Current Analytical Chemistry - Volume 9, Issue 1, 2013

Whilst a multitude of techniques have been employed to study the biology of tumour tissue and its response to chemotherapeutic reagents, most current methodologies do not capture the sophistication of the in vivo environment. Microfluidics however offers the ability to maintain and interrogate primary tissue samples in an environment with biomimetic flow characteristics. In this study head and neck squamous cell carcinoma (HNSCC) tumour biopsies have been used to investigate the performance of a microfluidic device for generating clinically-useful information. The response of fresh and cryogenically-frozen primary HNSCC or metastatic lymph node samples to chemotherapy drugs (cisplatin, 5-flurouracil or docetaxel), alone and in combination, were monitored for both proliferation (water-soluble tetrazolium salt metabolism) and cell death biomarker release (lactate dehydrogenase, LDH) “off-chip”. The frozen tissue showed no significant difference in terms of either proliferation or LDH release in comparison with the matched fresh samples. Administration of all drugs caused cell death, in a dose-response manner, with the combination showing the greatest amount of cytotoxicity particularly at days 8 and 9; correlating well with published clinical data. The system described here offers an innovative method for studying the tumour microenvironment in vitro and, through incorporation of relevant analytical modules, provides the basis of a pre-clinical device that can be used to define personalised treatment regimens.

Besides the internal genetic content and the chemical exchanges with external tissue environment, the physical interactions of cancer cells with its microenvironment also provide the guidelines for successful oncogenesis. During its metastatic journey from primary tumor to distant locations of body, a cancer cell is exposed to stresses of diverse origin and kind, of which fluid shear stresses of interstitial and haematogenic nature have been least studied in relation to their impact in cancer progression. Here, we have designed a biomicrofluidic system integrated with microfabrication compatible traction force microscopy facility. It is further compatible with high-resolution visualization by laser scanning confocal microscope. This system enables us to study the flow induced changes in plasma membrane of a representative cancer cell line HeLa as the function of imparted shear stress and importantly, cell-substrate adhesion strength. Using the device, we are able to reveal novel shear induced immediate and late changes in the fluidity of apical plasma membrane. We have shown while transient changes in membrane fluidity depend on the orientation of flow and are largely independent of cellsubstrate adhesion landscape, long term membrane fluidization is controlled by the cell-substrate adhesion strength that is represented by maximum traction stress that cell exerts on the microchannel wall. It is then probed that membrane fluidization probably occurs due to the endocytosis of lipid raft domains. The conclusions drawn from the work, in this respect, are anticipated to provide new directions in cancer research.

Tissue engineered bone grafts are among the most promising approaches to heal large bone defects. An in vitro culture phase prior to transplantation provides the opportunity to optimize the graft properties. In our study we developed computational models for the investigation of the deformation, perfusion and revascularization of a porous β-tricalcium phosphate (β-TCP) scaffold seeded with human bone marrow derived mesenchymal stem cells (MSCs). The deformation model allows predicting the resulting forces in the β-TCP scaffold due to different external loadings. In addition to pressure related forces, fluid convection within the scaffold also exerts shear stress onto the cells. However, perfusion is necessary for the supply of the cells with nutrients such as glucose. To find an optimal ratio between shear stress and nutrients supply, we develop fluidic models for the β-TCP scaffold. Furthermore to the scaffold specifications, also the cellular glucose consumption of osteogenic differentiated and undifferentiated MSCs was integrated in the computational model. Beyond the in vitro culture phase, bone grafts have to be supplied by the host’s vascular system. Therefore angiogenesis has to be induced, e.g. by preloading the graft with pro angiogenic factors such as VEGF-A. A stochastic model, based on the Fokker-Planck equation was developed to investigate the impact of a given cytokine gradient onto the endothelial cell migration. The stochastic model was parameterized by data derived from live cell imaging studies.

Surface engineering provides a powerful tool to create model systems to investigate the complex interactions between cells and the extracellular matrix (ECM). Cell adhesive peptide ligands (e.g. Arginine-Glycine-Aspatate (RGD) sequence) and carbohydrates are abundant in the ECM, and both are involved in mediating cell adhesion. However, how these interact is not well understood in vivo. Model systems to investigate their collective roles are rare. We have developed a robust surface patterning method allowing spatial control of carbohydrate (i.e. mannose) and RGD on glass while overcoming non-specific biomolecular interactions. The method effectively combines a traditional photolithographic process with two robust chemical pathways, namely click chemistry and silanization, in order to tailor the surface chemistry. This method is especially promising for the quantitative immobilization of different molecules and holds potential for the investigation of interactions between carbohydrates and common ECM proteins on cell adhesion.

The development of drug delivery systems in experimental therapy usually requires in vitro release models, that should posses specific characteristics including: low cost, simple procedure, high reproducibility and very importantly resemble as strictly as possible the in vivo behaviour. In this respect, the paper describes the effects of the use of different experimental procedures on the drug release profiles from controlled delivery formulations based on nano and micro systems. As examples of micro and nanosystems, microparticles constituted of poly-lactide-co-glycolide (PEM) or gelatine (GEM) and nanostructured lipid carriers (NLC) or cubosomes (CBS) were selected, respectively. All the analysed formulations contained bromocriptine (BC) that represents a poorly water-soluble drug. The influence of the experimental release method and of release media has been investigated using different experimental set-up including direct and reverse dialysis, flow-through cell, USP XXII paddle and Franz cell methods.

The microcirculation plays a key role in the delivery of essential substrates for oxidative processes in cells, the removal of products of cell metabolism, and the regulation of peripheral blood flow distribution. The functional properties of microvascular networks strongly depend on the rheological properties of blood and on the heterogeneity of their architecture. However, studying blood flow behaviour through in vivo microvascular systems is limited by ethical, economical and technical issues. Such limitations have opened the way to in vitro models, such as straight microcapillaries or network- like microchannel constructs, but current in vitro models present simplifications in the architecture design which result in the impossibility of faithfully reproducing key features of the in vivo microvascular haemodynamics. In the present study we report the development of a microfluidic-based in vitro model of the human arteriolar system, characterised by circular channel cross-section, network asymmetry and the presence of both bifurcation- and side-branches. The developed microdevice allows for the quantification of the velocity fields, cell-depletion layer thickness and haematocrit distribution within biomimetic microchannel networks. Results show the potential of our in vitro model in reproducing key features of blood flow behaviour which have been detected for microvascular systems in vivo, including the relationships between cell-depletion layer thickness, haematocrit and vessel diameter. The developed microdevices can find extensive applications in biological and biophysical research, where the mimicking of flow dynamics at the microcirculatory level is required.

This review provides an overview of the emerging trends in analysis of essential oils and indicates future trends. Since early human history the essential oils have been used in folk medicine, food and cosmetic industries in various parts of the world. GC-FID is the traditional method for essential oils quantification while GC-MS is the most common analytical method for qualitative analysis. Chiral GC allowed the identification of a great number of chiral essential oil constituents. An alternative to GC analysis is HPLC chromatography. The use of hyphenated techniques, such as LC-MS-MS provides important information about the structure of essential oils constituents. However, only a comparatively small number of reports on essential oil analyses by HPLC can be found in the literature. Multidimensional chromatography is an approach capable of providing greater resolution.13C-NMR spectroscopy is a complementary tool for analysis of essential oils. Advantage of this method compared to mass spectrometry is identification of stereoisomers and thermally unstable compounds while its disadvantage is inability to identify the minor oils constituents. 1H-NMR as an online tool for GC analysis showed promising results but requires further research to be applied on the analysis of essential oils. The range of information obtained from essential oils analysis enables the application of chemometrics.

The female flowers of hops (Humulus lupulus L.) are used as a flavoring agent in the brewing industry. There is growing interest in possible health benefits of hops, particularly as estrogenic and chemopreventive agents. Among the possible active constituents, most of the attention has focused on prenylated flavonoids, which can chemically be classified as prenylated chalcones and prenylated flavanones. Among chalcones, xanthohumol (XN) and desmethylxanthohumol (DMX) have been the most studied, while among flavanones, 8-prenylnaringenin (8-PN) and 6- prenylnaringenin (6-PN) have received the most attention. Because of the interest in medicinal properties of prenylated flavonoids, there is demand for accurate, reproducible and sensitive analytical methods to quantify these compounds in various matrices. Such methods are needed, for example, for quality control and standardization of hop extracts, measurement of the content of prenylated flavonoids in beer, and to determine pharmacokinetic properties of prenylated flavonoids in animals and humans. This review summarizes currently available analytical methods for quantitative analysis of the major prenylated flavonoids, with an emphasis on the LC-MS and LC-MS-MS methods and their recent applications to biomedical research on hops. This review covers all methods in which prenylated flavonoids have been measured, either as the primary analytes or as a part of a larger group of analytes. The review also discusses methodological issues relating to the quantitative analysis of these compounds regardless of the chosen analytical approach.

This review focuses on modern spectroscopic approaches to the investigations of the incorporation and organization of carotenoids in biological membranes and in experimental models of them. In particular, the application of electronic absorption, emission, Raman, resonance Raman and electron paramagnetic resonance spectroscopies are discussed in the light of the molecular organization of carotenoids in lipid membranes. The perspectives and prospects of exploiting the energy transfer processes involving carotenoids are discussed in this context. The incorporation and intermolecular interactions of carotenoids within lipid membranes depend on two major factors. One is the hydrophobic polyene backbone and the other the oxygen-containing polar side groups. They determine the miscibility of carotenoids with lipids and their tendency to form aggregates, and also affect their localization and orientation in lipid membranes.

This review deals with analytical techniques which are applied for the determination of mycotoxins in biological matrices. Mycotoxins are small (MW < 800) and naturally toxic secondary metabolites produced by a wide range of fungal species. Their common occurrence in food and feed poses a danger to the health of humans and animals. These secondary metabolites can act as mutagenic, carcinogenic, teratogenic, neurotoxic and genotoxic. On the basis of these fungal-related health risks, producers, scientific researchers and main regulatory and inspection authorities in the field of food and feed safety became very interested in establishing sensitive analytical methods for the detection of certain or multiple compound classes of the fugal species (e.g. aflatoxins, ochratoxins and trichothecenes). The aim of this work is to present analytical methods for monitoring mycotoxins including ultra- and high performance liquid chromatography (UPLC/HPLC) coupled with tandem mass spectrometry (MS/MS) compared with high resolution Orbitrap™ technology. Sample pre-treatment methods such as solid phase extraction (SPE), and liquid-liquid-extraction (LLE) are shortly discussed in this review, since they will influence the instrument choice in future experiments. Future prospects and current challenges in performing mycotoxins analysis are also outlined.

This paper demonstrates the special potential of Raman spectroscopy for the study of selected plant metabolites. Carotenoids, which are beneficial components in fruits and vegetables, have been shown to be a significant factor in lowering the risk of various types of cancer and ischemic heart diseases. On the other hand, alkaloids may have various effects on human health, e.g. caffeine is a mild stimulant of the central nervous system and as a result it can influence human behaviour. Polyacetylenes are highly cytotoxic against numerous cancer cell lines and demonstrate antifungal, anti-inflammatory and anti-platelet-aggregatory properties. In most cases, vibrational measurements can be performed directly on plant tissues as well as on fractions isolated from the plant material by hydro-distillation or solvent extraction. Raman spectroscopy techniques allow obtaining spectra which present some characteristic key bands of individual components. Based on such markers related to individual plant substances, spectroscopic analyses in principle allow the discrimination of different species, and even chemotypes among the same species. Moreover, Raman microspectroscopy provides 2- and 3-dimensional images of the investigated plant samples. These maps can be directly compared to the corresponding visual images obtained from a light microscope and offer additional detailed information regarding the local distribution of specific compounds in the surface layers of the analyzed plant tissue.

In order to investigate the enantioseparation property of the biselector chiral stationary phase derived from polysaccharide, a new biselector chiral stationary phase was prepared by coating a blend of two amylose derivatives on an aminated silica gel. The blend consisted of amylose tris(phenylcarbamate) and amylose tris(benzoate) at a glucose unit ratio of 1:1 (mol/mol). For the sake of enantioseparation comparison, the corresponding single selector chiral stationary phases were also prepared with the two individual amylose derivatives. The enantioseparation ability of these chiral stationary phases was evaluated by using structurally various chiral analytes. The biselector chiral stationary phase showed an overall improved enantioseparation ability in comparison with the two single selector chiral stationary phases. The effect of alcohols in mobile phase on the enantioseparation of the biselector chiral stationary phase was also discussed.

Resonance light scattering technique has been employed to investigate the interaction between anthracyclines and DNA in the presence of quercetin. Upon binding to DNA, anthracyclines undergo dramatic enhancement in their resonance light scattering intensities, while quercetin goes through no obvious change in its intensity. Once being added into anthracyclines- DNA system, quercetin can induce great difference in resonance light scattering intensities between anthracyclines- DNA-quercetin ternary system and anthracyclines-DNA binary system. Consequently, the DNA binding abilities of anthracyclines can be improved by quercetin. Improved efficiency of quercetin on the interaction between anthracyclines and DNA has been calculated. Under the experimental condition, the improved efficiency values of doxorubicin, epirubicin and mitoxantrone were 38.78%, 28.53% and 16.69%, respectively. Additionally, the weak resonance light scattering intensity of quercetin-DNA system is greatly enhanced by anthracyclines and the enhanced intensity is proportional to the concentration of anthracyclines in the range 0.06-9.0 μg mL-1 for doxorubicin, 0.08-12 μg mL-1 for epirubicin and 0.01-3.0 μg mL-1 for mitoxantrone. The detection limits are 4.7 ng mL-1 for doxorubicin, 5.4 ng mL-1 for epirubicin and 1.4 ng mL-1 for mitoxantrone.

We constructed nano-metal oxide (tin oxide, cerium oxide, cobalt oxide, iron oxide, nickel oxide, gold oxide, and silver oxide) -modified carbon paste electrodes (CPEs) for detecting flavonols (kaempferol, myricetin, and quercetin) and flavones (luteolin and apigenin) using high-performance liquid chromatography with an electrochemical detector (HPLC-ECD). Flavonoids are more efficiently catalytically oxidized on a gold oxid e-modified CPE than on a bare CPE. For direct current mode, with the current at a constant potential, and measurements with suitable experimental parameters, a linear concentration from 40 to 1600 ng mL-1 was found. The limits of quantification for flavonoids were below 0.5 ng. The developed method detected flavonoids in urine samples. Findings using HPLC-ECD and HPLC with an ultraviolet detector were comparable.

A combination of a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method and cloudpoint extraction (CPE) before spectrophotometric analysis has been developed for the determination of carbaryl residues in vegetable samples that include cucumber, cabbage, Chinese cabbage, kale, and yard long bean. The initial surfactantrich phase became colored upon diazotization with 2,4-dimethoxy aniline under alkaline conditions, and the product was monitored at 510 nm. The optimum conditions for CPE were 1.5% (w/v) Triton X-114 in the presence of 1.5% (w/v) NaCl, and 15-min equilibrated at 45 °C. The diazotization was carried out by adjustment using 0.1 mol L 1 DMA, 0.1 mol L 1 NaNO2, and 1.0 mol L 1 HCl followed by hydrolysis at pH 12 using 1.0 mol L 1 NaOH. Under these conditions, the proposed method gave a limit of detection (LOD) of 0.1 mg kg 1, which is 10-fold lower than analysis without preconcentration, and below the maximum residue limit (MRL) set in vegetable samples. Acceptable recoveries (> 79%) and precision with the relative standard deviation less than 11% were obtained, in good agreement with the results obtained from high performance liquid chromatography (HPLC), at P=0.05. No contamination by carbaryl in any vegetable samples studied was observed. The proposed method is simple, effective, reliable, and can be used for extraction and preconcentration as well as removal of sample matrix interferences.

A novel capillary zone electrophoresis (CZE) method is, for the first time, developed for effective separation and simultaneous determination of ceftriaxone sodium and levofloxacin in human urine. The electrophoresis conditions were investigated and optimized. A 25 mM disodium tetraborate buffer adjusted with sodium hydroxide or phosphoric acid to pH 9.2 was used and the peak area was determined with UV detection under 20 kV separation voltages. Under optimized conditions, the two drugs can be separated effectively. Good linearity of quantitative analysis was achieved in the range of 10-500 μg mL-1 for ceftriaxone sodium and 25-500 μg mL-1 for levofloxacin with the correlation coefficients (r) of >0.999. The limits of detection (LOD) for ceftriaxone sodium and levofloxacin were 2.5 and 7.5 μg mL-1, respectively. Their average recovery from human urine was 99.15 % with the RSD of 0.65-1.54 % for ceftriaxone sodium, and 99.57 % with the RSD of 0.2-2.0% for levofloxacin. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for selective and simultaneous analysis of the test drugs in human urine at clinically relevant concentrations.