Correlation coefficient, r Application of TLC and HPLC to quantification of protoporphyrin IX, Zn-protoporphyrin IX, and hemin in Parma ham INTRODUCTION Protoporphyrin IX plays a specific role among all tetrapyrroles, as it is a kind of a template for a wide variety of naturally occurring compounds, e.g., for the investigated Zn-protoporphyrin IX and hemin. All these compounds affect the colour properties of meat, and moreover, Zn-protoporphyrin IX contributes to the formation of a characteristic stable red colour of Parma ham. Fig. 1 protoporphyrin IX Zn-protoporphyrin IX hemin AIM The aim of this study was the quantification of three porphyrins, i.e., protoporphyrin IX, Zn-protoporphyrin IX, and hemin in Parma ham using Thin-layer chromatography (TLC) and High performance liquid chromatography (HPLC). EXPERIMENTAL TLC Stationary phase: glass plates, pre-coated with the 0.25 mm thick layer of RP-18 F254, 10 cm  20 cm (Merck, Darmstadt, Germany); Mobile phase: Methanol; RF value of protoporphyrin IX, 0.31±0.02; Zn- protoporphyrin IX, 0.57±0.02; hemin, 0.25±0.02; Detection: densitometric detection (CD 60 model DESAGA densitometer; Heidelberg, Germany) in fluorescence mode, at the wavelength λ = 400 nm. HPLC Stationary phase: Pursuit 5 C18 (5 m particle size) column; 250 mm  4.6 mm (Varian, Harbor City, CA, USA); Mobile phase: A + B, 9: 1 (v/v); A: Methanol + dichloromethane, 9:1 (v/v); B: Water + glacial acetic acid, 97:3 (v/v); Flow rate: 0.8 mL min-1; TR value of protoporphyrin IX, 9.0 min; Zn- protoporphyrin IX, 5.5 min; hemin, 2.6 min; Detection: Diode Array Detection (Varian model 330 DAD detector), at the selected wavelengths (λ) of 400, 414 and 397 nm, for protopophyrin IX, and Zn- protoporphyrin IX and hemin respectively. H. De Maere1,3 M. Jaros2 M. Dziewiecka² I. Fraeye1 M. Sajewicz2 H. Paelinck1 T. Kowalska² 1Research Group for Technology and Quality of Animal Products, KAHO Sint-Lieven, Gebroeders Desmetstraat 1, 9000 Ghent, Belgium 2 Department of General Chemistry and Chromato-graphy, Institute of Chemistry, University of Silesia, 9 Szkolna Street, Katowice, Poland ³ Food Quality Laboratory, Groupe ISA, 48 Boulevard Vauban, 59046 Lille-Cedex, France Contact: Hannelore.demaere@kahosl.be Extraction procedure Parma ham 10-g Parma ham sample was morcelated; The morcelated portion of meat was homogenized for 10 minutes and put on ice with the addition of 100 mL acetone + 0.2 g chloroacetic acid. The sample was filtered through a paper filter. To the filtrate, several drops of aqueous ammonia were added to obtain neutral pH value (pH 7). The purified samples were analyzed for the contents of protoporphyrin IX, Zn- protoporphyrin IX and hemin by means of TLC/densitometry and HPLC/DAD. Fig. 2 TLC chromatogram of the Parma ham extract. Signal intensities: 725 mAV for hemin; 1300 mAV for protoporphiryn IX; and 850 mAV for Zn-protoporphyrin IX Fig. 3 HPLC Chromatogram of Parma ham extract, with the 3 porphyrins indicated on it. RESULTS Table 1 Calibration curves obtained from TLC and HPLC for protoporphyrin IX, Zn-protoporphyrin IX and hemin, and the respective LOD and LOQ values (n=6) Compound Technique Calibration curve Correlation coefficient, r Standard deviation, SD LOD (ng) LOQ   Protoporphyrin IX TLC y = 39.12x + 839.92 0.9854 687.589 58 176 HPLC y = 0.1223x – 3.5765 0.9921 0.856 23 70 Zn-Protoporphyrin IX y = 57.689x + 492.46 0.9505 1051.218 60 182 y = 0.1785x -2.7965 0.9654 2.399 44 134 Hemin y = 0.89705x + 670.44 0.9730 798.418 2937 8900 y = 0.1235x – 2.8672 0.9966 0.517 14 42 MeCagrO2 07-021-Fr “The document reflects the author’s views. The interreg IVA 2 Seas Programme Authorities are not liable for any use that may be made of the information contained therein.” Table 2 Contents of protoporphyrin IX, and Zn-protoporphyrin IX and hemin in Parma ham, as established from the chromatographic peak heights by means of TLC and HPLC (n=6) Compound Technique Amount found (g per 1 g ham)   Protoporphyrin IX TLC 1.2 HPLC 15.2 Zn-Protoporphyrin IX 0.62 20 Hemin 12 16 CONCLUSION 1. By means of TLC and HPLC, the presence of the three porphyrins in Parma ham was demonstrated and their contents were quantified. 2. HPLC turned out to be the most adequate method to quantify the three porphyrins (e.g. better calibration curves, lower LOD and LOQ). 3. Using TLC 1.2 μg protoporphyrin IX, 0.62 μg Zn-protoporphyrin IX and 12 µg hemin was found in 1 g Parma ham. Using HPLC at the other hand results in respectively 15.2 µg, 20 µg and 16 µg per 1 g ham. REFERENCES: 1. J. Wakamatsu, Hiroko Odagiri, T. Nishimura, A.Hattori, Quantitative determination of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC. Meat Science 82 (2009) 139–142. 2. D.A. Skoog, Podstawy chemii analitycznej. Tom 2, PWN, Warszawa 2007. 3. I. Żak, Porfiryny i ich pochodne. W: (red.). I.Żak Chemia medyczna. ŚAM, Katowice 2001. 4. M. Trytek, M. Makarska, K.Polska, S. Radzki, J. Fiedurek, Porphyrins and phthalocyanines. Part I. Properties and some applications. Prace przeglądowe. Biotechnologia 2005, 4 (71) 109–127.