Current Analytical Chemistry - Volume 8, Issue 4, 2012

Reactive oxygen species (ROS) are very unstable molecules generated during the metabolism. They can be produced in excess, contributing to cellular dysfunctions. Antioxidants are substances that play an important role as a health-protecting factor, limiting the lesions caused by ROS. Two kinds of electrochemical biosensors based on ROS have been described to evaluate the antioxidant capacity in the food industry. The first one involves hydroxyl radicals and studies their effect on DNA alterations. The second one consists of the superoxide radicals scavenging ability, radicals being essentially generated by the xanthine/xanthine oxidase system. These sensors are commonly based on either cytochrome c or superoxide dismutase, even though recent strategies are emerging. Whatever the involved ROS, all the described biosensors possess similar advantages such as simplicity, rapidity and low cost and are successfully applied for the assessment of antioxidant properties in various foods, additives or beverages.

Phenolic compounds are important components of many fruits, vegetables and beverages, contributing to their flavor, color and sensory properties. Besides, they are associated with prevention of diseases thought to be induced by oxidative stress, such as cardiovascular diseases, cancer and inflammation. Due to their bioactive functions, phenolic compounds have received particular attention in recent years. Amongst the different analytical methods reported to determine the polyphenol content and composition of food and beverages, liquid chromatography (LC) is the preferred option compared to gas chromatography (GC) since no derivatization is needed. UV detectors have been commonly used for the determination of phenolic compounds, but they lack of sensitivity and/or specificity. LC coupled with electrochemical detection (ECD) or coupled to mass spectrometry (MS) detectors are the most sensitive, selective and reproducible methods for quantifying phenolic compounds. This manuscript reviews the main applications of LC coupled to ECD or MS for the analysis of phenolic compounds in food and beverages.

The introduction of instrumentation based on microfluidic technologies has revolutionised many areas of life sciences, including genomics and proteomics amongst others. As the role of reactive oxygen species has more recently been linked to an important number of pathologies, such as cancer or cardiovascular disease, this particular field of study has not been the exception in adopting these new technologies. Added interest has been generated thanks to the high compatibility of reactive oxygen species detection techniques in microfluidic devices and previously established ones e.g. fluorescence or electrochemistry. Moreover, innovation in the field has generated devices capable of detecting the state denominated oxidative stress (in vivo), produced by an unbalance between the production of reactive oxygen species and intrinsic antioxidant mechanisms, as well as assessing the capacity of other endogenous molecules to counter the harmful effects of these species. This review has focused on how miniaturisation enabled by microtechnologies and the understanding of microfluidics has offered researchers a unique insight into the cellular processes involving the production of reactive oxygen species in the cellular metabolism, as well as providing a tool for antioxidant capacity screening of compounds.

Polyphenolic compounds, which are widely distributed in plant-derived foods, recently attracted much attention because of their possible health benefits arising from their antioxidant activity, such as free radical scavengers and inhibition of lipoprotein oxidation. The detection of phenolic substances in food samples has been performed for many methods among them electrochemical sensors and biosensors approaches. Polyphenolic compounds are good substrates for oxidases enzymes, then biosensors modified with tyrosinase, laccase and peroxidase have been developed for detection of phenolic compounds since phenols can act as electron donors for these enzymes. Furthermore, as polyphenols are electroanalytically active compounds that can be easily oxidized at inert electrodes, electrochemical sensors have also been used as tools for estimating the total phenolic content (TPC). This paper critically reviews both electrochemical sensors and biosensors developed for the evaluation of polyphenolic compounds in foods and beverages. Due to the ability of these devices to perform simple, fast and reliable analysis, they are promising tools for the assessment of antioxidant properties.

Vascular generation of nitric oxide (NO) plays an important role in the regulation of blood flow, and is counterbalanced by the formation of radical oxygen species (ROS). Thus, an imbalance in vascular NO and ROS production contributes to endothelial dysfunction, which is associated with cardiovascular disease. Here we review the main and commonly used methods that have been applied to determine vascular NO and ROS. Only NO microsensors, electron spin resonance (ESR), and NO sensitive fluorescent probes allow real time measurement of NO levels in cell suspensions, cell cultures, and isolated vascular segments in vitro, and of these techniques only NO microsensors have so far been developed for real time detection of NO in vivo. ROS formation has been detected in cell cultures by lucigeninenhanced fluorescence, fluorescent probes e.g. 2',7'-dichlorodihydrofluorescein and dihydroethidium, by use of ESR, and also by electrochemical detection. The limitations of the electrochemical microsensors include interference from other substances, physical force on the sensors, and influence of temperature and changes in pH that may lead to artifactual changes in current. Therefore, specificity testing and calibration are pivotal, and in case of ROS, application of more than one technique is recommendable. Technical advances have allowed simultaneous detection of both NO and superoxide anion by use of electrochemical microsensors. Miniaturizing the sensors may also allow incorporation of those in lab-ona- chip and may lead to real time measurements of NO and ROS in the coronary circulation in situ, and hence provide direct evaluation of endothelial and vascular function in cardiovascular disease.

Recent advances in nanotechnology provide exciting opportunities for the development of novel and improved technologies for the detection of relevant molecules in food. Nanoparticle based analytical technologies are rapidly emerging at the intersection between analytical chemistry, nanotechnology and the food industry. This review discusses recent advances in the use of nanoparticles and nanostructures for developing analytical assays for the detection of food antioxidants. The assembly of nanoparticles in functional analytical devices, the detection principle of these assays in relation to the physicochemical properties of these nanoparticles and the analytical performance characteristics are reviewed. Selected examples from literature, which illustrate the state-of-the-art and practical applications, as well as hints at future research directions, are discussed.

Different degrees of glycosaminoglycan sulfation result in their different charge densities. The charge density differences impact their migration behavior in polyacrylamide gel electrophoresis and size exclusion chromatography, two of the most common methods for determining relative molecular masses of polysaccharides. In this study, we investigated the feasibility of using commercially available heparin oligosaccharides as calibrants for measuring the relative molecular masses of intermediates in a bioengineered heparin process that have different levels of sulfation. A size exclusion chromatography method was established that eliminates this charge density effect and allows the determination of relative molecular mass using a single calibration curve with heparin oligosaccharides calibrants. This is accomplished by overcoming the electrostatic interaction between the glycosaminoglycans and size exclusion chromatography stationary phase using high ionic strength mobile phase.

This work describes a simple and sensitive method for the concurrent determination of sumatriptan (SUM) and naproxen (NAP) in dosage forms and human plasma using LC/MS and internal standardization. The drugs were separated isocratically on a C18 column using a binary mobile phase composed of water and acetonitrile at 0.05 % acetic acid. The method was validated over a linearity range of 10-900 ng/mL and 0.1-10 μg/mL for SUM and NAP, respectively. The LOQ for SUM and NAP were 10 and 100 ng/mL, respectively. The proposed method was successfully used for the determination of SUM and NAP in plasma after a double liquid liquid extraction (LLE). The assay was successfully applied to the determination of both drugs in pharmaceutical dosage forms without interference from tablet excipients. Results obtained by the proposed method were statistically compared to those of a reported method, and no significant difference was observed.

This work describes an electroanalytical method for simultaneous determination of cadmium (II) and lead (II) ions using a carbon paste modified with carbon nanotubes and an ion exchange synthetic resin (Amberlite IR120) electrode under Differential Pulse Anodic stripping voltammetric (DPASV) conditions. The procedure is based on the effective preconcentration of metal ions on the electrode surface containing ion exchange resin under open circuit condition. Using optimized conditions of 600 s preconcentration time for an electrode composition of 5% w/w Amberlite IR 120 and 30 % w/w carbon nanotubes in the paste, a scan rate of 10 mVs-1, and a potential pulse amplitude of 75 mV, linear calibration graphs can be obtained by were monitoring the peaks at -0.75 V for cadmium (II) and -0.58 V for lead (II) from 0.07 - 2.50 μmol L-1 and 0.01 - 5.80 μmol L-1, respectively. The proposed method was applied for the determination of lead (II) and cadmium (II) in ethanol fuel, gasoline for aviation, and spiked water. For all samples analyzed, recoveries ranged from 95.8 to 106.3%. Interferences were also evaluated.

Gemifloxacin (GMF) is a broad-spectrum quinolone antibacterial agent used in the treatment of acute bacterial exacerbation of chronic bronchitis and mild-to-moderate pneumonia. The investigation of the GMF - double-stranded DNA (dsDNA) interaction using an electrochemical DNA-biosensor, showed for the first time clear evidence of interaction with dsDNA. The interaction between GMF and dsDNA onto pencil graphite electrode (PGE) was assessed at pH 4.80 acetate buffer using differential pulse voltammetry (DPV). The interaction mechanism was disclosed with the decrease of the guanine oxidation peak current. The changes in the experimental parameters such as the accumulation time and the concentration of GMF were studied. Linearity of the guanine oxidation signals was obtained in response to the range of 0.5 - 8.0 μg/mL GMF. Detection and quantification limits in the range of μg/mL were determined.

An electrochemical biosensor was developed based on formaldehyde dehydrogenase immobilized with Nafion membrane for determination of formaldehyde in fish. The enzyme was immobilized through the entrapment technique and measured based on the reduction of β-nicotinamide adenine dinucleotide. The response time of the formaldehyde biosensor was <1 min, with an optimum pH of 8. The optimum enzyme loading and NAD+ concentrations were found at 30 mg/mL and 0.5 mM, respectively. Using the formaldehyde biosensor, a linear response of formaldehyde showed a range of 0.1 to 10 ppm and a detection limit of 0.016 ppm. In application of Nash method, the samples were stored at 4°C ± 1 for 10 days. With the two combined methods, a linear correlation coefficient with R2 = 0.9982 (y = 0.956x - 0.014) was found. The developed formaldehyde biosensor showed a good reproducibility, long storage stability (more than 6 months stored at 4°C), and also effective monitoring of formaldehyde level in Indian mackerel (Rastrelliger kanagurta) fish.

A fast and effective microextraction technique is proposed for the determination of trace amounts of Cr(III) in water samples, using ultrasound-assisted emulsification-microextraction (USAEME) followed by flame atomic absorption spectrometry (FAAS). In the proposed approach, sodium diethyldithiocarbamate trihydrate solution (NaDDTC.3H2O) was used as a chelating agent and chloroform was selected as extraction solvent. After determination of the most suitable solvent and extraction time, some effective parameters such as an extraction solvent volume, temperature, and pH were investigated and optimized. The optimized USAEME procedure used 100 μL of chloroform, 20 min of extraction with no ionic strength adjustment at 50 °C and 5 min of centrifugation at 4000 rpm. The method was applied to the analysis of tap, well, dam on industrial waste water samples and the Trace Metals in Drinking Water standards CRM-TMDV. The detection limit for Cr(III) was 0.079 μg L-1with an enrichment factor of 95, and the relative standard deviation was 2.8-1.1% (n=8, c=500 μg L-1). The proposed USAEME procedure has been demonstrated to be viable, simple, rapid and easy to use for the residue analysis separation of Cr(III).

The retention behavior of homo-oligonucleotides was investigated in ion-pair reverse-phase high-performance liquid chromatography (IP-RPLC). Rectification of retention time was performed by introducing the concept of “standard column” with double-point retention time correction (DP-RTC) using two anchor oligonucleotides as internal standards to ensure the accuracy of the retention measurement. Through polynomial fitting, a quadric relationship was adopted between normalized retention time and chain length of (dA)10-21 and (dT)10-21. The retention times of homo-oligonucleotides depended on the number of bases and increased with increasing hydrophobicity, namely, A

Silver doped LiFePO4 powder was synthesized using the hydrothermal method, and was used as a sensing material. An Optical WaveGuide (OWG) sensor which reacted to Volatile Organic Compound (VOC) was developed by spin-coating a thin film of LiFe0.99Ag0.01PO4 onto a Tin-diffused glass optical waveguide. This OWG sensor was used to detect formaldehyde gas as a typical example of VOCs. The experimental result showed that the sensor easily and repeatedly detected 100ppt of formaldehyde, with a short response time (less than 2 s). At the low concentration (below 10ppm), the volatile organic compounds caused no interference with the detection of formaldehyde vapor. The sensor also had the characteristics of high sensitivity, a simple structure, and easy fabrication.

Molecularly imprinted polymers were synthesized to screen suitable functional monomers for the detection of organochlorine pesticides, heptachlor and DDT. Three functional monomers i.e., methacrylic acid (MAA), 4-vinyl pyridine (4VP) and styrene were investigated. Rebinding experiments show that while MAA is best for the detection of heptachlor, 4-vinyl pyridine suits well for DDT. The experimental findings are corroborated through density functional theory calculations. Molecular complexes of the monomer-pesticide were investigated for their binding energies. Computed binding energies agreed well with the experimentally obtained imprinting factors. The computed structures have been examined for the nature of interactions which stabilize the complex. Electrostatic interactions, studied through potential maps are able to explain the observed stabilization of the complexes.

This paper describes the development and validation of an isocratic LC-UV method for the assay of sitagliptin phosphate in tablets, a drug recently approved for the treatment of type 2 diabetes mellitus (DM). The method employs a Phenomenex® C18 column (150mm x 4.6mm I.D., 5μm) with 0.025 M phosphate buffer pH 6.8: acetonitrile (60:40, v/v) as mobile phase, at a 0.8 mL min-1 flow rate and UV detection at 267 nm. The chromatographic separation was obtained within a retention time of 3.7 min. The method was linear (r= 0.9999) in the range of 25-75 μg.mL-1. The method's specificity and stability-indicating capability were proved through force degradation studies, being the peak purity evaluated by PDA detector. Sitagliptin phosphate was exposed to oxidative, hydrolytic and photolytic stress conditions, and no interference from degradation products was observed. The method has shown good and consistent recoveries (mean value 100.0%) with low intra and inter-day relative standard deviation (RSD). In robustness study, it was observed that peak area was unaffected by small changes in critical factors. The validated method can be successfully applied to determine sitagliptin in tablets and in stability studies.

Reversed zirconia-based stationary phases belong among interesting alternatives of widely used RP silica phases. Zirconia displays specific surface properties, which are responsible for a unique retention mechanism. Furthemore, it is necessary especially for acidic analytes to add a strong Lewis base to the mobile phase mainly to improve the separation efficiency. However, commonly recommended strong Lewis base buffers are not volatile and therefore are incompatible with a mass spectrometric detection. A novel RP zirconia-based stationary phase with deactivated Lewis acid sites of zirconia was introduced to enable the use of volatile additives (acetate, formate) despite their low Lewis basicity. It is reported on the influence of surface deactivation on chromatographic behavior of acidic, basic and neutral analytes in this paper. The influence of variables such as pH levels, Lewis base type, its concentration and temperature were studied. The results were compared with the undeactivated stationary phase. In general, around 2.5-fold stronger retention of compounds was observed on the deactivated phase. Moreover, some variances in retention and selectivity were observed for both acidic and basic analytes when using different Lewis bases. For these reasons the properties of deactivated and unmodified phases under the same conditions were partly diverse and consequently the selectivity was similar but not identical.