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Simultaneous Solid Phase Extraction and Derivatization of Aliphatic Primary Amines Prior to Separation and UV-Absorbance Detection
... The LODs for amoxicillin and ampicillin were 20 and 19 ng/mL. An innovative strategy for the analysis of aliphatic amines from soil samples was shown by Felhofer et al. [33]. The immobilisation of a chromophore onto a SPE sorbent allowed sample clean-up, pre-concentration and derivatisation. ...
... An example of an ingenious labelling strategy was the use of magnetic beads for full sample preparation including derivatisation of N-glycans from therapeutic antibodies [18]. Also, the combination of sample clean-up and derivatisation on one single SPE cartridge demonstrated originality [33]. Moreover, the development of labelling agents which offer higher selectivity and improved reaction kinetics will play an important role for the development of future derivatisation reagents. ...
Derivatisation is a well-established and mature form of sample preparation for CE. The modification of the analyte can cause superior analysis characteristics such as better sensitivity and selectivity, however, derivatisation of the analyte introduces an additional step into the analytical work-flow. This review covers articles from January 2012 to January 2015 on derivatisation in CE. The main sections are on the derivatisation modes (i.e., pre-capillary, in-line, in-capillary and post-capillary), separation, and detection modes (i.e., laser induced fluorescence and others). Laser-induced fluorescence is discussed in more detail since this detection mode was most prevalent. A table of the common labelling agents and wavelengths for excitation and emission and the common derivatisation reactions are included. In addition, a comprehensive table which summarises all research articles is provided. This review is suitable for analytical chemists as a guide for "how to get started" with derivatisation for separation and detection in CE. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... Generally speaking, and among different protocols that can be applied to improve the preconcentration or separation of target analytes, perhaps the most common ones are off-line (also described as pre-column). These methods are generally manually performed by allowing researchers to control a wide range of conditions [523], including heating the sample and have been applied toward the analysis of acrylamide [524], mono-and oligosaccharides [525], sialylated N-glycan linkage isomers [526], 3-hydroxyaspartate [527], or acidic metabolites [528]. Off-line approaches have also allowed taking some advantage of advanced oxidation methods (combining H 2 O 2 and UV light) to follow the degradation of pharmaceuticals [529] or to convert alcohols into the corresponding acids, thus facilitating their separation by CE [350,530]. ...
This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. "Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications," pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.
... Based on evidence from in vitro and in vivo model of the purification method of methylglyoxal-putrescine adducts (Park et al., 2017), one fraction was fractionated to contain methylglyoxal-spermidine Schiff bases and was normalized in the unbound fractions after SPE separation to their hydrophobic selectivity (Felhofer et al., 2013). ...
Methylglyoxal regulates cell division and differentiation through its interaction with polyamines. Loss of their biosynthesizing enzyme causes physiological impairment and cell elongation in eukaryotes. However, the reciprocal effects of methylglyoxal and polyamine production and its regulatory metabolic switches on morphological changes in prokaryotes have not been addressed. Here, Bacillus subtilis methylglyoxal synthase (mgsA) and polyamine biosynthesizing genes encoding arginine decarboxylase (SpeA), agmatinase (SpeB), and spermidine synthase (SpeE), were disrupted or overexpressed. Treatment of 0.2mM methylglyoxal and 1mM spermidine led to the elongation and shortening of B. subtilis wild-type cells to 12.38±3.21μm (P<0.05) and 3.24±0.73μm (P<0.01), respectively, compared to untreated cells (5.72±0.68μm). mgsA-deficient (mgsA(-)) and -overexpressing (mgsA(OE)) mutants also demonstrated cell shortening and elongation, similar to speB- and speE-deficient (speB(-) and speE(-)) and -overexpressing (speB(OE) and speE(OE)) mutants. Importantly, both mgsA-depleted speB(OE) and speE(OE) mutants (speB(OE)/mgsA(-) and speE(OE)/mgsA(-)) were drastically shortened to 24.5% and 23.8% of parental speB(OE) and speE(OE) mutants, respectively. These phenotypes were associated with reciprocal alterations of mgsA and polyamine transcripts governed by the contents of methylglyoxal and spermidine, which are involved in enzymatic or genetic metabolite-control mechanisms. Additionally, biophysically detected methylglyoxal-spermidine Schiff bases did not affect morphogenesis. Taken together, the findings indicate that methylglyoxal triggers cell elongation. Furthermore, cells with methylglyoxal accumulation commonly exhibit an elongated rod-shaped morphology through upregulation of mgsA, polyamine genes, and the global regulator spx, as well as repression of the cell division and shape regulator, FtsZ.
... Moreover, although SPE using C18 resin was effective at isolating PUT-MG Schiff bases (Fig. S5), the loading efficacy of this adduct was approximately 0.41% (data not shown). Thus, we utilized the unbound fractions after the SPE separation, and the mixtures were normalized to their hydrophobic selectivity (Felhofer et al., 2013) to purify in vitro-and in vivo-formed PUT-MG Schiff bases. To determine whether PUT-MG adducts were composed of several types of enantiomeric compounds, the RIs of Schiff bases were measured by HPLC (Fig. 6B) as conducted in another study on enantioselectivity and enantiopurity of chiral Schiff bases (Angelaud et al., 2000). ...
Polyamines protect protein glycation in cells against the advanced glycation end product precursor methylglyoxal, which is inevitably produced during glycolysis, and the enzymes that detoxify this α-ketoaldehyde have been widely studied. Nonetheless, nonenzymatic methylglyoxal-scavenging molecules have not been sufficiently studied either in vitro or in vivo. Here, we hypothesized reciprocal regulation between polyamines and methylglyoxal modeled in Dictyostelium grown in a high-glucose medium. We based our hypothesis on the reaction between putrescine and methylglyoxal in putrescine-deficient (odc⁻) or putrescine-overexpressing (odcoe) cells. In these strains, growth and cell cycle were found to be dependent on cellular methylglyoxal and putrescine contents. The odc⁻ cells showed growth defects and underwent G1 phase cell cycle arrest, which was efficiently reversed by exogenous putrescine. Cellular methylglyoxal, reactive oxygen species (ROS), and glutathione levels were remarkably changed in odcoe cells and odc̄ cells. These results revealed that putrescine may act as an intracellular scavenger of methylglyoxal and ROS. Herein, we observed interactions of putrescine and methylglyoxal via formation of a Schiff base complex, by UV–vis spectroscopy, and confirmed this adduct by liquid chromatography with mass spectrometry via electrospray ionization. Schiff bases were isolated, analyzed, and predicted to have molecular masses ranging from 124 to 130. We showed that cellular putrescine–methylglyoxal Schiff bases were downregulated in proportion to the levels of endogenous or exogenous putrescine and glutathione in the odc mutants. The putrescine–methylglyoxal Schiff base affected endogenous metabolite levels. This is the first report showing that cellular methylglyoxal functions as a signaling molecule through reciprocal interactions with polyamines by forming Schiff bases.
A pretreatment method of dispersive solid‐phase extraction (DSPE) along with back‐extraction followed by capillary electrophoresis (CE)‐UV detector was developed for the determination of mercury species in water samples. Sulfhydryl‐functionalized SiO2 microspheres (SiO2−SH) were synthesized and used as DSPE adsorbents for selective extraction and enrichment of three organic mercury species namely ethylmercury (EtHg), methylmercury (MeHg) and phenylmercury (PhHg), along with L‐cysteine (L‐cys) containing hydrochloric acid as back‐extraction solvent. Several main extraction parameters were systematically investigated including sample pH, amount of adsorbent, extraction and back‐extraction time, volume of eluent and concentration of hydrochloric acid. Under optimal conditions, good linearity was achieved with correlation coefficients over 0.9990, in the range of 4−200 μg/L for EtHg, and 2−200 μg/L for MeHg and PhHg. The limits of detection (LOD) were obtained of 1.07, 0.34 and 0.24 μg/L for EtHg, MeHg and PhHg, respectively, as well as the limits of quantitation (LOQ) were 3.57, 1.13 and 0.79 μg/L, respectively, with enrichment factors ranging from 109 to 184. Recoveries were attained with tap and lake water samples in a range of 62.3−107.2%, with relative standard deviations of 3.5–10.1%. The results proved that the method of SiO2−SH based DSPE coupled with CE‐UV was a simple, rapid, cost‐effective and eco‐friendly alternative for the determination of mercury species in water samples.
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Volatile amines are among the most frequently used chemicals in organic and pharmaceutical chemistry. Synthetic route optimization often involves the evaluation of several different amines requiring the development and validation of analytical methods for quantitation of residual amine levels. Herein, a simple and fast generic GC-FID method on an Agilent J&W CP-Volamine capillary column (using either He or H2 as the carrier gas) capable of separating over 25 volatile amines and other basic polar species commonly used in pharmaceutical chemistry workflows is described. This 16min method is successfully applied to the analysis and quantitation of volatile amines in a variety of pharmaceutically-related drugs and synthetic intermediates. Method validation experiments showed excellent analytical performance in linearity, recovery, repeatability, and limit of quantitation and detection. In addition, diverse examples for the application of this method to the simultaneous determination of other amine-related chemicals in reaction mixtures are illustrated, thereby indicating that these GC-FID method conditions can be effectively used as starting point during method development for the analysis of other basic polar species beyond the validated list of amines described in this study.
Artemether–lumefantrine is the most widely used artemisinin based combination therapy for malaria. The present work aims to develop and validate simple, accurate, precise and rapid ratio first order derivative spectrophotometric method for the simultaneous estimation of artemether and lumefantrine in a fixed dose combination tablet. The first step in the method development was to derivatize artemether as it does not show absorption in UV region, hence it was derivatized using hydrochloric acid as the derivatizing agent. The derivatizing conditions further were optimized by full factorial multivariate approach, where independent variables selected were volume of concentrated hydrochloric acid and time taken for artemether derivatization at room temperature. Further, based on the statistical analysis derivatizing conditions were optimized i.e. 1.3 ml of conc. HCl at room temperature for 30 min. At this condition, the artemether was found to absorb satisfactorily in the UV region and the absorbance of lumefantrine was found to remain unaffected. The developed method showed good calibration data in the range of 5-30 μg/ml for artemether and 2-12 μg/ml for lumefantrine. The mean % recovery values were found to be 99.96-100.49% and 99.48-100.31% for artemether and lumefantrine, respectively. Also, the developed method was effectively applied in the estimation of artemether and lumefantrine in commercial tablet (ARH-L DS tablets) concluding that it can be practically applied for the quality control of the routinely examined drugs in combined dosage forms with reduced expenditure of time.
Capillary electrophoresis (CE) is presented as an efficient and potent alternative to other chromatographic techniques, such as liquid chromatography (LC) or gas chromatography (GC), to analyze organic pollutants in environmental samples. Despite the insufficient sensitivity associated with the use of CE with optical detection, the latest developments on the use of alternative detection systems and/or sample preconcentration techniques together with its efficiency and versatility have demonstrated the potential of CE to face the determination of pollutants at trace levels. Other interesting characteristics of CE in the field of environmental analysis include its enormous possibilities to achieve stereoselective analysis of chiral compounds and the low amount of reagents needed (green analytical technique). Nowadays, a large number of research works have been published dealing with the use of CE as a powerful technique for pollutants analysis. This article presents the fundamentals of CE techniques as well as the main developments and applications performed in environmental analysis during the past 10 years. The CE determination of a large variety of organic pollutants, including pesticides, phenols, pharmaceuticals, nitroaromatic compounds, amines, and so on, is described.
The Urey in situ organic compound analysis instrument, consisting of a subcritical water extractor (SCWE) and a portable microchip capillary electrophoresis instrument called the Mars Organic Analyzer (MOA), was field tested in the Atacama Desert, Chile, in June 2005. Soil samples from the most arid Yungay region were collected, biomarkers were extracted by the SCWE, and organic amine composition and amino acid chirality analysis was performed by the MOA. Samples collected from the top 1 cm of duracrust soil but shielded from the ambient environment by rocks were compared to the exposed duracrust. The shielded duracrust yielded amines and amino acids ranging from 50 to 100 ppb, while amino acid signals from the exposed duracrust were below blank levels. Samples from buried gypsum deposits located directly above a water flow channel contained amino acids ranging from 13 to 90 ppb. Chiral analysis revealed D/L ratios of 0.39 +/- 0.08 and 0.34 +/- 0.07 for alanine/serine and 0.78 +/- 0.06 for aspartic acid, indicating significant racemization of biologically produced amino acids. On the basis of the D/L ratios, we estimate sample ages ranging from 103 to 105 years. These results demonstrate the successful field testing of the Urey instrument, as well as the detection of biomarkers from past terrestrial life in one of the most arid and Mars-like regions on Earth.
Much attention has recently been directed to the development and application of online sample preconcentration and microreactions in capillary electrophoresis using selective adsorbents based on chemical or biological specificity. The basic principle involves two interacting chemical or biological systems with high selectivity and affinity for each other. These molecular interactions in nature usually involve noncovalent and reversible chemical processes. Properly bound to a solid support, an "affinity ligand" can selectively adsorb a "target analyte" found in a simple or complex mixture at a wide range of concentrations. As a result, the isolated analyte is enriched and highly purified. When this affinity technique, allowing noncovalent chemical interactions and biochemical reactions to occur, is coupled on-line to high-resolution capillary electrophoresis and mass spectrometry, a powerful tool of chemical and biological information is created. This paper describes the concept of biological recognition and affinity interaction on-line with high-resolution separation, the fabrication of an "analyte concentrator-microreactor", optimization conditions of adsorption and desorption, the coupling to mass spectrometry, and various applications of clinical and pharmaceutical interest.
To support the transfer of advances from R+D to routine analytical laboratories, we discuss recent developments in sample preparation for commercial capillary-electrophoresis (CE) equipment. We focus on practical considerations that permit the coupling or the integration of sample-treatment devices into commercial CE equipment. We describe and critically compare at-line and on-line coupling strategies. We also describe integrated in-capillary, in-vial or in-replenishment-system methodologies to perform solid-phase extraction or liquid-phase extraction.
Non-aqueous solutions of Ellman's reagent [5,5′-dithio(2-nitrobenzoic acid), DTNB] can be used to quantify thiols supported on macroporous polystyrene and TentaGel resins. Organic solutions of Ellman's reagent may also be used as a qualitative tests for thiols on a wider range of solid supports.
Preconcentration methodology based on adsorption chromatographies for enriching aliphatic amines (CI to C4 substituted primary, secondary, and tertiary) and alkanolamines in water was studied by free zone capillary electrophoresis (CZE) with indirect UV detection. The solid-phase extraction of amines from water (pH 5) as a preconcentration step was studied for ion exchange solid-phase extraction (SCX) cartridges, cation ion exchange extraction disks, and ion-pairing with C18 extraction disks. In the course of the investigations, the electrophoretic properties of the amines was studied in some detail in order to optimize separations and detection limits. The indirect mode is particularly powerful in being able to detect primary through quarternary amines without derivatization. Mobilities of amines were correlated with their Stokes′ radii. Increased selectivity for resolving closely related amines under CZE was explored using nonionic surfactants, pH adjustment, and optimized background electroyle. Techniques were developed for obtaining stable baselines in the indirect detection mode. Linearity, sensitivity and efficiency were explored for this mode of detection for a large set of amines.
Miniaturized, portable instrumentation has been gaining popularity in all areas of analytical chemistry. Capillary electrophoresis (CE), due to its main strengths of high separation efficiency, relatively short analysis time and low consumption of chemicals, is a particularly suitable technique for use in portable analytical instrumentation. In line with the general trend in miniaturization in chemistry utilizing microfluidic chips, the main thrust of portable CE (P–CE) systems development is towards chip-based miniaturized CE. Despite this, capillary-based (non-chip) P–CE systems have certain unmatched advantages, especially in the relative simplicity of the regular cylindrical geometry of the CE capillary, maximal volume-to-surface ratio, no need to design and to fabricate a chip, the low costs of capillary compared to chip, and better performance with some detection techniques. This review presents an overview of the state of the art of P–CE and literature relevant to future developments. We pay particular attention to the development and the potential of miniaturization of functional parts for P–CE. These include comp-onents related to sample introduction, separation and detection, which are the key elements in P–CE design. The future of P–CE may be in relatively simple, rugged designs (e.g., using a short piece of capillary fixed to a chip-sized platform on which injection and detection parts can be mounted). Electrochemical detection is well suited for miniaturization, so is probably the most suitable detection technique for P–CE, but optical detection is gaining interest, especially due to miniaturized light sources (e.g., light-emitting diodes).
Analysis of biogenic amines (BA) in foods was reviewed. Biogenic amines are natural antinutrition factors and are important from a hygienic point of view as they have been implicated as the causative agents in a number of food poisoning episodes, and they are able to initiate various pharmacological reactions. Histamine, putrescine, cadaverine, tyramine, tryptamine, β-phenylethylamine, spermine, and spermidine are considered to be the most important biogenic amines occurring in foods. Analysis of BA is important because of their toxicity and their usage as indicators of the degree of freshness or spoilage of food. Several methods have been developed for determination of biogenic amines in food. The analytical methods used for quantification of BA are mainly based on chromatographic methods: thin layer chromatography (TLC), gas chromatography (GC), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). HPLC is most often used for the analysis method of BAs. Due to low volatility and lack of chromophores of most BA, UV-spectrometric detectors cannot be used. The large majority of assays employs fluorimetric detection with precolumn or postcolumn derivatization techniques. This review shows that these methods allow quantitative determination of biogenic amines, individually or simultaneously in foods.
Biogenic amines are natural antinutrition factors and are important from a hygienic point of view as they have been implicated as the causative agents in a number of food poisoning episodes, and they are able to initiate various pharmacological reactions. Histamine, putrescine, cadaverine, tyramine, tryptamine, β-phenylethylamine, spermine, and spermidine are considered to be the most important biogenic amines occurring in foods. These amines are designated as biogenic because they are formed by the action of living organisms. Histamine has been implicated as the causative agent in several outbreaks of food poisoning, while tyramine and β-phenylethylamine have been proposed as the initiators of hypertensive crisis. The toxicity of biogenic amines to chicks in terms of loss of weight and mortality was also reported. The toxicity of histamine appeared to be enhanced by the presence of other amines such as cadaverine, putrescine, and tyramine. Biogenic amines may also be considered as carcinogens because of their ability to react with nitrites to form potentially carcinogenic nitrosamines. The biogenic amine content of various foods and feed have been widely studied and found in cheese, fish and meat products, eggs and mushrooms. Food substances that have been prepared by a fermentative process, or have been exposed to microbial contamination during aging or storage, are likely to contain amines. Alcoholic beverages such as beers can contain biogenic amines, as do some other fermented foods such as sauerkraut and soy bean products. Amines were also considered as endogenous to plant substance that is commonly used for food, where some fruits and vegetables were found to contain high concentrations of various amines.
This paper presents a fully automated method for determining ten primary amines in wastewater at ng/L levels. The method is based on simultaneous derivatization with pentafluorobenzaldehyde (PFBAY) and headspace solid-phase microextraction (HS-SPME) followed by gas chromatography coupled to ion trap tandem mass spectrometry (GC–IT-MS–MS). The influence of main factors on the efficiency of derivatization and of HS-SPME is described in detail and optimized by a central composite design. For all species, the highest enrichment factors were achieved using a 85 μm polyacrylate (PA) fiber exposed in the headspace of stirred water samples (750 rpm) at pH 12, containing 360 g/L of NaCl, at 40 °C for 15 min. Under optimized conditions, the proposed method achieved detection limits ranging from 10 to 100 ng/L (except for cyclohexylamine). The optimized method was then used to determine the presence of primary amines in various types of wastewater samples, such as influent and effluent wastewater from municipal and industrial wastewater treatment plants (WWTPs) and a potable water treatment plant. Although the analysis of these samples revealed the presence of up to 1500 μg/L of certain primary amines in influent industrial wastewater, the concentration of these compounds in the effluent and in municipal and potable water was substantially lower, at low μg/L levels. The new derivatization–HS-SPME–GC–IT-MS–MS method is suitable for the fast, reliable and inexpensive determination of primary amines in wastewater in an automated procedure.
A new support employing a reactive, N-hydroxysuccinimide ester derivative of silica, was developed for high-performance affinity chromatography. The support can be rapidly prepared from a stable precursor support and then used for coupling of ligands containing primary amino groups. Procedures for coupling to batch support or for coupling inside prepacked columns are described. In either case, ligand coupling was shown to be extremely rapid and efficient. This new support was used to prepare affinity chromatography columns using protein (protein A, concanavallin A), peptide (melittin), and low-molecular-weight [6-aminohexyl-Cibacron, and 2-(trifluoromethyl)-10-(3-aminopropyl)phenothiazine] affinity ligands. The various supports performed well, demonstrating that this new coupling reaction provides a rapid, efficient alternative for the synthesis of affinity chromatography columns.
This review highlights recent progresses in capillary electrophoresis (CE) analysis of amino acid enantiomers in the last decade. Various chiral selectors including cyclodextrins (CDs), bile salts, crown ethers, cinchona alkaloids, metal-chiral amino acid complexes, macrocyclic antibiotics and proteins have been employed to separate amino acid enantiomers. In the CE analysis of amino acids, the selection of the separation mode is one of the most important issues to obtain good resolution of target enantiomers. Among several separation modes, CD-modified capillary zone electrophoresis (CD-CZE), CD electrokinetic chromatography (CDEKC), micellar EKC (MEKC), CD-modified micellar electrokinetic chromatography (CD-MEKC), capillary electrochromatography (CEC), ligand-exchange CE (LE-CE), and nonaqueous CE (NACE) have been employed to the chiral analysis of amino acids. More than 160 published research articles collected from SciFinder Scholar databases from the year 2001 described the enantioseparations of amino acids by capillary-based electrophoresis. This review provides a comprehensive table listing the CE analysis of amino acid enantiomers with categorizing by the separation modes.
Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.
Over the last years, there has been an explosion in the number of developments and applications of CE and microchip-CE. In part, this growth has been the direct consequence of recent developments in instrumentation associated with CE. This review, which is focused on the contributions published in the last 5 years, is intended to complement the articles presented in this special issue dedicated to instrumentation and to provide an overview of the general trends and some of the most remarkable developments published in the areas of high-voltage power supplies, detectors, auxiliary components, and compact systems. It also includes a few examples of alternative uses of and modifications to traditional CE instruments.
Solvent bar microextraction (SBME) combined with gas chromatography-flame ionization detector (GC-FID), was used for preconcentration and determination of some aliphatic amines in waste water samples. The effect of different variables on the extraction efficiency was studied simultaneously using an experimental design. The variables of interest in the SBME were ionic strength, organic additive effect, sodium hydroxide concentration, stirring rate and extraction time and temperature. A Plackett-Burman design was performed for screening in order to determine the significant variables affecting the extraction efficiency. Then, the significant factors were optimized by a Box-Behnken design (BBD) and the response surface equations were derived. The optimum experimental conditions were sodium chloride concentration, 20% (w/v); sodium hydroxide concentration, 1 mol L(-1); stirring rate, 700 rpm; extraction temperature, 45 degrees C; extraction time, 30 min, and without addition of acetone. Under the optimum conditions, the preconcentration factors were between 260 and 1130. The limit of detections (LODs) ranged from 0.01 microg L(-1) (for dibutylamine) to 0.06 microg L(-1) (for N-ethyldiisopropylamine). The linear dynamic ranges (LDRs) of 0.05-800 and 0.1-600 microg L(-1) were obtained for most of the analytes. The performance of the method was evaluated for extraction and determination of aliphatic amines in waste water samples in the range of microgram per liter and satisfactory results were obtained (RSDs<13.6%).
This study describes the use of Tween 20-capped gold nanoparticles (Tween 20-AuNPs) for the selective extraction and enrichment of five aminothiols, including glutathione, gamma-glutamylcysteine, cysteine, homocysteine, and cysteineglycine, prior to analysis by capillary electrophoresis with UV detection. Tween 20-AuNPs are capable of extracting aminothiols from a complicated matrix because a Tween 20 capping layer can inhibit effectively the nonspecific adsorption. Moreover, Tween 20-AuNPs had better aminothiol loading compared to Zonyl FSN-100- and Triton X-100-capped AuNPs. The extraction efficiency of aminothiols was highly dependent on the number of Tween 20-AuNPs, the concentration of dithiothreitol, and the type of surfactant (i.e., capping agent). Under optimal extraction conditions, the limits of detection at a signal-to-noise ratio of 3 for five aminothiols were down to 10-65 nM. Total and free aminothiols in plasma were determined by varying the order of disulfide reduction with tris(2-carboxyethyl)phosphine. Efficient separation of total and free aminothiols with baseline resolution was successfully achieved by the addition of cationic polyelectrolyte, poly(diallyldimethylammonium chloride), to the background electrolyte. Because this extraction method provided no matrix effect, the concentrations of total and free aminothiols in plasma can be quantified with an external calibration method. On the basis of the advantages of simplicity, high selectivity, high sensitivity, and good reproducibility, this proposed method may have great potential for disease diagnosis.
A simple, precise, and accurate hydrophilic interaction liquid chromatographic (HILIC) method has been developed for the determination of five aromatic amines in environmental water samples. Chromatography was carried out on a bare silica column, using a mixture of acetonitrile and a buffer of NaH(2)PO(4)-H(3)PO(4) (pH 1.5, containing 10mM NaH(2)PO(4)) (85:15, v/v) as a mobile phase at a flow rate of 1 mL min(-1). Aromatic amines were detected by UV absorbance at 254 nm. The linear range of amines was good (r(2)>0.998) and limit of detection (LOD) within 0.02-0.2 mg L(-1) (S/N=3). The retention mechanism for the analytes under the optimum conditions was determined to be a combination of adsorption, partition and ionic interactions. The proposed method was applied to the environmental water samples. Aromatic amines were isolated from aqueous samples using solid-phase extraction (SPE) with Oasis HLB cartridges. Recoveries of greater than 75% with precision (RSD) less than 12% were obtained at amine concentrations of 5-50 microg L(-1) from 100mL river water and influents from a wastewater treatment plant (WWTP). The present HILIC technique proved to be a viable method for the analysis of aromatic amines in the environmental water samples.
A novel method is firstly presented for field and rapid analysis of short-chain aliphatic amines in water as their pentafluorobenzaldehyde (PFBAY) derivative using solid-phase microextraction (SPME) and portable GC. In the proposed method, short-chain aliphatic amines in water rapidly reacted with PFBAY, and then were headspace extracted and concentrated by SPME. The formed amines derivatives were analyzed by portable GC. The SPME parameters of fiber selection, extraction temperature, extraction time, and stirring rate were studied. The method validations including LOD, recovery, precision, and linearity were studied. It was found that the proposed method required the whole analysis time 22 min, and provided low LOD of 1.2-4.6 ng/mL, good recovery of 91-106%, good precision of RSD value 3.5-9.3%, and linear range 20.0-500 ng/mL (r(2) >0.99). The obtained results demonstrated that the SPME-portable GC is a simple, rapid, and efficient method for the field analysis of short-chain aliphatic amines. Finally, the proposed method was further applied to the quantification of ethylamine, propylamine, and butylamine in environmental water.
DTNB has been found to react quantitatively with sulfite ion liberating two moles of 3-carboxylato-4-nitro-thiophenolate ion. While this reaction provides a quantitative measurement of sulfite ion, it also provides quantitative interference with the determination of sulfhydryl groups in proteins. Thiosulfate also interferes with the sulfhydryl determinations using DTNB and the equilibrium constant for the reaction formulated as Eq. (3) is (4.8 ± 0.2) × 10−2 at pH 7.75 and at ionic strength of 0.4.
The reaction of the Ellman reagent [5,5′-dithiobis-(2-nitrobenzoic acid)] with a protein sulfhydryl group yields 1 mole of thiophenylated protein and 1 mole of thiophenylate anion, when the reaction is carried out above pH 7.0. The quantity of thiophenylate anion liberated in this reaction is then determined by light absorption measurement at 412 mμ. In the present paper, this technique is modified so that the thiophenylated protein is isolated from the reaction mixture. The amount of thionitrobenzoic acid bound to the protein is then determined. Using this principle, sulfhydryl analyses have been successfully made on (1) a sulfhydryl-containing protein in the presence of a reduced thiol; (2) heme proteins; (3) an insoluble protein; and (4) a protein rendered insoluble by denaturation. The classical Ellman procedure cannot be used for assays of free sulfhydryl groups on any of these proteins.
Capillary electrophoresis (CE) methods have been developed for the speciation and quantitation of thiols and disulfides of biological interest, including the endogenous compounds glutathione, glutathione disulfide, cysteine, cystine, homocysteine, and homocystine and the therapeutic agents penicillamine, penicillamine disulfide, N-acetylcysteine, and captopril. Good speciation and quantitation were achieved for the underivatized thiols and disulfides using a detection wavelength of 200 nm; detection limits were in the range 20-90 microM (1-4 pmol) using a 50-microns-diameter capillary. To achieve lower detection limits, thiols were derivatized with the thiol-specific probe molecule, 5,5'-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent). Good speciation and quantitation were achieved for the Ellman's derivatized thiols using a detection wavelength of 357 nm; detection limits were in the range 5-50 microM (0.03-0.3 pmol) using a 25-microns-diameter capillary. Both the underivatized and derivatized methods were applied to the determination of glutathione in human erythrocytes. Glutathione concentrations of 2-3 mM were obtained for the erythrocyte samples analyzed, with good agreement between results obtained by the two methods.
The analysis of biological samples (e.g., blood, urine, saliva, tissue homogenates) by capillary electrophoresis (CE) requires efficient sample preparation (i.e., concentration and clean-up) procedures to remove interfering solutes (endogenous/exogenous and/or low-/high-molecular-mass), (in)organic salts and particulate matter. The sample preparation modules can be coupled with CE either off-line (manual), at-line (robotic interface), on-line (coupling via a transfer line) or in-line (complete integration between sample preparation and separation system). Sample preparation systems reported in the literature are based on chromatographic, electrophoretic or membrane-based procedures. The combination of automated sample preparation and CE is especially useful if complex samples have to be analyzed and helps to improve both selectivity and sensitivity. In this review, the different modes of solid-phase (micro-) extraction will be discussed and an overview of the potential of chromatographic, electrophoretic (e.g., isotachophoresis, sample stacking) and membrane-based procedures will be given.
Methods of electromigration in laboratory apparatus of small-bore size have recently undergone development at a remarkably rapid pace, leading to a variety of new analytical techniques. One such technique is called "capillary electrophoresis" (CE), which is further classified on the basis of electromigration mode, viz., "capillary zone electrophoresis" (CZE), which, in turn, has several variations. This review aims to give a short overview of the various electromigration methods for amino compounds by using CE. Firstly, this review briefly summarizes the detection methods employed for detection of monoamines and polyamines by CE for both native and derivative forms. Next, current CE methods are described, and their applications to detection of amino acids, biogenic amines, aromatic amines, including heteroaromatic amines and their enantiomers, are introduced from representative papers. Finally, new methods for single-cell analysis and microchip CE techniques are focused on.
When working with capillary zone electrophoresis (CZE), the analyst has to be aware that the separation system is not homogeneous anymore as soon as a sample is brought into the background electrolyte (BGE). Upon injection, the analyte creates a disturbance in the concentration of the BGE, and the system retains a kind of memory for this inhomogeneity, which is propagated with time and leads to so-called system zones (or system eigenzones) migrating in an electric field with a certain eigenmobility. If recordable by the detector, they appear in the electropherogram as system peaks (or system eigenpeaks). However, although their appearance can not be forecasted and explained easily, they are inherent for the separation system. The progress in the theory of electromigration (accompanied by development of computer software) allows to treat the phenomenon of system zones and system peaks now also in very complex BGE systems, consisting of several multivalent weak electrolytes, and at all pH ranges. It also allows to predict the existence of BGEs having no stationary injection zone (or water zone, EO zone, gap, dip). Our paper reviews the theoretical background of the origin of the system zones (system peaks, system eigenpeaks), discusses the validity of the Kohlrausch regulating function, and gives practical hints for preparing BGEs with good separation ability not deteriorated by the occurrence of system peaks and by excessive peak-broadening.
Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD-CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.
This paper provides an overview on the current status of the analysis of biogenic amines by CE. The basic CE separation and detection strategies for the analysis of biogenic amines are briefly described. CZE and MEKC that provide highly efficient and reproducible analysis of biogenic amines are particularly surveyed. With respect to the detection of biogenic amines, we focus on LIF, UV-visible absorption, electrochemiluminescence, and MS. Derivatization strategies, indirect methods, and on-line concentration techniques such as field-amplified sample stacking, sweeping, and use of polymer solution are described. To show the practicality of CE, we highlight currently developed techniques for the determinations of biogenic amines in biological samples, including foods, beverages, cerebrospinal fluids, urine, and single cells.
An in-capillary sample preconcentration strategy based on solid phase extraction (SPE) technology coupled with capillary electrophoresis (CE) has been developed taking advantage of both techniques (SPE and CE). An in-line frit-free preconcentration device for capillary electrophoresis containing MCX beads, obtained from the corresponding Waters OASIS cartridges, was prepared. The retention of the particles was based on the relative diameters of the particles, carefully selected, and the capillaries. An experimental preconcentration factor of 100 was found for the system. Conditions were optimised for 3-nitrotyrosine measurement in rat urine being 4.4 microM spiked in the urine the lowest value detectable.
CE suffers from an inherent low concentration sensitivity. Analyte detection limits can be improved by combining CE with SPE. This paper presents an overview of coupled SPE-CE systems that have been reported in literature. Attention is paid to fundamental aspects of coupling SPE and CE, as well as to important SPE requirements. Interfaces for inline and online coupling with CE are critically discussed, and their mode of operation is outlined. Advantages and limitations of the interfaces are discussed and typical examples are selected. Finally, some future developments are discussed.
Sorbent preconcentration offers good strategies to overcome the poor detection limits of capillary electrophoresis (CE). The present review focuses on the recent trends of the coupling between sorbent preconcentration techniques, namely solid-phase extraction (SPE) and solid-phase microextraction (SPME), to capillary electrophoresis (CE). Special attention is given to their environmental and biological application. We also discuss the most important advantages and disadvantages of the different methodologies and briefly outline the new trends of the coupling between sorbent preconcentration and CE.
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