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PubMed2006

Determination of pesticides in water by cone-shaped membrane protected liquid phase microextraction prior to micro-liquid chromatography.

A new sample pre-treatment technique termed cone-shaped membrane liquid phase microextraction (CSM-LPME) was developed and combined with micro-liquid chromatography (micro-LC) for the determination of selected pesticides in water samples. Four pesticides (hexaconazole, procymidone, quinalphos and vinclozolin) were considered as target analytes. Several important extraction parameters such as types of extraction solvent, agitation rate, pH value, total exposure time and effect of salt and humic acids were optimized. Enrichment factors of > 50 folds were easily achieved within 20 min of extraction. The analytical data demonstrated relative standard deviations for the reproducibility of the optimized CSM-LPME method ranging from 6.3 to 7.5%. The correlation coefficients of the calibration curves were at least 0.9995 across a concentration range of 2-100 microg/L. The detection limits for all the analytes were found to be in the range of 1.1-1.9 microg/L.

Analytic Sample Preparation Methods↗
PubMed2006

Recent developments in the analysis of brominated flame retardants and brominated natural compounds.

This article reviews recent literature on the analysis of brominated flame retardants (BFRs) and brominated natural compounds (BNCs). The main literature sources are reviews from the last five years and research articles reporting new analytical developments published between 2003 and 2006. Sample pretreatment, extraction, clean-up and fractionation, injection techniques, chromatographic separation, detection methods, quality control and method validation are discussed. Only few new techniques, such as solid-phase microextraction (SPME) or pressurized liquid extraction (PLE), have been investigated for their ability of combining the extraction and clean-up steps. With respect to the separation of BFRs, the most important developments were the use of comprehensive two-dimensional gas chromatography for polybrominated diphenyl ethers (PBDEs) and the growing tendency for liquid-chromatographic techniques for hexabromocyclododecane (HBCD) stereoisomers and of tetrabromobisphenol-A (TBBP-A). At the detection stage, mass spectrometry (MS) has been developed as well-established and reliable technology in the identification and quantification of BFRs. A growing attention has been paid to quality assurance. Interlaboratory exercises directed towards BFRs have grown in popularity and have enabled laboratories to validate analytical methods and to guarantee the quality of their results. The analytical procedures used for the identification and characterization of several classes of BNCs, such as methoxylated polybrominated diphenyl ethers (MeO-PBDEs) (also metabolites of PBDEs), halogenated methyl or dimethyl bipyrroles (DBPs), are reviewed here for the first time. These compounds were generally identified during the routine analysis of BFRs and have received little attention until recently. For each topic, an overview is presented of its current status.

Analytic Sample Preparation Methods↗
PubMed2006

Determination of endocrine-disrupting compounds in cereals by pressurized liquid extraction and liquid chromatography-mass spectrometry. Study of background contamination.

A sensitive method based on pressurized liquid extraction (PLE) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has been developed for the determination in cereal samples of seven endocrine-disrupting compounds: bisphenol A (BPA), 4-tert-butylbenzoic acid (BBA), 4-nonylphenol (NP), 4-tert-butylphenol (t-BP), 2,4-dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP) and pentachlorophenol (PCP). For the PLE procedure, methanol was selected as the extraction solvent. An experimental design approach was applied to optimize other PLE parameters. The recoveries achieved for the all seven compounds were in the 81-104% range, with relative standard deviations of 4-9%. An additional preconcentration step, based on solid-phase extraction (SPE), after the PLE step proved to be a successful way for obtaining a more sensitive method. The detection limits achieved in corn breakfast cereals were in the 0.003-0.013 microg g(-1) range, except for BPA, with a detection limit of 0.043 microg g(-1), for a sample size of 2.5 g. These values are similar to or even lower than currently legislated limits for pesticides in cereals and cereal-based foodstuffs. We also investigated possible contamination during the experimental process by the target compounds released from purified water, plastics, syringes, peristaltic pump tubes, glassware and other laboratory materials in contact with the samples along the analytical process.

Analytic Sample Preparation Methods↗
PubMed2000

Pattern recognition analysis of endogenous cell metabolites for high throughput mode of action identification: removing the postscreening dilemma associated with whole-organism high throughput screening.

Although whole-organism HTS can give clear indications of in vivo activity, typically few clues are given as to the mechanism of action (MOA), and determining the MOA for large numbers of active compounds can be costly and complex-an alternative approach is required. This report demonstrates that it is possible to conduct relatively high throughput MOA characterization of HTS hits utilizing a single sample preparation and analytical method. By monitoring a wide range of endogenous cellular metabolites via (1)H nuclear magnetic resonance spectroscopy, the MOA of herbicides can be predicted using computational methods to compare the metabolite perturbation patterns. Herbicides that induce a characteristic pattern of metabolic perturbation in maize include inhibitors of acetolactate synthase, acetyl co-enzyme A carboxylase, protoporphyrinogen oxidase, 5-enolpyruvylshikimate-3-phosphate synthase, and phytoene desaturase. In soya, photosystem II inhibitors can also be detected, further demonstrating that this method is not limited to inhibitors of enzymes that directly act upon endogenous metabolites, or a single species. The methods, including data analysis, can be readily automated, enabling relatively high throughput MOA elucidation of whole-organism screen hits. Additionally, for compounds with a novel MOA, this approach may lead to MOA identification faster than traditional methods. It is envisaged that application of these data analysis methods to other data types-for example, transcription (mRNA) or translation (protein) profiles-is likely to permit higher throughput with smaller sample requirements, along with ability to discriminate MOAs that are not adequately discriminated based upon endogenous metabolite profiles.

Automation↗
PubMed2006

High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma.

The balance between nitric oxide (NO) and vasoconstrictors like endothelin is essential for vascular tone and endothelial function. L-Arginine is converted to NO and L-citrulline by NO synthase (NOS). Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. ADMA is degraded by dimethylamino dimethylhydrolases (DDAHs), while SDMA is exclusively eliminated by the kidney. In the present article we report a LC-tandem MS method for the simultaneous determination of arginine, ADMA, and SDMA in plasma. This method is designed for high sample throughput of only 20-mul aliquots of human or mouse plasma. The analysis time is reduced to 1.6 min by LC-tandem MS electrospray ionisation (ESI) in the positive mode. The mean plasma levels of l-arginine, ADMA, and SDMA were 74+/-19 (SD), 0.46+/-0.09, and 0.37+/-0.07 microM in healthy humans (n=85), respectively, and 44+/-14, 0.72+/-0.23, and 0.19+/-0.06 microM in C57BL/6 mice. Also, the molar ratios of arginine to ADMA were different in man and mice, i.e. 166+/-50 and 85+/-22, respectively.

Analytic Sample Preparation Methods↗
PubMed1997

[Determination of molybdenum in natural waters by direct current plasma atomic emission spectrometry with ion-exchange preconcentration].

A new method was developed for the determination of trace molybdenum in natural waters by direct current plasma atomic emission spectrometry after separating and preconcentrating with D301 anion exchange resin. Mo adsorbed on the resin column can be recovered quantitatively by using 20mL of 8mol/L HNO3 at a flow rate of 0.6mL/min with elution rate of (81.6 +/- 1.78)%. The behaviour of Mo on the resin has been discussed. Simulative water and natural water samples have been determined. It was found that the interference element Ca could be separated effectively from Mo; an enrichment factor of 50 was obtained for Mo in 1L water sample; the determination limit of Mo was improved to 0.5 microg/L and the recovery of 90-107% was obtained.

Analytic Sample Preparation Methods↗
PubMed2005

Application of single drop microextraction for analysis of chemical warfare agents and related compounds in water by gas chromatography/mass spectrometry.

Retrospective detection and identification of chemical warfare agents (CWAs) is important from the verification point of view of the Chemical Weapons Convention. In the present work, a novel method for determination of CWAs and their markers in water has been described. It is based on a single drop micro extraction (SDME) of analytes and gas chromatography/mass spectrometric identification. Extraction conditions, such as solvent selection, agitation, extraction time, and salt content, were found to have significant influence on SDME. The conditions optimized for extraction of CWAs were 1 microL CH2Cl2/CCl4 (3:1 v/v), 30-min extraction time, 300-rpm stirring rate, and with or without NaCl addition. Under optimized conditions, comparison of SDME, solid-phase microextraction, and liquid-liquid extraction was also made. The limit of detection by SDME ranged from 75 to 10 microg L(-1) at a signal-to-noise ratio of 10:1.

Analytic Sample Preparation Methods↗
PubMed2006

Development of an analytical method for organotin compounds in fortified flour samples using microwave-assisted extraction and normal-phase HPLC with UV detection.

The normal high-performance liquid chromatography with UV detection was applied for the determination of tributyltin chloride (TBT), triphenyltin chloride (TPhT), tetraphenyltin (TrPhT), triethyltin chloride (TET) and tetraethyltin (TrET) from flour samples. The separation was performed in the isocratic mode on cyanopropyl column with a mobile phase of hexane-acetonitrile-THF (97/1/2). Under the experimental conditions used, quantitative limit of TBT, TPhT, TrPhT, TET and TrET are 0.95, 0.46, 0.97, 0.75 and 0.96 microg/ml, respectively. Microwave-assisted extraction of organotin (OT) compounds at 100 degrees C with an extraction time of 3 min was described. The extraction of organotin can be finished in acetic acid-hexane (20/80) medium. The quantitative extraction of five organotin compounds was achieved with recoveries ranging from 88 to 101% R.S.D. 3-8%.

Analytic Sample Preparation Methods↗
PubMed2006

High-performance anion-exchange chromatography using on-line electrolytic eluent generation for the determination of more than 25 intermediates from energy metabolism of mammalian cells in culture.

In this work, we present an improved method for the determination of a wide range of intracellular metabolites from mammalian cells by anion-exchange chromatography. The analysis includes the measurement of intermediates from glycolysis and tricarboxylic acid cycle as well as several additional nucleotides and sugar nucleotides. The use of an electrolytic on-line eluent generation device made the method highly convenient, reliable and prone to errors. Due to short delay times of the eluent generator, rapid KOH gradient changes could be applied to improve separation and to speed up elution. Suppressed conductivity and UV in series was used for detection. The detection wavelength of the UV detector was switched from 220 to 260 nm during the elution for a more selective signal depending on the absorption of analytes. Standards from more than 50 metabolites of major cellular pathways were chromatographically tested and compared to chromatograms from extraction samples of Madin-Darby canine kidney (MDCK) and BHK21 cells. A validation for most substances was performed. Detection limits were below the micromolar range and the coefficient of correlation (R(2)) was above 0.99 for most analytes. Working ranges were between 0.125-3.875 and 4.5-139.5 microM. Sample pH had a major impact on the quantification of several metabolites, but measurements were robust within a pH range of 6.5-9.0.

Analytic Sample Preparation Methods↗
PubMed2006

Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry.

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that are growing on agricultural commodities. Their frequent presence in food and their severe toxic, carcinogenic and estrogenic properties have been recognised as potential threat to human health. A reliable risk assessment of mycotoxin contamination for humans and animals relies basically on their unambiguous identification and accurate quantification in food and feedstuff. While most screening methods for mycotoxins are based on immunoassays, unambiguous analyte confirmation can be easily achieved with mass spectrometric methods, like gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). Due to the introduction of atmospheric pressure ionisation (API) techniques in the late 80s, LC/MS has become a routine technique also in food analysis, overcoming the traditional drawbacks of GC/MS regarding volatility and thermal stability. During the last few years, this technical and instrumental progress had also an increasing impact on the expanding field of mycotoxin analysis. The aim of the present review is to give an overview on the application of LC-(API)MS in the analysis of frequently occurring and highly toxic mycotoxins, such as trichothecenes, ochratoxins, zearalenone, fumonisins, aflatoxins, enniatins, moniliformin and several other mycotoxins. This includes also the investigation of some of their metabolites and degradation products. Suitable sample pre-treatment procedures, their applicability for high sample through-put and their influence on matrix effects will be discussed. The review covers literature published until July 2006.

Aflatoxins↗
PubMed2006

Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns.

OBJECTIVES: To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl(-)) on plasma unbound bilirubin (B(f)) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns. DESIGN AND METHODS: B(f) was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with B(f) measured at a 2-fold sample dilution using a FloPro Analyzer. B(f) was measured at two peroxidase concentrations to determine whether the peroxidase steady state B(f) (B(fss)) measurements were significantly less than the equilibrium B(f) (B(feq)), in which case it was necessary to calculate B(feq) from the two B(fss) measurements. B(f) was also measured before and after adding 100 mmol/L Cl(-) to the UB Analyzer assay buffer. RESULTS: B(feq) at the 42-fold dilution was nearly 10-fold less than but it correlated significantly with B(feq) at the 2-fold dilution (mean 8.2+/-5.2 nmol/L versus 73.5+/-70 nmol/L, respectively, p<0.0001; correlation r=0.6). The two UB Analyzer B(fss) measurements were significantly less than B(feq) in 42 of 74 (57%) samples, and Cl(-) increased B(feq) in 66 of 74 (89%) samples by a mean of 82+/-67%. CONCLUSIONS: B(fss) measured by the UB Analyzer at the standard 42-fold sample dilution using assay buffer without Cl(-) and a single peroxidase concentration is significantly less than the B(feq) in undiluted plasma. Accurate B(f) measurements can be made only in minimally diluted serum or plasma.

Analytic Sample Preparation Methods↗
PubMed2006

Efficient design and analysis of biospecimens with measurements subject to detection limit.

Pooling biospecimens is a well accepted sampling strategy in biomedical research to reduce study cost of measuring biomarkers, and has been shown in the case of normally distributed data to yield more efficient estimation. In this paper we examine the efficiency of pooling, in the context of information matrix related to estimators of unknown parameters, when the biospecimens being pooled yield incomplete observations due to the instruments' limit of detection. Our investigation of three sampling strategies shows that, for a range of values of the detection limit, pooling is the most efficient sampling procedure. For certain other values of the detection limit, pooling can perform poorly.

Analytic Sample Preparation Methods↗
PubMed2006

Coupling of acetonitrile deproteinization and salting-out extraction with acetonitrile stacking for biological sample clean-up and the enrichment of hydrophobic compounds (porphyrins) in capillary electrophoresis.

A new sample pretreatment approach in CE was developed for concurrent biological sample clean-up and the concentration of hydrophobic compounds based on the combination of ACN deproteinization with salting-out extraction. Further enhancement in concentration detection sensitivity was achieved by coupling (offline) salting-out extraction with an online CE sample enrichment technique known as "ACN stacking". By optimizing the pH of salting-out extraction, a number of model compounds (hydrophobic porphyrins with clinical significances), i.e. zinc-protoporphyrin, protoporphyrin, and coproporphyrin (CP) III and I, can be efficiently extracted from the aqueous sample into a smaller volume organic solvent (ACN) phase and an enrichment factor of ca. 100 can be obtained. The pressure injection of the enriched ACN phase (containing ca.1% NaCl) into the CE capillary at 10% capillary volume resulted in additional concentration of the various hydrophobic porphyrins, allowing for a combined enrichment factor of ca.1000 to be obtained. Calibration curves obtained for the determination of a pair of positional isomers with significant diagnostic value, urinary CPIII and CPI, were found to be linear between 10-300 ng/mL (with R2 = 0.999), and LODs (absorbance detection at 400 nm) were ca. 0.8 ng/mL (1.1 nmol/L of CPIII or CPI). Based on a single salting-out extraction, intraday precisions (nine consecutive injections) for both CPIII and CPI (at spiked concentrations of 10-300 ng/mL into urine) in terms of migration time and peak area were found to be within the range of 0.2-0.5 and 0.8-2.9%, respectively.

Acetonitriles↗
PubMed2002

Fragmentation characteristics of peptide-metal ion adducts under matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometric conditions.

Fragmentation reactions of sodium-cationized enkephalin peptides generated by matrix-assisted laser desorption/ionization were studied using post-source decay (PSD) with a reflectron time-of-flight mass spectrometer. Several matrices and analyte-matrix sample preparation methods were evaluated for high-intensity ion currents that could last for the entire PSD analysis. A triple dried-droplet sample preparation procedure with 2,5-dihydroxybenzoic acid as the matrix was found to yield abundant longer-lasting ion signals of the peptide-Na(+) ion adducts. The principal decay product of these adduct ions is the [b(n-1) + Na + OH](+) ion, which provides an unambiguous identification of the C-terminal residue of a peptide. In some peptides, the loss of a second residue from the C-terminus is also observed. No other sequence-specific ions were observed.

Amino Acid Sequence↗
PubMed2007

Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon-limited yeast cultures, which reveals a transient activation of the purine salvage pathway.

In this study we have revised our original procedure of yeast metabolites extraction. We showed that: (a) less than 5% of intracellular metabolites leaks out during the step of rapid arrest of cellular metabolism by quenching yeast cells into a 60% methanol solution kept at -40 degrees C; and (b) with a few exception, the stability of metabolites were not altered during the 3 min boiling procedure in a buffered ethanol solution. However, there was a loss of external added metabolites of 5-30%, depending on the type of metabolites. This was mainly attributable to their retention on cellular debris after ethanol treatment, which prevented centrifugation of the cellular extracts before evaporation of ethanol. We further simplified our previous high-performance ionic chromatography (HPIC) techniques for easier, more reliable and robust quantitative measurements of organic acids, sugar phosphates and sugar nucleotides, and extended these techniques to purine and pyrimidine bases, using a variable wavelength detector set at 220 and 260 nm in tandem with a pulsed electrochemical or suppressed conductivity detector. These protocols were successfully applied to a glucose pulse to carbon-limited yeast cultures on purines metabolism. This study showed that glucose induced a fast activation of the purine salvage pathway, as indicated by a transient drop of ATP and ADP with a concomitant rise of IMP and inosine. This metabolic perturbation was accompanied by a rapid increase in the activity of the ISN1-encoded specific IMP-5'-nucleotidase. The mechanism of this activation remains to be determined.

Adenine Nucleotides↗
PubMed2005

Reconstitution and alignment by a magnetic field of a beta-barrel membrane protein in bicelles.

A protocol is described for the reconstitution of a transmembrane beta-barrel protein domain, tOmpA, into lipid bicelles. tOmpA is the largest protein to be reconstituted in bicelles to date. Its insertion does not prevent bicelles from orienting with their plane either parallel or perpendicular to the magnetic field, depending on the absence or presence of paramagnetic ions. In the latter case, tOmpA is shown to align with the axis of the beta-barrel parallel to the magnetic field, i.e. perpendicular to the plane of the bilayer, an orientation conforming to that in natural membranes and favourable to structural studies by solid-state NMR. Reconstitution into bicelles may offer an interesting approach for structural studies of membrane proteins in a medium resembling a biological membrane, using either NMR or other biophysical techniques. Our data suggest that alignment in the magnetic field of membrane proteins included into bicelles may be facilitated if the protein is folded as a beta-barrel structure.

Analytic Sample Preparation Methods↗
PubMed2006

Capillary scale monolithic trap column for desalting and preconcentration of peptides and proteins in one- and two-dimensional separations.

Monolithic columns based on poly-(styrene-divinylbenzene) (PS-DVB) were utilized both for preconcentration (in 10 mm x 0.20 mm I.D. format) and analytical separation (in 60 mm x 0.20 and 0.10 mm I.D. format) of peptides and proteins in column switching micro-scale high-performance liquid chromatography. A special holder for short monolithic preconcentration columns was designed and pressure durability tests approved long-term stability up to 400 bar. An 11-20% decrease in the average peak widths of nine peptides was obtained upon combining a preconcentration column with an analytical column as compared with a setup using an analytical column only. Trapping efficiency, especially for small and hydrophilic peptides, was optimized by using 0.10% heptafluorobutyric acid instead of 0.050% trifluoroacetic acid as solvent additive during sample loading. Using a 10 mm x 0.20 mm I.D. preconcentration column, loadabilities between 0.5 and 1.6 microg were determined by frontal analysis of proteins and bioactive peptides, respectively. A 100-fold concentration followed by direct on-line intact mass determination is demonstrated for diluted (3 micromolL(-1)) protein solutions. The applicability of the monolithic preconcentration column for multidimensional chromatography was tested by off-line two-dimensional separation, combining strong cation-exchange chromatography and ion-pair reversed-phase chromatography. Peptide identification data from digested protein mixtures demonstrated reproducibilities of 46-75% in triplicate analyses, and confident peptide identifications of low abundant peptides even in the presence of a 650-fold molar excess of high abundant peptides.

Analytic Sample Preparation Methods↗