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Keywords:

  • artemether;
  • dihydroartemisinin;
  • artesunate;
  • artesunic acid;
  • colorimetric;
  • malaria;
  • Fast red TR;
  • diazonium salt;
  • counterfeit

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusions
  7. References

The recent and widespread appearance of counterfeit antimalarial tablets in South–east Asia prompted the search for simple field assays to identify genuine drugs. In a recently described colorimetric assay for artesunate, Fast red TR salt reacted with an alkali-decomposition product of artesunate to produce a distinct yellow colour. However, that assay is specific for artesunate and it cannot be used to test for artemether. Because of potential concerns over artemether tablet counterfeiting, the colorimetric assay was modified to detect artemether, dihydroartemisinin and artesunate tablets. Other common antimalarials (artemisinin, chloroquine diphosphate, mefloquine HCl, sulphadoxine and pyrimethamine), as well as aspirin and acetaminophen, were negative in the assay, indicating its specificity for artemether, dihydroartemisinin and artesunate. The colorimetric method can be used to obtain a rapid visual assessment of tablet authenticity. The method can also be used to quantify the drug content of tablets, when used in conjunction with a spectrophotometer.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusions
  7. References

The artemisinins are increasingly being used because of their effectiveness against multi-drug resistant malaria. This family of drugs is derived from the natural product artemisinin and includes artemether (AM), artesunate (ARTS) and dihydroartemisinin. Reports of the distribution of counterfeit artesunate tablets in South–east Asia are becoming more frequent. In 1999, an investigation into the sale of counterfeit artesunate revealed that 71% of the drug vendors and pharmacies sampled in Cambodia sold the counterfeit drug (Rozendaal 2001). In response to this serious health threat, Green et al. (2000) developed and validated a colorimetric field test [ARTS-Fast red TR (FRTR)] to distinguish genuine artesunate from counterfeit tablets. In conjunction with organoleptic evaluation, the ARTS-FRTR test was used to survey the extent of the distribution of counterfeit artesunate tablets collected in South–east Asia. Initial testing has shown that 38% of artesunate tablets tested in South–east Asia contained no active ingredient (Newton et al. 2001). Distribution of counterfeit artemisinin derivatives, such as artemether and dihydroartemisinin has not been documented to date, but these drugs are similar in cost and appearance to artesunate and it is likely that they will become targets of counterfeiting. As artemether is not detected in the ARTS-FRTR test (Green et al. 2000), we modified this assay to identify the presence of artemether as well as dihydroartemisinin and artesunate.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusions
  7. References

Fast red TR salt (reagent grade; dye content approximately 20%) was purchased from Sigma-Aldrich Fine Chemicals (Milwaukee, WI, USA). Artemether and dihydro- artemisinin were a gift from Dr P. Olliaro, World Health Organization. Artesunate was produced by Mepha (Aesch-Basel, Switzerland). Arenax® tablets (100 mg β-artemether per tablet) were manufactured by Arenco Pharmaceutica, Belgium. Malaxin® (60 mg dihydroartemisinin) was manufactured by Dongsan Pharmaceutical Co. Ltd., South Korea and Arsumax® (50 mg artesunate) was produced by Guilin Pharmaceutical Works, China (the use of trade names is for identification only and does not imply endorsement by the Public Health Service or the US Department of Health and Human Services). The buffering reagent consisted of 0.1 M borate in 5% ammonia solution. FRTR solution (5 or 10 mg/ml water) was prepared immediately before sample addition. Absorbance measurements were recorded with a Spectronic-20 (Bausch and Lomb Inc., Rochester, NY, USA) spectrophotometer set at 420 nm. All reagents were prepared with de-ionized water and were analytical grade, unless noted otherwise.

Assay procedure (AM-FRTR test)

The assay procedure is as follows: scrape about 5% of the tablet mass into a glass tube, add 0.4 ml of methanol and mix for about 10 s, add 0.4 ml of 5 N HCl and incubate the sample at room temperature (22–27 °C) for at least 1 h, add 2.2 ml of buffering solution. Add 0.1 ml of FRTR solution and thoroughly mix. A yellow colour develops within 5 min if artemether, dihydroartemisinin or artesunate is present in the sample.

The specificity of the AM-FRTR test was assessed by testing other common antimalarial tablets (Table 1). Each tablet was weighed and 5% of the tablet mass was added to a glass tube. Methanol (0.5 ml) was added and the samples vortexed for 10 s. Insoluble tablet excipients were centrifuged (500 g; 10 min) and 0.4 ml of the supernatant was transferred to a clean glass tube. To perform the quantitative analysis, the tablet and scrapings were weighed and the insoluble excipient centrifuged. Subsequently, steps 3–5 were performed as described above. For quantitative assessment, standard curves were prepared from artemether, dihydroartemisinin and artesunate at concentrations of 0.5, 1 and 2 times the concentration determined for 5% of Arenax (100 mg artemether), Malaxin (60 mg dihydroartemisinin), or Arsumax (50 mg artesunate) in 0.5 ml of methanol. Absorbance (420 nm) values were recorded for the standard curve and tablet samples. Linear regression analysis of absorbance vs. concentration data from the standard curve samples was used to calculate the concentration of the active ingredient for each tablet. Two ml of ethyl acetate was added to the solution and the sample thoroughly shaken. After phase separation, quantitative analysis of the yellow reaction product extracted into the organic phase was assessed as described above.

Table 1.   Specificity of AM-Fast red TR (AM-FRTR) test with other commonly used antimalarials. Per cent accuracy is defined as the difference between the expected value and calculated value divided by the expected value times one hundred. Calculated amount of active ingredient (mg/tablet) and per cent accuracy are given before and after (*) ethyl acetate extraction Thumbnail image of

Formation of artemether acid decomposition product

The diazo-coupling reaction of FRTR with artemether depends on the formation of an acid decomposition product. This product, described by Thomas et al. (1992), is an α, β-unsaturated decalone that absorbs ultraviolet radiation at 254 nm. The optimum incubation period for its formation was assessed by adding 2 ml of 5 N HCl to 0.1 ml of artemether (1 mg/ml methanol) and monitoring the absorbance at 254 nm for 4 h.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusions
  7. References

A distinct yellow colour is apparent for the AM-FRTR test in the presence of artemether, dihydroartemisinin or artesunate (Table 1). A faint orange colour appeared in the presence of acetaminophen, while the other antimalarials appeared colourless. After several hours, a weak yellow colour appeared in the drugs containing the sulphonamides, i.e. Falcidin (500 mg sulphadoxine) and co-trimoxazole (400 mg sulphamethoxazole). Quantitative analysis using absorbance measurements showed the artemether content of Arenax and the dihydroartemisinin content of Malaxin to be within 15% of expected values, while the artesunate content of Arsumax showed a much greater deviation of 65% (Table 1). All samples appeared a bit cloudy, especially Arsumax, which may have contributed to a greater deviation. Therefore, extraction with ethyl acetate was employed in an effort to improve accuracy. After addition of 2 ml of ethyl acetate followed by vigorous shaking, the entire yellow reaction product migrated into the organic phase. From absorbance measurements, the active ingredients for each tablet were calculated (Table 1). Ethyl acetate extraction showed improved accuracy (% deviation from expected value) of artesunate content in the Arsumax tablet from 65% to 11%, while the Malaxin tablet showed a greater deviation (46%) of dihydroartemisinin from the expected value. This may by attributable to preferential solubility of the reaction products in ethyl acetate relative to the aqueous buffer. Although optimum conversion of artemether into its decomposition product occurs at about 4 h, colorimetric detection of a 5% tablet containing 100 mg artemether is easily achieved if incubated for at least 1 h. This observation was also evident with dihydroartemisinin and artesunate.

Discussion and conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusions
  7. References

The artemisinins do not have the particular chemical groups that easily react with certain reagents to yield coloured products, however, they can be transformed by acid or base treatment to more reactive compounds, i.e. enolate/carboxylates or α, β-unsaturated decalones (Zhao & Zeng 1986; Thomas et al. 1992). These compounds react readily with diazonium salts (Zollinger 1991). Green et al. (2000) demonstrated that the alkali-decomposition product of artesunate react with the diazonium salt, FRTR, to produce a yellow product correlating well with artesunate concentration. As the ARTS-FRTR test did not respond to artemether, a strategy of converting artemether to the reactive α, β-unsaturated decalone was implemented as a means of adapting the ARTS-FRTR test to detect and quantify genuine artemether as well as dihydroartemisinin and artesunate. A qualitative or semi-quantitative assessment of the drug can be determined visually, i.e. without the need for a spectrophotometer, by comparing the colour intensity of the sample to solutions of known drug content. The specificity and simplicity of this colorimetric method (AM-FRTR test) will certainly be useful in detecting counterfeit artemether, artesunate and dihydroartemisinin in the field.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusions
  7. References
  • 1
    Green MD, Mount DL, Wirtz RA, White NJ (2000) A colourimetric field method to assess the authenticity of drugs sold as the antimalarial artesunate. Journal of Pharmaceutical and Biomedical Analysis 24, 6570.
  • 2
    Newton P, Proux S, Green M et al. (2001) Fake artesunate in southeast Asia. Lancet 357, 19481950.
  • 3
    Rozendaal J (2001) Fake antimalaria drugs in Cambodia. Lancet 357, 890890.
  • 4
    Thomas CG, Ward SA, Edwards G (1992) Selective determination, in plasma of artemether and its metabolite, dihydroartemisinin, by high-performance liquid chromatography with ultraviolet detection. Journal of Chromatography 583, 131136.
  • 5
    Zhao S & Zeng M (1986) Application of precolumn reaction to high-performance liquid chromatography of qinghaosu in animal plasma. Analytical Chemistry 58, 289292.
  • 6
    Zollinger M (1991) Colour Chemistry: Synthesis, Properties and Applications of Organic Dyes and Pigments. VCH, New York, p. 119.