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Evaluation of Artemisia annua L. clean-up methods for artemisinin quantification by
HPLC
Celeghini, R. M. S.; Silva, A. P.; Sousa, I. M. O.; Foglio, M. A.
Centro de Pesquisas Químicas, Biológicas e Agrícolas da Universidade Estadual de Campinas (CPQBA/UNICAMP),
Campinas, SP, Brazil. E-mail: foglioma@cpqba.unicamp.br

ABSTRACT: Evaluation of Artemisia annua L. clean-up methods for artemisinin quantification by HPLC.
Artemisinin, an endoperoxide sesquiterpene lactone isolated from Artemisia annua L. (Asteraceae), has received
considerable attention due to its antimalarial activity. The limiting step in sample preparation is result of large
amounts of chlorophyll encountered in the crude extract. In this work we describe several sample preparation
methods using solid-phase extraction (SPE) and compare them with conventional liquid-liquid extraction method.
The aim of this work was to define the optimum conditions for a clean-up procedure of plant extract. Silica, and
Florisil cartridges were evaluated as solid supports for SPE and treatment with lead acetate for liquid-liquid
extraction. The data presented herein suggest that the use of Florisil in analysis clean-up procedures is a low
cost, rapid and efficient method with high yield recoveries.
Key words: Artemisia annua, artemisinin, clean up, High Performance Liquid Chromatography

INTRODUCTION
Artemisinin (1), an endoperoxide
sesquiterpene lactone isolated from Artemisia annua
L. (Asteraceae), has received considerable attention
due to its antimalarial activity. Sesquiterpene lactones
are common in most Asteraceae tribes, with more
than 4000 known structures. Several methods have
been reported for the measurement of artemisinin and
its main derivatives in plant material and biological
fluids (Edwards, 1994). However, most of them are
either not sufficiently sensitive and do not offer reliable
results, or are difficult to apply in routine analyses
(Christen & Veuthey, 2001). Therefore, new methods
for the determination of these compounds, such as
supercritical fluid extraction and chromatography,
pressurized solvent extraction, extraction, highperformance liquid chromatography coupled to mass
spectrometry scattering detection (Wang et. al., 2005).
A diversity of bioactivities has been reported
for this class of compounds, such as antiinflammatory, antitumoral, antiulcerogenic, cytotoxic,
diuretic and cardiotonic, among others. Previously
we demonstrated that the resulting enriched
sesquiterpene fraction from Artemisia annua L. crude
ethanolic extract inhibited the ulcerative lesion index
in all experimental models tested, in rats. The results
mentioned therein suggested that the antiulcerogenic
properties were afforded by cytoprotective
mechanisms as result of active principles that increase
the gastric mucous prostaglandin level (Foglio et. al,
2002).
A variety of new analytical methods for the
Recebido para publicação em agosto/2004
Aceito para publicação em julho/2006

determination of artemisinin and derivatives have
appeared during the last decade. New extraction
processes use mild operating conditions to avoid the
degradation of the analytes. They allow obtaining clean
extracts from complex matrices. In this context, SFE,
PSE and MAE are interesting alternatives to classical
solid-liquid extraction under reflux. Several
methodologies have been applied to the analysis of
artemisinin and its derivatives. Techniques such as
HPLC-ELSD, HPLC-EC, HPLC-MS, GC-MS, CE-UV
or SFC-ELSD have been developed with the goal of
analyzing metabolites in a faster, better, cheaper and
more efficient manner (Hussen et. al. 2006). Many
important herbal drugs (e.g. ginkgo, black cohosh,
soy) contain bioactive compounds possessing no
chromophore, thus detection techniques other UV are
needed for their identification and standardization.
Evaporative light scattering is one of the possible
alternatives, but despite the fact that this technique
has been developed more than 30 years ago, it is still
considered to be an exotic detection alternative, with
few applications at hand (Ganzera&Stupper 2005). The
refractive index detector is one of the least sensitive
LC detectors. It is very sensitive to changes in ambient
temperature, pressure changes, flow-rate changes and
can not be used for gradient elution. Despite these
many disadvantages, this detector is extremely useful
for detecting those compounds that are nonionic, do
not adsorb in the UV, and do not fluoresce, as
artemisinin.
Analysis of artemisinin [1] is difficult because
the compound is unstable and other compounds in
the crude plant extracts interfere in its detection. Due
to the lack of chromophores, artemisinin is suitable
for HPLC with UV detection only after derivatization.

Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.119-122, 2006.

Recovery Each sample was spiked with artemisinin standard in three different concentrations levels and subject to extraction and clean up procedure Rev. MATERIAL AND METHOD Plant material and fractionation Artemisia annua L. for the purpose of quality control. 80A) cartridge (6 mL volume) used in conjunction with vacuum Manifold. filtered and concentrated almost to dryness under vacuum and diluted to a final volume (10 mL) for analysis by HPLC-IR.5 ml). The cartridge was conditioned with hexane (6 mL) and not allowed to dry. The cartridge was conditioned with n-hexane (6 mL) and not allowed to dry. (3 x 5 mL).esp. 750. v. a column oven. dried over MgSO4. 6000 rpm at room temperature (25oC) with three portions of 5 mL methanol for 2 min .170 µm. Stock solutions of the standards of 550. The analytes were eluted with n-hexane-ethyl acetate (4:1. Chromatographic methodologies A modular Waters system comprised of a Waters 515 pump. filtered and concentrated almost to dryness under vacuum and diluted to a final volume (10 mL) for analysis by HPLC-IR.8. This material was allowed to dry under air circulation (40ºC) and grinded for use. identified in Artemisia annua L. leaves (hybrid CPQBA 2/ 39 x PL5) were collected from the experimental field of CPQBA/UNICAMP. detector and column temperature were 35 ºC. (1992) and with silicagel. p. Florisil Clean Up Procedure (Manirakiza et al. (2000). Clean-up procedure with 10% lead acetate solution (Lonergan et al. with lead acetate as described by Lonergan et al. Bras. extracted with chloroform (3 x 10 mL).. Med.Waters workstation was utilized. 2006. at 35ºC. A Phenomenex LC-CN column (25mm x 4mm x 5µm) was employed.120 This research is focused on validation of clean up methods for analytical analysis (HPLC-IR) the antimalarial. The crude extracts were submitted to clean-up procedures with Florisol Ò as described by Manirakiza et al.. Small air chambers were removed from the adsorbent by gentle tapping and the concentrated extract (1 mL) was then applied to the column. Voucher specimen is deposited at CPQBA/UNICAMP under registration number 229. concentrated under reduced pressure providing the crude extract (yield 20%). 1250 and 1500 µg/mL were used. The samples were filtered on Buchner funnels fitted with glass frit (porosity 4 mm). Small air chambers were removed from the adsorbent by gentle tapping and the concentrated extract (1 mL) was then applied to the column. Botucatu. filtered and concentrated almost to dryness under vacuum and diluted to a final volume (10 mL) for analysis by HPLC-IR. n. using methanol: water (60:40v/v) mL/min at a flow rate of 1 mL/min with 20µL injection volume . . washed with H2O: CH3CN Sílica (2g) were added to a 6 mL cartridge used with vacuum Manifold. Quantitative analysis Determination of the content of the artemisinin in plant material was performed by external standard method.. The analytes were eluted with dichloromethane (4 mL). The plant material was extracted using Ultra Turrax mixture during 2 min . 1000. 4 mL). Triplicate determination were carried out. In this work we describe several sample preparation methods using solid-phase extraction (SPE) and compare them with conventional liquid-liquid extraction method. artemisinin. 2000) Florisil (500 mg. Pl. and Florisil cartridges were evaluated as solid supports for SPE and treatment with lead acetate for liquid-liquid extraction. This mixture stood at room temperature for one hour and filtered. The filtrate was centrifugated at 10000 rpm. Sílica Clean Up Procedure [1] Artemisinin The limiting step in sample preparation is result of large amounts of chlorophyll encountered in the crude extract. Silica.119-122. 1992) A 10% lead acetate solution (2 mL) was added to the crude methanolic extract (1g) dissolved in ethanol (0. a Waters 2414 refraction index detector and an Empower. The aim of this work was to define the optimum conditions for a clean-up procedure of plant extract. Separations were made in the isocratic mode.

0 mL/min. 2006. n.8. Clean-up methods results of percent recovery of artemisinin reference standard added to Artemisia annua L. sample injection 20µL and detector and column temperature were 35 ºC. silica gel and lead acetate were studied. The low recoveries obtained with lead acetate clean up method are a result of low artemisinin yield recovery efficiency. The results obtained with Florisil â indicated good accuracy and. RESULT AND DISCUSSION The extraction of natural products is essential not only as an evaluation tool for raw materials. This is why in a plant development project it is important to have simple. The recovery of both methods was expressed as the percentagem recovery of standard artemisinin added to sample. but also for the quality control of products.119-122. The percent recovery of added artemisinin was calculated comparing peak areas of the resulting solutions with reference standard artemisinin solutions at the same concentration. TABLE 1. which allow the quantitative determination of the analytes and possibly of their precursors (Christen & Veuthey 2001). an agreement between the theorical value and the real value of concentration. v. creating delays in the flow of information from the analysis laboratory to the field or product line. Botucatu. rapid and specific extraction and analytical procedures. Rev. Standards mixture HPLC separation using a refractive index detector with Zorbax SB. However. Table 1 show that yields recoveries with standard Florisil â and clean up with lead acetate. traditional methods of extraction can be both time consuming and labour intensive.6mm x 5µm). flow-rate 1.CN column (150mm x4. Med. Artemisinin is stable in neutral solvents heated up to 150°C. Preliminary studies were carried out in order to develop a suitable clean-up method for the purification of crude methanolic Artemisia annua extracts obtained using Ultra Turrax mixture. The silicagel clean up method was discontinued as result of the large amounts of solvent required for separations of artemisinin and chlorophyll with low efficiency. according to method under test. sorbents such as Florisilâ. Pl. The crude extract was filtered on a column containing silica or Florisilâ that was preconditioned with appropriate solvents. consequentially. For this purpose. an extraction procedure of the plant material is required. . In fact. Bras.121 FIGURE 2. eluted with methanol: water (60:40v/v)..esp. Therefore this procedure was not considered suitable for sample procedures prior to analysis. p.. whatever the analytical method used.

7010-7013. G.M.119-122. that retain less polar material showing less efficiency. Chromatographia. v.Fluids. annua crude extract after clean-up procedure with Florisilâ cartridge obtained by High Pressure Liquid Chromatography (HPLC) separation using a refractive index detector. Planta Medica. p. supl. M. LONERGAN. v. Med. v.A.4: p. G. v. n 11/12.515-518.. 1827-1839. Single step clean-up and GC-MS quantification of organochlorine pesticide residues in spice powder. amara alkaloids by ion-pair chromatography. 2006. Antiulcerogenic activity of some sesquiterpene lactones isolated from Artemisia annua L..0 mL/min. HOSTETTMANN. A.2001. 88.. n. Isolation NMR studies andbiological activities of onopordopicrin from Centaurea sochifolia. New Trends in Extraction. Journal of Chromatography A. MANIRAKIZA. HUSSEN. p. et al Simultaneous determination of Ephedra sinica and Citrus aurantium var. 52. 1994.27. Development of a pressurized liquid extraction and clean-up procedure for the determination of á-endosulfan. v.esp. Botucatu. GANZERA.L. A Phenomenex LC-CN column (25mm x 4mm x 5µm) eluted with methanol: water (60:40v/v). et al.. larger amounts of impurities were trapped at the top of the cartridge avoiding pigment contamination compared to silica clean up procedure. p 787-790.18. Bras.M. The Royal Society of Tropical Medicine and Hygiene. 3739.1. and the volume of solvents required for the elution of the analytes were low. 2000. rapid and efficient method with high yield recoveries for large number of sample analysis. n. WANG. et al. p 225-228.p. by LC-MS with selected ion monitoring. The results obtained were highly reproducible and in good agreement with the certified values demonstrating the suitability of the developed analytical method for the extraction and clean-up of Artemisia annua L. CONCLUSION The data presented herein suggest hat the use of Florisilâ in analysis clean-up procedures is a low cost. Journal of Agricultural and Food Chemistry. v. 1992. 2002. Since recoveries achieved with Florisilâ were higher than those obtained with lead acetate. FIGURE 1. Analysis of artemisinin in Artemisia annua L.F. et al. 2006. flow-rate 1.. Talanta. 2005. Princípios ativos de plantas superiores. v. et al. Measurement of Artemisinin and its Derivates in Biological.15.55. K. VEUTHEY. sample injection 20µL and detector and column temperature maintined at 35 ºC. Pl. p 59-100. Current Medicinal Chemistry. J. ACKNOWLEDGMENT: FAPESP for research grant REFERENCE CHRISTEN. Identification and Quantification of Artemisinin and its Derivatives. fully activated Florisilâ was selected as the cleanup sorbent for all subsequent studies (Figure 1). 202210. EDWARDS. â-endosulfan and endosulfan sulfate in aged contaminated Ethiopian soils.66. n.8. p. FOGLIO.8.68. P. Journal of Natural Products. Chromatogram of A. p... v. São Carlos: Editora Edufscar. et al. et al. P. 2003. Rev. n. v. p.2005.889-894 .122 With Florisilâ more polar interfering materials were retained.53. .