Simultaneous LC-MS/MS quantification of free deoxyguanosines 2'-dG and 8-OHdG in ovine seminal plasma as biomarkers of oxidative DNA damage.

Oxidative stress is harmful for spermatic DNA, since high levels of reactive oxygen species lead to the formation of toxic adducts derived from DNA oxidation. This phenomenon is especially relevant under pathological conditions such as asthenozoospermia, necrozoospermia, oligozoospermia, or azoospermia. The nucleoside most susceptible to this damage is 2'-deoxyguanosine (2'-dG), which is oxidized to form 8-hydroxy-2'-deoxyguanosine (8-OHdG), one of the most widely studied biomarkers of oxidative stress. Previous studies have reported an increased liberation of cell-free DNA during impaired spermatogenesis and cellular necrosis, supporting the presence of free nucleosides in seminal plasma under altered physiological conditions. Consequently, the determination of both biomarkers not only provides information about the oxidative status, but also about cellular turnover. The aim of this study was, therefore, to develop, validate, and apply a reliable and complete analytical methodology for the simultaneous quantification of the mentioned biomarkers in ovine seminal plasma by solid phase extraction (SPE) off-line LC-MS/MS. The procedure includes the study and development of the SPE-based sample preparation protocol, as well as the optimization of instrumental parameters, including the description of the fragmentation pathways for both analytes and the internal standard. Validation followed US FDA guidelines for bioanalytical methods, fulfilling acceptance criteria for precision, linearity response, LOD and LOQ, recoveries, and matrix effect. In addition, sustainability was assessed in accordance with two green analytical chemistry metrics (GAPI and AGREE). The proposed method was applied to seminal plasma extracted from three healthy -pooled sample- and one ram with low sperm quality (LQ)-absence of sperm motility and severely low sperm concentration-. Both 2'-dG and 8-OHdG levels were markedly elevated in the affected ram, with approximately two-fold and four-fold increase, respectively. These promising preliminary results align with the proof-of-concept design of the study. This analytically validated approach represents the first SPE off-line LC-MS/MS method for the simultaneous quantification of free 2'-dG and 8-OHdG in seminal plasma, providing a robust analytical framework for future studies exploring these nucleosides as complementary biomarkers of oxidative DNA with potential applicability to human andrology upon validation in clinical cohorts.