Measurement of serum testosterone using high-performance liquid chromatography/tandem mass spectrometry

Abstract

Low levels of serum testosterone typically found in women and children cannot be reliably measured by immunoassay. We developed a simple and sensitive method using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Sample preparation involved protein precipitation of serum (1.0 mL) with acetonitrile containing the internal standard (testosterone-d3) followed by liquid extraction with methylene chloride. The chromatographic cycle per specimen was 10 min. The performance was evaluated according to the CLSI EP10-A2 protocol. Within- and between-run imprecision was 20.9%, 2.29% and 1.80%, and 1.81%, 3.58% and 2.97% at mean concentrations of 0.17, 14.1 and 28.8 nM, respectively, with no apparent carryover. The method was linear from 0.21 to 53.1 nM and the analytical recovery was 100.5-106.2% across the concentrations tested. There was no interference observed from other steroids that were tested. Correlation using de-identified patient specimens with a commercial HPLC-MS/MS method showed a slope of 0.991, an intercept of -0.017 and a correlation coefficient (R(2)) of 0.998 by linear regression over concentrations ranging from 0.21 to 16.7 nM. In conclusion, we report here an HPLC-MS/MS method suitable for clinical measurement of serum testosterone.

Importance of hypogonadism and testosterone replacement therapy in current urologic practice: a review

“…The most clinically important method of direct measurement of T, as opposed to indirect immunoassays, is liquid chromatography tandem mass spectroscopy (LC-MS) [13]. This assay is based on initial separation by liquid chromatography of extracted TT, followed by measurement of the area under the curve to reflect T concentration in the sample [26]. This approach avoids internal problems of specificity and linearity of detection that are observed in immunological assays-even low T concentrations can be detected-as long as an adequate volume of specimen is provided [27].…”

“…Using non-parametric transformation, the norms were calculated from 300 to 864 ng/dL [30]. Unfortunately, this study is rather limited because it focused on only one age group (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Nevertheless, the above studies illustrate that testosterone norms need to be based in populations who are in good general health and exhibit normal reproductive and sexual function.…”

The polycystic ovary syndrome: a position statement from the European Society of Endocrinology

“…The vast majority of laboratories performing a total serum testosterone assay use direct methods without extraction before performing an immunoassay (48,49). In fact, most immunoassays yield values higher than those obtained using gas chromatography coupled with mass spectrometry (GC-MS) or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) (50,51), which are recommended as gold standard methods for evaluating steroid hormones (52,53).…”

Liquid chromatography-tandem mass spectrometry applications in endocrinology

“…A chromatogram of Te extracted from the serum of a healthy woman is shown in Figure 6, and the method's performance characteristics are listed in Table 2. Considering the analytical sensitivity and small volume of sample used (100 mL), this method is one of the most sensitive of the published LC-MS/MS methods for the measurement of Te (Cawood et al, 2005;Vicente et al, 2006;Borrey et al, 2007;Thienpont et al, 2008). To address the issue of poor performance of commercial IAs for Te, it was suggested to use the isotope-dilution LC-MS/MS method for the routine measurement of Te in samples from women and children (Rosner et al, 2007).…”