USE OF ACCURACY PROFILE FOR THE VALIDATION OF THE DIRECT QUANTITATION OF TAGITININ C IN TITHONIA DIV

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 CO2 EXTRACTION TO FT-IR
SPECTROSCOPY BY MEANS OF OPTICAL FIBRESE.
Ziemons1, V. Barillaro2, E. Rozet1, N. Wandji
Mbakop1, R. Lejeune1, L. Angenot3, L. Thunus1
and Ph. Hubert11 Laboratory of Analytical
Chemistry, Bioanalytical Chemistry Research Unit,
Department of Pharmacy, University of Liège,
Avenue de lHôpital 1,Bât B36, 4000 Liège,
Belgium.2 Laboratory of Pharmaceutical
Technology, Department of Pharmacy, University of
Liège, Avenue de lHôpital 1, Bât B36, 4000
Liège, Belgium. 3 Laboratory of Pharmacognosy,
Department of Pharmacy, University of Liège,
Avenue de lHôpital 1, Bât B36, 4000 Liège,
Belgium.
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.

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 CO2 at 13.5 MPa, 40 C and at a
flow rate of 2 mL min-1.
Tithonia diversifolia
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.
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 CO stretching
vibration at 1668 cm-1 (Fig. 4. (1))
Figure 4. Infrared spectra of the extraction of
tagitinin C from T. diversifolia leaves over
time.
Quantitative analysis was performed by
integrating the area under the extractogram
absorption curve (i.e. absorbance of the highly
specific CO 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.
Figure 1. Tithonia diversifolia.
Figure 2. Tagitinin C.
EXPERIMENTAL
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.
Figure 5. Extractogram of tagitinin C from the
T. diversifolia leaves
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.
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 ß-expectation tolerance
limits and the dotted curves represent the
acceptance limit (15 )
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.
Figure 3. Schematic diagram of the on-line
SFE/FT-IR system.
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).
ACKNOWLEDGEMENTS
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.
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 n215269).