A hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the quantification of intracellular cystine and its application in cystinosis research.

≥ 0.9986) and a lower limit of quantification (LLOQ) of 12.5 nM, representing a > 8-fold improvement over reported reversed-phase LC methods. Addition of N-ethylmaleimide (NEM) prior to cell lysis effectively limited cysteine oxidation and maintained sample stability at 4 °C. Applicability was demonstrated in an isogenic laboratory HEK293T cell model, where CTNS-knockout (KO) cells exhibited significantly elevated intracellular cystine levels compared to wild-type (WT) cells. As additional controls, elevated levels were successfully restored following cysteamine treatment or lentiviral-vector (LV)-mediated CTNS protein re-expression. The developed method hence provides a sensitive and reliable analytical platform for in vitro evaluation of novel therapeutic strategies in cystinosis research.