Optimizing Serum Sample Preparation for Free Testosterone Determination: A Comparative Analysis Using BioSPME

by | Jan 17, 2024

Discover BioSPME: A faster, more reliable method for free testosterone measurement.

This article from issue 15 of the Analytix Reporter introduces a new method using BioSPME (Biocompatible Solid Phase Microextraction) for preparing serum samples to measure free testosterone. BioSPME is an alternative to traditional equilibrium dialysis and employs a 96-pin device and a robotic system that can be used for efficient sample processing within an hour. The study demonstrates that BioSPME offers a reliable and faster method for free testosterone measurement in serum samples, showing a strong correlation with the established equilibrium dialysis LC-MS/MS method.

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The industry’s gold standard for sample preparation of free hormones from serum has been associated with equilibrium dialysis. It is the free portion of hormones, including testosterone, that is responsible for biological activity. Free testosterone accounts for approximately 1-2% of total testosterone. In males, this generally falls in the range of 20–230 pg/mL, and for females in the range of 0.6–10 pg/mL (0.8-1.4% of total). Solid phase microextraction (SPME) is a relatively new method to employ for the measurement of free concentration and one that has recently been incorporated into a 96-pin device for use with conventional well plates. Biocompatible SPME, or BioSPME, has been shown to be a fast sample preparation technique.


The BioSPME sample preparation method for the 200 µL samples utilized a Supel™ BioSPME C18 (see Figure 1 in the article) 96-pin device with a Hamilton® STARlet system. Although the method includes multiple steps, it was developed to have only a total processing time. See complete experimental data in the article.


The instrumental limit of detection, LOD, and lower limit of quantification, LLOQ, of the derivatized testosterone, Te-NHOH, were determined by serial dilution and n=12 injections (see Table 3 in the article to view the full results).


A BioSPME extraction method prior to analysis by LC-MS/MS was developed, and the evaluated results showed a strong correlation (R2 = 0.92–0.96) for serum samples analyzed by externally validated equilibrium dialysis LC-MS/MS for free testosterone. The BioSPME method was automated by using a Hamilton® Starlet Robotic system and can be adapted to other robotic liquid handlers that have gripper functionality. The time to process one 96-well plate was approximately an hour. The developed LC-MS/MS detection method used derivatization of the final extract by hydroxylamine hydrochloride to increase the sensitivity for the detection of free testosterone.

Learn more about BioSPME at SigmaAldrich.com/biospme

*The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

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