A Sensitive and Selective UHPLC–MS/MS Method Based on Mercaptopropionic Acid Derivatization for Quantification of Juglone in Biological Matrices

In this study, a highly sensitive and selective ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for the quantification of juglone, a poorly ionizable naphthoquinone, in biological matrices. To enhance ionization efficiency, pre-column derivatization was employed using sulfhydryl nucleophiles (β-mercaptoethanol and 3-mercaptopropionic acid) via Michael addition. Derivatization with mercaptopropionic acid significantly improved mass spectrometric response (50-fold increase) and minimized matrix effects compared to mercaptoethanol. The method, validated per FDA guidelines, exhibited excellent linearity ( r ≥ 0.995) from 3 to 150 ng/mL, with accuracy within −10.6% to 4.3% and precision between 1.29% and 5.34%. The lower limit of quantification was 3 ng/mL. Extraction recovery and matrix effects ranged from 99.84% to 102.06% and 92.75% to 98.98%, respectively, with derivatives demonstrating good stability. Cellular uptake studies in MCF-7 cells revealed time-dependent but limited intracellular accumulation of juglone, with the majority retained in the culture medium. This robust derivatization-based UHPLC–MS/MS approach provides a reliable tool for investigating juglone's pharmacokinetics and toxicological mechanisms.