Development of a total serum testosterone, androstenedione, 17-hydroxyprogesterone, 11β-hydroxyandrostenedione and 11-ketotestosterone LC-MS/MS assay and its application to evaluate pre-analytical sample stability

Abstract

BackgroundClassically, serum testosterone (T) and androstenedione (A4) have been the mainstay for the biochemical assessment of hyperandrogenism. However, recent evidence suggests 11β-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) may also be important. Here, we describe the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitation of total serum T, A4, 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we applied the method to assess pre-analytical stability.MethodsAn isotopically labelled internal standard was added to samples prior to supported liquid extraction (SLE). Extracts were analysed using LC-MS/MS to detect T/A4/17OHP/11OHA4 and 11KT along with their corresponding internal standards. Samples (n = 7) were collected from healthy volunteers (n = 14) and left incubated at 20 °C for up to 72 h. Tubes were retrieved at select time points, centrifuged, separated and frozen prior to analysis.ResultsThe total run time was 4 min. For all analytes, intra- and inter-assay imprecision did not exceed 7.9% and 5.3%, respectively; matrix effects were negligible and mean recoveries ranged from 95.3 to 111.6%. The limits of quantitation (LOQs) were 0.25 nmol/L for T, A4 and 11OHA4, 0.50 nmol/L for 17OHP, and 0.24 nmol/L for 11KT. No significant change was observed in pre-centrifugation A4 or female T concentrations over 72 h. Significant increases (p < 0.01) in concentrations of 11KT, 17OHP, 11OHA4 and male T were observed after 2, 8, 12 and 24 h, respectively.ConclusionsWe developed a robust LC-MS/MS assay for the quantitation of total serum T/A4/17OHP/11OHA4 and 11KT. Applying the method to determine pre-analytical stability suggests samples requiring 11KT need separating from the cells within 2 h.

Peripheral blood mononuclear cells preferentially activate 11-oxygenated androgens

“…The preference of PBMCs to generate 11KT from 11KA4 via AKR1C3 activity is further reflected in the significant increases in 11KT serum concentrations, if cellular components were not removed from the whole blood samples in a timely fashion, confirming previous preliminary observations (23). We found that these changes became significant after two hours at room temperature without separation, suggesting that blood samples for the measurement of 11-oxygenated androgens should be processed within two hours of collection.…”

“…Of note, a number of studies have shown gradual increases in T measured in serum, if the blood sample is left unseparated from cellular components for an increased period of time, using either immunoassays (19,20,21) or ultra-high performance liquid chromatographytandem mass spectrometry (LC-MS/MS) for steroid quantification (22,23). One of those studies additionally investigated the effect of time before separation on 11KT concentrations measured in serum and observed a much more pronounced increase in 11KT than T over time (23), which could have a differential impact on the measurement of circulating 11-oxygenated androgen concentrations.…”

Elevated 11-oxygenated androgens are not a major contributor to HPG-axis disturbances in adults with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

“…Total testosterone, 17-OHP, androstenedione, 11-KT and 11OHA4 were quantified by LC-MS/MS. Steroid extraction and quantitation was carried out as previously described (20).…”

Steroid Ligands, the Forgotten Triggers of Nuclear Receptor Action; Implications for Acquired Resistance to Endocrine Therapy

“…Steroids [4AD, 11-keto-testosterone (11KT), 11b-hydroxyandrostenedione (11OHA4), testosterone (T), 17-OH-progesterone (17OHP), and dehydroepiandrosterone sulphate (DHEAS)] were quantified in serum samples from patients with breast cancer (n ¼ 6) using previously described LC/MS-MS methods (23,24). The same samples were analyzed for 17b-estradiol (E 2 ) using LC/MS-MS, the details of the method and its performance characteristics are provided in Supplementary Data (Supplementary Materials and Methods).…”