Ammonium Acetate Methanol Research Articles

Synthesis of 2-Methyl-4-aryl-4,6,7,8-tetrahydro-5(1H)-quinolone Derivatives and their Effects on Potassium Channels

In this study, twelve compounds having 2-methyl-4-aryl-4,6,7,8-tetrahydro-5(1H)-quinolone structure have been synthesized by the reaction of 4-aryl-3-butene-2-on derivatives with 1,3-cyclohexanedione analogs in the presence of ammonium acetate in methanol. The structures of the compounds have been elucidated by IR, 1H-NMR, 13C-NMR, mass spectroscopy and elementel analysis. Their potassium channel opener activities have been investigated on isolated rabbit bladder smooth muscle using pinacidil (CAS 85371-64-8) as standard. The test compounds and pinacidil caused concentration-dependent relaxation responses in bladder smooth muscle strips precontracted with 80 mmol/L KCl with the efficacy order: pinacidil > or = 3g > or = 3j > or = 3a > or = 3l = 3i > or = 3c = 3b > or = 3d > or = 3h > or = 3k. In bladder smooth muscle strips precontracted with 15 mmol/L KCl, the efficacy order was: pinacidil > 3h > or = 3c > or = 3j > or = 3g > or = 3l > or = 3i = 3b > or = 3k > or = 3f > or = 3a. The test compounds and pinacidil caused concentration-dependent inhibition of electrical field stimulation-evoked contractile responses in the bladder smooth muscle strips with the efficacy order: 3j > or = 3l pinacidil > or = 3k > or = 3h > or = 3a > or = 3g > or = 3c > or = 3i > or = 3b > or = 3f.

Determination of propiverine hydrochloride in human plasma by high performance liquid chromatographytandem mass spectrometry: application to the pharmacokinetic study of a sustained release formulation

A rapid, sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of propiverine hydrochloride (CAS 54556-98-8) in human plasma using cetirizine di-hydrochloride as internal standard (IS, CAS 8388-51-0). Following liquid-liquid extraction with ethyl acetate, the separation was performed on a reverse phase C18 column with a mobile phase consisted of methanol-ammonium acetate (pH 4.0; 10 mM) (70:30, v/v). The detection was performed by a triple-quadrupole mass spectrometer in the positive ion and multiple reaction monitoring (MRM) mode, m/z 368.3 --> 116.1 for propiverine and m/z 389.2 --> 201.0 for the IS. The calibration curve fitted well over the concentration range of 0.2-200 ng/mL (all the concentration data in this study are related to salt (propiverine hydrochloride)). The limit of detection (LOD) and lower limit of quantification (LLOQ) in human plasma were 0.05 and 0.2 ng/mL, respectively. The method was proved to be rapid, sensitive, specific, accurate and reproducible and has been successfully applied to a pharmacokinetic study of propiverine hydrochloride sustained release capsules (the 30 mg dose in this study is related to 30 mg of salt (propiverine hydrochloride)). The major pharmacokinetic parameters in healthy Chinese volunteers are given for the first time and the sustained release characteristics of the sustained release formulation are evaluated. [corrected].

A rapid and sensitive determination of aucubin in rat plasma by liquid chromatography–tandem mass spectrometry and its pharmacokinetic application

A sensitive, accurate, rapid and robust LC-MS-MS method for the quantification of aucubin, a major bioactive constituent of Aucuba japonica, Eucommia ulmoides and Plantago asiatica, was established and validated in rat plasma. Plasma samples were simply precipitated by adding methanol and the supernatant was chromatographed by a Diamonsil® C(18)(2) column with the mobile phase comprising a mixture of 10 mm ammonium acetate in methanol and that in water with the ratio of 50:50 (v/v). Quantification of aucubin was performed by mass spectrometry in the multiple-reaction monitoring mode with positive atmospheric ionization at m/z 364 → 149 for aucubin, and m/z 380 → 165 for catalpol (IS), respectively. The retention time was 2.47 and 2.44 min for aucubin and the IS, respectively. The calibration curve (10.0-30,000 ng/mL) was linear (r² > 0.99) and the lower limit of quantification was 10.0 ng/mL in the rat plasma sample. The method showed satisfactory results such as sensitivity, specificity, precision, accuracy, recovery, freeze-thaw and long-term stability. This simple LC-MS method was successfully applied in a pharmacokinetic study carried out in Sprague-Dawley rats after oral administration of aucubin at a single dose of 50 mg/kg. Herein the pharmacokinetic study of aucubin is reported for the first time.

Exploitation of detergent thermodynamics in the direct solubilization of myelin membrane proteins for two‐dimensional gel electrophoresis for proteomic analysis

Performing 2-DE of lipid-rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2-D-proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2-DE profiling of myelin proteins is very important for the detection of immuno-reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent-mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB-14-4 resulted in significant increase in protein representation of myelin in 2-DE compared with the addition of ASB-14-4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB-14-4 strategy than other strategies of myelin sample processing such as ASB-14-4 to myelin or ethanol or acetone or methanol-ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2-D gel is achieved.

In vivo screening of diarylimidazoles as anticonvulsant agents

In this study, the anticonvulsant activity evaluation of a series of new 2,4(1H)-diarylimidazoles, characterized by the presence of different substituents on the aryl rings, was described. The anticonvulsant activity profile of those compounds was determined by Maximal Electroshock Seizure (MES), subcutaneous Metrazol Seizure (scMet) tests, and 6-Hz seizure model, whereas their neurotoxicity was examined using rotarod test. The trifluoromethoxy derivative, obtained starting from phenylglyoxal, p-OCF3-benzaldehyde, and ammonium acetate in methanol (r.t.), exhibited a great ability to prevent the seizures induced in the 6-Hz test, becoming a new promising molecule to develop for the treatment of therapy-resistant partial seizures.

Determination of L-Ephedrine, Pseudoephedrine, and Caffeine in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry

A rapid and simple liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of L-ephedrine, pseudoephedrine, and caffeine in male Fisher-344 rat plasma at nanogram-per-milliliter concentrations for use in support of toxicology studies. Only 25 μL of plasma is required, and extraction is performed using a simple, single-step protein precipitation. The method was validated over a range of 2.09 to 5460 ng/mL for L-ephedrine, 2.09 to 5050 ng/mL for pseudoephedrine and 2.03 to 5340 ng/mL for caffeine. A binary gradient elution at 0.3 mL/min was used with a Waters XBridge Phenyl (2.1 × 150 mm, 3.5 μm) column and a Waters XBridge Phenyl 2.1- × 10-mm guard column at ambient temperature. The mobile phase consisted of 10 mM ammonium acetate in water (pH 5.0) and methanol. Caffeine trimethyl-(13)C(3) was used as the internal standard. The method was evaluated for linearity, recovery, precision, accuracy, and stability, and it was successfully applied in toxicokinetic studies of ephedrine, administered alone, in combination with caffeine, and in the herbal source Ma Huang.

Investigation of myorelaxant activity of 9-aryl-3,4,6,7-tetrahydroacridine-1,8-(2H,5H,9H,10H)-diones in isolated rabbit gastric fundus

In this study, twelve compounds having 9-aryl-3,4,6,7-tetrahydroacridine-1,8-(2H,5H,9H,10H)-dione structure were synthesized by reaction of 5-methyl-1,3-cyclohexanedione, the appropriate aromatic aldehydes, and ammonium acetate in methanol. The structures of the compounds were elucidated by infrared, 1H- and 13C-nuclear magnetic resonance spectroscopy (-NMR), mass spectroscopy, and elemental analysis. The maximum relaxant effects (Emax) and pD2 values of the compounds 3a–l and pinacidil were tested on isolated strips of rabbit gastric fundus smooth muscle.

Simultaneous Determination of Escin Ia and Its Isomer Isoescin Ia by LC–MS–MS: Application to a Pharmacokinetic Study of Escin Ia in Rats

A rapid and sensitive LC–MS–MS method for the simultaneous determination of escin Ia and isoescin Ia in rat plasma, urine, feces and bile samples was developed and validated. Analytes and telmisartan [internal standard (IS)] were extracted by solid-phase extraction on C18 cartridges. Components in the extract were separated on an HC-C18 column (5 μm, 150 × 4.6 mm i.d.) using 10 mM ammonium acetate–methanol–acetonitrile (40:30:30, v/v/v) as the mobile phase. The method demonstrated good linearity from 5 ng mL−1 (LLOQ) to 1,500 ng mL−1 for both escin Ia and isoescin Ia. Intra- and inter-day precision measured as RSD was within ±15%. Recoveries and matrix effects of both escin Ia and isoescin Ia were satisfactory in all four matrices examined. The method was successfully applied to a pharmacokinetic study in Wistar rats after a single intravenous administration of escin Ia at the dose of 1.0 mg kg−1.

Sensitive, selective and rapid determination of bupropion and its major active metabolite, hydroxybupropion, in human plasma by LC‐MS/MS: application to a bioequivalence study in healthy Indian subjects

A sensitive, selective and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of bupropion (BUP) and its major active metabolite hydroxybupropion (HBUP) in human plasma. Separation of both the analytes and venlafaxine as internal standard (IS) from 50 μL human plasma was carried out by solid-phase extraction. The chromatographic separation of the analytes was achieved on a Zorbax Eclipse XDB C(18) (150 × 4.6 mm, 5 µm) analytical column using isocratic mobile phase consisting of 20 mm ammonium acetate-methanol (10:90, v/v), with a resolution factor of 3.5. The method was validated over a wide dynamic concentration range of 0.1-350 ng/mL for BUP and 0.1-600 ng/mL for HBUP. The matrix effect was assessed by post-column infusion and the mean process efficiency was 96.08 and 94.40% for BUP and HBUP, respectively. The method was successfully applied to a bioequivalence study of 150 mg BUP (test and reference) extended release tablet formulation in 12 healthy Indian male subjects under fed conditions.

Study and Development of Reversed-Phase HPLC Systems for the Determination of 2-Imidazolines in the Presence of Preservatives in Pharmaceutical Preparations

Different HPLC chromatographic systems were investigated on a C18 ACE 5 pm, 150 x 4.6 mm id column for the determination of tymazoline, tramazoline, and antazoline, with either naphazoline or xylometazoline, in commercial preparations. For the development and optimization of the systems, a Response Surface Method (r=0.925-0.980) was used to illustrate the changes in k as a function of pH values and different salt concentrations. The simultaneous separation of 2-imidazolines was accomplished at 40 degrees C with 0.01 M ammonium acetate-methanol (50+50, v/v, pH 6.0) mobile phase at a flow rate of 1.2 mL/min. In order to deal with the usual coexistence of 2-imidazolines with benzethonium and benzalkonium chloride preservatives, it was necessary to use another chromatographic system, 0.01 M ammonium acetate-methanol (50+50, v/v) mobile phase on a cyano ACE 5 pm, 150 x 4.6 mm id column. As part of a more thorough theoretical investigation, a partial least-squares (PLS) technique was used for modeling the RP-HPLC retention data. The model was based on molecular structure descriptors of the analytes' X variables and on their retention time (Log K) Y. The goodness of fit was estimated by the PLS correlation coefficient (r2) and root mean square error of estimation values, which were 0.994 and 0.0479, respectively.

LC-MS/MS Assay for Quantification of a Novel Antidiabetic Flavone Derivative S002-857 in Rat Plasma, Urine and Feces: Application in Pharmacokinetic Studies

This study aimed to develop an LC-MS/MS assay for the quantitation of S002-857 in plasma, urine and feces to evaluate the pharmacokinetic parameters and elimination pathway for further development of S002-857. A sensitive and selective LC-MS/MS method was developed and validated for estimating a novel antidiabetic flavone derivative, S002-857, synthesized by Central Drug Research Institute, CSIR, Lucknow, India. Plasma and urine samples were prepared by two-step liquid-liquid extraction with diethyl ether and feces by protein precipitation using acetonitrile. The analyte was chromatographed on a cyano column with methanol-ammonium acetate buffer (pH 4.6, 10 mM; 90:10, v/v). The calibration curves were linear over a range of 0.78–400 ng mL−1 for plasma and 1.56–400 ng mL−1 for urine and feces samples. The accuracy and precision were 85%. S002-857 was stable in plasma for 8 h at ambient temperature, 30 days at −60 °C and after three freeze-thaw cycles. The assay was successfully applied to determine the pharmacokinetic parameters and excretion profile in male rats.

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC–ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 μL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 μm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10–10,000 ng/mL ( r > 0.99) for SASP and 10–1000 ng/mL ( r > 0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.

Determination of the content of granisetron in patch and its in vitro release rate

Objective: To establish a HPLC method for determination of the content of granisetron in patch,and to investigate its in vitro release characteristics.Methods: The separation of granisetron was performed on a Luna-CN column(150 mm×4.6 mm,5 μm) with column temperature at 30 ℃,and the mobile phase consisted of methanol-ammonium acetate buffer(49∶51) whose pH value was adjusted to 6.0 with triethylamine and acetic acid.The flow rate was 1.0 ml/min.The detecting wavelength was 302 nm.Results: The linear range of granisetron was 0.267-66.80 μg/ml(r=0.999 9).The average recovery rates of high,middle and low concentrations(16.46,13.72,10.98 μg/ml) were(99.03±0.54)%,(97.82±0.23)% and(100.11±0.43)%(n=3).The cumulative release amount of granisetron patch within 24 h was(90.52±2.14)%.Conclusion: The HPLC method can be used for the quality control of granisetron patch because of its accuracy,good reproducibility and high sensitivity.Granisetron is well released from the patch in vitro.

LC–MS–MS determination of ibuprofen, 2-hydroxyibuprofen enantiomers, and carboxyibuprofen stereoisomers for application in biotransformation studies employing endophytic fungi

The purpose of this study was the development and validation of an LC-MS-MS method for simultaneous analysis of ibuprofen (IBP), 2-hydroxyibuprofen (2-OH-IBP) enantiomers, and carboxyibuprofen (COOH-IBP) stereoisomers in fungi culture medium, to investigate the ability of some endophytic fungi to biotransform the chiral drug IBP into its metabolites. Resolution of IBP and the stereoisomers of its main metabolites was achieved by use of a Chiralpak AS-H column (150 × 4.6mm, 5μm particle size), column temperature 8°C, and the mobile phase hexane-isopropanol-trifluoroacetic acid (95: 5: 0.1, v/v) at a flow rate of 1.2mL min(-1). Post-column infusion with 10mmol L(-1) ammonium acetate in methanol at a flow rate of 0.3mL min(-1) was performed to enhance MS detection (positive electrospray ionization). Liquid-liquid extraction was used for sample preparation with hexane-ethyl acetate (1:1, v/v) as extraction solvent. Linearity was obtained in the range 0.1-20μg mL(-1) for IBP, 0.05-7.5μg mL(-1) for each 2-OH-IBP enantiomer, and 0.025-5.0μg mL(-1) for each COOH-IBP stereoisomer (r ≥ 0.99). The coefficients of variation and relative errors obtained in precision and accuracy studies (within-day and between-day) were below 15%. The stability studies showed that the samples were stable (p > 0.05) during freeze and thaw cycles, short-term exposure to room temperature, storage at -20°C, and biotransformation conditions. Among the six fungi studied, only the strains Nigrospora sphaerica (SS67) and Chaetomium globosum (VR10) biotransformed IBP enantioselectively, with greater formation of the metabolite (+)-(S)-2-OH-IBP. Formation of the COOH-IBP stereoisomers, which involves hydroxylation at C3 and further oxidation to form the carboxyl group, was not observed.

Basic drug analysis by strong cation‐exchange liquid chromatography–tandem mass spectrometry: simultaneous analysis of amisulpride, and of metamfetamine and amfetamine in serum/plasma

In the HPLC of basic drugs and metabolites, good efficiency and peak shape can often be attained using strong cation-exchange packings with isocratic 100% methanol eluents containing an ionic modifier at an appropriate pH* and ionic strength. Solvent extracts can be analysed directly, and use of ammonium acetate as modifier facilitates the use of atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry, selected reaction monitoring mode. For the analysis of amisulpride and of metamfetamine/amfetamine in plasma (200 µL) after single oral doses in man, a column packed with Waters Spherisorb S5SCX (5 µm average particle size, 100 × 2.1 mm i.d.) was used with methanolic ammonium acetate (40 mmol/L, pH* 6.0, flow rate 0.5 mL/min) as eluent (35°C). Deuterated internal standards were used for each analyte. Detection was by positive-mode APCI. Responses for all analytes were linear over the calibration ranges. Intra-assay precision (RSD) was 2-18%, and inter-assay precision was 2-12%. The limit of detection was 0.5 µg/L for all analytes. No significant matrix effects or isobaric interferences were noted. The total analysis time was 7 min. Similar methodology can be applied to a wide range of basic analytes using MS/MS detection.

Modeling of chromatographic lipophilicity of food synthetic dyes estimated on different columns

The retention behavior of some food synthetic dyes has been studied by RP-HPLC on chemically bonded C18, C8, C16 and CN stationary phases. Using methanol-ammonium acetate (0.08 mol/L, pH=6.76) as mobile phase, a linear behavior of retention parameters throughout the methanol fraction variance was obtained in all cases (r>0.99). The patterns of chromatographic behavior of the compounds illustrate high similarities between the C18, C8 and C16 columns, respectively. Highly significant correlations were obtained between experimental lipophilicity indices log k(w) and phi(0) estimated on C18 and C8 stationary phases and some computed log P-values. An extensive investigation made for quantitative structure-property (lipophilicity) relationships of studied dyes, using descriptors from Dragon software, multiple linear regression and genetic algorithm, revealed that the molecular descriptors appearing in the best models combine 2-D and 3-D aspects of the molecular structure. The most significant descriptors can be classified as radial distribution function, GETAWAY (autocorrelation), 3D-MoRSE signal, Burden eigenvalues and edge adjacency descriptors.

Chromatographic separation and sensitive determination of teriflunomide, an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid–liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200 μL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50 mm × 4.6 mm, 3 μm) analytical column using isocratic mobile phase, consisting of 20 mM ammonium acetate–methanol (25:75, v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for teriflunomide ( m/ z 269.0 → 82.0) and IS ( m/ z 434.1 → 350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1–4001 ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0 min and is applied to a bioequivalence study of 20 mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.

Optimization of Protein Extraction from Hypericum perforatum Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development

Sample preparation is crucial for obtaining high-quality proteins for the purpose of electrophoretic separation and further analysis from tissues that contain high levels of interfering compounds. Hypericum perforatum is a medicinal plant that contains high amounts of phenolic compounds, of which hypericins, hyperforins, and flavonoids contribute to the antidepressant activities of the plant. This study focuses on obtaining optimized amounts of high-quality proteins from H. perforatum, which are suitable for electrophoretic analyses. From the tested protein extraction solutions, sodium borate buffers at pH 9 and 10 gave the best protein yields from mature H. perforatum leaves. With these buffers, relatively high protein yields could also be obtained from roots, stems, and flower buds. The protein extracts of all organs were well resolved in SDS-PAGE after an efficient removal of non-protein contaminants with PVPP, phenol extraction, and methanolic ammonium acetate precipitation. The method was suitable for high-quality protein extraction also from other tested species of genus Hypericum. The applicability of the protocol for immunoblotting was demonstrated by detecting Hyp-1 in H. perforatum leaves at different stages of development. Hyp-1, which has been suggested to attend to the biosynthesis of hypericin, accumulated in high amounts in H. perforatum leaves at mature stage.

Determination of metoclopramide in human plasma by LC–ESI-MS and its application to bioequivalance studies

An LC–MS method for the determination of metoclopramide in human plasma was developed and validated. Sample preparation involved extraction with ethyl acetate. Chromatographic separation was performed on a Thermo Hypersil-Hypurity C 18 (150 mm × 2.1 mm, 5 μm) with the mobile phase consisting of 40 mM ammonium acetate–methanol–acetonitrile. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H] + ions at m/ z 300 for metoclopramide and at m/ z 384 for the internal standard (prazosin). The method was validated over 0.78–50.00 ng mL −1 for metoclopramide. The recovery was 67.8–83.1%, and the limit of quantitation (LOQ) detection was 0.78 ng mL −1 for metoclopramide. The intra- and inter-day precision of the method at three concentrations was 5.0–13.6% with accuracy of 99.2–104.0%. Stability of compounds was established in a battery of stability studies. The method was successfully applied to bioequivalence studies of metoclopramide hydrochloride tablets to obtain the pharmacokinetic parameters.

Determination of β-artemether and its main metabolite dihydroartemisinin in plasma employing liquid-phase microextraction prior to liquid chromatographic–tandem mass spectrometric analysis

A method for the determination of artemether (ART) and its main metabolite dihydroartemisinin (DHA) in plasma employing liquid-phase microextraction (LPME) for sample preparation prior to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed. The analytes were extracted from 1mL of plasma utilizing a two-phase LPME procedure with artemisinin as internal standard. Using the optimized LPME conditions, mean absolute recovery rates of 25 and 32% for DHA and ART, respectively, were achieved using toluene-n-octanol (1:1, v/v) as organic phase with an extraction time of 30min. After extraction, the analytes were resolved within 5min using a mobile phase consisting of methanol-ammonium acetate (10mmolL(-1), pH 5.0, 80:20, v/v) on a laboratory-made column based on poly(methyltetradecylsiloxane) attached to a zirconized-silica support. MS-MS detection was employed using an electrospray interface in the positive ion mode. The method developed was linear over the range of 5-1000ngmL(-1) for both analytes. Precision and accuracy were within acceptable levels of confidence (<15%). The assay was applied to the determination of these analytes in plasma from rats treated with ART. The two-phase LPME procedure is affordable and the solvent consumption was very low compared to the traditional methods of sample preparation.