3.3 Methods
Various conventional methods thin layer chromatography (TLC)
(Pras et al., 1991), Gas chromatography (GC) (Fulzele et al., 1991), GC-MS and
tandem mass spectroscopy MS\MS (Dhingra et al., 2000), HPLC with UV detection
(Pras et al., 1991) and with electrochemical detection (HPLC-EC) (Acton et al.,
1985) have been proposed and assessed to detect and quantify artemisinin. Radio
immuno assay (RIA) and Enzyme electrochemical detection (ELISA) comprise the
unconventional techniques to detect artemisinin (Dhingra et al., 2000).
In fact, TLC is not reliable technique to quantify artemisinin
due to the poor staining characteristics of the intact molecule and
interference with other constituents of the plants. TLC is useful as an assay
method only after a tedious chromatographic enrichement (Pras et al., 1991).
Gas chromatography also has been applied for the analysis of artemisinin.
However, ART is thermolabile compound (stable upto 150oC) and
decomposes on the column. High-pressure liquid has been used chromatography
with ultra violet detection but the plethora of crude extract constituents that
absorb in the low wavelength region required to detect artemisinin effectively
its peak. Moreover, artemisinin needs to be derivatized due to its lack of
chromophores (Pras et al., 1991). This process can hamper the result by
derivatizing the other compounds present in the crude extract. Moreover, ART is
sensitive to acid and base treatment. The most sensitive way for detecting an
quantifying artemisinin in crude plant extract without any molecular breakdown
or interference from other related compounds and which does not require any
derivatization or sample purifiction is High pressure liquid chromatography
with electrochemical detection (HPLC-EC). HPLC-EC measures ART directly because
the peroxide moiety undergoes electrochemical reduction. This method is highly
sensitive and can detect nanogram levels of artemisinin. However, the reductive
electrochemical detection involves very special precautions as molecular oxygen
is reduced at the low cathodic potentiel of -0.8 V (Acton et al., 1985).
The unconventional methods (RIA and ELISA) are sensitive and
highly specific than conventional methods to detect in artemisinin levels in
small samples of plant tissues from young seedlings and from cell or tissue
cultures. Although RIA is more sensitive, the use of radioactive compounds
present a series of problems of special acquisition and use requirements,
uncertain stability, high cost, health hazards and disposal difficulties
(Dhingra et al., 2000). Hence ELISA is as sensitive as RIA, safer and is based
on the peroxide bridge for antibody specificity to detect artemisinin and
closely related compounds in crude extracts of artemisia annua
(Dhingra et al., 2000).
In our study, we used UV-Vis spectroscopic and HPLC-MS with
UV detection for investigating the binding of hemin with antimalarial drugs.
3.3.1Ultraviolet/Visible molecular absorption spectrometry
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