Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry Yu Chen a,d , Mehrdad Yazdanpanah b , Xiao Yan Wang c , Barry R. Hoffman a,c , Eleftherios P. Diamandis a,b,c , Pui-Yuen Wong a,b, ⁎ a Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada b Laboratory Medicine Program, Toronto General Hospital/University Health Network, Toronto, Ontario, Canada c Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada d Department of Laboratory Medicine, Dr. Everett Chalmers Regional Hospital/Horizon Health Network, Fredericton, New Brunswick, Canada abstract article info Article history: Received 2 September 2009 Received in revised form 13 November 2009 Accepted 6 December 2009 Available online 21 December 2009 Keywords: Testosterone Serum free hormone LC-MS/MS Ultrafiltration Immunoassay Background: Currently there is no reliable method suitable for routine measurement of serum free testosterone (FT). Aim: To develop such a method involving liquid chromatography tandem mass spectrometry (LC-IDMS/ MS) that directly detects and quantifies the FT present in serum. Methods: Ultrafiltrate testosterone obtained from 0.5 mL of serum was partially purified by liquid/liquid extraction and quantified using an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source. Using split samples serum free testosterone was compared between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS. Results: Total imprecision determined over twenty runs was b 6% at 67 pmol/L and 158 pmol/L FT. The dynamic response was linear up to at least 2500 pmol/L while physical LLOQ (18 % CV) equaled 16 pmol/L. The UF method agreed poorly with analogue immunoassay (correlation coefficient 0.667; bias -81%), somewhat better against cFT when total testosterone was determined by immunoassay (correlation coefficient 0.816, bias 21% ) and still better yet against cFT when total testosterone was determined by LC- MS/MS (correlation coefficient 0.8996, bias 10%). Agreement was closest with ED method (correlation coefficient 0.9779, bias 2.4%). Conclusion: We present a relatively simple UF coupled LC-MS/MS definitive method that measures serum free testosterone. The method is relatively fast, reliable and is suitable for the routine clinical laboratory practice. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction Testosterone in the blood circulates in three forms—tightly bound to sex-hormone binding globulin (SHBG), loosely bound to albumin and unbound (free testosterone (FT)). Only the free fraction, amounting to 1–2% of the total, is able to penetrate the cell membrane to interact with the androgen receptor to regulate the expression of androgen-responsive target genes [1]. Because of this, free testoster- one is considered the most physiologically relevant fraction. However, FT is rarely measured in routine clinical practice since it is more technically difficult to determine for its very low concentration and similar structure molecule interference than either total testosterone or the indirect measures of the free fraction expressed in terms of the bioavailable testosterone (albumin-bound fraction), androgen index (total testosterone modulated by the SHBG-bound fraction) or mass action calculation (total testosterone and both protein bound fractions) [2]. Although total testosterone accurately measured frequently suf- fices [3-5], it is inherently less reliable than the direct measure of the free fraction because many factors including aging, obesity, pregnancy, testosterone/estrogen treatment, and polycystic ovary syndrome, affect the amount and affinity of the binding proteins, SHBG in particular, thereby leading to a mismatch between total testosterone and the free fraction. When this occurs, the total testosterone becomes inconsistent with the clinical status of the patient [6,7]. For example, a cohort study of American men over 65 years of age [8] and a cross- sectional analysis of Australian men over 70 years of age [9] have shown that the level of total testosterone remains relatively stable with age while the amount of free testosterone declines. The mismatch Clinical Biochemistry 43 (2010) 490–496 ⁎ Corresponding author. University Health Network, Toronto General Hospital, Room 3EB-362, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4. Fax: +1 416 340 3551. E-mail address: pui-yuen.wong@uhn.on.ca (P.-Y. Wong). 0009-9120/$ – see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.12.005 Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem