Development of ultrasound-assisted emulsification microextraction for the determination of seven pesticides in apple juice by gas chromatography–mass spectrometry

Α simple, relatively rapid, sensitive and cost-effective method based on ultrasound-assisted emulsification microextraction (USAEME) followed by gas chromatography coupled with mass spectrometry has been developed for the determination of seven endocrine disruptor pesticides (chlorpyrifos, deltamethrin, dimethoate, fenitrothion, malathion, pendimethalin and procymidone) in apple juice. This approach is based on the emulsification of organic extraction solvent in a diluted apple juice sample by ultrasound radiation and further separation of both liquids phases by centrifugation. The influence of the different parameters affecting the procedure (extraction solvent, extraction solvent volume, ultrasound time, centrifugation time, ionic strength and pH) was evaluated in order to optimise the efficiency of the extraction process. Target analytes were extracted from a 0.5 g apple juice sample that was diluted by 10 times with aqueous buffer solution (pH 7). The optimised USAEME procedure used 100 μL of chloroform as extraction solvent, 8 min of ultrasound extraction, ionic strength (2.5% w/v) and 7.5 min of centrifugation at 3800 rpm. The optimised method presented recoveries between 70 and 113% for the target analytes. Acceptable linearity for all target analytes was recorded with correlation coefficients (r) higher than 0.992. The limits of quantification were found between 1.1 and 4.6 μg kg^?1 ensuring compliance with the maximum residue limits established by the European Commission. The proposed method was applied for the determination of the endocrine disruptor pesticides in apple samples proving its suitability to the Commission Implementing Regulation (EU) no. 400/2014.     

Mercury speciation in fish tissue by eco-scale thermal decomposition atomic absorption spectrometry: method validation and risk exposure to methylmercury

A non-chromatographic method for Hg speciation in fish muscle using eco-scale thermal desorption atomic absorption spectrometry was validated according to the demands of the Decisions 2007/333/EC, 2011/836/EC and 2002/657/EC. The method is based on the determination of total Hg in solid sample and MeHg⁺, respectively, after double liquid–liquid extraction (47% HBr, toluene, 1% l-cysteine) according to a procedure recommended by the European Commission. The method was applied for the speciation of Hg in fish muscle to asses the weekly intake and health risk exposure to MeHg⁺. The method provided calibration coefficients better than 0.999, limits of detection of 0.2 µg kg^?1 total Hg and 3.0 µg kg^?1 MeHg⁺. The accuracy of the method assessed by the analysis of CRMs was 100 ± 9% for total Hg and 101 ± 13% for MeHg⁺ for a coverage factor k = 2. The figures of merit of the method comply with the requirements in the European legislation. Total Hg in the analyzed fish species was in the range 12–108 µg kg^?1 wet mass, of which 83–97% as MeHg⁺. Although significant variation in total Hg was observed among fish species, no differences in terms of distribution of organic and inorganic Hg species were identified. No risk exposure to MeHg⁺ was ascertained from muscle fish consumption from reliable sources, since weekly intake was below 30% of provisional tolerable weekly intake of 1.6 µg kg^?1 body weight. Speciation is recommended as MeHg⁺ is much more helpful than total Hg to evaluate the dietary risk exposure.     

Gas chromatography–triple quadrupole tandem mass spectrometry: a powerful tool for the (ultra)trace analysis of multiclass environmental contaminants in fish and fish feed

A new method for rapid determination of 73 target organic environmental contaminants including 18 polychlorinated biphenyls, 16 organochlorinated pesticides, 14 brominated flame retardants and 25 polycyclic aromatic hydrocarbons in fish and fish feed using gas chromatography coupled with triple quadrupole tandem mass spectrometry (GC–MS/MS) was developed and validated. GC–MS/MS in electron ionization mode was shown to be a powerful tool for the (ultra)trace analysis of multiclass environmental contaminants in complex matrices, providing measurements with high selectivity and sensitivity. Another positive aspect characterizing the newly developed method is a substantial simplification of the sample preparation, which was achieved by an ethyl acetate QuEChERS (quick, easy, cheap, effective, rugged and safe) based extraction followed by silica minicolumn clean-up. With use of this sample preparation approach the sample laboratory throughput was increased not only because six samples may be prepared in approximately 1 h, but also because all the above-mentioned groups of contaminants can be determined in a single GC–MS/MS run. Under the optimized conditions, the recoveries of all target analytes in both matrices were within the range from 70 to 120 % and the repeatabilities were 20 % or less. The method quantification limits were in the range from 0.005 to 1 μg kg^–1 and from 0.05 to 10 μg kg^–1 for fish muscle tissue and fish feed, respectively. The developed method was successfully applied to the determination of halogenated persistent organic pollutants and polycyclic aromatic hydrocarbons in fish and fish feed samples.     

Rapid Determination of Meropenem in Biological Fluids by LC: Comparison of Various Methods for Sample Preparation and Investigation of Meropenem Stability

A rapid and sensitive LC-UV method was developed and validated for the determination of meropenem, in human plasma and urine. Meropenem retention time was 4.8 min. Method development was based on comparative analysis of different extraction methods published as well as careful study of meropenem stability in biological samples under different conditions. Best results in plasma sample preparation were obtained from protein precipitation with methanol. LOQ was 0.1 µg mL^?1 for plasma and 1 µg mL^?1 for urine samples. Meropenem in plasma has low stability at room temperature (<20% of original content after 12 h), but had acceptable stability when the whole analysis procedure was designed to minimize the exposure of meropenem-containing samples and solutions to temperatures higher than 4 °C. The developed method was applied to a human pharmacokinetic study in patients with acute peritonitis.     

Bamboo Charcoal as Solid-Phase Extraction Adsorbent for the Determination of Organophosphorus Pesticides in Water Samples by High-Performance Liquid Chromatography

In this paper, bamboo charcoal was successfully developed for the solid-phase extraction adsorbent for the determination of six organophosphorus pesticides in water samples. After the bamboo charcoal was pretreated and packed in the solid-phase extraction cartridge, the organophosphorus pesticides in water samples were carried out the solid-phase extraction. To establish a perfect solid-phase extraction procedure, the experimental conditions including the eluent, eluent volume, pH of the sample, flow rate of the sample, and loading volume of the sample were all investigated. When 100 mL water samples in the pH range of 6–7 were loaded with the flow rate of 2.5 mL · min^?1 and then eluted with 10 mL acetonitrile, the proposed extraction method was validated by the recovery, correlation coefficient (R²), repeatability (RSD, n = 7) and LODs, which were 69.6–93.4%, 0.9982–0.9998, 2.9–5.6%, and 0.08–1.04 µg · L^?1, respectively. Furthermore, the analysis of the tap, snow, and river water samples demonstrated the feasibility of the proposed SPE method for real water samples. Based on the aforementioned factors, it could be concluded that bamboo charcoal was a good solid-phase extraction adsorbent, and this proposed solid-phase extraction method was suitable for the effective enrichment and determination of the organophosphorus pesticides in water samples.     

Determination of tributyltin in marine sediment and waters by pressurised solvent extraction and liquid chromatography-tandem mass spectrometry

In this study, tributyltin (TBT) was extracted from marine sediment matrix with the use of pressurised solvent extraction (PSE), which uses high-temperature and -pressure conditions to increase extraction efficiency. The analyte was chromatographically resolved using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system with a pentafluorophenyl (PFP) column and a methanol/aqueous formic acid mobile phase gradient, and was detected by MS/MS as product fragments after collisionally induced dissociation (CID) of the cationic parent molecule. This study represents the first application of PSE extraction combined with LC-MS/MS analysis for the determination of TBT in sediments. The method has been validated according to the International Organisation for Standardisation (ISO) 17025:2001 and affords automated extraction of sediment samples with high-sensitivity analysis. The full method limit of detection was established as 1.25 ng Sn g^?1 with an instrument detection limit of 0.01 ng Sn g^?1. The chromatographic procedure may also be applied for the direct analysis of water matrices without the need for sample manipulation, and therefore represents a combined analytical approach for the monitoring of TBT contamination in marine or estuarine ecosystems.     

Supramolecular microextraction of cobalt from water samples before its microsampling flame atomic absorption spectrometric detection

The supramolecular solvent system consists of tetrahydrofuran (THF) and 1-decanol, that was used as an extraction solvent for a microextraction procedure for the preconcentration and separation of Co(II). The proposed supramolecular-based procedure was combined with microsampling flame atomic absorption spectrometry for the determination of cobalt at trace levels in water samples. N-Benzoyl-N,N-diisobutylthiourea was used to chelate Co(II) in an aqueous solution. Quantitative extraction efficiency was obtained at pH 6.5. The effects of analytical parameters including pH, amount of ligand, type, ratio and volume of supramolecular solvent, sample volume and interfering ions were investigated for optimisation of the procedure. The proposed supramolecular solvent-based microextraction procedure (Ss-ME) exhibits a limit of detection (LOD) of 1.29 µg L^?1 and a limit of quantification (LOQ) of 3.88 µg L^?1. The procedure was validated by addition/recovery tests and by applying TMDA 64.2 and TMDA 53.3 water certified reference materials. The microextraction method was successfully applied for the preconcentration and determination of cobalt in water samples.     

Pre-concentration and quantification of aluminum in fish samples using a dispersive liquid–liquid micro-extraction–spectrofluorimetric method based on Al(III)–8-hydroxyquinoline complex’s fluorescence properties

A simple and fast dispersive liquid–liquid micro-extraction (DLLME)–spectrofluorimetric technique was developed and validated for the extraction and quantification of trace amounts of Al in fish samples, where 8-hydroxyquinoline was utilized as a spectrofluorimetric probe. In order to optimize the efficacy of the DLLME technique, the impact of experimental parameters on the extraction of Al(III) from fish samples was evaluated. Under the optimal conditions, the method was linear in the concentration range of 0.1–7.0 μg/g (r = 0.9996) with a LOD of 0.092 μg/g; additionally, the method was accurate (RE% of ? 3.0 to + 10.0%), precise (RSD% of 1.2–14.3%) and robust (RSD% of 3.8 and p value of 0.21) and its recovery was in the range of 97.0 ± 3.89–110.0 ± 12.5%; moreover, samples were stable before and during the analyses. Therefore, it can be claimed that the developed method could be successfully applied for the quantification of Al in fish samples.     

Rapid and simple method for determination of hexabromocyclododecanes and other LC–MS–MS-amenable brominated flame retardants in fish

In this study, a novel analytical approach for simultaneous determination of hexabromocyclododecane isomers (HBCDs), tetrabromobisphenol A (TBBPA), three brominated phenols, and four hydroxylated derivatives of polybrominated diphenyl ethers (OH-PBDEs) was developed and validated for muscle tissue of both lean and fatty fish. The rapid, simple, and high-throughput sample-preparation procedure was based on acetonitrile extraction then purification by dispersive solid-phase extraction (d-SPE) with a combination of C₁₈ and primary–secondary amine (PSA) sorbents. Ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS–MS) was used for identification and quantification of the analytes. Method recovery for both matrices ranged from 80 to 115 % with relative standard deviations (RSDs) <13 % for all analytes. Limits of quantification (LOQs) were in the range 0.1–1 μg kg^?1 wet weight. The validated method was used for analysis of brominated compounds in 32 fish and five bivalve samples collected from different European markets within the monitoring survey organized in the framework of the CONffIDENCE project. Of the 12 targeted analytes, only α-HBCD, 2,4-dibromophenol (2,4-DBP), and 2,4,6-tribromophenol (2,4,6-TBP) were quantified in the samples. α-HBCD was found in six fish samples (herring and mackerel) in the range of 0.8–2.5 μg kg^?1 wet weight. 2,4-DBP and 2,4,6-TBP were found in three blue mussel samples in the range of 19.6–43.5 and 2.3–7.5 μg kg^?1 wet weight, respectively.     

Determination of Carbon‐based Engineered Nanoparticles in Marketed Fish by Microwave‐assisted Extraction and Liquid Chromatography‐atmospheric Pressure Photoionization‐tandem Mass Spectrometry

An optimized method for the determination of two major carbon‐based engineered nanoparticles (C₆₀ and C₇₀) in marketed fish samples is described. The method involves the use of microwave‐assisted extraction (MAE) coupled with liquid chromatography ‐ tandem mass spectrometry with atmospheric pressure photoionization (LC‐APPI‐MS/MS). Factors affecting the extraction efficiency of the analytes from fish samples were optimized by a central composite design method. The optimal extraction temperature and time for MAE were found to be 233 °C for 22 min, and the extraction solution was composed of toluene and acetone in a ratio of 4.64:1. The limits of quantitation (LOQs) were 0.1 and 0.05 ng/g for C₆₀ and C₇₀, respectively. The precision for these analytes at two spiked levels, as indicated by relative standard deviations (RSDs), were less than 10% for both intra‐ and inter‐day analysis. Accuracy, expressed as the mean extraction recovery, was between 85 and 98%. The method was further validated based on EU Commission Decision 2002/657/EC, including a decision limit (CCα) and detection capability (CCβ) for marketed fish samples.     

Pesticide residues analysis in honey using ethyl acetate extraction method: validation and pilot survey in real samples

This paper presents a cost-effective and validated multi residue confirmatory method for the determination of 167 chemically different pesticides and a survey study on Cyprus honey samples. This method uses ethyl acetate for the extraction of pesticides from honey and the determination is performed with liquid chromatography (LC) coupled to mass spectrometry (MS) operating in tandem mode (MS/MS) and with GC–ECD (gas chromatography with electron capture detector) analysis. The LC-MS/MS analytical system is especially important in the analysis of polar and non-volatile pesticides. For the validation of the method, blank honey samples were spiked with 146 pesticides (organophosphorous, carbamates, triazoles, amides, neonicodinoids, strobilurines, phenylureas, bendimidazoles and others) for the LC-MS/MS analysis at three levels: 0.01, 0.05 and 0.1 mg kg^?1 and with 21 pesticides for the GC-ECD analysis at two levels: 0.01 and 0.05 mg kg^?1for organochlorines and 0.05 and 0.2 mg kg^?1for the pyrethroids. As blank sample, a sample of honey which did not contain detectable levels of the analytes sought was used. The validation study was in accordance to the DG SANCO guidelines. The scope of validation included recovery, linearity, limits of quantification and precision. Linearity is demonstrated all along the range of concentration that was investigated with correlation coefficients ≥0.98. Recoveries of the majority of compounds were in the 70%–120% range and were characterised by precision lower or equal to 20%. The validated method was used for a survey of 36 samples of honey produced in different areas of Cyprus and this is the first work on Cypriot honey samples investigating a broad range of pesticides. Only coumaphos was detected at concentrations higher than 0.01 mg kg^?1 in the 58.6% of the honey samples analysed for Coumaphos. The results were evaluated in accordance to the provisions of the Commission Regulation (EU) No 37/2010 on pharmacologically active substances and their classification regarding maximum residue limits (MRLs) in foodstuffs of animal origin. The concentrations of coumaphos in all positive samples were at levels much lower than the MRL.     

Analysis and validation of perfluorinated compounds in water,sediment and fish with LC-ESI–MS/MS

The analysis of perfluorinated compounds (PFCs) in environmental matrices is challenging, as the concentrations are generally low, but the risk of contamination is high. Sample preparation is a critical step and it is necessary to minimise the possibility of contamination. In this study, we successfully applied and validated a modified ion pair extraction method to quantify PFCs in sediment and fish samples. A large volume injection method was validated and used to quantify PFCs in different water matrices. Isotope internal standard of every analyte was applied to correct matrix effects. The recoveries of the analytes were 92–106% for water matrix, 93–119% for fish matrix and 86–103% for soil matrix whereas the achieved limit of quantitation values were 1.3–14.9 ng/L for water, 0.19–0.28 μg/kg for fish and 0.14–0.41 for soil samples. Thirty-one surface water, 8 stormwater and 41 sediment samples collected all over Estonia were analysed and 4 (out of 8 analysed) PFCs were found in quantitative amount. The most frequently detected analyte perfluorobutanoic acid (PFBA) was found in 26% of the water samples with a maximum concentration of 137 ng/L.     

A Sensitive Method for Methyl tert-Butyl Ether Analysis in Water Samples by a New HS-SPME Vial and GC

In this study, a new sample vial has been designed for the extraction and determination of methyl tert-butyl ether (MTBE) in water samples by headspace solid-phase microextraction method. The special feature of this new vial is cooling the HS above the aqueous sample by cold water stream for maximum analyte absorption on SPME fiber coating. The analysis was by a gas chromatograph equipped with flame ionization detector and a capillary column (CP-sil 13 CB). Some significant variables affecting the extraction procedure were optimized. By use of divinylbenzene/carboxen/polydimethylsiloxane fiber, a sample volume of 10 mL, stirring rate of 1,000 rpm, salt concentration of 24%, extraction time of 15 min and extraction temperature of 83 °C, detection limit of 0.022 μg L⁻¹ and a good linearity (R ² = 0.998) in a calibration range of 0.1–400 μg L⁻¹ were achieved. The relative standard deviation for triplicate runs ranged between 6 and 8%. The method could be applied to the analysis of trace levels of MTBE in various water samples.

     

A Sensitive Method for Methyl tert-Butyl Ether Analysis in Water Samples by a New HS-SPME Vial and GC

In this study, a new sample vial has been designed for the extraction and determination of methyl tert-butyl ether (MTBE) in water samples by headspace solid-phase microextraction method. The special feature of this new vial is cooling the HS above the aqueous sample by cold water stream for maximum analyte absorption on SPME fiber coating. The analysis was by a gas chromatograph equipped with flame ionization detector and a capillary column (CP-sil 13 CB). Some significant variables affecting the extraction procedure were optimized. By use of divinylbenzene/carboxen/polydimethylsiloxane fiber, a sample volume of 10 mL, stirring rate of 1,000 rpm, salt concentration of 24%, extraction time of 15 min and extraction temperature of 83 °C, detection limit of 0.022 μg L^?1 and a good linearity (R ² = 0.998) in a calibration range of 0.1–400 μg L^?1 were achieved. The relative standard deviation for triplicate runs ranged between 6 and 8%. The method could be applied to the analysis of trace levels of MTBE in various water samples.     

Determination of β2-Agonists in Porcine Urine by Molecularly Imprinted Solid Phase Extraction Followed Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Detection

A novel, sensitive, and robust method has been developed to detect 9 β₂-agonists in porcine urine to monitor illegal use of β₂-agonists in swine rearing. The method based on the molecular imprinted polymer (MIP) rapid extraction followed ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS) detection. The cleaning efficiency of MIP cartridges was demonstrated by comparing with common ion exchange solid phase extraction. The presented method was validated in accordance with the European Commission Decision 2002/657/EC. The linearity, decision limit (CCα), detection capability (CCβ), recovery, precision, robustness, and stability were studied in detail. CCα and CCβ values were from 0.006 ng/mL to 0.03 ng/mL and from 0.02 ng/mL to 0.08 ng/mL, respectively. The mean recoveries and repeatability varied from 68.8% to 94.2% and from 2.8% to 10.1%. The proposed method was applied to test 170 porcine urine samples from the Shaanxi province in China and two urine samples were confirmed as clenbuterol positive and the concentrations of clenbuterol in positive urine samples were about 0.08 ng/mL and 0.1 ng/mL, respectively. The developed method was demonstrated to be more sensitive and robust for the determination of 9 β₂-agonists in porcine urine. The method was proven to be simple and easy in operation with high selectivity and good reproducibility.     

GC/TOFMS Analysis of Endogenous Metabolites in Mouse Fibroblast Cells and Its Application in TiO2 Nanoparticle-Induced Cytotoxicity Study

In this paper, we present an optimized procedure for metabolomic analysis of endogenous metabolites in mouse fibroblast (L929) cell line using gas chromatography/time-of-flight mass spectrometry with multivariate statistics. The optimization of metabolite extraction was performed using three solvents: methanol, water, and chloroform, and then followed by methoxymation and silylation. This method was subsequently validated using 29 reference standards and cell line samples. The intra- and inter-day relative standard deviations (RSDs) of the standard compounds were lower than 15.0 and 25.0 %, respectively. As for most of the tested metabolites in cell line samples, RSDs were below 20.0 % for reproducibility and stability, respectively. We applied this approach in metabolomic study of L929 cells obtained from TiO₂ nanoparticle-induced cytotoxicity model samples (n = 5) and control samples (n = 5). Metabolite markers associated with TiO₂ nanoparticle-induced cytotoxicity were identified and validated by statistical methods and reference standards. Our work highlights the potential of this method for cell metabolomic study.

     

Enantioselective analysis of zopiclone in rat brain by liquid chromatography tandem mass spectrometry

A new high-performance liquid chromatographic method with triple quadrupole mass spectrometry detection was developed and validated for the quantification of zopiclone enantiomers in rat brain samples. Zopiclone enantiomers were resolved on a CHIRALPAK AD column with a mobile phase consisting of acetonitrile/ethanol/methanol (60:20:20, v/v/v) at a flow rate of 1.3 mL?min^-1. Moclobemide was used as internal standard. The sample treatment procedure was carried out employing solid-phase extraction, yielding mean absolute recoveries of 89.6 and 91.7 % for each zopiclone enantiomer. The validated method showed linearity in the range of 0.29–344.8 ng?g^?1, with quantification limits of 0.29 ng?g^?1 for both enantiomers. Precision and accuracy were within acceptable levels of confidence (<15 %). The method was applied in a pilot study of zopiclone kinetic disposition in rats. It could be observed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-(R)-zopiclone, confirming the stereoselective disposition of zopiclone.     

Development of an analytical procedure for quantifying the underivatized neurotoxin β-N-methylamino-l-alanine in brain tissues

The cyanotoxin β-methylamino-l-alanine (BMAA) has received renewed attention as an environmental risk factor for sporadic cases of amyotrophic lateral sclerosis (ALS) (Nunn et al., Brain Res 410:375–379, 1987). The aim of the present study was to develop and to validate an analytical procedure that allows the quantification of native BMAA and of its natural isomer, 2,4 diaminobutyric acid (DAB), in brain tissues. An analytical procedure was previously reported by our group for the determination of underivatized BMAA in environmental samples. It included a step of sample clean-up by solid phase extraction (SPE) with a mixed-mode sorbent and the analyses were performed by LC/MS-MS using hydrophilic interaction chromatography and multiple reactions monitoring scan mode. As brain tissues have a higher lipid content, the crucial step of sample clean-up had been optimized by evaluating the efficiency of the addition of a liquid/liquid extraction step prior to the SPE procedure or alternatively, of washing steps to the SPE extraction procedure. The efficiency was checked by visualizing the complexity of the resulting chromatograms in LC/MS and their performance by using spiked brain samples. The optimized analytical procedure, including a washing step with cyclohexane to the SPE with a recovery yield close to 100 %, was validated using the total error approach and allowed the quantification of BMAA in a concentration level ranging from 20 to 1,500 ng/g in brain samples. Finally, the feasibility of implementation of this procedure was verified in human brain samples from two patients who died of ALS.     

Analysis of paroxetine, fluoxetine and norfluoxetine in fish tissues using pressurized liquid extraction, mixed mode solid phase extraction cleanup and liquid chromatography-tandem mass spectrometry

Recent studies have shown that selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine are accumulated in the tissues of fish as a result of discharges of pharmaceuticals into surface waters from municipal wastewater treatment plants. In this study, an analytical method based on liquid chromatography with atmospheric pressure chemical ionization and tandem mass spectrometry (LC-APCI-MS/MS) was developed and validated for the determination of residues of paroxetine, fluoxetine and its active metabolite, norfluoxetine, in fish tissue. The procedure for sample preparation includes extraction of tissue by pressurized liquid extraction (PLE), followed by cleanup on a mixed-mode solid phase extraction (SPE) cartridge, Oasis MCX. With the optimized method, matrix interferences were reduced and recoveries >85% were obtained. The limits of quantitation (LOQ) determined by analysis of spiked fish tissue were 0.24, 0.07, and 0.14 ng/g wet weight for paroxetine, fluoxetine and norfluoxetine, respectively. This method was successfully applied to the analysis of samples of fish collected from Hamilton Harbour in Ontario, Canada, which is an urbanized and industrialized embayment of Lake Ontario. These analyses showed that the three analytes were present in fish tissues at concentrations up to approximately 1 microg/kg wet weight.     

Determination of Atovaquone in Human Plasma by LC-MS-MS and Its Application to a Bioequivalence Study

A sensitive and selective method for the determination of atovaquone in human plasma was developed and validated. The procedure employed the use of an internal standard (chlorothalidone) and a solvent extraction step. Detection was by electrospray ionization tandem mass spectrometry with multiple reaction monitoring. The method showed a linear range from 50 to 2,000 ng mL^?1. The extraction recovery was determined to be 84.91 ± 6.42% (SD), the intra- and inter-day assay accuracy (relative error) was within 7.57% and precision (RSD) was below 6.06%. The method was successfully employed to analyze plasma samples and evaluate the pharmacokinetics of atovaquone in human volunteers.