Mass Spectral Analysis of Sterols and Other Steroids in Different Ionization Modes: Sensitivity and Oxidation...

In the course of synthetic work and mass spectrometry (MS) with hydroxy steroids, we observed not only the loss of H₂O but prominent 2 and 4 amu losses using atmospheric pressure chemical ionization (APCI), leading to confusion of the structural assignments. This loss of 2 amu, which we attributed mainly to oxidation of hydroxyls, varied among 44 steroids and sterols analyzed; 36 showed losses of 2n amu in APCI MS analysis (17/22 Δ⁵ steroids, 17/19 Δ⁴ steroids, and 2/3 estrogens). With the Δ⁵ steroids and sterols, the precursor MH⁺ was either observed as a minor ion or (more frequently) not detected at all, constituting the base peak in 7/22 cases. With heated electrospray ionization (HESI) MS, 2n amu losses were detected (generally weakly) in 9/44 cases but constituted the base peak in 3/9. In general, the sensitivity (base peak intensity) of steroids correlated with conjugation of the of the steroid frame. Δ⁴ steroids generally performed best on HESI⁺ (up to a maximum factor of 8-fold), while Δ⁵ steroids and sterols performed better on APCI⁺ (up to >137-fold), except for two trihydroxypregnanes. Estrogens did not show a clear trend. Sensitivity generally increased with the use of NH₄F as a mobile phase additive in ESI⁺ (up to a maximum of 7-fold). We conclude that the prominence of 2n amu losses is variable among steroids and sterols but is more commonly an artifact of APCI MS. These m/z losses can constitute dominant ions that impede detection of the precursor MH⁺ and complicate structural assignments.