Biomedical Chromatography

Bartlett, Michael G.; Imai, Kazuhiro; Li, Famei; Lim, Chang Kee

doi: 10.1002/bmc.679pmid: N/A

Mikšík, Ivan; Sedláková, Pavla; Mikulíková, Kateřina; Eckhardt, Adam; Cserhati, Tibor; Horváth, Tibor

doi: 10.1002/bmc.640pmid: 16779791

This article gives an overview of uncommon replaceable matrices (gels) for capillary gel electrophoresis. This electrophoretic technique is useful mainly for the separation and analysis of biopolymers—nucleic acids and their fragments, and proteins/peptides. Commonly used gels are not reviewed. Those mentioned and discussed here are gels containing saccharides, newly developed acrylamide‐based gels and thermoadjustable viscosity polymers, namely triblock copolymers and grafted polyacrylamide. Copyright © 2006 John Wiley & Sons, Ltd.

Srinivas, Nuggehally R.

doi: 10.1002/bmc.680pmid: 16779774

The concepts of drug development have evolved over the last few decades. Although number of novel chemical entitities belonging to varied classes have made it to the market, the process of drug development is challenging, intertwined as it is with complexities and uncertainities. The intention of this article is to provide a comprehensive review of novel chemical entities (NCEs) that are substrates to cytochrome P450 (CYP) 2D6 isozyme. Topics covered in this review aim: (1) to provide a framework of the importance of CYP2D6 isozyme in the biotransformation of NCEs as stand‐alones and/or in conjunction with other CYP isozymes; (2) to provide several case studies of drug disposition of important drug substrates, (3) to cover key analytical perspectives and key assay considerations to assess the role and involvement of CYP2D6, and (4) to elaborate some important considerations from the development point of view. Additionally, wherever applicable, special emphasis is provided on chiral drug substrates in the various subsections of the review. Copyright © 2006 John Wiley & Sons, Ltd.

Gregg, Shonetta D.; Fisher, Jeffrey W.; Bartlett, Michael G.

doi: 10.1002/bmc.659pmid: 16779790

Jet propellant 8 (JP‐8) is a complex mixture of compounds that varies from batch to batch. Quantification of various compound classes of JP‐8, including BTEX, PAHs and VOCs, has been accomplished. Very few papers have tackled total JP‐8 quantification because of its complexity. The components in JP‐8 tend to co‐elute and present at low concentrations, often nondetectable. JP‐8 is the major source of chemical exposure for Department of Defense personnel and a potential hazard for civilians and marine animals. Some components of JP‐8 have been identified as possible human carcinogens and have been studied extensively. Development of analytical methods to analyze the components of this fuel are essential to measure the extent of exposure, as well as the short‐term and long‐term exposure in rodents, humans and marine life. To date, JP‐8 has been examined in urine, blood, contaminated water and fish tissue. This paper reviews methods currently utilized in the literature for the analysis of JP‐8 and its components. This paper also discusses extraction methods and detectors commonly used in JP‐8 and hydrocarbon analysis in general. Finally, the effects of exposure and the future of JP‐8 and petroleum analysis with respect to human health are discussed. Copyright © 2006 John Wiley & Sons, Ltd.

Dixon, Steven P.; Pitfield, Ian D.; Perrett, David

doi: 10.1002/bmc.672pmid: 16779789

‘Multi‐dimensional’ liquid separations have a history almost as long as chromatography. In multi‐dimensional chromatography the sample is subjected to more than one separation mechanism; each mechanism is considered an independent separation dimension. The separations can be carried out either offline via fraction collection, or directly coupled online. Early multi‐dimensional separations using combinations of paper chromatography, electrophoresis and gels, in both planar and columnar modes are reviewed. Developments in HPLC have increased the number of measurable analytes in ever more complex matrices, and this has led to the concept of ‘global metabolite profiling’. This review focuses on the theory and practice of modern ‘comprehensive’ multi‐dimensional liquid chromatography when applied to biomedical and pharmaceutical analysis. Copyright © 2006 John Wiley & Sons, Ltd.

Flanagan, R. J.; Morgan, P. E.; Spencer, E. P.; Whelpton, R.

doi: 10.1002/bmc.671pmid: 16779788

This paper discusses new developments in plasma micro‐extraction techniques in the context of established micro‐extraction and protein precipitation methodology. Simple liquid–liquid solvent extraction (LLE) of plasma with direct GC or HPLC analysis of the resulting extract has been used for many years. Butyl acetate and methyl t‐butyl ether (MTBE) give efficient extraction of many drugs and metabolites from small volumes of plasma or whole blood at an appropriate pH, and form the upper layer, thus simplifying extract removal. Butyl acetate does not interfere with NPD, ECD or MS in GC, whilst MTBE has a relatively low UV cutoff (220 nm). Thus, HPLC eluents that use a high proportion of an organic component allow MTBE extracts to be analysed directly. ‘Salting‐out’ and extractive derivatization using acetic anhydride or phenylboronic acid can be used with appropriate analytes. As regards protein precipitation, an important consideration is lowering the pH, although this is not feasible with acid‐labile analytes. More recent developments include sold‐phase micro‐extraction (SPME) and liquid‐phase micro‐extraction (LPME). This latter technique especially may prove invaluable as analytes that cannot easily be extracted with LLE can be isolated simply at low cost with a minimum of apparatus. Copyright © 2006 John Wiley & Sons, Ltd.

Lamari, Fotini N.; Theocharis, Achilleas D.; Asimakopoulou, Athanasia P.; Malavaki, Christina J.; Karamanos, Nikos K.

doi: 10.1002/bmc.669pmid: 16779785

Chondroitin sulfate (CS) is a linear heteropolysaccharide consisting of repeating disaccharide units of glucuronic acid and galactosamine, which is commonly sulfated at C‐4 and/or C‐6 of galactosamine. The administration of CS as a supplement or a drug for the treatment of osteoarthrosis, the prevention of subsequent coronary events, treatment of psoriasis and ophthalmic diseases has been suggested. Much debate on the metabolism of CS and therefore the effectiveness of these treatments, especially after oral administration, has arisen due to the macromolecular nature of CS. Difficulties in analysing CS in blood due to the low endogenous concentrations and the covalent and anionic complexes with proteins have hampered the resolution of these issues. In this review, the information on the pharmacokinetics of CS obtained from studies in experimental animals and in humans is presented. Emphasis has been given to the analytical methods used for the determination of glycosaminoglycans, intact CS and CS‐derived disaccharides in blood serum and plasma. Copyright © 2006 John Wiley & Sons, Ltd.

Johnson, Deborah‐Ann; Smith, Kevin D.

doi: 10.1002/bmc.641pmid: 16779786

One of the most ubiquitous plasma proteins, α‐1‐acid glycoprotein (AGP), has a high affinity, low capacity binding for basic drugs positively charged at physiological pH. Moreover, as an acute phase protein its level is increased in various disease states in a manner that is likely to influence the free plasma level of a drug, the ability to attain minimum effective concentration and overall in vivo effectiveness. AGP is a glycoprotein known to display disease specific changes in glycosylation and although this secondary modification is not directly involved in drug binding, it may influence the conformation of the binding site. Binding studies reveal that α‐1‐acid glycoprotein bind mainly to the tuberculosis drugs: rifampicin; isoniazid; pyrazinamide; p‐aminosalicylic acid; capreomycin; ethionamide; levofloxacin and ofloxacin out with the therapeutic plasma range tested. These results are however still considered significant as not only are α‐1‐acid glycoprotein levels increased during the acute phase response but specific α‐1‐acid glycoprotein from tuberculosis samples are subject to glycosylation changes which can increase the binding affinity and cause binding to occur at the therapeutic concentration. Copyright © 2006 John Wiley & Sons, Ltd.

Bhushan, Ravi; Agarwal, Rachna

doi: 10.1002/bmc.650pmid: 16779784

Arachin and its molecular species (arachin I and arachin II) were separated and isolated. The number and kind of subunits of arachin, arachin I and arachin II were determined. Studies were carried out under different experimental conditions using slab gel electrophoresis, size‐exclusion chromatography and reversed‐phase high‐performance liquid chromatography. Gel electrophoresis was done under varying concentrations of resolving gel. Tube gel as well as slab gel electrophoresis were used and continuous as well as discontinuous buffer systems were used for both types of electrophoresis. In addition, the subunits were separated by reversed‐phase HPLC using a gradient program. Arachin and arachin II were found to have 12 subunits each while arachin I showed six subunits. The subunits of arachin I were allowed to reconstitute by removing SDS. Eight combinations were tried for studying the reconstitution pattern. Molecular weight and weight ratio in each case were also determined. Copyright © 2006 John Wiley & Sons, Ltd.

Catimel, Bruno; Nice, Edouard C.; Kärrlander, Maria; Ross, Janine; Catimel, Jenny; Burgess, Antony W.; Faux, Maree

doi: 10.1002/bmc.648pmid: 16779787

Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high‐specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno‐affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies. Copyright © 2006 John Wiley & Sons, Ltd.