Optimization of a static headspace GC-MS method and its application in metabolic fingerprinting of the leaf volatiles of 42 citrus cultivars

Citrus leaves, which are a rich source of plant volatiles, have the beneficial attributes of rapid growth, large biomass, and availability throughout the year. Establishing the leaf volatile profiles of different citrus genotypes would make a valuable contribution to citrus species identification and chemotaxonomic studies. In this study, we developed an efficient and convenient static headspace (HS) sampling technique combined with gas chromatography-mass spectrometry (GC-MS) analysis and optimized the extraction conditions (a 15-min incubation at 100 ˚C without the addition of salt). Using a large set of 42 citrus cultivars, we validated the applicability of the optimized HS-GC-MS system in determining leaf volatile profiles. A total of 83 volatile metabolites, including monoterpene hydrocarbons, alcohols, sesquiterpene hydrocarbons, aldehydes, monoterpenoids, esters, and ketones were identified and quantified. Multivariate statistical analysis and hierarchical clustering revealed that mandarin (Citrus reticulata Blanco) and orange (Citrus sinensis L. Osbeck) groups exhibited notably differential volatile profiles, and that the mandarin group cultivars were characterized by the complex volatile profiles, thereby indicating the complex nature and diversity of these mandarin cultivars. We also identified those volatile compounds deemed to be the most useful in discriminating amongst citrus cultivars. This method developed in this study provides a rapid, simple, and reliable approach for the extraction and identification of citrus leaf volatile organic compound, and based on this methodology, we propose a leaf volatile profile-based classification model for citrus.