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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↗
PubMed2006

Quantification of 3R,3'R-zeaxanthin in plant derived food by a diastereomeric dilution assay applying chiral high-performance liquid chromatography.

For quantification of 3R,3'R-zeaxanthin in plant derived food, a new diastereomeric dilution assay (DIDA) was developed using chiral high-performance liquid chromatography coupled to diode-array detection (HPLC/DAD). As diastereomeric internal standard (I.S.), 3R,3'S-(meso)-zeaxanthin was applied. DIDA showed sufficient sensitivity with low limits of detection and quantification (18 microg100 g(-1) and 45 microg 100 g(-1)), a good inter-assay coefficient of variation (2.4%), determined by analysis of a certified BCR reference material, and a good recovery (94.7%). To prove the applicability of DIDA, various plant materials were analyzed, showing that DIDA is highly advisable if saponification is applied during work-up to hydrolyze xanthophyll esters.

Analytic Sample Preparation Methods↗
PubMed2006

Developments in single-drop microextraction.

Single-drop microextraction (SDME) has become a very popular liquid-phase microextraction technique because it is inexpensive, easy to operate and nearly solvent-free. Essentially, SDME combines extraction (and conceivably, cleanup) and concentration in a minimum number of steps, and thereafter, direct extract introduction into an analytical system. In this review, in order to encourage further development of SDME, we focus on its recent developments in its various guises. Its applications when used in combination with different analytical techniques, such as gas chromatography, high-performance liquid chromatography, inductively-coupled plasma mass spectrometry, capillary electrophoresis, mass spectrometry and electrothermal atomic absorption spectrometry, are summarized. SDME does have some limitations, and these are also discussed as well. Finally, an outlook on the future of the technique is given.

Analytic Sample Preparation Methods↗
PubMed2006

Terpene degradation and extraction from basil and oregano leaves using subcritical water.

In the first part of this study, the stability of five terpenes (alpha-pinene, limonene, camphor, citronellol, and carvacrol) under subcritical water conditions was investigated. The stability studies were carried out at four different temperatures (100, 150, 200, and 250 degrees C) with two different heating times (30 and 300 min). When water temperature was increased, the degradation of terpenes became more serious. Prolonged exposure time to each heating temperature also caused decreased terpene stability. The terpene recoveries were determined by conducting subcritical water extraction of sand spiked with terpenes. The recoveries are typically around 70 to 80% for extractions at 100 degrees C. Terpene recoveries were decreased with increasing water temperature due to poorer stability of terpenes. After the degradation and recovery studies, basil and oregano leaves were extracted using water at both 100 and 150 degrees C. The concentrations of each individual terpene in the water extract generally ranged from trace quantity to 65 microg terpene/g herb. However, the concentration of carvacrol in the oregano-water extract at 150 degrees C was found to be as high as 4270 microg carvacrol/g oregano.

Analytic Sample Preparation Methods↗
PubMed2006

Microwave-assisted extraction of organophosphate flame retardants and plasticizers from indoor dust samples.

A procedure for the determination of eight organophosphate flame retardants and plasticizers in dust samples is presented. Microwave-assisted extraction and gas chromatography (GC) with nitrogen-phosphorus detection (NPD) were used for sample preparation and analytes quantification, respectively. Influence of different variables (type and volume of organic solvent, temperature, time, agitation, etc.) on the yield of the extraction step was evaluated. The most important factor was the type of solvent, with the highest efficiencies corresponding to acetone. Under final conditions 10 mL of this solvent were employed. The extraction was carried out at 130 degrees C and satisfactory yields, similar to those obtained with the Soxhlet technique, were achieved. Due to the high content of organic carbon in dust samples, primary acetone extracts had to be subjected to intensive clean-up. Dilution with ultrapure water followed by concentration on a reversed-phase sorbent and further purification using silica, allowed a significant reduction of co-extracted interferences. Application of the developed methodology to indoor dust from private houses showed important concentrations of several organophosphate esters. The highest levels, up to 19 microg/g, corresponded to tris(butoxyethyl) phosphate; moreover, average values of two chlorinated compounds, used as flame retardants and considered as the most concerning species in the group, exceeded the 1 microg/g level.

Acetates↗
PubMed2007

Principles, developments and applications of on-line coupling of extraction with chromatography.

On-line coupling of extraction and chromatographic separation allows the whole analysis to be performed in a closed system. On-line systems are particularly useful when the analytes are labile, the amount of sample is limited, or very high sensitivity is required. Many on-line systems have been developed both for liquid and for solid samples. This review discusses the different instruments that have been constructed and the factors that need to be considered in the coupling. Selected illustrative applications are described to illustrate the potential of the on-line systems.

Analytic Sample Preparation Methods↗
PubMed2006

Superheated water extraction and phase transfer methylation of phenoxy acid herbicides from solid matrices.

Phase transfer catalytic methylation was applied to directly derivatise chlorophenoxy acid herbicides in superheated water extracts from sand and soil samples. The extractions were carried out at 120 degrees C statically for 5 min and then dynamically for 10 min at 1.0 mL min(-1) using water at pH 11.0 for a sand matrix and a flow rate of 0.5 mL min(-1) at pH 7.0 for soil samples. The methylation was carried out on-line on the extraction solution with ultrasonication at 80 degrees C, using either 0.05 mmol tetrabutylammonium bromide (TBAB) or 0.0125 mmol cetyltrimethylammonium bromide (CTAB) as phase transfer catalysts with 0.20 mmol methyl iodide in 2.0 mL dichloromethane trapping solvent. The former catalyst provided a higher yield but the latter gave fewer interfering peaks. The recoveries of most chlorophenoxy acids using the TBAB catalyst ranged from 67 to 105% for sand and from 82 to 114% for soil sample, except phenoxyacetic acid, 2-(2, 4-dichlorophenoxy)propanoic acid and 1-naphthaleneacetic acid, while those by using CTAB were slightly lower. Detection limits of all the analytes extracted from sand using TBAB catalyst were in a range of 5.3-16 microg g(-1) analysed by using gas chromatography with flame ionization detection (GC-FID).

Acetates↗
PubMed2006

Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry.

Nicotine is a major addictive compound in cigarette. Its smoke is rapidly and extensively metabolized to several metabolites in human. Cotinine as a major metabolite of nicotine is commonly used as a biomarker to determine active and passive smokers. Cotinine has a longer half-life ( approximately 20 h) compared to nicotine ( approximately 2h). A simple, sensitive, rapid and high throughput GC-MS method was developed for simultaneous quantification of urinary nicotine and cotinine in passive and active smokers. In the sample preparation method, the analytes and internal standard were first basified and followed by liquid-liquid extraction. Upon completion, anhydrous sodium sulphate was added to the solvent mixture to trap moistures. The clear extract obtained was directly injected into GC-MS, operating under selective ion monitoring (SIM) mode. Calibration curves in the range of 0.5-5000 ng/mL of the analytes in urine matrix were established with linear correlation coefficients (r(2)) greater than 0.997. The limit of detection for both nicotine and cotinine were 0.20 ng/mL. The mean recoveries for nicotine and cotinine were 93.0 and 100.4%, respectively. The within- and between-assay accuracies were between 2.1 and 7.9% for nicotine and between 0.7 and 11.1% for cotinine. Within- and between-assay precisions of 3.3-9.5% for nicotine and 3.4-9.8% for cotinine were also achieved. The method can be used in routine assessment and monitoring of active smoking and exposure to environmental tobacco smoke. The applicability of the assay was demonstrated in a small-scale comparison study between smokers and non-smokers.

Adult↗
PubMed2003

Quantitative analysis of acyl-lysophosphatidic acid in plasma using negative ionization tandem mass spectrometry.

Analysis of acyl-lysophosphatidic acids (LPAs) has clinical importance as a potential biomarker for ovarian and other gynecological cancers or obesity from the point of view of prevention. Here we report a simple sample preparation and analytical method with high sensitivity and specificity for the early detection of gynecological cancers to improve the overall outcome of this disease. We established a novel quantification method for acyl-LPAs in plasma by electrospray negative ionization tandem mass spectrometry (MS-MS) using multiple reaction monitoring mode without conventional TLC step. Protein-bound lipids, acyl-LPAs in plasma were extracted with methanol/chloroform (2:1) containing LPA C(14:0) as internal standard under acidic conditions. Following back-extraction with chloroform and water, the centrifuged lower phase was evaporated and reconstituted in methanol and then analyzed. Using ESI-MS-MS with negative ionization MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interference. For MRM mode, Q1 ions selected were m/z 409, 433, 435, 437 and 457 which corresponds to molecular mass [M-H](-) of C(16:0), C(18:2), C(18:1), C(18:0) and C(20:4) LPA, respectively. Q2 ions selected for MRM was m/z 79, phosphoryl product. Using MS-MS with MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interference. This method allowed simultaneous detection and quantification of different species of LPAs in plasma over a linear dynamic range of 0.01-25 micromol/l. The method detection limit was 0.3 pmol/ml with correlation coefficient of 0.9983 in most LPAs analyzed. When applied to plasma from normal and gynecological cancer patients, this new method differentiated two different groups by way of total LPA level.

Case-Control Studies↗
PubMed2006

[Developments of liquid-phase microextraction based on hollow fiber].

The liquid-phase microextraction (LPME), based on disposable hollow fiber, has been developed to be a new environmentally benign sample preparation technique which incorporate sampling, extraction and concentration into a single step. The novel technique can be easily manipulated in combination with high performance liquid chromatography, gas chromatography and capillary electrophoresis, and can provide excellent sample clean-up effect and high degree of extraction recovery and enrichment. It is proved to be simple, low-cost and virtually solvent-free. The extraction set-up, extraction mode, basic principles and recent applications of the hollow fiber-based liquid-phase microextraction are reviewed.

Analytic Sample Preparation Methods↗
PubMed1990

Target amplification for DNA analysis by the polymerase chain reaction.

The polymerase chain reaction (PCR) has become a standard laboratory technique. An enzymatic reaction, as simple to perform as it is satisfying to contemplate, the PCR solves two of the more universal problems in the chemistry of natural nucleic acids. It allows for the physical separation any particular sequence of interest from its context; and then provides for an in vitro amplification of this sequence which is virtually without limit. The surprising robustness of PCR derives from its fortuitous combination of three phenomena, each of which is intrinsically powerful. The first of these is the impressive ability of almost all oligodeoxynucleotides to bind tightly and specifically to their complementary nucleic acid sequences, discriminating easily between hundreds of thousands of sites. The second familiar phenomenon is illustrated by the notion that the probability for the occurrence of a compound action is the product of the individual probabilities for the occurrence of each of its components. The third phenomenon embodied in the polymerase chain reaction relates to the branching structure of its propagation and the inherent robustness attached to such a form. Consideration of the above leads to certain generalities regarding the relative utility of various protocols for carrying out the PCR. Specific conditions of time, temperatures, concentrations, etc. will be described, as well as sample preparation and analytical methods.

DNA Probes↗
PubMed1997

[Enzymatic analysis of the quality of foodstuffs].

Enzymatic analysis is an independent and separate branch of enzymology and analytical chemistry. It has become one of the most important methodologies used in food analysis. Enzymatic analysis allows the quick, reliable determination of many food ingredients. Often these contents cannot be determined by conventional methods, or if methods are available, they are determined only with limited accuracy. Today, methods of enzymatic analysis are being increasingly used in the investigation of foodstuffs. Enzymatic measurement techniques are used in industry, scientific and food inspection laboratories for quality analysis. This article describes the requirements of an optimal analytical method: specificity, sample preparation, assay performance, precision, sensitivity, time requirement, analysis cost, safety of reagents.

Enzymes↗
PubMed2001

Affinity chromatography as a method for sample preparation in gas chromatography/mass spectrometry.

Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometry (MS) technology, e.g., GC/MS and LC/MS and their variants, GC/tandem MS and LC/tandem MS, are best suited both for qualitative and quantitative analyses. GC/MS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GC/MS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentrations in complex matrices require further chromatographic techniques, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for sample purification. In recent years, affinity chromatography (e.g., boronate and immunoaffinity chromatography) has been developed to a superior technique for sample preparation of numerous classes of compounds in GC/MS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GC/MS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins.

Blood Proteins↗
PubMed1994

Analytical inverse supercritical fluid extraction of polar pharmaceutical compounds from cream and ointment matrices.

It has been shown that inverse supercritical fluid extraction (SFE) can be used to analytically isolate a polar analyte from its matrix even at low concentrations (0.016%). Inverse SFE has been shown to be successful not only for the semi-solid Neosporin ointments, but also for the semi-liquid Neosporin creams as well. The technique has been shown to be superior to solid-phase extraction in both cases and affords the analytical chemist a quicker and safer method of sample preparation of analytes from both creams and ointments.

Chemical Phenomena↗
PubMedDate not supplied

Comparison of an HPTLC and an HPLC procedure for the determination of chlorpropham, propham and thiabendazole residues in potatoes.

HPTLC was used to check for residues of chlorpropham, propham (sprout inhibitors) and thiabendazole (fungicide) applied to potatoes. The method used gave good precision and analytical sensitivity. The sample preparation method developed by the authors was simple and gave good recovery and selectivity as far as other components of the matrix were concerned. Comparison with liquid chromatography confirmed the validity of the results.

Carbamates↗