07/24/2026
Challenges in the development of a method for the detection of Free Testosterone
Testosterone is an androgen steroid hormone that circulates in blood predominantly bound to sex hormone-binding globulin (SHBG), weakly bound to albumin, and as a small unbound fraction known as free testosterone (FT). Testosterone exists in a dynamic equilibrium, with only ~1–2% of total testosterone being free in men and as little as 0.2–1% in women.
In the present study, for which we prepared a poster that was presented at the 12th EBF Young Scientist Symposium (please see below for your reference), we aim to optimize a competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of free testosterone within a regulated pharmacokinetic (PK) framework, with the objective of achieving a sensitivity in the range of 0.2–0.6 pg/mL. This poster presents key challenges encountered during method development and validation, and discusses the strategies implemented to overcome them.
Unlike conventional PK assays that quantify non-endogenous drug concentrations, this method focuses on the free fraction of a drug with an endogenous counterpart.
As FT is in equilibrium with binding proteins in the sample matrix, any modification to matrix conditions can alter this equilibrium. A major challenge was the establishment of reliable quality controls, as predicting the resulting free testosterone concentration in drug spiked samples proved difficult. Several diluents, buffers, and sample treatments were evaluated. Treated hormone-free matrix and freezing of quality controls allowed the system to reach equilibrium and improved assay performance.
The method is being evaluated using serum samples from post-menopausal women, a population with low expected free testosterone concentrations. This work supports the development of a non-conventional PK assay, for the quantification of biologically active hormone fractions at low-concentrations.
Get in touch with our team to discuss your upcoming bioanalytical requirements — we will be pleased to support you in moving your project forward.
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