Current Pharmaceutical Analysis - Volume 5, Issue 2, 2009

Frustration currently observed in most of the pharmaceutical companies as a result of the high attriction rate of finally selected drug leads and optimized drug candidates would be also be related to challenges and limitations of available analytical protocols in the development of any new drug. For example, typical detection limits reported in the pharmaceutical literature are 10 ppm of the label-free analyte or a biological activity level of 1/1000 of the normal therapeutic dose. One of the intensively appearing and most promising solutions demonstrated recently to overcome these shortcomings is increasing the use of the non-optical analytical and screening platforms in drug discovery programmes based on integrated microfluidic and electrodriven phenomena along with acoustic resonance, field effect nanowires, microcantilevers or differential microcalorimetry approaches. Especially, application of electrodriven separation and detection systems in both small- and large-molecule hit therapeutics development, evaluation and confirmation issues should be extremely attractive. The list of advantages offered by this last mentioned combination of separation and detection methods is quite extensive, and primarily includes primary the extensive miniaturization potential, enlarged scope of substances for direct analysis, reduced costs, increased high-throughput and significantly extended high-content information capacity of the electrodriven hit-to-lead discovery assays compared to traditional optical methodologies. Examples of such practically implemented and robust electrodriven microanalytical schemes for drug discovery and bioassay technologies are illustrated in this special issue of Current Pharmaceutical Analysis, which also highlights some key advances in resolving of enrolled encumbrances revealed by these approached. The review by Lund and Parviz opening this special issue presents the set of most promising future-generation approaches, including also innovative electrodriven and electronic procedures, to enable fast DNA sequencing at the target price of 1000 USD per genome in 2014 year. Involvement of sophisticated synthetic methods in fabrication of nanotubes mimicking peptide nanopores and their further integration with a chip containing detectors has been also discussed in this paper. In addition, the minireview by Vander Heyden et al. deals with the use of microfluidic devices and chip-based systems employing variety of laser-induced fluorescence, electrochemical and mass spectrometry detection modes in a high-throughput screening and ultra-fast analysis of chiral pharmaceuticals in picomolar range to reduce attrition rate of drug leads. Similarly, Yu et al. describe the recent advances in electrodriven microfluidic chips protocols devoted to deciphering of variable drugprotein, drug-DNA and biomolecules involved in highly specific interactions and the determination of ADMET properties of drug candidates. These considerations are also continued in review by Nagels et al., which presents the advantages and limitations in construction concepts of miniaturized potentiometric sensors for the highly sensitive detection of neutral and/or charged biomolecules, poly-ionic molecules, living microorganisms and drug dissolution studies in hydrodynamic conditions. Since the availability of various electrochemical procedures today enables the sensitive and robust analysis of pharmaceutical dosage forms and biological fluids, the range of such dedicated applications in batch conditions were critically addressed by Ozkan in his review paper. Similarly, Hu et al. summarized recent trends, pitfalls and developments in use of voltammetric procedures for direct drug determination in static and flow conditions at broad concentration range of up to 10-12 M and of obtained biologically through meaningful analytical result.

The sequencing of the human genome at the dawn of the twenty-first century marked the beginning of an ongoing race to make genome sequencing affordable enough to be incorporated into routine patient care. Genome sequencing is still prohibitively expensive, and while the next generation of sequencing techniques will no doubt have a substantial impact on genome sequencing, they have not been shown to be capable of achieving the aggressive goal established by the National Institutes of Health of a US$1000-genome by the year 2014. To achieve this goal, researchers will likely need to develop radically new approaches to DNA sequencing. Some of the most promising future-generation techniques attempt to sequence DNA using electronic mechanisms. This article describes the current state of approaches to sequence DNA using electronic techniques. It outlines existing techniques that have been used to sequence the human genome, nextgeneration techniques expected to substantially reduce the time and cost required to perform genome sequencing, and future electronic approaches.

Microfluidic devices on chip-based systems are becoming increasingly popular, because of many advantages, like low mobile phase consumption and greatly reduced analysis times. The ultimate goal is to design all-including labon- a-chip tools that can be employed in the field and that integrate all elements necessary for an analysis, e.g. one or more separation channels, (multiple) detectors, mixing chambers and sample pretreatment areas. The chip format permits to make any channel pattern, rendering the applications of lab-on-a-chip unlimited. However, overcoming the limitations inherent with the detection systems remains a challenging problem. Whereas UV-detection is predominant in the larger systems, its sensitivity is inadequate due to the minute pathlengths employed in a chip. This review summarizes the shortcomings of UV detection, and discusses some possible solutions. Besides, it presents an overview of the alternative detection methods that have been employed in pharmaceutical and chiral chip analysis, including their positive and negative aspects as well. Finally, the applications reported on the chips are discussed.

Identification of biomolecule interactions is crucial in understanding biological and biochemical processes. This review presents a brief outline of the advances in microfluidic chips devoted to biomolecule interactions. Various separation modes used for biomolecule interactions are discussed first, in which microchip isoelectric focusing, microchip zone electrophoresis, microchip electrophoresis frontal analysis, and T-sensor microfluidic channel are included. Detection methods on these microsystems are essential for the identification and quantification of chemical species and biomolecule that are being analyzed. Within the detection methods section, time-resolved resonance raman spectroscopy, mass spectrometry, surface plasmon resonance imaging, deep UV laser-induced fluorescence, indirect laser-induced fluorescence, contactless conductivity detection techniques are discussed. Applications including biomolecule interaction kinetics, protein-protein interaction, protein-DNA interaction, drug-protein interaction, and drug-DNA interaction are outlined. To our knowledge, biomolecule interactions studying microfluidic chip have not been reviewed before.

The miniaturization of potentiometric sensors is developing only slowly. ISFET technology has not yet brought great breakthroughs in this respect. Potentiometric microelectrode arrays are developed however for DNA analysis. As potentiometry moves into new and unexpected applications such as biomolecules, multiply charged molecules, DNA, marker proteins, viruses and bacteria, dissolution testing..., the need for miniaturized sensors and arrays will grow. Possibly, planar screen printed electrodes can be applied. Use of the electrodes in hydrodynamic conditions may be needed. The use of FIA and HPLC for rapid screening of electrode compositions is explained. This provides new insights into these chemically dynamic sensor systems, and into the interaction of organic ionic molecules with surfaces.

A review of the applications, principles, advantages of stripping voltammetric techniques, namely anodic, cathodic, abrasive, adsorptive stripping voltammetry and potentiometric stripping analysis is presented. Stripping voltammetric methods in pharmaceutical analysis are discussed and the generally applicable procedure is given. The use and advantages and disadvantages of these techniques at different electrodes are discussed. The analytical applications of stripping techniques to pharmaceutically active compounds are also discussed. Various selected determination studies on drugs by stripping techniques from pharmaceutical dosage forms and biological samples are reviewed.

This minireview summarized several examples to illustrate the application of electrochemical methods for pharmaceutical and drug analysis. Special attentions were devoted to the voltermmetric and potentiometric techniques. A section dedicated to the application of the electrochemical detectors coupled with flow system was also presented. Finally, we briefly outline future trends of the electrochemical methods for drug analysis.

Aptamers, like antibodies, have high molecular recognition specificity and selectivity. Coupled with microfluidic devices, apatamers and target-aptamer complexes can be rapidly separated while only a small sample volume is required. Trace protein detection using aptamer probes on a microchip platform was demonstrated using vascular endothelial growth factor 165 (VEGF165) and a selective aptamer probe as a model system. The fluorescently labeled aptamer was separated from the VEGF165-aptamer complex in 10 s. The equilibrium dissociation constant of the complex was determined to be 8.0 nM using frontal analysis on the microchip. Two calibration curves were constructed with a detection limit of 1.0 nM VEGF165 using pinched and gated injections. The blood plasma effects on the microchip electrophoretic separation and VEGF165-aptamer complex formation were investigated using rat blood plasma. Results demonstrate the power of microchip capillary electrophoresis in terms of assay speed, low reagent consumption and high separation efficiency. However, they also indicate the necessity of further improvements in the detection limit and/or the pretreatment of the plasma sample when measured by microchip CE.

Interleukin-6 (IL-6), an immune protein in the hematopoietins family, has been demonstrated as a prognostic indicator for survival in patients with gastric carcinoma. In this contribution, we introduced a new and high-throughput electrochemical immunosensor for the detection of IL-6 in human. The immunoassay protocol was fabricated by the immobilization of anti-IL-6 antibodies on the nanogold and partial ferrocenyltethered dendrimer (Fc-D)-functionalized interface. A sandwich-type immunoassay format was employed for the IL-6 determination using horseradish peroxidase (HRP)-labeled anti-IL-6 as secondary antibody. The properties and factors influencing the performance of the proposed immunosensor were evaluated in detail. Under optimal conditions, the current response of the formed sandwich-type immunocomplex relative to H2O2 system was proportional to IL-6 concentration ranging from 2.5 pg/mL to 120 pg/mL with a detection limit of 1.0 pg/mL (at 3δ). The precision, reproducibility and stability of the immunosensor were acceptable. Subsequently, the immunosensors were used to assay the IL-6 in human serum specimens. Analytical results were in agreement with those obtained by the standard chemiluminescence ELISA.

The present work illustrates possibilities of column coupling capillary electrophoresis (CE-CE) combined with fiber-based diode array detection (DAD) for the direct quantitative determination of trace drug (amlodipine, AML), in biological multicomponent ionic matrices (human urine). Capillary isotachophoresis (ITP) served as an ideal injection technique in CE-CE. Moreover, ITP provided an effective on-line sample pretreatment before a capillary zone electrophoresis (CZE) separation. Enhanced separation selectivity, due to the combination of different separation mechanisms (ITP vs. CZE, carboxyethyl-β-cyclodextrine as a complexing buffer additive in CZE), enabled to obtain a pure analyte zone, suitable for its detection and quantitation. The DAD, unlike single wavelength UV detection, enabled to characterize the purity of the analyte zone. A processing of the raw DAD spectra (the background correction and smoothing procedure) was essential, when a trace analyte signal was evaluated. Obtained results indicated pure zone of interest confirming effective ITP-CZE separation process. The proposed ITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and was successfully applied to a pharmacokinetic study of AML.

Cefoperazone sodium is irreversibly oxidized at the glassy carbon electrode in various buffer systems and at different pH values using cyclic, linear sweep, differential pulse and square wave voltammetric techniques. The oxidation of cefoperazone sodium was irreversible and exhibited diffusion controlled process depending on pH. A detailed oxidation mechanism was proposed and discussed. The dependence of peak current and potentials on pH, concentration, scan rate, nature of the buffer was investigated. According to the linear relationship between the peak current and the concentration, differential pulse (DPV) and square wave (SWV) voltammetric methods for cefoperazone sodium assay in pharmaceutical dosage forms and biological fluids were developed. The determination of cefoperazone sodium was proposed in phosphate buffer at pH 2.00, which allows quantitation over the 4x10-6 - 2x10-5 M and 4x10-6 - 6x10-5 M ranges in supporting electrolyte for DPV and SWV, respectively, and 4x10-6 - 2x10-5 M in spiked serum samples for both techniques. The repeatability, reproducibility, precision and accuracy of the methods in all media were investigated. Precision and accuracy of the developed method were used for the recovery studies. The standard addition method was used for the recovery studies. No electroactive interferences were found in biological fluids from the endogenous substances and additives present in pharmaceutical dosage form.

Cysteic acid, containing negatively charged sulfonic ligand, has been deliberately modified on the electrode by the electrochemical oxidation method. It could promote the accumulation of adenine on the electrode because of the electrostatic interaction between the negatively charged cysteic acid and the positively charged adenine at pH 6.0. After accumulation at the potential of 0.2 V for 300 s, adenine produced well-defined oxidation peaks at about +1000 mV. Cyclic voltammetric study indicated that the oxidation process was irreversible and adsoption-controlled. Based on the differential pulse voltammetric (DPV) procedure, the linear calibration curve for the detection of adenine over the range 2.0 x 10-7 -1.0 x 10-4 M was obtained. The detection limit (3σ) was down to 5.9 x 10-8 M. Such modified electrode was also successfully applied for the determination of adenine in Vitamin B4 tablet.

Cefotaxime sodium has an antibacterial effect and it is classified as a third-generation broad spectrum cephalosporin antibiotic. It is irreversible, oxidized at the glassy carbon electrode in various buffer systems and at different pH values. The detailed mechanism of the oxidation process is described in this manuscript. Differential pulse and square wave voltammetric methods were developed for its determination in pharmaceutical dosage forms and spiked human serum samples according to the linear relation between the peak current and cefotaxime sodium concentration. For analytical purposes, a very well resolved diffusion controlled voltammetric peak was obtained in Britton-Robinson buffer at pH 2.0 at 0.87 and 0.89V for differential pulse and square wave voltammetric techniques, respectively. The linear response was obtained within the range of 1x10-6 - 6x10-5 M with a detection limit of 2.83x10-7 M for differential pulse and 2x10-6 - 6x10-5 M with a detection limit of 3.61x10-7 M for square wave voltammetric techniques. The repeatability and reproducibility of the methods for both media (supporting electrolyte and serum sample) were determined. Precision and accuracy of the developed method were used for the recovery studies. The standard addition method was used for the recovery studies. No electroactive interferences were found in biological fluids from the endogenous substances and additives present in pharmaceutical dosage form.