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Analytical and Bioanalytical Chemistry (v.377, #5)

Wolfish behavior...the successful scientist as leader of a pack or a lone wolf by J. Fetzer (pp. 785-786).

Current trends in pharmaceutical analysis by Aldo Roda (pp. 787-787).

Fast LC/MS in the analysis of small molecules by Philip R. Tiller; Leslie A. Romanyshyn; Uwe D. Neue (pp. 788-802).

Liquid chromatography-mass spectrometry (LC/MS) has become one of the most widely used analytical techniques in both qualitative and quantitative analysis of small molecules. Recently, with the increasing demand for ever-higher sample throughput, the use of faster chromatographic separations has become popular, along with other LC/MS methods that decrease analytical cycle-time. The burgeoning use of LC/MS has meant that the primary expertise of many practitioners today is not in the field of LC/MS, which has been facilitated by the ease-of-use of modern LC/MS systems. An examination of the current state of the literature, relating to "fast LC/MS", should serve well to those new to LC/MS, and should help them in the development of fast LC/MS methods that are effective in terms of both the chromatography and the utilization of the mass spectrometer. This review paper focuses on fast LC/MS analyses of small molecules that have been reported in peer-reviewed publications.

Keywords: Fast-LC/MS; Gradient; Isocratic; Parallel-LC/MS

New approaches to chromatographic determination of lipophilicity of xenobiotics by Roman Kaliszan; Antoni Nasal; Michał Jan Markuszewski (pp. 803-811).

Liquid chromatography and capillary electrophoresis are unique tools for fast and efficient modeling of pharmacokinetic properties of drug candidates. Therefore numerous new separation methods and procedures have very recently been introduced to facilitate the high-throughput screening of biopartitioning features of xenobiotics. This report is a concise, up-to-date review of progress in the chromatographic assessments of data of importance for medicinal chemistry and molecular pharmacology.

Keywords: Lipophilicity; Gradient reversed-phase high-performance liquid chromatography; Micellar electrokinetic chromatography; Microemulsion electrokinetic chromatography; Micellar liquid chromatography; Modern stationary phases

Protein microarrays: new tools for pharmaceutical development by K. D. Kumble (pp. 812-819).

Protein microarrays are a relatively new technology, which will dramatically impact the pharmaceutical industry. The critical need for more rapid identification of novel drug targets, and for obtaining high-quality information early in the target validation process is a major driver for the industry. High-throughput protein analytical techniques are critical for obtaining biological information beyond that which transcript analysis can provide, given that proteins are the "worker bees" in cells. The vast complexity of proteins when compared to DNA and RNA in terms of sheer number, and structural and biochemical diversity requires a higher degree of sophistication in both assay design and data analysis. High-throughput microarray technology platforms allow for simultaneous, multi-parametric analysis of complex protein mixtures. Protein microarrays have tremendous potential as a tool for the study of protein–protein, enzyme–substrate, and antibody–antigen interactions among others. They can also be used for biomarkers and drug target identification via comparative proteomic analysis of healthy and disease tissues. More recently, cellular microarrays that enable identification of cell-surface receptors and other cell-surface proteins allowing rapid screening of cell-specific, novel drug targets, are being developed. This review will focus on the technical issues and potential applications of protein microarrays in pharmaceutical discovery.

Microchip PCR by L. J. Kricka; P. Wilding (pp. 820-825).

Miniaturization of genetic tests has become an important goal. This review surveys the current progress towards the miniaturization of tests based on the polymerase chain reaction (PCR). It examines the different types of PCR microchip designs, fabrication methods,and the components of a microchip PCR device. It also discusses the problems attributable to surface chemistry of microchip components (inhibition of PCR), and the static and dynamic surface passivation strategies developed for the solution of these difficulties

Keywords: PCR Microchip ; Miniaturization ; DNA

Bioluminescence and chemiluminescence in drug screening by Aldo Roda; Massimo Guardigli; Patrizia Pasini; Mara Mirasoli (pp. 826-833).

Drug screening, that is, the evaluation of the biological activity of candidate drug molecules, is a key step in the drug discovery and development process. In recent years, high-throughput screening assays have become indispensable for early stage drug discovery because of the developments in synthesis technologies, such as combinatorial chemistry and automated synthesis, and the discovery of an increasing number of new pharmacological targets.Bioluminescence and chemiluminescence represent suitable detection techniques for high-throughput screening because they allow rapid and sensitive detection of the analytes and can be applied to small-volume samples. In this paper we report on recent applications of bioluminescence and chemiluminescence in drug screening, both for in vitro and in vivo assays. Particular attention is devoted to the latest and most innovative bioluminescence and chemiluminescence-based technologies for drug screening, such as assays based on genetically modified cells, bioluminescence resonance energy transfer (BRET)-based assays, and in vivo imaging assays using transgenic animals or bioluminescent markers. The possible relevance of bioluminescence and chemiluminescence techniques in the future developments of high-throughput screening technologies is also discussed.

Keywords: Bio-chemiluminescence; Drug screening; High-throughput screening; Bioluminescence resonance energy transfer; Reporter gene; In vivo imaging

Label-free screening of bio-molecular interactions by Matthew A. Cooper (pp. 834-842).

The majority of techniques currently employed to interrogate a biomolecular interaction require some type of radio- or enzymatic- or fluorescent-labelling to report the binding event. However, there is an increasing awareness of novel techniques that do not require labelling of the ligand or the receptor, and that allow virtually any complex to be screened with minimal assay development. This review focuses on three major label-free screening platforms: surface plasmon resonance biosensors, acoustic biosensors, and calorimetric biosensors. Scientists in both academia and industry are using biosensors in areas that encompass almost all areas drug discovery, diagnostics, and the life sciences. The capabilities and advantages of each technique are compared and key applications involving small molecules, proteins, oligonucleotides, bacteriophage, viruses, bacteria, and cells are reviewed. The role of the interface between the biosensor surface (in the case of SPR and acoustic biosensors) and the chemical or biological systems to be studied is also covered with attention to the covalent and non-covalent coupling chemistries commonly employed.

Keywords: Biosensor; Screening; Label-free; Review; Acoustic; Surface plasmon resonance; Quartz crystal microbalance; Calorimetry

Methodological approaches for the study of GABA_A receptor pharmacology and functional responses by Alison J. Smith; Peter B. Simpson (pp. 843-851).

Inhibitory GABA_A receptor ion channels are the target for a wide range of clinically-used therapeutic agents. The complex structural diversity of these ligand-gated channels, revealed by molecular cloning studies, together with increasing requirements for higher-throughput functional assays in drug discovery, has led to the development of a wide range of techniques to examine GABA_A receptor pharmacology and function. In the current article we review some of the methodologies which have contributed to the expansion of knowledge in this field. The techniques include: molecular approaches, immunoprecipitation, and immunopurification to study receptor assembly, structure, and functional expression; in situ hybridization, immunocytochemistry, and autoradiography to examine receptor distribution in native tissues; radioligand binding, site-directed mutagenesis, and electrophysiology to examine pharmacology and allosteric modulation; and patch clamp, ion flux, microphysiometry, and a variety of novel fluorescence-based technologies to examine ion-channel function. The use of gene targetting approaches in transgenic mice has also provided important insights into the role of specific GABA_A receptor subtypes in vivo. The continuing evolution of novel technologies and assay approaches with appropriate sensitivity and resolution to measure subtle modulation of GABA_A ion channels will facilitate ongoing investigation of the physiological functions of these important inhibitory receptors.

Keywords: GABA_A receptor pharmacology; Review; Functional assays; Binding; Electrophysiology; Ion flux; Microphysiometry; Fluorescence

Chemical and analytical characterization of related organic impurities in drugs by Sándor Görög (pp. 852-862).

A system is proposed for the classification of related organic impurities in drugs and drug products including among others (separated and non-separated) intermediates, various kinds of by-products, among them products of different side reactions, epimeric/diastereomeric, enantiomeric impurities, impurities in natural products, and finally degradation products. Examples are taken mainly from the author's own experience and from among the named impurities in the European Pharmacopoeia with focus on impurities in hydrocortisone, prednisolone, enalapril maleate, lisinopril, ethynodiol diacetate, pipecuronium bromide, cimetidine, and ethynylsteroids. The methodological aspects of impurity profiling from the detection to the identification/structure elucidation and quantitative determination of impurities are briefly summarized.

Keywords: Impurity profiling; Hydrocortisone; Prednisolone; Enalapril maleate; Lisinopril; Ethynodiol diacetate; Pipecuronium bromide; Cimetidine; Ethynylsteroids

An overview of qualimetric strategies for optimisation and calibration in pharmaceutical analysis using flow injection techniques by J. M. Bosque-Sendra; L. Gámiz-Gracia; A. M. García-Campaña (pp. 863-874).

Flow Injection analysis represents an attractive tool because of its great advantages, such as versatility, speed, high sampling rate and wide applicability in the field of pharmaceutical analysis. However, due to the inherent characteristics of the technique, the choice of the best set of operational and chemical conditions is complicated and the conventional univariate optimisation method present some limitations, mainly due to fact that the interdependence of variables is not considered. In relation to the calibration process, because of the transient character of the signals obtained using FIA manifolds coupled with different detection techniques, different strategies can be used in calibration to solve some problems related to the nature of the signal thereby improving the performance characteristics of the method. This paper offers an overview of different methodologies used in optimisation based on the use of statistically designed experiments and some strategies developed for calibration applied to the analysis of pharmaceuticals.

Study of donepezil binding to serum albumin by capillary electrophoresis and circular dichroism by Roberto Gotti; Carlo Bertucci; Vincenza Andrisano; Romeo Pomponio; Vanni Cavrini (pp. 875-879).

The interaction between human serum albumin (HSA) and the acetylcholinesterase inhibitor donepezil, has been studied by means of capillary electrophoresis frontal analysis (CE/FA) and circular dichroism. CE/FA enabled rapid and direct estimation of the quantity of free donepezil present at equilibrium with a physiological level of serum albumin (600 μmol L⁻¹). Application of Scatchard analysis enabled estimation of the binding parameters of HSA towards donepezil, such as association constant and number of binding sites on one protein molecule. Furthermore, due to enantioseparation ability shown by HSA on donepezil in CE mode, displacement experiments were carried out using ketoprofen and warfarin as coadditives to the HSA based running buffer. The addition of these compounds reduced the enantioresolution of donepezil by HSA only when used at high concentration. These data were confirmed and corroborated by circular dichroism (CD) experiments. Using CD, bilirubin was also applied as a ligand specific to site III of HSA. The observed behaviour suggested that donepezil could be considered a ligand with independent binding to sites I and II; although site III is not the highest affinity site, indirect interaction (i.e. cooperative binding) can be assumed.

Keywords: Binding studies; Capillary electrophoresis; Circular dichroism; Donepezil; Human serum albumin

Rapid analysis of fluoxetine and its metabolite in plasma by LC-MS with column-switching approach by S. Souverain; M. Mottaz; S. Cherkaoui; J.-L. Veuthey (pp. 880-885).

A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography–electrospray ionisation–mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4 min. Method performances were evaluated. The method showed good linearity in the range of 25–1000 ng mL⁻¹ with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25 ng mL⁻¹ for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma samples.

Keywords: Fluoxetine; Norfluoxetine; Column-switching; On-line sample preparation; Large particles support; LC-MS

Determination of serum d-lactic and l-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization by Hisanori Hasegawa; Takeshi Fukushima; Jen-Ai Lee; Kazuhisa Tsukamoto; Kyoji Moriya; Yoshikazu Ono; Kazuhiro Imai (pp. 886-891).

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor (α) of 1.32 and a resolution (R _s) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5–101.2 and 89.0–100.7%, and the intra-day and inter-day precisions were 0.3–1.2 and 0.4–4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients (n=31) as compared with normal subjects (n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.

Keywords: d-Lactic acid; l-Lactic acid; NBD-PZ; Column-switching HPLC; Amylose-based chiral column; Diabetic patients

Monolithic silica columns with chemically bonded β-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals by D. Lubda; K. Cabrera; K. Nakanishi; W. Lindner (pp. 892-901).

An enantioselective silica rod type chiral stationary phase (CSP) is presented; a novel combination of the well known enantiomer separation properties of β-cyclodextrin and the unique properties concerning the flow behavior of silica monoliths. Two different synthesis routes are described, and it was found that the in situ modification of a plain silica rod column turned out to be the best. The chromatographic behaviour of the β-cyclodextrin silica rod was studied and compared with a very similar commercially available β-cyclodextrin bonded particulate material (ChiraDex^®). Even if the amount of β-cyclodextrin bound to the silica rod was only about half of the amount of β-cyclodextrin bound to ChiraDex^® particles, good resolutions were achieved for a set of chiral test components like Chromakalin, Prominal, Oxazepam, Methadone and some other drugs. By taking advantage of the unique features of the silica rods relating to their flat H/u (Van Deemter) curves, fast enantiomer separations could be demonstrated.

Keywords: Silica monolith; β-Cyclodextrin (β-CD); Enantioselective stationary phase; High-performance Liquid Chromatography (HPLC); Enantioselective separation

Non-particulate (continuous bed or monolithic) restricted-access reversed-phase media for sample clean-up and separation by capillary-format liquid chromatography by Reda Jarmalavičienė; Olga Kornyšova; Douglas Westerlund; Audrius Maruška (pp. 902-908).

Restricted-access reversed-phase non-particulate (continuous bed or monolithic) stationary phases of different hydrophobicity synthesized in 100 μm i.d. fused silica capillaries have been evaluated. A specific property of restricted-access media (RAM) is that they interact with small analytes and exclude big molecules, e.g. proteins, from access to the active sites and adsorption on the surface. This dual property facilitates direct injection of biological fluids for drug or drug-metabolite analysis. Different RAM and RAM-precursor capillary columns were tested to assess the influence of chromatographic bed morphology on loadability. Inverse size-exclusion chromatography was used for investigation of pore structural properties of the capillary-format continuous beds. The data obtained were used to discuss the mechanism of separation of the biological samples using capillary columns and to propose a model for the topochemical architecture of the RAM investigated. Different morphology of the non-particulate reversed-phase precursors resulted in two types of RAM material shielded with hydrophilic polymer, classified as homogeneous or heterogeneous topochemistry stationary phases. Capillary columns were applied for chromatography of biological fluids. High resolution was obtained, without the need for column switching, when capillary columns operated in gradient conditions. Extensive evaluation of the chromatographic properties (hydrophobicity, efficiency, separation impedance, and loadability) of the non-particulate reversed-phase materials was performed before and after shielding with hydrophilic polymer to generate restricted-access properties. Minor changes of hydrophobicity, efficiency, or separation impedance were observed after the shielding.

Keywords: Capillary liquid chromatography; Continuous beds; Monoliths; Restricted-access media; Biological fluids; Direct injection technique

Hyphenation of liquid chromatography to ion trap mass spectrometry to identify minor components in polypeptide antibiotics by C. Govaerts; E. Adams; Ann Van Schepdael; J. Hoogmartens (pp. 909-921).

The application of liquid chromatography–ion trap mass spectrometry for the characterization of linear and cyclic polypeptide antibiotics was investigated. The aim was on-line identification of impurities in those antibiotic complexes without recourse to time-consuming isolation and purification procedures. Hyphenated techniques, such as liquid chromatography coupled to mass spectrometry, are ideally suited for this purpose. Characterization was performed with an ion trap mass spectrometer offering MS n capability; this enables more structural information to be obtained. Liquid chromatography in combination with ion trap mass spectrometry was successfully applied for the characterization of impurities in gramicidin, polymyxin B, polymyxin E, and bacitracin and the study of the degradation products of polymyxins B and E.

Keywords: Polypeptide antibiotics; Cyclic peptides; Liquid chromatography; Mass spectrometry; Ion trap; MS n

Design of luminescent biochips based on enzyme, antibody, or DNA composite layers by Christophe A. Marquette; Daphné Thomas; Agnès Degiuli; Loïc J. Blum (pp. 922-928).

The use of beads bearing bioactive molecules to develop generic biochips based on chemi- and electro-chemiluminescent detection was evaluated. The biochips were composed of arrayed biosensors, including enzyme-charged beads, antigen-charged beads, or oligonucleotide-charged beads, entrapped in poly(vinyl alcohol) (PVA-SbQ) photopolymer. In each case the sensing layers were spotted at the surface of a glassy carbon electrode as 0.3 µL drops, generating 500–800 µm spots. The luminescent reactions were either catalysed by horseradish peroxidase or triggered by application of a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference. Enzyme biochips were designed for the concomitant detection of choline, glucose, glutamate, lactate, lysine, and urate, based on the corresponding oxidase-charged beads and the electro-chemiluminescent (ECL) reaction with luminol-immobilised beads of the hydrogen peroxide produced. Limits of detection of 1 µmol L⁻¹ for glutamate, lysine and uric acid, 20 µmol L⁻¹ for glucose, and 2 µmol L⁻¹ for choline and lactate were found with detection ranging over three decades at least. Use of the electro-chemiluminescent biochip was extended to a tri-enzymatic sensing layer based on kinase-oxidase activity for detection of acetate. A reaction sequence using acetate kinase, pyruvate kinase, and pyruvate oxidase enabled the production of H₂O₂ in response to acetate injection in the range 10 µmol L⁻¹ to 100 mmol L⁻¹. Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realised for the model detection of human IgG. Biotin-labelled anti-human IgG were used in a competitive assay, in conjunction with peroxidase-labelled streptavidin. Free antigen could then be detected with a detection limit of 25 pg (10⁸ molecules) and up to 15 ng. In a similar way, the use of oligonucleotide-immobilised beads enabled the realisation of DNA-sensitive biochips which could be used to detect a biotin-labelled sequence al a level of 5×10⁸ molecules.

Keywords: Biochip; Chemiluminescence; DNA array; Electrochemiluminescence; Enzyme array; Photopolymer; Protein array

Enhanced sensitivity electrochemical assay of low-molecular-weight heparins using rotating polyion-sensitive membrane electrodes by Wei Qin; Wei Zhang; Kang Ping Xiao; Mark E. Meyerhoff (pp. 929-936).

Use of a novel rotating polycation-sensitive polymer membrane electrode yields sensors that can serve as simple potentiometric titration endpoint detectors for the determination of three FDA approved low-molecular-weight heparin (LMWH) anticoagulant drugs (Fragmin, Normiflo, and Lovenox). The rotating electrode configuration dramatically improves the reproducibility and increases the sensitivity for LMWH determinations by protamine titration. At a rotation speed of 3000 rpm, electrodes with optimized thin (50 µm) polymer membranes doped with dinonylnaphthalene sulfonate (DNNS) respond to low levels of protamine (<2 µg mL⁻¹) with good precision (±1 mV, N=10), when protamine is infused continuously into a Tris-buffer solution, pH 7.4. When infusing protamine (at 5 µg min⁻¹) continuously into solutions containing Fragmin, a clear endpoint is obtained, with the amount of protamine required to reach this endpoint proportional to the level of Fragmin present. A detection limit of less than 0.02 U mL⁻¹ Fragmin can be obtained via this new method, approximately one order of magnitude lower than that previously reported based on a non-rotating polycation electrode. Similar low detection limits can be achieved for potentiometric titrations of Normiflo and Lovenox. Such titrations can also be carried out in undiluted plasma samples containing the various LMWH species. In this case, detection of the LMWHs at clinically relevant concentrations (>0.2 U mL⁻¹) can be readily achieved.

Keywords: Polyion sensor; Low-molecular-weight heparins; Rotating electrode

How experimental design can improve the validation process. Studies in pharmaceutical analysis by S. Furlanetto; S. Orlandini; P. Mura; M. Sergent; S. Pinzauti (pp. 937-944).

A critical discussion about the possibility of improving the method validation process by means of experimental design is presented. The reported multivariate strategies concern the evaluation of the performance parameters robustness and intermediate precision, and the optimisation of bias and repeatability. In particular, accuracy and precision improvement constitutes a special subset of experimental design in which the bias and the relative standard deviation of the assay are optimised. D-optimal design was used in order to plan experiments for this aim. The analytical methods considered were capillary electrophoresis, HPLC, adsorptive stripping voltammetry and differential pulse polarography. All methods were applied to real pharmaceutical analysis problems.

Keywords: Validation; Experimental design; Accuracy; Precision; Robustness testing

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Analytical and Bioanalytical Chemistry (v.377, #5)