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A Sensitive and Selective UHPLC-MS/MS Method Based on Mercaptopropionic Acid Derivatization for Quantification of
Xinyue Zheng1,2, Yue Deng2, Si Cheng3
1Central Hospital of Dalian University of Technology, Dalian University of Technology, Dalian, People's Republic of China.
Journal of Separation Science
|April 15, 2026
Summary
A new ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method uses derivatization to accurately quantify juglone. This technique improves detection of juglone in biological samples for pharmacokinetic and toxicological studies.
Area of Science:
- Analytical Chemistry
- Pharmacology
- Biochemistry
Background:
- Juglone, a naphthoquinone, is poorly ionizable, posing challenges for quantification in biological matrices.
- Accurate measurement of juglone is crucial for understanding its pharmacokinetics and toxicological effects.
- Existing analytical methods may lack the sensitivity or selectivity required for complex biological samples.
Purpose of the Study:
- To develop and validate a highly sensitive and selective UHPLC-MS/MS method for juglone quantification.
- To improve the ionization efficiency of juglone through pre-column derivatization.
- To investigate the cellular uptake and distribution of juglone in MCF-7 cells.
Main Methods:
- Development of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method.
- Pre-column derivatization of juglone using sulfhydryl nucleophiles (3-mercaptopropionic acid) via Michael addition to enhance ionization.
- Validation of the method according to FDA guidelines, assessing linearity, accuracy, precision, and limits of quantification.
- Cellular uptake studies using MCF-7 cells to determine juglone accumulation.
Main Results:
- Derivatization with 3-mercaptopropionic acid yielded a 50-fold increase in mass spectrometric response and reduced matrix effects.
- The validated method demonstrated excellent linearity (r ≥ 0.995) from 3 to 150 ng/mL.
- High accuracy (-10.6% to 4.3%) and precision (1.29% to 5.34%) were achieved, with a lower limit of quantification of 3 ng/mL.
- Cellular uptake studies showed limited intracellular accumulation of juglone, with most remaining in the culture medium.
Conclusions:
- A robust derivatization-based UHPLC-MS/MS method was successfully developed for sensitive and selective juglone quantification.
- The method overcomes ionization challenges, providing a reliable tool for pharmacokinetic and toxicological research.
- Juglone exhibits time-dependent but limited cellular accumulation, with a preference for remaining in the extracellular medium.
Keywords:
cytotoxicityjugloneliquid chromatography–tandem mass spectrometrypharmacokinetic studypre‐column derivatizationMore Related Videos
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