Mass defect filtering combined with molecular networking to profile flavonoids in citrus fruit based on liquid chromatography-high resolution mass spectrometry platform: Citrus sinensis (L.) Osbeck as a case study

Abstract

Citrus flavonoids are attracting great interest due to their well-known beneficial effects, but many of them have not been characterized. In this work, ultra-high liquid chromatography coupled to high resolution mass spectrometry (UHPLCHRMS) was used for profiling flavonoids in citrus fruit. We proposed a strategy combining mass defect filtering (MDF) and MS/MS–based molecular networking (MMN) to handle complex UHPLC–HRMS data. The proposed strategy was explained and validated in the fruit of Citrus sinensis (L.) Osbeck, and when specific mass and mass defect windows were pre-defined, MDF enable removal of considerable un-related and/or interference MS1 peaks. In citrus fruit, the number of MS1 peaks in positive and negative modes were reduced by 70.80% (from 15,113 to 4413) and 55.30% (from 5617 to 2511), respectively, and thus the potential MS features of flavonoids were retained and exposed. After MDF, an MS/MS similarity-based MMN map was constructed to cluster flavonoids with similar chemical structures. MMN facilitated the annotation of 65 unknown citrus flavonoids by using only 21 pre-identified flavonoids as references. The compounds comprised 42 polymethoxylated flavonoids, 17 flavones, 24 flavanones, and 3 flavonols. Eleven of them had not been previously reported in Citrus sinensis (L.) Osbeck to our knowledge. Results of the current work indicated that the combination of MDF and MMN is a useful strategy for removing interference MS1 peaks and performing the structural annotation of unkonwn compounds in complex samples.