title: Evaluation of a candidate reference measurement procedure for serum free testosterone based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry.
abstract: 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.
authors.affiliation: Laboratory for Analytical Chemistry and Laboratory for Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Gent, Belgium.
authors.forename: Katleen
authors.initials: K
authors.lastname: Van Uytfanghe
authors.forename: Dietmar
authors.initials: D
authors.lastname: Stöckl
authors.forename: Jean M
authors.initials: JM
authors.lastname: Kaufman
authors.forename: Tom
authors.initials: T
authors.lastname: Fiers
authors.forename: H Alec
authors.initials: HA
authors.lastname: Ross
authors.forename: André P
authors.initials: AP
authors.lastname: De Leenheer
authors.forename: Linda M
authors.initials: LM
authors.lastname: Thienpont
mesh_terms: D001798:Blood Proteins / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000592:standards; D006801:Humans; D007201:Indicator Dilution Techniques / Q000592:standards; D008297:Male; D011485:Protein Binding; D012015:Reference Standards; D015203:Reproducibility of Results; D013739:Testosterone / Q000097:blood* / Q000378:metabolism; D014462:Ultrafiltration / Q000592:standards
chemical_list: D001798:Blood Proteins; D013739:Testosterone