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Proceedinas - - - ~of t h e Analytical Division ofThe Chemical SocietyCONTENTS47 Presentation o f the Fifth andSixth SAC Silver Medals47 Presentation Dinner t o ProfessorL. S. Barka Retrospective View o fEuroanalysis 111, Dublin48 Education i n Analytical Chemistry:50 Summaries o f Papers50 'Recent Developments in Clinical83 Erratum83 Equipment News85 Theophilus Redwood Lecture85 Analytical Division Distinguished85 SAC Silver Medal86 Conference86 Course88 Analytical Division Diaryand Biological Analysis'Service AwardVolume 16 No 2 Pages 47-88 February 197PADSDZ 16(2)47-88(1979)ISSN 0306-1 396PROCEEDINGSFebruary 1979OF THEANALYTICAL DIVISION OF THE CHEMICAL SOCIETYOfficers of the Analytical Divisionof The Chemical SocietyPresidentR.BelcherHon. SecretaryP. G . W. CobbHon. Treasurer Hon. Assistant SecretariesJ. K. Foreman D. 1. Coomber, O.B.E.; D. C. M. Squirrel1Secretary Hon. Pubficity and Public Refations Officer Editor, ProceedingsDr. A. Townshend, Department of Chemistry,University of Birmingham, Birmingham, B15 2TTMiss P. E. Hutchinson P. C. WestonProceedings is published by The Chemical Society.Editorial: The Director of Publications, The Chemical Society, Burlington House, London, W1 V OBN.Telephone 01 -734 9864. Telex 268001.Subscriptions (non-members): The Chemical Society, Distribution Centre, Blackhorse Road,Letchworth, Hens., SG6 1 HN.Nonmembers can only be supplied with Proceedings as part of a combined subscription with The Anslystand Analytical Abstracts@ The Chemical Society 1979A MEETING ONHPLC STATIONARY PHASE TECHNOLOGYApril 4th 1979Colworth House, BedfordA joint meeting of the East Anglia and Midlands Regions and of theChromatography and Electrophoresis Group will be on the topic "RecentDevelopments in H PLC Stationary Phase Technology and Application toAnalytical Chemistry." The following have agreed to speak: Dr. D. C. Burgess,Glaxo Laboratories; Dr. G. B. Cox, Dupont Ltd.; A. D. Dale, Roche ProductsLtd.; A. D. Jones, Unilever Research; Dr. S. A. Matlin, the City University;and Dr. B. B. Wheals, the Metropolitan Police Forsenic Science Laboratory.For further details contact the secretary of the East Anglia Region, G.Telling, Unilever Research, Colworth House, Sharnbrook, Bedford, MK44 1 LO

ISSN:0306-1396    

DOI:10.1039/AD97916FX005

出版商:RSC    

年代:1979

数据来源: RSC

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February , 1979 ANALYTICAL DIVISION DIARY 87Analytical Division Diary, continuedMarch, continuedWednesday, 21st, 2.30 p.m.: LeedsNorth East Region and Chromatography andElectrophoresis Group on “Applications ofChromatography in the Textiles Field.”“Polyacrylamide Gel Electrophoresis of Suber-imidate Crosslinked Microfibrillar Proteinsfrom Wool,” by B. Ahmadi and P. T.Speakman.“Analysis of Fatty Amides by TLC,” byD. L. Connell.“Identification of the Internal Lipids of theWool Fibre by TLC,” by H. E. Crabtree,P. Nicholls and E. V. Truter.Department of Physical Chemistry, TheUniversity, Leeds.Wednesday, 28th, 2.15 p.m.: LeedsParticle Size Analysis Group on “Measure-ment of Fine Particles in the WorkingEnvironment. ”“Airborne Particles in the Urban Environ-ment,” by D.Ball.“Particle Size Distributions of FoundryDust,” by N. Vaughan and J. LeGuen.“Measuring the Respirable Fractions ofTalc,” by T. Ogden.One other paper.Department of Colour Chemistry and Dyeing,The University, Leeds.CHEMICAL SOCIETY, ANALYTICAL DIVISIONCHROMATOGRAPHY AND ELECTROPHORESIS GROUPAND EAST ANGLIA REGIONA Symposium onCHROMATOGRAPHY IN PHARMACEUTICALAND FORENSIC ANALYSISatSouthend College of Technology, CaernarvonRoad, Southend, 25th April, 1979The speakers will include D. W. Selby, J. Chamberlain, M. S. Moss andG. J. Dickes.The Registration Fees for the Symposium, to include morning coffee, lunchand afternoon tea, will be: CS members, f 7.00; non-members, f 12.00; RIC full-time student members and retired members, f4.00.For further details contactDr. D. Simpson, Analysis for Industry, Factories 2/3, Bosworth House, HighStreet, Thorpe-le-Soken, Essex, C016 OEAAnalytical Division DiaryFEBRUARYTuesday, 20th, 4.15 p.m.: LoughboroughJlidlands Region, jointly with the Lough-borough University of Technology Chemi-cal Society.“Optoacoustic Spectrometry and its Applica-tion to the Examination of Solid and LiquidSamples,” by G. F. Kirkbright.Lecture Theatre J O O l , University of Tech-nology, Loughborough, Leks.MARCHFriday, 2nd, 2 p.m.: SouthamptonAutomatic Methods Group on “Corrosion.”The University, Southampton.Wednesday, 21st, 2 p.m.: LondoniWicrochemica1 Methods Group : Meeting ofthe Elemental Analyser User Forum on“Determination of Low Levels of Carbonand Nitrogen.”Speakers: E. Hall and D. Mealor.Royal School of Mines, Prince Consort ICoad,London, S.W.7.Friday, 23rd, 9 a.m.: EdinburghScottish Region, Chromatography and Electro-phoresis and Joint Pharmaceutical A nalysisGroups, jointly with the Association ofClinical Biochemists and the Chromato-graphy Discussion Group on “High-performance Liquid Chromatography inClinical and Biological Chemistry.”“Recent Developments in Post-columnReactors for HPLC,” by Professor R. W.Frei.“HPLC Analysis of Drugs of Abuse,” byA. C. Moffatt.“HPLC Analysis of Antidepressants on SilicaGel,” by I. D. Watson.“The Contribution of HPLC in Cases ofAcute Poisoning,” by M. J . Stewart.“HPLC in Clinical and PharmacologicalStudies of Analgesic Drugs,” by L.F.Prescott.“Clinical Analysis of Steroids by HPLC, ”by P. F. Dixon.“The Development of HPLC Methods forthe Estimation of Enzymes of the HaemBiosynthetic Pathway,” by C. K. Lim.Department of Chemistry, The University,West Mains Road, Edinburgh.Tuesday, 27th, 4.30 p.m.: SwanseaWestern Region, jointly with the South WestWales Section of the CS/RIC.“Nuclear Microprobes : A New Technique forMaterials Examination,” by T. B. Pierce.Chemistry Department, University College,Swansea.Thursday, 8th, 6.45 p.m.: MoretonNorth West Region on “Preparative LiquidChromatography.”“Preparative Liquid Chromatography-Theory and Approaches,” by D. Neal.“Applications of Preparative Liquid Chroma-tography in the Pharmaceutical Industry, ”by F.Bailey.E. l i . Squibb & Sons Laboratories, ReedsLane, Moreton, Wirral.Friday, 9th, 6.30 p.m.: ChepstowWestern Region.A discussion on “Short Term Toxicity Test-ing, ’’ will be introduced by B. Hepburn.George Hotel, Chepstow.Wednesday, 14th, 6.30 p.m. : LondonMicrochemical Methods Group.A discussion on “The Analytical Poten-tialities of NMR,” will be introduced byA. G. Ferrige.The Savoy Tavern, Savoy Street, London,w.c.2.Wednesday, 14th, 7 p.m.: GlaspowScottish Region and Atomic SpectroscopyGroup.A discussion on “A4tomic Absorption Spectro-scopy: Flame versus Furnace,” will beintroduced by A. M. Ure and J. M. Ottaway.University of Strathclyde, Cathedral Street,Glasgow .Friday, 16th, 2 p.m.: BrightonAnalytical Division : Annual General Meeting,followed a t 2.30 p.m. by a meeting on“HPLC.”“Recent Advances and Future Developmentsin HPLC,” by Professor J. H. Knox.“Injection Technique in Liquid Chromato-graphy,” by C. F. Simpson.“Detection Systems for HPLC-CurrentStatus,” by T. J . N. Webber.“High Performance Size Exclusion Chromato-graphy-Recent Advances and Applicat-ions,” by B. B. Wheals.Old Ship Hotel, Brighton.[continued inside back coverPrinted by Heffers Printers Ltd Cambridge Englan

ISSN:0306-1396    

DOI:10.1039/AD97916BX007

出版商:RSC    

年代:1979

数据来源: RSC

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Vol. 16 No. 2 February 1979 Presentation Dinner for Professor L. S. Bark On November 17th, a Presentation Dinner in honour of Professor L. S. Bark was held a t the Kersal Cell, Salford. The dinner was arranged by the North West Region of the Analytical Division to celebrate the appointment of Stuart Bark to a Chair of Analytical Chem- istry a t the University of Salford and more recently to the Deanship of the Faculty of Science.The Kersal Cell is one of the oldest buildings in the North, having been founded in 1142 by King Stephen, and was associated with Henry vllI and Oliver crOmwell; John BYrom lived and wrote there. The guests at the dinner in Presentation by the President, Professor R. Belcher, to Professor L. S . Bark (R). 4748 PRESENTATION DINNER FOR PROFESSOR L.s. BARK Proc. AnaZyt. Div. Chem. SOC. Professor and M r s . Bark (R) with the Mayor and Mayoress of Salford. this historic setting included the Mayor of Salford, Councillor D. A. Dow, and Mrs. Dow, Professor R. Belcher, President of the Analy- tical Division, the Pro-Vice Chancellor of Salford University and Directors and Senior Managers from various industrial companies, including ICI, Lankro Chemicals, Pilkington Bros., Shell, Squibb, Turner and Newall and Unilever.The chair was taken by Mr. J. W. Ogleby, Chairman of the AD North West Region. During the dinner, Professor Belcher pre- sented Professor Bark with a Capo di Monte figure on behalf of the North West Region and colleagues and friends. In his speech, Professor BeIcher briefly traced the history of analytical chemistry in the UK and the reasons for the lack of Chairs in this subject in British Uni- versities.He pointed out that IUPAC and the CS have Divisions devoted to analytical chemistry on the same basis as other subjects, and that virtually all of the major analytical techniques had been developed within the Universities. He indicated that analytical chemistry is hardly recognised as an academic subject within the Universities, and con- sidered ways in which more Chairs could be set up and the important role that industry had to play.In his reply, Professor Bark stressed the need for the Universities to produce people who can solve real problems, rather than in- strumental specialists, and suggested that there is not a large enough supply of trained analy- tical chemists going into industry so that qualified chemists from other disciplines have t o move to analytical work. He considered that there is a need for more Chairs of Analytical Chemistry, with fairly large groups in order to be able to undertake viable research pro- jects. There was a need for support from in- dustry in this, and it was necessary for there to be a policy of reciprocal co-operation.

ISSN:0306-1396    

DOI:10.1039/AD979160047b

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

48 PRESENTATION DINNER FOR PROFESSOR L. s. BARK Proc. Analyt. Div. Chem. SOC. Education in Analytical Chemistry: a Retrospective View of Euroanalysis 111, Dublin The education sessions were organised by Professor D. Thorburn Burns and Dr. B. Woods on behalf of the Scientific Committee (Chairman D. Carroll), following a request from the Working Party on Analytical Chemistry (WPAC) of the Federation of European Chemi- cal Societies (FECS) for greater emphasis on educational matters, as a development of the discussions organised in Budapest (1975) and in Heidelberg (1972).Despite the title the conference was truly international with dele- gates from 37 nations and all continents; however, all of the invited and submitted contributions to the education sessions were of European origin.An extensive programme was organised and as most of the material is avail- able in abstract1 and will appear in more detail in a special issue of Zeitschrift f u r Analy- tische Chemie, only a brief outline of items in the programme is given and more attention is directed to the problems, omissions and Iessons learnt for the organisation of future meetings. The contributed papers session was chaired by the Analytical Division President, Pro- fessor R.Belcher, and contained papers by L. J . Ottendorfer, C. Whalley and W. P. Hayes. Dr. Ottendorfer discussed the likely impact of public health and environmental legislation on fundamental aspects and forward planning of relevent teaching programmes in tertiary edu- cation. Despite the abstract Mr.Whalley’s paper and the discussion were not as contentious as might have been expected. The assessment of industrial current and future needs in what is a constantly changing situation is of interest and i t is hoped that a full text will appear in Proceedings to allow further discussion. It is unlikely that TJK analytical chemistry academic departments and staff will be redundant,F e b wary, 1 9 79 RETROSPECTIVE VIEW OF EUROANALYSIS I11 49 particularly in view of the current Science Research Council interest in the future of analytical sciences and the recent RIC data indicating industrial manpower shortages in the subject by the 1980s.Dr. Hayes advocated the use of historical aspects of chemistry to stimulate and retain student interest using as illustration sources fabrication, technology and analysis of gold and silver objects.Examples and data were given ranging from ancient civilisations, through post-Restoration England to modern day Hallmarking and Coinage practice. The poster session (Chairman, Dr. B. Woods) was well supported and contained details of courses, syllabi and staffing levels from the following FECS members countries : Belgium, Czechoslovakia, Denmark, Finland, West Germany, The Netherlands, Norway, Poland, Republic of Ireland, Spain, Sweden, Switzer- land, UK and Yugoslavia.Material from Italy arrived too late for inclusion. In addi- tion, contributions were made on the teaching of atomic-emission (M. A. Leonard) and atomic- absorption spectroscopy (J . E. Cantle). The Analytical Division was represented by the Education and Training Group, which showed results of the three questionaires2-* carried out to monitor the state and requirements of education and training in both UK industry and academic institutions and details of the Robert Boyle Essay award^.^ The invited lecture session covered industrial, public service and academic aspects of the subject in a pan-European manner.The keynote lecture, shared jointly by Dr. D. Betteridge and Professor H. Malissa (Chairman WPAC), was a most successful and stimulating combination of pragmatic, empirical and theo- retical material. T. Miesel and E. Pungor outlined the structure, content and philosophy of courses in Hungary. W. H. C. Shaw summarised UK industrial analytical training requirements from the pharmaceutical in- dustries view and put forward a cogent case for the introduction of a number of specialised undergraduate courses to produce graduates in analytical chemistry.6 W.F. Smyth des- cribed problem-orientated analytical teaching and research in the life sciences based on his experiences at the University of Arhus. The teaching implications of analytical chemistry in the civil service in western Europe were out- lined by Y.Siegwart with particular reference to Switzerland. R. Visser described the possi- bilities and problems that can arise in nation- wide planning of analytical chemistry in universities. A recent graduate, W. F. Horak, discussed student reaction to course content, leadership and structure. The last paper ‘International Student Competitions in Analy- tical Chemistry,” was presented by V.Vajgand and contained data on feedback and profiling learning rates and abilities, besides details of the nature and practice of the competitions that have been organised since 1968. The attendance of more than 100 at the session is an indication of the interest in education in analytical chemistry ; the large numbers and shortage of time, however, detracting from the depth of the final public discussion.The over-all view is that despite the success and support of the education in analytical chemistry sessions too much was attempted within the formal conference programme. It is advised that future programme organisers resist pressure, from whatever source, to add to the initial outlined programme: the balance of the programme may well be improved but Osler’s edict should be remembered, “super- fluity of lecturing causes ischial bursitis.” Lecture sessions and topics should be restricted to allow more time for formal and informal discussion. From the number of times Pro- ceedings was cited as a reference it is clearly playing a useful role in the dissemination of educational data and information in analytical chemistry.Poster sessions were proved to be a suitable medium for the transmission of information on lecture syllabus content, assess- ment procedures and practical work. At the moment there is insufficient published material to derive maximum benefit from this system ; it is hoped that the material published as a result of Euroanalysis I11 will assist in filling this need.The almost total lack of material submitted at Euroanalysis I11 on the content and philosophy of practical courses is to be regretted as this is the easiest material to produce in a form suitable for adoption and transfer from one institution to another, and is of great value in view of the time and effort needed to develop good practical course material ab initio.The view was expressed several times that analytical conferences are not the place to discuss political topics such as the need for more Chairs of analytical chemistry, despite strong feelings on this item. Among the positive outcomes from Euroanalysis 111, in addition to those cited earlier, are the closer links between FECS member countries’ teachers of analytical chemistry and the awareness engendered in the host country, Ireland, of the role and importance of the correct level of support for the teaching of analytical chemistry, at least as judged by the lively correspondence50 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc.Analyt. Div. Chem. SOC. in the Irish Times. Euroanalysis IV in Helsinki will, it is felt, benefit from having a firmer base of published material on which to build and may see the start of European views and standards for syllabi and the like.References 1. Abstracts of Papers Presented a t the Third European Conference on Analytical Chem- istry, Institute of Chemistry of Ireland for the Working Party on Analytical Chemistry of the Federation of European Chemical Societies, Dublin, 1978. 2. Education and Training Committee of SAC, PYOG. SOC. Analyt. Chem., 1972, 9, 173. 3. Education and Training Group Committee, PYOG. Analyt. Div. Chem. SOC., 1977, 14, 1. 4. Education and Training Group Committee, PYOC. Analyt. Div. Chem. SOC., in the press. 5. Proc. Sac. Analyt. Chem., 1974, 11, 132. 6. Shaw, W. H. C., Proc. '4nalyt. Div. Chem. Soc., 1977, 14, 54. D. Thorburn Burns Department of Analytical Chemistry, Queen's University of Belfast, Belfast, BT9 5AG, Northern Ireland

ISSN:0306-1396    

DOI:10.1039/AD9791600048

出版商:RSC    

年代:1979

数据来源: RSC

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50 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Pmc. Analyt. Div. Chem. SOC. Recent Developments in Clinical and Biological Analysis The following are summaries of thirteen of the papers presented at a Meeting of the Scottish and North East Regions, the Automatic Methods, Microchemical Methods and Joint Pharma- ceutical Analysis Groups and the Northern Ireland Sub-committee held on June 22nd and 23rd, 1978, at the University of St.Andrews. Analytical Methods in Clinical Analysis - The Changing Scene P. D. Griffiths Department of Biological Medicine, t n i v e r s i t y of Dundee, Dundee, DD 1 4HN The purpose of this opening paper is first to familiarise analytical chemists with the discipline of clinical biochemistry, and secondly to provide a background for subsequent papers, which mostly cover recent advances in the analytical field.The work of the clinical biochemist is defined as: (i) carrying out complex assays for various constituents of biological fluids ; (ii) improving the technique and performance of such assays; (iii) developing new assays ; (iv) assisting clinical staff in understanding the meaning of the results obtained; and (v) research. Some of the inherent problems in carrying out this work are : (i) the material is potentially hazardous; (ii) only small amounts are available for analysis; (iii) the substance to be measured may have a very low concentration or activity; (iv) measurement usually has to be carried out in the presence of a multiplicity of unknown (v) usually results are required very rapidly; (vi) a laboratory may have a repertoire of more than 100 different analyses to perform; (vii) there has been an almost exponential increase in workload over the last 10-15 years and annual loads of in excess of 1 million assays are now to be found; and (viii) the interpretation of the results obtained can be difficult. There are both within- person and between-person variations, and analytical variation contributes to both.When the latter is a significant component of the total variation then an improved analytical technique is very desirable. The changing scene in clinical biochemistry is a result not only of progress in the range and nature of analytical methods, but also of technological advances enabling assay methods to be semi-automated.The advent of semi-automatic procedures began with the work of Skeggs (1957), who introduced the continuous-flow AutoAnalyzer. Although from the viewpoint of substances, which may directly or indirectly influence the assay result ;Febvzzavy, 1979 DEVELOPMENTS IX CLINICAL AND BIOLOGICAL ANALYSIS 51 the Technicon Corp. this and the subsequent series of multi-channel analysers has meant huge financial success, Skeggs’ work was a catalyst to other manufacturers, who have introduced a wide variety of different machines to permit the rapid and precise measurement of a variety of the commonly required analytes in biological fluids.The contrast between the laboratory of 20 years ago with its very limited range of equipment, e.g., EEL flame photometer, two EEL absorptiometers, Van Slyke apparatus for carbon dioxide combining power, two or three weighing balances, bench centrifuges, reversion spectroscope, an ultraviolet spectrophotometer and large amounts of glassware, having a total value of about @ 000, and a modern laboratory with its multi-channel analyser, automated spectrophoto- metric enzyme analysers, centrifugal analysers, at omic-absorpt ion spectrophot ome ters, gas and high-perf ormance liquid chromatographs, high-speed refrigerated centrifuges, ult racent ri- fuges, radiation counters and perhaps even a combined gas chromatograph - mass spectrometer system, is enormous.Although these various machines have been of great help in coping with the large workload and in opening up the repertoire of techniques available to a routine laboratory, they are not without their disadvantages.They tend to be inflexible; because it is often easier to do a group of tests simultaneously rather than a single test, they cause a falsely elevated workload; the ease by which a particular test can be automated tends to distort the pattern of the work- load; the data handling problem is greatly aggravated; the manufacturer, by his designs, imposes constraints on the analytical methods that can be used; a different type of staff skill is required ; and increasing dependence on machines raises problems in terms of rapid (and usually costly) maintenance and repair and what one does to sustain the rapid service while the machine is out of action.Earlier it was pointed out that there had been an increase in the range of tests required.This arises partly because of the hitherto unrecognised value of some assays for clinical pur- poses and partly by development of realistic techniques for measurement of substances known to be important but for which adequate methods were not available. An illustration given of the first category was the transaminase enzymes (aminotransferases), whose greatly increased activity in the circulation following breakdown of tissue cells could be used as an indicator of damage to heart muscle or liver.In the second category, radioimmunoassay of hormones was an example which was cited. Another important development in recent years has been the entry of manufacturers into the provision of ready-to-use reagents and, in particular, kits which require minimal staff effort to set up a batch of analyses.There is some evidence that the higher costs are more than offset by savings in the expensive local labour, and although this has rarely led to staff reductions, it has allowed the increase in load to continue without the need for additional staff. Although in theory one could expect greater reliability of reagents as a consequence, it cannot be ignored that, just as occurred with instruments, the manufacturer more and more is dictating the analytical methods used in the laboratory.Another major change has been the introduction of computers into clinical biochemical laboratories, for process control of instruments, to convert raw data into results and to handle the whole data processing of the laboratory, a task which has achieved mammoth proportions since analytical barriers to increased workloads have been removed.New instru- ments and new techniques will be absorbed, new assays will be required and perhaps tissue analysis will become more important. The scientific problems may be fewer than personnel problems arising from the changing industrial practices, which, together with the impact of the Health and Safety at Work Act, promise that, whatever else, the future will not be uneventful.It was predicted that the problems would not change much in the near future. Mass Spectrometry in a Biomedical Environment J. D. Baty Depavtment of Biochemical #!edicine, University of D m d e e , Dundee, O D 2 9s Y The use of mass spectrometry (ms) as an analytical tool in biomedical research is increasing, owing largely to the successful interfacing of the mass spectrometer with the gas chromato-52 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc.AnaZyt. Div. Chem. SOC. graph. This combination results in a sensitive and specific analytical instrument whose areas of application include clinical chemistry,l pharmacology2 and many other biomedical fields.3 A modern gas chromatograph - mass spectrometer system can constantly monitor the effluent from a gas chromatograph, generating a complete mass spectrum every few seconds and thereby obtaining data on every compound emerging from the chromatograph.An alternative mode of use is to monitor only certain pre-selected mass values known to be characteristic of a compound.In this technique, known by a plethora of titles, such as mass fragmentography, ion recording and ion monitoring, the mass spectrometer behaves as a selective gas chromatographic (GC) detector, recording the profiles of only a small number of mass values throughout the GC analysis. The technique of monitoring all the masses, i e ., obtaining a complete mass spectrum, has the advantage of allowing the structures of previously unknown compounds to be investigated. A data system is an essential requirement for the storage of mass spectra acquired by regular scanning of the GC effluent, as several hundred spectra can be acquired, for example, in a 1-11 capillary-column GC - MS run with a mass spectrum obtained every 2 s.Having stored all the mass spectral data in the computer, it is possible to select any mass value, trace the profile of this mass throughout the GC analysis and to determine if this value increases when a com- pound enters the mass spectrometer. Such a technique is illustrated (Fig. 1) by the work of Alme et aE.4 on the analysis of bile acids in urine. After extraction and derivatisation, the samples are analysed by the repetitive scanning technique and computer plots of selected mass values are obtained. A simultaneous rise in a series of mass values known to be charac- teristic of a compound indicates the presence of this material.A rise in those masses known to be diagnostic of a class of compounds can indicate a previously undetected member of the class.It is important to appreciate the distinction between the approach described above and the technique of mass fragmentography, in which only pre-selected mass values are monitored. This technique restricts the number of compounds that can be investigated in a single analysis but greatly increases the sensitivity and specificity of the analysis, as all of the mass spectro- meter time is devoted to monitoring relevant mass values.This approach has a great number Trihydroxy bile acid derivatives 12A A5-Cholenoic acid 623 548 533 458 443 Mass values 369 368 353 343 253 I I 0 50 1 00 Scan number Fig. 1. GLC - MS analysis of a monosulphate fraction from urine of a patient with intrahepatic cholestasis. The lower chromatogram is a partial ion current (PIC) chrom- atogram representing current of all ions from rnle 200 to 800. Adapted from reference 4.FebYIUYy, 1979 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL ANALYSIS 53 of applications in drug analysis, where a mass value characteristic of the drug is monitored together with an internal standard.The mass value selected for the latter can be the same as that chosen for the analyte (if the compounds are resolved by the gas chromatograph) or an isotopic analogue of the analyte (labelled with deuterium, carbon-13 or nitrogen-15) can be used.Several reviews of this technique have appeared.596 The method is often used as the reference technique in comparison with radioimmunoassay, etc.’ One problem that occasionally arises in using this approach is the appearance of background signals at the pre-selected mass values.If this cannot be circumvented by changing the mass value or using a different GC phase or a derivative of the compound, then the analysis can still be performed if a high-resolution instrument is available. The terms low resolution and high resolution in mass spectrometry refer to the ability of the instrument to resolve integral mass values (low resolution) or accurate mass values (high resolution).While the inherent sensi- tivity of the instrument is reduced when operating at a high resolving power, the freedom from chemical background contamination (only the analyte will show a signal at its accurate mass value) exerts a compensatory increase in the signal to noise ratio.An elegant example of this technique is the work of Millington et aL8 on the detection and analysis of picomole levels of androst anediols in prost ate tissue. An expanding area of interest in the biomedical field is the use of stable isotope-labelled compounds as probes in the investigation of enzymatic reactions.9 Such compounds can be used in human studies with none of the risk associated with radioactive labels, and mass spectrometry gives a specific analysis of the reaction products.l* This brief review has touched only the surface of the many exciting applications of mass spectrometry. Space does not permit a discussion on “soft” ionisation techniques, such as chemical and field ionisation, metastable ions or negative ion work. One of the most interest- ing developments that will be of great significance with regard to biochemical problems is the recent introduction of liquid chromatography directly interfaced with mass spectrometry.This technique promises to extend the types of compound amenable to mass spectroscopic analysis. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. References Lawson, A. M., Clin. Chem., 1975, 21, 803. Lawson, A. M., and Draffan, G.H., Prog. Med. Chem., 1975, 12, 1. Wallet, G. R., Editor, “Biochemical Applications of Mass Spectrometry,” Wiley-Interscience, New Alme, B., Bremmelgard, A., Sjovall, J., and Thommassen, P., J . Lipid Res., 1977, 18, 339. Holmstedt, B., and Palmer, L.. Adu. Biochem. Psychopharmac., 1973, 7, 1. Falkner, F. C., Sweetman, B. J., and Watson, J. T., AppZ. Spectrosc. Rev., 1975, 10, 51.Bjorkhem, I., Blomstrand, R., Lantto, O., Svensson, L., and Ohman, G., Clin. Chem., 1976, 22, 1789. Millington, D. S., Buoy, M. E., Brooks, G., Harper, M. E., and Griffiths, K., Biomed. Mass Spectrom., Baty, J. D., and Robinson, P. R., Clin. Pharmac. Ther., 1977, 21, 177. Baillie, T. A., Editor, “Stable Isotopes : Applications in Pharmacology, Toxicology and Clinical York, 1972.1975, 2, 219. Research,” Macmillan, London, 1978. Enzyme lmmunoassay in Clinical Practice Vi n ce n t M arks Department of Biochemistry, Division of Clinical Biochemistry, University of Surrey, Guildford, G U2 5XH Enzyme immunoassays (EIA), of which there are many varieties, are specific types of ligand assays, the best known example of which is radioimmunoassay (RIA). There are three essen- tial reagents in every immunoassayl: (i) a high avidity, specific antibody; (ii) a high specific- activity label; and (iii) a pure standard.There was no serious doubt, in the early days of immunoassay, that the label would invariably have to be a radioisotope in order to achieve the requisite sensitivity; and that separation of the free from the antibody-bound label would be necessary in order to quantitate the two fractions.After a short initial period one technique for preparing radiolabels emerged as pre-eminent, namely, the chloramine T method of Hunter and Greenwood.2 Much of the subsequent54 DEVELOPMENTS ITU’ CLINICAL AND BIOLOGICAL ANALYSIS PYOC. Annblt. Ilk). CIwz. SOC. methodological development in immunoassay in the 1960s was devoted to devising better, more rapid, more reliable and technically simpler methods of separating the bound from free label.The idea that substances other than radioisotopes might be used as labels in immunoassay, though recognised as a possibility, was not seriously entertained until almost a decade after the first description of radioimmunoassay. Thereafter, just as the technology of radioimmuno- assay expanded from the use of iodine-131 as the labelling material to encompass other isotopes, namely, iodine-125, carbon-14, hydrogen-3 and selenium-75, so did the ways in which enzymes could be employed in immunoassay. One important difference between the developmental history of radio- and enzyme immuno- assays is that whereas the principles of RIA [like those of competitive protein binding (CPE) and receptor assays] entered the public domain through the medium of the scientific literature, many of those relating to enzyme immunoassay entered via the Patent Office.In most types of enzyme immunoassay the enzyme merely replaces iodine-125 or hydrogen-3 as the label in a conventional radioimmunoassay. Conceptually, therefore, the principles are the same. These can be called substitutional enzyme immunoassays as they are, in reality, more akin to radioimmunoassay than to the second type of enzyme immunoassay which, because there is no need to separate free from bound ligand, are often referred to as homogeneous assays.In only two areas, so far, have enzyme immunoassays proved themselves clearly superior to radioimmunoassays : (i) the detection and measurement, if needs be, of circulating antibodies in patients suffering from infective, and especially tropical, diseases by means of enzyme-linked immunoabsorbant assays (ELISA) ; and (ii) the clinical measurement in the blood and urine by homogeneous enzyme immunoassay (EMIT) of drugs and other small molecules, such as thyroxine and cortisol, mainly as an aid to monitoring and regulating therapy but also for diagnosis.Principles Enzyme immunoassays have recently been extensively reviewed (Scharpe ct d. ,3 Wisdom4 and Schuurs and Van Weeman5) and are therefore described briefly. The sub- stitutional type of enzyme immunoassays are essentially the same as those of the correspond- ing radioimmunoassays right up to the final stage of quantitation, which is achieved by measuring enzyme activity rather than isotopic disintegrations.These are analogous to the now classical antigen-excess type of radioimmunoassays in which competition between labelled hapten and native hapten for a limited number of antibody binding sites takes place. Secondly, there are immunoenzymometric assays based on the principles of imniunoraclio- metry, proposed and developed by Miles and Hales6 in which it is the antibody that is labelled and in excess.The sandwich-type enzyme immunoassay, which employs the principles of yet another radioimmunoassay technique developed by Hales, namely his two-site immunoradio- metric assay, is a development of this and is particularly useful for detecting and measuring large molecules.ELISA is an enzyme substitute for the radioallergosorbent, or R,GT technique, developed by Wide and used mainly for the detection and measurement of anti- bodies. EMIT, the only homogeneous assay system7 to have progressed beyond the development stage, has no radioimmunoassay analogue and utilises a chemical property of the enzyme, which actually depends upon the fact that they are proteins with both shape and structure, rather than mere labels.Because of their geometry the rate of reaction between some enzymes and their substrates is altered by the proximity of the enzyme to another molecule, such as an antibody, either by interfering with access of the substrate to the active site or by steric hindrance. Firstly, there are competitive enzyme immunoassays.Substitution Enzyme Immunoassays Firstly the relative merits and demerits of substitutional enzyme immunoassays zcysz,6s radioimmunoassays will be considercd. Both types of assay can be specific and sensitive; specificity is, however, a charac- teristic of the antibody and not of the enzyme label as is sensitivity which, although often as Substitutional and homogeneous assays are best considered separately.Febvzmy, 1979 DEVELOPMENTS IN CLIXICAL AND BIOLOGICAL AKALYSIS 55 good in enzyme immunoassay as in radioimmunoassay, is generally no better and sometimes worse.Arguments that the equipment needed to carry out substitutional enzyme immunoassays is simple, cheap and widely available whereas that needed for RIA is sophisticated and expensive, are spurious.Automated enzyme analysers are just as expensive as automated isotope counters and there is little difference in cost between a single-well y-counter suitable for manual use in RIA and a spectrophotometer. Substitutional EIAs are no simpler, and often more complicated, to perform than analogous radioiinmunoassays. They are rarely quicker and enzyme labels are usually no cheaper to produce than radiolabels, mainly because of the inefficient coupling methods employed.Though generally less easy to prepare in the user’s laboratory than radiolabels, enzyme labels do not require special isotope facilities. Methods of preparing enzyme-labelled haptens that permit almost lOOq/, incorporation of the enzyme into the hapten - enzyme complex, with almost complete retention of catalytic activity, have been developed in the author’s laboratory and should eventually make self-production of enzyme labels for substitutional enzyme imniunoassays a practical proposition.Enz!.me labels often have a longer shelf-life than radiolabels although partial disruption of the covalent links made between some enzymes and haptens, antibodies or solid phase supports, can occur in some systems leading to unacceptably high blank values.Radiation hazards to users of radioimmunoassay kits have been grossly exaggerated ; the only rml risk from radiation, and that very slight, is to workers making the kits and only then if there has been gross carelessness or negligence. It must be recognised, however, that both emotional and legal obstacles exist in many parts of the world to the use of radioisotopes, even in infinitesimal amounts, and it is at this level of logic that enzyme immunoassay shows a considerable advantage.Substitutional type EIAs have been described for a wide range of compounds but few of them are currently of interest to clinicians. The reason for the small number of kits available commercially, more than eight years after introduction of the technique, is difficult to under- stand but may be related to the difficulty, and consequently the expense, of producing enzyme labels and the absence of clear advantages over RIAs.The one area in which substitutional enzyme immunoassays have proved themselves, in the author’s opinion, is in the detection and quantitation of antibodies using an ELISA technique, plastic microprecipitin templates and a chromogenic substrate. In this way it is possible to carry out large numbers of assays which can be read by eye, making the technique especially useful for investigations in remote areas without ready access to well-equipped clinical laboratories.Homogeneous Enzyme Immunoassays The impact of homogeneous enzyme immunoassays on clinical practice has been nothing short of revolutionary, especially in hospitals that have adopted them for drug measurement and therapeutic monitoring.The reasons are not hard to find. The commercially available systems are easy to use; as accurate and precise as other assay techniques of comparable sensitivity and generally faster, simpler and more amenable to performance in outpatient clinics, at the bedside or in coronary care units where speed may be essential. The major limitation of the currently available EMIT system is that its applications are restricted to the measurement of small molecules, e.g., drugs and hormones such as thyroxine, T3 and steroids.Because the reagents are difficult to produce and have to undergo rigorous testing and quality control before being assembled to produce a reliable analytical system, the cost tends to be high.Nevertheless, as the system has gained wider acceptance the cost of individual assays has fallen so that a true costing, i.e., one that takes into account labour, time, space and capital equipment, as well as the cost of the reagents, often compares favourably with most current in-house methods.EMIT was first introduced for detection, rather than quantitation, of drugs of abuse in urine. It is now mainly used for making blood drug measurements, or in the diagnosis of thyroid disease, and lends itself admirably to automation. In the authors’ laboratory EMIT has been used to measure serum thyroxine in a Rotochem centrifugal analyser with a speed and precision surpassing that of RIA but with lower sensitivity.Investigators with instruments56 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc. AnaZyt. Div. Chem. SOC. portable enough to use with EMIT reagents in the neurological outpatients clinic, for example, report that the benefit to patients of being able to have their plasma anticonvulsant levels measured whilst they are still in consultation with the doctor is considerable.The doctor can, on the basis of the analytical results obtained, adjust the dose of drug should it be necessary, immediately without the necessity of writing to the patient and getting him to attend again solely ior this purpose. Improvements in anticonvulsant therapy that began with the introduction of blood drug monitoring are thus likely to continue as a result of this new technology .Some use enzymes but others do not. One uses a fluorophore which ceases to fluoresce when the hapten to which it is linked binds to an antibody. I t seems likely that the various homogeneous assay systems including those under development, will eventu- ally make as big an impact upon clinical laboratory practice as radioimmunoassay has done in the past.Other types of homogeneous immunoassay are being developed. Others depend upon the removal of quenching. References 1. 2. 3. 4. 5. 6. 7. Yalow, R. S., and Berson, S. A., J . Clin. Invest., 1960, 39, 1157. Hunter, W. H., and Greenwood, F. C., Nature, Lond., 1962, 194, 195. Scharpe.S. L., Cooreman, W. M., Blomme, W. J., and Laekman, S. M., Clin. Chern., 1976, 22, 733. Wisdom, G. B., Clin. Chem., 1976, 22, 1243. Schuurs, A. H. W. M., and Van Weemen, B. K., Clinica Chim. A&, 1977, 81, 1. Miles, L. E. M., and Hales, C. N., Nature, Lond., 1968, 219, 186. Rubenstein, K. E., Schneider, R. S., and Ullman, E. F., Biochem. Biophys. Res. Cornrnun., 1972, 47, 846.Fluorimetry and Phosphorimetry in Clinical Analysis J. N. Miller Department of Chemistry, Loughborough University of Technology, Loughborough, Leicestershire, LEI 1 3T Z; Analyses in clinical and biological fields present problems that are unique in their severity and complexity. Analytes must often be determined at picogram or nanogram levels, and the samples studied are immensely complex mixtures of organic and inorganic species; frequently a number of these species are chemically very similar, as when a drug is to be analysed in the presence of its metabolites.A successful analytical method must therefore possess high sensitivity and excellent selectivity; in addition, it should be relatively simple to perform and easily automated. They are well known to be exceptionally sensitive: a strongly fluorescent material can generally be determined at concentrations of 1 ng ml-l or less, and non- or feebly fluorescent materials can be modified using labelling or other reactions to produce suitably fluorescent derivatives.Moreover, various advances in the design and performance of commercially available fluori- meters have led to continuing improvements in the limits of detection attainable.The selectivity of fluorimetric methods, however, is often less satisfactory, mainly because of the large spectral band width (typically 50-100 nm) of the fluorescence spectra of many organic compounds. Overlaps between the fluorescence spectrum of the compound under study and other fluorescence signals and with first- or second-order Rayleigh or Raman scattering bands of the solvent thus occur frequently. This paper summarises some of the many recent advances made in improving the selectivity of luminescence analyses, which can be divided into three groups as follows: (a) optical and electronic modifications of existing fluorimetric procedures ; (b) combinations of luminescence spectroscopy and chromatographic methods ; and (c) combinations of fluorimetry and specific biochemical interactions. Fluorimetric methods have now been in use for over 20 years.Optical and Electronic Modifications of Conventional Procedures A number of methods are available for improving the selectivity of fluorimetry, which These include the involve only minor modifications to existing equipment and techniques.February, 1979 DEVELOPMENTS IN CLISICAL AN L, BIOLOGICAL ANALYSIS 57 use of derivative fluorescence spectrxcopy, synchronous scanning spectroscopy and polarising films.Derivative fluorescence spectroscopy, which involves obtaining spectra showing dI,/dX, d21f/dA2, etc., plotted against A, rather than the conventional If 'uersus X spectra, was apparently introduced by Green and 0'Haver.l They demonstrated that derivative techniques are of value in the analysis of mixtures and that they enhance the resolution of minor spectral features. The derivative spectra can be generated optically or electronically: the latter method is simpler and a suitable unit is commercially available (Perkin-Elmer Ltd., Beacons- field, Bucks.).Until recently, this approach had been applied only in studies of hydrocarbons, the fluorescence spectra of which exhibit well defined vibrational fine structure.The principle can also be applied, however, to the resolution of species with broad, featureless spectra.2 Figs. 1 and 2 show its application to the solution of an old problem, the identification of a tyro- sine contribution in the fluorescence of a protein containing both tyrosine and trytophan.In such proteins the fluorescence of the latter amino acid is generally predominant and the identification and quantitation of a small overlapping tyrosine component is difficult .3 Fig. 2 shows that, while the tyrosine contribution to the fluorescence of human serum albumin is only just discernible in a conventional spectrum, it is readily apparent in first and, particu- larly, second-order derivative spectra.I I I I 250 300 350 400 4 Xlnm 0 Fig. 1. Conventional excitation (E) and fluores- cence (F) spectra of aqueous solutions of tyrosine (broken line) and tryptophen (solid line). An alternative approach to reducing spectral overlaps is the synchronous scanning method developed by Lloyd.4 The technique involves the simultaneous scanning of both monochro- mators of a spectrofluorimeter, with a fixed wavelength difference between them.This difference will depend on the conventional excitation and emission spectra of the sample under study. So far, this technique also has been applied mainly to the resolution of hydrocarbon mixture^,^ but it is equally effective in reducing the band widths of featureless spectra (Fig.3) and has been applied with success to the problem of tyrosine and tryptophen mixtures6 A further advantage of this approach is that discrete peaks due to solvent Rayleigh scattering never occur in synchronous spectra, although peaks due to Raman scattering may be detected.' Polarised fluorescence has long been used in the study of protein conformation, but the use of polarising films in analytical work has remained unjustly neglected.Suitably oriented polarisers can largely eliminate Rayleigh and Raman scattered light signals: this effect not only58 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL AKALYSIS YYUC. A Jzal_?Jt. nhl. CheWZ. SUC. h/nm Fig. 2 . Fluorescence emission spectrum (I,) of an aqueous human serum albumin solution, lvith its first and second derivatives.facilitates analyses where such signals seriously overlap the desired fluorescence signal, but also reduces the scattered-light background over a wide range of wavelengths, yielding improved detect ion limits. 8 Combinations of these modified techniques may also be of value. For example, derivative synchronous spectra have very low spectral band widths, and polarising films could be used to minimise scattering effects in synchronous or derivative spectroscopy.1 250 300 350 400 Xlcm Fig. 3. Excitation (E), fluorescence (F) and synchronous (S) spectra of an aqueous solution of tryptophan. The synchronous spectrum was ob- tained a t a constant wavelength difterence of 60 nin (see text).February, 1979 DEVELOPMEKTS IN CLINICAL AND BIOLOGICAL ANALYSIS 59 Combinations of Luminescence Spectroscopy and Separation Methods This field has seen many advances in recent years and numerous analyses based on the fluorimetric detection of high-performance liquid chromatographic (HPLC) separations, and in the fluorimetric scanning of thin-layer chromatographic (TLC) plates, are now well established. HPLCg is dealt with in other papers presented in this meeting and TLC - fluorimetry has also been thoroughly reviewed recently.10911 Noteworthy recent advances in the latter field have included the production of improved spectrodensitometers, and the development of high- performance and reversed-phase TLC plates.HPTLC is of particular value in conjunction with fluorimetry, providing rapid and efficient separations of picogram amounts of sample.12 Combinations of TLC and phosphorimetry may also be of value, and several recent advances have been made in this field.In some respects, phosphorimetry complements fluorimetry, as many non-fluorescent species are phosphorescent, and vice versa. Phosphorimetry has some additional advantages, however, including the elimination of scattered-light interference, and the extra selectivity provided by easily measured phosphorescence lifetimes and fluorescence to phosphorescence ratios.13 In this laboratory, a thin-layer phosphorimeter has been built (as an attachment to a spectrofluorimeter) that permits in sit% observations of phosphorescence on the surface of a TLC plate at 77 K.14 Nanogram amounts of phosphorescent materials can be detected, especially with the aid of a solvent enhancement effect.15 Table I shows the principal luminescence and chromatographic properties of 6-mercapto- purine and some related compounds, many of which can be detected at subnanogram levels using thin-layer phosphorimetry at 77 K.Fig. 4 shows scans of chromatograms of typical mixtures of these materials, and illustrates the advantages of spectroscopic selectivity.The precision of this method is excellent (coefficient of variation about 4%) and it is being applied to the analysis of a number of classes of drugs in serum. The availability of the thin-layer phosphorimeter also encourages the development of phosphorescent label molecules, which would be used in a manner analogous to that of dansyl chloride, fluorescamine and other fluorescent labels ; such studies are proceeding in this laboratory.16 TABLE I LUMINESCENCE OF BIERCAPTOPURINES AND RELATED COMPOUNDS Detection limits at 77 K Compound 2-Mercaptopurine 6-Mercaptopurine 2-Thioxanthine 6-Thioxanthine 6-Mercaptopurine riboside 6-Mercaptopurine riboside-5- 6-Methylmercaptopurine 2-Amino-6-mercaptopurine 6-Mercaptoguanosine 6-Mercaptopurine-2-deox yriboside Azathioprine phosphate Xexlnm 342 316 295 344 323 337 286 330 341 320 300 h*/nm 510 454 458 505 462 446 436 479 49 1 460 442 I I n ethanol/ On cellulose ng ml-l* thin layer/ng 1 50 104 50 50 1 1.4: 0.01 30: 2 20 25 1 - 50 $ 0.1 60: 4 120 0.05 104 100 RF 0.37 0.44 0.19 0.21 0.62 0.71 0.63 0.32 0.51 0.41 0.66 * In neutral ethanol, except those marked t On cellulose TLC plates with 0.1 M HC1 as eluting agent, except for 2-thioxanthine, for which the solvent analysed in ethanol containing 0.1 M NaOH.system was propan-2-01- methanol - water - ammonia (60 + 20 + 20 + 1). The thin-layer phosphorimeter can also be used for observations of room-temperature phosphorescence (RTP).This phenomenon was apparently first observed over 30 years ago, but has only recently been exploited as an analytical technique.17 In suitable conditions many materials probably show the RTP effect, especially in conjunction with the enhancing heavy- atom effect.l8,l9 As RTP retains most of the advantages of phosphorimetry, and eliminates the major drawback (viz., the need to use liquid nitrogen as a coolant), it seems likely to become widely used, though many aspects of the phenomenon remain poorly understood.Table I1 shows that several mercaptopurine derivatives can be studied using RTP with detection limits that, although less good than those at 77 K, may still be valuable.2060 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS PYOC. Anal$€. Div.Chem. SOC. (a 1 A D F Fig. 4. Thin-layer phosphorimetry : chromatograms of a mixture of (A) 2-amino-6-mercaptopurine, (13) 6-mercaptopurine, (C) 6-mercaptoguanosine and (D) 6-methylmercaptopurine, scanned a t 77 K. ( a ) A,, = 342 nm, A, = 485 nm; and (b) Aex = 320 nm, Ap = 448 nm. R, values on cellulose thin-layers using 0.1 M HC1 as eluting solvent: A, 0.32; B, 0.44; C, 0.51; and D, 0.63.TABLE 11 ROOM-TEMPERATURE PHOSPHORESCENCE OF MERCAPTOPURINE DERIVATIVES Limit of detection on cellulose thin Compound layerslng 6-Mercaptopurine 4 6-Methylniercaptopurine 10 2-Amino-6-mercaptopurine 5 6-Mercaptoguanosine 10 6-Thioxanthine 30 Combinations of Fluorimetry and Specific Biochemical Interactions This is probably the most elegant approach to the development of highly selective fluori- metric analyses.To the well established and widely reviewed methods of fluorimetric enzyme assayz1 ,22 have recently been added a number of fluorescence immunoassay techniques, in which the radioactive labels familiar in radioimmunoassay are replaced with fluorescent labels. At present, fluoroimmunoassays may sometimes be less sensitive than radioimmuno- assays, but they have a number of important advantages.The labels themselves are cheap and stable and do not interfere with the immunochemical reactions; no hazardous procedures are involved; and, most important, fluoroimmunoassays may be homogeneous, i.e., they may not require a discrete step to separate antibody-bound and unbound labelled antigen. This last advantage arises because the fluorescence properties of the label may change when the labelled antigen is bound to an antibody. Changes may occur in fluorescence intensity (quenching or enhancement) or polarisation, and assays based on these changes have been l e s ~ r i b e d .~ ~ - ~ ~ Fig. 5 shows the use of a fluorescence enhancement assay of serum thyroxine (T4) involving thyroxine labelled with 2-methoxy-2,4-diphenyl-3 (2H)-furanone (MDPF).The properties of this label are closely similar to those of fluorescamine, but MDPF derivatives are more stable.26 The fluorescence of MDPF - T4 is relatively feeble (probably because ofFebmavy, 1979 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS 61 quenching by the iodine atoms in T4), but when the conjugate is bound to T4 antibodies the fluorescence is substantially enhanced.A similar effect occurs with fluore~cein~~ and also with fluore~camine.~' When unlabelled T4 is added to a mixture of labelled T4 and anti-T4 antibodies, it displaces the labelled T4 from the antibody binding sites, with a reduction in the label fluorescence. This homogeneous assay can be used to determine nanogram amounts of 0 10 100 1 000 Concentration of thyroxine in serum/ng mi-' Fig.5 . Homogeneous fluorescence immunoassay of thyroxine using MDPF as the fluorescent label (see text). The phenomenon of energy transfer offers excellent prospects for the development of immunoassays. This effect occurs when a donor fluorophore transfers excitation energy to an acceptor fluorophore, the donor fluorescence being quenched and the acceptor fluorescence possibly enhanced.Energy transfer is possible only when the emission spectrum of the donor overlaps the excitation spectrum of the acceptor and, particularly important in immuno- assays, when the donor and acceptor groups are very close together. Thus, if an antigen is labelled with a donor and the corresponding antibody labelled with an acceptor, energy transfer will occur when they specifically combine.Addition of unlabelled antigen in a sample will displace the labelled antigen and the energy-transfer effects will be reduced. This principle has been applied to the homogeneous assay of both low and high molecular weight antigens.28 Fig. 6 shows its application to the determination of serum albumin.The fluorescence at 520 nm of albumin labelled with fluorescein isothiocyanate (donor) is quenched when it combines with antibody labelled with rhodamine isothiocyanate (acceptor) ; the rhoda- mine fluorescence at 580 nm is simultaneously enhanced. Addition of unlabelled albumin (e.g., in human serum diluted about 1 000-fold) displaces some of the labelled albumin, so producing an increase in fluorescence at 520 nm (Fig.7 ) . The precision of this method is acceptable (coefficient of variation about 7%), the results obtained agree well with other immunoassays for albumin and preliminary experiments show that the method can be adapted for automatic analysis by using the flow injection analysis method.29 I am profoundly grateful for the continued co-operation of Professor J.W. Bridges, and for the contributions of A. Al-Mousawi, C. S. Lim, G. Handley, E. U. Akusoba and J. I. Braith- waite. I also thank the Medical Research Council for several Project Grants in support of various aspects of this work.62 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc. Analyt. Div. Clzem. SOC. 90 80 - 7 0 - 0 cv v) Y \r 6 0 - \ 1. 2.3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. - - 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. /d’ - 1.41 0 , 0,’ / - 1.21 / / . L I I-‘ 0 2 4 6 8 1 0 ‘ M d a r ratio, Ab/Ag Fig. 6. Energy transfer between fluorescein-labelled human albumin and rhodamine-lalelled anti-albumin antibodies. The fluorescein emission a t 520nm is quenched and the rho- damine fluorescence a t 580nm is enhanced.I 1 50 t 4 O L - -10 -9 -8 -7 -6 -5 -4 log (albumin) Fig. 7 . Determination of human albumin using energy-transfer immunoassay. Addition of pure albumin or diluted serum t o a mixture of fluorescein-labelled albumin and rhodaniine- labelled antibody causes an increase in the fluorescein emission a t 520 nm. References Green, G. L., and O’Haver, T. C., Analyt. Chem., 1974, 46, 2191.O’Brien, I. P., M.Sc. Dissertation, Loughborough University, 1977. Miller, J . N., Prog. Biophys. Molec. Biol., 1974, 28, 41. Lloyd, J . B. F., Nature Phys. Sci., 1971, 231, 64. Lloyd, J . B. F., J . Forens. Sci. SOC., 1971, 11, 83. Miller, J . N., and McDonnell, J., unpublished work. Lloyd, J. B. F., Analyst, 1977, 102, 782. Lim, C. S., Miller, J . N., and Bridges, J.W., Analytzca Chim. Acta, 1978, 100, 235. Slavin, W., Rhys Williams, A. T. R., and Adams, R. F., J . Chromut., 1977, 111, 222. Pollak, V., and Boulton, A. A., J . Chromat., 1972, 72, 231. Lawrence, J . F., and Frei, R. W., J . Chromaa, 1974, 98, 253. Seiler, N., and Knodgen, B., J . Chromat., 1977, 131, 109. Miller, J . N., and Bridges, J. W., in E. Reid, Editor, “Assay of Biological Samples for Drugs and Gifford, L.A,, Miller, J. N., Burns, D. T., and Bridges, J. W., J . Chromat., 1975, 103, 15. Miller, J. N., Phillipps, D. L., Burns, D. T., and Bridges, J. W., AnaZyt. Chem., 1978, 50, 613. Akusoba, E. U., and Miller, J. N., Proc. Analyt. Dzv. Chem. SOC., in the press. Lloyd, J. B. F., and Miller, J. N., Talanta, in the press. Vo Dinh, T., Lue Yen, E., and Winefordner, J.D., AnoZyt. Chem., 1976, 48, 1186. Jakovljevic, I. M., Analyt. Chem., 1977, 49, 2048. Al-Mousawi, A., Miller, J. N., and Bridges, J. W., to be pablished. Roth, M., Methods Biochem. Anal., 1969, 17, 189. Leaback, D. H., “An Introduction t o the Fluorimetric Estimation of Enzyme Activities,” Second Watson, R. A. A., London, J., Shaw, E. J., and Smith, D.S., c‘linica Chim. A d a , 1976, 73, 51. Smith, D. S., F E B S Lett., 1977, 77, 25. Shaw, E. J., Watson, R. A. A., London, J., and Smith, D. S., J . Clin. Path., 1977, 30, 526. Handschin, U. E., and Ritschard, W. J., Analyt. Biochem., 1976, 71. 143. Handley, G., Miller, J. N., and Bridges, J, W., unpublished work. Ullman, E. F., Schwarzberg, M., and Rubenstein, K. E., J . Biol. Chem., 1976, 251, 4172.Miller, J. N., Bridges, J. W., Lim, C. S., and Braithwaite, J. I., t o be published. Other Trace Compounds,” Ellis Horwood, Chichester, 1978. Edition, Koch-Light, Colnbrook, 1976.Febrzttsvy, 1979 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS 63 Data-acquisition Procedures for Clinical Analyses Alan S. McLelland Department of Biochemistry, Royal Infirmary, Glasgow, G4 OSF Reporting computers have become a feature of clinical chemistry as laboratories have struggled to cope with the explosion in workload, which has seen, for example, the annual number of analyses processed in the Eastern District of Glasgow jump from 35 000 to 1 020 000 during 1958-1 978.Analytical equipment manufacturers have responded by introducing high-capacity analysers such as the Technicon 18/60 and SMAC and Vickers M300, but the paperwork associated with a workload of over a million analyses per annum is likely to be 600-700 request forms per working day and both laboratories and manufacturers have been much slower to automate the clerical aspects of a laboratory. All reporting computer configurations have two major problems, entry of patient identifica- tion information and of the analytical results.This process is known as data acquisition; it has been simply defined as “the transfer of information into the computer from outside.” Despite the variety of analytical techniques and instruments there are only a limited number of methods for data acquisition. These, in order of increasing sophistication are: (1) direct data entry, (2) chart reading, (3) data logging, (4) hard-wired calculators and (5) programmable calculators and/or computers.Direct Data Entry By this method data is typed either directly into the computer through, for example, a visual display unit, or on to a storage medium such as paper tape, using a standard teletype. Direct data entry is commonly used to enter patient identification information into the corn- puter.Various alternative methods have been tried (e.g., pre-punched patient identification on cards), but the rapidly changing nature of data on a patient’s request forms (change of drug therapy, etc.) together with the requirements to match current requests with a patient master file, and to perform simple checks on various fields within the request form (validate hospital number by check digit, exclude impossible dates of birth, etc.) has led to increasing use of on-line terminals.This technique is also used to input small batches of results into the system, as it is flexible and can enter results plus identification number, which may be self-checking, thus removing the need for sequential entry of results in worksheet order.Disadvantages of direct data entry are as expected, a 3-5% transcription error rate and no reduction in the number of transcription steps from analysis to final report. Chart Readers A common problem in analytical-data acquisition is the analogue output from a continuous- flow analyser. Two problems are apparent; these are location of the top of the peak, which defines the relationship between the unknown sample and the standard curve, and validation of the assay quality by the shape of the peak.A chart reader solves the first problem as the operator places a cursor on the top of the peak and transmits the X , Y co-ordinates to a computer file or storage medium. Advantages are flexibility (the system can handle both continuous-flow and reaction-rate output), price (a chart-reader system will cost of the order of k2 000 at present prices) and saving in the trans- cription step from chart to worksheet.Disadvantages are that results are identified by sequence number only, that peaks can be missed, that it can be tedious to align the chart on the reader and that batching of samples delays availability of interim results, Further, because analyses tend to end a t about the same times of the day, a single chart reader can be a bottle- neck. Data Loggers The instruments store data from detectors at fixed intervals on paper or magnetic tape.Clearly, this approach, applied to a typical con tinuous-flow analyser output, will require storage of large amounts of unnecessary data. However, lengthy sample aspiration on a continuous flow analyser will yield a flat “equilibrium” plateau.Sampling the detector voltage while on64 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc. AnaZyt. Div. CJzeiiz. SOC. the plateau will give a single voltage proportional to the concentration of analyte. Hence the Technicon SMA 6/60 and 12/60 and some discrete analysers like the Vickers D300, where the flow cell contents are held for a period by the photometer valve system, lend themselves best to this method.Disadvantages of data loggers are again identification of results by sequence number only and batching of peaks delaying results until data on the storage medium has been re-processed. Several independent analysers can be linked to one logger, but this usually implies that no results are obtained until the slowest channel has stopped.Hard-wired Calculators These instruments combine data logging with real-time calculation to give interim results quickly. An example is the LKB 8200 Reaction Rate Calculator, where the rate of change of the output voltage from an 8600 Reaction Rate Analyser is recorded, and fixed calculations are performed to give enzyme activity and a measure of the quality of the assay. Other instru- ments in this class, e.g., the Kemtronix amino-acid integrator, offer switch-selectable options but, here again, the logic within the options is unalterable.The main advantages of a real-time system are the fast output of interim results and the ability to monitor the quality of analyses.However, the calculation routines are inflexible, the calculators tend to be specific to a particular analyser or class of analysers and provision of dedicated calculators for each analyser can be expensive. Programmable Calculators and/or Computers Programs in these devices can be altered to cope with deficiencies in the original logic. Data-acquisition hardware may be offered as optional extras, but the larger analysers (Tech- nicon SMAC, Vickers M300, IL Multistat I and 111, Aminco Rotochem, etc.) at present feature integral miniprocessors, as they offer all the advantages of hard-wired calculators in addition to program-development potential.Laboratories that develop their own miniprocessors for data acquisition appreciate that they are independent of the analyser and so can be adapted to meet changes in analytical hardware.The only real disadvantage of this class is the price of the processor. Conclusion The present options for data-acquisition hardware have been outlined above. Future development is likely to occur in two areas. Firstly, more attention will be paid to positive automatic identification information reading at the sampler to allow flexibility of analysis and remove the rigid adherence to sequential analysis in worksheet order, and secondly, a sixth class of data-acquisition hardware, the microprocessors, will find an ever increasing place in clinical laboratories. These have the advantages of cheapness, while retaining much of the flexibility of the miniprocessors, and have already appeared in analysers such as the Union Carbide Centrifichem, the Technicon SMA I1 and various radioimmunoassay instruments. The SMA I1 development is particularly interesting because much of the program used was developed on SMAC over a period of years, and it is clear that this philosophy of using the flexibility of a mini-computer until a final version of the program has been proved, then replacing the miniprocessor with a microprocessor, thus freeing the flexible and more expensive instrument for further development elsewhere, is one that we will continue to see applied to the future data-acquisition problems of clinical chemistry laboratories.Developments in Atomic-absorption and Atomic-f luorescence Spectrometric Techniques for Biological Materials J.M. Ottaway Department of Pure and Applied Chemistry, University of Strathclyde, Cathedral Street, Glasgow, GI I X L The author’s laboratory maintains close collaboration with the Biochemistry Laboratory of the Glasgow Royal Infirmary with regard to the development and application of atomic spectro-February, 1979 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL ANALYSIS 65 metric techniques of relevance to clinical analysis.In another contribution to the programme of this meeting, Fell1 outlines some of the clinical applications of these studies, and the present paper is concerned principally with a review of developments in analytical techniques. Carbon Furnace Atomic-absorption Spectrometry Both flame atomic-emission and atomic-absorption spectrometry have become well estab- lished techniques in the clinical laboratory for the routine determination of elements such as sodium, potassium, calcium, magnesium, iron, copper, zinc, etc.Both methods are accepted as being reliable, suitable for technician but not fully automated use, and provide the fast sample throughput possible with flame methods. Although interferences are still not well understood, results of acceptable accuracy can be achieved if interferences are controlled by the use of suitable releasing agents.In recent years, the introduction of electrothermal or carbon furnace atomisers has become more widespread in clinical laboratories, principally for the determination of lead and cadmium but also less commonly for elements such as cobalt, nickel, manganese, aluminium, gold and chromium.Although the Delves' cup2 flame method has been used for blood-lead deter- minations for a number of years, it seems likely that most laboratories will find the use of automated furnace systems more convenient for this determination and less susceptible to the skill of the ~ p e r a t o r . ~ The main advantages of electrothermal atomisation, compared with flame atomisation, are the increase in sensitivity, which can be up to 1000 times and the relatively non-hazardous operation, which allows unattended automatic procedures to be adopted.The increase in sensitivity permits direct analysis of lead in blood and cadmium in blood and urine, without pre-concentration of the sample solution, which is necessary if the same analyses are to be carried out with a flame.Although electrothermal methods have sufficient sensitivity for these direct analyses, analysis times are much longer than with flames and the problem of interferences from the strong chloride matrix in clinical samples must be overcome. Molecules such as sodium chloride released into the vapour phase of a graphite furnace produce large non-atomic background signals and the use of simultaneous background correction is essential. Fortunately, recent improvements in the design of background correctors have helped to alleviate this problem, as has the introduction of rapid furnace heating, which helps to separate atomic and non-atomic signals with respect to time. The chloride matrix of typical clinical materials can also cause serious chemical inter- ferences.Reduced signals may be obtained owing to the release of the analyte as a molecular chloride species. In addition, reaction of hydrochloric acid, released during the decomposition of magnesium chloride, has been postulated to cause a decrease in analyte atom concentrations of a number of element^.^^^ Although analysis in the presence of chloride is possible by standard addition method^^.^ this can lead to a serious loss of sensitivity in some instances.8 For this reason analysis in a nitric and/or sulphuric acid medium is preferred9 and recent methods for the determination of lead in blood have recommended treatment of the sample with nitric acid before injection into the furnace.l0y1l Two recent suggestions may help to alleviate this problem.Regan and Warrenl29l3 have shown that addition of ascorbic acid to such solutions helps to remove the effects of interferences from a chloride solution. Other workers have investigated the advantages of applying coatings of materials such as moly- bdenum or tantalum carbide to the surface of graphite tubes.14-16 Apart from extended tube lifetimes,15 preliminary results indicate that many chloride-type interferences can be reduced or removed when atomisation is performed from a coated tube.l6 The mechanisms that cause these changes in atomisation behaviour are not yet clear but future work may permit the selection of releasing agents for use in furnace atomisation and allow interference free deter- minations of analytes in a chloride matrix.The increased sensitivity of the carbon furnace also allows the determination of metals in separate fractions of b l o ~ d ~ , ~ and in separate protein fractions of blood ser~m.~'JS Conibina- tion of the carbon furnace with separation techniques such as gel chromatography, electro- phoresis and liquid chromatography may provide information of great value to the clinical biochemist and further developments and applications of these combinations of techniques can be expected.In our laboratory we have been investigating the use of the carbon furnace as an atomic-emission source. At the present time, sensitivity would be sufficientlg for the deter- mination of elements such as sodium, potassium, calcium, magnesium, iron and copper in clinical samples but normal levels of lead, cadmium and zinc could not be determined.I t is66 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Pmc. Analyt. Div. Chenz. SUC. surprising that atomic-emission techniques have not found a more prominent role in the clinical field, in view of the possibilities for simultaneous multi-element analysis. If this ever becomes a serious possibility, the safe automatic operation of the carbon furnace may represent. an important advantage.Flame Atomic-fluorescence Spectrometry For a number of years, the application of flame atomic-fluorescence spectrometry (AFS) to clinical analysis has been under study in our laboratory2°,21 using modified atomic-absorption instruments. For elements such as cadmium and zinc, for which suitable intense line sources are available, the sensitivity is such that direct analysis of blood and urine is possible with minimum sample treatment.The wider application of this technique is, however, limited by the lack of commercially available equipment designed for AFS. We have recently construc- ted an AFS instrument specifically designed for the analysis of clinical materials. This is illustrated diagrammatically in Fig.1 and has so far been applied successfully to the analysis of cadmium.22 The instrument uses cadmium electrodeless discharge lamps prepared by a rigorously defined p r o c e d ~ r e ~ ~ , ~ ~ and a xenon arc, which permits background correction for the light scattered by undissociated particles in the flame. A separated air - acetylene flame is used with a commercial pneumatic nebuliser.A double monochromator has been incorporated into the instrument to reduce stray light25 and signal retrieval is by means of a photon counter. Determination of cadmium in blood is achieved following a 1 + 4 dilution to give a solution 0.04 M in hydrochloric acid and 0.5% in Triton X. After centrifuging, the supernatant is nebulised directly. Urine samples are simply acidified with a few drops of hydrochloric acid and nebulised.The full paper should be consulted for details of the method and results.22 The significance of the AFS method is that measurements of the same sensitivity as carbon furnace AAS can be obtained, but with the speed, reproducibility and freedom from inter- ference (for chloride matrices) possible with flame atomisation.The xenon-arc source, incorporated into the instrument for background correction, can be used at higher powers to generate AFS signals giving the same sensitivity as flame AAS.26 The same instrument could therefore be used for the analysis of many elements of clinical interest and with suitable modification could be operated in a simultaneous or rapid sequential multi-element procedure. Further work designed to demonstrate these possibilities is in progress. With the number of elements now routinely determined in the clinical laboratory i t is surprising to the author that techniques involving simultaneous multi-element analysis have not been more widely adopted. It would be even more surprising if such developments did not take place in the future. A & Mechanical ~ chopper Electrodeless Heater d isc harge Fan {d!) -lamp , , 'Cavitv i r m o noch r o m a t or Spherical mirror Fig. 1. Instrumentation. The support of the Scottish Home and Health Department towards the construction of the flame atomic fluorescence spectrometer is gratefully acknowledged.Fehvmvy, 1979 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS References 67 1 . 3. 4. 5. 6. 7 . 8. 9. 10. 1 1 . 12. 13. 14. 1.5. 16. 17. 1s. 19. 20. 21. 22. 23. 24. 25. 26. 0 -. Fell, G. S., Proc. Analyt. Div. Cheni. SOC., 1979, 16, 77. Delves, H. T., Analyst, 1970, 95, 431. Sabet, S., Ottaway, J. M., and Fell, G. S., PYOC. Analyt. Div. Chern. SOC., 1977, 14, 300. Ottaway, J . M., Proc. Analyt. Div. Chem. SOC., 1976, 13, 185. Hutton, R. C., PhD Thesis, University of Strathclyde, 1977. Kamel, H., Brown, D. H., Ottaway, J. M., and Smith, W. E., Analyst, 1976, 101, 790. Iiamel, H., Teape, J., Brown, D. H., Ottaway, J . M., and Smith, W. E., AnaZyst, 1978, 103, 921. Campbell, W. C., and Ottaway, J . hf., Analyst, 1977, 102, 495. Ottaway, J . M., P ~ o c . Analyt. Div. Chem. SOC., 1975, 12, 176. Rains, T. C., unpublished NBS method. Stoeppler, M., Brandt, K., and Rains, T. C., Analyst, 1978, 103, 714. Regan, J . G. T., and Warren, J., Analyst, 1976, 101, 220. Regan, J . G. T. and Warren, J . , Atom. Absorption Newsl., 1978, 17, 89. Runnels, J . H., Merryfield, R., and Fisher, H. B., AnaZyt. Chem., 1975, 47, 1258. Zatka, V. J., Analyt. Chem., 1978, 50, 538. Manning, D. C., and Slavin, W., Analyt. Chem., 1978, 50, 1234. Iiamel, H., Brown, D. H., Ottaway, J . M., and Smith, W. E., Analyst, 1977, 102, 645. Delves, H. T., Clinica Chim. Acta, 1976, 71, 495. Ottaway, J . XI., and Shaw, F., Appl. Spectrosc., 1977, 31, 12. Hough, D. C., and Ottaway, J . M., Proc. SOC. Analyt. Chem., 1974, 11, 223. Fell, G. S., Hough, D. C., Hussein, F. E. R., and Ottaway, J . M., Proc. Analyt. Div. Chern. SOC., 1976, Jlichel, R. G., Hall, M. L., Ottaway, J . M., and Fell, G. S., Analyst, in the press. JIichel, R. G., Coleman, J., Winefordner, J . D., Spectrochim. Acta, 1978, 33B, 195. JIichel, R. G., Ottaway, J. M., Sneddon, J. S., and Fell, G. S., Analyst, 1978, 103, 1204. Rlichel, R. G., Hall, M. L., Rowland, S. A. K., Sneddon, J., Ottaway, J . M., and Fell, G. S., Analyst, Johnson, D. J., Plankey, F. W., and Winefordner, J . D., Analyt. Chem., 1975, 47, 1739. 13, 271. i n the press.

ISSN:0306-1396    

DOI:10.1039/AD9791600050

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

Fehvmvy, 1979 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS 67 Measurement of Plasma Drug Concentrations-A Clinician’s Viewpoint D. H. Lawson Clinical Pharmacology Service, Glasgow Royal Infirnaary, Glasgow, G4 OSF It has recently become appreciated that the safety and effectiveness of many drugs can be improved by tailoring their dosage to suit individual needs. One of the first to emphasise this was Sjoqvist, who demonstrated some 10-fold differences in steady-state plasma levels of the anti-depressant nortriptyline after standard dosing to a series of patients and indicated that on theoretical grounds this may account for some of the variation in effectiveness of this drug.More recently Koch-Weser, working in Boston, reported that of 200 patients receiving phenytoin at an anticonvulsant clinic 90% received 300 mg, whereas of 200 patients receiving warfarin, doses ranging from less than 2 to over 11 mg daily were common. These differences stem from the fact that although both drugs have a narrow therapeutic range and are quite toxic if given to excess, with warfarin a clear-cut, measurable end-point is in view (a given degree of prolongation of prothrombin time) whereas with phenytoin no such measurable end- point can be reached.Thus it is easy to tailor doses of warfarin to the individual’s need but not doses of phenytoin. There is a wide range of drugs: hypotensives, hypoglycaemics, hypolipidaemics, diuretics, hypnotics, etc., where such dosage tailoring according to a measur- able end-point is feasible and in these instances the need to measure plasma levels to help in adjusting dosage is minimal.Where, however, no such end-point is definable, under certain circumstances measuring drug levels may be helpful in reducing toxicity and in improving efficacy. Before attempting to indicate when blood levels should be monitored, it is wise to recall that many factors may explain the observed individual variations in blood levels after a standard dose of drug is administered (Table I).Because of these many variables, the interpretation of any given blood concentration of a drug can prove to be extraordinarily difficult. Keeping this in mind, there are certain guide- lines that should be followed before the plasma concentration of a drug is measured in order to improve its efficacy.These are : the drug should have a reversible action ; the concentration of68 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL ANALYSIS P?’oc. Analyt. Diu. Ch??ir. sot. TABLE I FACTORS EXPLAINING VARIABILITY I N DRUG RESPONSE Dose prescribed . . .. .. . . . . Medication errors Compliance Dose consumed . . . . . . . . . . Formulation Absorption Distribution Biotransformation Excretion Serum concentration .. .. . . . . Passive diffusion Active transport Tissue concentration . . .. .. . . Responsiveness Co-administered drugs unbound drug in plasma should reflect its concentration at receptor sites; tolerance sliould not develop a t receptor sites ; there should be a measurable pharmacological response wherever possible ; and accurate, reproducible and specific assays should be available to measure the drug and/or its active metabolites.For this purpose phenytoin is an appropriate choice. This drug is well absorbed if given as the sodium salt, it is approximately 90% bound to plasma protein (mainly albumin) and is large1~- nieta- bolised by a saturable enzyme system in the liver to the parahydroxy derivative, which is inactive and is secreted in the bile or glucuronated and excreted by the kidney.l\’ork in neurosurgical units has demonstrated that the concentration of free (unbound) warfarin in the brain is similar to that in the plasma. Because of its high level of protein binding, care should be exercised in administering the drug to patients with low plasma albumin concentrations. This is not merely a theoretical hazard, as the Boston Drug Surveillance Program have reported a more than 10-fold difference in the frequency of adverse effects to this drug in patients with low serum albumin levels as compared with normal subjects.It cannot be assumed that the extent of protein binding is constant and indeed Lunde and co-workers emphasised that in patients with renal impairment greatly reduced protein binding was present, perhaps as a result of displacement by other substances.For these reasons measuring total phenytoin in plasma may not give sufficiently meaningful information and free levels should be measured directly from, e.g., saliva samples. The major problem with interpreting phenytoin levels is not, however, protein binding but rather the difficulties that arise in a drug with saturable metabolism in which a small dose increase can lead to a greatly disproportionate blood level increase. Thus, for example, a patient n-hose plasma level is below the therapeutic range at 300 mg daily could well have a level in the toxic range if the dose is sharply increased to 400 mg daily.Clearly drug levels cannot be monitored in all patients on phenytoin, however it is policy in the author’s unit to measure such levels when fits are uncontrolled at 400 mg daily, where signs of possible neurotoxicity are present, where previously good control has deteriorated or where co-existing disease or potentially interacting drugs are administered. I t should be emphasised however that the difficulties in interpreting the resultant levels are substantial, even for experienced workers dealing with reliable patients.Other drugs that should be monitored at regular intervals include most of the other anti- convulsants : digoxin, particularly in patients with any degree of renal impairment, lithium, aminophylline, lignocaine, procainamide, possibly quiiiidine, salicylates and phenylbutazone.Perhaps the drugs whose levels are most commonly monitored in most laboratories at the present time are the antibiotics, particularly the aminoglycosides such as gentamicin. Although there are many patient factors such as variable absorption from intramuscular injection sites, variable distribution in those with ascites or obesity, or variable excretion in those with renal impairment, perhaps the greatest problem with these drugs is to ensure the accuracy of the assay employed. In the author’s view this is one facet that requires more thought than perhaps we have given in the past.In this paper the author has tried to emphasise the many factors which are involved in determining the blood levels attained after a given dose of a drug and to indicate that n-liile the accuracy and specificity of the assay employed is of major importance, there are many other variables that have to be taken into account before meaningful information can be obtained Consideration of a particular drug is the best way to see these principles in action.February, 1979 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS 69 from these measurements.Discussion has been confined to the therapeutic use of drugs and overdoses have not been considered as this topic is covered in a paper by Prescott.In con- clusion, it should be emphasised that with notable exceptions, such as paracet amol, salicylate and digoxin poisoning, the measurement of plasma levels of drugs in patients experiencing intentional overdosage is rarely, if ever, helpful in their management.Measurement of Antidepressant Drugs Robin Braithwaite Poisons Unit, Guy's Hospital, London, S.E.1 The first tricyclic antidepressant drug to be introduced into medicine was imipramine in 1957. Following its success, the synthesis of chemically related tricyclic compounds yielded many new antidepressants, the most important and widely used being amitriptyline.The use of this group of drugs has increased dramatically over the last 20 years and more recently a number of new tetracyclic antidepressants (e.g . , maprotiline and mianserin) have been introduced. *At present there are 16 different tricyclic and tetracyclic antidepressants in clinical use in about 30 separate preparations. Approximately 10 million prescriptions are now issued each year in the UK for this group of drugs.With the increased usage of this group of drugs there has been a greater need to investigate their therapeutic and toxic effects in man. Consequently, the development of suitable analyti- cal methods for their qualitative and quantitative determination in biological fluids has been necessary. As can be seen, there are many structural similarities within this group of compounds, which may present problems in their analysis.A further complication is that these drugs are extensively meta- bolised by the liver and tertiary amine-type antidepressants (e.g., amitriptyline , imipramine and clomipramine) undergo demethylation to produce secondary amine products as pharnia- cologically active metabolites (nortriptyline, desipramine and desmethylclomipramine , respec- tively).An added difficulty in the analysis of this group of drugs is their high lipid solubility. Following therapeutic and toxic doses, plasma concentrations are extremely low, with most of the drug being distributed in the tissues and major organs. The chemical structure of some of these drugs is shown in Fig. 1. ,CH3 CH-CH,-CH, -N \ CH3 Amitriptyline ,CH3 'H CH,-N Nortriptyline I CH,-CH,- lmipramine CH3 / CH3 CH,-CH,-CH2-N I / CH3 CH,-CH,-CH,-N \ \ CH3 H Maprotiline Mianserin Clomipramine Fig.1. Structures of some important antidcpressants. In recent years, many methods for the analysis of different tricyclic antidepressants have been described. Most of these have been based on gas chromatography using a variety of detection systems, the most favoured being nitrogen-specific detection. Our own methodology is based on this technique and has recently been described in detai1.l This procedure has many advantages over previously described methods and has been in routine use for 2 years.'70 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL ANALYSIS PYOC.AnaZyt. D i V . CheWZ. SOC.The extraction procedure employed can be summarised as follows: (1) plasma sample (0.5- 1.0 m1) + buffer (pH 10) (1.0 ml) + internal standard (maprotiline or nortriptyline); (2) extract with 5 ml of hexane for 20 min, then centrifuge ; (3) transfer the hexane layer to a clean tube containing 1.0 ml of 0.05 M sulphuric acid; (4) extract for 10 min and centrifuge, then discard the hexane layer and transfer the acid layer to a micro-tube; (5) re-extract the acid phase with 100 pl of 4 M sodium hydroxide solution and 50 pl of butyl acetate by vortex mixing for 30 s, then centrifuge; (6) inject 1-2 pl of butyl acetate phase on to the chromatograph.This procedure can be applied to the quantitative analysis of all of the currently available tricyclic and tetracyclic antidepressants and their active metabolites.The chromatographic system has recently been modified and improved on that previously described.l The present system is based on a Hewlett-Packard 5710A gas chromatograph with dual nitrogen - phosphorus detectors. The column used is glass (8 f t x 2 mm i d . ) , packed with 3% SP2250 on Supelcoport, 80-100 mesh.l The column temperature is 250 "C with a detector temperature of 300 "C.The flow-rate of the carrier gas (helium) is 40 ml min-l and those of air and hydrogen are 50 and 3 ml min-l, respectively. The resolution of several closely related antidepressants is shown in Fig. 2. Each peak on this chromatogram represents 10 ng of drug (free base) on the column. As can be seen, it is possible to resolve clearly both imipramine and clomipramine and also their demethylated metabolites.Fig. 3 shows typical Time/min Fig. 2. Resolution of closely related antidepres- sants : imipramine (1), de- sipramine (2), maprotilinc (3), clomipramine (4) and desmethylclomipramine (5). Each peak repre- sents 10 ng of drug (free base) on injection. Chro- matographic conditions as described in the text.$1 r 1 I I l l 1 Y 8 4 0 8 4 0 T i me/m in Fig. 3. Chromatograms obtained from the analysis of spiked plasma (1.0 ml) containing: (a) 50 ng of amitriptyline (1) and nortriptyline (2) with maprotiline as internal stan- dard (3) ; and ( h ) 50 ng of imipramine (1). and desipramine (2) with mapro- tiline as internal standard (3). Chro- matographic conditions as described in the text.February, 1979 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS 71 chromatograms obtained from the analysis of spiked plasma (1 .O ml) containing low concentra- tions of either amitriptyline and nortriptyline or imipramine and desipramine.The detection system used has been found to be extremely stable with a small solvent detector response and a high sensitivity towards all of the antidepressants investigated.The method is capable of the accurate analysis of antidepressant concentrations in plasma down to 5 ng ml-l. The analytical techniques described1 have been applied successfully to the determination of antidepressant concentrations in plasma in two different situations. The first of these applica- tions has been in the diagnosis and treatment of poisoning.With the increased usage of this group of drugs, overdosage has become more common and now accounts for 10% of all deaths due to poisoning in the UK.2 The number of admissions to hospital for treatment due to poisoning with these drugs has also become very considerable. The severely poisoned patient may show serious toxic effects, such as coma, convulsions, cardiac arrhythmias and myocardial depression.Treatment is, however, largely supportive and most patients who are treated in hospital do survive. Nevertheless, diagnosis and treatment may sometimes present many problems. In an early review of the clinical features of tricyclic antidepressant poisoning in adults by Noble and matt he^,^ it was reported that neither the active drugs nor their nieta- bolites were detected in the plasma of any of their patients.They therefore concluded that analysis of plasma concentrations was not of any practical value. However, using more refined techniques, studies by our~elves*~~ and other groups6 have shown that in both adults and children the con centrations of tricyclic antidepressants in plasma correlate well with the severity of the symptoms.Plasma concentrations above 1000 ng ml-l are generally asso- ciated with severe symptoms of p~isoning.~-~ Experience in our own department has shown that it is impracticable and unnecessary to measure drug concentrations in every patient who is poisoned, particularly in those who show no serious symptoms or where the diagnosis is certain. The measurement of plasma concentra- tions is, however, of great value in atypical or severe poisoning, particularly if there is no clear history of overdosage.The real value in drug level measurements is in confirming the diagnosis and giving some idea of the likely duration of symptoms. An example of the useful- ness in carrying out such determinations is illustrated by the following case h i ~ t o r y .~ A 5-year-old boy was admitted to his local hospital with convulsions on the day following his first diptheria vaccination. He was treated with intravenous diazepam and intubated and given cardiac massage. Encephalitis was suspected and the patient was transferred to a neurological centre. On arrival he was in deep coma; the ECG showed serious cardiac arrhythmias and a pacemaker was inserted.The child had been prescribed imipramine for enuresis but the mother denied the possibility of overdose. The plasma concentration of imipramine plus desipramine was found to be 2 500 ng ml-l, which confirmed the later diagno- sis of antidepressant poisoning. The child remained unconscious for 2 days but eventually recovered. The second major application of the measurement of antidepressant concentrations in plasma is in therapeutic monitoring.There is now general acceptance that for many important drugs in clinical use, plasma concentrations are more closely related to pharmacological and clinical effects than drug dosage. In major areas of drug treatment (e.g., therapy with drugs such as lithium, phenytoin, digoxin and theophylline) measurement of drug plasma concen- trations is an accepted way of improving response by way of tailoring drug dosages to suit individual requirements.Thus, in the treatment of depressive illness, it is common clinical experience to find that a significant proportion of patients treated with tricyclic antidepressants fail to show a satisfactory response, whereas others complain of side-effects.Obvious important considerations in this context, as with any drug treatment, are the diagnostic criteria used for the selection of patients for treatment and the dosage prescribed. Another important factor, which is often overlooked, is the extent to which patients take the drugs they are prescribed. A number of studies have shown that drug compliance in patients receiving antidepressant medication is alarmingly p0or.7~8 The actual relationship between plasma antidepressant concentrations and clinical response is still under active investigation.Some studies have demonstrated a clear relationship between drug levels and therapeutic effects and others have n0t.~-1~ However, much of the conflict in findings of these studies can be accounted for by the difference in design of the various trials as well as the criteria used in the selection of patients.13 In a complex heterogeneous disorder such as “depression,” where there is no simple universally acceptable classification Non-accidental poisoning was subsequently suspected.72 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc.AnaZyt. Div. Chem. SOC. system, and where there is no “physiological” measure of success or failure to treatment, the problem is difficult.One is entirely reliant on an empirical diagnosis and a purely clinical assessment which can be very unreliable. Despite these misgivings, optimum therapeutic ranges have been proposed for a number of tricyclic antidepressants, e.g., nortriptyline, ami- triptyline and imi~ramine.~-l~ Individual variation in plasma antidepressant concentra- tions is considered to be an important (but not the only) factor in determining therapeutic response and toxicity.From a knowledge of the antidepressant plasma concentration it is possible to guide the clinician towards a more rational approach to treatment. Those situa- tions where measurement of drug plasma concentration is of value are as follows (for ami- triptyline, nortriptyline and imipramine) : (1) after receiving a seemingly adequate dosage the patient shows little response; (2) the patient complains of or sustains troublesome or serious side effects; (3) suspected poor compliance or abuse; (4) the control of long-term prophylaxis of recurrent depression; (5) patients receiving other medication when there is a risk of a drug interaction; (6) patients who have a complicating medical condition.In many patients receiving antidepressant medication, treatment could be made much more effective if more attention were paid to drug plasma concentrations. In summary, the development of gas-chromatographic methods for the routine determina- tion of antidepressant drugs has proved invaluable in understanding their therapeutic and toxic effects in man.1. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 1 Y . References Dawling, S., and Braithwaite, R. A , , J . Chromat. Biomed. Appl., 1978, 146, 449. Office of Population, Censuses and Surveys, Series DH4 No. 2, HM Stationery Office, London, 1977. Noble, J., and Matthew, H., Clin. Toxic., 1969, 2, 403. Eraithwaite, K.A., PYOC. Eur. SOC. Toxic., 1977, 18, 231. Crome, P., and Braithwaite, R. A., Arch. Des. Childh., 1978, 53, 902. Petit, J. M., Spiker, D. G., Ruwitch, J. F., Ziegler, V. E., Weiss, A. N., and Riggs, J . T., Clin. Pharmac. Wilcox, D. R. C., BY. Med. J., 1965, ii, 790. Johnson, D. A. W., B r . Med. J., 1973, ii, 18. Kragh-S~rensen, P., Asberg, M., and Eggert-Hansen, C., Iancet, 1973, i, 113.Ziegler, V. E., and Biggs, J. T., Arch. Gen. Psychiut., 1977, 34, 607. Montgomery, S., Braithwaite, R., Dowling, S., and McAuley, R., Clzn. PhaYmac. They., 1978, 23, 309. Montgomery, S., McAuley, R., Rani, S. J., Montgomery, D. B., Braithwaite, R. A, and Dawling, S., Barnes, T., and Braithwaite, R. A., in Gaind, R., and Hudson, B., Editors, “Current Themes in Ther., 1977, 21, 47.BY. Med. J., 1979, in the press. Psychiatry,” Macmillan, London, 1978, p. 333. Determinations of Paraquat in Clinical Practice Using Radioimmuno- assay T. Levitt Poisons Unit, New CYOSS Hospital, Auonley Road, London, SE14 The herbicide paraquat (l,l’-dimethyl-4,4’-bipyridylium ion) is being used increasingly in suicide attempts in the UK. Although in absolute terms the numbers have remained small, it has become one of the major causes of death, within hospital, owing to chemical or drug ingestion.We decided therefore to raise antibodies to this herbicide as a basis for a radio- immunoassay.1 Methods The hapten 6- (1, I ’-methyL4,4’-bipyridyliurn) hexanoic acid, prepared from 6-bromohexanoic acid and monoquat, was coupled with bovine serum albumin using carbodiimides.This antigen produced antibodies in rabbits within 4 months. The assay procedure is based on the charcoal separation technique and the resultant inhibi- tion curve is shown in Fig. 1. As plasma volumes up to 100 p1 can be used without altering the inhibition, the formal sensitivity2 of the assay is 0.6 pg 1-1 of paraquat ion. A range of compounds, including the two structurally related herbicides diquat and morfamquat, do not cross-react with the antiserum.Serial dilution of paraquat standards and patient samples give parallel response curves, suggesting that the assay is in fact measuring paraquat. FurtherFebruary, 1979 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS 73 evidence for both the specificity and accuracy of the assay was obtained by measuring the blood concentrations of paraquat from 40 patients both by radioimmunoassay and by the gas - liquid chromatographic method of Draffen et aZ.3 The slopes of the resulting regression lines (1.06 and 0.95), together with the correlation coefficient (0.991), indicate that both techniques are measuring the same moiety.Recoveries from plasma are complete, with coefficients of variation lying between 2.8 and 8.0yo throughout the assay range.1 I lO'0.5 i.0 215 5.0 7.5 10.0 Paraquathg per tube Fig. 1 . Inhibition curve. An important requirement of the assay is that it should be rapid. It was shown that the equilibrium between the antigen and the antibody is reached within a few minutes a t room temperature. Similarly, charcoal contact times could be reduced to 1 min without an appre- ciable loss of precision.Moreover, as a graph on semilogarithmic paper of logit (counts bound divided by counts bound with zero standard) v e m m concentration is linear, only three or four single standards need to be used. Using these conditions, results can be obtained within 30 min. The inter-assay variation, determined by analysing a sample from a patient on five consecutive days, was 973 & 63 pg 1-1 for the neat sample and 95 2.8 pg 1-1 when the sample was diluted by a factor of 10.The assay is sensitive enough for the range of concentrations of paraquat in blood and urine that are of clinical importance. For the determination of paraquat in tissues, there are advantages in using less tissue and a more sensitive assay.The apparent equilibrium constant as determined by a Scatchard plot was 2.4 X lo9 1 mol-l. Using this figure, together with the guide-lines set out by Albano and Ekins,2 approximations of the optimum concentrations of antibody and radioactive paraquat could be made that gave rise to an assay with a sensitivity of 120 pg ml-1 and a reproducibility of 7% at the 1 pg 1-1 level.Results and Discussion The radioimmunoassay was developed because it is believed that paraquat intoxication is one of the surprisingly few instances of poisoning where the rapid determination of blood concen- trations can be of clinical use. Although there is a high mortality (30-50y0) associated with the ingestion of the concentrated formulations used in industry, many patients who present have taken dilute solutions of the weaker preparations of the weedkiller and they are unlikely to be in any serious danger.The treatments being advocated a t present are expensive and in some instances they are not without risk to the ~ a t i e n t . ~ As the clinical symptoms may take several days to develop, there is an obvious need to provide some means of determining the severity of the poisoning.74 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc..4naZyt. Div. Ckcm. Soc. The relationship between the plasma concentrations of paraquat, the estimated time after ingestion and the outcome are shown in Fig. 2. It can be seen that a single blood level on a sample taken before treatment may be an aid to prognosis.At present, similar blood level data is being accumulated at the Central Toxicology Laboratory, ICI, Macclesfield, and in the Department of Therapeutics, The Royal Infirmary, Edinburgh, and it is hoped that in con- junction with these and other centres the areas of risk can be defined more closely in the future. Blood level profiles of patients who have not undergone therapy designed to enhance the elimination of paraquat remain scarce.Of the six such patients admitted to a hospital within an estimated 4 h of ingestion, all had been treated with Fuller’s earth or bentonite and none showed signs of further absorption. The decline in the blood levels in all patients appeared to be biphasic within the initial 24-36 h after inge~tion.~ The half-lives for the first phase showed no gross differences between survivors (3.8 2.3 h, .rz = 11) and non-survivors (4.2 1.8 h, n = 4) and this phase continued for approximately 12 h for both groups.Therefore, at present, despite the disadvantages of relying on estimations of the time of ingestion, there seems to be little to be gained by delaying therapy until a series of blood samples can be collected.The aforementioned techniques, which are designed to aid in the removal of paraquat from the body, include haemodialysis, haemoperfusion, forced diuresis and plasmaphaeresis. At present they all suffer from the disadvantage that the evidence for their efficacy relies heavily upon the patients’ own estimations of the amounts ingested. The haemoperfusion columns contained charcoal, either coated or uncoated, as the adsorbent material. The blood concentrations on both the arterial and venous sides of the column are shown in Fig.3. In agreement with i n vitro studies,6 the uncoated charcoal column was the Two examples are given here in which the use of haemoperfusion was examined. 3 000 - 2500 - m rl ‘5 F 2000 Cl 0 C + 2 1500 P + 4- C u s 1000 500 0 A A A A A 0 0 0 .0. \: A 0 P O , . , 4 12 20 28 36 Period post-ingestion/h Fig. 2. Concentration of paraquat in the plasma of patients related to time of ingestion. A, Died; and 0, survived. 2 000 I 1 O o o b it Time/h I , 4 1 I h 1 Time/h 0 2 4 6 8 1 0 1 2 Fig. 3. Paraquat levels in plasma during haemoperfusion. (a) Patient D.S. ; and (b) patient M.W. Blood samples taken from inlet (arterial) side (0) and outlet (venous) side (a).Febyllnry, 1979 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL ANALYSIS 75 more efficient and less erratic of the two.As paraquat is not taken up into the red cells, the clearance data (Fig. 4) mean that in effect between 70 and 85% of the blood was being cleared at a flow-rate of 300 ml min-l for a period of 30 h.Clearance values of this order, if confirmed, would make haemoperfusion more effective than haemodialysis in removing paraquat . j 1 7 a a - D .S I I I I I 1 0 4 8 12 16 20 24 Period of haemoperfusion/h Fig. 4. Clearance of paraquat by haemoperfusion . Clearance = ([arterial] - [venous])/[arterialj x plasma flow. The cumulative amounts of paraquat removed by haemoperfusion (Fig.5) emphasise that elimination therapy should be started as quickly as possible if significant amounts are to be removed. Extensive haemoperfusion may also be of use because almost half of the paraquat was removed (from D.S.) after the first 4 h of perfusion at times when the blood concentrations were comparatively low. 0 4 8 12 16 20 24 28 32 4 8 12 Period of perfusiodh Fig. 6.Paraquat removed by haemoperfusion. ( a ) A, Amount removed by Patient D.S.; and (b) patient N.W. kidney excretion ; 0, amount removed by haemoperfusion. In this particular case, the fact remains that although the haemoperfusion was instituted within 5 11 of ingestion, and although the perfusion period was extensive with high clearance values, the amounts of paraquat removed seemed to be low compared with the total body load.Indeed, it can be seen that the amount of paraquat excreted by the kidneys during the period of perfusion was far higher than that removed by the column, despite the fact that the patient's renal clearances of paraquat and creatinine had both fallen below 10 ml min-l after 10 h of perfusion. Patient N.W. presented a similar picture, although the renal failure was more gradual with the clearances of paraquat declining from an initial figure of 240 to 4 ml min-l after 3 d.In both cases, as soon as the perfusion was stopped the blood concentrations of paraquat rose. As it was unlikely that further absorption of paraquat from the gastrointestinal tract was taking place, the tissue The same can be true for patients undergoing haemodialysis.76 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc.Analyt. Div. Chem. SOC. concentrations must have exceeded those in the blood at this stage. This could have been due either to clearances by various tissues that were greater than those of the columns or because by the time the perfusions were started the tissues already contained high concentrations of paraquat that were not in rapid equilibrium with the blood.In either case the role of assisted elimination would seem to be a limited one for the more severely poisoned patients who present more than a few hours after ingestion. Nevertheless, the clearance values being obtained with uncoated charcoal columns are not insignificant when compared with renal clearances of paraquat and it remains to be seen whether in cases of moderate intoxication haemoperfusion could help relieve the burden upon the kidney if instituted quickly enough and thereby help to maintain the all-important renal function.Obviously a great deal more information needs to be collected before any definitive state- ments can be made about the use of these or any other techniques for treating paraquat poisoning.I t is to be hoped that the radioimmunoassay will assist in this task. References 1. 2. 3. 4. Lancet, 1976, i, 1057. 5 . 6. Widdop, B., personal communication. 7. Levitt, T. E., Clin. Chern., submitted for publication. Albano, J., and Ekins, R. P., in “Proceedings of Symposium on In Vitro Procedures with Radio- Draffen, G. H., Clare, R. A,, Davies, D.L., Hawksworth, G., Murray, S., and Davies, D. S., J , Chcwmt., isotopes in Clinical Medicine and Research,” IAEA, Vienna, 1969, p. 491. 1977, 139, 311. Davies, D. S., Hawksworth, G. &I., and Bennett, P. N., Proc. Eur. SOC. Toxic., 1977, 18, 21. Okonek, S., and Hofmann, A,, in Fletcher, K., Editor, “Clinical Aspects of Paraquat Poisoning,” Imperial Chemical Industries Limited, Manchester, 1977, p.55. Essential and Toxic Trace Elements in Animals C. F. Mills Rowett Research Institute, Bucksburn, A berdeen Thirteen trace elements are now known to be essential for animals and preliminary evidence suggests that at least two more may soon be regarded as essential. Among these, instances of clinical disease, impaired growth, poor viability or poor reproductive performance, are most commonly caused by deficiencies of the elements copper, cobalt, iron, iodine, selenium, manganese and zinc.Among the many potentially toxic elements, the most frequent problems arising from chronic (as distinct from acute) exposure, originate from excessive con- sumption of lead, molybdenum, fluorine, cadmium, zinc and mercury. The recognition of disorders, arising from trace-element deficiency or excess in animals, at a sufficiently early stage to minimise adverse effects on growth or health, necessitates reliable diagnosis, often at stages when no overt signs of clinical disease may be apparent. Most frequently, the only tissue available for routine chemical examination upon which a diagnosis must be based is whole blood or plasma.This account will consider, briefly, some of the factors that have been found to influence the validity of determinations of the trace element content of blood for such a purpose. The analytical techniques now available are, in most instances, adequate for the routine monitoring in blood of most of the essential trace elements. Notable exceptions are the elements manganese, nickel and, in some instances, selenium, for which either improvements in sensitivity or the development of analytical techniques that are more suitable for routine application would be welcome.Most of these arise from the often unjustified assumption that changes in the trace-element content of blood or plasma closely reflect the concentrations of these elements at their functionally active sites at which metabolic defects are arising during deficiency or intoxication.Indirect evidence suggests that the extent to which this is the case differs greatly between individual essential or toxic elements. For example, differences in relationships between plasma and tissue trace-element content are evident on comparing the responses to zinc and copper The interpretation of analytical data is a more frequent cause of problems.February, 1979 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL ANALYSIS 77 depletion.When a low dietary intake provokes copper deficiency, plasma copper usually declines slowly as the element is progressively withdrawn from hepatic reserves and reaches a low equilibrium value, the magnitude of which bears no close relationship either to the interval that will elapse before metabolic lesions develop or to their severity.In contrast, zinc deple- tion, especially in the rapidly growing young animal, causes an almost immediate decline in plasma zinc, even though no change may be detectable in the total content of zinc in most major organs and tissues despite the rapid development of clinical signs of deficiency.Relation- ships between plasma and tissue zinc or copper during the development of deficiency are also influenced markedly by concurrent infection or other stress and, in the instance of zinc, by the extent of tissue anabolism or catabolism, the former tending to decrease and the latter increase plasma zinc concentrations. In some instances where the functional roles of essential trace elements are known, it has proved possible to monitor the development of deficiency disorders by assaying the blood or plasma activity of trace element dependent enzymes.This approach has several advantages. Hence the measurement of plasma ferroxidase I or glutathione peroxidase activity, for monitoring changes in copper and selenium status, respectively, provides indications of changes in the metabolisable tissue reserves rather than the total tissue contents of these elements.The importance of being able to distinguish between total tissue content and the functionally active component of an essential metal in the body is illustrated well by the syndrome of molybdenum intoxication, where the development of a systemic copper deficiency is indicated clearly by a marked decline in (copper-dependent) ferroxidase I activity, despite the develop- ment of supranormal concentrations of copper in plasma.The progressive development of trace-element deficiency or intoxication is often accompanied by characteristic changes in the plasma content of several elements, whose retention and tissue distribution are influenced by metabolic processes sensitive to the element in deficit or excess.Thus, prolonged exposure to high intakes of cadmium or zinc provokes a fall in plasma copper and, ultimately iron, such changes reflecting the development of competitive metabolic inter- actions during the absorption or incorporation of these elements into their functional sites. It is worth noting that, in the instance of cadmium or molybdenum intoxication, such secon- dary changes in plasma composition often arise before the concentration of the toxic element in plasma or whole blood has changed sufficiently to be of diagnostic significance.It must be emphasised, in conclusion, that the detection of incipient trace-element deficiency or toxicity and the resolution of the aetiology of these conditions in animals and man is facilitated if analytical data are available for tissues other than blood and if the trace element composition of the diet is known. Data for plasma composition alone can be misleading in the absence of such information, as is illustrated in several recent studies of clinical problems arising from trace-element imbalance in the diet of farm animals and from occasional errors in the formulation of trace-element supplements used for preparation of parenterally administered foods for human subjects.The aetiology of several such situations has only been resolved by careful consideration of the nature of clinical symptoms, of anomalies in the composition of both blood plasma and diet and by awareness of the metabolic defects likely to arise from deficiency, excess or antagonistic imbalance between the trace elements.Essential and Toxic Elements in Human Medicine G. S. Fell Department of Pathological Biochemisty, Glasgow Royal Infirmary, Glasgow, G4 OSF As was demonstrated by Mills in the Plenary Lecture, knowledge of the biological role of a wide range of inorganic elements is growing rapidly.Applied research in the subject is of proven economic benefit in agriculture, but in human medicine clinical interest tends to be limited to those elements where deficiency or excess gives rise to recognisable clinical effects. Thus, elements such as sodium, potassium, calcium, magnesium, phosphorus, iron, iodine and cobalt are of established clinical relevance and the toxic effects of lead and mercury are known to be important. Recent developments in instrumental analytical chemistry could allow the hospital biochemist the possibility of investigation into a wider range of biologically active78 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL *4NALYSIS PYOC. Analyt.Cl'em. D i V . SOC. elements than was hitherto possible. below. Areas of potential clinical importance are discussed Primary Changes in Specific Diseases Certain rare but clinically important inborn errors of metabolism are directly linked to trace metals.Thus, copper metabolism is disturbed in Wilson's disease and in Menke's syndr0me.l In the instance of Wilson's disease or hepatolenticular disease, copper determinations in blood serum and in urine are of importance in order to establish the diagnosis and to regulate chelation therapy.In zinc metabolism a primary failure of intestinal absorption is thought to be important in the development of the childhood condition of acrodermatitis enteropathica. This previously fatal condition manifests itself as widespread skin lesions and intestinal dis- orders, with a serious reduction of resistance to infection.The demonstration of a low plasma zinc content is of diagnostic value and regular determinations of plasma zinc are required during oral zinc sulphate therapy. This treatment can dramatically relieve the disorder. These two rare conditions, together with the commoner disorder of iron metabolism, namely haemochromatosis, have allowed detailed investigation of the metabolic pathways of copper, zinc and iron in man and have given much valuable information.Secondary Changes Caused by Common Clinical Conditions llajor alterations to trace metal biochemistry can occur as a secondary consequence of malnutrition, in common diseases such as diabetes, liver disease or myocardial infarction and during infectious illness. Also, the complex endocrine events making up the metabolic response to injury necessarily bring about profound changes in essential inorganic element homeostasis, along with that of other body constituents.Not all of the changes in levels of zinc, copper, manganese, nickel, chromium and other metals that are reported in various combinations of such common clinical conditions are in themselves significant. However, in instances of injury and infection, detailed studies of zinc metabolism are of value in relation to the state of protein synthesis and catabolism.2 Recently it has been demonstrated that zinc deficiency per se can impair the cell-mediated immune system in protein-energy maln~trition.~ Numerous other examples of disturbances of trace metal biochemistry could be given and these show that the more widespread ability to determine essential trace metals in common clinical conditions has revealed and is revealing many hitherto unsuspected changes, which are as yet of unknown importance.Changes Caused by Medical Treatment Direct therapy using metal salts such as lithium carbonate in the prophylaxis and treatment of manic depression or of gold sols given by injection in the symptomatic relief of arthritis are well recognised as effective treatments, but as being associated with toxic side effects unless dosage is carefully controlled. Certain clinical procedures by-pass the normal regulatory function of the intestinal tract, and can result in the delivery of nutrients or other products directly into the bloodstream, causing unintended imbalances of trace element metabolism with serious clinical consequences.Complete Parenteral Nutrition Patients unable to feed normally can be kept alive almost indefinitely by intravenous infu- sion of highly purified amino-acid, carbohydrate and fat solutions. I3alanced additions of vitamins and all known essential trace elements are also required if a variety of deficiency diseases are to be avoided.Clinical signs and symptoms that are reversible upon the addition of supplemental zinc, copper, chromium and manganese have been reported. The period of rapid body weight gain that follows recovery from serious illness is the most likely period when acute deficiencies of the essential elements will be enco~ntered.~ Regular determination of the concentration of the trace elements in blood serum and urine is of value when adjusting the dosage of additive mixtures, which are at best empirical guesses as to the true requirements.FebTUaTy, 1979 DEVELOPMENTS I N CLINICAL AND BlOLOGlCAL ANALE-SIS 79 Haemodialysis The hospital and home renal dialysis programmes have allowed patients with terminal renal failure to be maintained for many years until a suitable renal transplant can be arranged.believed that a significant amount of illness is associated with trace elements delivered in inappropriate amounts directly into the peripheral bloodstream during the dialysis procedure. Lacking any renal function, the patients are thereafter unable to excrete any excess in their urine. Direct toxicity, resulting in acute intravascular haemolysis, from excess of copper and zinc concentrations in the water used to prepare the dialysis bath fluid, is known, and this requires checks upon the purity of the supply.In general it can be shown that during the dialysis process there is a significant transfer of a wide range of metals across the dialj.sis membrane. Recently, however, unsuspected and serious adverse effects from aluminium have beeu reported to be associated with brain damage, bone disease and severe anaemia in renal dialysis patient^.^ In at least two areas of Britain epidemiological evidence links the fre- quency of illness in dialysis patients with the level of aluminium in the domestic water supply.There is a direct correlation between serum aluminium levels, the incidence of brain damage and aluminium concentration in the water used to prepare dialysis fluid.An increased cerebral spinal fluid concentration of aluminium is found in affected patients and the analysis of brain tissue, post mortem, shows a high concentration of aluminium in the grey matter. Unfortunately, various serious complications are associated with this treatment and it i: now Changes Caused by Drugs and Intravenous Fluids Such action, however, is rarely truly metal-specific and desired effects such as copper removal during penicillamine therapy in Wilson’s disease may be accompanied by unintended zinc loss in urine that is severe enough to cause dramatic clinical effects of hair loss and skin rash.l Where drugs or other products are given intravenously imbalances of trace element metabolism are particularly likely.Certain amino acid - sugar mixtures given in intravenous feeding promote zinc loss in urine and certain antibiotics may also have a similar effect. A illore general problem exists in relation to the accidental contamination with metals of drugs, protein solutions or other products that have to be given by injection.An example of this potential problem is that of the aluminium content of sterile plasma protein solution (SPPS), widely used as a plasma volume expander or in plasmapheresis procedures. The author has found that this product contained an average of 500 pg 1-1 of aluminium (compared with the expected value of less than 100 pg 1-l). Similarly, the coagulation factor, Factor VIII, has up to 2000 pg 1-1 of aluminium; this is presumably added during the manufacturing process. It is unlikely that these products at present constitute a serious clinical hazard insofar as normal renal function in the patients given these preparations will ensure the prompt excretion of any excess of aluminium in the urine. However, some studies are required to prove that this is so and this example of aluminium should serve to remind the pharmaceutical manufacturing industry of the need to extend the range of metals that they monitor in their products, using methods with sufficient analytical sensitivity to give reliable information at low detection limits.Some pharmaceutical products act by chelation of various essential or toxic metals. Environmental and Occupational Hazards Much publicity is given to environmental pollution by metals such as lead, mercury and recently cadmium.These metals are strongly tissue-bound and therefore accumulate in the body during periods of relatively low-level exposure. Definite health risks are also known to exist for certain groups of industrial workers.6 Direct analysis in blood, urine or tissues is now possible for lead, mercury and cadmium as well as for other potential hazards.Serious problems of interpretation of these results as well as some doubts as to the analytical reproducibility of methods, however, remain and make it important that all such metal analyses are considered alongside other clinical evidence and, where possible, independent biochemical measurements of cellular toxicity.Thus, it is well established that in early lead toxicity there will be detectable changes in enzymes and meta- bolites of the haem biosynthetic pathway and that for cadmium early changes in urinary protein excretion can give some evidence of impending toxic damage.80 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS Proc. Analyt. Div. Chenz.SOC. Laboratory Investigation of Trace Metal Disorders Most analyses for metal are carried out on blood, blood serum or plasma and urine samples. These are by far the most convenient materials to obtain clinically and on which the hospital biochemist has had much experience of interpretation. In certain circumstances tissue biopsy or analysis of hair, nail or other materials are appropriate but these will always remain a small proportion of the total number of investigations.Partial or complete metabolic balance studies are also feasible, whereby estimates of the total input (in food, intravenous fluids, etc.) and the total output (urine, faeces, etc.) can be attempted. Studies using neutron activation allowed some 20 elements to be determined during intravenous feeding.' The use of suitable radioisotopes can give useful information as to the absorption, transport, tissue distribution and excretion of some important metals.Hence studies with iron-59, copper-67 and zinc-65 and other radioisotopes, when used together with a whole-body counter, are of considerable research value. However, for the initial assessment of disorders and in routine patient care, direct deter- mination of metals in blood or urine is needed in order to identify severe disorders and indicate the need for clinical action.A list of proposed action limits for ten elements of clinical interest is shown in Table I. These concentration values for blood and urine indicate levels beyond which some clinical consequence is to be expected imminently, and the table also gives further suggestions as to parallel biochemical and clinical investigations.TABLE I BLOOD AND URINE ACTION LIMITS FOR TEN INORGANIC ELEMENTS OF CLINICAL IMPORTANCE Values lying outside these limits may be associated with acute clinical symptoms. Values persistently close to the limits may be associated with chronic clinical conditions. Blood Element concentration* Urine excretion Lithium Magnesium Aluminium Phosphorus (as PO,3-) lron . .Copper . . Zinc . . Cadmium Mercury.. Lead . . . . 0.5 < S > 2.0 mmol-l -90% of dosage excreted per 24 h, Li clearance 0.6 < S > 2.0 mmoll-1 2.0 mmol < 24 h urine > input dependent A1 renal clearance < 1 .O ml min-1 . . .. S > 4.0 pmol 1-1 . . 0.5 < S > 2.0 mmol 1-1 5.0 mmol < 24 h > input dependent 24 h > 10 pmol .. 10 < S > 30 pmol 1-1 . . . . 10 < P > 30 pmoll-I 10 < P > 30 pmol 1-1 24 h > 3.0 pmol 5 < 24 h > 20 pmol . . WB > 80 nmoll-1 (method dependent) dependent) . . WB > 0.5 pmol 1-1 (as methylmercury) mercury) . . WB > 2.0 pmoll-1 >0.5 pmoll-1 > 80 nmol 1-1 (method >0.5 pmol 1-1 (as inorganic * WB = whole blood, S = serum and P = plasma.Future Developments Other investigations Dosage. Renal function tests, plasma urea-electrolytes, dehydration. I.V. input, losses. Serum Ca, P, proteins, blood pH, renal function. Renal dialysis water supply. Al(OH), dosage. Renal function. I.V. input, insulin dosage, serum Ca, Mg, protein, blood pH, P50, red cell ATP.Haematology ; serum trans- ferrin, ferritin, red cell proto- porphyrin, urine haemosiderin. L.F.T., caeruloplasmin, renal tubular function. Plasma albumin, cx2 macro- globulin, serum alkaline phosphatase, I.V. input, losses. Total urine protein, urine ,!$. microglobulin renal function, renal calculi, lung function. Total urine protein, nephrotic syndrome. Haematology, red cell por- phyrins, ALA-D.Urine porphyrins, urine ALA. Continuing improvements to the reliability, speed and the range of existing methods for the determination of metals will allow the hospital biochemist to investigate hitherto neglected areas of the periodic table. Much basic study of physiology and biochemistry of the less commonly investigated trace elements is required if full clinical advantage is to be gained from this new analytical capacity.More attention should be paid to inter-element interactions andFcbruary, 1979 DEVELOPMENTS IN CLINICAL AND BIOLOGICAL ANALYSIS 81 the importance of the nutritional status of individual subjects exposed to toxic metal hazards. Modern medical treatment involving direct introduction of materials into the circulating blood- stream also require the clinician to be aware of potential problems that can arise from distur- bances of trace metal biochemistry. References 1.Prasad, A. S., and Oberlease, D., Editors, “Trace Elements in Human Health and Disease, Volume 1, 2. Wilkinson, A., and Cuthbertson, D. P., Editors, “Metabolism and the Response to Injury,” Pitman 3. Golden, M. H. N., Golden, B.E., Harland, P. S. E., and Jackson, A. A., Lancet, 1978, 1226. 4. Johnstone, I. D. A,, Editov, “Advances in Parenteral Nutrition,” MTP Press Ltd., Lancaster, 1978, 5. Elliot, H. L., Dryburgh, F., Fell, G. S., Sabet, S., and MacDougall, A,, Br. ,Wed. J . , 1978, 1101. 6. Brown, S. S., Editor, “Clinical Chemistry and Chemical Toxicology of Metals,” North-Holland, 7. Zinc and Copper” Academic Press, London, 1976.Medical, Bath, 1976, pp. 307-317. pp. 241-263. Amsterdam, 1977. Jacobson, S., and Wester, P.-O., BY. J . Nutr., 1977, 37, 107. Complex Concentrations that are too Dilute to Analyse-The Dilemma and a Solution David R. Williams DeFartment of Chemistry, University of Wales Institute of Science and Technology, Cardiff, CF1 3NU The problems associated with determining the concentrations of very dilute low molecular weight complexes used as enzyme co-factors or as precursors for metalloprotein synthesis, under in vivo conditions, are manifold; biosolutions contain thousands of other complexes of each metal, protein-bound metal species are present in excess and analyses using isolation techniques or pre-concentration approaches often displace the equilibrium involving the complex in question.Nevertheless, if meaningful biological response veysus low molecular weight concentration relationships are to be established, a knowledge of such concentrations is necessary.lS2 An acceptable solution stems from the cognisance that all concentrations, no matter how dilute, are dependent upon analyses of the total concentrations of metals and of ligands present and are consonant with all equilibrium constants, solubility products and kinetic parameters pertinent to that solution. Computerised approaches are now available to unravel these relationships and thus establish the concentration patterns that dictate biological phenomena. Our primary concern has been with speciation studies of blood plasma investigated using the ECCLES computer ~ r o g r a m , ~ but there have also been subsidiary projects simulating the modus operandi of a stomach ulcer preparation4 and the bio-availability of zinc from soya bean protein.The blood plasma sim~lations~~~-s have produced tables of the relative concentrations of low molecular weight complexes of calcium(II), magnesium(II), manganese(II), iron(III), nickel(II), copper(II), zinc(I1) and lead(I1) ions.The predominant species present in addition to metallo- protein were ternary (i.e., mixed-ligand) metal complexes, viz., [Cu.cystinate.histidinate]-, [Fe.citrate.hydroxide]-, [Zn.(cysteinate)J2- and [Zn.cysteinate.histidinate.H]O or [Zn.(histi- dinate),]O tended to exist at lower concentrations. In general, the charged complexes usually remained circulating in plasma or were “desalin- ated” through the kidneys and thus excreted in the urine.Low charge density or neutral complexes, on the other hand, are capable of passing through lipid - protein cell membranes and of being assimilated into tissues. Computer simulation, being based on total concentrations and stability constants, can calculate such species distributions during health and both diseased and medicated states, provided that total concentration analyses are available.Such calculations have been used to quantify the new low molecular weight complexes formed when ligand drugs such as EDTA (and derivatives), desferrioxamine, penicillamine and triethylenetetrammine are administered to p l a ~ m a , ~ , ~ the redistribution of naturally occurring complexes necessary, the charge density82 DEVELOPMENTS I N CLINICAL AND BIOLOGICAL ANALYSIS P?’OC.Annlyt. D~zJ. CJ~EWI. SOC. dependent probabilities that such complexes will pass through cell membranes into tissues and dose - response relationships. Recent researches using computer simulation have examined the therapy of Il-ilson’s disease,g the source of copper excreted as a result of D-penicillaminelo treatment and tlie inflammation dependence upon tissue copper in cases of rheumatoid arthritis.ll Animal screens have shown that the greater the concentration of copper passed into inflamed tissue, the better is the reduction in swelhg.Speciation studies using computer simulation are helping to design agents to administer copper orally and to manipulate membrane soluble species irc i i v o .New projects being undertaken include a study of more iron(II1) complexes12 and tlie design of agents for removing radi0nuc1ides.l~ 1. 2. 3. 4. 5. 6 . 7. 8. 9. 10. 11. 12. 13. References Fiabane, A. M., and Williams, D. K., “The Principles of Bio-inorganic Chemistry,” Royal 172stifi[tr of Williams, D.R , Edztov, “An Introduction to Bio-inorganic Chemistry,” Thomas, Illinois, 1876. May, M., Linder, 1’. W., and Williams, D. R., J . Chem. Soc., Dalton Tvans., 1977, 588. Williams, D. R., J . Inovg. Nucl. Chem., 1977, 39, 711. Makar, G. I<. I<., and Williams, D. R., J . Inovg. Nucl. Chem., 1977, 39, 201. May, P. M., and Williams, 1). K., Expevaentza, 1976, 32, 1492.May, P. M., and Williams, 1). K., FEBS Lett., 1977, 78, 134. Berthon, G., May, P. M., and Williams, 1). R., J . Chem. Soc., Dalton Trans., submitted for publication. May, P. &I., and Williams, I). R., Proc. R. SOC. Med., 1977, 19. Jackson, G. E., May, P. M., and Williams, 1). K., FERS Lett., 1978, 90, 173. May, P. M., Williams, D. K., and Jackson, G. E., I . 1 ~ o v g . Nucl. Chem., 1978, 40, 1227.May, P. M., Linder, P. W., and Williams, D. K., zn Sigel, H., E d i t o r , “Metal Ions in Biological Systems,” TVilliams, D. K., Chemy Brzt., 1978, 14, 282. Chemzstvy Monogyafih fov Teachevs, No. 31, Chemical Society, London, 1977. Marcel Dekker, New York, 1978, Volume 7, p. 29. Industrial Toxic Hazards A. W. Baird Employmemt Medical Advisory Sevvice, Health and Safety Executive, Guavdian Royal Excharigc Biiilditig, 314 Vmcent Stveet, Glasgow Industrial disease has long been recognised, the “Rules of the Tavern” commonly seen repro- duced in public houses and hotels were drawn up to prevent the general public from having contact with undesirables who might have contracted industrial disease.The examples given are the knife grinder who could have suffered from silicosis overlaid with tuberculosis, and the tinker or tinkler who tinned copper vessels with a lead compound, absorbed this, became lead poisoned and was an undesirable bedfellow.Modern practice is more scientific but we must recognise that people are all different and can be young or old, male or female, intelligent or otherwise, in good nutrition or bad nutrition, and all these factors will vary the effect that an industrial environmental exposure will have on a particular person. I t has been recognised recently that babies may have their intelligence affected by the level of lead in drinking water, which may or may not be directly attributed to industry. The workplace has also to be considered. The clean, well kept, tidy workplace, whether in old or new factories, is probably safer than the old, dirty, poorly lit shed, yet the description of occupation may appear identical unless the actual working conditions have been examined. The influence of management and supervision, as well as of the worker himself, can influence his liability to contract a particular industrial disease. This is exemplified by the contrast between a well kept lead refinery, where the process is clearly visible to supervision, and the old, dirty refinery where bad practice can be carried on without it being so obvious. Unusual causes of lead absorption can be found, as occurred recently in a firm re-finishing baths in Glasgow local authority houses, where lead glaze was being removed by a rotating disc. A modern development of this hazard lies in the lead solder applied to motor car bodies, this also being removed by a lead disc, precautions being taken to prevent breathing in of the lead dust but the effectiveness of the precautions depending on good layout, good lighting andFebruary , I979 EQUIPMENT NEWS 83 good protective equipment, i e . , giving conditions under which a man can work comfortably and yet be protected from the hazard to which he is exposed. A particular problem was the demolition of St. Enoch Station, a former British Rail property maintained for almost 90 yrs with heavy applications of lead paint, which had to be demolished by oxy-acetylene cutting by men over 100 ft above the ground, where the individual was more concerned with the hazard of falling than with the possible effect of the inhalation of lead fume, and where biological monitoring showed a progressive increase in the lead content of the workers, leading to suspension and the necessity for treatment in hospital. The fact that all the burners worked on slightly different jobs, in different ways, and using different techniques, had to be taken into account when assessing the risk and the progression of the lead absorption. The analysts’ figures cannot be used in isolation, as there may be a built-in error in the result. The results from different laboratories may vary through deficiencies in the methods used that cannot be overcome. The identification of intoxication depends on an assessment of working conditions, the clinical condition of workers and the biochemical results available. This requires inter-professional discussion in order to achieve the object to which we all aspire, that is, the prevention of ill health.

ISSN:0306-1396    

DOI:10.1039/AD9791600067

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

February , 1979 Equipment EQUIPMENT NEWS 83 Scanning Electron Microscope The Alpha 9 scanning electron microscope just announced has a guaranteed resolution of 120 A (12.0 nm). The magnification range is from 30 x to 80 000 x . Rapid scan a t 0.2 s per frame for focusing and stigmator compensations, full area rapid scan a t 0.7 s per frame, normal scan full area a t 5 s per frame and photoscan full area a t 40 s per frame, are attainable.The standard stage allows specimens up to 32 mm diameter x 15 mm thick to be examined, while larger specimens up to 80 mm in diameter can be examined by fitting a large universal stage. International Scientific Instruments Inc. (UK), Waterwitch House, Exeter Road, New- market, Suffolk. Detector for Scanning Electron Microscope A detector for scanning electron microscopes known as the Robinson Backscattered Electron Detector is now available.Using this detector no specimen preparation is needed, and porous, wet or frozen specimens can be imaged. The detector shows a high degree of atomic number contrast sensitivity in addition to the topo- graphic contrast evident in the usual SEM image. Differences of about 1% in average atomic number have been clearly distinguished.The environmental cell modification allows samples to be examined in the range from 1 to 0.1 Torr. International Scientific Instruments Inc. (UK), Waterwitch House, Exeter Road, New- market, Suffolk. Power Supply for Atomic Absorption A two-lamp power supply is available for EDLs and atomic-absorption spectrophotometers.The two-lamp EDL power supply is offered in two models, a single channel for operation of a single lamp a t a time, or a dual channel for simultaneous use of two individual EDLs. The power supply is operable with all EDLs and with all Perkin-Elmer atomic-absorption spectro- photometers except Models 290 and 290(B). Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 1QA.Graphite Furnace for Atomic Absorption The Model HGA-500 furnace is programmable through its built-in microcomputer for autom-84 EQUIPMENT NEWS Proc. Analyt. Div. Chew. SOC. ated atomic-absorption spectroscopy, accommo- dating seven complete analytical programmes with six in memory and one working. The temperature range is from ambient to 3 000 "C. Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 1QA.Automatic Syringe Sampler for Gas Chromatography The MS-1 automatic syringe is designed to simplify the injection process and improve pre- cision. The syringe accepts a sealed vial con- taining the sample, which is injected at the push of a button. Between sample injections, solvent flush is accomplished by forcing the cleaning flush through the system under pressure.Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 1QA. Purge and Trap Sampler An automatic purge and trap sampler, which concentrates traces of volatile organic com- pounds to levels detectable by gas chromato- graphs and gas chromatograph - mass spectro- meters, has been introduced. This device is the H-P7675A sampler, which uses the dynamic headspace technique.Volatile organic compounds in the sample are transferred to the gaseous phase and adsorbed on a trap, which is then rapidly heated in order to desorb the volatile organics and flush them into the analytical column; this last can be a packed or capillary column. While the chro- matographic analysis proceeds the trap is heated to a high temperature and back-flushed to re- move any residues before the next adsorption cycle.The sampler's standard purge-cap as- sembly for gas-chromatographic applications is threaded to accommodate standard laboratory glassware, including 15-ml centrifuge tubes. A larger purge-cap assembly for 50-ml tubes and 25 x 200 mm culture tubes is also available. Hewlett-Packard, King Street Lane, Winnersh, Wokingham, Berkshire, RG11 5AR.Elemental Analyser An improved version of the Model 240 ele- mental analyser is now available. The Model 240B includes a faster heating combustion furnace, a new detector design with better temperature control for increased stability, an external programmer index wheel, a redesigned autosampler, a programmable calculator - data analysis system for on-line calculation and printout of results.A Model AD-2 autobalance is also available. Perkin-Elmer Ltd., Post Office Lane, Beacons- field, Buckinghamshire, HP9 IQA. Radiochromatogram Scanner A range of radiochromatogram scanners (con- sisting of three instruments is announced. The 7220 is a strip scanner for paper chromatograms with a thin-layer plate accessory, the 7221 is a strip scanner for thin-layer plates and the 7230 is a bi-dimensional scanner for paper or thin- layer plates up to a maximum size of 20 x 20 cm.Packard Instrument Ltd., Caversham Bridge House, 13-17 Church Road, Caversham, Berk- shire, RG4 7AA. Monochromator The Model H-25, a quarter-metre monochroma- tor, replaces the older M-25. The new mono- chromator is a compact instrument covering a spectral range from 180 nm in the near ultra- violet to 30 pm in the far infrared.The 58 x58 mm gratings are interchangeable on pre- focused mountings and a wide range of gratings is available. The focal length is 250 mm and the aperture f/3. With 0.05-mm slits the resolution is 0.3 nm at 500 nm while the dispersion is 3 nm mm-l with a 1 200 lines mm-l grating.EDT Research, 14 Trading Estate Road, London, NWlO 7LU. Acrylonitrile Monitoring An ambient air monitoring system for acrylo- nitrile is announced, comprised of a process chromatograph analyser unit, a multi-stream sample aspiration system, microcomputer con- trollers and a data processor. The system is expanded to include monitoring of other un- desirable compounds associated with acrylo- nitrile manufacture or processing, e.g., styrene, butadiene and hydrogen cyanide.Anacon (Instruments) Ltd., St. Peters Road, Maidenhead, Berkshire, SL6 7QA. Flatbed Chart Recorders A range of flatbed chart recorders, the BS900 series of multi-channel recorders, is now avail- able in either three-channel or four-channel versions. Input amplifiers for each channel can be selected from modules having 12 or 18 voltage ranges or a 24 range module with 12 voltage and 12 current ranges; the chart speed module has 16 ranges.The pens, which are rechargeable and fibre tipped, have a dynamic response of 0.65 s for full-scale deflection. Bryans Southern Instruments Ltd., Willow Lane, Mitcham, Surrey, CR4 4UL.February, 19 79 THEOPHILUS REDWOOD LECTURE 85 Fan-fold Chart for Recorders A fan-fold chart cassette for PlOOL Series chart recorders, carrying a 7.5 m length of chart, is announced.The instruments remain un- changed and will accept either the new fan-fold cassette or the original roll-type cassette. The fan-fold chart offers instant readability and easy removal of the traced portion without interrupting the run.The visible portion of trace on the folds of the stacked chart can show the trend of the over-all trace at 80-mm intervals. Foster Cambridge Ltd., Howard Road, Eaton Socon, Huntingdon, Cambridgeshire, PE 19 3EU. Electronic Balance A fully electronic, top-loading, analytical balance is announced. The new A30 model features readability to within 0.1 mg, even by inexperienced operators without special trah- ing. A.Gallenkamp & Co. Ltd., P.O. Box 290, Technic0 House, Christopher Street, London, EC2P 2ER. Translation Stages A new line of rack and pinion and ball-bearing translation stages for precise and repeatable positioning of lenses, mirrors, reticles, pinholes, slides and any other application requiring pre- cise coordinate positioning of optical and opto- mechanical components or systems is now avail- able.Melles Griot B.V. Industrial & Scientific Optics, Nieuwe Kade 10, Postbus 567, Arnhem, The Netherlands. Valves with Replaceable Seats and Non- rotating Stem Angle pattern valves for high pressure and severe service are now available with replaceable seats and non-rotating stems. The one-piece, replaceable seat threads into the valve’s female high pressure inlet port, allowing the heart of the valve to be replaced with a saving in down- time and maintenance costs.The non-rotating stem prevents possible galling or damage to the stem tip. The standard material of manufac- ture is 316 stainless steel. End connections are in, 8 in and in straight-thread female ports. The service rating is 45 000 p.s.i.g.Techmation Ltd., 58 Edgware Way, Edgware, Middlesex, HA8 8 JP. oxytocin in solution and to maintain this dose at the precise rate for the controlled induction of labour. Pye Dynamics Ltd., Sales Department, 459 Park Avenue, Bushey, Watford, Hertfordshire. Induction and Monitoring System The system includes apparatus to monitor and display foetal heart rates and intra-uterine con- traction pressure, plus a programmed infusion pump to administer synthetic oxytocin at accurately controlled dose rates.The infusion pump is similar to the MP 40. Pye Dynamics Ltd., Sales Department, 459 Park Avenue, Bushey, Watford, Hertfordshire. Theophilus Redwood Lecture At its meeting on December 13th, 1978, the Council of the Analytical Division, on the recommendation of its Honours Committee, agreed that the 1980 Theophilus Redwood Lecture should be given by Professor L. S. Bark, Professor of Analytical Chemistry at the University of Salford . An a I yt i ca I Division Distinguished Service Award On the recommendation of its Honours Com- mittee, at its meeting on December 13th, 1978, the Council conferred the fourth Analytical Division Distinguished Service Award on Mr. K. L. Smith formerly of The Boots Company Limited, Nottingham. Infusion Pump The MP 40 has been designed to administer a progressively increasing or decreasing dose of

ISSN:0306-1396    

DOI:10.1039/AD979160083b

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

February, 19 79 THEOPHILUS REDWOOD LECTURE 85 SAC Silver Medal Nominations are invited for the award of the SAC Silver Medal, which is for the encourage- ment of young scientists working in any field covering the practice and teaching of analytical chemistry. The award is accompanied by a cash prize and is normally made annually to the candidate who, in the opinion of the AD Council, has made the greatest contribution and whose work has made the most significant impact in any branch of analytical chemistry.In addi-86 CONFERENCE tion, the future promise of the candidate is taken into consideration. It is hoped to provide an opportunity for the successful candidate to deliver a lecture to the Division on a suitable occasion subsequent to the presentation of the Medal. The rules are as follows- 1.The award of the Silver Medal will normally be considered annually by the Honours Committee, acting on behalf of the Council of the Division, but an award may not be made if it is considered that the work of no candidate reaches the required standard. 2. Candidates must be British subjects of 38 years of age or under in the year in which the award is considered. Evidence of age will be required.3. The merits of the candidate’s work may be brought to the notice of the Council by any person (being a member of the Analytical Division of the Chemical Society) who desires to recommend the candidate by letter addressed to The President, Analy- tical Division, The Chemical Society, Burlington House, London, W 1V OBh’. The letter should be accompanied by a short statement on the candidate’s career (date of birth, education and experience, degrees and other qualifications, special awards, etc., with dates, and any other relevant information) and a list of titles of, and references to, papers or other works published by the candidate, independently or jointly.One reprint of each paper (or other work) for which reprints are available should be submitted.4. The award will be made on an over-all assessment of the candidate’s contribution, the impact of his/her work and his/her future promise in any field covered by the principles, teaching and practice of the analytical sciences. No restriction is placed as to where the work is conducted. 5. The Committee assessing the applications shall be at liberty to call any candidate for interview. 6. The successful candidate will receive the sum of L l O O in addition to the Medal. 7. The decision of the Council shall be final. 8. Any alteration to these Rules shall be sub- ject to the approval of the Council. Proc. Analyt. Div. Chem. SOC. Recommendations for the next award should be made to The President, Analytical Division, The Chemical Society, Burlington House, London, W1V OBN, by March 31st, 1979.

ISSN:0306-1396    

DOI:10.1039/AD9791600085

出版商:RSC    

年代:1979

数据来源: RSC

摘要:

86 CONFERENCE Proc. Analyt. Div. Chem. SOC. Course Asbestos Sampling and Safety April 24, 1979, London This one-day seminar is intended for those frequently involved with asbestos problems who have little or no scientific training in industrial hygiene. It will take place a t the Bloomsbury Centre Hotel, Coram Street, London, W.C. 1. Further details can be obtained from The Registrar, McCrone Research Institute, 2 McCrone Mews, Belsize Lane, London, NW3 5BG.

ISSN:0306-1396    

DOI:10.1039/AD979160086b

出版商:RSC    

年代:1979

数据来源: RSC