采用超滤法结合液质联用技术直接测定血清游离睾酮。
Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry.
机构信息
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
出版信息
Clin Biochem. 2010 Mar;43(4-5):490-6. doi: 10.1016/j.clinbiochem.2009.12.005. Epub 2009 Dec 21.
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 <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.
背景
目前尚无适用于常规测量血清游离睾酮（FT）的可靠方法。
目的
开发一种涉及液相色谱串联质谱（LC-IDMS/MS）的方法，该方法可直接检测和定量血清中的 FT。
方法
使用 Agilent 1200 系列 HPLC 系统和配备大气压化学电离离子源的 API 5000 质谱仪，对来自 0.5 mL 血清的超滤睾酮进行部分纯化和定量。使用拆分样本，比较了直接超滤（UF）与 LC-MS/MS、模拟 FT 免疫测定、根据质量作用方程计算的游离睾酮（cFT）和平衡透析（ED）与 LC-MS/MS 。
结果
在 20 次运行中，在 67 pmol/L 和 158 pmol/L FT 时，总不精密度<6%。动力学响应至少线性至 2500 pmol/L，而物理 LLOQ（18%CV）等于 16 pmol/L。UF 方法与模拟免疫测定的相关性较差（相关系数 0.667；偏倚-81%），与免疫测定测定总睾酮时的 cFT 相关性稍好（相关系数 0.816，偏倚 21%），与 LC-MS/MS 测定总睾酮时的 cFT 相关性更好（相关系数 0.8996，偏倚 10%）。与 ED 方法的一致性最好（相关系数 0.9779，偏倚 2.4%）。
结论
我们提出了一种相对简单的 UF 与 LC-MS/MS 相结合的明确方法，可测量血清游离睾酮。该方法相对快速、可靠，适用于常规临床实验室实践。
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