Quantifying Testosterone by UHPLC-MS/MS

Testosterone quantification was determined using Agilent’s UHPLC Infinity II and 6495 triple quadrupole mass spectrometer, and MassHunter workstation software (8.0.8.23.5). Briefly, plasma samples containing 0.5 ng/mL ¹³C₃-testosterone (Cerilliant, Round Rock, TX) were extracted with tert-butyl methyl ether (Sigma 34875), dried, and derivatized using hydroxylamine hydrochloride (Sigma 431362). The recovered ketoxime steroids were reconstituted in methanol/water (1:1 v/v) and injected into the Infinity II UHPLC. Ketoxime steroids were separated using a Chromolith reverse phase column (RP-18 endcapped 100–2mm, Sigma 152006) and introduced into a JetStream source (Agilent) for triple quadrupole analysis. Data were analyzed and quantified using MassHunter software (Agilent) and GraphPad PRISM (version 8.0) software was used to graph and perform statistics (two-sided Students t-test)^{61 (link),62 (link)}.

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Vellano C.P., White M.G., Andrews M.C., Chelvanambi M., Witt R.G., Daniele J.R., Titus M., McQuade J.L., Conforti F., Burton E.M., Lastrapes M.J., Ologun G., Cogdill A.P., Morad G., Prieto P., Lazar A.J., Chu Y., Han G., Khan M.A., Helmink B., Davies M.A., Amaria R.N., Kovacs J.J., Woodman S.E., Patel S., Hwu P., Peoples M., Lee J.E., Cooper Z.A., Zhu H., Gao G., Banerjee H., Lau M., Gershenwald J.E., Lucci A., Keung E.Z., Ross M.I., Pala L., Pagan E., Segura R.L., Liu Q., Borthwick M.S., Lau E., Yates M.S., Westin S.N., Wani K., Tetzlaff M.T., Haydu L.E., Mahendra M., Ma X., Logothetis C., Kulstad Z., Johnson S., Hudgens C.W., Feng N., Federico L., Long G.V., Futreal P.A., Arur S., Tawbi H.A., Moran A.E., Wang L., Heffernan T.P., Marszalek J.R, & Wargo J.A. (2022). Androgen receptor blockade promotes response to BRAF/MEK-targeted therapy. Nature, 606(7915), 797-803.

Publication 2022

MassHunter workstation software 13C3-testosterone tert-butyl methyl ether hydroxylamine hydrochloride Chromolith reverse phase column (RP-18 endcapped 100–2mm, JetStream source MassHunter software GraphPad PRISM (version 8.0)

Hydroxylamine hydrochloride Ketoxime Methanol Plasma Prism Steroids Students Tert butyl methyl ether Testosterone

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, European Institute of Oncology, Tris Pharma (United States), Novartis (Switzerland), University of Milano-Bicocca, Moffitt Cancer Center, Royal North Shore Hospital, Melanoma Institute Australia, University of Sydney, Oregon Health & Science University

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Testosterone quantification

control variables

0.5 ng/mL 13C3-testosterone (Cerilliant, Round Rock, TX)

controls

Positive control: 13C3-testosterone as an internal standard
Negative control: Not explicitly mentioned

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