Abstract
Background Recent evidence suggests that 11-oxygenated C19 steroids may have an important role in the assessment of hyperandrogenism [1]. Two in particular, 11β-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) have been implicated in polycystic ovarian syndrome [2] and congenital adrenal hyperplasia [3]. At present there are limited methods available to quantitate these potentially useful analytes and no data is available on their stability. Objectives Here, we sought to develop an LC-MS/MS assay to quantify total serum testosterone (T), androstenedione (A4), 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we aimed to apply the method to evaluate the stability of 11OHA4 and 11KT. Method An LC-MS/MS assay that measured total serum T, A4, 17OHP, 11OHA4 and 11KT was developed and validated in accordance with FDA guidelines. The method was applied to assess the stability of each analyte in serum over a 3 day period. Seven BD Vacutainer® SST™ tubes were collected from 14 volunteers (6 males, 8 females) into. The tubes were stored unseparated at 20°C (68°F) and retrieved at the following times points: 0, 2, 8, 12, 24, 48 and 72h. Once retrieved, samples were centrifuged, aliquoted and stored at -20°C (-4°F) prior to analysis. Results The performance characteristics of the method were acceptable when assessed against an industry standard. Although 11OHA4 concentrations did not significantly change over the 3 day period, we observed a progressive and statistically significant increase in 11KT concentrations between day 0 to day 3 (p<0.001). This increase was most pronounced between time 0 and 8h with concentrations rising on average 26% (95% CI 18 – 33). Summary We have developed a robust LC-MS/MS assay to quantify T, A4, 17OHP, 11OHA4 and 11KT. Application of the method to assess stability has shown that 11KT concentrations increase in-vitro. This change is likely to be clinically significant and underpins the necessity to standardise sample collection before conclusions can be made about the clinical utility of 11KT. References [1] Pretorius E, Arlt W, Storbeck KH. A new dawn for androgens: Novel lessons from 11-oxygenated C19 steroids. Mol Cell Endocrinol. 2017;441:76-85. [2] O’Reilly MW, Kempegowda P, Jenkinson C, Taylor AE, Quanson JL, Storbeck KH, Arlt W. 11-Oxygenated C19 steroids are the predominant androgens in polycystic ovary syndrome. J Clin Endocrinol Metab. 2017;102:2016-3285. [3] Turcu AF, Nanba AT, Chomic R, Upadhyay SK, Giordano TJ, Shields JJ, Merke DP, Rainey WE, Auchus RJ. Adrenal-derived 11-oxygenated 19-carbon steroids are the dominant androgens in classic 21-hydroxylase deficiency. Eur J Endocrinol. 2016;174:601-609.