Combinatorial Chemistry & High Throughput Screening - Volume 10, Issue 7, 2007

The growing need for microfluidic systems in high throughput screening (HTS) urgently demands the development of compatible detection systems, especially for the analysis of non-labeled and ionizable analytes. Direct detection employing electrochemical processes offers remarkable advantages such as reduced cost, low power requirements, enhanced portability, independence of sample turbidity, subnanomolar sensitivity, reproducibility, and compatibility with micromachining and microfabrication procedures. More than 70% of current drugs are ionizable under physiological conditions and have the potential to be measured using electrochemical detection. The papers in Part 3 of this special issue of Combinatorial Chemistry & High Throughput Screening summarize and highlight some recent advances, directions and key concepts in the development of new supramolecular electrode materials, ion recognition modes, detection schemes, operational principles, applications and future prospects for electrochemical HTS devices and sensors. The review by Uslu and Ozkan opening this issue presents the status and recent achievements in the use of native and chemically modified solid noble metal and carbon nanotube electrodes for HTS of drugs and pharmacologically active compounds. In addition, the review by Gulaboski et al. describes the use of new four-electrode and three-phase electrode voltammetry at the interface of immiscible electrolyte solutions mimicking biomembrane systems which enables precise determination of unknown standard partition coefficients for ionized drugs-of-abuse, peptides, nutrients, and examples of ubiquitous environmental pollutants. Similarly, Ganjali et al., review the use of podand, macrocycle or transition metal complexed Schiff bases as highly selective ionophores in the construction of disposable electrodes for HTS of inorganic cations and anions, as well as ionizable drugs and nutrients. In the minireview by Wang et al., recent progress is discussed concerning the sensitive and specific electrochemical detection of drugs and their metabolites under the continuous flow conditions of HPLC or ultra-fast flow-injection analysis. The practical implementation and unique properties of lipophilic podand ionophore supported potentiometric detection in the construction of DNA sensitive arrays coupled with HPLC is reported in the research paper by Nagels et al. In addition, the construction and characteristics of lipophilic calix[4]resorcinarene based potentiometric sensor enabling selective determination of positional isomers of phenylenediamine type endocrine disrupting agents and allergens are reported in the paper by Radecki et al. Spectacular examples of selective electrochemical determination of drugs used in mono and combined therapies of metabolic, degenerative and infectious human diseases such as a non-insulin-dependent diabetes mellitus, beginning prostatic hyperplasia, atherosclerosis, rheumatoid arthritis, and ascariasis are described in the series of research papers by Gupta et al., Dogan-Topal et al., Wang et al., and Aboul-Enein et al. Innovative potentiometric and voltammetric procedures were developed and described in these research papers using a boron doped diamond, a carbon nanotubes and liquid membrane electrodes. By reducing the size of such working electrodes, it will be possible to enhance their performance and sensitivity even further. Thus, it is quite reasonable to say that complete self-contained HTS microsystems equipped with electrochemical detection may be realized soon through an on-chip and proteochip integration of the potentiostatic circuitry with supramolecular versions of nanovalves, switchers, transducers, digital multiplexers, logic networks, sequential and combinational circuits as well as memory that mimic the operation of currently used semiconductor logic gates. Hoping that our publishing efforts will help transform such promising views into reality, I would like to extend my grateful thanks, as the Guest Editor of this special issue of Combinatorial Chemistry & High Throughput Screening, to all authors who kindly contributed in all three parts of this exceptional project.

This review summarizes recent progress in the development and application of solid electrodes to the screening of pharmaceutical dosage forms and biological fluids. Recent trends and advances in the electroanalytical chemistry of solid electrodes, microelectrodes and electrochemical sensors are reviewed. The varieties of solid electrodes and their basic physico-chemical properties and some specific characteristics including some supramolecular phenomena at their surface are surveyed. This review also includes some selected designs and their applications. Despite many reviews about individual solid electrodes in the literature, this review offers the first comprehensive report on all forms of solid electrodes. Special attention is paid to the possibilities of solid electrodes in high throughput electroanalytical investigation of drug dosage forms and biological samples using modern electroanalytical techniques. Various selected studies on these subjects since 1996 are reviewed in this paper.

The latest results of voltammetric research on the ionic transfer process of ionisable drugs across bare and lipid-modified liquid-liquid interfaces are reviewed. In recent years, two voltammetric methods have been extensively applied to this purpose, i.e. the classical four electrode voltammetry at the interface between two immiscible electrolyte solutions, and the “three-phase electrode.” Thus, a brief background of the methodologies and some successful examples of their application are highlighted in this work. Particular attention is given to the ionic transfer kinetics and to the electrochemical characterization of the drug-membrane interactions between the ionized drugs and lipid-modified interfaces. Future trends in this area are also mentioned in connection with high-throughput assessment of ADMET properties of drugs.

Schiff base compounds refer to the branch of supra-molecules and can be used as sensing material in the construction of potentiometric ion selective electrodes (ISEs). This relatively modern field has been subject to extensive research in the period of 1999-2007 when more than 100 ISEs employing Schiff bases were constructed. The quantitative high-throughput detection of 29 cations and 7 anions has been demonstrated in various scientific branches, such as biomedicine, pharmacy, biochemistry, pharmacology, environmental chemistry, food technology, and agriculture. This review discusses Schiff base compounds and their applications in the design and development of ion selective sensors and microsensors.

Recent advances in electrochemical detection techniques coupled with high-performance liquid chromatography (HPLC-ECD) in pharmaceutical and biomedical analysis are reviewed. ECD classification and modes including common amperometric, coulometric, conductimetric, and potentiometric detector, are outlined and the some typical examples of determinations in pharmaceutical and biomedical analysis are described. The electrochemical detection system can offer superior merits over other detectors commonly used with HPLC. These techniques have great potential owing to their prominent characteristics in high-throughput screening procedures of drugs in various matrices. Fundamental 67 references from last 5 years related with a field are cited in this review.

Potentiometric sensors are studied as viable candidates for the construction of high throughput DNA arrays. For preliminary investigations, such sensors were used in an HPLC setup in the present work. This avoided errors due to ionic contaminants or additives in the commercial samples. The oligonucleotides dT10, dT20 and dT30 were used as test substances. The potentiometric sensors were of the coated wire type, containing PVC, DOP, MTDDACl and a synthetic podand urea receptor. The HPLC system consisted of a reversed phase column eluted with a phosphate buffer, triethylammoniumacetate (TEAA), and an acetonitrile gradient. Molar responses and sensitivities increased with increasing chain length of oligonucleotides, yielding detection limits as low as 10-6M (dT30, injected concentration). The slopes of the calibration graphs were at least 23 mV/decade (dT10), which was much higher than expected. The results are discussed in view of the potential use of this sensor type in high throughput microarrays.

The construction and characterization of potentiometric membrane electrodes are described for the quantification of alfuzosin, a drug used in a mono- and combined therapy of benign prostatic hyperplasia (BPH). The membranes of these electrodes consist of alfuzosin hydrochloride-tetraphenyl borate, (Az-TPB), chlorophenyl borate (Az-ClPB), and phosphotungstate (Az3-PT) ion associations as molecular recognition reagent dispersed in PVC matrix with dioctylpthalate as plasticizer. The performance characteristics of these electrodes, which were evaluated according to IUPAC recommendations, revealed a fast, stable and liner response for alfuzosin over the concentration ranges of 8.3 x 10-6 to 1.0 x 10-2 M, 3.8 x 10-6 to 1.0 x 10-2 M, 7.5 x 10-7 to 1.0 x 10-2 M AzCl with cationic slopes of 57.0, 56.0 and 58.5 mV/decade, respectively. The solubility product of the ion-pair and the formation constant of the precipitation reaction leading to the ion-pair formation were determined conductometrically. The electrodes, fully characterized in terms of composition, life span and usable pH range, were applied to the potentiometric determination of alfuzosin hydrochloride ion in different pharmaceutical preparations and biological fluids without any interference from excipients or diluents commonly used in drug formulations. The potentiometric method was also used in the determination of alfuzosin hydrochloride in pharmaceutical preparations in four batches with different expiration dates. Validation of the method showed suitability of the proposed electrodes for use in the quality control assessment of alfuzosin hydrochloride. This potentiometric method offers the advantages of high-throughput determination, simplicity, accuracy, automation feasibility, and applicability to turbid and colored sample solutions.

The electrochemical behavior of atorvastatin calcium at glassy carbon and boron-doped diamond electrodes has been studied using voltammetric techniques. The possible mechanism of oxidation was discussed with model compounds. The dependence of the peak current and potentials on pH, concentration, scan rate and nature of the buffer were investigated for both electrodes. The oxidation of atorvastatin was irreversible and exhibited a diffusion-controlled fashion on the diamond electrode. A linear response was obtained within the range of 9.65 x 10-7 - 3.86 x 10-5 M in 0.1 M H2SO4 solution for both electrodes. The detection limits of a standard solution are estimated to be 2.11 x 10-7 M with differential pulse voltammetry (DPV) and 2.05 x 10-7M with square wave voltammetry (SWV) for glassy carbon electrode, and 2.27 x 10-7 M with DPV and 1.31 x 10-7M with SWV for diamond electrodes in 0.1 M H2SO4 solution. The repeatability of the methods was found good for both electrodes. The methods were fully validated and successfully applied to the highthroughput determination of the drug in tablets, human serum and human urine with good recoveries.

The electrochemical response characteristics of poly(vinyl)chloride (PVC) based membrane sensors for determination of tetramisole hydrochloride (TmCl) is described. The membranes of these electrodes consist of tetramisoletetraphenyl borate (Tm-TPB), chlorophenyl borate (Tm-ClPB), and phosphotungstate (Tm3-PT) ion associations dispersed in a PVC matrix with dibutylpthalate as a plasticizer. The electrodes were fully characterized in terms of composition, life span, usable pH range, and working concentration range and ionic strength. The electrodes showed Nernstian response over the concentration ranges of 7.4 x 10-7 to 1.0x10-2 M, 1.7 x 10-6 to 1.0x10-2 M, and 5.6 x 10-6 to 1.0x10-2 M TmCl, respectively, and were applied to the potentiometric determination of tetramisole ion in pure solutions and pharmaceutical preparations. The potentiometric determination was also used in the determination of tetramisole in pharmaceutical preparations in four batches of different expiration dates. The electrodes exhibited good selectivity for TmCl with respect to a large number of excipients such as inorganic cations, organic cations, amino acids, and sugars. The solubility product of the ion-pair and the formation constant of the precipitation reaction leading to the ion-pair formation were determined conductometrically. The new potentiometric method offers the advantages of high-throughput determination, simplicity, accuracy, automation feasibility, and applicability to turbid and colored sample solutions.

Polycysteic acid based electrochemical oxidation of L-cysteine (CySH) and carbon nanotubes (CNTs) formed a composite thin film material at a glassy carbon electrode (GCE) that was used a novel modifier for electroanalytical determination of sinomenine which is used for rheumatoid arthritis treatment. The determination of sinomenine at the composite modified electrode was studied by differential pulse voltammetry (DPV). The peak current obtained at + 0.632 V (vs SCE) from DPV was linearly dependent on the sinomenine concentration in the range of 1.0 x 10-7 to 6.0 x 10-5 M in a B-R buffer solution (0.04 M, pH 1.81) with a correlation coefficient of 0.998. The detection limit (S/N = 3) was 5.0 x 10-8 M. The electrochemical reaction mechanism of sinomenine was also discussed. This new method was then applied to the high-throughput determination of sinomenine in human serum samples with satisfactory results. This polycysteic acid/CNTs composite film may be considered to be a promising, low-cost, durable, and biocompatible material for the modification of sensors in applications to pharmaceutical and biomedical analysis.

The PVC supported liquid membrane electrodes incorporated with undecylcalix[4]resorcinarene (UDC[4]Rene) generates a cationic potentiometric response after stimulation by neutral (unprotonated) form of diaminobenzene isomers. The potentiometric signals were generated upon the formation of supramolecular complex between the UDC[4]Rene (host) and diaminobenzene (guest) at the organic-aqueous interface. In this paper for the first time we report the generation of cationic potential signal by electrode incorporated with host molecule possessing oxygen as a heteroatom, after stimulation with neutral guest containing nitrogen in its structure We also broaden the family of receptor molecules, which are able to generate charge separation at the organic/aqueous interface after interaction with uncharged molecules. The results obtained confirm the possibility of applying this potentiometric method for the screening of diaminobenzene isomers identified recently as allergens and endocrine disrupting chemicals.

An inexpensive, rapid and reproducible capillary electrophoretic method has been developed and validated for the determination of metformin in pharmaceutical preparations. The method was developed utilizing a fused silica capillary (60 cm x 50 μm I.D.), phosphate buffer (50 mM, 3.0 pH)-acetonitrile (95:5, v/v) as background electrolyte (BGE), 20 kV applied voltage with UV detection at 254 nm and at a working temperature of 23±1°C. Linearity was observed in the concentration range from 100 ng/L to 5 μg/L, with a correlation coefficient (R2) of 0.9998. The limits of detection and quantification achieved were 60 and 100 ng/mL, respectively. The recovery of metformin from pharmaceutical preparations was 99.1%. These validation parameters demonstrate the precision of the method and its suitability for the determination of metformin in pharmaceutical tablet formulations.

Born in Wroclaw, Poland, in 1953, Grzegorz Bazylak studied food chemistry (1974-1979) at the Technical University of Lodz (Lodz, Poland), where he also received his Ph.D. degree (1990) in technical sciences with a thesis entitled, “Studies on the association processes of amines and multidentate tricyclic Schiff base nickel(II) chelates.” Subsequently, he joined as an Adjunct Professor the Hygenics Department (1991-1997) and the Institute of Physiology & Biochemistry (1997-2001) at the Faculty of Medicine, Medical University of Lodz (Poland). In 1996, with a fellowship from The British Council, he was a Short Term Visiting Scientist at the Zeneca SmithKline Beecham Centre for Analytical Science, Department of Chemistry, Imperial College of Science, Technology & Medicine (London, UK) where he collaborated with Professor Colin F. Poole and Professor Andreas Manz. In years 2001 - 2003, with a scholarship from the Ministerie van de Vlaamse Gemeenschap (Flanders, Belgium), he moved to the University of Antwerpen (Belgium) as a Postdoctoral Fellow in the research groups of Prof. Luc J. Nagels, Prof. Herman J. Geise, Prof. Achiel Haemers, and Prof. Koen Augustyns. In 2003, he returned for his D.Sc. degree (habilitation) in pharmaceutical sciences at the Medical University of Gdansk (Poland) with a thesis entitled, “Chromatographic screening and potentiometric microdetection of adrenolytics employing supramolecular effects.” In 2002, he was appointed Adjunct Professor in the Department of Drug Chemistry, Faculty of Pharmacy, The Ludwik Rydygier Medical University in Bydgoszcz (Poland). In July 2004, he accepted a position as an Associate Professor at the Department of Pharmaco-Bromatology & Molecular Nutrition in the same institution and, after fusion of local universities in November 2004, has been continuing this appointment until today at the Collegium Medicum Nicolaus Copernicus University (Bydgoszcz, Poland). In years 2003-2006, he was an Institutional Coordinator of the European Socrates-Erasmus Programs in both universities. In conjunction with Prof. Jan Biernat, Prof. Renata Bilewicz, Prof. Marek Pietraszkiewicz, Prof. Jerzy Radecki, Prof. Zbigniew Brzozka, Prof. Wanda Radecka-Paryzek, Prof. Lucjan Piela, and Prof. Grzegorz Schroeder, he co-founded in 1997 the Polish Supramolecular Chemistry Network Foundation (Warsaw, Poland). He has served as a Guest Editor of leading international journals including Combinatorial Chemistry & High Throughput Screening (2000, 2004, 2007), Frontiers in Bioscience (2007) and Current Drug Discovery Technologies (2007). He is also the recipient of the Individual Scientific Award of the Polish Ministry of Health (2003) and the Polish Pharmaceutical Society (2004). He is (co-)author of approximately 150 scientific publications. His current research efforts focus on the development of supramolecularly driven high-throughput screening nanoseparation and nanodetection techniques for drugs and food components, supramolecular farmacochemistry, farmacobromatology, nutreogenomics and molecular nutrition. SELECTED PUBLICATIONS [1] H. Chaberska, H. Kaczmarek, G. Bazylak: Viability of murine 3T3 fibroblasts on the poly(methyl methacrylate) surface modified by constant UV irradiation, Polim. Med., 2007, 27(3), 13-19. [2] A. Jaworowska, G. Bazylak: Residential factors affecting nutrient intake and nutritional status of female pharmacy students in Bydgoszcz, Rocz. Panstw. Zakl. Hig., 2007, 58, 245-251. [3] Jaworowska, G. Bazylak: Obesity development associated with viral infections, Postepy Hig. Med. Dosw., 2006, 60, 227-236. [4] Jaworowska, A. Malak, G. Bazylak: Slimming diets, food intake and nutritional status of pharmacy students in Bydgoszcz, Bromat. Chem. Toksykol., 2006, 39 Suppl. 581-585. [5] G. Bazylak, L.J. Nagels: Potentiometric detection of beta-adrenolytic and beta-adrenergic drugs in HPLC systems, Postepy Hig. Med. Dosw., 2005, 59, 554-569...