LC-MS Evaluation of the Redox Trypanothione Balance in Leishmania infantum Parasites

Leishmaniases are neglected tropical diseases caused by protozoan parasites of the <i>Leishmania</i> genus, with a significant global health burden, particularly in low-income regions. The parasites rely on a unique thiol-based redox system centered on trypanothione, which is essential for survival under oxidative stress encountered during their life cycle in both insect vectors and mammalian hosts. Given the absence of mammalian analogs, the trypanothione system represents an attractive target for antileishmanial drug development. However, accurate quantification of the reduced and oxidized forms of trypanothione has been challenging due to its instability and structural similarity between redox states. Here, we developed and validated a rapid, sensitive liquid chromatography-mass spectrometry (LC-MS) method for assessing the trypanothione redox state in <i>Leishmania infantum</i>. By incorporating N-ethylmaleimide as a thiol-blocking agent during sample preparation, the native redox state was preserved, enabling precise measurement of the reduced-to-oxidized ratio. Our approach demonstrated high sensitivity (nanomolar range), a rapid analysis time (5 min/sample), and robustness across various conditions. Moreover, we validated the method's relevance in detecting oxidative stress and response to the trypanothione reductase inhibitor auranofin. This LC-MS technique provides a valuable tool for exploring <i>Leishmania</i> redox biology and supports the discovery of redox-targeting therapies against leishmaniasis.