Chromatographic fingerprint analysis of secondary metabolites in citrus fruits peels using gas chromatography–mass spectrometry combined with advanced chemometric methods

Multivariate curve resolution (MCR) and multivariate clustering methods along with other chemometric methods are proposed to improve the analysis of gas chromatography–mass spectrometry (GC–MS) fingerprints of secondary metabolites in citrus fruits peels. In this way, chromatographic problems such as baseline/background contribution, low S/N peaks, asymmetric peaks, retention time shifts, and co-elution (overlapped and embedded peaks) occurred during GC–MS analysis of chromatographic fingerprints are solved using the proposed strategy. In this study, first, informative GC–MS fingerprints of citrus secondary metabolites are generated and then, whole data sets are segmented to some chromatographic regions. Each chromatographic segment for eighteen samples is column-wise augmented with m/z values as common mode to preserve bilinear model assumption needed for MCR analysis. Extended multivariate curve resolution alternating least squares (MCR-ALS) is used to obtain pure elution and mass spectral profiles for the components present in each chromatographic segment as well as their relative concentrations. After finding the best MCR-ALS model, the relative concentrations for resolved components are examined using principal component analysis (PCA) and k-nearest neighbor (KNN) clustering methods to explore similarities and dissimilarities among different citrus samples according to their secondary metabolites. In general, four clear-cut clusters are determined and the chemical markers (chemotypes) responsible to this differentiation are characterized by subsequent discriminate analysis using counter-propagation artificial neural network (CPANN) method. It is concluded that the use of proposed strategy is a more reliable and faster way for the analysis of large data sets like chromatographic fingerprints of natural products compared to conventional methods.

► A chemometrics-based strategy is developed for chromatographic fingerprinting.
► Combination of multivariate curve resolution and clustering methods is proposed.
► Extended MCR-ALS followed by PCA and KNN are used for GC–MS fingerprints of citrus samples.
► Different chromatographic problems are solved using the proposed strategy.
► The main secondary metabolite(s) (chemotype) for each cluster are characterized using CPANN method.