The effect of mephedrone on human neuroblastoma and astrocytoma cells.

Mephedrone is an illegal drug that is used recreationally. Few studies have been conducted to investigate the mechanisms by which mephedrone is harming cells. In this research, we investigated the effect of mephedrone using toxicology coupled with LC-MS/MS based metabolomics in the two CNS derived cell lines. Methods of assessment such as neutral red (NR) assay, dimethylthiazolyl diphenyltetrazolium bromide (MTT), lactose dehydrogenase (LDH) measurement, and morphology were performed to identify the effect on cell viability and to identify the best concentration to be used in a metabolomics study. A concentration of 100 µM of mephedrone was used in the metabolomic experiment because at this concentration mephedrone had induced several intracellular changes. Although there no clear indicators of cellular damage caused by mephedrone. In astrocytes there was a clear indication that cell membrane function might be impaired by depletion of ether lipids.

LC/MS-based discrimination between plasma and urine metabolomic changes following exposure to ultraviolet radiation by using data modelling.

INTRODUCTION: This study sought to compare between metabolomic changes of human urine and plasma to investigate which one can be used as best tool to identify metabolomic profiling and novel biomarkers associated to the potential effects of ultraviolet (UV) radiation. METHOD: A pilot study of metabolomic patterns of human plasma and urine samples from four adult healthy individuals at before (S1) and after (S2) exposure (UV) and non-exposure (UC) were carried out by using liquid chromatography-mass spectrometry (LC-MS). RESULTS: The best results which were obtained by normalizing the metabolites to their mean output underwent to principal components analysis (PCA) and Orthogonal Partial least squares-discriminant analysis (OPLS-DA) to separate pre-from post-of exposure and non-exposure of UV. This separation by data modeling was clear in urine samples unlike plasma samples. In addition to overview of the scores plots, the variance predicted-Q2 (Cum), variance explained-R2X (Cum) and p-value of the cross-validated ANOVA score of PCA and OPLS-DA models indicated to this clear separation. Q2 (Cum) and R2X (Cum) values of PCA model for urine samples were 0.908 and 0.982, respectively, and OPLS-DA model values were 1.0 and 0.914, respectively. While these values in plasma samples were Q2 = 0.429 and R2X = 0.660 for PCA model and Q2 = 0.983 and R2X = 0.944 for OPLS-DA model. LC-MS metabolomic analysis showed the changes in numerous metabolic pathways including: amino acid, lipids, peptides, xenobiotics biodegradation, carbohydrates, nucleotides, Co-factors and vitamins which may contribute to the evaluation of the effects associated with UV sunlight exposure. CONCLUSIONS: The results of pilot study indicate that pre and post-exposure UV metabolomics screening of urine samples may be the best tool than plasma samples and a potential approach to predict the metabolomic changes due to UV exposure. Additional future work may shed light on the application of available metabolomic approaches to explore potential predictive markers to determine the impacts of UV sunlight.

The Activity of Red Nigerian Propolis and Some of Its Components against Trypanosoma brucei and Trypanosoma congolense.

Propolis is a resin that is gathered by bees from exudates produced by various plants. Its exact chemical composition depends on the plants available near the hive. Bees use propolis to coat the surfaces of the hive, where it acts as an anti-infective. Regardless of the chemical composition of propolis, it is always anti-protozoal, probably because protozoan parasites, particularly Lotmarium passim, are widespread in bee populations. The protozoa Trypanosoma brucei and T. congolense cause disease in humans and/or animals. The existing drugs for treating these diseases are old and resistance is an increasingly severe problem. The many types of propolis present a rich source of anti-trypanosomal compounds-from a material gathered by bees in an environmentally friendly way. In the current work, red Nigerian propolis from Rivers State, Nigeria was tested against T. brucei and T. congolense and found to be highly active (EC50 1.66 and 4.00 µg/mL, respectively). Four isoflavonoids, vestitol, neovestitol, 7-methylvestitol and medicarpin, were isolated from the propolis. The isolated compounds were also tested against T. brucei and T. congolense, and vestitol displayed the highest activity at 3.86 and 4.36 µg/mL, respectively. Activities against drug-resistant forms of T. brucei and T. congolense were similar to those against wild type.

The Antiprotozoal Activity of Papua New Guinea Propolis and Its Triterpenes.

Profiling a propolis sample from Papua New Guinea (PNG) using high-resolution mass spectrometry indicated that it contained several triterpenoids. Further fractionation by column chromatography and medium-pressure liquid chromatography (MPLC) followed by nuclear magnetic resonance spectroscopy (NMR) identified 12 triterpenoids. Five of these were obtained pure and the others as mixtures of two or three compounds. The compounds identified were: mangiferonic acid, ambonic acid, isomangiferolic acid, ambolic acid, 27-hydroxyisomangiferolic acid, cycloartenol, cycloeucalenol, 24-methylenecycloartenol, 20-hydroxybetulin, betulin, betulinic acid and madecassic acid. The fractions from the propolis and the purified compounds were tested in vitro against Crithidia fasciculata, Trypanosoma congolense, drug-resistant Trypanosoma congolense, Trypanosoma b. brucei and multidrug-resistant Trypanosoma b. brucei (B48). They were also assayed for their toxicity against U947 cells. The compounds and fractions displayed moderate to high activity against parasitic protozoa but only low cytotoxicity against the mammalian cells. The most active isolated compound, 20-hydroxybetulin, was found to be trypanostatic when different concentrations were tested against T. b. brucei growth.

Activity of Compounds from Temperate Propolis against Trypanosoma brucei and Leishmania mexicana.

Ethanolic extracts of samples of temperate zone propolis, four from the UK and one from Poland, were tested against three Trypanosoma brucei strains and displayed EC50 values < 20 µg/mL. The extracts were fractionated, from which 12 compounds and one two-component mixture were isolated, and characterized by NMR and high-resolution mass spectrometry, as 3-acetoxypinobanksin, tectochrysin, kaempferol, pinocembrin, 4'-methoxykaempferol, galangin, chrysin, apigenin, pinostrobin, cinnamic acid, coumaric acid, cinnamyl ester/coumaric acid benzyl ester (mixture), 4',7-dimethoxykaempferol, and naringenin 4',7-dimethyl ether. The isolated compounds were tested against drug-sensitive and drug-resistant strains of T. brucei and Leishmania mexicana, with the highest activities ≤ 15 µM. The most active compounds against T. brucei were naringenin 4',7 dimethyl ether and 4'methoxy kaempferol with activity of 15-20 µM against the three T. brucei strains. The most active compounds against L. mexicana were 4',7-dimethoxykaempferol and the coumaric acid ester mixture, with EC50 values of 12.9 ± 3.7 µM and 13.1 ± 1.0 µM. No loss of activity was found with the diamidine- and arsenical-resistant or phenanthridine-resistant T. brucei strains, or the miltefosine-resistant L. mexicana strain; no clear structure activity relationship was observed for the isolated compounds. Temperate propolis yields multiple compounds with anti-kinetoplastid activity.

The Strong Anti-Kinetoplastid Properties of Bee Propolis: Composition and Identification of the Active Agents and Their Biochemical Targets.

The kinetoplastids are protozoa characterized by the presence of a distinctive organelle, called the kinetoplast, which contains a large amount of DNA (kinetoplast DNA (kDNA)) inside their single mitochondrion. Kinetoplastids of medical and veterinary importance include Trypanosoma spp. (the causative agents of human and animal African Trypanosomiasis and of Chagas disease) and Leishmania spp. (the causative agents of the various forms of leishmaniasis). These neglected diseases affect millions of people across the globe, but drug treatment is hampered by the challenges of toxicity and drug resistance, among others. Propolis (a natural product made by bees) and compounds isolated from it are now being investigated as novel treatments of kinetoplastid infections. The anti-kinetoplastid efficacy of propolis is probably a consequence of its reported activity against kinetoplastid parasites of bees. This article presents a review of the reported anti-kinetoplastid potential of propolis, highlighting its anti-kinetoplastid activity in vitro and in vivo regardless of geographical origin. The mode of action of propolis depends on the organism it is acting on and includes growth inhibition, immunomodulation, macrophage activation, perturbation of the cell membrane architecture, phospholipid disturbances, and mitochondrial targets. This gives ample scope for further investigations toward the rational development of sustainable anti-kinetoplastid drugs.

Deletion of TSPO Resulted in Change of Metabolomic Profile in Retinal Pigment Epithelial Cells.

Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD.

Isolation of a Novel Flavanonol and an Alkylresorcinol with Highly Potent Anti-Trypanosomal Activity from Libyan propolis.

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans, showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4'-methyl ether (4), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans, while the purified compounds had low activity against P. falciparum. Anti-trypanosomal activity (EC50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol (10) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction.

Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

Phosphoenolpyruvate carboxylase identified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism.

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10(Δpepc) ), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10(Δpepc) had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using (13)C-U-D-glucose and (13)C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10(Δpepc) and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of (13)C,(15)N-U-glutamine was similar in both parasite lines, although the flux was lower in D10(Δpepc); it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery.

An evaluation of Minor Groove Binders as anti-lung cancer therapeutics.

A series of 47 structurally diverse MGBs, derived from the natural product distamycin, was evaluated for anti-lung cancer activity by screening against the melanoma cancer cell line B16-F10. Five compounds have been found to possess significant activity, more so than a standard therapy, Gemcitabine. Moreover, one compound has been found to have an activity around 70-fold that of Gemcitabine and has a favourable selectivity index of greater than 125. Furthermore, initial studies have revealed this compound to be metabolically stable and thus it represents a lead for further optimisation towards a novel treatment for lung cancer.

Chemical characterisation of Nigerian red propolis and its biological activity against Trypanosoma Brucei.

INTRODUCTION: A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), with regards to chemical composition and activity against Trypanosoma brucei in comparison with other African propolis. OBJECTIVE: To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti-trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis. METHODOLOGY: Profiling was carried out using HPLC-UV-ELSD and HPLC-Orbitrap-FTMS on extracts of two samples collected from RSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucidation of the compounds with a purity > 95% was performed by 1D/2D NMR HRMS and HRLC-MS(n) . RESULTS: Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlated with literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanones, liquiritigenin (2) and pinocembrin (5); an isoflavan, vestitol (3); a pterocarpan, medicarpin (4); two prenylflavanones, 8-prenylnaringenin (7) and 6-prenylnaringenin (8); and two geranyl flavonoids, propolin D (9) and macarangin (10). The tenth was elucidated as a previously undescribed dihydrobenzofuran (6). The isolated compounds were tested against Trypanosoma brucei and displayed moderate to high activity. Some of the compounds tested had similar activity against wild type T. brucei and two strains displaying pentamidine resistance. CONCLUSION: Nigerian propolis from RSN has some similarities with Brazilian red propolis. The propolis displayed anti-trypanosomal activity at a potentially useful level.

LC-MS metabolomics of psoriasis patients reveals disease severity-dependent increases in circulating amino acids that are ameliorated by anti-TNFα treatment.

Psoriasis is an immune-mediated highly heterogeneous skin disease in which genetic as well as environmental factors play important roles. In spite of the local manifestations of the disease, psoriasis may progress to affect organs deeper than the skin. These effects are documented by epidemiological studies, but they are not yet mechanistically understood. In order to provide insight into the systemic effects of psoriasis, we performed a nontargeted high-resolution LC-MS metabolomics analysis to measure plasma metabolites from individuals with mild or severe psoriasis as well as healthy controls. Additionally, the effects of the anti-TNFα drug Etanercept on metabolic profiles were investigated in patients with severe psoriasis. Our analyses identified significant psoriasis-associated perturbations in three metabolic pathways: (1) arginine and proline, (2) glycine, serine and threonine, and (3) alanine, aspartate, and glutamate. Etanercept treatment reversed the majority of psoriasis-associated trends in circulating metabolites, shifting the metabolic phenotypes of severe psoriasis toward that of healthy controls. Circulating metabolite levels pre- and post-Etanercept treatment correlated with psoriasis area and severity index (PASI) clinical scoring (R(2) = 0.80; p < 0.0001). Although the responsible mechanism(s) are unclear, these results suggest that psoriasis severity-associated metabolic perturbations may stem from increased demand for collagen synthesis and keratinocyte hyperproliferation or potentially the incidence of cachexia. Data suggest that levels of circulating amino acids are useful for monitoring both the severity of disease as well as therapeutic response to anti-TNFα treatment.

A short review of applications of liquid chromatography mass spectrometry based metabolomics techniques to the analysis of human urine.

The applications of metabolomics as a methodology for providing better treatment and understanding human disease continue to expand rapidly. In this review, covering the last two years, the focus is on liquid chromatography-mass spectrometry (LC-MS) profiling of metabolites in urine. In LC-MS based metabolomics there are still problems with regard to: chromatographic separation, peak picking and alignment, metabolite identification, metabolite coverage, instrument sensitivity and data interpretation and in the case of urine sample normalisation. Progress has been made with regard to all of these issues during the period of the review. Of particular interest are the increasing use of orthogonal chromatographic methods for optimal metabolite coverage and the increasing adoption of receiver operator characteristic (ROC) curves for biomarker validation.

Chromatographic and spectroscopic analysis of the components present in the phenanthridinium trypanocidal agent isometamidium.

The chromatographic isolation and characterisation of the four compounds present in the quaternary phenanthridine veterinary trypanocidal agent, isometamidium chloride hydrochloride (ISM), is reported. The isolated compounds were unambiguously characterised using spectroscopic (NMR, UV, IR and MS) methods as 3-amino-8-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium (1a) and related isomers, 8-amino-3-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium, 3,-8-diamino-7-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium and 3,-8-bis[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium. During the course of this study, it was realised that the nature of the solvent used in the NMR study was critical as in DMSO-d6 the quaternary group in the compounds was reduced to dihydro forms (e.g. 2a).

An LCMS method for the assay of melittin in cosmetic formulations containing bee venom.

There is a growing interest in the potential of bee venom in cosmetics as a rejuvenating agent. Products currently on the market do not specify exactly their content of bee venom (BV). Therefore, we developed a method for the detection and quantification of melittin, as a marker of bee venom content, in selected commercial creams which contained BV according to their marketing claims, in order to gauge the relative quality of such formulations. A quantitative method was achieved following a rigorous extraction procedure involving sonication, liquid-liquid extraction and solid phase extraction since carryover of excipients was found to cause a rapid deterioration in the chromatographic performance. The method employed a standard additions approach using, as spiking standard, purified melittin isolated from bee venom and standardised by quantitative NMR. The aqueous extracts of the spiked creams were analysed by reversed phase LCMS on an LTQ Orbitrap mass spectrometer. The purity of the melittin spiking standard was determined to be 96.0%. The lowest measured mean melittin content in the creams was 3.19 ppm (±1.58 ppm 95% CI) while the highest was 37.21 ppm (±2.01 ppm 95% CI). The method showed adequate linearity (R (2) ≥ 0.98) and a recovery of 87.7-102.2% from a spiked blank cream. An assay precision of <20% RSD was achieved for all but one sample where the RSD value was 27.5%. The method was sensitive enough for use in routine assay of BV-containing cosmetic creams. Differences in the melittin content of the commercial products assayed were nearly tenfold.

The abundant dietary constituent ferulic acid forms a wide range of metabolites including a glutathione adduct when incubated with rat hepatocytes.

1. The metabolism of ferulic acid (FA) has been studied in a number of different systems and several metabolites of FA have been characterised. No previous work has been carried out using hepatocytes to characterise the metabolism of FA. 2. A metabolomics approach in combination with high resolution mass spectrometry was used to characterise the metabolites of FA formed in isolated rat hepatocytes. FA was incubated with rat hepatocytes and the metabolites formed were profiled at 30 and 120 minutes. The metabolites were characterised according to their accurate mass at <2 ppm using Fourier transform mass spectrometry (FT-MS). 3. Sixteen metabolites of FA were identified. The most abundant metabolite was the sulphate of FA and this was followed by FA glucuronide and glycine conjugates. A wide range of low level metabolites were produced in the hepatocyte incubations. Novel metabolites resulted from side chain oxidation. 4. In addition, a glutathione (GSH) adduct of FA was formed. Incubation of a solution of FA with GSH also resulted in formation of this adduct indicating that it could be formed purely by a chemical reaction. Thus the metabolism of FA in rat hepatocytes is more complex than previously described.

The isolation of antiprotozoal compounds from Libyan propolis.

Propolis is increasingly being explored as a source of biologically active compounds. Until now, there has been no study of Libyan propolis. Two samples were collected in North East Libya and tested for their activity against Trypanosoma brucei. Extracts from both samples had quite high activity. One of the samples was fractionated and yielded a number of active fractions. Three of the active fractions contained single compounds, which were found to be 13-epitorulosal, acetyl-13-epi-cupressic acid and 13-epi-cupressic acid, which have been described before in Mediterranean propolis. Two of the compounds had a minimum inhibitory concentration value of 1.56 µg/mL against T. brucei. The active fractions were also tested against macrophages infected with Leishmania donovani, and again moderate to strong activity was observed with the compounds having IC50 values in the range 5.1-21.9 µg/mL.

Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase.

Several mitochondrial proteins are tumor suppressors. These include succinate dehydrogenase (SDH) and fumarate hydratase, both enzymes of the tricarboxylic acid (TCA) cycle. However, to date, the mechanisms by which defects in the TCA cycle contribute to tumor formation have not been elucidated. Here we describe a mitochondrion-to-cytosol signaling pathway that links mitochondrial dysfunction to oncogenic events: succinate, which accumulates as a result of SDH inhibition, inhibits HIF-alpha prolyl hydroxylases in the cytosol, leading to stabilization and activation of HIF-1alpha. These results suggest a mechanistic link between SDH mutations and HIF-1alpha induction, providing an explanation for the highly vascular tumors that develop in the absence of VHL mutations.

Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software.

Background: Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is considered a first-line antimalarial agent. Artemether is extensively metabolized in vivo to its active metabolite DHA, and therefore its determination offers considerable difficulties. In the present study, DHA identification and estimation were accurately performed by the mass spectrometric analysis, using a high-resolution liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) LTQ Orbitrap hybrid mass spectrometer. Methods: The plasma samples were taken from healthy volunteers, and the spiked plasma was extracted by adding 1 mL of a mixture of dichloromethane and tert.-methyl butyl ether (8:2 v/v) to 0.5 mL of plasma. The internal standard solution (artemisinin 500 ng/mL) was added to the plasma samples. After vertexing and centrifugation, the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100 µL of acetonitrile and was injected onto the LC-MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A consisted of 0.1% v/v formic acid in water, Mobile phase B consisted of acetonitrile only, and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500 µL/min. The ESI interface was operated in a positive ion mode with a spray voltage of 4.5 kV. Results: Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water, respectively, in the source of the mass spectrometer. The ions observed were (MH-H2O) m/z 267.15 for DHA and (MH-m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines. Discussion: The validated method was applied successfully for the determination and quantification of DHA in plasma samples. This method works well for the extraction of drugs, and the Orbitrap system with the help of Xcalibur software accurately and precisely determines the concentration of DHA in spiked as well as volunteer's plasma.