*4.3. Dihydroethidium*

Dihydroethidium (DHE), also called hydroethidine (HE), has been branded as a superoxide indicator and, when combined with the hexyl triphenylphosphonium cation (MitoSOXTM Red), it can specifically detect mitochondrial ROS. Oxidation of DHE by intracellular O2•− forms 2-hydroxyethidium (2-OH-E+), that emits a red fluorescence with excitation at 510 nm [133] (Figure 5). However, DHE is also susceptible to non-specific oxidation by other oxidants (e.g., H2O2, •OH), generating ethidium (E+), a compound with fluorescence characteristics similar to those of 2-OH-E+ [134]. For this reason, because both by-products of specific (2-OH-E+) and non-specific (E+) oxidation of DHE have overlapping fluorescence, quantification of O2•<sup>−</sup> by this means is not possible when only fluorescence-based techniques are used. Of concern, many reports have used these methods to assess O2•<sup>−</sup> in sperm cells, thus not considering the potential contribution of the non-specific oxidation of DHE via alternative pathways [135–138].

**Figure 5.** The chemistry behind the ROS detection methods based on dihydroethidium and MitoSOX Red oxidation. The non-specific oxidation, which forms ethidium, is predominant over the superoxide anion-induced reaction that results in the formation of 2-hydroxyethidium. Notably, both oxidized by-products present overlapping fluorescence properties.

An alternative to unambiguously confirm the presence of intracellular O2•<sup>−</sup> is to separately identify both 2-OH-E+ and E<sup>+</sup> with techniques such as high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS) [139,140]. Using HPLC and a reversed-phase column, the 2-OH-E+ and E<sup>+</sup> peaks can be separated and resolved, allowing O2•<sup>−</sup> quantification [140]. To the best of our knowledge, this methodology has only been used to analyse menadione-treated spermatozoa [33] and has never been used to compare the level of ROS spontaneously generated by normal and pathological sperm cells. Recently, we have used the LC–MS/MS approach to investigate sperm O2•<sup>−</sup> generation during in vitro incubation [141]. As previously reported, we also observed an increase in DHE over time. However, this was not accompanied by an increment in 2-OH-E+ levels but was rather a consequence of an increase in the level of E<sup>+</sup> (i.e., not related to O2•<sup>−</sup> generation). Our finding clearly shows the importance of distinguishing 2-OH-E+ from E<sup>+</sup> when assessing O2•<sup>−</sup> production.
