1. USE OF ACCURACY PROFILE FOR THE VALIDATION OF THE DIRECT QUANTITATION OF TAGITININ C IN TITHONIA DIVERSIFOLIA LEAVES BY ON-LINE COUPLING OF SUPERCRITICAL CO 2 EXTRACTION TO FT-IR SPECTROSCOPY BY MEANS OF OPTICAL FIBRES USE OF ACCURACY PROFILE FOR THE VALIDATION OF THE DIRECT QUANTITATION OF TAGITININ C IN TITHONIA DIVERSIFOLIA LEAVES BY ON-LINE COUPLING OF SUPERCRITICAL CO 2 EXTRACTION TO FT-IR SPECTROSCOPY BY MEANS OF OPTICAL FIBRES E. Ziemons 1, V. Barillaro 2, E. Rozet 1, N. Wandji Mbakop 1, R. Lejeune 1, L. Angenot 3, L. Thunus 1 and Ph. Hubert 1 1 Laboratory of Analytical Chemistry, Bioanalytical Chemistry Research Unit, Department of Pharmacy, University of Liège, Avenue de l’Hôpital 1,Bât B36, 4000 Liège, Belgium. 2 Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Liège, Avenue de l’Hôpital 1, Bât B36, 4000 Liège, Belgium. 3 Laboratory of Pharmacognosy, Department of Pharmacy, University of Liège, Avenue de l’Hôpital 1, Bât B36, 4000 Liège, Belgium. Tithonia diversifolia EXPERIMENTAL RESULTS AND DISCUSSION Figure 3. Schematic diagram of the on-line SFE/FT-IR system. CONCLUSIONS SFE/FT-IR using chalcogenide optical fibres has proved to be a suitable method for the quantification of dynamic extractions of tagitinin C from Tithonia diversifolia leaves in a rather short time. SFE/FT-IR process was successfully validated using the accuracy profile concept. The use of this single decision tool allowed to visually grasp the ability of the SFE/FT-IR method to fulfil its objective and control the risk associated with its further use in routine analysis. Tithonia diversifolia (Hemsley) A. Gray (Asteraceae) is a shrub which is native to Mexico and also grows in parts of Africa and Asia. Extracts of this plant have been used traditionally for the treatment of diarrhea, fever and malaria. Recently, the antimalarial properties of the plant against Plasmodium falciparum were investigated in vitro by Goffin et al. [1]. Tagitinin C was identified as an active compound against Plasmodium. An additional work of Gu et al. showed significant antiproliferative activity of tagitinin C [2]. Figure 1. Tithonia diversifolia.Figure 2. Tagitinin C. Supercritical fluid extraction with carbon dioxide as extraction medium was on-line coupled to a FT-IR spectrometer equipped with a Mercury Cadmium Telluride detector (MCT) using a home-made high-pressure fibre optic flow cell. The high-pressure fibre optic flow cell was a 1/8” stainless steel cross cell with an optical path length of 1.5 mm. Chalcogenide-glass infrared fibers were used as input and ouput fibres in order to couple the IR cell located into the SFE system to the FT-IR spectrometer. Plano/convex AMTIR lenses were used to focus the infrared beam into the fibre and the optical path of the instrument. [1] GU, J. et al, Journal of Natural Products, 65 (2002) 532. [2] GOFFIN, E. et al, Planta Medica, 68 (2002) 541. [3] ZIEMONS E. et al, Talanta, 62 (2004) 383. [4] ZIEMONS E. et al, Journal of Supercritical Fluids, 33 (2005) 53. [5] HUBERT Ph. et al., Journal of Pharmaceutical and Biomedical Analysis, 38 (2005) 370. ACKNOWLEDGEMENTS The work was funded by grants from the Belgian National Fund for Scientific Research (3.4.510.04.F). Research grant from the Walloon Region and the European Social Fund to one of the author (E. Rozet) is also gratefully acknowledged (First Europe Objective 3 project n°215269). PURPOSE To develop a method for the direct quantitation of tagitinin C in T. diversifolia leaves by on-line coupling SFE/FT-IR by means of optical fibres. To validate the SFE/FT-IR method using the accuracy profile. The aerial parts of T. diversifolia were collected at the Democratic Republic of São Tomé e Principe in 1997. Only the plant leaves were used in this work after being thoroughly grounded and sieved under 63 µm size. The validation data were processed using the enoval internet validation package (version 1.1a.; Arlenda, Liège, Belgium). Figure 4. Infrared spectra of the extraction of tagitinin C from T. diversifolia leaves over time. Figure 5. Extractogram of tagitinin C from the T. diversifolia leaves Figure 6. Accuracy profiles of added amounts of tagitinin C (µg) using a linear regression model. The continuous line is the relative bias, the dashed lines are the β-expectation tolerance limits and the dotted curves represent the acceptance limit (15 %) Fig. 4 shows infrared spectra of the extraction of tagitinin C from T. diversifolia leaves over time after an automatic subtraction of water spectrum based on the band at 1608 cm -1. As can be seen, well defined absorbance spectra were obtained between 1600-1850 cm -1 using supercritical CO 2 at 13.5 MPa, 40 °C and at a flow rate of 2 mL min -1. Previous works in our laboratory [3,4] have shown that the extract consisted in a mixture of unidentified products strongly absorbing between 1800 and 1700 cm -1 (Fig. 4. (2)(3)) and tagitinin C which has a highly specific C=O stretching vibration at 1668 cm -1 (Fig. 4. (1)) Quantitative analysis was performed by integrating the area under the extractogram absorption curve (i.e. absorbance of the highly specific C=O stretching vibration at 1668 cm -1 versus time). As illustrated in Fig. 5, the response of tagitinin C reaches rather quickly a maximum value and then decreases slowly with time. For the validation of the SFE/FT-IR method, the recent concept of accuracy profile based on two-sided 95 % expectation tolerance intervals for total measurement error of tagitinin C was used [5]. The standard addition method was first performed using leaves powder spiked with three known amounts of tagitinin C. In order to build the accuracy profile based on a linear regression model, a data treatment was carried out each day: the intercept of the standard addition regression equation was subtracted to the analytical response of tagitinin C.