ESPE Abstracts (2015) 84 P-1-147

A New LC-MS/MS Assay for the Analysis of Sulfated Steroids in Human Serum: Quantification of Cholesterol Sulfate, Pregnenolone Sulfate, 17-Hydroxypregnenolone Sulfate and Androgen Sulfates

Alberto Sánchez-Guijoa, Vinzenz Ojib, Michaela F Hartmanna, Heiko Traupeb & Stefan A Wudya

aJustus-Liebig-University, Giessen, Germany, bUniversity of Münster, Münster, Germany

Background: Steroids are found in human blood predominantly as sulfated steroids. Conjugation of steroids increases their solubility in blood, facilitating their physiological regulation and excretion. Chromatographic separation and quantification of an extensive number of sulfated steroids is challenging. For instance, androgen sulfates are structurally related and their signals are very similar in mass spectrometry.

Objective and hypotheses: Some of the most abundant sulfated steroids are cholesterol sulfate (CS), dehydroepiandrosterone sulfate (DHEAS) and androsterone sulfate (AnS). So far, no assay has been developed for the simultaneous quantification of such compounds present in higher concentrations.

Method: We developed a novel assay for the quantification of the aforementioned compounds and other eight sulfated steroids in human serum by LC-MS/MS. The method uses 300 μl volume of serum, and the sulfated steroids are analyzed independently from free steroids, which are isolated during the sample preparation too. The method allows for the quantification of CS, DHEAS, AnS, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, androstenediol sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate and dihydrotestosterone sulfate.

Results: The performance of the method has been studied at three different concentration levels for each compound, allowing for the study of a broad range of concentrations. The method is sensitive and the quantification parameters are reliable. The average recovery was 97.6%. Averaged intra and inter-assay accuracies were 8.0 and 6.3%, respectively, and precision was always lower than 20% at all the concentration levels.

Conclusion: We present a reliable method to quantify sulfated steroids in human serum. The study of serum from patients with steroid sulfatase deficiency has proven the utility of this assay for the diagnosis of this condition by analysing the levels of CS.

Funding: This work was supported by the Selbsthilfe Ichthyose e. V., the Medical Faculty (OJ111409) of the University of Münster, and by the German Research Foundation (DFG) within DFG Research Group 1369 “Sulfated Steroids in Reproduction” to subproject 7.

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