Recent progress in carbohydrate separation by high-performance liquid chromatography based on hydrophilic interaction

This article surveys recent developments in the separation and analysis of carbohydrates by high-performance liquid chromatography, in adsorption or partition modes, on polar sorbents with less polar eluents, a technique that is now termed hydrophilic interaction chromatography. A variety of chromatographic methods are included under this generic heading, the most important being adsorption chromatography on silica and partition chromatography on silica-based sorbents bearing bonded polar phases. Examples are given of the applications of these stationary phases, as well as the newer polymer-based polar sorbents, in high-performance liquid chromatography of carbohydrates and their derivatives.     

Sensitive determination of carbohydrates labelled with p-nitroaniline by capillary electrophoresis with photometric detection using a 406 nm light-emitting diode

p-Nitroaniline was explored as a derivatising reagent for UV absorbance detection of carbohydrates after separation by CE. This derivatising agent has three advantages: first, it has excellent water solubility; second, it has high molar absorptivity; and third, it is possible to obtain sensitive detection using a UV or blue light-emitting diode (LED) as the light source. The labelling reaction took less than 30 min to complete with high reaction yield. The separation process was modelled and optimised using an artificial neural network. Nine carbohydrates were separated by a CE system within 16 min using a 0.17 M boric acid buffer at pH 9.7. On-column LED detection at 406 nm allowed the detection of carbohydrates with good detection limits (<1.1 microM or 8.8 fmol) and reproducible quantification in the concentration range of 2.6-200 microM. This method was applied successfully to the determination of component carbohydrates in some food samples.     

Analysis of carbohydrates by anion exchange chromatography and mass spectrometry

A versatile liquid chromatographic platform has been developed for analysing underivatized carbohydrates using high performance anion exchange chromatography (HPAEC) followed by an inert PEEK splitter that splits the effluent to the integrated pulsed amperometric detector (IPAD) and to an on-line single quadrupole mass spectrometer (MS). Common eluents for HPAEC such as sodium hydroxide and sodium acetate are beneficial for the amperometric detection but not compatible with electrospray ionisation (ESI). Therefore a membrane-desalting device was installed after the splitter and prior to the ESI interface converting sodium hydroxide into water and sodium acetate into acetic acid. To enhance the sensitivity for the MS detection, 0.5 mmol/l lithium chloride was added after the membrane desalter to form lithium adducts of the carbohydrates. To compare sensitivity of IPAD and MS detection glucose, fructose, and sucrose were used as analytes. A calibration with external standards from 2.5 to 1000 pmole was performed showing a linear range over three orders of magnitude. Minimum detection limits (MDL) with IPAD were determined at 5 pmole levels for glucose to be 0.12 pmole, fructose 0.22 pmole and sucrose 0.11 pmole. With MS detection in the selected ion mode (SIM) the lithium adducts of the carbohydrates were detected obtaining MDL's for glucose of 1.49 pmole, fructose 1.19 pmole, and sucrose 0.36 pmole showing that under these conditions IPAD is 3-10 times more sensitive for those carbohydrates. The applicability of the method was demonstrated analysing carbohydrates in real world samples such as chicory inulin where polyfructans up to a molecular mass of 7000 g/mol were detected as quadrupoly charged lithium adducts. Furthermore mono-, di-, tri-, and oligosaccharides were detected in chicory coffee, honey and beer samples.     

Design and applications of coupled SPE-CE

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.     

Derivatization Reaction of Carbohydrates with Urea as the Reagent and Fluorimetric Determination of Carbohydrates

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Electroanalytical Methods for the Determination of Pharmaceuticals: A Review of Recent Trends and Developments

Electroanalysis is a powerful analytical technique that is increasing in utility in the pharmaceutical industry. It is used as an alternative or complementary technique to spectrophotometric and separation techniques due to its high sensitivity, speed of analysis, reduction in solvent and sample consumption, and low operating cost compared to other analytical methods. A review of the principles and application of modern electroanalytical techniques, namely, cyclic, linear sweep, differential pulse, square wave and stripping voltammetric techniques, is presented. This review gives recent pharmaceutical analysis applications used for each mode of electroanalytical chemistry. The review will also describe recent developments for enhancing concentration limits of detection, electrode types, and so forth. Selected studies on these subjects are given as examples.     

Analysis of inorganic and small organic ions by CE with amperometric detection

Capillary electrophoresis has become a widely useful analytical technology. Amperometric detection is extensively employed in capillary electrophoresis for its many inherent virtues, such as rapid response, remarkable sensitivity, and low cost of both detectors and instrumentations. Analysis of inorganic and small organic ions by capillary electrophoresis is an important research field. This review focuses on the recent developments of capillary electrophoresis coupled with amperometric detection for analysis of inorganic and small organic ions. Advancements in electrophoresis separation modes, amperometric detection modes, working electrodes, and applications of inorganic ions, amino acids, phenols, and amines are discussed.     

HPAE-PAD – a sensitive method for the determination of carbohydrates

High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was used for the determination of eleven monosaccharides. Three analytical columns with different selectivities were tested, and the resulting separations were compared calculating precision and detection limits. The monosaccharides could be separated on CarboPAC PA10 in one analysis run with the lowest detection limits and a high precision. For the determination of polysaccharides and humic bound carbohydrates in natural organic matter, an hydrolysis step had to be carried out. With the exception of fructose, the recoveries varied between 56% and 83%. The described methods were applied for the determination of bound carbohydrates in a bog lake water and a soil extract without preconcentrating the samples.     

Sensitive, indirect photometric detector for high-performance liquid chromatography using a light-emitting diode

A low-noise detector for indirect photometric detection has been constructed using a highly stable source--a light-emitting diode (LED). Use of the detector is demonstrated for reversed-phase liquid chromatography by adding methylene blue to the mobile phase to make a background signal. The indirect determination of alcohols by their effect on methylene blue concentration distribution is demonstrated, and an investigation is made into the conditions for high sensitivity. Because the source exhibits low noise, the detection limits for alcohols are as low as more complex and expensive detection methods, despite the lower radiant power of the LED. Detection limits for nine alcohols are below micrograms injected amounts.     

The determination of reducing carbohydrates using a cation-exchange column and potentiometric detection with a metallic copper electrode

Summary A metallic copper electrode is employed as a potentiometric detector for reducing carbohydrates after their separation on a cation-exchange column. A post-column reactant solution comprising 1 mM copper (II) and 35 mM ammonia is mixed with the column effluent and the metallic copper electrode provides a steady baseline potential by responding in a Nernstian manner to the level of copper ions present. This in turn is affected by the presence of eluted reducing carbohydrates, leading to an indirect detection method for these species. The method is applied to the detection of maltose, lactose, xylose, glucose, sorbose, fructose and arabinose. The electrode response mechanism is discussed and detection limits in the low nanomole range are reported. The potentiometric detector is shown to be more sensitive than refractive index detection.     

Drug detection in hair by chromatographic procedures.

This article reviews the analysis of 31 drugs and drug metabolites in human hair by thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry and mass spectrometry. The most important detection method after chromatographic separation of the components is the mass spectrometry because of its sensitivity and specificity. Washing steps to exclude external contamination, extraction, derivatization, stationary phases, detection modes and detection limits of the mass spectrometric and gas chromatographic-mass spectrometric procedures are presented in five tables. Additionally, a method for a gas chromatographic-mass spectrometric screening procedure is presented.     

Theoretical investigation of low detection sensitivity for underivatized carbohydrates in ESI and MALDI

Electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) mainly generate protonated ions from peptides and proteins but sodiated (or potassiated) ions from carbohydrates. The ion intensities of sodiated (or potassiated) carbohydrates generated by ESI and MALDI are generally lower than those of protonated peptides and proteins. Ab initio calculations and transition state theory were used to investigate the reasons for the low detection sensitivity for underivatized carbohydrates. We used glucose and cellobiose as examples and showed that the low detection sensitivity is partly attributable to the following factors. First, glucose exhibits a low proton affinity. Most protons generated by ESI or MALDI attach to water clusters and matrix molecules. Second, protonated glucose and cellobiose can easily undergo dehydration reactions. Third, the sodiation affinities of glucose and cellobiose are small. Some sodiated glucose and cellobiose dissociate into the sodium cations and neutral carbohydrates during ESI or MALDI process. The increase of detection sensitivity of carbohydrates in mass spectrometry by various methods can be rationalized according to these factors. Copyright © 2016 John Wiley & Sons, Ltd.     

Ultra sensitive trace gas detection for biomedical applications

We present an overview of our recent progress on spectroscopic trace gas detection for biomedical applications. The latest developments of cavity-enhanced spectroscopy as well as magnetic rotation spectroscopy lead to unprecedented sensitivity and specificity. The current detection limits of our laser spectroscopic approaches are in the picomolar to nanomolar range, depending on the molecular compound. The time resolution of the measurements is down to the sub-second range. This very high sensitivity and time resolution open up exciting perspectives for novel analytical tasks in biomedical research and clinical diagnosis.     

Analysis of glycosaminoglycan monosaccharides by capillary electrophoresis using indirect laser-induced fluorescence detection

Two methods for monosaccharide analysis by capillary electrophoresis (CE) using counterelectroosmotic and coelectroosmotic modes with indirect laser-induced fluorescence detection were optimised and compared. A mixture of seven glycosaminoglycan-derived hexoses was separated in alkaline fluorescein-based electrolytes and detected in both counterelectroosmotic and coelectroosmotic conditions. The fluorescein concentration and pH of the background electrolyte, and the influence of the reversal of electroosmotic flow by addition of hexadimethrine bromide on the separation were studied. Coelectroosmotic CE conditions provided better resolution and limits of detection. A 10(-6) M fluorescein solution at pH 12.25 containing 0.0005% (w/v) hexadimethrine bromide was used as background electrolyte. Quality parameters such as run-to-run, day-to-day precision and limits of detection were calculated, and better figures of merit were obtained for the coelectrooosmotic conditions than for the counterelectroosmotic mode. The coelectroosmotic method was applied to the quantitation of the hexosamine contents in glycosaminoglycans after acid hydrolysis. The method proved to be suitable for the determination of dermatan sulfate in heparin down to 2% (w/w).     

Comparison of Pulsed Electrochemical Detection Modes Coupled with Microchip Capillary Electrophoresis

There is a need to develop analytical methods that are capable of rapidly measuring small biological markers in the field of metabolomics. Among others, carbohydrates play an important role biologically yet are traditionally hard to detect since they have no chromophore or fluorophore. In the present report, the first application of integrated pulsed amperometric detection (iPAD) coupled with microchip electrophoresis to the analysis of glucose, mannose, sucrose, maltose, glucosamine, lactose, maltotriose and galactose is demonstrated. iPAD is an electrochemical detection mode that can be used for direct detection of carbohydrates, amines and sulfur containing compounds. The effect of different solution parameters, including the buffer concentration, pH and the concentration of SDS on both separation and detection response was analyzed. In addition, a comparison study between PAD and iPAD was performed using glucose, glucosamine, sucrose and maltose as model carbohydrates.     

Recent developments in capillary electrophoresis and capillary electrochromatography of carbohydrate species

This review article is concerned with the recent developments in capillary electrophoresis (CE) and capillary electrochromatography (CEC) of carbohydrates. The literature shows that CE possesses impressive potential in the analysis of carbohydrates. On the other hand, CEC has just started to show promise in the analysis of carbohydrates. Advances in separation and detection approaches of derivatized and underivatized carbohydrates are discussed based on the available literature. In addition, important applications are illustrated.     

Persistent Organic Pollutants – Are Our Methods Sensitive and Selective Enough?

Due to the exponential growth of the world population and a strong worldwide economic development, humans are increasingly exposed to chemicals. This is particularly true for persistent, toxic chemicals that accumulate in organisms including humans. The presence of these persistent organic pollutants (POPs) has been reported since the 1960s. Analytical methods developed for the determination of POPs in wildlife, food, feed, sediments, dust, air, and humans have seen a tremendous improvement in sensitivity and selectivity. Detection limits of 0.1 mg/kg for pesticides in the 1960s have decreased down to < 0.1 ng/kg for dioxins presently. On the other hand, due to new toxicological insights, authorities nowadays often demand much lower detection limits than in the early days of discovery. This paper compares the lowest effectual levels of a number of POPs with the analytical capabilities of modern laboratories in terms of sensitivity and selectivity. Methods discussed are gas chromatography (GC), including multi-dimensional GC and liquid chromatography (LC), combined with electron capture detection (for GC) or with different mass spectrometric detectors. The conclusion is that the still improving sensitivity of current methods is sufficient to allow detection of POPs at a level clearly below the lowest observable adverse effect levels. Selectivity has also improved, but given the high complexity of some of the POPs and the multitude of POPs and other chemicals to which we are exposed, further developments in selectivity are still badly needed. To limit and focus the information for regulators, an effect-directed analysis is proposed as an alternative approach.     

Phosphorimetric Detection in HPLC VIA Trivalent Lanthanides: High Sensitivity Time-Resolved Luminescence Detection of Tetracyclines Using Eu3+ in a Micellar Post Column Reagent

Abstract

The detection of tetracyclines in HPLC by sensitized Europium phosphorescence has been reinvestigated using a dedicated LC detector with time-resolved luminescence capability. A micellar Eu³⁺ post column reagent was developed which contained 1% Triton X-100 and 200uM tri-n-octyl phosphine oxide buffered at pH 10.5. This reagent provided optimized time-resolved detection and was found to be compatible with a several mobile phases used in reversed phase chromatography of tetracyclines. The sensitivity and quantitative linearity of the technique was determined using standard fluorometric and time-resolved modes of detection with both grating and filter emission monochromation. Peak areas were linear with concentration from 2 ug on column to the limit of detection. Subnanogram limits of detection were obtained for both tetracycline and oxytetracycline. The relative sensitivity of the technique for compounds tested was oxytetracycline=tetracycline > doxycycline > chlortetracycline > minocycline. The high selectivity of this method promises to be useful in     

Carbohydrate and alditol analysis by high-performance anion-exchange chromatography coupled with electrochemical detection at a cobalt-modified electrode

A cobalt oxyhydroxide film dispersed on a carbon electrode surface was characterized and proposed as an amperometric sensor for determination of alditols and carbohydrates in flowing streams. Complex mixtures of carbohydrates were separated by anion-exchange chromatography using a moderately alkaline solution as mobile phase. The cobalt modified electrode (GC-Co) was employed under a constant applied potential of 0.5 V (vs Ag/AgCl). Under these experimental conditions the detection limits (S/N=3) for all analyzed electroactive molecules ranged between 0.3 micromol L(-1) and 1.5 micromol L(-1) and the dynamic linear ranges spanned generally three orders of magnitude above the relevant detection limits. Analytical determinations of carbohydrates and alditols in red and white wines, are reported.     

INAA with gamma-gamma coincidence for selenium determination in food

Selenium detection limits of INAA are normally above its concentration in most biological materials. Gamma-gamma coincidence methodology can be used to improve the detection limits and uncertainties in the determination of selenium. Here, some edible parts of plants were measured using a HPGe detector equipped with a NaI(Tl) active shielding, producing spectra both in normal and coincidence modes. The results presented the reduction of the detection limits of selenium by a factor of 2 to 3 times and improvement in the uncertainty of up to 2 times.