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-theart technology. Because FTe is defined as the hormone fraction in serum water in equilibrium with the proteinbound 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 withinday, between-day, and total CVs of 3.0%, 3.1%, and 4.3%

The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report

“…Alternatively, methods of directly assessing the quantity of free T in serum, such as equilibrium dialysis, are more accurate, correlating well with mass spectrometry (114). However, this method is not widely available given its relative technical complexity and high economic cost.…”

Liquid chromatography-tandem mass spectrometry applications in endocrinology

“…The free fraction of steroids can be measured with dialysis or ultrafiltration of the samples (Sinha-Hikim et al, 1998;Van Uytfanghe et al, 2004), followed by instrumental analysis; alternatively it could be calculated using methods based on the dissociation constants and the law of mass action (Vermeulen et al, 1999;Ly & Handelsman, 2005).…”

State-of-the-Art of Serum Testosterone Measurement by Isotope Dilution–Liquid Chromatography– Tandem Mass Spectrometry

“…Details of the ID-GC-MS RMP are described elsewhere (17,(19)(20)(21)(22) (for a summary see Table 1). Calibration was done with testosterone/ [3,[4][5][6][7][8][9][10][11][12][13] C 2 ] testosterone mixtures at a 1:1 isotope ratio (Ϯ15%) prepared from 3 independent working solutions, demonstrated beforehand to not differ by Ͼ1% from each (n ϭ 6, probability 95%) (20 ).…”

“…sample clean-up (as used in the reference ID-GC-MS measurement procedure) and assessment of interference from other steroid hormones as part of the method validation (22 ). Our study showed that the ID-LC-MS/MS procedures for measurement of testosterone in serum have sufficient analytical sensitivity to distinguish between normogonadal, hypogonadal, and female testosterone concentrations, proper specificity, even in the low concentration range, and fairly good accuracy within the biologically derived total error limit.…”