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

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.