Origin of indigo colorants revealed by ion mobility spectrometry coupled to mass spectrometry followed by supervised classification

Identification of colorants is a key theme in art history and restoration. Indigo, the famous blue dye and pigment, was historically produced from four biological sources cultivated in specific parts of the world: woad in Europe, true indigo in Indian region, anil in South and Central America, and Japanese indigo in Eastern Asia. Therefore, identification of the biological source of indigo could reveal the provenance of pigments and dyes used in historical artworks, assist evaluation of their age, and help to trace ancient trade routes. To date, none of the previous reports achieved confident identification of indigo sources. This is the first systematic comprehensive untargeted workflow for discrimination of indigo colorants from different biological sources. Besides the new analytical procedure based on flow injection analysis and high-resolution mass spectrometry coupled to ion mobility spectrometry, the original multi-method screening scheme for feature selection, classification model training, and validation is presented. Reference indigo pigments were analyzed neat or embedded into appropriate binders to simulate oil painting or egg tempera mural. Combinations of eight feature selection methods (information gain, information gain ratio, Gini decrease, ANOVA, Chi squared, ReliefF, fast correlation based filter, and significance analysis of microarrays) and five classification algorithms (k-nearest neighbors, supportvector machines, random forest, neural network, and naive Bayes) were evaluated via double (nested) crossvalidation. The best performance was achieved with random forest across all feature selection methods (average classification accuracy 94%, negative ionization mode data). Tentative substance classes were proposed for the most important features based on their mass fragmentation patterns. A methylated flavonoid (diosmetin) and an indole alkaloid (2,2 '-biindolyl) were identified in woad and true indigo pigments, respectively, and confirmed by chromatography. Plant sources of colorants were determined in historical samples of oil paintings and textiles.