22


August/September 2010


Using GC-MS/MS for Superior Sensitivity, Specificity and Precision in Free Testosterone Analysis


By Rohan A. Thakur, Clark Williard and Agnita Rajasekaran Taylor Technology - a subsidiary of PharmaNet Development Group, Princeton, NJ


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 estrogen 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 ionization (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 analyzing 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.


Estrogens (estradiol and estrone) and androgens (testosterone and androstenedione) are a class of biologically important endogenous compounds that routinely require reproducible results at very low detection limits (attogram level). Endogenous and exogenous estrogens are associated with the pathogenesis of breast cancer [4]


biological functions and is used as a therapeutic agent for the treatment of


hypogonadism, the stimulation of erythropoiesis and hereditary angioneurotic edema [5]


. As a consequence,


measurements of estrogens and androgens is of high biological importance, determining for example testosterone levels in women and children and estrogen levels in post-menopausal women [6,7]


.


Analytical sensitivity and specificity play major roles in the interpretation of steroid measurements. Sample preparation is critical for the sensitivity, precision and accuracy of results. Ion suppression can be directly related to inadequate sample preparation and is a major problem with ESI based LC-MS/MS techniques [8]


.


The limitations of LC-MS/MS related to accuracy, precision, specificity and sensitivity have been significant enough to initiate an inter-laboratory comparison study of serum total testosterone measurements performed by mass spectrometric methods [6]


. Recent whereas testosterone affects major


data indicates that GC-MS/MS offers three key advantages over LC-MS/MS when it comes to this type of analysis. The derivatization technique improves sensitivity (Neg Ion CI dramatically reduces chemical noise), the high number of theoretical plates of a GC column results in highly focused chromatographic peaks (approximately 3 sec or less), and the entire column eluent enters the ion source (unlike LC-MS/MS ion sources which sample 5% of the LC eluent) . As a


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