Ozonation of ethinylestradiol in aqueous-methanolic solution: direct monitoring by electrospray ionization mass spectrometry

Abstract

The ozonolysis of ethinylestradiol (1), a synthetic steroidal estrogen, in an aqueous-methanolic solution was investigated. HPLC-UV analyses revealed that 1 was completely consumed after a 20 min reaction time either at pH 5 or 8. ESI(-)-MS (electrospray ionization mass spectrometry in the negative ion mode) monitoring also revealed the continuous consumption of 1 (detected as [1 – H]- of m/z 295) concomitantly with the emergence of oxidation products. Chemical structures were proposed for these products based on the data of MS and MS/MS (the m/z values and fragmentation profiles of the anionic specie, respectively). These data, in conjunction with the well-established knowledge about the reactivity of organic molecules toward ozone in aqueous solution, were evaluated and an unprecedented reaction route for the ozonation of 1 could thus be suggested. Hence, the first step in this reaction sequence was ascribed to involve a 1,3-dipolar cycloaddition of ozone at the phenolic ring of 1 yielding the di-hydroxylated product 2 (detected as [2 – H]- of m/z 311). The loss of acetylene as the unique dissociation channel for [2 – H]- thus confirmed that such hydroxylation occurred at the phenolic ring rather than the acetylenic moiety of 1. Subsequent oxidations were proposed to be the origin of a number of other products, all of them bearing the COOH functionally (this was verified by the characteristic loss of CO2 during the dissociation of the related deprotonated molecules). The ESI(-)-MS records also revealed notable differences between the reaction conducted at pH 5 and 8, i.e. at slightly acid or basic media, respectively.