MSACL 2024 Abstract

Establishment of an LCMS/MS Assay to Measure 11-Oxygenated Androgens Tatiana C Coverdell, Ning Pan Bernhardt, Caroline Nottingham Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland Tatiana Coverdell, PhD (Presenter) National Institutes of Health

Presenter Bio: Tatiana Coverdell is a clinical chemistry fellow at the National Institutes of Health in Bethesda, Maryland. She completed her doctoral studies with Professors John Campbell and Stephen Abbott at the University of Virginia in 2022, where her research was focused on nucleus ambiguus neurons in the hindbrain and their control over swallowing, speech, and cardiorespiratory function.

INTRODUCTION: 11-oxygenated androgens are important biomarkers for many disorders, including polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH). Unfortunately, testing of these compounds is limited due to a lack of assay availability, especially in a clinical setting. Future studies utilizing an LCMS/MS method for measuring these compounds could allow for the characterization of these biomarkers that accurately reflect disease control and facilitate treatment monitoring.

OBJECTIVES: The primary objective of this study was to establish an in house assay using liquid chromatography tandem mass spectrometry (LCMS/MS) to quantitatively measure the following 11-oxygenated androgens in serum: 11-ketotestosterone, 11-hydroxytestosterone, 11-hydroxyandrostenedione, and 11-ketoandrostenedione.

METHODS: Serum was obtained from healthy and affected patient populations. Serum was then combined with internal standards, diluted with deionized water, and then loaded on a supported liquid extraction cartridge. Methyl-tert-butyl-ether (MTBE) was used to elute the steroids from the column. The eluate was then concentrated under nitrogen and reconstituted with methanol and water. Samples were then injected via an autosampler and resolved with a C18 column on an Agilent 1290 binary pump and Agilent 6490 Triple Quadrupole LCMS/MS equipped with an electrospray ionization (ESI) source. Mobile phases consisted of aqueous ammonium fluoride and ammonium fluoride in methanol. Steroids were quantified using dynamic multiple reaction monitoring mode.

RESULTS: We determined retention times and qualifier ions for each compound. We also built calibration curves for each compound with R squared values greater than 0.98. For each compound, we determined an analytical measurement range that was relevant to values that would be expected for affected and healthy individuals. We also determined lower limits of quantification.

CONCLUSION: We established an in house LCMS/MS method for quantification of the following four 11-oxygenated androgens in serum: 11-ketotestosterone, 11-hydroxytestosterone, 11-hydroxyandrostenedione, and 11-ketoandrostenedione. This assay can be further used to diagnose and aid in treatment for individuals with PCOS, CAH, and other disorders resulting from excess adrenal androgens.