Innovative stability-indicating LC-Corona CAD method for simultaneous determination of assay in Artesunate and Mefloquine hydrochloride fixed-dose combination product

This work describes for the first time a stability-indicating HPLC-Corona CAD method for content determination of Artesunate (AS) and Mefloquine hydrochloride (MQ) in coated tablets 100 + 220 mg (ASMQ) developed by Farmanguinhos-Fiocruz. The chromatographic separation was carried out on two Promosil C18 columns in sequence. Chromatography was done using 0.05% formic acid/acetonitrile (80:20) in gradient at flow rate of 1 mL min^?1, flow 0.6 mL/min for the right pump, and 0.3 mL/min for the left pump (acetonitrile 100%). The temperature was set at 25 °C for the oven and the detection. The elution time of AS and MQ was found to be 40.5 ± 0.5 min and 10.5 ± 0.5, respectively. The method was validated for system suitability, selectivity, linearity, precision, accuracy, and robustness. The forced degradation studies indicated that AS instability is the major trigger for product degradation, especially under heat and oxidative conditions. In conclusion, the method validation was in agreement with ICH guideline Q2(R1) and AOAC acceptance criteria. Our findings prospected the Corona-CAD detector as a quality control solution regarding the challenges of stability-indicating methods for fixed-dose products.     

Identification of impurities in artemisinin, their behavior in high performance liquid chromatography and implications for the quality of derived anti-malarial drugs

Previous work [1] on the HPLC analysis of artemisinin tentatively identified the two impurities present above trace levels. This identification was based on LC-MS results and NMR of impurities isolated from artemisinin. In this work the impurities have been synthesized allowing verification of their identity by LC-MS. It is found that the previously suggested elution order is incorrect. A determination of relative response factors strongly impacts suggested limits on impurity levels and explains the erroneous peak assignment. The fates of the identified impurities are explored in the transformation of artemisinin to its derivative active pharmaceutical ingredients. A survey of a wide variety of artemisinin samples isolated from different geographical regions, different growing seasons, different plant backgrounds and using different extraction and purification approaches showed that artemisinin has sufficient purity for its intended use as a raw material for anti-malarial drug products.     

Voltammetric behaviour of antimalarial drug artesunate in solubilized systems

The voltammetric behaviour of artesunate is studied at glassy carbon electrode in different buffer systems using square wave, differential pulse and cyclic voltammetric techniques. The peak current is linear with the drug concentration in the range 4.0-40 μg mL(-1) for serum, plasma and urine. The mean percentage recoveries of the drug, urine, plasma and serum samples are 98.6-100.2%. No electroactive interferences from the excipients and endogenous substance could be observed in the pharmaceutical dosage forms and in biological samples.     

Phosphate imbalance conducting by BPs-based cancer-targeting phosphate anions carrier induces necrosis

As a vital nutrient closely related to the cancer-cells proliferation, phosphate anions have been paid great attention as a promising anticancer agent. Generally, the transport of phosphate anions depends on a protein transport system which is regulated by ion homeostasis regulations. Herein, we designed a reactive anionic nanocarrier based on black phosphorus nanosheets (BPs) and artesunate (ART), which could enter cells through endocytosis to generate phosphate anions, avoiding the regulation of cell homeostasis. The ionic nanocarrier was coated by polydopamine to defend BPs and ART and functionalized by folate (FA) and hyaluronic acid (HA) for targeting factor. With the anchoring groups FA/HA targeted the carrier into cells, polydopamine coating decomposed to expose ART for further generating reactive oxygen species (ROS) in cancer cell microenvironment, providing oxidation conditions. Next, ROS generated by ART makes BPs decompose to phosphate anions with effectively speed, giving rise to the destruction of ion homeostasis to induce necrosis and inhibit the proliferation for cancer cells. In consequence, this research provides novel idea and direction for the ionic carriers and tumor therapeutics.     

High performance liquid chromatographic evaluation of artemisinin,raw material in the synthesis of artesunate and artemether

A barrier to the development of artemisinin derivative based combination treatment of malaria is the lack of defined specifications and purity test methods for the raw material artemisinin. An HPLC method previously published in the International Pharmacopoeia to evaluate purity of artemisinin as an active pharmaceutical ingredient is adapted for use. Excellent method precision and linearity are demonstrated along with observations of robustness. In support of the development of specifications major impurities are identified using high resolution HPLC–MS, isolation via preparative HPLC followed by NMR. The identified impurities differ from those previously claimed.     

Determination of artesunate using reversed‐phase HPLC at increased temperature and ELSD detection

Artesunate (ART) determination can be performed by evaporative light scattering detection with mobile phase composed of CH₃CN/HCOOH 0.01 M (40:60 v/v; pH 2.85). Evaporative light scattering detection instead of UV detection allowed to improve the sensitivity and the LOD. However, the evaporative light scattering detection response of dihydro‐artemisinin appears weaker than for ART, whereas with UV detection the response of ART and dihydroartemisinin seemed similar. Constant analysis time was obtained on using the mobile phase with a flow rate of 0.5 mL/min and column temperature at 60°C instead of 0.7 mL/min at room temperature. This led to less solvent consumption. Moreover, decrease in the flow rate and increase in the column temperature were advantageous for higher sensitivity with both evaporative light scattering detection and UV detection. ART determination in rectal gel and suppositories were compared with these different detection modes and similar results were obtained.     

Characterization of genuine and fake artesunate anti-malarial tablets using Fourier transform infrared imaging and spatially offset Raman spectroscopy through blister packs

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.     

Combined Fourier-transform infrared imaging and desorption electrospray-ionization linear ion-trap mass spectrometry for analysis of counterfeit antimalarial tablets

This paper reports use of a combination of Fourier-transform infrared (FTIR) spectroscopic imaging and desorption electrospray ionization linear ion-trap mass spectrometry (DESI MS) for characterization of counterfeit pharmaceutical tablets. The counterfeit artesunate antimalarial tablets were analyzed by both techniques. The results obtained revealed the ability of FTIR imaging in non-destructive micro-attenuated total reflection (ATR) mode to detect the distribution of all components in the tablet, the identities of which were confirmed by DESI MS. Chemical images of the tablets were obtained with high spatial resolution. The FTIR spectroscopic imaging method affords inherent chemical specificity with rapid acquisition of data. DESI MS enables high-sensitivity detection of trace organic compounds. Combination of these two orthogonal surface-characterization methods has great potential for detection and analysis of counterfeit tablets in the open air and without sample preparation.     

Investigation of porous graphitic carbon at high-temperature liquid chromatography with evaporative light scattering detection for the analysis of the drug combination artesunate—Azithromycin for the treatment of severe malaria

Artesunate combined therapies represent the best option for the treatment of malaria and require the development of new methods of analysis. Retention, selectivity and detection with high-temperature liquid chromatography-porous graphitic carbon-evaporative light scattering detection was studied for artesunate and azithromycin separation. Organic solvent, concentration of organic modifiers, temperature and flow rate were all relevant parameters to optimize this separation. The behaviour of artesunate in the tested conditions appeared close to a neutral compound. In CH₃OH, only azithromycin retention was dramatically altered depending on the [triethylamine]/[formic acid] ratio and on the temperature, whereas in CH₃CN, azithromycin, artesunate, artemisinin and dihydroartemisinin retentions decreased with the temperature increase whatever the organic modifier ratio. The best efficiency was obtained with CH₃CN. 25% variation of the concentration values of the organic modifiers did not significantly influenced the retention. The sensitivity of ELSD increased with the flow rate decrease. Peak area and S/N ratio dramatically decreased with the flow rate increase by 10- and 5-fold for artesunate and azithromycin, respectively. Non-linear calibration curves were obtained for both artesunate and azithromycin.     

Simultaneous Determination of Artesunate and Dihydroartemisinin Concentrations in Human Plasma by High Performance Liquid Chromatography-Mass Spectrometry with Electro-Spray Ionization

An analytical procedure was developed/validated for the quantification of artesunate (ARTS) and a metabolite, dihydroartemisinin (DHA), in human plasma. Plasma samples were processed by solid-phase extraction and analyzed by LC-MS. The detector response was specific and linear for ARTS and DHA concentrations in the range of 5–1000 ng/mL. Intra-/inter-day precision/accuracy were determined to be within the acceptance criteria for assay validation guidelines. The analytes were stable under various processing/handling conditions. The ARTS/DHA concentrations were readily measured in plasma samples up to 8 hrs after an oral administration of an ARTS formulation in humans, suggesting that the assay is practically useful.