Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry.
Level 4 - case-series / case-control
Analytical validation and split-sample comparative study using laboratory specimens
PubMed 20026023 · doi:10.1016/j.clinbiochem.2009.12.005
What was done
Ultrafiltrate obtained from 0.5 mL of serum was partially purified by liquid-liquid extraction and quantified using an HPLC system coupled to an API 5000 mass spectrometer with atmospheric pressure chemical ionization. Total imprecision of the direct ultrafiltration LC-MS/MS method was assessed over twenty runs. Using split serum samples, results from direct ultrafiltration coupled LC-MS/MS were compared against analogue free testosterone immunoassay, free testosterone calculated from mass action equations (using total testosterone measured by immunoassay or LC-MS/MS), and equilibrium dialysis coupled to LC-MS/MS.
What was found
Total imprecision was under 6% at 67 pmol/L and 158 pmol/L free testosterone over 20 runs. The dynamic response was linear up to at least 2500 pmol/L with a lower limit of quantitation of 16 pmol/L (18% CV). Method comparison showed poor agreement with analogue immunoassay (correlation coefficient 0.667, bias -81%), better agreement against calculated free testosterone with total testosterone by immunoassay (correlation coefficient 0.816, bias 21%), higher agreement against calculated free testosterone with total testosterone by LC-MS/MS (correlation coefficient 0.8996, bias 10%), and closest agreement with equilibrium dialysis coupled LC-MS/MS (correlation coefficient 0.9779, bias 2.4%).
Why it matters
Direct ultrafiltration coupled to LC-MS/MS provides a practical and accurate method for measuring serum free testosterone that avoids the substantial bias of analogue immunoassays and closely matches equilibrium dialysis.
Limits
The abstract does not disclose the sample size (n), the demographic or clinical characteristics of the patient cohort, or assay performance across populations with abnormal sex hormone-binding globulin concentrations.
Cited by
- supports Equilibrium dialysis and equilibrium ultrafiltration are direct measurement assays for free testosterone rather than calculated estimates.