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Quantitative High-Performance Thin Layer Chromatography
Category: Pharmaceutical Chromatography | 25/09/2007 - 15:26:30
Determination of artemisinin in Artemisia annua leaf
Worldwide, there are 400 million new cases of malaria and over one million deaths every year, most of them in Africa. A child dies of malaria every 30 seconds despite the fact that the disease is curable. Increasingly widespread resistance to conventional malaria treatment has necessitated the search for alternatives.
HPTLC: High Performance Thin Layer Chromatography
Derivatives of artemisinin, an active substance in the Artemisia annua leaf, have enormous potential. High-performance thin layer chromatography (HPTLC) can be used to provide a swift, reliable determination of the artemisinin content of dried plant material.
Fig.1: Structure of artemisinin
Malaria, a tropical disease caused by parasites, is transmitted by mosquitoes of the Anopheles genus. Since no vaccination is available at present, the most effective way of avoiding infection is through physical protection in the form of suitable clothing, mosquito nets, repellents and insecticides.
Various medicinal treatments have been available for the prevention and treatment of malaria since the mid-20th century, but increasing resistance to formerly effective treatment has once again made malaria therapy a major problem. In future we will need to find new ways of dealing with the rapid spread of resistant strains. There is also an urgent need for new agents that are both effective and affordable.
Artemisinin-based Combination Therapy
A new and very promising approach based on powerful artemisinin derivatives has been explored in China. Used as part of an artemisinin-based combination therapy (ACT), they are recommended by the WHO as the drug of choice for severe forms of malaria. Artemisinin (Fig.1) is a compound isolated from Artemisia annua, a plant that grows mainly in Vietnam and China, where its medicinal properties have been known for centuries. For use in drugs, artemisinin is extracted from the leaves of Artemisia annua. Because of the substance's complex molecular structure, chemical synthesis is still laborious and expensive, which is the reason why no synthetic alternatives are available. The demand for artemisinin and ACTs rapidly exceeded supply once the new therapeutic approach began to establish itself. In December 2004, the WHO took up a stance on the threat of short supplies and organised an international conference in Tanzania to secure the availability of ACTs. The delegates represented the entire production chain, from seed development and artemisinin processing all the way through to the manufacture of saleable drugs. The choice and cultivation of varieties with a high artemisinin content is especially important, as is pinpointing the best moment to harvest, minimum-damage storage and a suitable form of extraction.
CAMAG Laboratory was brought in to develop a reliable method of determining the content of artemisinin while the plant is growing and throughout the processing phase. Analysing artemisinin, however, is not without difficulty. The lack of a chromophoric group in the molecule calls for HPTLC detection using MS or ELSD, or the integration of a derivatisation step, which can rapidly become complex and time consuming. In thin-layer chromatography, derivatisation is a relatively simple matter.
HPTLC Chromatogram for Pharmaceutical Analysis
Fig.2: HPTLC chromatogram after derivatisation with modified anisaldehyde reagent. From left to right seven tracks with artemisinin reference solution in increasing con-centrations, then two tracks with Artemisia extract.
The method developed in the CAMAG Laboratory is practicable and uses high-performance thin layer chromatography (HPTLC) to identify the artemisinin content of dried Artemisia annua leaves quickly, reliably and affordably. To achieve this, the plant material is extracted for ten minutes using toluene. The extract is then applied to an HPTLC plate coated with silica gel and the chromatogram is developed. When derivatised with modified anisaldehyde reagent, the artemisinin zone will turn red. The quantitative densitometric evaluation was elegantly solved: the red fluorescence of the artemisinin is measured at its optimum excitation wavelength of 520 mm. By using a combination with a 540 nm cut-off filter, which only permits the passage of red fluorescent light, the specificity of the measurement is optimised. The signal thus obtained is three times higher than conventional absorption measurement techniques at 535 nm allow.
|Fig.3: Identification of eight Artemisia samples||Fig.4: Typical densitogram of an Artemisia sample|
The HPTLC method suggested here has also demonstrated its advantages in practice. It permits the parallel screening of nine samples in one hour. Plant material with an unknown artemisinin content can be analysed reliably over a wide concentration range of 0.05 - 3.25% artemisinin (measured on the basis of the dry material).
Dilution of the extracts permits precise determination of content in a linear working range of 30 - 100 ng absolute. The method was validated in the CAMAG Laboratory and results in overall accuracy rates of 5%. With values between 100.2 and 104.2%, the recovery rate is likewise good. The details of the method were published in the Journal of Liquid Chromatography and Related Technologies and in an application note.