Spectral analysis of extracts from red hot pepper (Capsicum annuum L.) Jana Simonovska1, Denitsa Yancheva2, Bozhana Mikhova2, Žejko Knez3, Mateja Primožić3, Zoran Kavrakovski4, Vesna Rafajlovska1 1Institute of Organic Technology, Faculty of Technology and Metallurgy, University “Ss. Cyril and Methodius” 2Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences 3Laboratory of Separation Processes and product design, Faculty of Chemistry and Chemical Engineering, University in Maribor 4Institute of applied chemistry and pharmaceutical analyses, Faculty of Pharmacy, University “Ss Cyril and Methodius”, Pepper is an excellent source of proteins, vitamins, minerals, fats and oils, phenolic compounds, aromatic substances and other biologically active compounds . The importance of the red hot pepper varieties and their oleoresin extracts in the food and pharmaceutical industry is caused by the characteristic compounds, capsaicinoids and carotenoids . Mainly, for determination of the compounds in the sweet or hot pepper varieties, the chromatographic methods and UV-VIS spectrometric method were used. On the other hand, 1H NMR spectroscopy has been extensively used to provide information about the composition and relative content of fatty acid units in triglycerides . For analysis of the capsaicinoids and carotenoids, NMR spectroscopy is one of the most informative methods applied . The degree of unsaturation of vegetable oils can be effectively studied by IR spectroscopy based on the changes observed in the frequency data of some bands and in the ratios of absorbances of the IR spectra. Therefore, the aim of this study is to evaluate the possibility of applying spectroscopic techniques (NMR and IR) in the characterization of extracts obtained from red hot pepper. ATR-IR spectra of extracts of Capsicum annuum seeds: ATR-IR characterization: The oil composition affects the exact position of the band for the C-H stretching of the cis-double bond: In the ATR-IR spectra of the extracts of Capsicum annuum the band for ν(=C-H) is found at 3009 cm–1, which is typical for vegetable oils, rich in linoleic acid, such as soybean and corn oil. Futhermore, the ratio A1/A2 (peak height for ν(=C-H) at 3009 cm–1 vs. peak height for νasym(C-H) at 2923 cm–1) could be used for quantitative estimation of the degree of unsaturation: According to the ratio A1/A2, the highest degree of unsaturation is found for the seed extracts. The ATR-IR technique provides also a fast and reliable identification of capsaicinoids in Capsicum annuum extracts. The typical frequencies of the ν(C=O)am (at 1647 cm-1) and δ(N-H)am of capsaicin (at 1514 cm-1) do not overlap with the IR band for ν(C=O) of triglycerides (at 1740 cm-1), and therefore allow identification of capsaicinoids even at low concentrations: Among the studied extracts, those obtained from placenta had the highest content of capsaicinoids. ATR-IR spectra of extracts of Capsicum annuum seeds: Sample A1* A2** A1 / A2 Extracts of Capsicum annuum seeds N1 0.022 0.130 0.169 N2 0.012 0.066 0.182 N3 0.027 0.159 0.170 N4 Extracts of Capsicum annuum pericarp N5 0.010 0.104 0.096 N6 0.007 0.049 0.143 N7 0.024 0.160 0.150 Extracts of Capsicum annuum placenta N8 0.023 0.162 0.142 N9 0.167 0.132 N10 0.019 0.152 0.125 Extracts of Capsicum annuum stalks N11 0.161 0.137 N12 0.028 0.158 0.177 red: N1 – extracted by supercritical CO2 at 400 bar, 40oC, 0.5 mm; blue: N2 – extracted by hexane via Soxhlet extraction, 1 mm; pink: N3 – extracted by hexane via Soxhlet extraction, 0.5 mm; black: N4 – extracted by supercritical CO2, 1 mm. ATR-IR spectra of extracts of Capsicum annuum placenta: * Peak height for ν(=C-H) at 3009 cm–1; ** Peak height for νasym(C-H) at 2923 cm–1; NMR spectra of extracts of Capsicum annuum seeds N1-N4: green: N8 – extracted via Soxhlet extraction, 0.25 mm; blue: N9 – extracted via Soxhlet extraction, 0.5 mm; red: N10 – extracted by hexane, 0.25 mm. NMR characterization : It was shown that the samples of extracts from the seeds contain exclusively triglycerides (TG). The calculated percent of unsaturated fatty acids was estimated about 80%. The ratio linoleic:oleic acid was estimated to be about 2:1. N-polyunsaturated fatty acids (linolenic acid) were not observed. In this way the composition of TG in the seeds was shown to be similar to the corn and soybean oil. Traces of capsaicinoids were detected. Quantitative determination was not possible. Carotenoids were not detected. The spectra of the samples from pericarp showed similar composition as these from the seeds. Additionally, the presence of about 25% of n-polyunsaturated fatty acids was detected. In the samples from the placenta a substantial amount of capsaicinoids was present. The proportion TG:capsaicinoids was estimated as 1:2. In the region   1.2 - 1.4 ppm intense signals appear for long CH2 chains, probably waxes. In the samples from stalk intensive signals were obtained due to presence of waxes. The proportion of the TG:capsaicinoids was estimated to be approximately 1:0.15. NMR spectra of extracts of Capsicum annuum seeds: Conclusion Extracts from the seeds, pericarp, placenta and stalk of hot red pepper (Capsicum annuum L., ssp. microcarpum longum conoides, convar. Horgoshka) were studied by NMR and IR spectroscopy. It was shown that both spectral techniques provide useful information on the triglyceride content and degree of unsaturation of the hot red pepper extracts. The IR spectroscopy could serve as a fast tool for identification of capsaicinoids in the extracts, while the NMR analysis could be successfully applied for determination of the proportion tryglycerides:capsaicinoids.