ORIGINAL PAPER Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum) V. A. Huck-Pezzei & J. D. Pallua & C. Pezzei & L. K. Bittner & S. A. Schönbichler & G. Abel & M. Popp & G. K. Bonn & C. W. Huck Received: 11 May 2012 / Revised: 9 July 2012 / Accepted: 23 July 2012 / Published online: 12 August 2012 # Springer-Verlag 2012 Abstract In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. John's wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological in- formation gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients. Keywords Bioanalytical methods . Biological samples . Drug monitoring . Drug screening . IR spectroscopy . Raman spectroscopy Abbreviations API Active plant ingredients ATR Attenuated total reflection FFPE Formalin-fixed paraffin-embedded FPA Focal plane array FTIR Fourier transform infrared HE Hematoxylin and eosin HCA Hierarchical cluster analysis HPLC High-performance liquid chromatography IR Infrared KM k-means LM Light microscopy MCT Mercury cadmium telluride MIA Multivariate image analysis MIR Mid-infrared NIR Near-infrared NIRS Near-infrared spectroscopy PCA Principal component analysis ROI Region of interest TLC Thin-layer chromatography Introduction St. John's wort (Hypericum perforatum)[1] belongs to the best-selling herbal medicines, and according to the German Prescription Report 2010, 27.7 M defined daily doses with a market value of approximately 13 M euros were sold last 2009 in German pharmacies. In 1996, German physicians prescribed 131 million daily doses of herbal medicines prepared from St. John's wort herb [2], which are used in the treatment of mild and moderate depressions [3]. Quality control plays a crucial role in the phytopharmaceutical V. A. Huck-Pezzei, J. D. Pallua, and C. Pezzei contributed equally to this work. V. A. Huck-Pezzei : J. D. Pallua : C. Pezzei : L. K. Bittner : S. A. Schönbichler : G. K. Bonn : C. W. Huck (*) Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck, Austria e-mail: christian.w.huck@uibk.ac.at G. Abel : M. Popp Bionorica SE, Kerschensteinerstrasse 11-18, 92318 Neumarkt, Germany Anal Bioanal Chem (2012) 404:1771–1778 DOI 10.1007/s00216-012-6296-9