A Novel Four-step Approach for Systematic Identification of Naphthoquinones in Juglans cathayensis Dode using Various Scan Functions of Liquid Chromatography-Tandem Mass Spectrometry along with Data Mining Strategies.

Introduction: Systematic analyses of naphthoquinones in Juglans cathayensis have not yet been reported. It is very challenging to identify naphthoquinones with various structural diversities, especially those at trace levels.

Objective: To develop an efficient analytical approach for systematic discovery and identification of naphthoquinones in Juglans cathayensis.

Methodology: A novel four-step approach was evaluated by utilizing various scan functions of liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (LC-QTRAP-MS/MS) and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) along with data mining strategies. First, MS/MS fragmentation behaviors of naphthoquinones were investigated. Second, multiple ion monitoring triggered enhanced product ion scan (MIM-EPI) with specified ions was conducted to identify targeted naphthoquinones. Third, other scan functions of QTRAP-MS/MS and data mining strategies were explored to identify untargeted naphthoquinones. Fourth, structural rationalization and confirmation of naphthoquinones were performed using QTOF-MS/MS via its accurate mass measurement and MS/MS fragmentation functions.

Results: Optimal scan methods and data mining strategies using QTRAP-MS/MS were obtained for identification of targeted and untargeted naphthoquinones. Consequently, 48 naphthoquinones including 24 novel ones were identified or tentatively identified from Juglans cathayensis.

Conclusion: A novel four-step approach for efficient discovery and identification of naphthoquinones was developed by exploring various scan functions of current LC-MS/MS technologies and data mining strategies, providing an example for systematic characterization of certain classes of phytochemicals, especially trace analytes in complex samples.