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The direct quantitation of active ingredients in solid pharmaceutical tablets by desorption electrospray ionization mass spectrometry (DESI MS) is complicated by the dependence of the DESI signal on variables such as spray angles and distances, morphological sample properties, and the difficulty of properly incorporating an internal standard. Here, a DESI MS method for the direct quantitative screening of widely counterfeited antimalarial tablets containing artesunate is presented. This method is based on reactive DESI, where analyte desorption and ionization occur by the formation of noncovalent complexes between alkylamine molecules in the DESI spray solution and artesunate molecules exposed on the sample surface in the open air. For quantitation purposes, the internal standard d4-artesunic acid was synthesized by esterification of d4-succinic anhydride and dihydroartemisinin, and homogeneously dispersed on the tablet surface via a controlled deposition procedure. The analyte-to-internal standard signal intensity ratio was observed to be largely independent of all DESI variables, only showing dependence on tablet hardness. Analysis of artesunate tablet standards prepared with known amounts of the active ingredient in the 0.02 to 0.32 mg artesunate mg(-1) tablet range resulted in a calibration curve with good linearity (r = 0.9985). Application of this method to the direct quantitation of genuine artesunate tablets from Vietnam showed a 6% (n = 4) precision and 94% accuracy after the spectral data were corrected for tablet hardness.

In support of the efforts to combat the illegal sale and distribution of counterfeit anti-malarial drugs, we evaluated a new analytical approach for the characterization and fast screening of fake and genuine artesunate tablets using a combination of Raman spectroscopy, Spatially Offset Raman Spectroscopy (SORS) and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) imaging. Vibrational spectroscopy provided chemically specific information on the composition of the tablets; the complementary nature of Raman scattering and FTIR imaging allowed the characterization of both the overall and surface composition of the tablets. The depth-resolving power of the SORS approach provided chemically specific information on the overall composition of the tablets, non-invasively, through a variety of packaging types. Spatial imaging of the tablet surface (using ATR-FTIR) identified the location of domains of excipients and active ingredients with high sensitivity and enhanced spatial resolution. The advantages provided by a combination of SORS and ATR-FTIR imaging in this context confirm its potential for inclusion in the analytical protocol for forensic investigation of counterfeit medicines.

Background and Objective: There are several reports of sub-standard and counterfeit antimalarial drugs circulating in the markets of developing countries; we aimed to review the literature for the African continent., Methods: A search was conducted in PubMed in English using the medical subject headings (MeSH) terms: 'Antimalarials/analysis'[MeSH] OR 'Antimalarials/standards'[MeSH] AND 'Africa'[MeSH]' to include articles published up to and including 26 February 2007. Data were augmented with reports on the quality of antimalarial drugs in Africa obtained from colleagues in the World Health Organization. We summarized the data under the following themes: content and dissolution; relative bioavailability of antimalarial products; antimalarial stability and shelf life; general tests on pharmaceutical dosage forms; and the presence of degradation or unidentifiable impurities in formulations., Results and Discussion: The search yielded 21 relevant peer-reviewed articles and three reports on the quality of antimalarial drugs in Africa. The literature was varied in the quality and breadth of data presented, with most bioavailability studies poorly designed and executed. The review highlights the common finding in drug quality studies that (i) most antimalarial products pass the basic tests for pharmaceutical dosage forms, such as the uniformity of weight for tablets, (ii) most antimalarial drugs pass the content test and (iii) in vitro product dissolution is the main problem area where most drugs fail to meet required pharmacopoeial specifications, especially with regard to sulfadoxine-pyrimethamine products. In addition, there are worryingly high quality failure rates for artemisinin monotherapies such as dihydroartemisinin (DHA); for instance all five DHA sampled products in one study in Nairobi, Kenya, were reported to have failed the requisite tests., Conclusions: There is an urgent need to strengthen pharmaceutical management systems such as post-marketing surveillance and the broader health systems in Africa to ensure populations in the continent have access to antimalarial drugs that are safe, of the highest quality standards and that retain their integrity throughout the distribution chain through adequate enforcement of existing legislation and enactment of new ones if necessary, and provision of the necessary resources for drug quality assurance.

A simple and rapid liquid chromatographic-mass spectrometric assay for the evaluation of artesunate in vials for injection has been developed and validated. The content of each vial was dissolved in 3.0 mL of methanol using a SGE analytical syringe (1.0 mL). Each sample was diluted to a theoretical concentration of 1000 ng/mL and analysed in triplicate. Three replicates of calibration standards at concentrations 500, 1000 and 1500 ng/mL were used to construct a calibration curve. Artesunate was analysed by liquid chromatography with atmospheric pressure chemical ionisation (APCI) mass spectrometric (MS) detection on a Hypersil Gold column (100 mm x 4.6 mm) using a mobile phase containing methanol-ammonium acetate 10 mM pH 5.3 (70:30, v/v) at a flow rate of 1 mL/min. The assay was implemented for the analysis of artesunate for injection purchased from Guilin Pharmaceutical Company in China.

A monoclonal antibody (MAb) 1C1 against artemisinin was prepared by a cell fusion with splenocytes and aminopterin-sensitive myeloma cells, SP2/0. An artesunate-BSA conjugate was used as immunogen to raise antibodies specific to artemisinin. The prepared anti-artemisinin MAb-1C1 have a novel characteristic which shows a specificity to compounds that are structurally related to artemisinin such as artesunate (630%) and dihydroartemisinin (29.9%). By using MAb-1C1, a specific and reliable ELISA was developed for the detection of compounds structurally related to artemisinin. The system shows a full measuring range from 2 to 20 microg/mL in the case of artemisinin and from 4 to 125 ng/mL in the case of artesunate in the competitive ELISA, and was validated to be of use for surveying and breeding of Artemisia annua containing a high amount of artemisinin as well as for the characterization of the pharmacokinetics of artemisinin and its derivatives.

Stability of artesunate (ART) was established in three pharmaceutical solvents. The chromatographic conditions developed for this study were acetonitrile:potassium phosphate buffer 10 mM (40:60, v:v; pH 2.9) at 0.7 mL min(-1) with UV detection at 220 nm using a short X-Terra RP C18 column (50 mm x 3 mm, 3.5 microm). This isocratic condition led to the separation between ART and its main degradation products (i.e. alpha-DHA and beta-DHA) with analysis time of less than 4 min. The retention factors are 1.49, 2.26 and 2.79 min for alpha-DHA, beta-DHA and ART, respectively. This method was proved linear (r(2)=0.9995), accurate (R.S.D.=0.20), precise (R.S.D.=0.74) and robust. The system performance remained unaffected by pH variation from 2.6 to 3.2 and variation of acetonitrile percentage from 38 to 42. Stability of ART was assessed in ethanol, propylene glycol (PG) and polyethylene glycol 400 (PEG 400). Unfortunately none of these solvents prevented ART from degradation longer than 3 months. In ethanol, significant degradation of ART occurred after 3 months at room temperature and this degradation was characterised by numerous degradation products. In PEG 400, significant degradation was observed after only 1 month, however DHA was the unique degradation product, which is also an efficient anti-malarial drug.

Artemisia annua L. (Qinghao) is a promising and potent antimalarial herbal drug. This activity has been ascribed to its component artemisinin, a sesquiterpene lactone that is very effective against drug-resistant Plasmodium species with a low toxicity. Our studies indicate that several flavonoids of A. annua can promote and enhance the reaction of artemisinin with hemin. These data are in good agreement with previous investigations on the in vitro potentiation of antimalarial activity of artemisinin by such flavonoids. As a consequence, in view of a possible use of the phytocomplex rather than pure artemisinin, an HPLC/DAD/MS method is proposed for the simultaneous detection and quantification of both flavonoids and artemisinin. Different extracts, obtained from two different herbal drugs, a commercial sample and a selected cultivar, were analyzed in order to determine which solvents provide the best yields of both artemisinin and flavonoids. Qualitative and quantitative results obtained using an HPLC method are described, which will be useful for developing highly effective herbal drug preparations.

Artemisinin is a sesquiterpene lactone endoperoxide and an important antimalarial drug produced in Artemisia annua. To unravel the diverse processes determining artemisinin yield in A. annua crops, artemisinin accumulation during the development of individual leaves was studied in two field experiments. During the life cycle of a leaf, artemisinin was always present. Quantities were low at leaf appearance and increased steadily. In leaves studied until after senescence, maximum quantities and concentrations were achieved after the leaf had turned brown. The total quantity of possible artemisinin precursors per leaf (dihydroartemisinic acid and other upstream precursors) was highest early in the leaf cycle when the leaf was still expanding. Dihydroartemisinic acid was more abundant than the other compounds and its quantity declined during leaf development whereas that of artemisinin increased. Dihydroartemisinic acid was not converted directly into artemisinin, because on a per leaf basis the decline in molar quantity of precursors in the earliest formed leaves was not compensated for by a simultaneous increase in artemisinin. Our results suggest that a (putative) intermediate such as dihydroartemisinic acid hydroperoxide temporarily may have accumulated in considerable quantities. The number of mature, capitate trichomes on the adaxial leaf side increased after leaf appearance until the end of leaf expansion, and then decreased, probably due to collapse of trichomes. Artemisinin production thus (also) occurred when trichomes were collapsing. Later formed leaves achieved higher concentrations of artemisinin than earlier formed leaves, because of a higher trichome density and a higher capacity per trichome.

A rapid and simple RP-TLC method for simultaneous quantification of pharmacologically important sesquiterpene artemisinin (AM) together with its precursors arteannuin-B (AB) and artemisinic acid (AA) in the inflorescence part of Artemisia annua plant has been developed. The RP-TLC of sesquiterpenes was performed on RP-18 F254 S thin-layer chromatographic plates by developing in mobile phase, containing 0.2% TFA in water/ACN (35:65, v/v). The densitometric determination of AM, AB and AA was carried out after derivatization with anisaldehyde reagent at 426 nm in absorption-reflectance mode.

Heme alkylation by the antimalarial drug artemisinin is reported in vivo, within infected mice that have been treated at pharmacologically relevant doses. Adducts resulting from the alkylation of heme by the drug were characterized in the spleen of treated mice, and their glucuroconjugated derivatives were present in the urine. Because these heme-artemisinin adducts were not observed in noninfected mice, this report confirms that the alkylating activity of this antimalarial drug is related to the presence of the parasite in infected animals. The identification of heme-artemisinin adducts in mice should be considered as the signature of the alkylation capacity of artemisinin in vivo.

A liquid chromatography--mass spectrometry (LC-MS) method with selected ion monitoring (SIM) was developed and validated for the analysis and standardization of artemisinin in Artemisia annua L. This method is simple and accurate and requires only an 11 min per sample running time for the direct detection and quantification of artemisinin, a sesquiterpene lactone with antimalarial activity. To accurately analyze artemisinin, SIM was used to record the abundance of the [M - 18 + H]+ ion peak at m/z 265.3, with a scan range between m/z 250 and 270. Quantification was based on the LC-MS peak area of artemisinin, and the standard curve was used for calculation. This method was then validated and applied to several populations of A. annua to assess the population means and the diversity within a selected population. The distribution of artemisinin was found to vary by plant origin with population means ranging from 0.03 to 0.71% artemisinin dry leaf weight. Individual plants within the selected population that exhibited the highest artemisinin mean were then sampled, and the contents ranged from trace amounts to 1.5% artemisinin dry leaf weight basis, making these single plants accumulating artemisinin in concentrations >1.0% promising candidates for plant breeding and varietal development for high-yielding artemisinin.

A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay method was developed and validated for the simultaneous quantitation of alpha-,beta-arteether (alpha-,beta-AE) and its metabolite alpha-dihydroartemisinin (DHA) in monkey plasma using the propyl ether analogue of beta-arteether (PE) as an internal standard. The method involves a simple two-step liquid-liquid extraction with hexane. The analytes were chromatographed on a C(18) reversed-phase chromatographic column by isocratic elution with methanol-ammonium acetate buffer (pH 4) (92 : 8, v/v) and analysed by mass spectrometry in the multiple reaction monitoring mode. The chromatographic run time was 7 min and the weighted (1/x(2)) calibration curves were linear over the range 0.78-200 ng ml(-1). The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in monkey plasma were 0.39 and 0.78 ng ml(-1) respectively for all the analytes. The intra- and inter-batch precision and accuracy were found to be well within acceptable limits (<15%). All three analytes were stable even after three freeze-thaw cycles (deviation < 15%). The average absolute recoveries of alpha-,beta-AE, DHA and PE, used as an internal standard, from spiked plasma samples were 85.85 +/- 6.56, 70.10 +/- 7.06, 54.37 +/- 3.39 and 93.90 +/- 6.9%, respectively. The assay method described here could be applied to study the pharmacokinetics of alpha-,beta-AE and DHA in rhesus monkeys., (Copyright 2003 John Wiley & Sons, Ltd.)

A novel, rapid, and simple capillary electrophoresis (CE) method has been developed for the determination of antimalarial artemisinin by on-line treatment with alkaline. By on-line reaction, artemisinin was automatically and reproducibly converted to the strongly UV-absorbing compound, Q292, by treating it with 0.20 mol/L NaOH solution for 3 min at 40 degrees C. Analysis was carried out in less than 12 min after conversion of artemisinin in a flow injection (FI) system that was coupled to CE equipment via a split-flow interface cell, and a sampling frequency of 8 h(-1) is achievable. The on-line conversion method has been applied to the determination of artemisinin in the traditional Chinese herbal drug Artemisia annua L., and the results are satisfactory.

Egg phosphatidylcholine-cholesterol liposome formulations containing the antimalarial drug beta-artemether have been prepared and analyzed for their encapsulating capacity, chemical stability, leakage, in vitro release and their therapeutic efficiency against Plasmodium chabaudi infection. A HPLC-UV analysis of beta-artemether liposomes without derivatisation was achieved. A good linearity of y=4437.7 x+469.01 (R(2)=0.9999) with a detection limit of 2 microg ml(-1) was reached. Prior to this, liposomal formulations composed of different molar ratios of EPC-CHOL were prepared to select beta-artemether crystal-free liposome preparations. The formulation corresponding to 4:3 and a total concentration of 300 mg lipids ml(-1) buffer (pH 7.2), which could incorporate as much as 1.5 mg beta-artemether was selected for therapy. A trapping efficiency of nearly 100% was reached, the drug being located in the lipid bilayers. A dialysis test demonstrated that the drug could be reversibly released from the liposomes, reaching equilibrium within 24 h. After 3 months storage at 4 degrees C, no leakage of beta-artemether had occurred indicating a high stability of the liposomes. These liposomes were used to treat mice infected with the virulent rodent malaria parasite Plasmodium chabaudi chabaudi, with a 100% cure by clearing the recrudescent parasitaemia.

Artemisinin, a sesquiterpene lactone endoperoxide, and a number of its precursors, metabolites and semisynthetic derivatives have shown to possess antimalarial properties. Several methods have been reported for the measurement of artemisinin and its main derivatives in plant material and biological fluids. However, most of them are either not sufficiently sensitive and do not offer reliable results, or are difficult to apply in routine analyses. Therefore, new methods for the determination of these compounds, such as supercritical fluid extraction and chromatography, pressurized solvent extraction, microwave-assisted extraction, high-performance liquid chromatography coupled to mass spectrometry or evaporative light scattering detection, will be presented. Applications to plant material, pharmaceutical formulations and biological fluids will also be reviewed.

Artesunate is the most widely used of the artemisinin derivatives. These drugs are being used increasingly throughout the tropical world, and are an essential component of the treatment of multi-drug resistant malaria. The recent and widespread appearance of counterfeit artesunate tablets in several countries in Southeast Asia poses a serious threat to health in this region. We have developed a simple, inexpensive colorimetric test to determine artesunate authenticity in tablets. The test is based on a reaction between an alkali decomposition product of artesunate and a diazonium salt, fast red TR (FRTR). The appearance of a yellow color indicates the presence of artesunate. The specificity of the test is dependent on the pH of the reaction. Among other antimalarials tested, (i.e. artemisinin, artemether, chloroquine, quinine, primaquine, sulfadoxine, and pyrimethamine) only artesunate produced a positive color reaction at pH 4. The assay requires only 1% of the total weight of a standard tablet containing 50 mg of artesunate and can be completed within 10 min. The method was tested on six genuine artesunate tablets and six counterfeit artesunate tablets obtained in Southeast Asia. The average amount of artesunate in the genuine tablets was determined to be 50.8 +/- 2.9 mg while the counterfeit tablets were found to contain no artesunate.

A previously established HPLC system with post-column derivatization for the analysis of artemisinin was coupled to an ADS (alkyl-diol silica) pre-column, allowing direct and repetitive injection of protein-rich fluids such as plasma. The limit of quantitation for 100 microl of plasma was 10 ng/ml (CV=10.5%) while concentrations down to 2 ng/ml could be quantified for 1.00 ml saliva samples (CV=11.1%). The system was linear in the tested range of 10-2000 ng/ml for plasma and 2-240 ng/ml for saliva samples, respectively. This paper introduces coupled column HPLC as a simplified method for the routine analysis of artemisinin in biological fluids.

Artemisinin and its analogues are a class of compounds of current interest in the treatment of drug-resistant malaria. These antimalarials are preferentially taken up into malaria infected erythrocytes as compared to uninfected erythrocytes, a fact that may represent an important parameter in drug potency. Numerous methods for the analysis of specific artemisinin analogues have been developed, but most are not widely adaptable to a large range of analogues. In this paper we describe a high-performance liquid chromatographic method developed and validated for artemisinin and several analogues of artemisinin using a readily available evaporative light scattering detector. This quantitation method was found to be straight forward, rapid, inexpensive and reproducible. Standard calibration curves constructed for six artemisinin compounds were linear with the detection limit determined between 6 and 60 ng. The intra- and inter-day accuracy were found to be 2.75% and 4.15%, respectively with less than 3% variation in precision. The validated assay was applied to a mixture of artemisinin derivatives, where they were easily separated and quantitated.

A simple, rapid, sensitive, selective and reproducible high-performance liquid chromatographic method with reductive electrochemical detection is described for the simultaneous quantification of artesunate (ARS) and dihydroartemisinin (DHA) in plasma. The procedure involved the extraction of ARS, DHA and the internal standard (artemisinin, ARN) with a mixture of dichloromethane and tert.-methyl butyl ether (8:2, v/v). Chromatographic separation consisted of the mobile phase (acetonitrile-water containing 0.1 M acetic acid, pH 4.8; 45:55, v/v) running through the column (Nova-Pak C18, 150 cm x 3.9 mm I.D., 5 microm particle size). The retention times of alpha-DHA, beta-DHA, ARS and ARN were 2.9, 4.2, 4.5 and 6.0 min, respectively. The average recoveries of ARS, alpha-DHA and ARN in the concentration range of 10-800 ng/ml were 81.9, 88.2, 101.1 and 84.3%, respectively. The coefficients of variation (precision and repeatability) were below 10% for all three compounds at concentrations of 50, 200, 400 and 800 ng/ml, and below 20% at a concentration of 10 ng/ml. The limits of quantification for both ARS and alpha-DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for application to pharmacokinetic studies of both ARS and DHA.

Four experiments were run to test the anticoccidial activity of dried Artemisia annua leaves and several of their chemical constituents for possible use as prophylactic feed additives. When fed over a period of 3 wk at a level of 5%, a dried leaf supplement of A. annua provided significant protection against lesions due to Eimeria tenella but not Eimeria acervulina or Eimeria maxima. When fed over a period of 5 wk at a level of 1% to chicks undergoing immunization with a live vaccine, it provided significant protection in partially immunized chicks against E. acervulina and E. tenella lesions from a dual species challenge infection. It also afforded lower mean lesion scores in challenged chicks immunized over a period of 5 wk. Artemisinin, an antimalarial component of A. annua, was present at a level of 0.034% in the dried leaf preparation. A 5% supplement thus afforded about 17 ppm artemisin. When the pure compound was fed at that level for a period of 3 wk, it protected weight gains and significantly reduced lesion scores attributable to E. tenella but not E. acervulina. Other components of A. annua, camphor and 1,8-cineole, at 119 ppm also protected weight gains, and reduced E. tenella lesion scores. Camphor reduced E. acervulina lesions. Artemisinin fed for 4 wk at levels of 2, 8.5, and 17 ppm significantly reduced oocyst output from separate E. acervulina and E. tenella infections and a dual species infection. Pure artemisinin thus appears to be effective against at least two coccidia species when used as a feed additive, and its activity may depend, in part, on the length of time it is administered before a challenge infection.

Artemisinin was derivatized to dihydroartemisinin carboxymethylether in three steps, without disturbing the peroxide bridge, and then linked to either thyroglobulin (TGB) or bovine serum albumin (BSA). The artemisinin-TGB and -BSA conjugates were injected in female New Zealand rabbits but only the artemisinin-TGB conjugate generated polyclonal antibodies. An enzyme-linked immunosorbent assay (ELISA) was developed and the specificity of the antibodies was confirmed by comparison with pre-immune serum and by competitive assays using different dilutions of artemisinin standards. Although anti-artemisinin antibodies cross-reacted with artemisitene and dihydroartemisinin at all dilutions used, cross-reaction with deoxyartemisinin, artemisinic acid, and arteannuin B occurred only at high concentrations. ELISA successfully detected artemisinin from crude extracts in concentrations as low as 1.5 ng ml-1; and was epsilon 400-fold more sensitive than the HPLC-EC. The ELISA successfully detected and quantified artemisinin in different organs of greenhouse-grown plants and in eight clones of Artemisia annua grown in tissue culture but artemisinin was overestimated owing to cross-reactivity of the antibodies with artemisinin-related compounds present in the samples. Despite overestimation of artemisinin content, the correlations between ELISA and HPLC-EC were r = 0.92 when samples were diluted 100 times, and r = 0.90 when samples were diluted 500 times, indicating that ELISA is a potential tool for screening large A. annua populations.

The development of selective analytical methods for the determination of artemisinin and its analogues and metabolites in biological fluids poses challenging problems. All are thermally labile, lack ultraviolet (u.v.) absorbent or fluorescent chromophores and do not possess functional groups for derivatization. Two chemical approaches have been used; acid (or alkali) catalysed decomposition to u.v. absorbing compounds followed by high-performance liquid chromatography (HPLC) of the decomposition products, and HPLC with reductive electrochemical detection. Each has its difficulties. The first, although sensitive, may lack specificity if drug metabolities are also converted to identical products, but a recently developed method surmounts this problem. The second, while providing good sensitivity and specificity, requires rigorous deoxygenation of the sample and mobile phase and an electrochemical detector, which is expensive, and may prove difficult to operate under field conditions. As an alternative to a chemical method, a bioassay may be suitable for plasma level monitoring in such circumstances. However its lack of selectivity means it is of questionable value in the determination of the pharmacokinetic parameters for individual drug-related species.

Ultrastructural changes induced in Plasmodium falciparum by artemisinin were studied in vitro. Electron microscopic autoradiography was performed on infected erythrocytes that were exposed in vitro to 3H-dihydroartemisinin and 14C-artemisinin. These drugs consistently were located in food vacuoles and mitochondria. Two hours after administration, changes were observed in parasite mitochondria, rough endoplasmic reticulum, and nuclear envelope. At four hours, in addition to the earlier changes, nuclear membranes and, to a lesser extent, some plasma membranes formed myelin figures. In addition, there was a disappearance of ribosomes, and a destruction of food vacuole membranes. These changes may lead to the total disorganization of the parasites. Approximately 30% of the parasites manifested these alterations.

beta-Arteether (1) is a new antimalarial drug derived from artemisinin (2, quinghaosu). Compound 1 is quite water insoluble, but very soluble in a variety of organic solvents. Solubilization in a variety of surfactants was investigated to obtain higher concentrations of 1 in aqueous solutions. Anionic and cationic surfactants exhibited dramatic solubilizing ability for 1, while nonionic surfactants showed significantly lower solubilizing ability. The solubilization data are analyzed on the basis of a pseudo-phase model with 1 exhibiting a high partition coefficient into the micellar phase. An empirical model is presented to identify the possible sites of solubilization of 1 in the micelle.