Addressing the analytical hurdles associated with Artemisia annua

extracts

Josh L Pilkington, Chris Preston and Rachel L Gomes

Department of Chemical and Environmental Engineering, University of Nottingham, University Park, Nottingham NG7 2RD

Malaria 247 million cases in 2008

Almost one million deaths annually

Concentrated in sub-Saharan Africa

Artemisinin Precursor to Artemisinin-based Combination

Therapies (ACTs), considered the most effectivetreatment against Plasmodium falciparum1

Primarily obtained through solvent extraction from the leaves of Artemisia annua, grown in temperate climates

Total synthesis is possible but prohibitively expensive

Production (Africa and Asia) requires improvements in efficiency (currently 50-60%) and cost-effectiveness

1 WHO (2011) Global Plan for Artemisinin Resistance Containment [online] http://www.who.int/malaria/publications/atoz/artemisinin_resistance_containment_2011.pdf [accessed 11/07/2011]

Artemisinin Solvent Extraction

Solvent extraction ConcentrationSolvent decantedCrystals harvested(Crude artemisinin)

Purification through multiple ethanol re-crystallisations

Artemisinin Detection In order to assess the efficiency of each processing stage, robust

analytical techniques are required to determine the relationship between processing parameters and overall processing efficiency

Most methods in the literature only analyse high purity artemisinin and are unsuited to earlier processing stages

Analytical techniques involving LC-MS or NMR require capital investments or expertise that are not available to producers

Liquid Chromatography (LC) HPLC-UV is likely to demonstrate the most favourable balance

between equipment cost, simplicity of operation and accuracy

Lapkin et al.[2] recently undertook a substantial investigation into various different HPLC methods:

-Compared various detection methods- Analysed extracts produced with different solvents- Concluded that HPLC-UV was not suitable for A. annua extracts

due to the high presence of impurities.

2 Lapkin, A. A., Walker, A., Sullivan, N., Khambay, B., Mlambo, B., Chemat, S. (2009) Development of HPLC analytical protocols for quantification of artemisinin in biomass and extracts. Journal of Pharmaceutical and Biomedical Analysis, 49, 908-915.

Aims and Objectives Develop a functional HPLC-UV analytical method to analyse the

artemisinin content of crude A. annua extracts

-Identify problems with Lapkin method-Overcome difficulties with impurities

- Evaluate against a range of extraction solvents

Utilise the HPLC-UV method to compare the extraction efficiency of artemisinin using different solvents

- Inform on optimum processing conditions

Producing Extracts Followed the procedure detailed in the recent

publication by Lapkin et al.[2]

Static, 4 hour extractions of 10g of leaf with 100ml solvent (ethyl acetate, hexane, ethanol and a 95:5 hexane:ethyl acetate mixture) in triplicate

An additional, agitated extraction with ethyl acetate was performed for comparison

Extracts strained through muslin fabric to separate leaves from extract (miscella) before vacuum filtration

2 Lapkin, A. A., Walker, A., Sullivan, N., Khambay, B., Mlambo, B., Chemat, S. (2009) Development of HPLC analytical protocols for quantification of artemisinin in biomass and extracts. Journal of Pharmaceutical and Biomedical Analysis, 49, 908-915.

Repeating Lapkin Method Co-elution of artemisinin and impurities was

observed for all but the ethanol extracts

Greater separation of impurities from artemisinin required for quantification

New HPLC-UV Method New method has a longer duration

but provides good separation of artemisinin from impurities

Limit of Quantification (LOQ) at λ=210nm was found to be 12μg/ml

Extract concentrations were between 430 and 920μg/ml depending on the extraction solvent, so artemisinin could be readily quantified

Observation To analyse the artemisinin content of an extract, the extraction solvent

(e.g. hexane) is usually evaporated off to leave a dry residue

The residue is then re-dissolved into another solvent (often acetonitrile) to be analysed by HPLC. This is sample reconstitution

During sample reconstitution, it was observed that most of the residue remains in the vial. Any solid residue remaining after reconstitution cannot be detected

Could artemisinin remain trapped inside the residue after reconstitution? This would lead to an underestimate of the actual artemisinin content of the extract

Extract ReconstitutionAliquots of each extract were evaporated to dryness and the residue weight recorded.

Four methods of reconstitution in acetonitrile were then undertaken:

1. Thirty seconds on vortex (2800rpm)

2. Ten minutes mixing (350rpm)

3. Thirty minutes mixing

4. Twenty four hours mixing1 2 3 4

Results: Effect of reconstitution method

30 Sec Vortex 10 Min Mixing 30 Min Mixing 24 Hour Mixing0

2

4

6

8

10

12Ethyl Acetate (Mixed)

Ethyl Acetate (Static)

Hexane-Ethyl Acetate (95:5, v/v)

Hexane

Ethanol

Method of Reconstitution

Tota

l Art

emisi

nin

Dete

cted

in E

xtra

ct

Resid

ue(m

g)

Summary: Effect of reconstitution method Increasing the duration of reconstitution increased the total

amount of detectable artemisinin in all extract residues

The impurity profile of extracts is also affected by reconstitution duration

The amount of artemisinin reconstituted after 24 hours is the same as after 48 hours, indicating the maximum amount of artemisinin attainable has been achieved

Results: Effect of reconstitution method

30 Sec Vortex 10 Min Mixing 30 Min Mixing 24 Hour Mixing0

2

4

6

8

10

12Ethyl Acetate (Mixed)

Ethyl Acetate (Static)

Hexane-Ethyl Acetate (95:5, v/v)

Hexane

Ethanol

Method of Reconstitution

Tota

l Art

emisi

nin

Dete

cted

in E

xtra

ct

Resid

ue(m

g)

Hypothesis - MechanismFor 30 seconds of reconstitution, acetonitrile is only able to contact artemisinin contained on the surface of the extract residue

Extract Residue

Reconstituted

Acetonitrile

KEY

TRIPLICATE VIALS

Hypothesis - MechanismFor intermediate reconstitution durations (10 and 30 minutes), the penetration of acetonitrile into wax is variable.

Extract Residue

Reconstituted

Acetonitrile

KEY

TRIPLICATE VIALS

Application: Scaling factors Possible to correlate the amount reconstituted in 30 seconds to

that reconstituted in 24 hours with scaling factors

Can be predicted to a precision of ~3% for the commonly encountered extraction solvents

Leaf Extraction Solvent Scaling Factor CV (%)

Ethyl Acetate (Mixed) 5.77 8.69

Ethyl Acetate (Static) 2.35 9.40

Hexane-Ethyl Acetate (95:5, v/v) 3.44 3.26

Hexane 2.43 3.14

Ethanol 1.46 3.08

Application: Solvent Selectivity Ethanol showed the highest selectivity for artemisinin and had

the greatest extraction extent under the conditions investigated

Leaf Extraction Solvent Extract Artemisinin Purity Total Artemisinin Extracted

Mean (wt%) CV (%) Mean (mg) CV (%)

Ethanol 16.91 0.74 10.34 0.08

Ethyl acetate (no mixing) 12.66 1.86 7.34 0.43

95:5 hexane:ethyl acetate 11.29 1.32 6.90 0.09

Ethyl acetate (mixing) 10.71 4.24 9.32 0.27

Hexane 9.82 1.10 6.00 0.07

Conclusions Waxy extract residues provide a barrier that hinders artemisinin re-

solubilisation. This has a critical impact on the detected amount of artemisinin and could lead to large underestimates- Literature needs to clarify method and duration of reconstitution for

clarity and repeatability- Industry needs set reconstitution procedures

Scaling factors can be used as a method to reduce long reconstitution times

Ethanol was found to be the optimal extraction solvent in terms of total artemisinin extracted and purity of the extract under the conditions investigated

Further Work Investigate more thoroughly the use of ethanol as an extraction

solvent and develop methods to purify extracts efficiently

Examine improved methods of overcoming the extended reconstitution times

Examine whether similar effects are observed during column chromatography for purification. Waxy residues are observed to form on the surface of the solid phase

Acknowledgements:Dr Chris Preston, Bio Project Consulting Ltd

Dr Rachel L Gomes, Department of Chemical and Environmental Engineering, University of Nottingham

Afro Alpine Pharma Ltd, Kabale, Uganda

More Information:Pilkington, J. L., Preston, C. and Gomes, R. L. (2012) The impact of

impurities in various crude A. annua extracts on the analysis of artemisinin by liquid chromatographic methods. Journal of Pharmaceutical and Biomedical Analysis. Article in Press.