candidate Reference Measurement Procedure for Serum Free Testosterone Based on Ultrafiltration and Isotope Dilution–Gas Chromatography–Mass Spectrometry

¹ Laboratory for Analytical Chemistry and ² Laboratory for Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Gent, Belgium.
³ Laboratory for Hormonology and Department of Endocrinology, Ghent University Hospital, Gent, Belgium.
⁴ Department of Chemical Endocrinology, University Medical Centre, Nijmegen, The Netherlands.

^aAddress correspondence to this author at: Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium. Fax 32-9-264-8198; e-mail linda.thienpont{at}ugent.be.

Background: To assess the analytical validity of free testosterone (FTe) measurements, a reference measurement procedure (RMP) is required. For steroids, isotope dilution–mass spectrometry is accepted as state-of-the-art technology. Because FTe is defined as the hormone fraction in serum water in equilibrium with the protein-bound fraction, the RMP should include a physical separation step. The use of equilibrium dialysis (ED) or ultrafiltration (UF) is advocated. Our objective was to develop such a candidate RMP.

Methods: We selected UF combined with isotope dilution–gas chromatography–mass spectrometry (ID-GC/MS) for direct measurement of Te in the ultrafiltrate. After optimization of the UF process, the complete procedure was validated by use of split-sample comparisons with indirect ED (iED) and symmetric dialysis (SyD).

Results: The candidate RMP gave maximum within-day, between-day, and total CVs of 3.0%, 3.1%, and 4.3%. The Deming regression equations for the respective method comparisons were: UF-ID-GC/MS = 0.98(iED) – 53 pmol/L (r = 0.94; S_y|x= 42 pmol/L) and UF-ID-GC/MS = 0.92(SyD) + 21 pmol/L (r = 0.97; S_y|x= 31 pmol/L).

Conclusions: We achieved the objective of a state-of-the-art candidate RMP, which agreed well with iED and SyD. However, we also demonstrated that a degree of discordance remains, which may require a decision from an authoritative organization on the recommended procedure to measure free hormone concentrations.

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