A sharp, robust, and quantitative method by liquid chromatography tandem mass spectrometry for the measurement of EAD for acute radiation syndrome and its application

- A sensitive LC-MS/MS approach was successfully developed and validated for EAD.
- The method was applied for PK study of EAD after a dose of 100 mg kg−1 administration.
- It was the first time to study PK of EAD, a steroid, by LC-MS/MS method.

17-Ethinyl-3,17-dihydroxyandrost-5-ene (EAD) is an agent designed for the treatment of acute radiation syndrome (ARS). Given its vital role played in the prevention and mitigation of ARS, the development of a sharp, sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor the metabolism of EAD in vivo was crucial. A new method was constructed and validated for the determination of EAD with the internal standard of androst-5-ene-3β,17β-diol (5-AED). The blood samples were precipitated with methanol, centrifuged, from which the supernatant was separated on UPLC with C18 column and eluted in gradient with acetonitrile and Milli-Q water both containing 0.1% formic acid (FA). Quantification was performed by a triple quadrupole mass spectrometer with electro spray ionization (ESI) in multiple reactive monitoring (MRM) positive mode.A good linearity was obtained with R > 0.99 for EAD within its calibration range from 5 to 1000 ng mL−1 with a lowest limit of quantification (LLOQ) of 5 ng mL−1. Inter- and intra-day accuracy and precision of three levels of quality control (QC) samples were within the range of 15%, while the LLOQ was within 20%. Samples were stable under the circumstances of the experiments. The method was simple, accurate and robust applied to determine the concentrations of EAD in Wistar rat after a single administration of EAD orally at the dose of 100 mg kg−1.