Performance Characteristics of a Novel Tandem Mass Spectrometry Assay For Serum Testosterone

Abstract

Background: Commercial immunoassays for testosterone (Te) may give inaccurate results for samples from women and children, leading to misdiagnosis and inappropriate treatment. We developed a sensitive and specific tandem mass spectrometric assay for measurement of Te at the concentrations encountered in women and children. Methods: Te was extracted with methyl tert-butyl ether from 100 L of serum or plasma, derivatized to form an oxime, and reextracted by solid-phase extraction. Instrumental analysis was performed on an API 4000 HPLC tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode. The MRM transitions (m/z) were 3043124 and 3043112 for Te and 3073124 and 3073112 for d 3 -Te. Results: Within-and between-run CVs were <12% and 7.9%, respectively. The limit of quantification was 0.0346 nmol/L (1 ng/dL). Reference intervals for sex hormone-binding globulin and total, free, and bioavailable Te were established for children of Tanner stages 1 through 5 and adult males and females. Conclusions: The sensitivity and specificity of the method are adequate for analysis of Te in samples from women and children. The method requires small sample volumes, has adequate precision, and is not subject to interferences.

Domestication effects on behavioural and hormonal responses to acute stress in chickens

“…Briefly, steroids were extracted from samples and CORT, DHEA, A4, Pregn, and PROG were derivatized with hydroxylamine to form oxime derivatives; E1, E2 and E3 were derivatized with dansyl chloride to form dansyl derivatives. Limit of quantification (LOQ) was 0.05 ng/ml for Pregn, 1 ng/ml for PROG, 0.5 ng/mL for CORT, 0.01 for Te and A4 and 0.05 ng/ml for DHEA,1 pg/ml for E1, E2 and E3 [45]. The intra-assay and inter-assay CV were < 8 and 11%, respectively [47].…”

“…Plasma samples were analysed for Pregn, CORT, DHEA, A4, Te, PROG, E1, E2 and E3 using liquid chromatography tandem mass spectrometry methods (LC-MS/MS) as previously described [45][46][47]. Briefly, steroids were extracted from samples and CORT, DHEA, A4, Pregn, and PROG were derivatized with hydroxylamine to form oxime derivatives; E1, E2 and E3 were derivatized with dansyl chloride to form dansyl derivatives.…”

Liquid chromatography-tandem mass spectrometry applications in endocrinology

“…Functional groups commonly targeted for the derivatization of endogenous steroids are keto and hydroxyl groups (Anari et al, 2002;Xu et al, 2004;Kushnir et al, 2006aKushnir et al, ,b, 2008; and the gain in the sensitivity in methods that use chemical derivatization can be up to few orders of magnitude.…”

“…Derivatization proved to be useful in the high-throughput screening of acylcarnitines (Millington et al, 1989), amino acids (Chace et al, 1993), and organic acids (Johnson, 1999); high-throughput quantitative analysis of methylmalonic acid (MMA) (Kushnir et al, 2001;Kushnir, Rockwood, & Nelson, 2004); to reduce the sample volume required for the analysis of endogenous steroids (Kushnir et al, 2006a(Kushnir et al, ,b, 2008; and in cases when very limited samples are available for analysis (Kushnir et al, 2009). …”

Steroid measurement with LC–MS/MS in pediatric endocrinology

“…Several LC-MS/MS methods have been described for the quantification of testosterone in serum/plasma to overcome this problem (Cawood et al, 2005;Ismail et al, 2002;Kushnir et al, 2006a;Moal et al, 2007;Rauh et al, 2006;Singh, 2008;Taieb et al, 2003).…”