Testosterone, Free, Mass Spectrometry/Equilibrium Dialysis (Endocrine Sciences)

CPT 84402; 84403

Test Details


Free testosterone is the fraction of total testosterone that is not bound to circulating proteins. The free fraction is available to diffuse into target cells to interact with receptors or enzymes. The concentration of free testosterone is low relative to total testosterone, typically <3% of the total testosterone concentration. Total circulating testosterone is bound to sex hormone-binding globulin (SHBG) or albumin. Measurement of total and free testosterone is key in diagnosis of androgen excess or deficiency. Free testosterone is best measured by equilibrium dialysis, while total testosterone is measured by HPLC with tandem mass spectrometry.1-3 Free testosterone is estimated in this profile using these recommended techniques.

Tritiated testosterone is added to the sample and allowed to dialyze across a membrane. Once equilibrium has been achieved, the solutions on both sides of the membrane are sampled to determine the percent free testosterone. Total testosterone is measured by HPLC with tandem mass spectrometry. Serum free testosterone is then calculated by multiplying the percent free testosterone by the total testosterone concentration with unit correction.

Special Instructions

This method is recommended for testing women, children, and hypogonadal men.


This method is recommended for testing women, children, and hypogonadal men.


Testosterone: high-pressure liquid chromatography (HPLC)/tandem mass spectrometry; free testosterone: equilibrium dialysis

Specimen Requirements

Information on collection, storage, and volume


Serum, frozen


2 mL

Minimum Volume

1 mL (Note: This volume does not allow for repeat testing.)


Gel-barrier tube or red-top tube

Storage Instructions


Causes for Rejection

EDTA plasma; citrated plasma; hemolysis >5%


Serum must be separated from cells within 45 minutes of venipuncture. Send serum in a plastic transport tube. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested.