ISSN:0003-2654    

DOI:10.1039/AN99318FX005

出版商:RSC    

年代:1993

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN99318BX007

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, FEBRUARY 1993, VOL. 118 1SN Editorial Changes to Editorial Staff on Analytical Journals: The Analyst, Analytical Proceedings and Journal of Analytical Atomic Spectrometry At the end of February 1993 Judith Egan, who is currently the Editorial Manager (Analytical) and Editor of JAAS is leaving, after 14% years at the RSC, the last 4% as Manager. Her replacement will be Janice Gordon, who has been with the RSC since 1979, from 1981 onwards her work being on Analytical Abstracts. However, Judith is not disappearing from the analytical chemistry community altogether. She is getting married and moving to Southern Germany and has been appointed European Associate Editor for J A A S , with a role similar to that of Dr. Julian Tyson in the USA for The Analyst. Also, in order to maintain and improve times to publica- tion, she will be in a position to respond to fluctuating work loads by assisting the full-time staff with editorial work, on The Analyst and J A A S , as and when the need arises.Hence, the future arrangements for the journals appear favourable. We are in an excellent position to reduce our times to publication, and with US and European Associate Editors plus the experi- enced full-time staff in Cambridge, can continue to offer a competent and valuable service to all authors world-wide. If you have any queries or comments about the journals, or the new editorial arrangements, please do not hesitate to contact the editorial office. Cover illustration: Three-dimensional representation of a sample using a coulometric array EC detector.ANALYST, FEBRUARY 1993, VOL.118 1SN Editorial Changes to Editorial Staff on Analytical Journals: The Analyst, Analytical Proceedings and Journal of Analytical Atomic Spectrometry At the end of February 1993 Judith Egan, who is currently the Editorial Manager (Analytical) and Editor of JAAS is leaving, after 14% years at the RSC, the last 4% as Manager. Her replacement will be Janice Gordon, who has been with the RSC since 1979, from 1981 onwards her work being on Analytical Abstracts. However, Judith is not disappearing from the analytical chemistry community altogether. She is getting married and moving to Southern Germany and has been appointed European Associate Editor for J A A S , with a role similar to that of Dr. Julian Tyson in the USA for The Analyst. Also, in order to maintain and improve times to publica- tion, she will be in a position to respond to fluctuating work loads by assisting the full-time staff with editorial work, on The Analyst and J A A S , as and when the need arises. Hence, the future arrangements for the journals appear favourable. We are in an excellent position to reduce our times to publication, and with US and European Associate Editors plus the experi- enced full-time staff in Cambridge, can continue to offer a competent and valuable service to all authors world-wide. If you have any queries or comments about the journals, or the new editorial arrangements, please do not hesitate to contact the editorial office. Cover illustration: Three-dimensional representation of a sample using a coulometric array EC detector.

ISSN:0003-2654    

DOI:10.1039/AN993180015N

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, FEBRUARY 1993, VOL. 118 17N Book Reviews Atomic Absorption Spectrometry. Theory, Design and Applications Edited by S. J. Haswell. Analytical Spectroscopy Library. Volume 5. Pp. xx + 530. Elsevier. 1991. Price US$177.00; Df1345.00. ISBN 0-444-8821 7-0. This book is based on the successful book of similar title, edited by John Cantle, which appeared some ten years ago and has a not dissimilar format. However, this volume has been extensively re-written (over SO%, according to the preface) and is not just an updated version of the earlier work, but a new book. The first two chapters provide a brief, but clear introduction to the basic theory and the instrumental requirements for the practice of atomic absorption spectrometry, whereas the third chapter deals with the basic practical aspects of the technique.These chapters justify the claim that ‘the book represents a complete text on AAS’, but in view of the depth of coverage that can be given in the relatively short space available (71 pp. for the three chapters), the absence of references in Chapters 2 and 3 is both puzzling and disappointing. In some cases the absence of references could be misleading to the less experienced user; for example the table of sensitivities, which is highly instrument dependent, is presented with no descrip- tion of the equipment used to generate the data. Figures for salt tolerance of burner systems are also presented with a similar lack of detail and are much poorer than can be reached with better designed nebulizer-burner systems. The following chapters deal with applications and com- mence with an excellent and well-referenced chapter on the analysis of waters, sewage and effluents, which covers the entire field from sampling to quality control protocols and is essential reading for anyone entering this field of analysis.The use of AAS and other methods of atomic spectroscopy in marine analysis is covered in Chapter 5 , which is an excellent summary of this field of analysis but is marred by the inclusion of a table of detection limits based on material that is not only 15-20 years old, but incorrectly referenced for good measure! The analysis of airborne particles in workplace atmospheres and the analysis of foods are covered by two readable and comprehensive chapters, which contain sufficient information for the procedures described to be implemented without recourse to the extensive references.The use of AAS in metallurgy is detailed in chapters dealing with ferrous metallurgy, which is very comprehensive with references up to 1989 and one on non-ferrous metallurgy, which is somewhat shorter, but still most informative and worth reading. Applied geochemistry is dealt with in a chapter that is a little less up-to-date than most of the others, but this does not detract from its usefulness, as the whole analytical process is described in detail and most of the more recent developments in this area have been in the field of ICP-OES (or ICP-MS), rather than in AAS. The chapter on the use of AAS in the petroleum industry clearly illustrates the limitations as well as the advantages of the technique and contains references as recent as 1991.The analysis of glass and ceramics is described in a short chapter, which details a thoroughly competent method for such analysis. Clinical analysis is dealt with in an informative chapter describing the rapid FAAS methods used in the clinical laboratory and this is followed by a long chapter dealing with body fluids and tissues using ETAAS. The two chapters compliment each other well and are referenced up to 1988. The chapter on forensic analysis is written in an entertaining style but is still informative to the non-specialist. The analysis of a wide range of chemicals and related products is dealt with in a chapter with nearly 300 refs., so that even if there is insufficient detail to implement a procedure described in the text, the reader should have little difficulty in finding additional reading.The final chapter on the analysis of polluted soils gives a general introduction to the subject, followed by useful notes on individual elements. Despite of some minor criticisms this is a well-produced book that should be useful to anyone working in the field of trace metal analysis even if they are not an AAS specialist, as great care has been taken to describe both the procedures that can be used along with limitations that are found in practice. It will also be useful to the specialist working in one field who is sometimes called upon to perform work outside of their normal expertise, an increasingly common situation these days! By current standards for scientific books, this one is not unreasonably priced and I have no hesitation in recommend- ing that it should be added to the library of anyone who uses atomic absorption spectrometry.Colin Watson Liquid Chromatography Column Theory By R. P. W. Scott. Separation Science Series. Series Edited by R. P. W. Scott and C. F. Simpson. Pp. x + 279. J. Wiley and Sons. 1992. Price f34.95. ISBN 0-471-93305- 8. A well written book should provide for the reader a comprehensive and lucid coverage of the title subject. Ray Scott has provided here all the algebra and calculus that chromatographers interested in column design and perfor- mance could want in a logically developed format. Following a brief introductory chapter on concepts and nomenclature, Chapter 2 introduces Plate Theory to define solute retention volumes, capacity and separation ratios.No modern text on column chromatography would seem to be complete without a discussion on dead volumes, the subject of Chapter 3. Whilst not resolving the controversies, a useful discussion of the different volumes is provided. Chapters 4 and 5 return to Plate Theory to provide a detailed description of the chromatographic processes themselves, including peak shape and column efficiency, resolving power and peak capacity. These chapters are heavily loaded with mathematics, which I assumed to be correct, and I found them somewhat unrewarding. The generality of Plate Theory is, however, well demonstrated by its extension to the description of the properties of the heat of absorption detector, although J suspect this section will have only a minority appeal.Chapter 6 introduces Rate Theory, which can be used to explain the dispersion processes that occur during a separation and to explore factors that can be optimized so as to maximize efficiency. This, and the subsequent chapters describing the van Deemter equation and alternative equations for peak dispersion (e.g. , Giddings and Knox) provided for me a much more practical insight into prospects for improving chromato- graphic performance. Here we find a lucid explanation of why higher efficiencies require larger, not smaller, particles-a concept many chromatographers still struggle with. Chapter 9 examines the experimental validity of the various Rate Theory equations, leading to the conclusion that the van Deemter equation remains the most appropriate for column design purposes.Chapter 10 concludes the examination of dispersion processes by looking at extra-column dispersion. This is a very readable chapter with good practical information on items such as connecting tubing parameters and configurations. The final four chapters discuss the practical processes of designing liquid chromatography columns; packed, open18N ANALYST, FEBRUARY 1993, VOL. 118 tubular and preparative. These are valuable chapters for the practical chromatographer as well as the column designer. Equations and graphs describe the relationships between separation difficulty and variables such as particle size, column length and radius, and solvent consumption.I looked in vain, however, for a discussion on tapered columns. It is interesting to recognize that, with an optimized column system, even the most difficult separation should require no more than 7.5 cm3 of mobile phase. For each type of column a BASIC computer program is presented that incorporates the essential design equations and which will provide the design and operating conditions to enable a given separation to be achieved in the minimum time and with the minimum solvent consumption. The book concludes with a list of symbols (but be careful as the symbols are not unique) and three appendices of physical data. References are provided at the end of each chapter although I found them to be not particularly up-to-date. The extensive mathematics inevitably will make the book heavy reading for many.It is then even more unfortunate that there are many errors of spelling, grammar, syntax and typography, no more evident than in Chapter 1. The presentation of the book is also spoiled by the use of a dot matrix printer, with varying numbers of characters per inch and lines per page. Nevertheless, there is much useful information to be discovered and the book should appeal to those enthusiasts interested in designing their own columns or selecting optimum column or operating parameters for a particular separation. John C. Berridge Analyses of Hazardous Substances in Air. Volume 1 Edited by A. Kettrup. Pp. 224. VCH. 1991. Price DM98.00. ISBN 3-527-2701 5-9 (VCH, Weinheim); 0-89573-901 -1 (VCH, New York). Almost thirty years ago, the Deutsche Forschungsgemein- schaft created the Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area. Part of the work of the Analytical Chemistry Working Group established by the Commission has been to recommend methods for the analysis of hazardous substances in air.Criteria for the selection of methods include suitability for routine use, reliability, and meeting requirements for quality control, and for these reasons the methods are recommended under German legislation. The volume under review results from an initiative of the Commission to make its methods available to an international audience. The preface to the book makes clear that the Working Group’s philosophy has not necessarily been to seek simple, cheap and quick methods.Preliminary chapters cover the theory of diffusion samplers, although I would have liked to see more information on their practical use, in what is essentially a practical text. Indeed, the subsequent analytical methods include details. of active sampling techniques exclu- sively. It appears that the Working Group require more tests of diffusion sampling in environments where mixtures may compete for adsorption. The first part of the book also includes details on statistical evaluation of the methods and quality indexes. Most of the book is devoted to practical methods. Infrared spectrophotometric determination of gases and vapours using portable long-pathlength cuvettes is described, with details of sampling options, calibration and calculation of results.The important role of atomic absorption spectrometry and gas and high-performance liquid chromatography are highlighted in the Preface, and three-quarters of the book is devoted to descriptions of their use. Curiously, only one method (for hexamethylene diisocyanate) using the last-named technique is described in the book, with the main emphasis being on gas chromatographic methods. Methods for the determination of ten individual or groups of substances are described, including dimethylamine, phenol, ethylene glycol derivatives and poly- cyclic aromatic hydrocarbons. Inevitably chapters of this type appear repetitive, but are comprehensive in detail. Finally, methods for the measurement of Pb, Ni, Co and Cr by atomic absorption spectrometry are described. By present day standards, the price of this book is not excessive and justifiably will find its way onto the bookshelves of many involved with the analysis of atmospheres.R. S. Barratt Gas-Liquid-Solid Chromatography By V. G. Berezkin. Chromatographic Science Series. Volume 56. Marcel Dekker. New York. 1991. Pp. viii + 231. Price US$99.75 (US and Canada); US$114.50 (all other countries). ISBN 0-8247-8425-1. Many practitioners of gas-liquid chromatography (GLC) are aware that the stationary liquid is not the only important item in a GLC column. The supposedly inert solid support can also have several major effects on analyses. Firstly, retention contributions often arise from adsorption by the support surface or gas-liquid interface. Secondly, the structure of the support controls the liquid distribution and hence the column efficiency.Thirdly, the support sometimes causes irreversible adsorption or catalytic activity. Finally, the supported liquid tends to age with time. These factors can all interfere with peak identification, qualitative and quantitative analysis, preparative and production scale separations, and with the measurement of equilibrium and other physico-chemical properties by gas chromatography. The cloud, however, also has several silver linings. The selectivity of a liquid phase can be improved by combining it with the powerful selectivity of a solid surface in the form of a modified active adsorbent. Adsorption at gadliquid and liquid/solid interfaces can advan- tageously be studied in GLC systems.In the first chapter of the book, Berezkin argues that these effects are so prevalent in GLC that the technique should be renamed ‘gas-liquid-solid chromatography (GLSC)’. In the remaining eight chapters he reviews the present state of knowledge of each effect in turn, including both solutions to problems caused by support effects and the exploitation of the effects for selectivity and physico-chemical study. Both packed and capillary columns are considered. The author is an established authority in the field; coverage of work over many years in the Soviet Union is comprehensive, of that in the West a little less so. The content is sound, except for the neglectful omission of the sorption effect in finite concentration equa- tions. The material is well organized and presented, though some repetition could have been avoided.Despite inadequate proofreading the English is good on the whole and only occasionally a barrier to comprehension. It is a pity the index is so sketchy and a list of symbols absent. The book emphasizes the fundamentals of GLSC but the practical implications are made clear. Each chapter normally begins at the level of basic chromatographic principles, with standard equations recapitulated where necessary. The book will be of much interest to specialists in the GLSC area of GC for whom it provides an excellent survey of an important and extensive body of work that is less widely known outside Eastern Europe than it should be. More generally the book should also be comprehensible to all chromatographers with a good basic understanding of GC wishing to study the role of the support in depth.J . R. ConderANALYST, FEBRUARY 1993, VOL. 118 19N FT-ICR/MS: Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Edited by Bruce.Asamoto. Pp. x + 306. VCH. 1991. Price DM134.00; f47.00. ISBN 0-90573-767-1 (VCH Publishers); 3-527-27919-9 (VCH Verlagsgesellschaft). Most people who make use of mass spectrometry to solve problems in analytical chemistry are aware of the advantages offered by FT-ICWMS. The technique can provide high mass analysis, high resolution and mass accuracy, fast scan times, positive and negative ion capability and compatibility with pulsed ionization techniques-almost a wish list for the analyst. Yet very few of us have ever seriously considered it as a routine analytical technique presumably because of its perceived difficulties. There has been an obvious need for those workers using FT-ICWMS to describe how the tech- nique is proving of benefit in their work in order that the rest of the analytical community can assess its utility, versus other MS methods, for specific applications.This publication, edited by Asamoto, attempts to fill this gap by detailing some of the applications to which FT-ICR/MS has been applied. Two introductory chapters cover, respec- tively, the historical development of FT-ICR and other techniques based on the cyclotron principle of mass analysis, and a detailed review of the range of FT-ICR instrumentation currently available. These chapters are well written, manage to explain the technique and the equipment without the use of a highly mathematical approach and provide an excellent introduction to FT-ICR.As the stated aim of this book is to emphasize work being done to solve real problems, most of the authors of the eight following chapters on the applications of FT-ICWMS are, like the editor, involved in industrial analysis. This occasionally means that the amount of detail given about any analysis is less than would be provided by an academic author but this reviewer did not find this to be a problem. One chapter covers the linking of modern sample handling and ionization tech- niques with FT-ICWMS, and there are specific chapters on GUMS, laser desorption using a pulsed C 0 2 laser, surface analysis by ultraviolet laser microprobe, chemical ionization and on peptide analysis.The final chapter by a group from American Cyanamid describes the use of their laboratory- built instrument to solve problems arising from the agrochem- ical and biotechnology areas. These chapters stress the advantages to be gained from the unique features of FT-ICW MS but do not neglect to point out where difficulties have been encountered. Despite the excellent appearance of the book (it is fully typeset), there are errors in certain of the tables and figures. I would point specifically to Tables 3.1 and 9.5 where several of the ionic formulae are clearly wrong, and to Fig. 7.4 where the ion at mlz 325 is incorrectly assigned in the legend. These are, however, only minor quibbles.This is a first-rate book and I recommend it without reservation both to the general analytical chemistry community and to all users of mass spectrometry. The editor and his co-authors should be congratulated on achieving the aims they set themselves and on producing such a readable and timely publication. John J . Monaghan Principles of Polarography By R. C. Kapoor and B. S. Aggarwal. Pp. xi + 185. Wiley. 1991. Price f28.50. ISBN 0-470-21732-4. The University Grants Commission, New Delhi, provided the incentive and a grant for the writing of this book, which was a collaborative effort between a senior author and a young chemist for the benefit of honours and postgraduate students of chemistry in Indian Universities. It contains 13 chapters and some 180 pages.It has been published by Wiley Eastern Ltd. in a compact 9 X 6 inch edition and overall, I found it written in an easy-to-read style and of value to students of the subject whether in higher education or in industry. The majority of the text is concerned with polarography (€15 pp.). Chapter 1 introduces the subject, Chapter 2 is concerned with polarographic apparatus, Chapter 3 with currents in polarography such as charging, migration, diffu- sion, kinetic, catalytic and adsorption currents. Reversible systems are dealt with in Chapter 4, and irreversible systems in Chapter 6 together with their associated mathematics. Chapter 5 deals with the polarography of complex (as in metal complex) systems and derives mathematical relationships from which for example overall stability constants can be calculated.This is an area of study in which the authors themselves have been actively involved, e.g. , Kapoor and Aggarwal, Indian J. Chem., 1972, 11, 71, on Ce”’ and Ce’” complexes of N , N-dihydroxyethylglycine. Chapters 7 and 8 concern themselves further with kinetic, catalytic and adsorp- tion currents whereas Chapters 9 and 10 deal with polaro- graphy in non-aqueous solvents and polarography of organic compounds, respectively. Chapter 11 is entitled Other Indicator Electrodes and is involved with the construction and use of the hanging mercury drop electrode, platinum, gold and carbon electrodes, etc. Special polarographic techniques are the subject of Chapter 12 with pulse and differential-pulse polarography receiving only four pages of consideration, square-wave polarography only one and stripping techniques only three.This is hardly in proportion to their current usage. The book concludes with a chapter on amperometry. Pages 164 and 165 mention the older polarographic literature and little mention is made of more recent publications, particularly in the applications area. The Appendix on polarographic characteristics of metal ions, anions and organic compounds could have been added together with the addition of a table concerned with stripping voltammetry of metal ions and organic molecules. W. F. Smyth HPLC in the Pharmaceutical Industry Edited by Godwin W. Fong and Stanley K. Lam. Drugsand the Pharmaceutical Sciences. Volume 47. Pp. viii + 309. Marcel Dekker. 1991. Price US$99.75 (US and Canada); US$114.50 (all other countries).ISBN 0-8247-8499-5. This volume has been prepared in 4 parts each containing between two and four chapters. Part One is entitled ‘Contemporary LC Techniques in Pharmaceutical Analysis’ and has three chapters. The opening chapter by P. Kucera and N. Licato, ‘High Speed HPLC using Short Columns Packed with 3 vm Particles’ discusses theoret- ical aspects and design of instrumentation for high-speed separations. The importance of equipment specifically desig- ned for high-speed separations is particularly emphasized by considering the dispersion processes that operate within different parts of a system. The title of this chapter might be considered misleading since the content includes much material concerned with microbore columns. However, since this does not interfere with Chapter 2 and it certainly enhances this chapter, this reviewer would only suggest a change of title.Chapter 2 by T. V. Raglione and R. A. Hartwick concerns ‘Microbore HPLC in Pharmaceutical Analysis’. This chapter essentially reviews the advantages of microbore systems such as improved mass sensitivity for bioanalytical applications and the facility to interface with other detection systems. Chapter 3 by F. K. Chow concerns ‘Column Switching Techniques in Pharmaceutical Analysis’. This is an unusual but worthwhile topic to include in such a publication on HPLC20N ANALYST, FEBRUARY 1993, VOL. 118 and discusses various column and valve geometries that can be used for chromatographing analytes in complex matrices.Part Two is entitled ‘Specialized Detection Techniques’ and includes four chapters. Chapter 4 by P. T. Kissinger and D. M. Radzik discusses and provides several examples of applica- tions using electrochemical detectors and Chapter 5 similarly describes radiochemical detectors. In Chapter 6, L. Huber and H. P. Fielder describe the photodiode array detector and using antibiotic examples they discuss the various data manipulations that are possible with the information collected by this detector. Chapter 7 by K. S. and V. F. Kalasinsky concerns HPLC coupled with FTIR. Interface designs are described that involve solvent removal, especially water, from eluate frac- tions and deposit analytes on a suitable substrate for diffuse reflectance spectroscopy.Part Three includes two chapters on ‘Automation in Pharmaceutical Analysis’. Chapter 8 by W. A. Hanson concerns ‘Applications of HPLC to Dissolution Testing of Solid Dosage Forms’. Advantages of monitoring dissolution samples by HPLCp. are provided, such as for products containing low concentrations of active ingredients, products with excipients that can interfere with other analytical methods and multicomponent products. Chapter 9 by R. A. Felder concerns ‘Robotic Automation of HPLC Labora- tories’. Different types of laboratory robot are defined and, in particular, their use in sample preparation is discussed. Part Four includes two chapters on ‘HPLC of Peptides, Proteins and Enantiomeric Drugs’. Chapter 10 by K. H. Bui concerns LC resolution of enantiomers.Various column and mobile phase combinations are reviewed, the advantages and disadvantages of systems based both on chiral and non-chiral stationary phases being discussed. Chapter 11 by K. Benedek and J. K. Swadesh concerns HPLC of proteins and peptides. This chapter relates method development with peptide structure including conformation and presence of prosthetic groups. In addition, problems of denaturation and peptide heterogeneity are discussed. This book provides excellent reviews of several aspects and applications of HPIX of pharmaceutical interest many of which are not normally to be found within textbooks on HPLC. Criticisms include: ( a ) some chapters contained several typographical errors, some of which could be quite misleading; (b) many of the figures are taken from previous publications and in some cases the legends are inadequate to satisfactorily explain them (this is presumably because addi- tional explanatory details were provided elsewhere in their original publications); and (c) in some chapters references are not sufficiently up to date.In spite of these shortcomings it is considered that this volume will be of great value particularly to analysts with experience of HPLC as an introduction to some specialized techniques used in pharmaceutical analysis. G. P. R . Carr A Handbook of Silicate Rock Analysis By P. J. Potts. Pp. x + 622. Blackie. 1991. Price f45.00. ISBN 0-21 6-93209-2. The fact that P. J. Potts’ ‘A Handbook of Silicate Rock Analysis’ has appeared in paperback, some five years after the original hardcover edition, probably says more about its merits than any review.Those geoanalysts who baulked at the hardcover price now have no excuse but to acquire this outstanding volume. Potts’ subject matter is extensive and his coverage encyclo- paedic. He is able to convey a general message without sacrificing a sense for detail. The result is a wcll-balanced work, conveying the concepts of geoanalytical techniques on the one hand, whilst providing a wealth of procedural detail on the other. Potts is a diligent writer, able to communicate scientific principles clearly without resorting to simplification. He prefaces the book by noting that the introduction of microcomputers and the resultant lack of interaction between user and machine has encouraged a ‘black box’ attitude towards analytical chemistry.His stated purpose has therefore been to provide an appreciation of what happens between ‘samples in’ and ‘results out’. He claims: ‘All analytical techniques available for routine silicate rock analysis are discussed. Sufficient detail is included to provide practitioners of geochemistry with a firm base from which to assess current performance, and in some cases, future developments’. This is no mean claim and the book lives up to it admirably. It is a remarkable foundation for those involved in the analysis of silicate rocks and is ideal as a course text. However, this is primarily a book for the analyst, written from a laboratory perspective. As such it is eminently practical. Its focus is on contemporary geoanalytical tech- niques, and 17 of its 20 chapters deal with specific instrumental techniques.In general the level of detail is appropriate to the subject, although in some cases I would have hoped for more. For example, in the wavelength-dispersive XRF section, the measurement of background is discussed but not the problem of needing to know the background at the peak position, but being unable to measure this in the sample itself. Potts’ approach necessarily results in an emphasis on specific procedures at the expense of a more holistic approach to methodologies. The choice is logical, given the ‘Handbook’ format, and Potts does devote 40 pages to general concepts, including sampling, contamination, reference materials and analytical data reporting in his opening chapter. There is an excellent expusk on ‘detection versus determina- tion limits’, which should be requircd reading for all who set foot in a geoanalytical laboratory. Typically, this focuses on the limits associated with an individual technique rather than those associated with a complete ‘method’ ( i e . , including sample preparation, preconcentration, measurement and data manipulation). In terms of production quality, the book has a clean presentation style for its text and the large, 8 X 11 in, format reduces the amount of page-turning required by the reader within each section. The binding could be stronger for a work that will be referenced frequently. The figures are generally well-presented; however, despite the large format, the layout of tables is frequently poor. Considering that this is essentially a second printing it is disconcerting to find a number of typographical errors. Generally speaking, the five years since this book first appeared have done little to date it. However, it is unfortunate that there are no references to ICP-MS work later than 1983 and that the text is necessarily light on recent advances in robotics. Of all the recently published books with relevance to geoanalysis, I can think of none to equal Potts. Chris Riddle

ISSN:0003-2654    

DOI:10.1039/AN993180017N

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

21N ANALYST, FEBRUARY 1993, VOL. 118 Conference Diary Date Conference March 8-1 2 9-10 16-17 19 23-25 23-26 25 29-30 29-30 30 30-1/4 April 3-4 4-8 5-7 5-8 5-9 6-7 Location PITTCON ’93,44th Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy Atlanta, GA, USA Industrial Waste Water Treatment Esher, UK Groundwater Pollution London, UK Symposium on Possibilities and Limitations of Antwerp, Chiral Separation Techniques Belgium International Symposium on Advanced IR Spectroscopy (AIRS) Japan Tokyo? 7th International Symposium on Instrumental Brighton, Planar Chromatography Sussex, UK Instrumental Technique Developments: Cambridge , Atomic Spectrometry Updates Atomic UK Spectroscopy Group Good Automated Laboratory Practice Barcelona, Symposium Spain 1993 European Seminar on Offshore Water and Environmental Management UK The Laboratory Environment: Working with Dangerous Substances London, UK 12th Pharmaceutical Technology Conference Elsinore, Denmark VDI-Meeting on Progress in Thermic, Catalytic and Sorptive Exhaust Cleaning Mannheim, Germany XIIIth World Congress on Occupational Safety New Delhi, and Health India 3rd International Conference on Ion-Beam and Namur, Surface Specific Analysis Techniques Belgium Annual Chemical Congress, ‘New Materials: New Toxicology’ UK Southampton , 4th International Meeting on Trace Elements in Chamonix, Medicine and Biology France BTS Colloquium (of the British Toxicology Society) on Early Markers of Carcinogenesis Canterbury, Kent, UK Contact Mrs.Alma Johnson, Program Secretary, Pittsburgh Conference, 300 Penn Center Boulevard, Suite 332, Pittsburgh, PA 15235-5503, USA Tel: +1 412 825 3220.Amanda Wright, IBC Technical Services Ltd., 57/61 Mortimer Street (1st Floor), London, UK WIN 7TD Jane Worman, IBC Technical Services Ltd., 57/61 Mortimer Street (1st Floor), London, UK W1N 7TD Royal Flemish Chemical Society (KVCV), Working Party on Chromatography, c/o Dr. R. Smits, BASF Antwerpen N.V., Central Laboratory, Scheldelaan, B-2040 Antwerp, Belgium Tel: +32 3 568 2831. Fax: +32 3 568 3250 Hirokazu Toriumi, AIRS Organizing Committee, Department of Chemistry, College of Arts and Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153, Japan Tel: +813 3467 1171, ext. 309. Fax: +813 3485 2904 Mrs J. A. Challis, Chromatographic Society, Suite 4, Clarendon Chambers, 32 Clarendon Street, Nottingham, UK NG1 5JD Tel: +44 602 500596.Fax: +44 602 500614 Ms J. M. Cook, Analytical Geochemistry Group, British Geological Survey, Keyworth, Nottingham, UK NG12 5GG Tel: +44 602 363330. Fax: +44 602 363200 Juan Sabater, Laboratorio Dr. J. Sabata Tobella, Calle de Londres 6, 08029 Barcelona, Spain Lisa Bilby, Business Seminars International, 56-60 St. John’s Street, Farringdon, London, UK EClM 4DT Pauline A. Sim, Gascoigne Secretarial Services, 24 Southfield Drive, Hazlemere, High Wycombe, Buckinghamshire, UK HP15 7HB Tel: +44 494 713664. Fax: +44 494 714516 The 12th Pharmaceutical Technology Conference, 24 Menlove Gardens North, Liverpool, UK L18 2EJ VDI, Verein Deutscher Ingenieure (Kommission Reinhaltung der Luft), Graf Recke-Strasse 84, P.O.Box 1139, D-W-4000 Dusseldorf 1, Germany National Safety Council, P.O. Box 26754, Siom, Bombay 400022, India Professor G. Demortier, Facultes Universitaires, N-D de la Paix, 22 rue Muzet, B-5000 Namur, Belgium Mervyn Richardson, BASIC, 6 Birch Drive, Maple Cross, Rickmansworth, Hertfordshire, UK WD3 2UL Tel: +44 923 774187. Fax: +44 494 714516 A. Favier, Laboratoire de Biochimie C., Hopital A. Michallon, B.P. 217X, F-38943 Grenoble Cedex 09, France Tel: +33 76 76 54 07. Fax: +33 76 42 66 44 Dr. E. S. Harpur, Sterling Winthrop Research Centre, Willowburn Avenue, Alnwick, North Cumberland, UK NE66 2JH22N ANALYST, FEBRUARY 1993, VOL. 118 Date 13-17 18-21 19-21 19-22 19-23 20-23 20-23 25-29 26-2/5 27 May 2-7 3-5 3-5 4-5 4-6 6-12 9-13 9-14 10-13 Conference Location International Meeting on the Effects of War Zadar, Activities in the Environment Croatia 4th International Symposium on Pharmaceutical and Biomedical Analysis USA Baltimore, MD, Anakon ’93 Baden-Baden, Germany Annual Physics Congress: Spectroscopy, The Changing Face of Physics UK Brighton, Focus ’93: The Association of Clinical Biochemists Annual National Scientific UK Meeting and Exhibition International Symposium on Electroanalysis in Loughborough, Biomedical, Environmental and Industrial Leicestershire, Sciences UK Birmingham, 5th European Congress on Biopharmaceutics Brussels, and Pharmacokinetics Belgium Eurolab 93, 12th SFBC National Meetingloth Nice, IFCC European Congress of Clinical France Chemistry Exhibition and Congress ‘Wasser’, AMK Germany Berlin’93 Validating Multicomponent Analysis London, 1 JK CLEO ’93, Conference on Lasers and Electro- Baltimore, MD, Optics USA Euroresidue 11, International Conference on Residues of Veterinary Drugs in Food Veldhoven , The Netherlands 5th International Symposium on the Analysis of Szombathely , Steroids Hungary ASTM Symposium On Quality And Statistics: Atlanta, GA, Total Quality Management USA Deauville Conference SAS 93 Deauville, France Interpack’93 (Environmentally Justified Diisseldorf , Packaging) Germany EMAS ’93-Modern Developments and Rimini, Applications in Microbeam Analysis Italy HPLC ’93, 17th International Symposium on Column Liquid Chromatography Germany Hamburg, International Environment ’93 and Analysis ’93 UK London, Contact Mervyn Richardson, BASIC, 6 Birch Drive, Maple Cross, Rickmansworth, Hertfordshire, UK WD3 2UL Tel: +44 923 774187.Fax: +44 494 714516 Shirley E. Schlessinger (Symposium Manager), Suite 1015,400 East Randolph Drive, Chicago, IL 60601, USA Tel: +1 312 527 2011. Gesellschaft Deutscher Chemiker, Abteilung Tagungen, Postfach 90 04 40, Varrentrappstasse 40-42, D-6000 Frankfurt am Main 90, Germany Spectroscopy, The Changing Face of Physics, The Conference Department, The Institute of Physics, 47 Belgrave Square, London, UK SWlX SQX Tel: +44 71 235 6111. Fax: +44 71 259 6002 Pat Nielsen, Pipers, Main Street, Akeley, Buckingham, UK MK18 5HW Dr. Arnold Fogg, Electroanalysis Conference, Chemistry Department, Loughborough University of Technology, Loughborough, Leicestershire, UK LEll3TU Mrs. F.Rey, 3/17 Avenue de I’Observatoire, B-1180 Brussels, Belgium Tel: +31 2 375 1648. Fax: +31 2 375 3299 Groupe SEPFI, Technoexpo, 8 rue de la Michodihre, 75002 Paris, France Tel: +33 1 47 42 92 56. Fax: +33 1 42 66 14 28 Company of Exhibitions, Fairs and Congresses, Messedamm 22, P.O. Box 191740, D-W-1000 Berlin 19, Germany Mr. T. Frost, The Wellcome Foundation, Dartford, UK DA1 5AH Meetings Department, Optical Society of America, 2010 Massachusetts Avenue, NW, Washington, DC 20036, USA Tel: +1 202 223 9037. Fax: +1 202 416 6140 Dr. N. Haagsma, Section of Food Chemistry, Department of Food of Animal Origin, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80.175, 3508 TD Utrecht, The Netherlands Tel: +31 30 535 365,535 367.Fax: +31 30 532 365 Professor S. Gorog, c/o Chemical Works of Gedeon Richter Ltd., P.O. Box 27, H-1475 Budapest, Hungary Tel: +36 1 157 4566. Fax: +36 1 157 1578 Scott Orthey, ASTM, 1916 Race Street, Philadelphia, PA 19103, 215/299-5507, USA Sabine Lauras, Nicko & Cri Associes, 7 rue d’Argout , F-75002 Paris, France Tel: +33 1 42 33 47 66. Fax: +33 1 40 41 92 41 Dusseldorfer Messegesellschaft mbH ‘NO WEA’ , P.O. Box 32 02 03, Stockumer Kirchstrasse 61, D-W-4000 Diisseldorf 30, Germany Abraham Boekestein, Mansholtlaan 12, Postbus 356, D-6700 Wageningen, Germany Gesellschaft Deutscher Chemiker, Abteilung Tagungen, Postfach 900440, Varrentrappstrasse 40- 42, W-6000 Frankfurt am Main 90, Germany Tel: +49 69 7917 360. Fax: +49 69 7917 475 Eileen Davies, IE ’93,12 Alban Park, Hatfield Road, St Albans, Hertfordshire, UK AL4 OJJ Tel: +44 727 855574.Fax: +44 727 841694ANALYST, FEBRUARY 1993, VOL. 118 23N Date 11-15 23-28 24-26 24-27 24-29 25-27 25-27 27-28 27-28 June 2-4 3 3-4 7-9 8-1 1 13-17 13-17 14-16 14-20 Conference IV Encontro de Usuarios de RMN Location Contact Rio de Janeiro, Brazil The Associacao de Usuarios de Ressonancia Magnetica Nuclear (Auremn), A/C Sonia Maria Cabral de Menezes, Petrobras/Cempes/Diquim, Radial 2, Quadra 7,21910.240 Cidade Universitaria, Ilha Do Fundao, Rio de Janeiro, RJ, Brazil American Society for Mass Spectrometry, P. 0. Box 1508, East Lansing, MI 48826, USA Tel: +1 517 337 2548. Dechema, P.O. Box 970146, D-W-6000 Frankfurt am Main 97, Germany Professor Dr. P.Sandra, IOPMS, Kennedypark 20, B-8500 Kortrij k, Belgium Tel: +32 56 204960. Fax: +32 56 204859 Dr. V. N. Makatun, Organizing Committee of the Mendeleev Congress, Presidium of Byelorussian Academy of Sciences, 66, F. Scorina Avenue, Minsk, Byelorussia Professor Dr. Willy R. G. Baeyens, University of Ghent, Pharmaceutical Institute, Harelbekestraat 72, B-9000 Ghent, Belgium Stephen Ward, Enterprise Public Relations, 165 Kensington High Street, London, UK W8 6SH Professor Dr. P. Sandra, IOPMS, Kennedypark 20, B-8500 Kortrijk, Belgium Tel: +32 56 204960. Fax: +32 56 204859 Dr. V. M. Bhatnagar, Alena Chemicals of Canada, P.O. Box 1779, Cornwall, Ontario, Canada K6H 5V7 Tel: +1 613 932 7702. 41st ASMS Conference on Mass Spectroscopy Las Vegas, NV, USA 11th Dechema Annual Meeting on Biotechnology 15th International Symposium on Capillary Chromatography (ISCC) Frankfurt, Germany Riva del Garda, Italy XV Mendeleev Congress on General and Applied Chemistry Minsk, Byelorussia Vth International Symposium on Quantitative Luminescence Spectrometry in Biomedical Sciences Control and Instrumentation Exhibition ’93 Ghent, Belgium Birmingham, UK Riva del Garda, Italy 2nd European Symposium on Analytical Supercritical Fluid Chromatography and Extraction (ESASF) European Conference on Environmental Pollution, Aquatic and Atmospheric Environment, AirNater Quality, Hazardous Wastes and Hydrology Helsinski, Finland International Symposium on Analysis of Peptides Stockholm, Sweden The Swedish Academy of Pharmaceutical Sciences Symposium on ‘Analysisof Peptides’, P.O.Box 1136, S-111 81 Stockholm, Sweden Tel: +46 8 24 50 85. Fax: +46 8 20 55 11 Professor J. Mattinen, Abo Akademi, Institution for Organisk Kemi, Akademig 1, SF-20500 Abo SO, Finland Dr. V. M. Bhatnagar, Alena Chemicals of Canada, P.O. Box 1779, Cornwall, Ontario, Canada K6H 5v7 Tel: +1 613 932 7702. G. Lachenal, Universite Lyon 1, Laboratoire des Materiaux Plastiques et des Biomateriaux, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France Tel: +33 72 43 12 11. Fax: +33 78 89 25 83 John Boother (Programme Chairman), JB Scientific, P.O. Box 5 , Riseley, Reading, UK RG7 1YL Tel: +44 734 883125. Fax: +44 734 885604 Congress Secretariat, c/o Professor Laura Frontali, Department of Cell and Developmental Biology, University of Rome ‘La Sapienze’, P.le Aldo Moro 5, 00185 Rome, Italy Tel: +39 6 445 3950. Fax: +39 6 499 12351 SSC3 Secretariat, Department of Chemical Technology, Danish Technological Institute, Teknologiparken, DK-8000 Arhus C, Denmark Tel: +45 86 14 24 00. Fax: +45 86 14 74 45 Ms. Janet Cunningham, Barr Enterprises, P.O. Box 279, Walkersville, MD 21793, USA Tel: +1 301 898 3772. Fax: +1 301 898 5596 Mrs. Tarja Jalasto, Finnish Society for Quality Control, Laaksolahdentie 41, P.O. Box 1, SF-02730 Espoo , Suomi-Finland NMR Symposium Turku, Finland European Conference on Analytical Chemistry, Chromatography and Spectroscopy and Thermal Analysis Brno, Czechoslovakia European Seminar on Infrared Spectroscopy LY on , France 7th International LIMS Conference Egham, Surrey, UK 6th European Congress on Biotechnology Firenze, Italy 3rd Scandinavian Symposium on C hemometrics Arhus, Denmark PREP-93, 10th International Symposium on Preparative Chromatography Arlington, VA, USA European Organization for Quality Control Helsinki, Suomi-Finland24N ANALYST, FEBRUARY 1093, VOL.118 Date 17-18 27- 117 29-47 July 4-7 4-8 11-14 11-15 12-14 19-21 19-23 25-29 Conference International Conference on Analytical Chemistry & Applied Chromatography/ Spectroscopy Fullerenes ’93, 1st International Interdisciplinary Colloquium on the Science and Technology of the Fullerenes XXVIII Colloquium Spectroscopicum Internationale XXVIII CSI Post-Symposium: Graphite Atomizer Techniques in Analytical Spectroscopy 6th International Conference on Indoor Air Quality and Climate, Indoor Air’93 International Symposium on Polymer Analysis and Characterization Chemometrics 111, 3rd Czechoslovak Chemometric Conference R & D Topics Meeting 1993 6th Symposium on Handling of Environmental and Biological Samples in Chromatography 12th International Symposium on Nuclear Quadrupole Interactions 107th AOAC Annual International Meeting and Exposition Location Contact Toronto, Canada Dr.V. M. Bhatnagar, Alena Chemicals of Canada, P.O. Box 1779, Cornwall, Ontario, Canada K6H SV7 Tel: + 1 613 932 7702. Gill Spear, Pergamon Seminars, c/o Elsevier Advanced Technology, Mayfield House, 256 Banbury Road, Oxford, UK OX2 7DH; Tel: +44 865 512242. Fax: +44 865 310981 or for North America, Kim Cavellero, Pergamon Seminars, 660 White Plains Rd.Tarrytown, Dr. B. L. Sharp, Loughborough University of Technology, Department of Chemistry, Loughborough, Leicestershire, UK LE11 3TU Santa Barbara, CA, USA NY 10591-5153, USA York, UK Durham, UK Chemistry, CSI Secretariat, Loughborough XXVIII CSI Post-Symposium, Department of University of Technology, Loughborough, Leicestershire, UK LEll 3TU Tel: +44 509 22575. Fax: +44 509 233163 Professor Olli Seppanen, SF-02150 Espoo, Finland Helsinki, Suomi-Finland Crete Brno, Czechoslovakia Bradford, W. Yorkshire, UK Guildford, Surrey, UK Zurich, Switzerland Washington, DC, USA August 9-11 3rd Soil and Sediment Residue Analysis Winnipeg, Workshop Manitoba, Canada 9-13 Asianalysis I1 Changchun, China 22-25 EUROTOX’93 (32nd Congress of Toxicology) Uppsala , Sweden Judith A.Sjoberg, Professional Association Management, 815 Don Gaspar, Sante Fe, NM, USA Dr. Josef Havel, Department of Analytical Chemistry, Masaryk University, Kotlarska 2, CS- 61 137 Brno, Czechoslovakia Tel: +42 5 712984. Fax: +42 5 740108 Miss P. Hutchinson, Analytical Division, The Royal Society of Chemistry, Burlington House, Piccadilly , London, UK W1V OBN Tel: +44 71 437 8656. Fax: +44 71 734 1227 M. Frei-Hausler, IAEAC Secretariat, Postfach 46, CH-4123 Allschwil 2, Switzerland Tel: +41 61 632789. Fax: +41 61 4820805 Professor D. Brinkmann, Physik-Institut, University of Zurich, Schonberggasse 9, CH-8001 Zurich, Switzerland Margaret Ridgell, AOAC, 2200 Wilson Boulevard, Suite 400, Arlington, VA 22201-3301, USA Dr. G. R. Barrie Webster, Pesticide Research Laboratory, Department of Soil Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Tel: + 1 204 474 6039. Fax: + 1 204 275 6019; or Professor Dr. Joseph Tarradellas, IGE, Federal Technical Institute EPF-L, CH-1015 Lausanne Ecublens, Switzerland Professor Erkang Wang, Asianalysis 11, Changchun Xnstitute of Applied Chemistry, Chinese Academy of Sciences, P. 0. Box 1022, Changchun, Jilin 130022, China Tel: +86 431 682 801 (ext. 562). Fax: +86 431 685 6.53 Dr. R. A. Ettlin, EUROTOX Secretary General, Sandoz Pharma Ltd., Toxicology, Building 881, P.O. Box, CH-4002 Basle, Switzerland Entries in the above listing are at the discretion of the Editor and are free of charge. If you wish to publicize a forthcoming meeting please send full details to: The Analyst Editorial Office, Thomas Graham House, Science Park, Milton Road, Cambridge, UK CB4 4WF. Tel: +44 (0)223 420066. Fax: +44 (0)223 420247.

ISSN:0003-2654    

DOI:10.1039/AN993180021N

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

1248 ANALYST, OCTOBER 1993, VO12. 118 ng ml-l iodide. The iodide comparator standards were prepared by transferring microlitre portions of the iodide standard solution onto pre-cleaned Gelman Sciences GA-6S membrane filters (0.45 pm pore size, 47 mm diameter) placed in pre-cleaned 1.2 ml polyethylene irradiation vials. The comparator standards and samples were prepared to have identical geometry. Reference Materials A number of CRMs and Standard Reference Materials (SRMs) obtained from the IAEA and the National Institute of Standards and Technology (NIST) , respectively, were analysed to evaluate the accuracy of the methods at the low levels of iodine that might be present in some biological and diet samples. The RMs used were: NIST SRM 1571 Orchard Leaves and SRM 1577 Bovine Liver, and IAEA A-11 Milk Powder.H-4 Animal Muscle and H-9 Mixed Human Diet. Irradiation and Counting All samples and comparator standards were irradiated in the Dalhousie University SLOWPOKE-2 research reactor at a maximum integral neutron flux of 1 x 1012 n cm-2 s-1 or a maximum epi-cadmium neutron flux of 1 x lo'* n cm-2 s - I . They were counted on a 25 ml active volume Aptec hyperpure Ge detector connected to a Link high count rate pulse processor and a Nuclear Data ND-66 analyser. The detector had a resolution of 2.08 keV (full-width at half-maximum) at the 1332 keV photopeak of 6OCo. The 443 kcV y-ray of 12*l was free from interference and was therefore used for assaying iodine. Microwave Acid Digestion of Samples Microwave acid digestion bombs (Parr Instrument Co.) were used for the dissolution of samples.The bomb contained a chemically inert Teflon sample cup of 45 ml capacity. Details of the development of the sample digestion procedure are given clsewhere.29 Briefly, 200-250 mg of a sample were accurately weighed into a pre-cleaned Teflon sample cup, 5 ml of ultrapure concentrated nitric acid were added, and the mixture was heated for 35 s at a power of 675 W in a microwave oven. The digestion step was repeated after a 20 rnin cooling period, if necessary. This procedure yielded sufficiently complete decomposition of the sample with respect to iodine. The contents of the cup were poured into a pre-cleaned 250 ml beaker containing 1 g of hydrazine sulfate. The cup and its lid were rinsed successively with 3 x 5 ml aliquots of each of a 5% hydrazine sulfate solution and DDW.The washings were added to the sample solution. The resulting solution was diluted to 100 mi, maintaining a final acidity of 0.2 rnol 1-l. Preconcentration of Iodine by Bismuth Sulfide Coprecipitation Details of the development of this method have recently been published.'" Bricfly, it involved the sequential dropwise addition of 1 ml of each of bismuth nitrate and thioacetamide stock solutions to a 100 ml digested sample solution at an acidity of 0.2 rnol I-'. The dark brown precipitate formed was allowed to settle for 20 rnin at room temperature and then filtered through a pre-cleaned Gelman membrane filter under vacuum suction. The precipitate was washed three times with 5 ml aliquots of a 0.2 mol I-' nitric acid solution containing 0 .I% hydrazine sulfate. The filter containing the precipitate was folded, placed in a 1.2 ml polyethylene vial and heat- sealed. The vial was irradiated in an epi-cadmium neutron flux of 1 .0 x 1010 n cm-2 s- for 30 or 60 min and counted for either 30 or 60 rnin after a decay period of I min. Preconcentration of Iodine by Toluene Extraction The digested sample solution was poured into a 250 ml beaker. The sample cup and lid of the microwave digestion bomb were thoroughly rinsed with 2 x 5 ml portions of a 5% hydrazine sulfate solution and subsequently with 2 x 5 ml portions of DDW. The washings were added to the sample solution and the final acidity of the resulting solution was adjusted to between 1 and 2 rnol I-', maintaining a total volume of 30 ml.This solution was transferred into a 125 ml Pyrex separating funnel. Then, 10 ml of toluene and 5 ml of a 10% NaN02 solution were added. Iodine was extracted into the organic phase by shaking on a wrist-action mechanical shaker for 10 min. The organic phase was transferred into another separat- ing funnel. The extraction procedure was repeated three times with 10 ml of toluene and 2 ml of a 10% NaN02 solution. The extraction recovery of iodine was checked by irradiating 1 mi aliquots of the organic phasc after each extraction and calculating the yield of iodine. Preliminary experiments with spiked standard iodine and with 1251 tracer revealed that three extractions were sufficient for the complete recovery of iodine (>99%).The organic phases were combined in a separating funnel. The iodine was reduced to iodide and back-extracted into the aqueous phase by equilibrating with 2 x 10 ml portions of a 5% hydrazine sulfate solution. The aqueous phases containing iodide were combined and diluted to 100 ml, maintaining a final acidity of 0.2 mol I-' with rcspect to nitric acid. Samples containing relatively large amounts of chlorine and bromine were first treated by this toluene cxtraction method to isolate iodine. Then, the bismuth sulfide coprecipitation procedure described above was used to concentrate the iodide further and collect it in a precipitate which is suitable for NAA. The iodine content of the precipitate was measured by irradiating in an epi-cadmium neutron flux of 1 x 1OlO n cm-? s-I for 60 rnin and counting for 60 rnin after a decay period of 1 min.Radiochemical Separation of Iodine by Bismuth Sulfide Coprecipitation About 250 mg of a sample were accurately weighed into a pre- cleaned polyethylene vial and irradiated for 60 min in an integral neutron flux of 1 X 1012 n cm-? s-I. The vial was opened and the sample was transferred into a Teflon sample cup of the microwave digestion bomb. The vial was rinsed three times with 1 ml portions of ultrapure concentrated nitric acid. The irradiated sample was spiked with 100 pI of 1251 tracer solution and digested according to the procedure described under Microwave Acid Digestion of Samples. To the digested sample solution, 2 ml of bismuth nitrate stock solution, 1 ml of ammonium iodide (1 mg ml-I I - ) and 2 ml of thioacetamide stock solution were added sequentially while stirring the solution with a magnetic stirrer.The resulting dark brown precipitate was allowed to settle for 20 min and then filtered through a Gelman membrane filter under vacuum suction. The entire procedure was completed within 50 min from the end of the irradiation. The filter was folded, placed in a new 1.2 ml polyethylene vial and counted for 30 min. The recovery of iodine was checked by measuring the activity of the 1251 tracer. Radiochemical Purification by Palladium Iodide Precipitation Thc sample was digested according to the procedure described under Microwave Acid Digestion o f Samples. This solution was taken through the bismuth sulfide coprecipitation proce- dure.The precipitate was collected and irradiated for 60 rnin in an integral neutron flux of 1 X 1012 n cm-2 s-I. The irradiated precipitate was washed off the filter into a beaker with 5 ml of concentrated nitric acid. The filter was rinsed thoroughly with 4 mol I-' nitric acid and subsequently with 10 ml of a 5% hydrazine sulfate solution. The sample solution was diluted to SO ml with DDW, maintaining a final acidity between 2 and 4 rnol I-'. To this solution, 0.5 ml aliquots of1248 ANALYST, OCTOBER 1993, VO12. 118 ng ml-l iodide. The iodide comparator standards were prepared by transferring microlitre portions of the iodide standard solution onto pre-cleaned Gelman Sciences GA-6S membrane filters (0.45 pm pore size, 47 mm diameter) placed in pre-cleaned 1.2 ml polyethylene irradiation vials.The comparator standards and samples were prepared to have identical geometry. Reference Materials A number of CRMs and Standard Reference Materials (SRMs) obtained from the IAEA and the National Institute of Standards and Technology (NIST) , respectively, were analysed to evaluate the accuracy of the methods at the low levels of iodine that might be present in some biological and diet samples. The RMs used were: NIST SRM 1571 Orchard Leaves and SRM 1577 Bovine Liver, and IAEA A-11 Milk Powder. H-4 Animal Muscle and H-9 Mixed Human Diet. Irradiation and Counting All samples and comparator standards were irradiated in the Dalhousie University SLOWPOKE-2 research reactor at a maximum integral neutron flux of 1 x 1012 n cm-2 s-1 or a maximum epi-cadmium neutron flux of 1 x lo'* n cm-2 s - I .They were counted on a 25 ml active volume Aptec hyperpure Ge detector connected to a Link high count rate pulse processor and a Nuclear Data ND-66 analyser. The detector had a resolution of 2.08 keV (full-width at half-maximum) at the 1332 keV photopeak of 6OCo. The 443 kcV y-ray of 12*l was free from interference and was therefore used for assaying iodine. Microwave Acid Digestion of Samples Microwave acid digestion bombs (Parr Instrument Co.) were used for the dissolution of samples. The bomb contained a chemically inert Teflon sample cup of 45 ml capacity. Details of the development of the sample digestion procedure are given clsewhere.29 Briefly, 200-250 mg of a sample were accurately weighed into a pre-cleaned Teflon sample cup, 5 ml of ultrapure concentrated nitric acid were added, and the mixture was heated for 35 s at a power of 675 W in a microwave oven.The digestion step was repeated after a 20 rnin cooling period, if necessary. This procedure yielded sufficiently complete decomposition of the sample with respect to iodine. The contents of the cup were poured into a pre-cleaned 250 ml beaker containing 1 g of hydrazine sulfate. The cup and its lid were rinsed successively with 3 x 5 ml aliquots of each of a 5% hydrazine sulfate solution and DDW. The washings were added to the sample solution. The resulting solution was diluted to 100 mi, maintaining a final acidity of 0.2 rnol 1-l. Preconcentration of Iodine by Bismuth Sulfide Coprecipitation Details of the development of this method have recently been published.'" Bricfly, it involved the sequential dropwise addition of 1 ml of each of bismuth nitrate and thioacetamide stock solutions to a 100 ml digested sample solution at an acidity of 0.2 rnol I-'.The dark brown precipitate formed was allowed to settle for 20 rnin at room temperature and then filtered through a pre-cleaned Gelman membrane filter under vacuum suction. The precipitate was washed three times with 5 ml aliquots of a 0.2 mol I-' nitric acid solution containing 0 . I% hydrazine sulfate. The filter containing the precipitate was folded, placed in a 1.2 ml polyethylene vial and heat- sealed. The vial was irradiated in an epi-cadmium neutron flux of 1 .0 x 1010 n cm-2 s- for 30 or 60 min and counted for either 30 or 60 rnin after a decay period of I min.Preconcentration of Iodine by Toluene Extraction The digested sample solution was poured into a 250 ml beaker. The sample cup and lid of the microwave digestion bomb were thoroughly rinsed with 2 x 5 ml portions of a 5% hydrazine sulfate solution and subsequently with 2 x 5 ml portions of DDW. The washings were added to the sample solution and the final acidity of the resulting solution was adjusted to between 1 and 2 rnol I-', maintaining a total volume of 30 ml. This solution was transferred into a 125 ml Pyrex separating funnel. Then, 10 ml of toluene and 5 ml of a 10% NaN02 solution were added. Iodine was extracted into the organic phase by shaking on a wrist-action mechanical shaker for 10 min.The organic phase was transferred into another separat- ing funnel. The extraction procedure was repeated three times with 10 ml of toluene and 2 ml of a 10% NaN02 solution. The extraction recovery of iodine was checked by irradiating 1 mi aliquots of the organic phasc after each extraction and calculating the yield of iodine. Preliminary experiments with spiked standard iodine and with 1251 tracer revealed that three extractions were sufficient for the complete recovery of iodine (>99%). The organic phases were combined in a separating funnel. The iodine was reduced to iodide and back-extracted into the aqueous phase by equilibrating with 2 x 10 ml portions of a 5% hydrazine sulfate solution. The aqueous phases containing iodide were combined and diluted to 100 ml, maintaining a final acidity of 0.2 mol I-' with rcspect to nitric acid.Samples containing relatively large amounts of chlorine and bromine were first treated by this toluene cxtraction method to isolate iodine. Then, the bismuth sulfide coprecipitation procedure described above was used to concentrate the iodide further and collect it in a precipitate which is suitable for NAA. The iodine content of the precipitate was measured by irradiating in an epi-cadmium neutron flux of 1 x 1OlO n cm-? s-I for 60 rnin and counting for 60 rnin after a decay period of 1 min. Radiochemical Separation of Iodine by Bismuth Sulfide Coprecipitation About 250 mg of a sample were accurately weighed into a pre- cleaned polyethylene vial and irradiated for 60 min in an integral neutron flux of 1 X 1012 n cm-? s-I.The vial was opened and the sample was transferred into a Teflon sample cup of the microwave digestion bomb. The vial was rinsed three times with 1 ml portions of ultrapure concentrated nitric acid. The irradiated sample was spiked with 100 pI of 1251 tracer solution and digested according to the procedure described under Microwave Acid Digestion of Samples. To the digested sample solution, 2 ml of bismuth nitrate stock solution, 1 ml of ammonium iodide (1 mg ml-I I - ) and 2 ml of thioacetamide stock solution were added sequentially while stirring the solution with a magnetic stirrer. The resulting dark brown precipitate was allowed to settle for 20 min and then filtered through a Gelman membrane filter under vacuum suction. The entire procedure was completed within 50 min from the end of the irradiation. The filter was folded, placed in a new 1.2 ml polyethylene vial and counted for 30 min. The recovery of iodine was checked by measuring the activity of the 1251 tracer. Radiochemical Purification by Palladium Iodide Precipitation Thc sample was digested according to the procedure described under Microwave Acid Digestion o f Samples. This solution was taken through the bismuth sulfide coprecipitation proce- dure. The precipitate was collected and irradiated for 60 rnin in an integral neutron flux of 1 X 1012 n cm-2 s-I. The irradiated precipitate was washed off the filter into a beaker with 5 ml of concentrated nitric acid. The filter was rinsed thoroughly with 4 mol I-' nitric acid and subsequently with 10 ml of a 5% hydrazine sulfate solution. The sample solution was diluted to SO ml with DDW, maintaining a final acidity between 2 and 4 rnol I-'. To this solution, 0.5 ml aliquots of

ISSN:0003-2654    

DOI:10.1039/AN99318025Nb

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

26N ANALYST, FEBRUARY 1993, VOL. 118 Future Issues Will lnclude- Detection of Aluminium(1n) Binding to Citrate in Human Blood Plasma by Proton Nuclear Magnetic Resonance Spec- troscopy-Peter J. Sadler, Gina Kubal, Stojan RaduIovic, Alan Tucker and Jimmy D. Bell Flow Injection Chemiluminometric Determination of Epinephrine, Norepinephrine, Dopamine and L-Dopa- Antony C. Calokerinos, Nikolaos T. Deftereos and Constanti- nos E. Efstathiou Development of an Antibody-based Amperometric Biosensor to Study the Reaction of 7-Hydroxycoumarin With its Specific Antibody-Malcolm R. Smyth, Eithne Dempsey, Ciara O’Sul- livan, Denise Egan, Richard O’Kennedy and Joseph Wang Novel Electrochemical Device for the Detection of Choles- terol and Glucose-John F. Cassidy, Cathriona Clinton, William Breen, Robert Foster and Eilish O’Donoghue Data Processing by Neural Networks in Quantitative Chem- ical Analysis-Martinus Bos, A.Bos and W. E. van der Linden Addition and Measurement of Water in Carbon Dioxide Mobile Phase €or Supercritical Fluid Chromatography- Dongjin Pyo and Doweon Ju Sensor-Tissue interactions in Neurochemical Analysis with Carbon-paste Electrodes In Vivo-Robert D. O’Neill Flow-through (Bio)chemical Sensors-Miguel Valcarcel and Maria Dolores Luque de Castro Analysis of Steroids: Qualitative and Quantitative Character- ization of Bulk Cholesterol by Gas Chromatography and Gas Chromatography-Mass Spectrometry-Anna Lauk6, Eva CsizCr and Sandor Gorog Organic-phase Biosensors for Assays of Pharmaceutical Products-Joseph Wang, Yuehe Lin and Liang Chen Total Phosphate Determination in Waste Waters by On-line Microwave Digestion Incorporating Colorimetric Detection -Stephen J. Haswell, Kathleen E. Williams, David A. Barclay and Gaynor Preston Development of an International Chemical Measurement System-Bernard King Low-level Determination of Formaldehyde in Water by High-performance Liquid Chromatography-Evangel0 Cot- saris and Brenton C. Nicholson 2nd National Symposium on Planar Chromatography: Modern Thin-Layer Chromatography Co-Chairmen: Professor Harold M . McNair and P r o f e s s o r Colin F. P o o l e September 1 9 - 2 2 , 1993 Research Triangle P a r k , N o r t h C a r o l i n a J J S A Further information may be obtained from: Janet E . Cunningham, Barr Enterprises, P.O. Box 279, Walkersville, M D 21793 USA Phone: (301) 898-3772 - Fax: (301) 898-5596

ISSN:0003-2654    

DOI:10.1039/AN993180026N

出版商:RSC    

年代:1993

数据来源: RSC

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ANALYST, FEBRUARY 1993, VOL. 118 123 Tutorial Review Multi-electrode Array Detectors in High-performance Liquid Chromatography: A New Dimension in Electrochemical Analysis C. N. Svendsen" ESA Analytical Ltd., 7 Cromweli Mews, St. Ives, Huntingdon, Cambridgeshire, UK PE 17 4HJ Liquid chromatography has been evolving rapidly over recent years and now provides a fast, accurate and reliable method for determining the identity and concentration of a wide range of compounds. Recent advances in detector systems include the introduction of a multi-electrode high efficiency electrochemical sensor, which enables compounds to be resolved as a function of their individual oxidation potential. Combining such array sensors with gradient chromatography increases both the range of compounds in samples that can be identified in a short period of time and the confidence with which they can be matched to known standards.This paper introduces the field of three-dimensional chromatography using electrochemical array detectors and provides examples of where this new technology is currently being used. Keywords: Electrochemistry; array detector; coulometry; high-performance liquid chromatography Introduction High-performance liquid chromatography (HPLC) was estab- lished over 15 years ago and has now become the method of choice for measuring a broad spectrum of compounds in a wide variety of samples. This is a result of the speed of the technique, its versatility and the ease with which it lends itself to automation.' A schematic diagram of the basic HPLC system is shown in Fig.1 and consists of: (i) a pump which circulates buffer (mobile phase) under high pressure through the system, (ii) an injector, either automatic or manual, which is used to introduce samples into the mobile phase flow, (iii) an analytical chromatography column which is packed with a material that interacts with compounds in the sample and selectively affects the speed at which they pass through it and (iv) a detection system of a type that can quantify the separated compounds as they elute from the column. Known pure compounds of interest (standards) are injected into the HPLC system and elute from the column as peaks at a specific retention time that is a function of the column packing, the mobile phase and the size and structure of the compounds. Unknown compounds in samples injected into the HPLC system can then be identified and quantified based on matching their elution time and peak area with that of the standards.The most commonly used detector for HPLC is the ultraviolet (UV) photometer. Ultraviolet detectors work by passing light of a specific wavelength through a cell connected to the HPLC system and measuring the absorbance in real I Usample 1- - Solvent Recorder U t Detect0 n U Fig. 1 Schematic diagram of a typical HPLC system * Present address: Department of Experimental Psychology, Downing Street, Cambridge University, Cambridge, UK CB2 3EB. time .2 Compounds which absorb light (chromophores) will increase the absorbance measured by the detector as they pass through it, creating a peak that has an area directly related to the concentration of the chromophores [Fig.2(a)]. The amount of light absorbed is also dependent upon the wavelength used, with each compound having a specific wavelength at which it exhibits maximum absorption [Fig. 2(c)]. The popularity of UV detection stems from its ease of use (UV detectors are generally very stable) and the fact that many compounds absorb light at some wavelength. This Time - Time - 1, , I +80 225 325 Wavelengthtnm +0.40 0 -0.40 Pote ntia IN Fig. 2 A comparison between UV and EC detection. ( a ) Three Compounds detected using a UV photometer where absorbance peaks are measured in real time (0.05 a.u.f.s., 254 nm). Note the large level of noise and small peak height. (b) The same three compounds detected using EC (4 nA full scale, -0.85 V).Note the small noise level and large peak height due to the increased sensitivity of EC detection. (c) A UV spectrum for hydroquinone showing a specific wavelength at which it maximally absorbs light. (d) An EC current voltage curve for hydroquinone which also has a specific voltage at which it oxidizes and produces a current124 Oxidation d B + e Reduction OH 0 Hydroqu i none Uuinone Fig. 3 The principle of EC detection is bascd on oxidation or reduction of compounds under study where, at a certain applied voltage, electrons are either donated or accepted thus producing a current which can be measured (4 Inlet w Reference electrode Outlet electrode Gasket Working electrode (b) 1 [Test electrodes Reference Counter electrodes electrodes Fig.4 The two major types of EC detector. (a) The flat-plate flow-over amperometric detector which oxidizes approximately 5% of the compounds flowing over it. ( h ) The porous flow-through coulometric electrode with a large surface area that makes it coulometrically efficient and enables 100% oxidation of compounds flowing through it review focuses on another type of detection system for HPLC known as electrochemical (EC) detection. Instead of passing light through a cell, EC detectors apply a voltage at an electrode surface over which the HPLC eluent flows. Elec- troactive compounds eluting from the column either donate electrons (oxidize) or acquire electrons (reduce) generating a current peak in real time [Fig. 2(6) and Fig. 31. Importantly, the amount of current generated depends on both the concentration of the analyte and the voltage applied, with each compound having a specific voltage at which it begins to oxidize [Fig.2(d)]. Although fewer compounds will oxidize at a working electrode surface than will absorb light, EC detectors are extremely sensitive, a feature often of primary importance to the analyst using HPLC. There are two types of EC detectors: (i) flat-plate ampero- metric detectors which generally have a glassy carbon surface which oxidizes or reduces only 5% (approximately) of compounds passing over it [Fig. 4(a)] and (ii) porous flow-through amperometric detectors which have a large surface area and oxidize close to 100% of the compound flowing through them [Fig. 4(b)]. When 100% of the compound is oxidized this is referred to as coulometry and in this paper amperometric detectors operating at close to 100% efficiency are termed to be coulometric.This difference in the amount of analyte oxidized at the electrode surface leads to a number of advantages with regard to sensitivity and selectiv- ity394 and is significant when considering EC array detectors as discussed below. ANALYST, FEBRUARY 1993, VOL. 118 Fig. 5 Three-dimensional representations of UV and EC array detector chromatograms. ( a ) A typical chromatogram from a UV diode array detector where peaks can be identified by both their retention time and absorbance characteristics at various wavelengths. Note that there is not true separation in the wavelength axis. ( h ) How the same hypothetical sample may look using an amperometric EC array detector.Note that once the oxidation potential for the compound is reached, all subsequent electrodes in the array give a similar peak thus obscuring resolution in the z-axis. ( c ) Finally how the same sample would look using a coulometric array EC detector. Note that due to the 100% conversion of compounds at their oxidation potentials there is now full resolution in the electrode axis, revealing some compounds which were previously obscured in both ( a ) and ( h ) Array Cell Concepts in HPLC When matching standard peaks with unknown peaks in complex sample matrices, there are often a large number of endogenous interfering substances present which elute at the same time as, or close to, the peaks of interest.Changing the mobile phase, column, flow rate or sample preparation technique may help to change the elution time or remove interfering compounds, but there is the consistent worry that a single peak may be contaminated by closely related sub- stances. For many years, the idea of using two different wavelengths and assessing the absorbance ratio between these for both standards and unknown cornpounds in samples has been of great interest as it would give more information on the purity of a single peak eluting from the column .s This idea led to the development of diode array UV detectors whichANALYST, FEBRUARY 1993, VOL. 118 125 perform multiwavelength scanning in real time6 resulting in a third chromatographic dimension; the wavelength (z) axis [Fig.5(a)]. Thus, in addition to using retention time, ratioing across different wavelengths in the z-axis and comparing standards with unknowns is a powerful tool for confirming the identity of a compound. Recent technology advances in the design of UV detectors have increased both their sensitivity7 and, by using algorithms8 or certain types of spectral suppression,9 the accuracy with which compounds can be identified. However, compounds with different absorbance wavelengths eluting from the column at the same time cannot generally be resolved in the z-axis and, therefore, the separation power of the column is not enhanced by using diode array systems [Fig. 5 ( a ) ] . A similar type of array detector has been developed using EC detection where voltage, rather than wavelength, is the third dimension.10 A typical chromatogram from an amper- ometric EC array detector is shown in Fig. S(b) where, following elution from the columm, compounds are intro- duced to a series of electrodes set at incrementally higher voltages. Although there is a specific potential, and thus electrode, where compounds begin to oxidize, they continue to be detected on all subsequent electrodes, because only 5% of the analyte is oxidized at each point of the array. This reduces the resolution of amperometric detectors and other methods of detection currently under development which use low efficiency sensors, such as microelectrode array systems. 11 An alternative approach is to use coulometric electrodes which, as mentioned previously, convert close to 100% of the analyte passing through them to the oxidized state.Therefore, in a serial array of coulometric electrodes set at incrementally higher voltages, compounds will be detected at a certain electrode depending on their individual oxidation potentials and once fully oxidized will be ‘invisible’ to electrodes further up the array. In contrast to the UV diode array and amperometric EC detectors described above, the coulometric array allows full resolution in the electrode axis enabling the separation of compounds co-eluting from the column but with different oxidation potentials [Fig. 5(c)]. Other EC technologies are also being developed for HPLC using a single electrode, which consistently performs sweeps through a specific voltage range (rapid scanning voltammetric detection), thus increasing the confidence of peak confirma- tion.” Very recently, a microelectrode array has been developed where up to 80 individual electrodes in a space of a few millimetres can be held at incremental voltages and the current measured at each in real time.13 Although rapid scanning voltammetry has exciting possibilities when used in situ to measure endogenous levels of electroactive com- pounds, both this method and the microelectrode array use amperometric detection and so are unable fully to resolve co-eluting compounds from the HPLC column in the electrode axis.Coulometric electrodes have a further advantage over amperometric electrodes with regard to their use in array systems. The flow-through porous-carbon graphite coulo- metric electrodes are contained in a sealed unit and require no maintenance for extended periods of time.As they have far more surface area than is required to oxidize normal levels of compounds passing through them, up to 95% of their surface can be contaminated with no loss in response. Should they eventually become contaminated by more than 95% (which may occur any time between one and three years of continual use provided multiple filters are used), they can be removed from the system and flushed with nitric acid to remove the contamination. In contrast, amperometric detectors are far less efficient and require frequent cleaning (sometimes every week) of their glassy carbon surface to avoid a loss in response. Although this may be practical when a single electrode is used, the time taken to clean multiple electrodes is obviously prohibitive.Fig. 6 How ratioing across the electrode axis can confirm compound identity. (a) A typical standard EC array chromatogram with two compounds (1,2) showing different oxidation potentials and a specific response at three different voltages. (b) In an unknown sample run under the same conditions as the standard above, two peaks were found with similar retention times to 1 and 2 in the standard. Although peak 1 has an identical relative response at each electrode in the array to that of peak 1 in the standard, peak 2 shows a very different response in the electrode axis. Using this method the analyst is immediately notified that peak 2 in the sample represents either a different compound from that of the standard or a co-elution with another compound in the sample Coulometric electrodes are unique in possessing such selectivity, sensitivity and stability and are the only type of EC detector that have been fully developed for commercial use, currently available as a fully computer controlled package (The Couloehem Electrode Array System or CEAS; ESA, Bedford, MA, USA, and ESA Analytical, Cambridge, UK).The remainder of this review uses the CEAS to describe in more detail the advantages of E C array systems. Benefits of EC Array Detectors There are a number of advantages in using EC array detectors over conventional single-channel detectors relating mainly to their increased resolution and accuracy. Enhanced Resolving Power In HPLC the column and mobile phase should be chosen such that there is maximum separation of all compounds in the sample.However, there are often circumstances where even after optimizing the chromatographic conditions, some con- taminants in the sample co-elute with peaks of interest. Under, these conditions further resolution can often be obtained using coulometric E C array detectors provided the co-eluting compounds have different oxidation potentials. Confirmation of Compound Purity When compounds pass through the E C array they are normally detected on three contiguous electrodes. The first electrode will oxidize a small portion of the compound, the second or dominant electrode oxidizes a large portion of the126 ANALYST, FEBRUARY 1993, VOL. 118 Table 1 Electrode versus time (ET) map and final data for three compounds in a standard and brain sample.( a ) A typical ET map where the response in peak height from each electrode (El-E16) is listed in columns for peaks found at specific retention times (RT) in the standard and the concentration and peak identification (Peak ID) can be entered. Standard ET maps are then merged with sample ET maps (brain) and peaks in the sample can then be matched with known peaks in the standard based on both retention time and oxidation potential. ( b ) Final data reports are generated from the ET maps and include concentration, retention time, peak height and ratio across the dominant and two sub-dominant electrodes (see text for details on ratios) ( a ) Electrode versus time map for three compounds in a standard and brain sample- Sample ID Peak ID RT/min Concentratiodng E l E2 E3 E4 E5 E6 E7 E8 E9 El0 El1 El2 El3 El4 El5 El6 Standard 4.56 TYr 10 - - - - - - - - - 170 2 134 10 324 5 032 204 - - ( b ) Final data report for sample brain- Concentration/ Compound ng ml-1 Tyrosine 21.48 Dopamine 0.108 HVA 0.510 Brain Peak 1 4.52 21.49 - - - - - - - - - 356 4 865 22 190 4 890 398 - - RT/ min 4.52 6.33 12.44 Standard DA 6.33 1 - 20 1 320 4 700 2 234 269 - - - - - - - - - - Brain Peak 4 6.39 0.108 - 120 508 240 23 - Standard HVA 12.31 1 - - - - - - 45 2 320 9 870 2 500 120 - - - - - Brain Peak 7 12.44 0.501 - - - - - - 20 2 300 5 043 1 022 L30 - - - - - Ratio accuracy Height/ E13/E12 pixels Ell/E12 22 190 94% 91% E3/E4 EYE4 E8/E9 E10/E9 508 84% 99% 5 043 52% * 82% * Although the ratios for tyrosine and dopamine were close to loo%, one of the ratios for homovanillic acid (HVA) was 52%, suggesting that either this was not pure HVA or there was co-elution with another compound using this particular method.Table 2 Areas of study currently under investigation using EC array detectors Area of study ( a ) Group I . Compound related areas- Analysis of neurotransmitters and Analysis of pharmaceuticals Explosive analysis Analysis of phenolic compounds in Analysis of isocyanates in polymers Amino acid analysis Analysis of microdialysates Alzheimer’s disease Huntington’s disease Parkinson’s disease Stroke and excitotoxicity Alcohol abuse Arthritis Epilepsy Motion sickness (c) Group 3. General- Invertebrate biology Circadian rhythms Pediatric neurology modulators beverages ( b ) Group 2.Disease related areas- References 14,15,16,17,18,19 20 21 22 23 24 25,26 27,28,29,30 31,32,33 34,35,36,37 38,39,40,41 42 43 44,45 46 47,48,49 50 51.52 compound and the third electrode oxidizes the remainder of the compound. Pure standards eluting at a given retention time will give a predictable response at all three electrodes and the ratio across these three electrodes remains constant and is independent of concentration [Fig. 6(a)]. In real samples, compounds eluting at a retention time matching that of the standard can also be ‘ratioed’ across three electrodes. If the ratio of the unknown matches the ratio of the standard this can help to confirm the identity of the compound as well as that of the standard [Fig.6(b), peak 11. Any difference in the ratio signifies that either there is some contamination (co-elution) of this compound with another unknown compound or that this compound is not the same as the standard compound [Fig. 6(b), peak 21. Using ratioing methods, accurate peak identifi- cation is possible which gives the analyst a higher degree of confidence in the identity of the compound under study. Increased Stability at High Voltages Working at high oxidation potentials often results in high background currents and decreased sensitivities. Unfortu- nately, some important compounds will only oxidize at high potentials. However, with an EC array, the electrodes set at high voltages are ‘buffered’ by the lower electrodes in the series which results in reduced background current which in turn increases sensitivity and stability. Simplified Sample Preparation Certain samples ( e .g . , plasma and urine samples) require extensive and time consuming clean-up procedures t o be carried out before being introduced into the HPLC system in order to remove interfering compounds. Although it is always important to remove protein and other possible contaminants, which may affect the HPLC system, from samples before injecting them, the added resolving power of EC array detectors can often separate interfering compounds from the compound of interest. Hence, in many instances sampleANALYST, FEBRUARY 1993, VOL. 118 127 clean-up procedures can be simplified and the possibility of artificially changing the concentration of compounds under investigation can be minimized.Data Processing With EC Array Detectors Although having a large number of detectors in series dramatically increases the amount of information about a compound of interest, it also produces escalating amounts of data which have to be simultaneously monitored and inter- preted. In single-electrode systems the information from the detector is normally fed into an integration system, often PC based, using a hard disk for permanent storage. Once stored on the hard disk chromatograms can then be analysed later using peak detection algorithms. The CEAS system allows between 8 and 16 electrodes to be connected in series. The signal from each electrode is stored simultaneously in real time on a computer hard disk and is later integrated using standard peak detection algorithms.Finally, each peak is converted into an electrode versus time (ET) format where its position is plotted with regard to time and oxidation potential. An ET map from a standard and unknown sample can then be merged Fig. 7 gradient from 1% mcthanol to 40% methanol in a phosphate (0.1 mol I - l ) buffer with ion pairing (pH 3.43. For abbreviations see Table 3 EC array chromatogram of a 30 component standard used to measurc simultaneously a wide ran e of neuroactivc substances using a Table 3 List of compounds and abbreviations for the standard chromatogram shown in Fig. 7 Compound name Compound Oxidation abbreviation pontential/mV Dihydroxyphenylacetic acid DOPAC 150 Dihydroxyphenylethyleneglycol DOPEG 180 L-Dopa LD 150 Dopamine DA 150 Epinephrine E 180 Guanine GAN 700 Guanosine GSN 840 Homovanillic acid HVA 450 Hydroxyindoleacetic acid HIAA 180 Hydroxyphenylacetic acid HPAC 650 Hydroxyphcnyllactic acid HPLA 650 Hydroxybenzoic acid HBAC 700 Hydroxytryptophan HTP 300 Kynurenine KYN 800 Melatonin MEL 600 Metenephrine MN 480 Mcthoxyhydroxyphenyl glycol MHPG 450 Methoxyt yramine MT 450 N-Mct h y lserotonin NMET 300 Norepinephrine NE 180 Normetanephrine NMN 480 Salsolinol SA 180 Octopamine OCT 620 Serotonin HT 180 Tryptophan TRP 600 Tyrosine TYR 650 Uric acid UA 300 Vanillic acid VA 480 Xanthine X 700 Vanillylmandelic acid VMA 300 Retention time/min 8.91 1.95 5.61 12.13 8.35 5.65 5.89 12.97 9.95 10.75 13.32 9.32 11.42 13.82 17.36 10.98 4.95 16.48 12.13 5.16 12.87 12.98 7.83 15.46 19.11 8.94 1.62 12.03 2.97 2.02128 ANALYST, FEBRUARY 1993, VOL.118 and peaks matched based on both retention time and oxidation potential [Table l(a)]. The concentration and confidence of matched peaks can then be established and reported in a final format as shown in Table l(6) where peak purity is defined as the ratio of the standard peak divided by the ratio of the unknown sample peak multiplied by 100%. A ratio of 100% indicates that the standard and unknown sample peaks matched perfectly across the electrodes (identical ratios) and any lower percentage gives an indication of the degree of confidence in the match. Practical Applications for EC Array Detectors Any HPLC application with EC detection would benefit from using an array of coulometric cells, but in particular those requiring either a high level of confidence in peak purity or the simultaneous separation of a large number of compounds are most suitable.It would be beyond the scope of this review to cover extensively all the areas of analytical science currently using EC array cell techniques. Therefore, some of the major fields are summarized in Table 2 with relevant references. This final section summarizes the use of the E C array in the neurosciences and the pharmaceutical industry where it is often necessary to measure accurately trace amounts of neuroactive compounds or drugs in various tissues (for an extensive review see ref. 53). Neurosciences The brain is a chemical factory constantly manufacturing, amongst many other things, neurotransmitters, neuromodula- tors and hormones.Views into this world of neurochemistry using HPLC are limited by the resolution of current instru- ments which are often only able to separate and detect one or two compounds simultaneously. Once separated, the only criterion on which the identitity of the compound is based is its retention time which may be identical with another closely related compound eluting from the column at the same time. Fig. 7 shows a chromatogram generated using the CEAS array system which resolves and provides ratio values for 30 compounds of general interest to the neuroscientist (see Table 3) within 35 min and provides oxidation potentials for each. Using this standard method, tissue samples, including brain, cerebrospinal fluid and microdialysates, can be rapidly analy- sed for all of these compounds where they are above the sensitivity limits of the detector (normally around 2 pg on- column).Note that many of the primary metabolites of dopamine and noradrenaline have oxidation potentials higher than their parent compounds and can, therefore, be separated in the z-axis even when co-eluting in time (for example noradrenaline and methoxyhydroxyphenyl glycol). Recently, more advanced techniques have been used, which permit the concurrent measurement of monoamines, their metabolites and derivatized amino acids using column switching coulome- tric array te~hnology.-~4 Pharmaceuticals Fig. 8(a) shows a drug standard which had previously been analysed using both a radioimmunoassay (RIA) and a single-channel HPLC assay with differing results.The HPLC data suggested that the concentration of the drug in patient plasma was twice as high as suggested in the same plasma using an RIA technique. The pure drug standard had a very clear spread across three of the EC electrodes which enabled a ratio to be generated [Fig. 8(a)]. When the patient plasma was measured there was only one peak at approximately the correct retention time but the ratio was very different to that of the standard, suggesting that there may be co-elution. Using a sharp gradient (where the mobile phase constituents are changed during the run) it was possible to separate a 0 2 4 6 b) D I,/ I/ 0 2 4 6 8 Time/m in Fig. 8 EC arra detectors and drug analysis in complex matrices. ( a ) Drug standard [D) showing a characteristic and specific distribution over three electrodes in the z-axis (1.2,3).(b) Drug eak in a patient’s plasma showing the separation of a metabolite (My from the parent compound (D). Note how the ratio across the electrode axis is identical with that seen for the standard confirming that no other metabolites were co-eluting under these chromatographic conditions (see text for details). The response (peak height) is measured as currentANALYST, FEBRUARY 1993, VOL. 118 hidden metabolite (M) away from the parent compound (D), which now had the correct ratio when compared with the standard [Fig. 8(b)]. The ratioing feature of coulometric EC array systems can immediately identify situations where drug metabolites may be interfering with the accurate monitoring of the parent compound.Furthermore, complex mixtures of drugs with different oxidation potentials can be resolved in a short period of time using EC array systems and in some cases both drug and neuroactive compound can be measured simultaneously, significantly enhancing the quality of data in pharmacokinetic studies of drug metabolism. Conclusions Electrochemical array detectors provide a new analytical tool for resolving and accurately detecting trace amounts of any electroactive compound in a wide range of samples. 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T., and Matsumiya, T., J. Pharmacobio- Dyn., 1989, 12, 3. Volicer, L.. Matson. W. R., Schnepper, P., Gamache, P., Datz, D. I., and Wolf, N., Brain Res., 1990, 526, 169. Rizzo, V.. Melzi D’Eril. G., Achilli, G., and Cellerino, G. P., J. Chromatogr., 1991, 536, 229. Gamache, P., CEAS Report 1050, 1992, Available from ESA. Achilli, G., and Cellerino, G. P., CEAS Report 1037, 1991, available from ESA, Bedford, MA. Achilli, G., and Cellerino, G. P., CEAS Report 2033. 1992, available from ESA, Bedford, MA. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 129 Achilli, G., and Cellerino, G.P., CEAS Report 1012, 1991, available from ESA, Bedford, MA. Langlais, P. J., Mair, R., Anderson, C. D., and McEntee, W. J., Neurochem. Res., 1988, 13, 43. Acworth, I. N., Biol. Psychiatry, 1991, 2, 329. Maruyama, W., Nakahara, D., Ota, M., Takahashi, T., Takahashi, A., Nagatsu, T., and Naoi, M., J. Neurochem., 1992, 59, 395. Volicer, L., Langlais, P. J., Matson, W. R., Mark, K. A., and Gamache. P. H., Arch. Neurol., 1985, 42, 1158. Matson, W. R., Bouckoms, A., Svendsen, C. N., Beal, M. F., and Bird, E. D., in Basic Clinical and Therapeutic Aspects of Alzheimer’s and Parkinson’s Disease, Plenum Press, New York, 1990, vol. 1, p. 513. Conner, D. J., Dietez, S . , Langlais, P. J., andThal, L. J., Exp. Neurol., 1992, 116, 69.Beal, M. F., MacGarvey, U., and Swartz, K. J., Ann. Neurol., 1990, 28, 157. Beal, M. F., Matson, W. R., Swartz, K. J., Gamache, P. H., and Bird, E. D., J. Neurochem., 1990, 55, 1327. Swartz, K. J., Matson, W. R., MacGarvey, U., Ryan, E. A., and Beal, M. F., Anal. Biochem., 1990, 185, 363. Beal, M. F., Matson, W. R., Storey, E., Milbury, P., Ryan, E. A., Ogawa, T., and Bird, E. D., J. Neurol. Sci., 1992, 108, 80. Wolf, J. A., Fisher, L. J., Xu, L., Jinnah, H. A., Langlais, P. J . , Iuvone, M., O’Malley, K. L., Rosenberg, M. B., Shimohama, S . , Friedmann, T., and Gage, F. H . , Proc. Natl. Acad. Sci, USA, 1989, 86, 9011. Ogawa, T., Saso, S . , Beal, M. F., Swartz, K., Matson, W. R., and Bird, E. D., in Basic Clinical and Therapeutic Aspects of Alzheimer’s and Parkinson’s Disease, Plenum Press, New York, 1990, vol. 1, p. 456. Tohgi, H., Abe, T., Kikuchi, T., Takahashi, S., and Nozaki, Y . , Neurosci. Lett., 1991, 132, 19. Ogawa, T., Matson, W. R., Beal, M. F., Myers, R. H., Bird, E. D., Milbury, P., and Saso, S . , Neurology, 1992, 32, 127. Uemura, Y., Miller, J., Matson, W. R., and Beal, M. F., Stroke, 1991, 22, 12. Langlais, P. J., and Mair, R. G., J . Neurosci., 1990, 10, 1664. Conncr, D. J., Langlais, P. J., and Thal, L. J., Brain Res., 1991, 555, 84. Beal, M. F., Swartz, K. J., Finn, S. F., Mazurek, M. P.. and Kowell, N. W., J. Neurosci.. 1991, 11, 147. Makino, Y., Ohta, S . , Tasaki, Y., Tachikawa, O., Kashi- wasakc, M., and Hirobe, M., J. Neurochern., 1990, 55, 963. Godefroy, F., Matson, W. R., Gamache, P. H., and Weil- Fugazza, J., Brain Res, 1990, 526, 169. Mori, A., Suzuki, S . , and Kabuto, H., Med. Sci. Res., 1991,19, 445. Suzuki, S., and Mori, A., Neurochem. Res., 1992, 17, 693. Lucot, J. B., Campton, G. H., Matson, W. R., and Gamache, P. H., Life Sci., 1989, 44, 1239. Shimizu, T., and Takeda, N., 2. Naturforsch., 1991,46, 127. Takeda, N., 2nd Svendsen, C. N., Hydrobiologica, 1991, 216, 554. Shimizu, T., Mihara, M., and Takeda, N., J. Chromatogr., 1991,539, 193. Siuciak, J. A., Gamache, P. H., and Dubocovich, M. L., J. Neurochem., 1992, 58, 722. Langlais, P. J., Wardlow, M. L., and Yamamoto, H., Pediatr. Neurol., 1991,7, 6. Yamamoto, H., Pediatr. Neurol., 1991, 7, 406. Gamache, P., Ryan, E., Svendsen. C. N., Murayama, K . , and Acworth, I. N., J. Chromatogr., in the press. Naoi, M., Murayama, W., Acworth, I. N., Nakahara, D., and Parvez, H., in Techniques in the Behavioral and Neural Sciences, Elsevier, Amsterdam, vol. 10, ch. 1, in the press. Paper 21061 1 OD Received November 17, 1992 Accepted January 8, I993

ISSN:0003-2654    

DOI:10.1039/AN9931800123

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, FEBRUARY 1993, VOL. 118 131 Use of a Synthetic Detergent to Partition Protein Mixtures Wakako Tsuzuki, Hanae Kasumimoto and Shouichi Kobayashi National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2- 1-2 Kannondai, Tsukuba, lbaraki 305, Japan A previously reported method for the preparation of organic solvent soluble lipase in high yield has been applied to the partitioning of a protein mixture using a mixture of six known proteins as a model system. The organic solvent soluble complex of these proteins was obtained according to the previously reported method. In order to extract the proteins from the complex, the latter was dissolved in dichloromethane followed by the addition of buffer and triethylamine. By using this procedure, the proteins could be recovered from the complex formed with the detergent.It was found that the composition of the solvent used to prepare the complex influenced the ability of each protein to form a complex with the detergent. By making use of the differences in the efficiency of each protein to form a complex, several crude lipases could be successfully purified; in addition, their activities were retained during the purification procedure. The results suggest that the application of a synthetic detergent may be effective for the purification of proteins and enzymes. Keywords: Protein; purification; detergent; partition The amphiphilic properties of detergents have been utilized by a number of workers to purify proteins. In particular, insoluble proteins, such as membrane binding proteins, have been purified with detergents.1-6 The mechanism by which the detergent dissolves the proteins has also been s t ~ d i e d . ~ - ~ It was found that the properties of the detergent, including its co-operative binding to the proteins, its micellization with proteins and its dissolution of membranes, were useful for protein purification. Recently, methods in which a detergent is used to modify an enzyme so that it becomes soluble in organic solvents have been developed.I(b12 The aim of these studies was to conduct the enzymic reaction in an organic solvent and to modify the properties of the enzyme itself. In previous work,12 lipase was modified using a synthetic detergent (Fig. 1). The complex formed between lipase and the detergent was soluble in several organic solvents and retained its activity.The complex was designated as organic solvent soluble lipase. This paper describes the application of a synthetic detergent to the purification of proteins. A mixture of six proteins was used as the model system in order to determine the conditions for partitioning. Several crude lipases were successfully purified under the specified partition conditions. The par- titioning of proteins is based on the differences in the efficiency of each protein to form a complex with the detergent. In comparison with previous purification pro- cedures using detergents, the method described here is based on the formation of an insoluble complex between the proteins and the detergent and on the extraction of the proteins from the complex by organic solvents.Furthermore, partitioning using the detergent can be applied not only to insoluble proteins but also to soluble proteins, as it is thought that the surface of the proteins is bound to the hydrophilic region of the detergent in the complex12 (Fig. 2). Experimental Reagents A mixture of six proteins (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin CH2-CH-CH-CH-CH-CO-NH-CH-CO-O-( CH2)l q-CH3 I I l l 1 I OH OH OH OH OH CH2 I CH2 --CO-O-(CH2)71-CH3 Molecular structure of the synthetic detergent, didodecyl- Fig. 1 glucosyl glutamate inhibitor and a-lactalbumin) was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden) as an electrophoresis calibration kit, which was dialysed before use to remove sucrose. The detergent, didodecylglucosyl glutamate, used in the preparation of the complex was synthesized according to the method described previously.11 Several lipases were purchased commercially, viz., lipase P and lipase A from Amano Pharmaceutical (Nagoya, Japan), lipase PN from Wako Pure Chemicals (Osaka, Japan) and pancreatic lipase (porcine Type 11) €rom Sigma (St. Louis, MO, USA). Tetrahydrofuran (THF) without stabilizer, dichloromethane and triethylamine were purchased from Wako Pure Chemi- cals. All the other chemicals were of analytical-reagent grade and were purchased from Wako Pure Chemicals or Kanto Chemicals (Tokyo, Japan). Preparation of the Complex Between the Model Proteins and the Detergent The six dialysed proteins (the amount of phosphorylase b, bovine serum albumin, ovalbumin , carbonic anhydrase, soy- bean trypsin inhibitor and a-lactalbumin was fixed at 64, 83, 147, 83, 80 and 121 pg, respectively) in water were added to a solution of didodecylglucosyl glutamate (2.9 mg) in THF, followed by the addition of THF and water to give a final volume of 300 pl with a specific THF concentration.The complex between the protein mixture and the detergent was prepared as described previously. 12 Preparation of the Complex Between Lipase and the Detergent Lipase (100 mg) in water (2 ml) was added to the detergent (500 mg) in THF (4 ml) and the complex was prepared as described previously. 12 Lipase Detergent Organic solvent soluble lipase Fig. 2 Organic solvent soluble lipase132 ANALYST, FEBRUARY 1993, VOL.118 Extraction of Proteins From the Complex The complex was dissolved in the organic solvent, with the addition of the buffer [0.2 mol 1-1 Tris-HCI buffer (pH 7.5)] [Tris = tris(hydroxymethyl)methylamine]. After addition of triethylamine, the mixture was stirred vigorously using a vortex mixer and centrifuged at 15000 rev min-1 for 10 min. The aqueous phase was separated and analysed for the extracted proteins. Determination and Electrophoresis of Proteins The content of the proteins in the aqueous phase was determined using a protein assay kit (Bio-Rad Laboratories, Richmond, CA, USA). The extracted proteins were charac- terized by sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) using the Phast System (Pharmacia Fine Chemicals).Lipase Assay The lipase activity was determined according to the method of Shimura et al. 13 4-Methylumbelliferyl oleate was used as the substrate. Results Partition of Six Known Proteins A flow chart illustrating the application of the synthetic detergent to the partitioning of a protein mixture is shown in Fig. 3, This rapid and simple procedure involves two main steps: the preparation of the complex with the detergent and the extraction of the proteins from the complex. The conditions for the preparation of the organic solvent soluble proteins were studied in detail and the results are described below. The effect of the THF concentration on the yield of the complex was studied. As shown in Fig. 4, the proteins extracted from the complex, which was prepared at different Protein-water Detergent-THF I Stir at 4 "C Evaporate I PreciDitate 1- Wash with water I Precipitate Lyop h i I ize t I Complex 'powder Dissolve with organic solvent Buffer Triethylamine Mix vigorously Protein in i aqueous phase Fig.3 Flow chart of the protein purification procedure using the detergent THF concentrations, were characterized by SDS-PAGE. A marked difference was found in the characteristics of the extracted proteins depending on the THF concentration. When the THF concentration of the solution was 33%, five proteins were able to form a complex with the detergent. On the other hand, only three proteins, bovine serum albumin, soybean trypsin inhibitor and a-lactalbumin, formed a com- plex when the THF concentration was 67%.These findings demonstrated that the THF concentration of the solution affected the yield of the complex formed by each protein with the detergent. The yield of each protein purified in a solution of THF at a concentration of 67% was measured using the gel reader. The contents of bovine serum albumin, soybean trypsin inhibitor and a-lactalbumin were 28, 7 and 65%, respectively. Considering that the total recovery of protein was 42%, the yield of a-lactalbumin was close to 60%. The time course of the formation of the complex was studied. The THF concentration of the solution was fixed at 67%. As shown in Fig. 5 , the yield of the total proteins - B +C 4--D -E -F Fig. 4 SDS-PAGE of the proteins. Characteristics of the proteins extracted from the complex prepared at different concentrations of THF.A mixture of six proteins (d) (A. phosphorylase b; B, bovine serum albumin; C, ovalbumin; D, carbonic anhydrase; E. soybean trypsin inhibitor; and F, a-lactalbumin) was used to form a complex with the detergent. The com lex was prepared in a solution of THF at a concentration of ( a ) 33; 6) 50; and (c) 67%. The proteins were extracted from the complex according to the method described under Experimental - 0.4 E 2 0.3 E .- >. C 0.2 (u 0. .- +- 2 0.1 0 4 8 12 16 20 24 Time/h Fig. 5 Time course of the yield of the complex prepared in a solution of THF at a concentration of 67%. The total protein yield is expressed as a mass percentage based on the mass of the starting materials, which was taken as 100%ANALYST, FEBRUARY 1993, VOL.118 (a) ( b) (4 (d) 133 +B A- B- C- - E + F Fig. 6 SDS-PAGE of proteins extracted from the complex depend- ing on the time allowed for the formation of the complex between the detergent and the proteins. The mixture of six proteins (d) was added to the detergent for the formation of the complex in a solution with THF at a concentration of 67% for ( a ) 4; (b) 8; and (c) 24 h. For definitions of A-F see Fig. 4 Table 1 Effect of the solvent on the extraction efficiency of proteins. The yield of the extracted proteins is expressed as a percentage of the yield obtained by using dichloromethane containing 1 YO triethyl- amine. which was taken as 100% Solvent + 1% Solvent Solvent alone triethylamine Lauryl alcohol 1 69 Chloroform 0 18 Dichloromethane 3 100 Dichloroethane 0 42 Benzene 0 36 reached 42% compared with the original value after mixing with the detergent for 16 h.Further mixing did not increase the yield of the extracted proteins. Even for a mixing time of 8 h, 38% of the proteins had already formed a complex. The time course of the formation of the complex was also followed by SDS-PAGE (Fig. 6). As shown in Fig. 6, no differences could be detected in the characteristics of the proteins which formed a complex with the detergent for various periods of time. This observation suggests that the time required for the formation of the complex does not affect the efficiency of the proteins in forming the complex but does influence the yield of the complex. As a result, the time required for the formation of the complex was fixed at 16 h.The effect of the ratio of the proteins to the detergent on the yield of the complex was investigated. The proteins were treated with the detergent at protein : detergent ratios of 1 : 1, 1 : 2, 1 : 4 and 1 : 8. A slight difference was detected in the yield of the proteins extracted from the complex. The yield of the proteins extracted from the complex prepared with a protein : detergent ratio of 1 : 1 was 80% of that extracted from the complex preparcd with a protein : detergent ratio o€ 1 : 4. A 4-fold increase in the ratio of detergent to protein did not induce an increase in the yield of the proteins extracted from the complex. According to the analysis by SDS-PAGE, the characteristics of the extracted proteins did not change in the range of protein : detergent ratios studied (data not shown) and a protein:detergent ratio of 1 : 5 was used in the preparation of the complex.E- F - Fig. 7 Characteristics of the proteins extracted from the complex using different organic solvents. Six proteins (a) were used to form the complex. The complex was dissolved in chloroform ( b ) , dichloro- methane (c), dichloroethane (d), lauryl alcohol ( e ) and benzene cf). The proteins were extracted after the addition of triethylamine and buffer. For definitions of A-F see Fig. 4 I I I I I 0.5 0.4 - E g 0.3 -0 2 1 .- ). a 0, 0.2 .- .I- ? 0.1 0 1 2 3 4 5 Concentration of triethylamine (%) Fig. 8 Effect of the concentration of triethylamine on the extraction efficiency of proteins from the complex, which was prepared in a solution of THF at a concentration of 67%.The total protein yield is expressed as a mass percentage based on the mass of the starting materials, which was taken as 100% Hence, it was found that only the THF concentration influenced the efficiency of the proteins in forming a complex with the detergent. On the other hand, it was observed that the time required for the mixing of the proteins with the detergent and the ratio of the proteins to the detergent for the preparation of the complex were correlated with the yield of the complex but not with the ability of the detergent to become attached to the proteins. The complex formed by the proteins and the detergent could not be dissolved in an aqueous solution but was readily soluble in several organic solvents.The conditions for extraction of the proteins from the complex were investigated and the results are described below. The organic solvent in which the proteins could be efficiently extracted from the complex was selected. As shown in Table 1 , five organic solvents were used for the extraction of the proteins from the complex, which was prepared in solution at a THF concentration of 67%. The complex was dissolved in each organic solvent, followed by the addition of the buffer (0.2 moll-' Tris-HC1 buffer pH 7.5). After mixingvigorously, the proteins extracted into the buffer phase were analysed.134 ANALYST, FEBRUARY 1993, VOL. 118 Table 2 Purification of several lipases using the detergent Specific activity* Lipase (A) Lipase P 12.11 Lipase A I .98 Lipase PN 0.22 Pancreatic lipase 5.13 Yield of Specific extracted activity* Recovery of protein of extracted total (YO) protein (B) B/A activity (%) 0.8 778.23 64.3 51.2 1 .5 58.38 29.5 44.3 29.2 1.55 7.1 69.0 1.9 31.86 6.2 12.3 * The units of specific activity are expressed as the amount of 4-methylumbelliferonc released in nanomoles per milligram of protein per minute (nmol mg-1 min-1).- 94000 - 67000 c- 43000 - 30000 - 20000 f- 14400 Fig. 9 SDS-PAGE of lipase P. ( a ) Untreated lipase P available commercially. ( h ) Characteristics of the pattern of the proteins extracted from the complex between lipase P and the detergent. The values adjacent to the gels indicate the relative molecular masses of the calibration proteins Proteins were not released from the complex without the addition of triethylamine to each organic solvent, whereas the addition of triethylamine induced the release of the proteins from the complex.When the final concentration of triethyl- amine was fixed at 1%, the extraction efficiency depended on the organic solvent. The use of dichloromethane containing 1% triethylamine resulted in the most efficient extraction of the proteins (Table 1). After the extraction of the proteins from the complex with the dichloromethane containing 1% triethylamine, the absorbance of the complex was monitored at 250 nm and no absorption was detected. Dichloromethane including the complex exhibited some absorbance of proteins at 280 nm. The results suggested that the efficiency of the extraction of the proteins from the complex by using dichloro- methane containing 1% triethylamine was almost 100%.The characteristics of the proteins extracted from the complex with each of the organic solvents tested are presented in Fig. 7. Few differences were detected in the characteristics of the extrac- ted proteins, suggesting that the extraction efficiency, which depends on the type of protein, was not influenced by the organic solvent used. As a result, dichloromethane containing triethylamine was considered to be the most suitable solvent for extracting the proteins from the complex. The effect of the concentration of triethylamine in dichloro- methane was studied. The complex, which was prepared in a solution with a THF concentration of 67%, was dissolved in dichloromethane. After the addition of the buffer to the dichloromethane phase, triethylamine was added at various concentrations.As shown in Fig. 8, the efficiency of the extraction of the proteins from the complex into the aqueous phase was affected by the triethylamine concentration. The highest efficiency of protein extraction was achieved at a triethylamine concentration of 1-2%. Therefore: the concen- tration of triethylamine was fixed at 1% for the extraction of the proteins from the complex with dichloromethane. Application to the Purification of Crude Lipases The proposed method was applied to the purification of several commercially available lipases. The complex of each lipase with the detergent was prepared in a solution with a THF concentration of 67%. The proteins were extracted from the complex with the buffer and dichloromethane containing 1% triethylamine.The yields of the proteins extracted from the complex and their specific activities are listed in Table 2. A comparison of the specific activities of the extracted lipases with those of the untreated lipases indicates that all the lipases can be purified by about 6- to 64-fold. For lipase P, which appeared to be highly purified, the properties of the proteins obtained by SDS-PAGE revealed that the main protein extracted from the complex corresponded to lipase P itself (relative molecular mass 33000 Da) (Fig. 9). No marked inactivation of any of the lipases appeared to occur during the purification process using the detergent. These findings indicated that the lipases were not denatured by the organic solvent due to the formation of the complex with the detergent.These results suggest that the proposed partition method might also be effective for the purification of enzymes. Discussion In previous work, 12 a lipase could be modified in high yield to the organic solvent soluble lipase by adding THF to the solution used for preparing the complex. For lipase B, about a 4-fold increase in the organic solvent soluble lipase could be obtained at a THF concentration of 67% compared with a solution containing 100% buffer, suggesting that the addition of THF to the solution used to form the complex resulted in the efficient production of the organic solvent soluble lipases. The THF concentration appeared to be an important factor for the efficiency of the formation of the complex between the proteins and the detergent.The results suggested that the method used for the preparation of the organic solvent soluble lipase might be applicable to the partitioning of a protein mixture, if there was a difference in the efficiency of the formation of the complex among the proteins. This is the first report to demonstrate that each protein has a different efficiency in forming a complex with a detergent at a specified THF concentration depending on the surface property of the protein. This finding has allowed the develop- ment of a novel technique for partitioning proteins, although the phenomenon is not yet fully understood. It has been shown here that there are specific properties of the interaction between the interface of the protein and the detergent.The first is that the concentration of THF affects the efficiency of complex formation between the protein and the detergent. Based on this property, it should be possible to separate a specific protein from other proteins by adjusting the THF concentration to obtain the most efficient production of the complex of the protein. The second property of the interactionANALYST, FEBRUARY 1993, VOL. 118 135 between proteins and the detergent is the requirement for amines in the extraction of proteins from the complex. The addition of triethylamine also induces the release of the proteins from the complex. Little protein could be extracted from the complex using an organic solvent and buffer. These results suggest that amines might sever a bond between the surface of the proteins and the detergent.These findings are based on the establishment of a novel method for the purification of proteins using a synthetic detergent. The principle of the partitioning of the proteins is different from other current purification methods using detergents. Further investigations using various detergents and other proteins are required in order to elucidate the mechanism of the binding in the complex and the types of protein that could be purified by this method. It may then be possible to develop a very effective method for the purification of proteins. During the partitioning procedure used here, the denatura- tion of the proteins is considered to be minimal, because the proteins rcmain coated with the detergent throughout the process. In fact, the application of the method to the purification of several crude lipases did not result in their inactivation, suggesting that this method might also be suitable for the purification of enzymes. The purification of other enzymes and the use of a commercially available detergent are currently under investigation in this laboratory. 1 2 3 4 5 6 7 8 9 10 11 12 13 References Fowler, L. R., Richardson, S. H., and Hatefi, Y., Biochim. Biophys. Actu, 1962, 64, 170. Jacobs, E. E., Andrews, E. C., Cunningham, W.. and Crane, F. L., Biochem. Biophys. Res. Commun., 1966, 25, 87. Nakagawa, H., and Asano, A., J . Biochem., 1970, 68, 737. Tzagoloff, A., and Penefsky, H. S., Methods Enzymol., 1971, 22, 219. Marchesi, V. T., and Andrews, E. P., Science, 1971,174,1247. Gulik-Krzywicki, T., Biochim. BiophyJ. Actu, 1975, 415, 1. Nozaki, Y., Raynolds, J. A . , and Tanford, C., J. Biol. Chem., 1974, 249,4452. Helenius, A., and Simons, K . . Biochim. Biuphys. Actu, 1975, 415, 29. Takagi, T., Tsujii, K., and Shirahama, K., J. Biochem., 1975, 77, 939. Okahata, Y . , and Ijiro, K., J. Chem. Soc., Chem. Commun., 1988, 1392. Takahashi, K . , Saito, J . , and lnada, Y., J . Am. Oil Chem. Soc., 1988, 65, 911. Tsuzuki, W., Okahata, Y . , Katayama, O., and Suzuki, T., J. Chem. Soc., Perkin Trans. I , 1991, 1245. Shimura, S . , Tsuzuki, W., and Suzuki, T., Anal. Sci., 1991, 7, 15. Paper 2/04 7961 Received September 7, 1992 Accepted October 12, 1992

ISSN:0003-2654    

DOI:10.1039/AN9931800131

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, FEBRUARY 1993, VOL. 118 137 Development of a Radioimmunoassay for the Determination of Buprenorphine in Biological Samples Lode Debrabandere, M. Van Boven and P. Daenens Laboratory of Toxicology, University of Louvain (KULeuven), E. Van Evenstraat, 4, B 3000 Leuven, Belgium The development of a specific and sensitive radioimmunoassay for the detection of buprenorphine in urine samples is described. With minor adjustments, the assay was also applied to the analysis for buprenorphine in plasma samples. The 2-diazobenzoic acid derivative of buprenorphine has been prepared as a hapten. The immunization of rabbits with the hapten-bovine serum albumin conjugate resulted in the production of antibodies, which cross-reacted with N-dealkylbuprenorphine up to about the 90% level.The antibodies showed very low cross-reactivities with the 3-O-glucuronides and with the structural analogue etorphine. The assay was mainly used to pre-screen for buprenorphine in urine samples of persons suspected of Temgesic misuse and to determine buprenorphine in plasma samples. A linear calibration graph for buprenorphine was obtained after logit-log regression [ Y = 0.383 (s, 0.059) - 0.535 X(s, 0.025); r = 0.997 (s, O.OOl)]. The spiking recovery study showed a linear regression of Y (observed) = 0.94 + 0.84 X (expected); r = 0.997. Intra- and inter-assay relative standard deviations were <4.35 and <6.36%, respectively. A comparison study of the high-performance liquid chromatographic determination ( X ) to the radioimmunoassay (v) resulted in the following regression equation for the urine samples: Y = 1.44 + 1.64X(n = 32; r = 0.910) and Y= 0.007 + 1.58 X ( n = 10; r = 0.930) for plasma specimens. The minimum detectable dose of the immunoassay was calculated to be 10 pg ml-1 (Student’s t-distribution, p = 0.01, degrees of freedom = 8).Keywords: Buprenorphine detection; drug abuse; biological sample; radioimmunoassa y Buprenorphi ne , (2S)-2-[ ( - ) (5R ,6R ,7R, 14S)-9a-cyclopropyl- methyl-4,5-epoxy-3-hydroxy-6-methoxy-6,14-ethanomorphin- an-7-yl]-3,3-dimethyIbutan-2-ol, is a very potent oripavine derivative with mixed agonist and antagonist opiate p receptor activities. 1 4 Buprenorphine can be administered sublingually (Temgesic Sublingualis; buprenorphine 0.2 mg), or by intravenous or intramuscular injection (Temgesic 10 ampoules for injection; buprenorphine 0.3 mg).Relatively soon after the introduction of buprenorphine, some cases of misuse were reported.5 13 An immunological method for the determination of buprenorphine in plasma samples was developed by Bartlett et al. 13 The antisera were obtained by immunizing rabbits with, respectively, 3-O-car- boxymethylbuprenorphine and N-hemisuccinylnorbuprenor- phine. The antibodies raised against the first hapten show an important cross-reaction with buprenorphine 3-O-glucuro- nide, while the antibodies against the second hapten cross- react with N-dealkyl buprenorphine and the structurally re- lated product etorphine. Diagnostic Products (DPC; Los Angeles, CA, USA) recently introduced the buprenorphine 1251 radioimmunoassay (RIA) kit, which was designed for the qualitative and quantitative determination of buprenorphine in equine urine.It was the purposc of this work to prepare a more specific and sensitive immunoassay. This RIA was developed, on the basis of antibodies elicited by 2-diazobupre- norphine-bovine serum albumin (BSA). The spacer between buprenorphine and BSA is established at C-2, and therefore, the C-3, C-6 and C-7 substituent and the nitrogen are free to serve as potential antigenic determinants. A similar spacer and linkage position has already been examined for the development of a highly specific RIA for the determination of morphine in brain tissue.15 The test has been applied to the determination of buprenorphine in human urine samples, and the results have been compared with those obtained using the commercially available kit.Experimental Materials and Methods Iodine-125 in NaOH, IMS 30 (606.8 Mbq pg-1 of iodine), was obtained from Amersham International (Amersham, Buck- inghamshire, UK). Buprenorphine hydrochloride and N-dealkylbuprenorphine were synthesized in the laboratory according to a modified method of Kleeman and Engel.16 Etorphine and diprenorphine hydrochlorides were obtained from C-Vet (Bury St. Edmunds, Suffolk, UK). Other reagents were obtained from the following sources: ammonium sulfate, ethanol, 4-aminobenzoic acid, chloramine-T, sodium metabi- sulfite, sodium nitrite and poly(ethy1ene glycol) (PEG) Type 6000 from Merck (Darmstadt, Germany); and BSA (fraction 5 ) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (98%) from Janssen Chimica (Beerse, Bel- gium).Goat antiserum to rabbit y-globulin, normal rabbit carrier serum (NRS) and Freund’s complete adjuvant were obtained from Calbiochem Biochemicals and Immuno- chemicals (San Diego, CA, USA). Norit Supra A , Norit Supra B and Norit Extra C were gifts from Norit (Amersfoort, The Netherlands). The SpectdPor membranes [relative molecular mass ( M , 12 000-14000)] for the dialysis of the BSA conjugate were obtained from Spectrum Medical Industries (Los Angeles, CA, USA). To determine the cross-reactivity of the glucuronides of buprenorphine and N-dealkylbuprenor- phine in the assay, the samples were analysed before and after hydrolysis with (3-glucuronidase (40 U ml-1; 1 U = 16.67 nkat)-arylsulfatase (20 U ml-1) from Helix pornatia (Merck).Prior to analysis, samples (100 pl) from patients were diluted 1 + 1 with 0.2 moll-1 citrate buffer (pH 5.0) and 100 p1 of P-glucuronidase-arylsulfatase, followed by incubation over- night at 37 “C. Purification of the hapten was carried out by high-perfor- mance liquid chromatography (HPLC) on a semi-preparative silica column (15 g of LiChrosorb Si-60, 10 pm, suspended in carbon tetrachloride and compressed at 350 x 102 kPa in a 25 X 1.25 cm stainless-steel column) using a Merck-Hitachi 6002 pump and an ultraviolet (UV) detector (Model 440, Waters Associates, Milford, MA, USA). The structure of thc synthesized buprenorphine and of N-deal kylbuprenorphine, as well as that of the synthesized hapten, was confirmed by nuclear magnetic resonance (NMR) spectrometry (JEOL FX90Q NMR spectrometer, Tokyo, Japan) and mass spec- trometry (MS) (HP 5995A; Hewlett-Packard, Avondale, PA, USA).The radioactivity of the tracer was counted on a y-counter (Berthold BF 5300, Wildbad, Germany).138 ANALYST, FEBRUARY 1993, VOL. 118 Preparation of the Hapten The hapten was synthesized by coupling buprenorphine at the C-2 position with the diazonium salt of 4-aminobcnzoic acid. To a 0.1 mot I-' HCI solution (15 ml) of 4-aminobenzoic acid (89 mg) a solution of NaN02 (45.6 mg) was added drop by drop until the reaction mixture immediately coloured blue on a freshly prepared iodine-starch paper. All solutions were kept at 0 "C throughout the reaction. Buprenorphine (300 mg) was added to 0.1 mol 1-1 NaOH, and the pH of the solution was adjusted to 11 by adding 0.1 mol 1-l HCI.The cold solution of diazobenzoic acid was added to the buprenorphine solution, and the mixture was stirred for 2 h. The pH was kept constant during the whole synthesis. After all the diazoben- zoic acid solution had been added, the mixture was kept at 0 "C for a further 30 min. The pH of the reaction mixture was adjusted to 4.5. The precipitate formed was filtered off, dried and purified by preparative HPLC. The mobile phase for the HPLC system consisted of a mixture of dichloromethane, methanol and ammonia solution, which was pumped at 5 ml min--l, in accordance with the following program. Starting from 100% dichloromethane, the mobile phase was changed in 30 min to 70% dichloromethane and 30% methanol-ammonia solution (99 + I ) .The hapten eluted at about 27 min. After each injection of the reaction mixture the hapten fraction was collected. The compound exhibited UV absorption at 280 and 360 nm. The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated at 40°C under a stream of nitrogen, and the residue was identified by MS and NMR. The total yield of the synthesis was 19%. Immunization A 750 pl aliquot of the dialysed hapten-BSA conjugate (15 nmol) was emulsified with an equal volume of complete Frcund's adjuvant, and two New Zealand rabbits were immunized by multiple intramuscular injections of the water- oil emulsion. The first two booster injections were given at a 2 week interval, while the remaining injections were given at intervals of approximately 4 weeks.Blood was collected from the central ear artery. The first blood samples were collected 10 d after the second booster injection; the second blood samples 1 month later and afterwards at intervals of 14 d. The sera from the two rabbits were tested for their ability to bind [12sI]iodobuprenorphine and for their cross-reactivity against the major metabolite N-dealkylbuprenorphine and the struc- tural analogue etorphine. The titre of the serum was obtained by treating diluted antiserum (1 + 50-1 + 800) as described in the method of second antibody separation of bound and free ligand. The dilution that results in 50% binding of the tracer was selected to generate calibration curves for buprenorphine. Preparation of the Immunogen The hapten (6.6 mg) was dissolved in 600 pl of dimethyl sulfoxide (DMSO) and 400 pl of phosphate buffer (pH 7.4; 0.05 rnol I-') was slowly added.In another tube, 15 mg of BSA was dissolved in 5 ml of phosphate buffer and a solution of 30 mg of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide in 0.5 ml of phosphate buffer was added. The hapten solution was mixed in portions with the BSA-carbodiimide solution over 20 h, under stirring at room temperature. Previous experiments had shown that in order to keep the hapten in solution, the addition of at least 60% of DMSO was necessary. The compounds of low relative molecular mass were removed from the solution by dialysis with a cellulose membrane having a cut-off value of 12000 to 14000. The mixture was dialysed in phosphate buffer (0.05 mol I-'), changing the buffer three times a day.After the first day a 0.005 mol 1 - 1 buffer was used. The dialysis was stopped after 3 d. Degree of Incorporation The degree of incorporation of the hapten was ascertained by UV spectroscopy. 17 The diazobenzoic acid derivative of buprenorphine is yellow with a maximum absorption at 360 nm; BSA does not exhibit any absorption at this wavelength. Synthesis of the Tracer The iodination of buprenorphine with chloramine-T has already been described in detail.18 To a solution of 100 pl of phosphate buffer (0.066 mol 1 - 1 , pH 7.0) was added 10 p1 (3.7 MBq; 0.5 nmol) of a sodium [1~sI]iodidc solution and 10 pl of a buprenorphine hydrochloride solution (30% v/v methanol, 1 mg in 10 ml); 10 pl (0.25 nmol) of a freshly prepared chloramine-T solution was added and the reaction mixture was vortex-mixed.After 4 and 8 min, a further 10 pl of the chloramine-T solution was added. The chloramine-T was neutralized after 10 min with 10 pl of a sodium metabisulfite solution (1 mg in 10 ml of distilled water). Assessment of the Optimum Conditions for the Assay Pro- cedure In order to optimize the immunoassay conditions, several methods for the separation of bound and free antigen were examined. Other parameters that influence the immunoassay, such as pH, incubation time and Concentrations of BSA and tracer, were also tested. No remarkable effects were observed when the pH of the reaction mixture ranged between 6 and 8 and when the final concentration of BSA was kept between 0.2 and 5%.The equilibrium between antigen and antibody was reached after 90 min. These optimum incubation condi- tions were kept constant for all remaining experiments. To 300 ELI of BSA solution (2% BSA in phosphate buffer, 0.1 moll-1, pH 7.4) were added, 100 pl of tracer (diluted in 2% BSA solution to approximately 30000 counts min-1) and 100 pl of antibody (dilution 1 + 199 in phosphate buffer). The mixture was allowed to equilibrate at room temperature for 90 min. Only for the separation of bound and free ligand with goat anti-rabbit y-globulin (GARGG) was an antiserum dilution of 1 + 399 used. Different separation techniques were tested and the effects o f the separation procedures on the apparent titre of the antiserum were examined.First, the fractional precipitation of bound and free antigen with ethanol, ammonium sulfate and PEG 6000 was attempted. Different concentrations of ethanol (10,20,50,80 and 90%), ammonium sulfate (0.5, 1, 1.5,2 and 2.5 mol 1 - 1 ) and PEG 6000 (2.5, 5, 10, 15 and 20%) were applied. The NRS was used as carrier protein for the PEG precipitation, and different concentrations of the carrier protein were checked. Second, the non-specific adsorption of the tracer to charcoal particles, as described by Herbert et al. , I 9 was attempted. Three types of charcoal (Norit Supra A, Norit Supra B and Norit Extra C), were examined. For the Separation of bound and free buprenorphine, 200 pl aliquots (1% suspension) of the three types of charcoal were added to the incubation mixture.After different time intervals, the matrix was centrifuged at 188Og for 15 min, and the supernatant phase was counted for 1 min. Furthermore, the influence of coating the charcoal with dextran was tested. As a third separation technique, a second antibody method using GARGG was evaluated. This technique was first introduced by Utigcr et ~ 1 . 2 ~ ) and Morgan and Lazarow." To optimize this procedure, several parameters were investi- gated, such as the incubation time and the optimum amount of GARGG and NRS. After the initial incubation (90min) at room temperature, bound and free buprenorphine could be separated by adding 50 p1 of NRS (1 + 100 in distilled water) and 50 pl of GARGG (1 + 11.5 in phosphate buffer, 0.1 mol I-', pH 7.4). After vortex-mixing, the mixture wasANALYST, FEBRUARY 1993, VOL.118 139 incubated for a further 6 h. The precipitate was centrifuged for 15 min at 300013 and counted for 1 min. Finally, a combination of the second antibody technique and fractional precipitation was examined. Different concen- trations of ammonium sulfate (0.5, 1.0 and 2.0 mol I-*), ethanol (12.5, 25 and 50%) and PEG 6000 (1, 4.5 and 9%) were added and mixed with 50 pl of GARGG and 50 p1 of NRS. At different time intervals (0.25,1,2,6,12 and 24 h) the precipitates were centrifuged and counted. The second antibody separation technique (GARGG) was selected for the derivation of the calibration graph. The logit-log transformation22 was used to linearize the calibration graph. For the derivation, buprenorphine hydrochloride was diluted in drug-free urine to produce a concentration range of 100-25000 pg ml-1, and 100 p1 of the spiked urine samples were analysed by the RIA procedure.When the determina- tion of very low buprenorphine concentrations is necessary, a calibration graph between 10 and 10000 pg ml-l is more appropriate. Non-specific binding (NSB) was determined by replacing the antiserum by an equal volume of phosphate buffer. For each batch of samples a calibration graph was obtained within the same day. Human Samples To one healthy human volunteer (male, 27 years), 0.3 mg of buprenorphine (Temgesic injection formulation) was ad- ministered by intramuscular injection. Urine samples were collected over 72 h. The same volunteer was treated several months later with 0.2 mg of buprenorphine sublingually (Temgesic sublingual compr.0.2 mg). For the sublingual administration, a 0.2 mg tablet was placed under the tongue until dissolved (8-10 min). The urine was collected over 10 d. Several urine samples from persons suspected of Temgesic misuse were also analysed by the immunoassay. The RIA has also been applied to the analysis of plasma samples, using a slightly modified procedure. The standards are dissolved in blank drug-free plasma, and the reaction matrix consists of a 0.5% (instead of 2%) BSA solution in buffer. Results Preparation of the Tracer, Hapten and Immunogen After isolation by solid-phase extraction (reversed-phase C-18) and determination by HPLC with electrochemical detection (ECD), the specific activity of the tracer was counted to be 34 TBq mmol--l. The label could be used for 3 or 4 weeks without any loss of binding to the antiserum. Thereafter, the antibody-binding capacity declined rapidly.The hapten-BSA conjugate was prepared by the carbodi- imide technique, which was first described by Sheehan and Hess.2’ The spacer (diazobenzoic acid) was placed in the ortho position to the phenolic group. It was expected that the CH 5 18 - 14 N- 6 -J;9 8 CH,- -OH 20; 3 0 -CH2 24 26 27 Fig. 1 buprenorphine Structural formula of the hapten used for the immunization of antibodies would recognize the most important functional groups of buprenorphine, such as the phenolic, methylcyclo- propyl and tert-butyl groups. The HPLC procedure described in the preparation of the antigen allowed the isolation of the hapten (Fig.1). The collected fraction was dried with sodium sulfate, and the solvent was evaporated at 40°C under a stream of nitrogen. The structure of the hapten was confirmed by NMR and MS. The major fragments (mlz) of the methylated hapten are: 55,57,83, 101, 135,542,554,586 and 643. ‘3C NMR (CDC13/DMSO): 6 166.8 (C-7’), 151.4 (C-l’), 144.6 (C-4), 138.0 (C-2), 136.8 (C-3), 135.1 (C-12), 132.7 (C-4’), 129.4 (C-3’ + C-5’), 125.5 (C-ll), 121.3 (C-l), 120.3 (C-2’ + C-6‘), 95.4 (C-5), 79.2 (C-6), 77.7 (C-20), 57.9 (C-24), (C-22), 34.4 (C-14) 34.1 (C-15), 32.0 (C-19), 28.1 (C-lo), 25.1 56.6 (C-9), 51.1 (OMe), 45.1 (C-13), 42.1 (C-7 + C-16), 38.9 (3 x Me), 21.2 (C-8), 18.7 (C-21), 16.7 (C-18), 8.1 (C-25), 2.8 (C-26), and 2.0 (C-27).The values of the aromatic ring indicate that the diazo substituent is attached at the 2-position as could be expected from the reaction conditions. It was observed that the sequence of adding the hapten solution to a mixture of BSA and carbodiimide was important because of the poor solubility of the hapten. A precipitation was observed if carbodiimide was first added to the hapten solution. By using the latter sequence of mixing, almost no hapten was coupled with BSA. Hapten-BSA Ratio By using UV spectroscopy, a hapten-protein ratio (pmol) of 4.3 : 0.214 was calculated, corresponding to the coupling of about 20 mol of hapten with 1 mol of BSA. An average of 15 molecules of hapten per molecule of BSA has been recom- mended for an optimal immuno-resp0nse.2~ Titre of the Antibodies Results for serial dilutions of the antiserum, examined for their ability to bind 50% of the tracer, are shown in Fig.2. It was observed that rabbit 1 was likely to produce sera with a titre; after 2 months however, the animal generated ‘elephant teeth’ and had difficulties in eating. From that moment on, the titre decreased and stabilized at Ifi200. Although the titre of the antibodies was sufficient (600) 2 months after the first injection of the conjugate, the cross-reaction of the antibodies towards N-dealkylbuprenorphine and etorphine was high (100 and 12%, respectively) in comparison with the selected antibodies of rabbit 2 after the third bleeding. The titre of the latter antibodies reached only 400, but the specificity towards the two compounds was superior when compared to the antibodies of rabbit 1 (90 and +4%, respectively).The quality of the antibodies remained constant while the titre fluctuated around 300. For their superior cross-reactivity characteristics, the antisera of rabbit 2 (third collection) were lyophilized and used for the development of the immunoassay. They were stored at 4°C. .- r) c 300 - 0 50 100 150 Timeld Fig. 2 Antiscra dilutions necessary to obtain 50% binding of the tracer. The arrows indicate the time intcrvals where the BSA-hapten conjugate was injectcd140 ANALYST, FEBRUARY 1993, VOL. 118 Determination of Optimum Conditions for the RIA Precipitation with salts or organic solvents was among the earliest methods of fractionation of biological molecules.The major problem with fractional precipitation methods is that they tend to yield a high assay blank. In the buprenorphine assay, comparable results were obtained for the precipitation with ammonium sulfate and ethanol in a final optimum concentration of 1.5 and 7.4 mol I - l , respectively; 4045% of the tracer was bound with an antiserum dilution of 1 + 199. The blank values ranged from 12 to 17%. At least 40 pl of carrier protein (NRS) were necessary to precipitate the antibodies with PEG 6000. The blank value with this method reached 18%. The precipitation with ammonium sulfate and ethanol occurred instantly, whereas the PEG 6000 precipita- tion was only complete after 30 min of incubation at 4 "C. Different types of charcoals of the Norit range were examined for their separation properties.The types Supra A and Supra B have similar adsorption characteristics. When the separation step was carried out with the Extra C quality, however, a lower value for the percentage binding of the tracer was obtained. Coating the charcoal with dextran ( M , 60 000-90 000) had n o influence on the adsorption characteris- tics of the charcoal. It was shown that incubation for 15 min with 200 p1 of a 1% freshly prepared charcoal suspension was sufficient to capture the free tracer. The NSB value was high, but acceptable (8.5%), and the percentage binding of the tracer reached 45% (antiserum dilution 1 + 199). In comparison with the results of the adsorption and precipitation techniques, the GARGG second antibody method resulted in a lower NSB value and in higher dilutions of the antiserum for a 50% binding of the tracer.An antiserum dilution of 1 + 399 could bind 50% of the tracer. To separate bound from free label, the optimum ratio of GARGG antiserum to NKS was investigated. Final dilutions of GARGG (1 + 124) and NRS (1 + 999) resulted in the highest percentage binding of the tracer. The optimum second incubation time for the precipitation of the antibody bound tracer was 6 h at room temperature. The rate of immunoprecipitation could be enhanced by addition of precipitation reagents to the second antibody.25 It was possible to reduce the total analysis time to about 2 h by using the combination method (fractional precipitation and GARGG). Although a higher NSB value and a lower maximum binding (+40%) were observed, it was still possible to obtain reproducible results.The three precipitation rcagents were mixed in different concentrations for the second antibody separation technique. The initial incubation condi- tions (titre 1 + 399) were the same as for the second antibody method. The best results were obtained with the combination of PEG (final concentration 9%) and GARGG. The major advantage of this method is the short second incubation time (15 min), permitting substantial reduction of the total analysis time. From the different methods studied, the second antibody method was selected for the assessment of the performance 95 2: 90 - 10 - 5 - I I I I I I I I I -1-4 0.1 0.25 0.50 1 2.5 5 10 25 50 Dose/ng rn I ~~ 1 Fig. 3 regression Calibration graph for buprenorphine based o n logit-log characteristics of the immunoassay , the longer analysis time not being a major criterion for the laboratory tests.The immunoassay with the latter separation technique resulted, after logit-log transformation, in a linear calibration graph for the concentration range 100-25000 pg ml-* (Fig. 3). The parameters of five RIA calibration graphs are summarized in Table 1. The calibration graph is expressed as: (Y) = PO + PI ( X ) with Y = log, y/l - y ( y = R/Bo; counts bound for arbitrary dose relative to that for zero dose); X = log, x (x = buprenorphine standard concentration); PO = intercept of the curve; and PI = slope of the curve. The 95% confidence limits for and PI are, respectively, 0.118 and 0.059. Minimum Detectable Dose (MDD) The MDD of the RIA can be defined as the dose level that results in an expected response, which is significantly different from the expected responsc for a zero-dose tube.26 Two populations (zero-dose and 10 pg ml-1 tubes) were compared with a 'one-sided' Student's t-distribution (p = 0.01, degrees of freedom = 8).The two populations were first checked for the validity of the equal variance distribution with an F-test. It was found that a concentration of 10 pg ml-1 is, for 99% of the examples, significantly different from the blank value. For routine analysis, however, only the concentration range between 0.1 and 25 ng ml-1 is considered. Precision The intra- and inter-assay RSDs for the immunoassay are illustrated in Table 2. The results are the mean of five replicate determinations.The intra- and inter-assay precision was calculated after the analysis of six different positive urine samples. Spiking Recovery To determine the recovery of buprenorphine, 900 p1 of a randomly chosen positive urine sample with an apparent Table 1 Pararncters o f the RIA calibration graph ( n = 5 ) Parameter Mean S Total countsldisintegrations min-' 28 050 384 Maximum binding, Po (%) 45 2.8 Intercept (Po) 0.383 0.059 Slope (PI) -0.535 0.025 Mid-point ((3501K)/ng ml- 1 2.3 0.30 Non-specific binding 6.6 0.8 r 0.997 0.001 Table 2 The intra- and intcr-assay prccision for six randomly selected urine samples Buprenorphine concentrationhg ml-' RSD" (%) n I n tra-assay- 5. 10 2.55 5 9.80 3.06 5 20.29 4.34 5 In fer-assay- 2.18 10.66 24.11 5.02 5 5.16 5 6.35 5 * RSD = relative standard deviation. Table 3 Spiking recovery of buprenorphine in the assay Addedlng Observedlng m l ~ 1 Expected1 Observed1 1 5.6 f 0.16 5.77 97 5 0.6 f 0.25 9.77 98 10 13.2 t 0.139 14.77 89 (n = 3) (mean f s) ng m l - 1 expected (%)ANALYST, FEBRUARY 1993, VOL.118 141 Table 4 The specificity of the RIA method for the major metabolite and the structurally related compounds etorphine and diprenorphine Compound Chemical structure Apparent added/ concentration/ reactivity Concentration buprenorphine Cross- ng ml-1 ng ml-1 (Yo) Etorphinc Diprenorphine 100 10 1 93 9 0.9 93 90 90 250 'CH2-CH3 25 2.5 6 1.1 0.15 2.4 4.4 6.0 250 0.8 0.3 25 0.3 1.2 2.5 0.0s 2.0 buprenorphine concentration of 5.3 ng ml-1 was spiked (n = 3) with 100 pl of three standard dilutions of buprenorphine ( I , 5 and 10 ng in 100 vl) in order to obtain a 1 : 10 spiking ratio, leaving the matrix of the spiked samples relatively intact.A 100 pl aliquot of the mixture was analysed by the assay procedure. To calculate the expected value, 90% of the non-spiked value was added to the added amount of buprenor- phine standard (Table 3). This recovery study resulted in the following linear regression equation: Y (observed) = 0.94 + 0.84 X (expected); Y = 0.997. Specificity The antibody specificity was assessed by measuring the cross-reactivity to other compounds that could be present in urine samples from persons suspected of drug misuse. The following compounds were found to be not detectable by the immunoassay procedure at a level of 10 pg ml-1: acetylsalicy- lic acid, amobarbital, Dr*-amphetamine, barbital, bezitramide, caffeine, chlorpromazine, cocaine, codeine, cotinine, dextro- moramide, diazepam, ethylmorphine, fentanyl, flunitraze- Pam, haloperidol, heroin, imipramine, lidocaine, lysergide (LSD), metamphetamine, methaqualone, morphine, nalox- one, normethadone, pentazocine, pethidine, phencyclidine, phenobarbital , pholcodine, propoxyphene and zolpidem.The specificity of the antiserum towards the major metabol- ite, N-dealkylbuprcnorphine, and towards t h c structural analogues etorphine and diprenorphine, is demonstrated in Table 4. The intermediate products of the synthesis of buprenor- phine, together with the 3-O-methyl derivative of buprenor- phine, N-ethylbuprenorphine and N-propylbuprenorphine, were also tested for their cross-reaction with the antibodies.The results are shown in Table 5 . Although the spacer was placed in the C-2 position, the antibodies were not able to recognize the alkyl group on the nitrogen. The major metabolite, N-dealkylbuprenorphine cross-reacts at the 90% level over a large concentration range, while the N-ethyl- and N-propyl-derivatives of buprenorphine show an even larger cross-reactivity (106 and 197%, respec- tively). This pattern of cross-reactivity was also observed for morphine by Catlin et al. 1s In this study, rabbits were injected with the 2-diazomorphine-BSA conjugate. Normorphine, the N-dealkylation product of morphine, was observed to be the most effective inhibitor of the 3H-labelled morphine.It was observed that the specificity of the antibodies was directed particularly to the region adjacent to the point of attachment to the carrier. This was indeed confirmed by the low cross-reaction of codeine and morphine 3-glucuronide and the high cross-reaction of normorphine. A 50% reduction in [3H]morphine binding with 4.4 pmol of morphine was obtained, whereas 225 and 350 pmol of morphine glucuronide and codeine, respectively, were required for a SO% binding reduction of the tracer. In the present study, the 3-0-methyl derivative of buprenorphine also shows a relatively low cross-reaction (12%). This substituent is positioned adjacent to the point of attachment of the hapten to BSA. Further- more, when urine samples were hydrolysed with (3-glucoroni- dase, a remarkable increase in apparent buprenorphine concentration was obtained, indicating a low cross-reaction of the glucuronides.Indeed, when the same urine samples werc analysed with use of the DPC RIA kit for buprenorphine, roughly the same results as those obtained by Hand et ~ 1 . 2 7 were observed. Hand etal. found an average increase in apparent buprenorphine concentration of only 50% after enzymic hydrolysis. With the newly developed assay, the apparent buprenorphine concentration increased 20-fold after enzymic hydrolysis. This clearly demonstrates a specificity of the antibodies towards the substituent at the 3-0-position higher than that obtained with the antibodies of the DPC kit.142 ANALYST, FEBRUARY 1993, VOL. 118 Table 5 Specificity of thc antibodies towards synthesized analogues of buprenorphine Concentration Cross- Chemical structure added/ ngml-L reactivity (Yo 1 Chemical Structure Concentration Cross- added/ reactivity ng ml-1 (Yo) 20 12 r Y-cH7-cH3 20 106 20 197 1000 0.02 ‘COCH3 1000 0.008 1000 0,009 1000 0.5 1000 0.04 Determination of Unchanged Buprenorphine in Urine Samples Several urine samples (n = 32) were analysed by both the RIA (Y) and HPLC-ECD ( X ) methods.The HPLC method allows the detection of 0.2 ng ml-1 of both unchanged buprenorphine and N-dealkylbuprenorphine.28 Results by the two methods were related by the regression line Y = 1.44 + 1.64 X (Y = 0.910). Determination of Unchanged Buprenorphine in Plasma Samples The immunoassay was also used for the determination of buprenorphine in plasma samples.Quantitative results for the analysis of 10 plasma samples by both RIA (Y) and HPLC- ECD (X) are correlated by the following regression line: Y = 0.007 + 1.58 X (Y = 0.930). Parallelism The logit-log method for RIA dose interpolation permits testing of parallelism of dose-response curves for the standard and unknown.29 An unknown urine sample, contain- ing 16.1 ng ml-1, of the drug, was assayed three times, both undiluted and diluted with blank urine (1 + 2, 1 + 4 and 1 + 8). The observed (Y) and expected ( X ) values yielded the following linear regression equation: Y = 0.72 + 0.989 X (Y = 0.996). Analysis for Buprenorphine-like Material in Human Samples Apparent buprenorphine concentrations in urine samples collected from a volunteer, following intramuscular injection of 0.3 mg of buprenorphine, ranged from 2.5 ng ml-1 (10 h after administration) to 0.3 ng ml-I (48 h).Lower values were determined in urine samples after sublingual administration of 0.2 mg of buprenorphine (0.9 ng mi-I 3 h after intake of the tablet and 0.03 ng ml-1 after 4 d). When the urine samples were submitted to enzymic hydrolysis prior to analysis, it was still possible to detect buprenorphine up to 7 d after administration. In urine samples of persons suspected of Temgesic misuse, buprenorphine concentrations as high as 50 ng ml-1 were measured. These high figures, when com- pared with those obtained after therapeutic administration, clearly suggest a misuse of the drug. Discussion This paper describes the development of an RIA for bupre- norphine, which depends on the synthesis of [ 1”IJiodobupre-ANALYST, FERKUARY 1993, VOI,.118 143 norphine and of the diazobenzoic acid derivative of buprenor- phine. The diffcrcnt results obtained with the four separation techniques clearly demonstrate the importance of the separa- tion procedure. The best results were obtained with the second antibody method (GARGG). Howcver, a long incuba- tion time is a disadvantage of this method. On the other hand, thc second incubation time could bc reduced to 15 min, when using the combination method of second antibody and PEG It is known that the immunological specificity of the hapten is determined by the position whcre the spaccr is attached to the antigen.15.10-7’ I t was therefore expected that, by attaching the spacer at the C-2 position of buprenorphine, the most important antigenic determinants of buprenorphine (the N - 17-methylcyclopropyl, the substituent on C-7 and the hydroxyl group on position 3) would still be accessible to the immunogenic system. The results, however, have shown that the antibodies obtained can recognizc thc hydroxy group and the substitutent on C-7, but cannot distinguish buprenorphinc from N-dealkylbuprenorphine.A comparison of the results on urine samples, obtained by the assay developcd ‘in-house’ and using a commercially available kit from DPC, indirectly shows a remarkably lower cross-reactivity of the formcr assay towards the 3-0 glucuronide of buprenorphine. This can also be concluded from the regression line obtained by comparison of HPLC and RIA results for the same urine samples.The exact value for the cross-reactivity, however, could not be determined, thc glucuronidc not being available. The purification of [ ~~~I]iodobuprenorphine by HPLC, as described previously,l3 resulted in a high radiochemical purity and specific activity of the tracer, thereby allowing a very sensitive immunoassay. The sensitivity of the assay was superior to that of the recently introduced commercial RIA for buprenorphinc. The latter assay had a limit of detection of 1 ng ml- 1. The proposed RIA has been used in the first place to screen a large number of urine samples from persons suspected of Temgesic misuse. Furthermore, thc test allows the determination of low levels of buprenorphine in plasma samples.Its applicability to the analysis of forensic spccimens (also containing other drugs) and to the analysis of plasma samples, collected from addicts during a detoxification pro- gramme with buprcnorphine, is under study. 6000 (9%). We thank Dr. J . Heykants, R. Woestenborghs and 1. Geuens of Janssen Pharmaceutica (Beerse, Belgium) for their help and interesting discussions. Dr. R. Busson and L. Laruclle arc gratefully acknowledged for recording the 13C NMR and mass spectra. References Cowan, A , . Lewis, J . W.. and McFarlane, J . R., Br. J . Pharmucol., 1977, 60, 537. Dum. J . E., Herz, A , , Br. J . Plzurrnucd., 1981, 74, 627. Hanbrock, J . M., and Rance. M. 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ISSN:0003-2654    

DOI:10.1039/AN9931800137

出版商:RSC    

年代:1993

数据来源: RSC