Endocrine Abstracts (2013) 32 P338 | DOI: 10.1530/endoabs.32.P338

TurboFlow-LC-MS/MS method for quantification of DHEA, DHEAS, 17[alpha]-hydroxyprogesterone, [delta]-androstenedione and testosterone in children

Tue Søeborg, Hanne Frederiksen, Trine Holm Johannsen, Anders Juul & Anna-Maria Andersson

Department of Growth and Reproduction, Rigshospitalet, Faculty of Health and Medical Sciences, Copenhagen University Hospital, University of Copenhagen, Copenhagen, Denmark.

Diagnosis and management of infants and children with sex steroid disorders requires fast and simultaneous assessment of several sex steroid metabolites in serum at low concentrations and on small sample volumes. Therefore, we developed a sensitive and selective TurboFlow-LC-MS/MS method for quantification of DHEA, DHEAS, 17α-hydroxyprogesterone, Δ4-androstenedione and testosterone in serum from pre-pubertal children.

Total run time was 10.75 min with the effluent being directed to the mass spectrometer for 2.6 min. Limits of quantification were determined as described by the International Conference on Harmonisation (ICH) with the following values: DHEA, 0.88 nM; DHEAS, 48 nM; 17α –hydroxyprogesterone, 0.19 nM; Δ4-androstenedione, 0.18 nM and testosterone, 0.10 nM. Intra-day relative S.D. ranged from 4.6 to 13.8% and inter-day relative S.D. ranged from 5.7 to 15.7%.

Steroid concentrations in 186 serum samples from children (8.4–14.8 years old) were compared with results obtained by immunoassays for DHEAS, Δ4-androstenedione and testosterone. DHEAS and testosterone gave overall similar results with mean values 19 and 18% higher, respectively by LC-MS/MS, while levels of Δ4-androstenedione on average were found to be 83% higher when analysed by immunoassay. DHEAS was quantified in all samples with both methods, while Δ4-androstenedione and testosterone were quantified in 78 and 61% of the samples, respectively using immunoassay and in 98 and 94% of the samples, respectively using the LC-MS/MS method. Concentrations of the five steroids determined by LC-MS/MS were similar to previously published results.

The presented method is suitable in a clinical setting for simultaneous quantification of five steroids important for management of children with disorders of sex development and steroid biosynthesis defects. Our study illustrates the importance of LC-MS/MS technology for quantification of – at least – Δ4-androstenedione and testosterone at low levels in children as an alternative to conventional immunoassays.

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