Analytical Platforms for Mass Spectrometry-Based Metabolomics of Polar and Ionizable Metabolites.

Metabolomics studies rely on the availability of suitable analytical platforms to determine a vast collection of chemically diverse metabolites in complex biospecimens. Liquid chromatography-mass spectrometry operated under reversed-phase conditions is the most commonly used platform in metabolomics, which offers extensive coverage for nonpolar and moderately polar compounds. However, complementary techniques are required to obtain adequate separation of polar and ionic metabolites, which are involved in several fundamental metabolic pathways. This chapter focuses on the main mass-spectrometry-based analytical platforms used to determine polar and/or ionizable compounds in metabolomics (GC-MS, HILIC-MS, CE-MS, IPC-MS, and IC-MS). Rather than comprehensively describing recent applications related to GC-MS, HILIC-MS, and CE-MS, which have been covered in a regular basis in the literature, a brief discussion focused on basic principles, main strengths, limitations, as well as future trends is presented in this chapter, and only key applications with the purpose of illustrating important analytical aspects of each platform are highlighted. On the other hand, due to the relative novelty of IPC-MS and IC-MS in the metabolomics field, a thorough compilation of applications for these two techniques is presented here.

Innovative Approaches to Assess Intermediate Cardiovascular Risk Subjects: A Review From Clinical to Metabolomics Strategies.

Current risk stratification strategies for coronary artery disease (CAD) have low predictive value in asymptomatic subjects classified as intermediate cardiovascular risk. This is relevant because not all coronary events occur in individuals with traditional multiple risk factors. Most importantly, the first manifestation of the disease may be either sudden cardiac death or acute coronary syndrome, after rupture and thrombosis of an unstable non-obstructive atherosclerotic plaque, which was previously silent. The inaccurate stratification using the current models may ultimately subject the individual to excessive or insufficient preventive therapies. A breakthrough in the comprehension of the molecular mechanisms governing the atherosclerosis pathology has driven many researches toward the necessity for a better risk stratification. In this Review, we discuss how metabolomics screening integrated with traditional risk assessments becomes a powerful approach to improve non-invasive CAD subclinical diagnostics. In addition, this Review highlights the findings of metabolomics studies performed by two relevant analytical platforms in current use-mass spectrometry (MS) hyphenated to separation techniques and nuclear magnetic resonance spectroscopy (NMR) -and evaluates critically the challenges for further clinical implementation of metabolomics data. We also discuss the modern understanding of the pathophysiology of atherosclerosis and the limitations of traditional analytical methods. Our aim is to show how discriminant metabolites originated from metabolomics approaches may become promising candidate molecules to aid intermediate risk patient stratification for cardiovascular events and how these tools could successfully meet the demands to translate cardiovascular metabolic biomarkers into clinical settings.

Singlet oxygen generation by the reaction of acrolein with peroxynitrite via a 2-hydroxyvinyl radical intermediate.

Acrolein (2-propenal) is an environmental pollutant, food contaminant, and endogenous toxic by-product formed in the thermal decomposition and peroxidation of lipids, proteins, and carbohydrates. Like other α,ß-unsaturated aldehydes, acrolein undergoes Michael addition of nucleophiles such as basic amino acids residues of proteins and nucleobases, triggering aging associated disorders. Here, we show that acrolein is also a potential target of the potent biological oxidant, nitrosating and nitrating agent peroxynitrite. In vitro studies revealed the occurrence of 1,4-addition of peroxynitrite (k2 = 6 × 103 M-1 s-1, pH 7.2, 25 °C) to acrolein in air-equilibrated phosphate buffer. This is attested by acrolein concentration-dependent oxygen uptake, peroxynitrite consumption, and generation of formaldehyde and glyoxal as final products. These products are predicted to be originated from the Russell termination of •OOCH=CH(OH) radical which also includes molecular oxygen at the singlet delta state (O21Δg). Accordingly, EPR spin trapping studies with the 2,6-nitrosobenzene-4-sulfonate ion (DBNBS) revealed a 6-line spectrum attributable to the 2-hydroxyvinyl radical adduct. Singlet oxygen was identified by its characteristic monomolecular IR emission at 1,270 nm in deuterated buffer, which was expectedly quenched upon addition of water and sodium azide. These data represent the first report on singlet oxygen creation from a vinylperoxyl radical, previously reported for alkyl- and formylperoxyl radicals, and may contribute to better understand the adverse acrolein behavior in vivo.

Reprogramming of Trypanosoma cruzi metabolism triggered by parasite interaction with the host cell extracellular matrix.

Trypanosoma cruzi, the etiological agent of Chagas' disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.

Stereochemical and structural effects of (2R,6R)-hydroxynorketamine on the mitochondrial metabolome in PC-12 cells.

BACKGROUND: Impairment in mitochondrial biogenesis and function plays a key role in depression and anxiety, both of which being associated with changes in fatty acid and phospholipid metabolism. The antidepressant effects of (R,S)-ketamine have been linked to its conversion into (2S,6S;2R,6R)-hydroxynorketamine (HNK); however, the connection between structure and stereochemistry of ketamine and HNK in the mitochondrial homeostatic response has not yet been fully elucidated at a metabolic level. METHODS: We used a multi-platform, non-targeted metabolomics approach to study the change in mitochondrial metabolome of PC-12 cells treated with ketamine and HNK enantiomers. The identified metabolites were grouped into pathways in order to assess global responses. RESULTS: Treatment with (2R,6R)-HNK elicited the significant change in 49 metabolites and associated pathways implicated in fundamental mitochondrial functions such as TCA cycle, branched-chain amino acid biosynthetic pathway, glycoxylate metabolic pathway, and fatty acid ß-oxidation. The affected metabolites included glycerate, citrate, leucine, N,N-dimethylglycine, 3-hexenedioic acid, and carnitine and attenuated signals associated with 9 fatty acids and elaidic acid. Important metabolites involved in the purine and pyrimidine pathways were also affected by (2R-6R)-HNK. This global metabolic profile was not as strongly impacted by treatment with (2S,6S)-HNK, (R)- and (S)-ketamine and in some instances opposite effects were observed. CONCLUSIONS: The present data provide an overall view of the metabolic changes in mitochondrial function produced by (2R,6R)-HNK and related ketamine compounds and offer an insight into the source of the observed variance in antidepressant response elicited by the compounds.

Effects of four antiplatelet/statin combined strategies on immune and inflammatory responses in patients with acute myocardial infarction undergoing pharmacoinvasive strategy: Design and rationale of the B and T Types of Lymphocytes Evaluation in Acute Myocardial Infarction (BATTLE-AMI) study: study protocol for a randomized controlled trial.

BACKGROUND: Early reperfusion of the occluded coronary artery during acute myocardial infarction is considered crucial for reduction of infarcted mass and recovery of ventricular function. Effective microcirculation and the balance between protective and harmful lymphocytes may have roles in reperfusion injury and may affect final ventricular remodeling. METHODS/DESIGN: BATTLE-AMI is an open-label, randomized trial comparing the effects of four therapeutic strategies (rosuvastatin/ticagrelor, rosuvastatin/clopidogrel, simvastatin plus ezetimibe/ticagrelor, or simvastatin plus ezetimibe/clopidogrel) on infarcted mass and left ventricular ejection fraction (LVEF) (blinded endpoints) in patients with ST-segment elevation myocardial infarction submitted to fibrinolytic therapy before coronary angiogram (pharmacoinvasive strategy). All patients (n = 300, 75 per arm) will be followed up for six months. The effects of treatment on subsets of B and T lymphocytes will be determined by flow-cytometry/ELISPOT and will be correlated with the infarcted mass, LVEF, and microcirculation perfusion obtained by cardiac magnetic resonance imaging. The primary hypothesis is that the combined rosuvastatin/ticagrelor therapy will be superior to other therapies (particularly for the comparison with simvastatin plus ezetimibe/clopidogrel) for the achievement of better LVEF at 30 days (primary endpoint) and smaller infarcted mass (secondary endpoint) at 30 days and six months. The trial will also evaluate the improvement in the immune/inflammatory responses mediated by B and T lymphocytes. Omics field (metabolomics and proteomics) will help to understand these responses by molecular events. DISCUSSION: BATTLE-AMI is aimed to (1) evaluate the role of subsets of lymphocytes on microcirculation improvement and (2) show how the choice of statin/antiplatelet therapy may affect cardiac remodeling after acute myocardial infarction with ST elevation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02428374 . Registered on 28 September 2014.

Recreational use of marijuana during pregnancy and negative gestational and fetal outcomes: An experimental study in mice.

The prevalence of marijuana use among pregnant women is high. However, the effects on gestation and fetal development are not well known. Epidemiological and experimental studies present conflicting results because of the route of administration, dose, time of exposure, species used, and how Cannabis toxicity is tested (prepared extracts, specific components, or by pyrolysis). In this study, we experimentally investigated the effects of maternal inhalation of Cannabis sativa smoke representing as nearly as possible real world conditions of human marijuana use. Pregnant mice (n=20) were exposed (nose-only) daily for 5min to marijuana smoke (0.2g of Cannabis) from gestational day (GD) 5.5 to GD17.5 or filtered air. Food intake and maternal weight gain were recorded. Ultrasound biomicroscopy was performed on 10.5 and 16.5dpc.On GD18.5, half of the dams were euthanized for the evaluation of term fetus, placenta, and resorptions. Gestation length, parturition, and neonatal outcomes were evaluated in the other half. Five minutes of daily (low dose) exposure during pregnancy resulted in reduced birthweight, and litter size was not altered; however, the number of male pups per litter was higher. Besides, placental wet weight was increased and fetal to placental weight ratio was decreased in male fetuses, showing a sex-specific effect. At the end of gestation, females from the Cannabis group presented reduced maternal net body weight gain, despite a slight increase in their daily food intake compared to the control group. In conclusion, our results indicate that smoking marijuana during pregnancy even at low doses can be embryotoxic and fetotoxic.

Effects of four antiplatelet/statin combined strategies on immune and inflammatory responses in patients with acute myocardial infarction undergoing pharmacoinvasive strategy: Design and rationale of the B and T types of lymphocytes evaluation in acute myocardial infarction (BATTLE-AMI) study: study protocol for a randomized controlled trial

BACKGROUND: Early reperfusion of the occluded coronary artery during acute myocardial infarction is considered crucial for reduction of infarcted mass and recovery of ventricular function. Effective microcirculation and the balance between protective and harmful lymphocytes may have roles in reperfusion injury and may affect final ventricular remodeling. METHODS/DESIGN: BATTLE-AMI is an open-label, randomized trial comparing the effects of four therapeutic strategies (rosuvastatin/ticagrelor, rosuvastatin/clopidogrel, simvastatin plus ezetimibe/ticagrelor, or simvastatin plus ezetimibe/clopidogrel) on infarcted mass and left ventricular ejection fraction (LVEF) (blinded endpoints) in patients with ST-segment elevation myocardial infarction submitted to fibrinolytic therapy before coronary angiogram (pharmacoinvasive strategy). All patients (n = 300, 75 per arm) will be followed up for six months. The effects of treatment on subsets of B and T lymphocytes will be determined by flow-cytometry/ELISPOT and will be correlated with the infarcted mass, LVEF, and microcirculation perfusion obtained by cardiac magnetic resonance imaging. The primary hypothesis is that the combined rosuvastatin/ticagrelor therapy will be superior to other therapies (particularly for the comparison with simvastatin plus ezetimibe/clopidogrel) for the achievement of better LVEF at 30 days (primary endpoint) and smaller infarcted mass (secondary endpoint) at 30 days and six months...

Development and validation of a CE-MS method for the targeted assessment of amino acids in urine.

A CE-ESI-MS method was developed and validated for the separation and quantitative analysis of amino acids (AA) in urine. Experimental parameters related to the CE-MS interface, BGE, and mass spectrometer (MS) settings were optimized providing a good separation of 27 AA, including the isomers L-leucine, L-isoleucine, and L-alloisoleucine, in less than 30 min. The sheath liquid was composed by 0.50% formic acid in 60% (v,v) methanol-water delivered at a flow rate of 5 µL/min. The BGE consisted of 0.80 mol/L formic acid at pH 1.96 and 15% methanol. A pH stacking procedure was implemented to enhance sensitivity (a 12.5% NH4 OH solution was injected at 0.5 psi/9 s prior to samples injected at 0.6 psi/20 s). The proposed method was validated according to FDA and ICH protocols exhibiting acceptable parameters. Analytical curves presented coefficients of determination from 0.996 to 0.9997 (with large F statistics and low p-values). LODs and quantification ranged from 0.63 to 29 µmol/L and from 1.9 to 86 µmol/L, respectively. Practical repeatability was obtained for all AA with coefficients of variation better than 0.55% CV (migration time) and 1.7% CV (peak area ratios; methionine sulfone as internal standard). Recoveries of AA in spiked urine ranged from 92.0 to 123% with few exceptions. Moreover, a successful quantification of AA in pooled control and test urine samples, which compose a vesicoureteral reflux cohort, was achieved showing the potential applicability of the proposed method for targeted metabolomics studies using CE-ESI-MS with an Ion Trap as mass analyzer.

Development and validation of a liquid chromatography method for anthocyanins in strawberry (Fragaria spp.) and complementary studies on stability, kinetics and antioxidant power.

A RPLC-DAD method for the analysis of eight anthocyanins was developed, validated and applied to strawberry extracts. The chromatographic method was conducted under gradient elution in acidulated water-methanol mobile phase and octadecyl-silica columns. An ultrasound extraction procedure was optimized by a 3(2) factorial design (%HCl in methanol, temperature, and time) and response surface methodology. Method validation was performed according to the following parameters: linearity (R(2)>0.99, p-value<10(-4), F>725), LOD (3-7 µmol L(-1)) and LOQ (9-22 µmol L(-1)), selectivity/specificity (baseline separation of all analytes and peak purity), instrumental precision (<6.4%CV), repeatability (<6.3%CV) and intermediate precision (<9.9%CV), recovery (83-99%), robustness (mobile phase pH, column temperature and flow rate) and stability (high temperatures and storage; 1st order kinetics). The antioxidant power of anthocyanins was measured on-line (ABTS(+) reaction; Trolox as reference). Ten strawberry extracts were quantified (average values: 24.2 µg/g for cyanidin-3-glucoside and 49.1 µg/g for pelargonidin-3-glucoside).

A Multiplatform Metabolomic Approach to the Basis of Antimonial Action and Resistance in Leishmania infantum.

There is a rising resistance against antimony drugs, the gold-standard for treatment until some years ago. That is a serious problem due to the paucity of drugs in current clinical use. In a research to reveal how these drugs affect the parasite during treatment and to unravel the underlying basis for their resistance, we have employed metabolomics to study treatment in Leishmania infantum promastigotes. This was accomplished first through the untargeted analysis of metabolic snapshots of treated and untreated parasites both resistant and responders, utilizing a multiplatform approach to give the widest as possible coverage of the metabolome, and additionally through novel monitoring of the origin of the detected alterations through a 13C traceability experiment. Our data stress a multi-target metabolic alteration with treatment, affecting in particular the cell redox system that is essential to cope with detoxification and biosynthetic processes. Additionally, relevant changes were noted in amino acid metabolism. Our results are in agreement with other authors studying other Leishmania species.

Neglected diseases prioritized in Brazil under the perspective of metabolomics: A review.

This review article compiles in a critical manner literature publications regarding seven neglected diseases (ND) prioritized in Brazil (Chagas disease, dengue, leishmaniasis, leprosy, malaria, schistosomiasis, and tuberculosis) under the perspective of metabolomics. Both strategies, targeted and untargeted metabolomics, were considered in the compilation. The majority of studies focused on biomarker discovery for diagnostic purposes, and on the search of novel or alternative therapies against the ND under consideration, although temporal progression of the infection at metabolic level was also addressed. Tuberculosis, followed by schistosomiasis, malaria and leishmaniasis are the diseases that received larger attention in terms of number of publications. Dengue and leprosy were the least studied and Chagas disease received intermediate attention. NMR and HPLC-MS technologies continue to predominate among the analytical platforms of choice in the metabolomic studies of ND. A plethora of metabolites were identified in the compiled studies, with expressive predominancy of amino acids, organic acids, carbohydrates, nucleosides, lipids, fatty acids, and derivatives.

Determination of herbicides paraquat, glyphosate, and aminomethylphosphonic acid in marijuana samples by capillary electrophoresis.

In this work, two methods were developed to determine herbicides paraquat, glyphosate, and aminomethylphosphonic acid (AMPA) in marijuana samples by capillary electrophoresis. For paraquat analysis, sample was extracted with aqueous acetic acid solution and analyzed by capillary zone electrophoresis with direct UV detection. The running electrolyte was 50 mmol/L phosphate buffer (pH 2.50). For glyphosate and AMPA, indirect UV/VIS detection was used, as these substances do not present chromophoric groups. Samples were extracted with 5 mmol/L hydrochloric acid. The running electrolyte was 10 mmol/L gallic acid, 6 mmol/L TRIS, and 0.1 mmol/L CTAB (pH = 4.7). The methods presented good linearity, precision, accuracy, and recovery. Paraquat was detected in 12 samples (n = 130), ranging from 0.01 to 25.1 mg/g. Three samples were positive for glyphosate (0.15-0.75 mg/g), and one sample presented AMPA as well. Experimental studies are suggested to evaluate the risks of these concentrations to marijuana user.

Multi-analytical platform metabolomic approach to study miltefosine mechanism of action and resistance in Leishmania.

Miltefosine (MT) (hexadecylphosphocholine) was implemented to cope with resistance against antimonials, the classical treatment in Leishmaniasis. Given the scarcity of anti- Leishmania (L) drugs and the increasing appearance of resistance, there is an obvious need for understanding the mechanism of action and development of such resistance. Metabolomics is an increasingly popular tool in the life sciences due to it being a relatively fast and accurate technique that can be applied either with a particular focus or in a global manner to reveal new knowledge about biological systems. Three analytical platforms, gas chromatography (GC), liquid chromatography (LC) and capillary electrophoresis (CE) have been coupled to mass spectrometry (MS) to obtain a broad picture of metabolic changes in the parasite. Impairment of the polyamine metabolism from arginine (Arg) to trypanothione in susceptible parasites treated with MT was in some way expected, considering the reactive oxygen species (ROS) production described for MT. Importantly, in resistant parasites an increase in the levels of amino acids was the most outstanding feature, probably related to the adaptation of the resistant strain for its survival inside the parasitophorous vacuole.

Separation and determination of chlorophenylpiperazine isomers in confiscated pills by capillary electrophoresis.

A simple capillary electrophoretic method with spectrophotometric UV detection at 236 nm has been developed for the selective separation and determination of 1-(2-chlorophenyl)piperazine (oCPP), 1-(3-chlorophenyl)piperazine (mCPP) and 1-(4-chlorophenyl)piperazine (pCPP) in confiscated pills. Several cyclodextrin derivatives were tested to compose the background electrolyte (BGE). The optimized BGE contained 20 mmol/L phosphoric acid adjusted to pH 2.5 with triethylamine and 10 mmol/L α-cyclodextrin, which provided acceptable resolution of analytes and candidate interferents in less than 15 min. The analyses were performed at constant voltage of 25 kV in 60 cm (effective length 50 cm; 50 µm i.d.) uncoated fused-silica capillary maintained at 25°C with sample injection at 4,826 Pa for 8s. Procaine at a concentration of 0.1mg/mL was used as internal standard (IS). Possible interference from other drugs such as amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 1-(3-trifluoromethylphenyl)piperazine and cocaine was also examined. The analytical curves were linear (R(2)=0.9994-0.9995) in the range of 10-200 µg/mL (for oCPP and mCPP) and 20-200 µg/mL for pCPP. Limits of detection (LODs) were 2.0 µg/mL (oCPP), 2.5µg/mL (mCPP) and 3.5 µg/mL (pCPP). Intraday precision at three concentration levels and six replicates of each level (10, 100, 200 µg/mL of each analyte; n=18) was evaluated for the corrected peak area ratio of analyte to IS and the migration times giving RSDs ≤ 4.9%. The accuracy was estimated for mCPP by a recovery test at the same three concentration levels and recoveries varied from 101.0 to 101.6%. The method has been successively applied to the analysis of 17 confiscated pills based mostly on mCPP.

HPLC-DAD-ESI/MS identification and quantification of phenolic compounds in Ilex paraguariensis beverages and on-line evaluation of individual antioxidant activity.

"Chimarrão" and "tererê" are maté (dried, toasted and milled Ilex paraguariensis leaves and stemlets) beverages widely consumed in South America. This paper describes the application of HPLC-DAD-ESI/MS method for the identification and quantification of caffeoylquinic acids (CQA), flavonol glycosides and purine alkaloids in these beverages. The beverage samples were prepared from commercial lots of maté from Southern Brazil. The caffeoylquinic acids, 4,5-diCQA, 3-CQA, 5-CQA, and 4-CQA were the major compounds, having 238-289, 153-242, 183-263, and 123-188 µg/mL, respectively, for chimarrão and 206-265, 122-218, 164-209, 103-169 µg/mL, respectively, for tererê. Caffeine also had high amounts while glycosides of quercetin and kaempferol were found at much lower levels. The individual antioxidant activity was also determined by an on-line system that measured their ABTS•+ radical scavenging activity, showing that the antioxidant capacity was not proportional to the concentrations of the phenolic compounds. 3-CQA, quercetina-3-O-ramnosylglucoside, and quercetina-3-O-glucoside were the major contributors to the antioxidant capacity, although the quercetin glycosides had concentrations less than 10 times that of 3-CQA.

Systematic evaluation of extraction methods for multiplatform-based metabotyping: application to the Fasciola hepatica metabolome.

Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica , using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability.

Sensitive and fast determination of Sudan dyes in chili powder by partial-filling micellar electrokinetic chromatography-tandem mass spectrometry.

A fast and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in food samples was developed for the first time using partial filling micellar electrokinectic chromatography-mass spectrometry (MEKC-MS). The use of MEKC was essential to achieve the separation of these neutral analytes, while the partial filling technique was necessary to avoid the contamination of the ion source with non-volatile micelles. MEKC separation and MS detection conditions were optimized in order to achieve a fast, efficient, and sensitive separation of the four dyes. Filling 25% of the capillary with an MEKC solution containing 40 mM ammonium bicarbonate, 25 mM SDS, and 32.5% (v/v) acetonitrile, a baseline separation of the four azo-dyes was obtained in 10 min. Tandem MS was investigated in order to improve the sensitivity and selectivity of the analysis. Limits of detection (LOD) values 5, 8, 15, and 29 times better were obtained for Sudan III, I, II, and IV, respectively, using partial filling MEKC-MS/MS instead of partial filling MEKC-MS. Under optimized conditions, LOD from 0.05 to 0.2 µg/mL were obtained. The suitability of the developed method was demonstrated through the fast and sensitive determination of Sudan I, II, III, and IV in spiked chilli powder samples. This determination could not be achieved by MEKC-UV due to the existence of several interfering compounds from the matrix.

Fast determination of Sudan dyes in chilli tomato sauces using partial filling micellar electrokinetic chromatography.

A new method based on partial filling micellar electrokinetic chromatography (MEKC) for the quantitative determination of Sudan dyes (I, II, III, and IV) in chilli sauces is presented. The separation is achieved filling 25% of the capillary with a MEKC buffer composed of 40 mM NH(4)HCO(3), 25 mM sodium dodecyl sulfate, and 32.5% (v/v) acetonitrile (ACN). The rest of the capillary is filled using a capillary zone electrophoresis (CZE) buffer composed of 40 mM NH(4)HCO(3) and 32.5% (v/v) ACN. Under optimized conditions, the azo dyes are baseline separated in less than 8 min with limits of detection ranging from 0.57 to 0.71 µg mL(-1) (S/N > 3). Using an internal standard, the repeatability of the quantitative determination is improved almost four times. The applicability of the method for rapid screening and determination of Sudan dyes is corroborated by analyzing spiked chilli sauce samples with recoveries from 85 to 99%. The reported conditions are demonstrated to be compatible with mass spectrometry detection.

Detection of paraquat in oral fluid, plasma, and urine by capillary electrophoresis for diagnosis of acute poisoning.

The aim of this work was to develop a simple and fast method for the detection of paraquat in oral fluid, plasma, and urine by capillary electrophoresis with diode-array detection (CE-DAD), to diagnose of acute poisoning related to this herbicide. The use of oral fluid in analytical toxicology has been established for drugs of abuse, but not for diagnosis of pesticides poisoning. Oral fluid was collected without stimulation using absorbent cotton swabs. Sample preparation included diluting, vortex mixing, and centrifuging of biological fluid, using ethyl paraquat as internal standard. CE-DAD analyses were performed in a fused-silica capillary, and separation was performed under constant voltage condition of 21 kV, with detection at 195 nm. The electrolyte was a 40 mmol/L phosphate buffer at pH 2.50. The proposed method provided a fast and simple assay for the determination of paraquat in human oral fluid, plasma, and urine. To our knowledge, this is the first mention of use of oral fluid as a biological fluid diagnosis of quaternary ammonium herbicide poisoning. After validation, the method was applied to two cases of acute poisoning by this herbicide.