Using GC-MS/MS for Superior Sensitivity, Specificity and Precision in Free Testosterone Analysis - Rohan A. Thakur, Clark Williard and Agnita Rajasekaran

This article discusses biologically important steroid measurements which have, in the past, seen a lack of analytical sensitivity. Examples include determination of
testosterone levels in women and children and oestrogen levels in post-menopausal women. The article gives insight into the analysis of free testosterone at physiologically relevant levels in plasma ultra-filtrate, concentrating on the sensitivity, specificity, precision and accuracy advantages offered by GC-MS/MS.

INTRODUCTION
Until 1955, mass spectrometry (MS) was most commonly used for the direct analysis of volatiles. Gas chromatography (GC) was coupled to MS for the first time
at Dow Chemical Company with the aim to expand the analytical capabilities of MS to cover complex mixtures of unknowns [1]. Subsequent technological developments involved the introduction of hybrid mass analyzers such as the triple stage quadrupole (TSQ) mass spectrometers and the use of tandem MS (MS/MS or SRM) as a high specificity technique for routine quantitative analysis of complex mixtures. GC-MS/MS was quickly established as the technology of choice for bioanalytical applications.

Although highly efficient for the analysis of complex mixtures, GC-MS/MS could not address the requirement of the industry for the analysis of larger non-volatile
molecules. To overcome this new challenge, high performance liquid chromatography (HPLC) was coupled to MS/MS (LC-MS/MS) leading to the development of the
atmospheric pressure based ionisation (API) technique of electrospray (ESI). This new method soon was soon adopted as the preferred technology for bioanalysis [2].

In addition, LC-MS/MS was seen as offering a number of advantages that allowed for easy, uncomplicated bioanalysis of small molecules. Compared to GC-MS/MS, the technique requires very little sample preparation while delivering good detection limits and is also capable of analysing a wider range of compound classes and molecular weights. Latest advances have seen the introduction of TurboFlow, on-line solid phase extraction (SPE) and other chromatographic techniques that facilitate direct injection of biological fluids into LC-MS/MS systems, eliminating sample preparation [3]. These methods are particularly beneficial for high-throughput laboratories, where rapid analysis of thousands of samples is needed. However, in applications where assay precision and accuracy are of utmost importance, LC-MS/MS may not always deliver the required results. Indeed for the highly sensitive and specific detection of steroids for example, it has been suggested that GC-MS/MS might actually be the preferred technique.