The First 1H NMR Total Assignment and a Quantum-Mechanically Driven Full Spin Analysis of the Steroid Hormone Equilenin

Equilenin is an equine estrogen constituting the basis of a highly-prescribed pharmaceutical preparation. Although routine 1H and 13C NMR data for it have been reported, complete assignments and a full analysis of the proton spin system have not been established. In the present study, equilenin was examined by solution NMR in deuterochloroform, employing conventional spectral analysis in conjunction with quantum-mechanical techniques to achieve a 1H iterative full spin analysis (HiFSA). The resulting model reproduces the experimental spectrum with high fidelity and permits the determination of true chemical shifts and scalar coupling constants for this complex spin system. In addition, the 13C NMR spectrum was fully assigned using a combination of one- and two-dimensional experiments. The obtained data constitute a robust spectroscopic reference set for equilenin and the analytical value of the Cosmic Truth software for resolving spin systems in steroids. The results provide a valuable source of data for researchers seeking to implement NMR-based assays relevant to analytical, regulatory, and forensic applications.