Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry

Abstract

Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stable-isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether-ethyl acetate from 250 microL or 500 microL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode after separation on a reversed-phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d(2) testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2-100 nmol/L. Interassay CVs were 4.2-7.6 % at mean concentrations of testosterone of 3.3-45 nmol/L. Total measurement uncertainty (U, k = 2) was 12.9 % and 13.4 % at testosterone levels of 2.0 nmol/L and 20 nmol/L, respectively. The limit of detection was 0.05 nmol/L (signal-to-noise ratio = 3) and the overall method recovery of testosterone was 95 %. Correlation (r) with our in-house extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18-30, 31-50, 51-70 and over 70 years were established. Sensitivity and specificity of the LC-MS/MS method offer advantages over immunoassay and make it suitable for use as a high-throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.

The regulations about eligibility for women with hyperandrogenism to compete in women′s category are well founded. A rebuttal to the conclusions by Healy et al.

“…Lean body mass (LBM): We believe that this is the key to the difference in strength and performance between men and women as well as to the reason why this new regulation relating to circulating testosterone levels in the blood introduced by IOC/IAAF is based on loose thinking and false pseudo-science. Men have a LBM that is on average 10 kg higher than that of a woman of the same height 3 and this supports Dr Ritzen and colleagues' recent published statement that: 'The world records for men in sports that rely on strength or size are on average 10% better than for women'. 4 Lean body mass is largely determined by height (Fig.…”

“…The authors do not mention the publications that, under resting conditions, demonstrate no overlap in testosterone levels between healthy men and women. 3 While the IAAF/IOC Regulations state that blood samples must be drawn under resting conditions, Healy et al obtained blood within two hours after completing national or international competitions. Others have shown that following strenuous exercise, testosterone levels in men decrease, while in women, T is unchanged or moderately increased.…”

Muscle function in monozygotic female twin pairs discordant for hormone replacement therapy

“…Sex hormone binding globulin (SHBG) levels were measured using solid-phase, chemiluminescent immunometric assays (Immulite 1000, Diagnostic Products Corporation, Los Angeles, CA, USA). Serum 17β-estradiol (E2) was determined in duplicate by extraction RIA as previously described 17 and testosterone levels as previously described 18 . E2 and testosterone were utilized together with SHBG in calculating the free E2 and free testosterone levels, respectively, according to a previously presented method 19 .…”

Combination of hormone replacement therapy and high physical activity is associated with differences in Achilles tendon size in monozygotic female twin pairs

“…17␤-Estradiol levels were utilized together with SHBG in calculating the free E 2 levels, according to a previously presented method (3). Testosterone was measured as previously described (21).…”