Instant CMQT stabilization coupled UHPLC-MS/MS for the quantification of reduced and total forms of cysteine, homocysteine, and glutathione in blood plasma.

BACKGROUND: Reduced and total forms of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are key players in redox homeostasis, and their dysregulation is linked to diseases such as sepsis. However, accurate quantification of reduced thiols in plasma remains challenging due to rapid air oxidation of native thiols. In this work, a thiol-specific reagent (2-chloro-1-methylquinolinium tetrafluoroborate, CMQT) that reacts rapidly under mild conditions was coupled with UHPLC-MS/MS to develop a method for the simultaneous determination of reduced and total forms of Cys, Hcy, and GSH in plasma samples.

RESULTS: CMQT rapidly stabilizes native thiols in whole blood without inducing hemolysis. The proposed method was validated for linearity, precision, accuracy and sensitivity. Calibration curves showed excellent linearity (R2 > 0.9993). Intra- and inter-day precisions ranged from 0.14% to 7.23%, and accuracy from -5.62% to 4.15%. The limits of detection for the reduced and total forms of Cys, Hcy and GSH ranged from 0.051 to 0.88 μM. The method was successfully applied to a lipopolysaccharide-induced endotoxemia mouse model, revealing that reduced Hcy and reduced GSH may serve as potential biomarkers for early and late oxidative stress, respectively. The method was also validated on human plasma samples, confirming its applicability across species and revealing sex-related thiol differences.

SIGNIFICANCE: The combination of rapid and hemolysis-free thiol stabilization with MS-based structural characterization provides a robust analytical tool for thiol redox studies and could be extended to other biological matrices.