ISSN:0144-557X    

DOI:10.1039/AP98219FX011

出版商:RSC    

年代:1982

数据来源: RSC

ISSN:0144-557X    

DOI:10.1039/AP98219BX013

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

ANPRDI 19(4) 159-220 (1982) April 1982 Hon. Secretary R. Sawyer Analvtical Proceedinos I D Proceedings of the Analytical Division of The Royal Society of Chemistry AD President L. S. Bark Hon. Treasurer D. C. M. Squirrel1 Hon. Assistant Secretary D. 1. Coomber, O.B.E. Hon. Publicity Secretary Dr. A. Townshend, Department of Chemistry, University of Hull, Hull, HU6 7RX Secretary Miss P. E. Hutchinson Editor, Analyst and Analytical Proceedings P. C. Weston Senior Assistant Editor Assistant Editors R. A. Young Mrs. J. Brew, Mrs. P. A. Fellows Publication of Analytical Proceedings is the responsi- bility of the Analytical Editorial Board: J. M. Ottaway (Chairman) J. M. Skinner G. J. Dickes J. D. R. Thomas 'G. W. Kirby A. M. Ure J. N. Miller 'P. C. Weston G. E. Penketh J. Whitehead T.B. Pierce "Ex officio members All editorial matter should be addressed to: The Editor, Analytical Proceedings, The Royal Society of Chemistry, Burlington House, Piccadilly, London, W1 V OBN. Telephone 01 -734 9864. Telex 268001. Advertisements: Advertising Department, The Royal Society of Chemistry, Burlington House, Piccadilly, Analytical Proceedings (ISSN 01 44-557X) is pub- lished monthly by The Royal Society of Chemistry, Burlington House, London W I V OBN, England. All orders, accompanied by payment, should be sent to The Royal Society of Chemistry, The Distribution Centre, Blackhorse Road, Letchworth, Herts., SG6 1 HN, England. 1982 Annual Subscription price if purchased on its own: UK €40.00, Rest of World €42.00, US $95.00, including air speeded delivery. Air freight and mailing in the USA by Publications Expediting Inc., 200 Meacham Avenue, Elmont, N.Y.11 003. USA Postmaster: Send address changes to: Analytical Proceedings, Publications Expediting Inc., 200 Meacham Avenue, Elmont, N.Y. 11 003. Second class postage paid at Jamaica, N.Y. 11431. All other despatches outside the UK by Bulk Airmail within Europe, Accelerated Surface Post outside Europe. PRINTED IN THE UK. @ The Royal Society of Chemistry 1982 London, W1 V OBN. Telephone 01 -734 9864. Editorial SAC 83 Organising SAC conferences is rather like painting the Forth Bridge using a different team of painters each time. It is probably true to say that the only time when planning stops is during the actual period of the Conference, when the organisers have other problems on their minds.It is interesting to reflect on the changes that have taken place since the first SAC Con- ference, which was held in Nottingham in July 1965. Although sections of the Society had previously organised international symposia at Oxford, St. Andrews, Birmingham and Edin- burgh, this was the first to be organised wholly by the Society. A total of 340 delegates from 25 countries attended the Conference and 55 papers were presented in two streams. There was also a well supported Trades Exhibition consisting of 40 stands. The next Conferences were held a t Notting- ham (1968), Durham (1971) and Birmingham (1977), 1974 being the year of the Centenary Conference in London. The Birmingham Con- ference marked the introduction of poster and workshop sessions, which greatly increased the number of papers that could be presented.The most recent Conference was held a t Lancaster (1980) and was attended by 425 delegates from over 30 countries. Some 150 papers were presented in poster sessions and three lecture streams and there were 7 work- shop sessions. Unfortunately, the industrial recession affected the exhibition, which was smaller than a t previous conferences. For the first time, the' SAC Conference is to leave England as the next one will be held a t Edinburgh University in July 1983. The Steering Committee has been hard a t work developing new ideas and several of these will be introduced to SAC Conferences for the first time. As an alternative to the cultural and scientific visits normally held on the Wednesday, five update courses are being organised on Flow Injection Analysis, Narrow-column HPLC, Microcomputers in Analytical Chemistry, New Techniques in Fluorescence and Inductively 159160 ANALYTICAL DIVISION ANNUAL GENERAL MEETING Anal. Proc.Coupled Plasma Emission Spectrometry. The purpose of these is to provide a concise sum- mary of the state of the art in each technique and practical demonstrations will be included. The Groups of the Division have been invited to take a more positive part in the Conference and instead of the hour-long discussion period at the end of each day provided a t previous Conferences, lecture periods of up to one day have been offered. Among the Groups that have accepted this invitation are Thermal Methods, Electroanalytical, Atomic Spectro- scopy, Joint Pharmaceutical Analysis and Chromatography and Electrophoresis. It is hoped that the Analytical Methods Committee will also take part.Organisations outside the Society have been invited to take part and these will probably include the British Steel Corporation/British Independent Steel Producers Association Chemists’ Conference, the Association of Clini- cal Biochemists, the Association of Public Analysts and the Inorganic Mass Spectrometry Group. The participation of these organisa- tions will bring many new people to the Conference and increase the attendance at all lectures and functions. As well as these innovative features the traditional aspects of the Conference will continue, and in particular the social aspects are receiving careful attention. Evening events are being organised for Sunday, Monday, Tuesday and Friday and the banquet will be held on the Thursday. Day tours, including the opportunity of playing golf a t St. Andrews, and industrial visits are being arranged for the Wednesday and there will be a full programme throughout the week for accompanying persons. There is no doubt that throughout the week delegates will experience the full delights of Scottish hospitality. The number of enquiries and promises of contributions already approach 200, and those people intending to participate in the Con- ference and who have not already registered should do so without delay. Forms of applica- tion can be obtained from the Analytical Division Secretary a t the RSC, Burlington House, Piccadilly, London, W 1V OBN. J . WHITEHEAD

ISSN:0144-557X    

DOI:10.1039/AP9821900159

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

160 ANALYTICAL DIVISION ANNUAL GENERAL MEETING Anal. Proc. Annual General Meeting of the Analytical Division The tenth Annual General Meeting of the Analytical Division of the Royal Society of Chemistry was held at 2.30 p.m. on Friday, March 12th, 1982, a t the Scientific Societies Lecture Theatre, 23 Savile Row, London, W.l. The Chair was occupied by the President, Professor L. S. Bark, DSc, CChem, FRSC. The Report of the Council for the year ending March, 1982, was presented by the Honorary Secretary and adopted. New Rules for the Analytical Division were ratified. The Scrutineers, Mrs. R. L. Jordan and Mr. D . J . Willis, reported that no ballot had been required for the Officers of the Division and the following would therefore serve for the coming year- President-S. Greenfield, PhD, CEng, MIM, Immediate Past President-L.S . Bark. Vice-Presidents-P. G. W. Cobb and D. I. Honorary Treasurer-D. C. M. Squirrell. Honorary Secretary-R. Sawyer. Honorary Assistant Secretary-D. I. Coomber Other Members of Council-The Scutineers further reported that 645 valid ballot papers had been received and that votes had been cast in the election of Ordinary Members of Council as CChem, FRSC. Coomber . (Programmes Secretary). follows: E. Bishop, 374; C. Burgess, 379; L. C. Ebdon, 32.2; M. C. Finniear, 367; D. G. Porter, 377; D. Simpson, 461; P. B. Smith, 392; A. Townshend, 453; T. S. West, 490; D. 13. Williams, 304; D. W. Wilson, 368. The President declared the following to have been elected Ordinary Members of Council for the ensuing 2 yea.rs: E.Bishop, C. Burgess, D. G. Porter, D. Simpson, P. B. Smith, A. Townshend, T. S. West, and D. W. Wilson. H. E. Brookes, G. B. Crump, G. J . Dickes, A. F. Fell, R. C. Mackenzie, T. B. Pierce, J. F. Tyson and C. A. Watson, having been elected Members of the Council in 1981, will, by the Rules of the Division, remain Members of the Council for 1982. H. J . Cluley (Chairman of Analytical Abstracts Editorial Committee), A. Dyer (Chairman of the North West Region), R. A. HalljChairman of the Northern Ireland Region), S . J . Lyle (Chairman of the South East Region), J. G. Jones (Chairman of the Western Region), C. J . Keattch (Chairman of the Group Liaison and Policy Committee), J . N. Miller (Chairman of the Midlands Region), J. M. Ottaway (Chairman of the Analytical Editorial Board and SAC 83 Steering Committee), J.Vallance (Chairman of the Novth East Region), J. Whitehead (Chairman of the Programmes Committee), R. J . Whiteoak (Chairman of theApril, 1982 REPORTS OF MEETINGS 161 East Anglia Region), J. E. Whitley (Chairman of the Scottish Region) and D. R. Williams (Chair- man of the Analytical Methods Committee) will be ex oficio Members of the Council for 1982. D. T. Burns (Honorary Secretary of the International Affairs Committee) is an additional Member of Council. The Honorary Officers and new Members of Council will assume their duties on July 14th. The Annual General Meeting was followed by the Address of the Retiring President, Professor L. S. Bark, entitled “Reflections on Analytical Chemistry.” In the evening the Biennial Formal Dinner was held in the Rooms of the Royal Society of Chemistry, Burlington House, Lon- don, W.l. The seventh RSC Analytical Division Distinguished Service Award was presented to Dr. H. J. Cluley. A fuller report will appear in t h e May issue of Analytical Proceedings, while a short biography of Dr. Cluley may be found elsewhere in this issue (p. 162).

ISSN:0144-557X    

DOI:10.1039/AP9821900160

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

April, 1982 REPORTS OF MEETINGS 161 Reports of Meetings North West Region The fifty-seventh Annual General Meeting of the Region was held a t 6.30 p.m. on Friday, January 15th, 1982, at UMIST, Sackville Street, Manchester. The Chair was taken by the Chairman of the Region, Dr. A. Dyer. The following office bea.rers were elected for the forthcoming year: Chairman-Dr. A. Dyer. Vice-Chairman-Dr. A. Mathias. Honorary Secretary-Mr. G. Davison, Kodak Ltd., Chemical Division, Research Department, Acornfield Road, Kirkby, Merseyside. Honor- ary Treasurer-Mr. P. Morries. Honorary Assistant Secretary-Mr. T. Hanley. Members of Committee-Mr. G. B. Crump (ex oficio), Dr. D. Dollimore, Mr. B. S. Eastwood, Dr. M. L. Hitchman, Professor G. F. Kirkbright, Mr. B. Taylor and Dr. F. Vernon. Mr.G. W. Earnshaw and Mr. M. S. Green were re-appointed as Honorary Auditors. Western Region The twenty-seventh Annual General Meeting of the Region was held a t 6 p.m. on Friday, December 4th, 1981, a t the Two Rivers Hotel, Newport Road, Chepstow. The Chair was taken by the Chairman of the Region, Dr. G. Nickless. The following office bearers were elected for the forthcoming year : Chairman- Mr. J. G. Jones. Vice-Chairman-Mr. E. B. Reynolds. Honorary Secretary and Treasurer- Mr. F. W. Sweeting, Wessex Water Authority, P.O. Box 95, The Ambury, Bath, BA1 2YP. Members of Committee-Dr. K. W. C. Burton, Dr. A. Craggs, Dr. E. J. Newman, Dr. G. Nickless (ex o$cio), Mr. B. Sanders and Dr. P. Takla. Mr. E. A. Hontoir and Mr. E. Marshall were re-appointed as Honorary Auditors. Midlands Region The twenty-seventh Annual General Meeting of the Region was held a t 6.30 p.m.on Tuesday, November 3rd, 1981, in the Department of Chemistry, The University, Birmingham. The Chair was taken by the Chairman of the Region, Professor J . N. Miller. The following office bearers were elected for the forthcoming year: Chairman-Professor J. N. Miller. Vice- Chairman-Dr. P. B. Smith. Honorary Secretary-Mr. H. E. Brookes, 35 Dunster Road, West Bridgford, Nottingham, NG2 6 JE. Honorary Treasurer-Dr. J . F. Tyson. Honor- ary Assistant Secretary-Dr. A. Braithwaite. Members of Committee-Dr. S. Greenfield, Dr. A. M. G. Macdonald, Mr. D. M. Peake and Mr. J. E. W. Tillman. Mr. W. G. Harris and Mr. H. Pugh were re-appointed as Honorary Auditors. Northern Ireland Region The first Annual General Meeting of the Region was held a t 5.15 p.m.on Thursday, October 22nd, 1981, a t the Queen’s University, Belfast. The Chair was taken by the Chairman of the Region, Mr. R. A. Hall. The following office bearers were elected for the forthcoming year: Chairman-Mr. R. A. Hall. Vice-Chairman- Dr. M. A. Leonard. Honorary Secretary and Treasurer-Mr. W. J. Swindall, Department of Chemistry, David Keir Building, Queen’s University, Belfast, BT9 5AG. Members of Committee-Professor D. T. Burns, Mr. E. L. Donaldson and Dr. G. Svehla. Mr. C. Wilson and Mr. V. L. Beavis were appointed as Honorary Auditors. Atomic Spectroscopy Group The seventeenth Annual General Meeting of the Group was held at 6.30 p.m. on Thursday, January 21st, 1982, at the University of Strath- Clyde, Glasgow.The Chair was taken by the Chairman of the Group, Dr. L. C. Ebdon. The162 ANALYTICAL DIVISION DlSTINGUISHED SERVICE AWARD Anal. Proc. following office bearers were elected for the forthcoming year : Chairman-Dr. L. C. Ebdon. Vice-Chairman-Dr. E. J. Newman. Honorary Secretary-Mr. D. J. Willis, Hilger Analytical Ltd., Westwood Industrial Estate, Ramsgate Road, Margate, Kent, CT9 4 JL. Honorary Treasurer-Dr. G. B. Marshall. Honorary Assistant Secretary-Mr. C. A. Watson. Members of Committee-Dr. J. F. Alder, Dr. D. Littlejohn, Dr. W. J. Price (ex oficio), Dr. P. B. Smith, Dr. R. D. Snook, Dr. K. C. Thompson and Mr. R. W. Williams. Mr. R. P. Blakemore and Mr. D. B. Ratcliffe were appointed as Honorary Auditors. J oi nt P ha rmaceut ica I Ana i ysis Group The twelfth Annual General Meeting of the Group was held a t noon on Thursday, January Zlst, 1982, at the Pharmaceutical Society of Great Britain, 1 Lambeth High Street, London SEl 7JN.Owing to industrial action on British Railways, the Chairman and the Im- mediate Past Chairman were unable to be present and the chair was taken by Mr. J . C. Deavin. Mr. Deavin announced that 1981 had been a successful year for the Group. The Annual Report for 1981 was presented by the Honorary Secretary. Mr. Deavin reported that Mr. C. A. Johnson had been elected by the Committee as the new Chairman for 1982/83. The Committee had proposed that a member of the Hospital Pharmacists Quality Control Group should be invited and Mr. T. M. French had accepted nomination. The Committee of Management for 1982 would therefore be: Chairman-Mr. C. A. Johnson. Honorary Secretary-Miss I. Ladden. Immediate Past Chairman-Mr. H. E. Brookes. Ordinary Members-Dr. A. H. Andrews, Dr. B. V. Fisher, Mr. T. M. French, Dr. R. G. Hopkins, Dr. A. Sinclair, Mr. A. G. Stewart. Co-opted member-Dr. D. C. Garratt. Members nominated by the Sponsoring Bodies-Mr. A. Wade (Pharmaceutical Society of Great Britain), Mr. P. G. W. Cobb (RSC, Analytical Division) and Professor W. B. Whalley (RSC Professional Affairs Board).

ISSN:0144-557X    

DOI:10.1039/AP9821900161

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

162 Ana I yt ica I ANALYTICAL DIVISION DlSTINGUISHED SERVICE AWARD Anal. Proc. Division Distinguished Service Award As announced in the February issue of AnaZyticaZ Proceedings (p. go), the seventh Analytical Division Distinguished Service Award has been conferred on Dr. H. J. Cluley. H. J. Cluley was born in Wolverhampton and educated a t Wolverhampton Grammar School. In 1941 he gained a University of London honours degree in Chemistry. His first post was in the chemistry laboratory of Chance Brothers Ltd., Smethwick, where he gained analytical and investigatory experience on glasses, re- fractories, glass-making raw materials and fuels. In 1946 he transferred to the electrical industry in which he has remained, joining the Chemistry Group of the then GEC Research Laboratories a t Wembley.His early work included studies on germanium, in the dawn of the semiconductor era, although he later special- ised in radiochemistry and gas chromatography techniques. At the same laboratories, now GEC Hirst Research Centre, he is currently Chief Chemist and Head of the Materials Science Division. He was awarded University of London MSc and PhD degrees in 1950 and 1955, respectively, for studies in analytical chemistry, and has published a number of papers, mostly on analytical topics.April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS IN HPLC 163 Dr. Cluley joined the SAC in 1948 and subse- quently served in various capacities. These included an initial period of membership of Council (1964-5) and a term as Vice-president (1970-l), but then, as later, his greatest involve- ment was with publications.He was a member of The Analyst Editorial Committee (1963-70), and also served as a member of the Analytical Abstracts Editorial Committee (196&70) with a period as Chairman (1967-70). Through the period of successive changes arising from the semi-amalgamation and final amalgamation of SAC with the Chemical Society he had a pro- longed stint as Chairman of The Analyst Publications Committee (1971-8). He has continued to serve the successively enlarged Society in various roles, particularly on the publications side, being Chairman of the Ana- lytical Abstracts Editorial Committee since 1978, and is currently a member of the Publications and Information Board and of the Secondary Services Committee. In various capacities he has been a member of the SAC or Analytical Division Council since 1967. Dr. Cluley’s other activities have included membership of The Chemistry Consultative Committee of Thames Polytechnic, of which he is currently Chairman, and membership of the Chemical Testing Committee of the National Laboratory Accreditation Scheme (NATLAS) , on which he is the RSC representative.

ISSN:0144-557X    

DOI:10.1039/AP9821900162

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 163 Clinical and Biological Developments in High- performance Liq u id C h ro matog ra p hy The following are summaries of eight of the papers presented at a Joint Meeting of the Scottish Region, the Chromatography and Electrophoresis and Joint Pharmaceutical Analysis Groups and the Association of Clinical Biochemists held on February 17-18, 1981, at the University of Edinburgh. Application of High-performance Liquid Chromatography in the Biological Analysis of Drugs. Plenary Lecture Goran Schill Department of Analytical Pharmaceutical Chemistry, Univevsity of Uppsala, S-751 23 Uppsala, Sweden High-performance liquid chromatography (HPLC) is today one of the most commonly used methods in the biomedical field, as it can combine highly selective and sensitive quantitation with technical simplicity. The biological material often has a very low content of drug or metabolite, and the endogen- ous components are usually present in much higher concentrations. The quantification of the drug usually must be preceded by an isolation procedure, and the greater the demands for sensitivity the more efficient isolation procedures must be used.The nature and concentration of the disturbing compounds can vary widely and it is necessary to have access to a variety of procedures with known selectivity for isolation, derivatisation and quantitation. By a suit- able choice of technique it is often possible to avoid multi-stage analytical methods. Quantitation The fluorescence detector has a higher maximum sensitivity, but the fluorescence yield is highly dependent on the structure of the solute and the properties of the solvent.Electrochemical detectors are mainly applied to phenols. The ultraviolet detector dominates in bioanalysis owing to its wide applicability. Liquid - Solid HPLC: Principles Most bioanalytical HPLC separations are carried out in reversed-phase systems with surface- The retention of a solute in this system modified, hydrophobised silica as the packing.164 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC Anal. Proc. depends on its binding to the adsorbing surface, but the adsorbent has a limited binding capacity and other components of the mobile phase will compete for the adsorption sites. When the solute is an uncharged compound, its retention can be regulated by the nature and concentration of an uncharged competing agent in the aqueous mobile phase.A charged solute can be transferred between the phases if it is accompanied by an equivalent amount of a counter ion. When this ion-pair distribution principle is applied to a cation, HA+, in a reversed-phase liquid - solid system, its capacity ratio, k ' H - 4 , increases with increasing hydro- phobicity and concentration of the counter ion, X-, in the mobile phase. The retention of HA+ is counteracted by the adsorption of ion pairs of mobile phase components of the same chaIge (Qf), as summarised in the equation1 where K,,, and K,, are extraction constants of HA+ and Q+ as ion pairs with X- and KO is the binding capacity of the adsorbent, q = Wstat/vmob. Uncharged components in the mobile pkiase may also counteract the retention of HA+.The competing effect can be used for regulation of the retention and for changing the separation selectivity when the sample components are affected differently by the competing agent. Of even greater importance is its use for preventing overloading of the adsorbent, which destroys the separating efficiency of the s y ~ t e m . ~ , ~ Liquid - Solid HPLC with Alkyl-bonded Phases The popularity of these systems in bioanalysis is due to their flexibility and compatibility with aqueous samples. The separation selectivity is good and can be sufficient to allow direct injection of unpurified samples such as urine, if the demand for sensitivity is moderate and the detector has sufficient sensitivity. The main metabolites of acetylsalicylic acid (salicylic, salicyluric and gentisic acids) can thus be quantified in therapeutic concentrations after direct injection of undiluted urine in an ion-pairing system with tetrabutylammonium as counter ion and fluorescence detection.The same detection principle can also be used for determination of quinidine and its main metab- olites after injection of urine in a system with perchlorate as counter When no selective detection is available, disturbances from endogenous con?,pounds can often be avoided by a change in the separation properties of the mobile phase. In a method for the determination of two polar pyridine derivatives, 5-fluoro-3-hydroxymethylpyridinium and 5-fluoro-3-pyridinecarboxylic acid, in plasma, the compounds are isolated from each other and from disturbing sample components by optimising the pH of the mobile phase.5 An ion-pairing reagent is used to prevent disturbances from endogenous compounds in a method for the determination of ampicillin in urine.6 Ampicillin is an amine and can be retained as an ion pair with an alkyl sulphate.Hexyl sulphate gives incomplete isolation, but a change to decyl sulphate increases the retention of ampicillin, while the endogenous compounds are unaffected, with complete separation as a result. A competing ion is used to improve separation in a method for the determination of an amine, zimelidine, and its metabolites in plasma.' The ion-pair chromatographic procedure gives an incomplete separation of one of the amines and an endogenous compound.Addition of a competing ion, dimethyloctylammonium, decreases the retention of the amine whereas the disturbing compound, which obviously is not cationic, is unaffected and a good separation is obtained. The use of an ion-pairing reagent to improve the separation between charged and uncharged compounds is demonstrated in a method for the determination of dopamine and its metabolites in rat brain.8 The compounds are amines and carboxylic acids and contain phenol functions, which make them suitable for amperometric detection. The separation is made with an acidic mobile phase and it is optimised by addition of hexyl sulphate, which increases the retention of the amines whereas the acids are unaffected. The minimum detectable amount of dopamine, DOPAC and homovanillic acid (HVA) is about 0.1 pmol in spite of the fact that the only pre- treatment of the sample is homogenisation with perchloric acid.Determination of catecholamines in plasma can be performed according to the same prin- ciple, but a pre-concentration by adsorption in alumina is usually necessary. This is performed in the presence of glutathione and EDTA to avoid oxidation and is followed by elution of theApril, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 165 catecholamines with perchloric acid. Isolation can be effected with an ion-pair system or on a cationic exchanger, as demonstrated in Fig. l.9 C I I I 1 I Unmodified Silica as Packing Ion-pair chromatographic separations can also be performed with unmodified silica as the stationary phase, in combination with aqueous phases containing the counter ion.Studies on catecholamines and mcre hydrophobic icns have shown that the silica acts as the more hydro- phobic phase.10y1l Unmodified silica has also been used for the isolation of sulphadiazine and its metabolites in plasma and urine with direct injection of the diluted or partly deproteinised biological sample. The solutes are retained in acidic form and separation from disturbing endogenous components is made by optimisation of pH.12 Liquid - Liquid HPLC Liquid - liquid systems often have considerable advantages in bioanalysis owing to the high separation selectivity and capacity of the liquid stationary phase. Problems with long-term stability can be solved by a suitable technique for preparation of the systems.Apomorphine can be isolated from plasma and brain tissue by an ion-pair chromatographic technique with unmodified silica as the packing and perchloric acid in methanol - methylene chloride as the mobile phase.13 The amine is extracted from the biological material as an ion pair with a large arylalkylsulphonate, but separation from this counter ion is easily achieved in the chromatographic system where the amine migrates as an ion pair with perchlorate while the sulphonate migrates in acidic form. Reversed-phase liquid - liquid systems with a strongly hydrogen-bonding agent in the stationary phase are suitable for the isolation of highly hydrophilic compounds from biological material. Tributyl phosphate (TBP) as the stationary phase gives highly selective separations of hydrophilic carboxylic acids, amino acids and dipeptideslP with regulation of the retention by pH of the aqueous mobile phase.The system can be applied to the determination of 5- hydroxyindoleacetic acid in urine. A chromatogram obtained after direct injection of un- purified urine is given in Fig. 2.15 10 5 0 Timelmin Fin. 2. 5-Hvdroxvindoleacetic acid I5-HIAA) in urine. Mobile phase: phosphate b;ffer (pH 5) -methanol (9 + 1) + 0.03% tributyl phos- pha.te. Stationary phase : tributyl phosphate on LiChrosorb RP-18. Detection : fluorescence, 280/370 nm. Sample: 10 pl of urine diluted 1 + 25. Fig. 1. Catecholamines in plasma. Mobile phase: buffer (pH 5) - methanol (9 + 1). Packing : Nucleosil SA 10.Electrode : carbon paste (CP-0), potential 0.6 V. Sample: nor- adrenaline (NA) , 2.1 nmol 1-1 ; adrenaline (A), 0.16 nmol 1-l; dopamine (DA), 0.22 nmoll-l. Internal standard (IS), methyldopamine. Highly selective separations of carboxylic acids and phenols can also be obtained in systems with an aqueous mobile phase and a stationary phase consisting of trioctylphosphine oxide (TOPO) dissolved in decane applied on a hydrophobic solid phase.16 Hydrogen-bon ded complexes are formed with one TOPO molecule bound to each hydrogen-donating group in the acids. A change in the TOPO concentration gives different changes in k’ depending on the166 CLINICAL AND BIOLOGICAL DEVELOPMENTS IN HPLC Anal. Proc. number of hydrogen-donating groups, which gives excellent possibilities for separations, The change in the TOPO concentration in the mobile phase can be made by adding a hydrogen- bonding agent, e.g., a hydrophobic carboxylic acid, to the mobile phase, which decreases the concentration of TOPO available for binding of sample components.Both the TBP and TOPO systems are highly stable and insensitive to limited temperature changes owing to the low solubility of TBP and TOPO in the mobile phase. Influence of Biological Materials on Column Performance Systems with TBP as stationary phase can withstand injections of more than 1 ml of undiluted plasma without a significant change in ~r0perties.l~ A packing of unmodified silica showed a slow decrease in capacity ratio and separation efficiency on repeated injection of partly deproteinised plasma, but it could be kept in good working condition for several months by washing with water and methanol after each working day.12 The use of a short pre- column is often recommended for retaining column performance in automated liquid chroma- tographic procedures.A 5-mm pre-column can withstand 100-300 injections of 200 pl of partly deproteinised plasma samples. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. References Tilly Melin, A., Askemark, Y., Wahlund, K. G., and Schill, G., Anal. Chem., 1979, 51, 976. Tilly Melin, A., Ljungcrantz, M., and Schill, G., J . Chromatogr., 1979, 185, 225. Sokolowski, A., and Wahlund, K. G., J . Chromatogr., 1980, 189, 299. Lagerstrom, P. O., J . Chromatogr., 1981, 225, 476. Henke, B., and Westerlund, D., J .Chromatogr., 1980, 187, 189. Carlqvist, J., and Westerlund, D., J . Chromatogr., 1979, 164, 373. Westerlund, D., and Erixson, E., J . Chromatogr., 1979, 185, 593. Magnusson, O., Nilsson, L. B., and Westerlund, D., J . Chromatogr., 1980, 221, 237. Eriksson, B. M., and Persson, B. A., J . Chromatogr., in the press. Crommen, J., J . Chromatogr., 1979, 186, 705. Crommen, J., Thesis, Universitk de Likge, 1980. Westerlund, D., Wijkstrom, A., and Carlqvist, J., J . Pharm. Sci., in the press. Eriksson, B. M., Persson, B. A,, and Lindberg, M., J . Chromatogr., 1979, 185, 575. Wahlund, K. G., and Edlkn, B., J . Liq. Chromatogr., 1981, 4, 309. Wahlund, K. G., and Edlkn, B., Clin. Chim. A d a , 1981, 110, 71. Stuurman, H. W., Wahlund, K. G., and Schill, G., J . Chromatogr., 1981, 204, 43. Advances in Detector Technology in High-performance Liquid C h r o m a t og r a p h y , PI e n a r y Lect u re John H.Knox Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3 J J Detectors for high-performance liquid chromatrography (HPLC) are, with few exceptions, concentration monitoring devices. Although any statement of the over-all performance of a liquid chromatograph should include a value for the smallest detectable injection of a solute, this quantity can never be a proper measure of the ultimate detection limit of the detector. For concentration monitors this has to be the concentration of solute which, when present with- in the detector, provides a signal equal to the detector noise (this is normally given by four standard deviations of the random short-term variations of the defector signal).This “noise equivalent concentration,’’ Cne, is readily related to the smallest sample that can be detected, Omin, defined as that sample which on elution from the column gives a peak height equal to twice the noise. The appropriate relationship can be stated in several ways, as in equations (1)-(4) - Qmjn = (base peak volume) x Cne . . .. - - (1) = 4 V m ( 1 + K’) x C n e / d N . . .. . . . * (2) .. . . .. ‘ - (3)April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 167 These equations show clearly that the minimum detectable sample depends on the column void volume, Vm, the plate count, N , and the column capacity ratio, k’, as well as on Cne. It is therefore not generally possible to deduce Cne from Qmin unless considerable additional infor- mation about performance of the column is given, or one can deduce the “base peak volume” from a chromatogram. Accordingly, it cannot be emphasised too strongly that, when describing new detector developments, authors must state the noise equivalent concentration, not simply the minimum detect able samp1e.l In the field of photochemical devices, an exciting development is that of the sheathed-flow micro-laser - fluorimeter of Herschberger et a1.2 By sheathing the eluate flow with an auxiliary flow of eluent they achieved an exceedingly small detection volume of a few nano1itres;jthe otherwise adverse effect on signal was compensated by using an intense laser source for illumination.The device thereby provided a noise equivalent concentration of around 10-lo g ml-l for strongly absorb- ing and fluorescing compounds. Another group of developments, which will undoubtedly be extended, is that of the diode array spectrophotometric detector^.^-^ At present such devices allow rapid scanning of the ultraviolet absorbance of the eluate and with suitable computer interfacing a three-dimensional output of detector signal against wavelength and time. This can be sectioned either in the time direction at any wavelength or in the wavelength direction at any time. Current devices are more noisy than single-wavelength photometers, giving a noise equivalent concentration of around 10-8 g ml-l for solutes with a molar absorptivity of lo4 1 mol-l cm-l.New forms of solid electrodes for amperometric detectors make one wonder whether the widely used carbon paste and glassy carbon electrodes are indeed the only usable forms of carbon for this application. Thus Wightman et aL6 described the use of basal plane graphite as an electrode. With suitable electrochemical conditioning this material gave a noise level about five times lower than carbon paste, corresponding to a noise equivalent concentration of 2 x 10-12 g ml-l. Perhaps the most exciting area in current detector research is HPLC - MS interfacing. Several ingenious devices have been developed with the object of coupling the HPLC column directly to the ion source of the mass spectrometer without the intermediate evaporation step involving a travelling wires or belt.s~10 Fig.1 shows outline diagrams of those devices for which the authors gave sufficient detail for Cn, to be calculated. The figure shows that single ion monitoring (SIM) provides about 100 times lower detection limits than does total ion monitor- ing (TIM), and that the simplest devices using either a constricted capillaryl1,l2 or a pinhole 0 r i f i c e ~ ~ 9 ~ ~ give as low or lower values of Cne than the more complex interfaces, sulch as the heated block orifice of Blackley et aZ.15 or the ingenious micro-vacuum nebuliser of Tsuge et aZ.16 A problem that inevitably arises with HPLC - MS interfacing is that the optimum flow-rate of liquid into the mass spectrometer is in the range 3-3Op1 min-l, whereas the standard HPLC flow-rate is around 1000 p1 min-l.The desirability of providing eluate at the lower flow-rates for mass spectrometers has considerably stimulated the development of what are widely miscalled “microbore columns.” These are simply packed columns of bore around 1 mm. Typically they are 100-500 mm long and are packed with conventional packing materi- als 5-20 pm in diameter. In theory they should provide the same plate efficiency and elution times as conventional 5 mm bore columns packed with the same materials: no special advan- tages in performance are expected from the narrower bore other than a reduction in volume flow-rate. In addition to their suitability for use with mass spectrometers they will prove attractive where expensive eluents or packing materials are required, and where the sample availability is extremely limited, as in some clinical and biochemical applications.The major problems still to be solved with regard to narrow-bore packed columns concern methods of packing, sample injection and sample detection. These will almost certainly be solved within the next year. Although it has proved possible to pack 10- and 20-pm particles into the narrower tubes to give reduced plate heights of 2, this goal has still to be achieved for 5-pm particles. Injection and detection are beset by the very serious extra-column dispersion caused by currently available devices. In general, the dispersion, oV, which is contributed by any part Several new detection systems have recently been described in the literature. Pyrolytic graphite has also been used successfully by Helper et a,?.’168 Restricted ca pi I lary CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 75pm 10pm Flow-rate/ Reference pI min-' McLaff erty Henion12 5-20 et a/." Anal.Proc. 10-7 TIM 10-9 SIM Orifice plate 5-15 pm 3-30 Arpino et TIM Kenyon et SIM Vapor- iser & skimmer 0dH2 75 pm Skimmer / \ 1 000 B I ackl ey et a/.15 SIM Vacuum He \-&A 2-20+ nebuliser '/V"c\ 50 helium ml min-' Tsuge et a/.'6 SIM 130pm 150pm wire tube Skimmer Fig. 1. Outline details of direct HPLC - MS inlet systems. of a chromatographic system is obtained from equation ( 5 ) , which relates the total variance (or second moment) of a detected band to the individual variances produced by the independent components : As a general rule, if undesirable peak broadening is to be avoided, the dispersion contributed by the injector and detector combined should not exceed about 50% of the dispersion produced by the column for an unretained solute, oVo.l The severity of this condition is shown by Table I, which gives oVo values for optimum operation (reduced plate height = 2) of packed columns of various diameters and plate efficiencies (for method of calculation, see ref.17). The data in Table I should be considered in relation to those in Table I1 which compare the dispersions obtained with three commercial photometers containing 8-pl flow cells (data obtained by Reese and Scott1*); Table I1 also gives the dispersion obtained by Knox and co-workers1 using an 8-pl flow cell, very short narrow-bore connecting tubing and a syringe injection head. Under the very best conditions (svo for an 8-pl flow cell can be reduced to about 150y0 of the cell TABLE I ov(total)2 = ov(colurnn)2 -k ov(detector)2 ov(injector)2 + .- . .. - (5) DISPERSION OF UNRETAINED SOLUTE BANDS BY PACKED COLUMNS Assumptions: h = 2, AP = 200 bar, eluent = water, flow resistance parameter 4 = 500. uvo/mm3 for column bore - Column N t,/s dp/pm L/mm 5mm 1 mm 50dp Good conventional column . . .. 10000 100 5 100 15 0.6 0.01 Column of optimum particle diameter 90 2.7 170 15 0.6 0.01 10000 10 1.6 30 5 0.2 0.001 100000 1000 5 1000 50 2.0 0.1April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 169 volume, very far from the theoretical value of (cell volume)/dl2 derived for plug flow. With bad design of the flow cell and the connecting tubing the value of avo can be up to four times larger.Evidently, for the full performance of even a good standard-bore column (100 mm long, 5 mm bore, 10000 plates, giving oVo = 15 pl) a flow cell of not more than about 4 p1 volume is required. However, for 1 mm bore packed columns, we require detector cells giving dispersions in the range 0.1-1 pl, approximately 100 times lower than those currently available. If we were to move towards columns with bores of 50 particle diameters in order to achieve geometries closer to those of GC columns, the requirements would be still more extreme. TABLE I1 DISPERSION BY COMMERCIAL PHOTOMETERS avo values in microlitres. Flow-ratelm1 min-l r Photometer 0.1 0.5 1.0 Reference Model A . . .. 16 40 Model B .. . .20 30 :: } Reese and Scottls Model C . . .. . . 14 20 22 Home assembled . . - - 12 Knox and co-workers1 The detectors that seem to have the greatest potential for miniaturisation would seem to be electrochemical detectors (see, for example, the rapid dropping electrode system of Hanekamp et aZ.19), microfluorimetric devices (e.g., ref. 2) and HPLC - MS devices. Low-volume ultra- violet photometers have also been developed 2 o y 2 1 but inevitably the path length is reduced with a consequent decrease in sensitivity. The goal for manufacturers of detectors must now be to provide devices whose sensitivity is not significantly impaired but which have flow-cell volumes in the range 0.1-1 pl and give peak dispersions below 1 pl. Glossary of Terms Noise equivalent concentration.Mean particle diameter. Volume flow-rate. Reduced plate height = absolute plate height/&. Column capacity ratio. Column length. Number of theoretical plates to which column is equivalent. Pressure drop across column. Quantity of injected solute which gives peak height in detector equal to twice noise. Retention time of unretained fully permeating solute. Retention time of solute with capacity ratio k’. Void volume of column. Standard deviation of band measured in volume units. Standard deviation of infinitesimal solute sample peak eluted through column alone. Column flow resistance parameter. . References 1. 2. 3. 4. Knox, J . H., Editor, “High Performance Liquid Chromatography,” Edinburgh University Press, Hershberger, L. W., Callis, J. B., and Christian, G.D., Anal. Chem., 1979, 51, 1444. Milano, M. J., Lam, S., Savonis, M., Paulter, D. B., Par, J. W., and Grushka, E., J . Chromatogr., Pardue, H. L., Anal. Chem., 1977, 49, 1171. Edinburgh, 1980. 1978, 149, 599.170 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC Nunn, W. G., Dessy, R. E., and Reynolds, W. R., A C S Symp. Ser., 1979, No. 102, 135. Wightman, R. M., Park, E. C., Borman, S., and Dayton, M. A., AnaZ. Chem., 1978, 50, 1410. Helper, B. R., Weber, S. G., and Purdy, W. C., AnaZ. Chim. A d a , 1978, 102, 41. Scott, R. P. W., Scott, C. G., Munroe, M., and Hess, J., J . Chromatogr., 1974, 99, 395. McFadden, W. M., Schwartz, H. L., and Evans, S., J . Chromatogr., 1976, 122, 389. Vacuum Generators Ltd., Technical Bulletin, 1979.McLafferty, F. W., Knutti, R., Venkataraghavan, R., Arpino, P. J., and Dawkins, B. G., Anal. Henion, J. D., Anal. Chew.., 1978, 50, 1687. Arpino, P. J., Kreis, P., Vajta, S., and Devant, G., J . Chromatogr., 1981, 203, 117. Kenyon, C. N., Melera, A., and Erni, F., J . Chromatogr. Sci., 1980, 18, 103. Blackley, C. R., Carmody, J. J., and Vestal, M. L., Anal. Chem., 1980, 52, 1636. Tsuge, S., Hirata, Y . , and Takeuchi, T., Anal. Chem., 1979, 51, 166. Knox, J. H., J . Chvomatogr. Sci., 1980, 18, 453. Reese, C. E., and Scott, R. P. W., J . Chromatogr. Sci., 1980, 18, 479. Hanekamp, H. R., Voogt, W. H., Bos, P., and Frei, R. W., Anal. Lett., 1979, 12(A2), 175. Scott, R. P. W., Kucera, P., and Munroe, M., J . Chromatogr., 1979, 186, 475.Ishii, D., Asai, K., Hibi, K., Jonokuchi, T., and Nagaya, M., J . Chromatogr., 1977, 144, 157. AnaL. Proc. Chem., 1975, 47, 1503. Problems of Sample Preparation for High-performance Liquid Chromatography Richard Gill and Anthony C. Moffat Home Ofice Centval Research Establishment, A Idevmaston, Reading, Berkshire, RG7 4PN The analysis of a drug in a biological fluid by high-performance liquid chromatography (HPLC) can often be troubled by interferences from endogenous compounds and several different approaches can be applied to tackle the problem. The solution may involve the use of a more selective detector, a change of the HPLC eluent or column packing material, or even a chemical derivatisation in order to enhance the relative detector response of the drug.An alternative approach involves the modification of the sample preparation procedure such that the interfering compounds are eliminated before chromatography. This aspect of HPLC analysis does not always receive sufficient attention and this paper outlines the methods avail- able for sample preparation that are applicable to the analysis of drugs in biological materials. Various problems arising from the management of the sample from collection through to injection will be considered, with reference to their significance in HPLC analysis. Sampling The importance of sampling is well illustrated by various problems associated with the analysis of blood. Firstly, a choice has to be made as to whether whole blood, plasma or serum should be analysed. At present, clinical and pharmacokinetic laboratories favour plasma ; however, the forensic scientist can be faced with no choice, as specimens are frequently haemolysed on arrival at the laboratory.Most drugs are concentrated in the plasma yet there are a significant number that are concentrated in the erythrocytes, e.g., the concentration of chlorthalidone in erythrocytes is 50-100 times that in plasma.1 Assays for such drugs will show enormous differences depending on whether whole blood or plasma is chosen for analysis. Further, haemolysis, even when undetectable to the eye, can release these drugs from the erythrocytes and cause significant changes in plasma concentrations.2 Even those drugs which are known to be concentrated in the plasma can present analytical problems for the unwary.The distribution of the drug in the plasma can involve many com- plex equilibria with binding to several types of protein (e.g., albumin, a-acid glycoprotein, lipoprotein). Any factor that disturbs these equilibria may lead to transfer of drug between plasma and red cells. Temperature can have a marked effect and hence the separation of plasma must be conducted at a controlled temperature. For example, plasma phenytoin levels increase by 10% when a sample is equilibrated and centrifuged at 4 "C rather than 24 oC.2 Contaminants can also influence the equilibria and recent studies have shown that a plasticiser present in the stoppers of some blood This is clearly significant when plasma is analysed. Crown Copyright.April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 171 collection containers can cause a decrease in the plasma concentration of several basic drugs as a result of displacing the drugs from a-acid glyc~protein.~,~ Hence the analyst must be aware of the pitfalls associated with the fractionation of blood before he proceeds with the analysis.Blood must be viewed not as a homogeneous entity, but more as a series of compartments. From an analytical viewpoint the analysis of whole blood is to be recommended as it avoids many of the problems; however, few data on drug levels in whole blood have been published, making the interpretation of such results very difficult at present. Many types of contaminants can be encountered in samples (e.g., antioxidants, pesticides, food additives, plastics additives, vulcanising agents) and such problems can be minimised only by careful manipulation of the sample.The need for close liaison between the analyst and the person taking the sample is also most important. A typical problem is the contamination of a blood sample by a local anaesthetic used whilst a catheter is inserted. During work on the metabolism of LSD in rhesus monkeys4 a metabolite of ketamine was encountered that was later discovered to have arisen from the sedation of the monkey with this drug at the animal house prior to the administration of the LSD. Storage Considerable time may elapse between the collection of a sample and the analysis, and changes occurring during the storage can be most important. A common source of error is the adsorption of drug by the storage container.Further, some drugs may undergo decomposition; temperature, pH, salt concentration and light can be involved. However, steps can sometimes be taken to prevent such decomposition. Succinylcholine is rapidly hydrolysed under alkaline conditions and hence it is always advisable to acidify a urine sample before storage when analy- sis of this drug is required.5 Metabisulphite is sometimes added as a preservative to prevent the oxidation or photo-oxidation of drugs during storage (e.g., the photodecomposition of LSD),6 yet has the drawback that N-oxides also present may be chemically r e d ~ c e d . ~ Changes to the biological material can also occur as a result of putrefaction duiing storage, with the appearance of endogenous compounds that can interfere with the analysis.Tyramine is often a particular problem with decaying liver.8 Drugs themselves can also be subject to changes and the reduction of the nitro group in nitrazepam to an amino group by bacteria has been ob~erved.~ Protein Treatment Putrefaction can, however, have some beneficial effects in the preparation of a sample for chromatography; the auto-digestion of protein tends to release the drugs in a form suitable for extraction. Usually, the production of a protein-free solution from a biological fluid or tissue is the first step in the preparation of a sample for HPLC. Such treatment not only releases bound drug but, when no extraction is performed, can protect the HPLC column from irrevers- ible contamination. Classical protein treatments in toxicology have been of two types : acid digestionlo+ and protein pre~ipitation.1~9~~ However, no single procedure works well with all types of drug.The protein precipitation methods give poor recoveries with drugs that are strongly bound to protein, whereas the acid digest methods often lead to decomposition of the drug. The search for a more general procedure has led to the use of the proteolytic enzyme subtilisin Carlsberg with which to degrade t i s s u e ~ . ~ ~ - ~ ~ The method is very simple and involves blending the tissue (e.g., liver) with Tris buffer a t pH 10.5 and adding the enzyme. Incubation at 60 "C for 1 h is followed by filtration through glass-wool to give a clear filtrate ready for extraction. Results for liver analyses have shown that this method gives good recoveries for most classes of drugs and does not lead to problems when used in the preparation of samples for HPLC.Further, the method has now been used for blood analysis17 and im- proved recoveries are indicated for all drugs, particularly those which show strong protein binding (e.g., phenylbutazone, phenytoin). The major problems with the enzyme method have arisen with those drugs which are sensitive to hydrolysis at high pH. Nevertheless, recent work has shown that the enzymic digestions proceed smoothly under less alkaline conditions (pH 7.5) with considerable improvement in the recovery of these drugs.17 Other procedures have been used for the removal of proteins from biological fluids for HPLC analysis. One of the simplest methods is the addition of a miscible organic solvent, such asCLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC Anal.PYOC. 172 methanol or acetonitrile, which precipitates the proteins, so allowing their removal by centri- fugation. Ultrafiltration of biological fluids through porous membranes has also been used. Both methods are unsuitable for drugs that are bound strongly to proteins and further problems can arise with the latter method as a result of drug adsorption on the ultrafiltration membrane. Protein-free or de-proteinised biological fluids can be introduced directly on to an HPLC column but this usually requires that the drug is either present in high concentration (typically micrograms per millilitre levels) or that a very selective detection system is used.Direct sample introduction can rapidly destroy the performance of an HPLC column (especially if the samples are not fully de-proteinised) and pre-columns are to be recommended when such methods are used to eliminate the need for frequent column repacking. Drug Conjugates HPLC, unlike gas - liquid chromatography, offers the possibility of handling intact drug conjugates without derivatisation. Paracetamol is a good example where the conjugates can be detected after therapeutic doses of the drug by direct injection of urine with ultraviolet detection.l* Generally, however, low concentrations require the use of more selective detec- tors. Often there is little possibility of extracting the conjugates from the sample before chroma- tography owing to their high polarity.In such instances, immunoassay techniques can serve as particularly useful ultra-selective detectors ; the HPLC eluate is collected in small fractions and each fraction subjected to the immunoassay. Such methods have been applied to conjugates arising from opiateslg and cannabisz0 If such selective detectors are not available it may be desirable to hydrolyse the conjugates, hence releasing the drug for extraction. The mild conditions offered by enzymatic methods appear to offer the best approach, yet sulphatase is not able to hydrolyse all ethereal sulphates but only aryl sulphates. Further, glucuronidase can be inhibited by high salt concentrations ; hence, it is always advisable to dilute samples and run control experiments (e.g., with phenol- phthalein glucuronide) to ensure that the enzyme is active. Conjugates can also be hydro- lysed by chemical methods (i.e., strong acids and alkalis) but care must be taken as the severe conditions can lead to the chemical modification of some drugs (e.g., transesterification under alcoholic conditions) .20 Extraction When drug concentrations are low and selective HPLC detectors are not available, extraction procedures are required to isolate the drug or metabolite from the endogenous material to which the detector also responds.Generally, such procedures can be classified as either liquid - liquid or liquid - solid extractions. Liquid - Liquid Extraction Solvent extraction is still the most widely used technique for sample clean-up before HPLC owing to its wide versatility.Polarity is usually the most important factor in the choice of the extraction solvent and generally as this increases the range of compounds extracted also in- creases. The solvent should be selected with minimum polarity consistent with high recovery of the drug. Thus, hexane is a good solvent for lipids and some bases but will not extract barbiturates. Diethyl ether will extract a wide range of drugs but not some alkaloids, whereas chloroform can be used for most alkaloids but not amphoteric alkaloids such as morphine where a mixture of chloroform and isopropanol is required. Other factors can influence the choice of solvent depending on the circumstances of the extraction (e.g., boiling-point, purity, flammability, toxicity, cost). I t is clearly important that the chosen solvent and the biological fluid should be easy to separate after mixing.Polar solvents that are miscible with water (e.g., n-propanol) can be forced to form a separate layer by saturating the aqueous phase with an inorganic salt. Salts can be important in other ways, as they help to form better phase boundaries (Le., avoid emulsions) and can also reduce the water content of the organic phase. Further, the addition of salts can be used to increase the selectivity of an extraction by aiding the transfer of a drug from the aqueous to the organic phase. Barbiturates can be completely extracted from acidi-April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 173 fied urine with diethyl ether, leaving the hydroxylated metabolites which can later be extracted with diethyl ether after saturating the urine with sodium chloride.Modification to the pH of the aqueous phase is also most important in the development of an extraction scheme. It is the uncharged form of the drug that is soluble in the organic solvent. Hence, acidic drugs are extracted under acidic conditions, basic drugs under alkaline conditions, neutral drugs are independent of pH and amphoteric drugs show an optimum pH for extraction. A further useful modification involves the addition of specific ions, enabling ionic drugs of the opposite charge to transfer from the aqueous to the organic phase as neutral ion pairs. Quatern- ary ammonium drugs can only be extracted by this method (e.g., extraction of tubocurarine with iodide).21 Successful application of solvent extraction depends on careful optimisation of conditions with modifications to both aqueous and organic phases.Fig. 1 shows HPLC traces for the analysis of baIbiturates after extraction from whole blood with a mixture of hexane and diethyl ether (1 + 1) at pH 7.5. Barbiturates are extracted with recoveries greater than 94% while allowing detection down to therapeutic levels. The use of a more polar extraction sol- vent or a lower extraction pH increases the background absorbance considerably.22 0 Timeimin Fig. 1. Analysis of whole blood by HPLC22: ( a ) , drug-free blood spiked with internal standard (T = talbutal, 5 pg ml-l) ; ( b ) , sample from a volunteer about 2 h after an oral dose of pentobarbitone (182 mg). Column : Hypersil- ODS (10 cm).Eluent: 40% methanol, pH 8.5. Flow-rate: 2 ml min-l, Liquid - Solid Extraction Liquid - solid extraction methods offer one important advantage over solvent extraction by avoiding the problems associated with the phase separation of two liquids. One approach is to convert the biological fluid into a solid residue (i.e., freeze-drying) followed by solvent extrac- tion. Very little work of this type has been conducted in drug analysis23 yet the method could certainly have potential for water-soluble drug conjugates. The more general approach to liquid - solid extraction is the adsorption of the drug from the aqueous fluid on to a solid adsorbent. A vast range of materials have been used, including silica, alumina, Florisil, octadecylsilica, charcoal, ion-exchange resins, ion-exchange paper, XAD resin and Celite.Simple procedures involve the addition of the solid t o the. fluid, agitation and separation by centrifugation (e.g., the extraction of anticonvulsants from serum with charcoal).24 XAD resins have also been used in porous nylon bags, hence avoiding the centrifugation step.25 However, the most satisfactory approach is probably passing the fluid over the adsorbent packed in a short column. Extractions of this type are often conducted with home-made columns ; however, several commercial disposable pre-packed columns con- taining a wide range of adsorbents are now available. Selectivities of liquid - solid extractions can be controlled by similar approaches to those used in solvent extraction.Anal.PYOC. 174 CLINICAL AND BIOLOGICAL DEVELOPMENTS IN HPLC Future Developments The future will no doubt see further moves towards the automation of sample preparation procedures. From a technical viewpoint the automation of column extraction appears to be the most attractive approach. However, liquid - solid extractions are not always able to match the selectivities that can be obtained with solvent extractions and, consequently, the automation of solvent extraction has not been totally ignored.26 Future research will probably lead to methods of increasing selectivity in liquid - solid extraction by the use of new types of adsorbent and by surface modifications to present adsorbents. Nevertheless, much work has already been directed towards the automation of column extraction methods.A commercial instrument for off-line batch extraction of samples has recently been intr~duced.~’ Further, several groups have applied column switching techniques for on-line handling of complex biological samples in HPLC28-30; essentially, one HPLC column acts as the extraction column, performing a partial fractionation of the sample, and then the appropriate fraction is switched to a second column to complete the separation. 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. 30. References Osselton, M. D., Hammond, M. D., and Moffat, A. C., J . Forensic Sci. Soc., 1980, 20, 187. Borg%, O., Petters, I., and Dahlqvist, R., Acta Pharm. Suecica, 1978, 15, 459. Borg%, O., Piafsky, K.M., and Nilsen, 0. G., Clin. Pharmacol. Ther., 1977, 22, 539. Sullivan, A. T., Twitchett, P. J., Fletcher, S. M., and Moffat, A. C., J . Forensic Sci. SOC., 1978, 18, Curry, A. S., “Poison Detection in Human Organs,” Third Edition, Thomas, Illinois, 1976, p. 304. Hellberg, H., Acta Chem. Scand., 1957, 11, 219. Whelpton, R., Acta Pharm. Suecica, 1978, 15, 458. Stevens, H. M., and Evans, P. D., Acta Pharmacol. Toxicol., 1973, 32, 525. Stevens, H. M., unpublished work. Dubost, P., and Pascal, S., Ann. Pharm. Fr., 1953, 11, 615; 1955, 13, 56. Stevens, H. M., J . Forensic Sci. Soc., 1967, 7, 184. Valov, P., Ind. Eng. Chem., Anal. Ed., 1946, 18, 456. Nickolls, L. C., “The Scientific Investigation of Crime,” Butterworths, London, 1956, p. 348. Osselton, M. D., Hammond, M.D., and Twitchett, P. J., J . Pharm. Pharmacol., 1977, 29, 460. Osselton, M. D., J . Forensic Sci. Soc., 1977, 17, 189. Osselton, M. D., Shaw, I. C., and Stevens, H. M., Analyst, 1978, 103, 1160. Hammond, M. D., Osselton, M. D., and Moffat, A. C., unpublished work. Knox, J . H., and Jurand, J., J . Chromatogr., 1978, 149, 297. Nelsen, P. E., Fletcher, S. M., and Moffat, A. C., J . Forensic Sci. SOC., 1980, 21, 195. Williams, P. L., Moffat, A. C., and King, L. J., J . Chromatogr., 1979, 186, 595. Stevens, H. M., and Fox, R. H., J . Forensic Sci. Soc., 1971, 11, 177. Gill, R., Lopes, A. A. T., and Moffat, A. C., unpublished work. Broich, J . R., Hoffman, D. B., Goldner, S. J., Andryauskas, S., and Umberger, C . J., J . Chromatogr., Adams, R. F., and Vandemark, F.L., Clin. Chem., 1976, 22, 25. Bogusz, M., Gierz, J., and Bialka, J., Forensic Sci. Int., 1978, 12, 73. MacDonald, J. C., Pharm. Technol., 1978, 2, 1. Williams, R. C., and Viola, J . L., J . Chromatogr., 1979, 185, 505. Dolphin, R. J., Willmott, F. W., Mills, A. D., and Hoogeveen, L. P. J., J . Chromatogr., 1976, 122, Lankelma, J., and Poppe, H., J . Chromatogr., 1978, 149, 587. Gfeller, J. C., and Stockmeyer, M., J . Chromatogr., 1980, 198, 162. 89. 1971, 63, 309. 259. liquid Chromatography in Drug Pre-formulation and Formulation Science H. H. van Rooij and E. Tomlinson” Subfaculty of Pharmacy, University of Amsterdam, Plantage Muidergracht 24, 1018 T V Amsterdam, The Netherlands Takeru Higuchi has often remarked that a cornerstone of excellent pharmaceutical drug formulation is the excellence of those analytical systems available to advance, refine and check the quality of that science.* To whom correspondence should be addressed.April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 175 This paper is concerned with examining the role that high-performance liquid chromato- graphy (HPLC) has, or could have, in helping to develop drug formulation science. We begin by distinguishing between the drug ( i e . , the active component) and the drug delivery system (i.e., the dose form in which the potent component is given). Table I indicates the various types of delivery systems in modern use. (Although the biological component, i.e., the patient, is often included in the definition of drug delivery systems, the area of bioanalysis lies largely outside the scope of this work.) Further distinction needs to be made between pre-formula- tion and formulation.The latter refers here to the manufacturing process per se, where assay purity profiles and product stability information are necessary; the former is that part of the drug development programme concerned with optimising and characterising the release of drug from an appropriate delivery system. Fig. 1 suggests how these differing areas of formulation are linked to the analytical (and in particular the HPLC) establishment. Pharmaceutical decision cell I I I I Analytical decision cell 4 Formulation 1 Pre-formulation Assay Quality control Purity profile Bioavailability Stability Sol u bi I ity Hydrophobicity HPLC, analytical LIJ 1 - H PLC, ph ysico-chemica I Fig.1. Inter-relationships between drug formulation and its analytical aspect. Certainly, use of HPLC in the analysis of drugs in dose forms is common in modern pharma- ceutical development ,l with its popularity still rising.2 However, analytical problems in this area are often being solved using over-sophisticated and complicated apparatus. Such redundancy results from the fact that what may be needed for one branch of pharmaceutical analysis (e.g., bioanalysis), may not be required for drug assay/qua.lity control of formulations. We contend that the physico-chemical structure of most drugs and the nature(s) of drug deliv- ery system(s) make the use of simple HPLC separation systems in drug pre-formulation and formulation studies eminently suit able.i. Cost ii. Precisionldetection limit iii. Accuracy/separation Fig. 1 iv. Analysis time v. Compatibility with Type of delivery system Natureiconcentration of I drug I phase system vi. Sampling procedure Fig. 2. Analytical considerations in determining the HPLC mode in pre-formulation and formulaticn. In determining the separation approach to be taken, the object of the test (Fig. 1) has first to be considered in relation to the nature of the drug and the type of dose form (Table I), as illustrated by Fig. 2. Each analytical point listed in Fig. 2 is now considered.176 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC Anal. Proc. This would appear to be insignificant in relation to total budgets. (i) Cost. (ii) Precision and detection limit and (vi) sampling.Precision in the analysis of drug dose form constituents is of prime relevance in the formulation (i.e., manufacturing) stage, and is given by 02total = 02manufacturing + 02samp1ing + (~'mw - .. - * (1) process process method where o is the standard error. Considering the variabilities encountered in the rLanufacturing process, we see that a simple sampling procedure coupled with reliable HPLC (retention times > 1% and assay +0.5% coefficient of variation) will give most information on the manufactur- ing process. As pointed out by bas to^,^ at the manufacturing stage it is essential (Fig. 1) to have a stability-indicating (HPLC) assay, as there are many types of processing that can result in decomposition of components, the major ones being granulation of tablets, heating to effect solution and terminal sterilisation by heating.A recent example of the usefulness of HPLC has been given by Walter~,~ who presented a simple separation method for the anorectic agent diethylpropion hydrochloride and its hydrolysis product l-phenylpropane-l,2-dione for use in stability studies on several manufactured tablets of the drug. TABLE I DRUG DELIVERY SYSTEMS Type Examples Solid . . . . . . Powders; capsules; tablets Semi-solid . . . . Suppositories ; pessaries ; suspensions; creams and ointments (oily Liquid . . . , Solutions ; syrups ; emulsions (water-in-oil, oil-in-water, multiple) ; Novel . . . . Devices/polymeric and aqueous) injections (oily and aqueous) An essential consideration in the precision of the HPLC assay is that most dose forms today contain between 5 and 500 mg of drug, which, for tablets, often corresponds to ratios of drug to excipients of 0.05: 1 (or 104-106 p.p.m.) (cf, bioanalysis, about 10-100 p.p.m.).Further, if we consider the physico-chemical nature of drug formulation excipients (Table 11), which are present as dispersing agents, disintegrants, lubricating agents, colours, taste maskers, solu- bilkers, buffers, etc., these ratios are often more favourable, as most of the adjuvants have very different physico-chemical characteristics than the drug, which is reflected in a more faveur- able signal to noise ratio for drug detection, and hence a more precise result. This advantage- ous signal to noise ratio means that larger volumes of solvent-extraction fluids can be used without a decrease in precision in the final detection step, which makes for easier sampling (Fig.2). I TABLE I1 COMMON FORMULATION EXCIPIENTS Cellulose Talc NaCl Pectin Silica NaH,PO, Saccharin Mannitol Carbowax Lactose Magnesium oxide Ionic and non-ionic surfactants Glycerin NaHCO, (v) Compatibility and (iv) analysis time. It was estimated5 some years ago that more than 80% of HPLC analyses were being performed using the reversed-phase HPLSC mode; undoubtedly this is due to the ready manipulation of the solvophobic effect6 and the incorporation of such exciting and flexible techniques as ion-pair chr~matography.~ However, if one examines the nature of drugs and the complex physical milieu in which they occur (Table I), it has to be concluded that reversed-phase HPLC is not the preferred separation mode for all drugs in theirApril, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 177 dose form.If we are concerned with fast analysis (Fig. 2 ) , the immediate drug extraction fluid should be compatible with the HPLC mobile phase (and hence with the total phase system). We suggest that for quality control/purity profiles and stability-indicating assays that, as many modern drugs are lipophilic (and often of extremely low aqueous solubility), straight-phase HPLC is the mode of choice. (It must be re-emphasised that this argument is being expanded for the analysis of drugs in the drug delivery system because of the already discussed character of these drugs.) This approach is exemplified by the HPLC determination of the chemical prodrug bezitramide and its hydrolysis product in tablets.* This is a highly lipophilic drug (oil - water liquid - liquid distribution coefficient >5 log units), which is almost insoluble in aqueous solutions.Simple extraction with chloroform of the drug from finely ground tablets, followed by either direct injection or rapid evaporation and then dissolution in the mobile phase, followed by injection into a straight-phase system (Fig. 3), results in a fast, accurate and stability-indicating analysis. Additionally, two objectives of quality control, viz., purity and assay of the main constituent, have been combined within one pro- cedure. A further example could be the analysis of triamcinolone acetonide cream, which could be analysed according to the US Pharmacopeia XIX using a straight-phase HPLC system, combined with a simple sampling procedure, making this a fast and reliable method.Drugs are generally formulated (Table I) as solid, semi-solid and oily preparations because of their low aqueous solubility, and it would thus appear that this suggestion of employing the more reliable straight-phase methodologies will suffice for most drug products. For aqueous preparations, however, fast analyses can be achieved by direct injection into a reversed-phase system. For example, Fig. 4 shows the reversed-phase HPLSC analysis of the drug fentanyl (a morphinomimetic) in an aqueous injection fluid containing stabilisers. Interestingly, use has been made here of the knowledge that fentanyl has a maximum absorbance at 210 nm, which argues for the use of variable-wavelength detectors.Significantly, making use of the differing spectral characteristics of drug delivery system components has been reported9 recently for the simultaneous reversed-phase HPLC determination of aspirin and salicylic acid in plain, buffered and enteric-coated tablets, in which ultraviolet (aspirin) and fluorescence (salicylic acid) linked detectors were employed. 1 Inj. I 0.01 6 A.U. I - I II L I $ 1 I 0 60 90 120 150 180 Time'k Fig. 3. Straight-phase HPLC of a non- aqueous extract of a tablet containing 5mg of bezitramide (I) a t low detector sensitivity (left), and a t high sensitivity (right), with which the hydrolysis product of the drug (11) is readily detected.Stationary phase, silica gel Si 60; mobile phase dichloromethane - hexane ( 1 + 20). Fig. 4. Reversed-phase HPLC of aqueous injections of fentanyl (I), showing the drug together with some formulation additives : citric acid (II), methyl P-oxybenzoate (111) and propyl 9-oxybenzoate (IV) . Stationary phase, octylsilica; eluent, sodium dodecyl- sulphate/perchloric acid - methanol (3 + 1).178 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC Pre-Formulation In Vitro Bioavailability Testing Highly lipophilic drugs Tablets Capsules Oily preparations Anal. Proc. Hydrophilic drugs Formulation Syrups adjuvant Aqueous Table I I ions - Solutions injections Although drug delivery systems may have the correct content of drug, this dose may or may not be totally biologically available to the patient.To simulate and examine drug release in vitro, bioavailability testing is a necessary part of pre-formulation studies. Here the drug is released from its dose form into a simulated (aqueous) gastric/intestinal fluid (at various pHs). Hence, for fast analyses irrespective of the nature of the drug, either main separation mode can be used. That is, either the simulated fluid is injected (via an automated sampling device) into a reversed-phase system, or, following a one-step extraction (off- or on-line), into a s t raight-phase system. Solubility and Hydrophobicity It is common to include in pre-formulation (and indeed drug design) studies determinations on the solubility and hydrophobicity of any potential drug candidate (Fig. 1).We have shown that reversed-phase HPLSC is eminently suitable for assessing both of these parameters because, in the case of drug hydrophobicity,lO the retention process is due to the solvophobic effect ,6 and, for solubility, liquid chromatograhic capacity ratios, being indicators of activities in aqueous environments,ll can be used in the Yalkowsky - Valvani equation to predict readily aqueous solubilities. Formulation Adjuvants Final mention should be made of the analysis of the other constituents of the delivery system, i.e., the adjuvants (Table 11). Although often given scant (analytical) attention, it is sug- gested that as many of these are inorganic and small organic ions the use of ion chromatography12 (ion exchange using conductimetric detection) can be of use, as this method readily selectively resolves and detects these ions in solution.Recommendations In addition, we may add that this scheme should be followed using minimal sample manipulation [equation (l)], simple, reliable HPLC separations and with selective detection systems. Fig. 5 outlines the above arguments in analysing drugs in their formulations. Fig. 5 . Recommendations. References 1. 2. 3. 4. Bailey, F., J . Chromatugr., 1976, 122, 73. Fell, A. F., Br. J . Pharm. Pract., 1980, February, 7 . Bastow, R. A., Technicon Int. Pharm. Seminar, Paris, October 4-5, 1973, 15. Walters, S. M., J. Pharm. Sci., 1980, 69, 1206.April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 179 5. 6. 7. 8. 9. 10. 11. 12. HorvAth, Cs., and Melander, W., Am. Lab., 1978, October, 17. Horvith, Cs., Melander, W., and MolnAr, I., J .Chromatogr., 1976, 125, 129. Tomlinson, E., Riley, C. M., and Jefferies, T. M., J . Chromatogr., 1978, 159, 315. Van Rooij, H. H., Doctoral Thesis, University of Amsterdam, 1980. Kirchhoeffer, R. D., J . Pharm. Sci., 1980, 69, 1188. Tomlinson, E., Poppe, H., and Kraak, J . C., Int. J . Pharm., 1981, 7 , 225. Hafkenscheid, T. L., and Tomlinson, E., Int. J . Pharm., 1981, 8, 331. Small, H., Stevens, T. S., and Bauman, W. C., Anal. Chem., 1975, 47, 1801. High-performance Liquid Chromatography in the Measurement of Antineoplastic Drugs M. J. Stewart,* Y. Y. Z. Farid and I. D. Watson Department of Biochemistry, Royal Infirmary, Glasgow, G4 OSF There is an increasing demand for the measurement of antineoplastic drugs in the plasma and urine of patients undergoing treatment with a variety of cytotoxic agents. This demand is associated with the growing awareness of the capabilities of high-perfmmance liquid chromato- graphy (HPLC), as many of the analytical problems that have hitherto prevented the quantita- tive measurement of this group of drugs are now amenable to solution.Antineoplastic drugs are by definition cytotoxic and their use is complicated by their effects on normal tissues. These vary from the merely unpleasant, e.g., alopaecia, to the potentially fatal cardiomyopathy which may occur in patients treated with anthracyclines. The rational use of chemotherapy in malignant disease has always been less than satisfactory owing to the lack of objective measurements.Many drugs are assessed only after the survival rate of treated patients is compared with controls. Thus, until recently, there has been no way of assessing the effectiveness of a drug in real time with a few exceptions where tumour shrinkage may be observed. The recent explosive growth of the science of pharmacokinetics has fuelled the demand by clinicians to know more about the handling of these drugs by their patients and the way is now open for the answering of this demand.l Unfortunately, there are still major difficulties associated with the provision of such a service, as the clinical demand, pharmacokinetic expertise and analytical facilities and knowhow are seldom available on one site. The questions posed are similar to those for any other group of drugs: is the particular tumour sensitive to the drug selected?; what is the active form of the drug? ; what is the active site of the drug? ; what is the necessary concentration at the active site?; is knowledge of the plasma/red cell/CSF drug concentration of value?; how should the drug be administered in order to achieve an effective concentration (route + duration)?; and what is the effect of this dose on normal tissues? In such a case, all of the above questions have to be answered for both drugs and the degree of synergy established.The answers to these questions require a wide variety of techniques, including in vivo trials, tissue culture studies, animal experiments with radioisotopes and epidemiological surveys. How- ever, in order to obtain objective data at many of these stages, analysis of drug and/or meta- bolite concentration can be of use.Two examples may illustrate this wide application of chemical analyses. (a) In drug sensitivity studies, cells from a malignant tumour may be removed from the host, cultured in vitro and treated with different concentrations of the drug concerned. “Kill curves” can be constructed to determine the concentration of drug that is most effective. However, cells in culture take up and metabolise drugs and, in addition, the in vitro studies may not reflect the in vivo situation in which tumour cells may be exposed to metabolites produced, e g . , in the liver of the host. Much information is therefore becoming available as sensitive techniques for the quantitative analyses of metabolites are developed.Two drugs that we are currently investigating in this way are cis- platinum and methotrexate. Some antineoplastic drugs are given in combination. * To whom correspondence should be addressed.Anal. Proc. (b) Once a regime has been applied to patients, the handling of some drugs in individual patients should be monitored in order to confirm that effective concentrations are being achieved. This view is simplistic, because for many drugs the plasma level may not reflect the dosage nor be related to effectiveness. This was shown to be the case for 5- fluorouracil, but only after efficient methods using HPLC had been developed and applied to the study of both drug and metabolites. HPLC is particularly suited to the analyses of antineoplastic drugs, for three reasons: they are mostly water-soluble and non-volatile (indeed many are thermolabile) and not easily analysed by gas - liquid chromatography; they may be difficult to extract from the biological matrix in the active form, e g . , the hydroxy metabolites of adriamycin associate using con- ventional techniques; and the metabolism may be complex and several metabolites may require to be measured. A listing of the drugs for which HPLC analyses are now available would be out of date before publication, such is the rate of development. However, it is worthwhile to consider another particular advantage of HPLC when compared with immunological methods, many of which have recently become popular, and one of which (for methotrexate) is in widespread use. Methotrexate is an effective antineoplastic drug that is given as a high-dose infusion over a period of hours. The concentration achieved must be controlled and thus is effectively managed by on-site optical immunoassay. However, methotrexate has several metabolites and these are not well differentiated by using optical immunoassay, although the interference is small duiing the high-dose infusion. At a later stage the hydroxy metabolite may be present at a higher concentration than the parent drug and at this stage HPLC is the only technique that may effectively quantitate this toxic metabolite. We are currently using HPLC in all instances where speed is not the primary consideration and where the selectivity and sensitivity of the technique give rise to major advantages over immunoassay. For a final example, of the improvements now available with the advent of HPLC, the laborious work of Takanashi and Bachur2 on the isolation and identification of the metabolites of the anthracyclines adriamycin and daunorubicin should be referred to. The exhaustive thin-layer chromatographic techniques, which suffered owing to the light sensitivity of some of the metabolites, could have been carried out and in a fraction of the time using HPLC with fluorescence detection. There is no shortage of drugs, specimens or problems in this exciting field and HPLC users are well equipped to tackle some of the latter to the benefit of the patient. 180 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC References 1. 2 . Bender, R. A., Zwelling, L. A., Doroshow, J . H., Lockyer, G. Y., Hande, K. R., Murinson, D. s., Cohen, M., Myers, C. E., and Chabner, B. A., Drugs, 1978, 16, 46. Takanashi, S., and Bachur, N. R., Drug Metab. Dispos., 1976, 4, 79.

ISSN:0144-557X    

DOI:10.1039/AP9821900163

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

180 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC Anal. Proc. High-performance Liquid Chromatography as an Aid to the Rational Use of Antimicrobial Agents Ian D. Watson, David J. Platt," Stuart J. Mclntosh, Michael J. Stewart and Howard N. Cohent Department of Biochemistvy, Royal Infirmary, Glasgow, G4 OSF An antimicrobial agent is a compound that inhibits the growth or leads to the destruction of microorganisms. In the clinical context the term is usually restricted to those compounds which exhibit selective toxicity and thereby exert maximum effect on the microorganism while producing a minimal or no adverse effect on the human host. Antimicrobials are structurally and functionally diverse and their activity is often character- ised by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) .l * Department of Bacteriology. t Department of Medicine.April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS IN HPLC Clinical 181 A knowledge of the concentration of an antimicrobial in vivo is of value in circumstances such as the investigation of pharmacokinetics, control of toxicity and compliance and assess- ment of the relationship between in vitro MIC/MBC and the in vivo effect.Many methods for determining such concentrations depend on the inhibition of growth of a control organism by the biological sample containing the drug; such assays determine the microbiologically active fractions, but are error prone and can be imprecise if other antimicrobials are present.2 To be of clinical value results may be needed rapidly and up to 24 h may elapse before the results of microbiological assay become available.Chemical methods may be used, e.g., for sulphonamides, although extraction may be required to improve acc~racy.~ These methods may fail to differentiate between the drug, its metab- olites and related compounds unless lengthy purification procedures are carried out. High-performance liquid chromatography (HPLC) offers a solution as most antimicrobials are organic, non-volatile, polar, ionisable compounds. It also facilitates discrimination between the parent drug, metabolites and other compounds; assay times are typically less than 1 h and yield accurate, precise and reliable data with a resultant improvement in clinical service and patient care. As with all drugs, the serum concentration is used as an index of the concentration at the site of action, but for antimicrobials a more relevant concentration may be that at the site of infection.HPLC can often achieve this, e.g., determination of trimethoprim in sputum (unpublished work). Most antimicrobials are relatively non-toxic, but in the investigation of hypersensitivity reactions and some more overtly toxic drugs such as gentamicin and chloramphenicol, a knowledge of serum concentrations has been claimed to be usefuL4,5 If levels are monitored with a view to adjusting the dosage, then swift turnround becomes essential. Pharmacological Statutory licensing requires that pharmacokinetic data be submitted to show that formula- tion and dosage will result in appropriate concentrations, i.e., greater than the MIC but below toxic levels.Such studies assist in the achievement of rational guidelines for treatment ; assessment is enhanced by accurate, precise methods, e.g., HPLC. Most drugs, including antimicrobials are given in multiple doses in an attempt to achieve “steady-state” conditions; this can be adversely affected by the vagaries of absorption, distribution, metabolism and excretion. Inadequate concentrations may result in failure to eradicate the pathogen and sub-inhibitory concentrations contribute to the emergence of drug resistance. Inappropriate levels may disturb the balance of the commensal flora and precipitate secondary (opportunistic) infections. Hence therapeutic monitoring of compliance at day 4 or 5 would be useful, but a rapid turn- round of results would be essential.The potential of HPLC for the assay of antimicrobials is becoming recognised for the reasons outlined above and may be illustrated by the following examples. Metronidazole and its metabolites have been assayed as a result of differences in metabolism between individualse; gentamicin is routinely monitored because of the risks of otoxicity4; some tetracycline ana- logues can cause phototoxic reactions with high doses’ ; amoxycillin and ampicillin are virtually non-toxic. However, unpredictable hypersensitivity reactions do occur and may be related to breakdown product s.8 Nalidixic acid is used in urinary tract infection; toxicity is exhibited as CNS disturbances possibly related to the concentration of the drug or its metabolite^.^ Chloramphenicol is highly toxic and may give rise to blood dyscrasias.As a result, it is used only rarely but is particularly effective in neonatal meningitis caused by Haernophilus injuenxae. However, neonates are very susceptible to chloramphenicol toxicity so that in addition to haematological monitoring it has been suggested that chloramphenicol levels should be measured.lO Pharmaceutical Antimicrobial production processes often involve a fermentation and require quality control HPLC has been used for a variety of pharmaceutical preparations, e.g., of the product.182 CLINICAL AND BIOLOGICAL DEVELOPMENTS IN HPLC Anal. PYOC. kanamycin,ll maridornycin,l2 mitomycin,13 neomycin,14 polymixins,15 octapeptins,15 spectino- mycin16 and spiramycin.17 The role of HPLC in assisting in the rational use of antimicrobials is further illustrated below by its application in two situations.Experimental Investigation of Third Generation Cephalosporins We wished to obtain a preliminary assessment of cephalosporin pharmacokinetics ; one of the problems of cefotaxime was rapid acetylation to a less active metabolite,18 which precluded the use of microbiological assay. We were interested in cefoperazone and, to avoid the problems associated with cefotaxime, developed an HPLC separation (Fig. 1). A 100-pl volume of serum was precipitated by 100 p1 of 6% copper sulphate solution and 100 p1 of 5% sodium tungstate solution ; the sensitivity was sufficient for measurements at a therapeutic level. Ultraviolet detection was just sufficiently sensitive, although electrochemical detection seemed to be more promising.E 0.002 +I 8 2 a (0 .0.001 0- Elution vol u me/m I Fig. 1. HPLC of cefoperazone (CP) in serum. CT = Cefotaxime (internal standard). Eluent, 0.01 M sodium dihydrogen phosphate (pH 4.6) - methanol - dioctylamine (60 + 40 + 0.05) ; flow-rate, 1 ml min-I. Investigation of Trimethoprim - Sulphonamide Preparations Trimethoprim - sulphonamide combinations have been in use for some time for the treatment of a variety of infections. We investigated a new trimethoprim - sulphonamide combination, co-trifamole (sulphamoxole - trimethoprim, 400 and 80 mg, respectively, per tablet) and com- pared it with the well established combination of co-trimoxazole (sulphamethoxazole - trimethoprim, 400 and 80 mg, respectively, per tablet, 2 tablets twice daily).After an initial dose of co-trifamole comparable to that of co-trimoxazole, a maintenance dose of co- trifamole equivalent to half that of co-trimoxazole is used. Urinary trimethoprim was measured by HPLClg and urine sulphonamides spectrophotornetrically.3 The two drugs sequentially block bacterial folate metabolism and synergistic action has been claimed20 with an optimum trimethoprim to sulphonamide ratio of between 1 : 10 and 1 : 40.21 Co-trimoxazole is formulated to give this ratio in serum.22 We have confirmed this, but found that co-trifamole gave ratios of between 1 : 40 and 1 : 75, i.e., outside the optimum range; how- ever, these drugs are not used to treat bacteraemia.I t is the concentration at the site of infection that is important. The MIC of sulphonamides for common urinary pathogens is ca. 200 ,ug ml-l and that of trimethoprim is ca. 2 pg ml-l, independent of synergy.22 During a 5-day period of study on a group of volunteers with these drugs we found that neither sulphonamide consistently exceeded the MIC, but the MIC for These drugs are often used to treat urinary tract infections.April, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS IN HPLC 183 trimethoprim was consistently exceeded by both preparations. The ratio of trimethoprim to sulphonamide for both in urine was ca. 1 : 2, a range of substantially reduced synergy. In the treatment of urinary tract infections both preparations are reported to have comparable efficacy23; moreover, the efficacy of trimethoprim alone is equal to that of co-trimoxazole.24 Our results in conjunction with the above reports suggest that the efficacy of these preparations may be due to trimethoprim alone. The examples given above illustrate the value of HPLC for the assay of antimicrobials and we suggest that such assays are a valuable adjunct to microbial investigations, HPLC can thus assist in the rational approach to the use of antimicrobials at all levels: clinical, pharmaco- logical and pharmaceutical. 1.2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. References Waterworth, P., in Reeves, D. S., Phillips, I,, Williams, J. D., and Ulise, R., Editors, “Laboratory Reeves, D. S., in Reeves, D. S., Phillips, I., Williams, J.D., and Ulise, R., Editors, “Laboratory Rieder, J., Chemother., 1972, 17, 1. Xnhalt, J. P., Antimicrob. Agents Chemother., 1977, 11, 651. Gal, J., Marcell, P. O., and Tarascio, C. M., J . Chromatogr., 1980, 181, 123. Wheeler, L. A., de Meo, M., Halula, M., George, L., and Heseltine, P., Antimicrob. Agents Chemother., Nilsson-Ehle, I., Yoshikawa, T. T., Schotz, M. C., and Guze, L. B., Antimicrob. Agents Chemother., Vree, T. B., Hehster, Y . A., Baars, A. M., and Van der Kleijn, E., J . Chromatogr., 1978, 145, 496. Cuisinaud, G., Ferry, N., Seccia, M., Bernard, N., and Sassard, J., J . Chromatogr., 1980, 181, 399. Crechiola, J., and Hill, R. E., J . Chromartogr., 1979, 162, 480. Mays, 0. L., Van Aperloorn, R. J., and Laubock, R. G., J . Chromatogr., 1976, 120, 93.Kando, K., J . Chromatogr., 1979, 169, 329. Srivastava, S. C., and Hornemann, V., J . Chromatogr., 1978, 161, 393. Tsuji, K., Geotz, J . F., Van Meter, W., and Gusciora, K. A., J . Chromatogr., 1979, 175, 141. Terabe, S., Konaka, R., and Shoji, J., J . Chromatogr., 1979, 173, 313. Myers, H. N., and Rindler, J . V., J . Chromatogr., 1979, 176, 103. Mourat, D., Delepine, B., Bovisseau, J., and Gayat, G., J . Chromatogr., 1978, 161, 386. Wise, R., Mills, P. J., Andrew, J . M., and Bedford, K. A., Antimicrob. Agents Chemother., 1980, 17, Watson, I. D., Shenkin, A., McIntosh, S. J., and Cohen, H. N., Clin. Chem., 1980, 26, 1791. Seydel, J. K., R. SOC. Med. I n t . Congv. Symp. Ser., 1979, No. 15, 65. Bushby, S. R. M., and Hitchings, G. H., BY. J .Pharmacol., 1968, 33, 72. Brumfitt, W., Hamilton-Miller, J . M. T., and Kasmidis, J., J . Infect. Dis., 1973, 128 (Suppl.), S778. Peters, H. J., Nuri, M., and Popa, G., Munch. Med. Wochenschr., 1977, 119, 409. Lacey, R. W., Lord, V. L., Gunasekara, H. K. W., Liebermann, P. J., and Luxton, D. E., Lancet, Methods in Antimicrobial Chemotherapy,” Churchill Livingstone, Edinburgh, 1978, p. 30. Methods in Antimicrobial Chemotherapy,” Churchill Livingstone, Edinburgh, 1978, p. 168. 1978, 13, 205. 1976, 9, 754. 84. 1980, i, 1270. Routine Assay of Catecholamines by High-performance Liquid Chromatography with Fluorimetric Detection G. Clough Biochemistry Department, General Hospital, Harrogate, HG2 7ND The assay of urinary catecholamines is of proven value in the investigation of patients with a suspected tumour of neurological origin, neuroblastoma, neuroganglioma and phaeochromo- cytoma.The incidence of phaeochromocytomas in hypertensive patients is reported to be between O.lyol and 0.6y0.2 The results from this Department yield an incidence of 0.3%. The need for the screening of many urine samples from hypertensives for catecholamine excre- tion is therefore apparent. To establish a firm diagnosis it is essential to measure the excretion of adrenaline, noradrenaline and dopamine and this can easily be done by using high-perform- ance liquid chromatography (HPLC). The procedure described is essentially that of Me11 and Gustafson3 and is suitable for routine handling of considerable numbers of samples, is reliable at the nanogram level, is relatively inexpensive, simple in operation and can be automated.184 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC Experimental Apparatus Anal.Proc. A Laboratory Data Control (LDC) Constametric IIG pump was used with manual injection through a Rheodyne 7125 valve. The native fluorescence of the eluate was excited at 286 nm and monitored at 312 nm using a Perkin-Elmer 204s spectrofluorimeter fitted with an 80-pl flow-through cell. Separation of the catecholamines was obtained through an hplc Technology Partisil 5 ODS 25-cm column fitted with a Whatman Co-Pel1 ODS guard column. The fluorimeter signal was fed into a Spectrum electronic filter and amplifier and the smoothed amplified output was then read in an LDC 308 integrating computer and the trace charted on a Servoscribe RE-541 recorder.Reagents Acetic acid (0.17 M) was used as the mobile phase and was degassed prior to use. Stock standards of noradrenaline, adrena- line and dopamine were obtained from Sigma and stock solutions of l mg ml-l were prepared in 0.01 III hydrochloric acid; these were diluted to a concentration of 1 pg ml-l with alumina eluate. The alumina (aluminium oxide, Camag MFC, pH 4.5, Brockman activity 1) was obtained from Hopkin & Williams and was activated by heating at 200 "C for 1 h. All solutions were made up using doubly glass-distilled water. Procedure The sample is 24-h urine collected directly into 50 ml of 30% hydIochloric acid. Two aliquots, each equal to l/40th of the 24-h sample, are taken and each aliquot placed in a 400-ml beaker.Ten micrograms of noradrenaline and 10 pg of dopamine are added to one beaker and the beaker labelled Test + internal sample, the remaining beaker being labelled Test. The samples are diluted to 150 ml with distilled water and 10 ml of 0.1 M EDTA solution and 2 g ol' activated alumina are added. The mixture is stirred just sufficiently to keep the alumina suspended and the pH is adjusted to 8.4 and maintained for 10 min. The alumina is allowed to settle within 3-5 min. the supernatant is poured off and the residue is centrifuged and washed twice with 50 ml of distilled water. The alumina is drained, stirred with 5 ml of 0.2 M hydrochloric acid on a vortex mixer, centrifuged and the clear eluate is retained. The extraction is repeated with 4 ml of 0.2 M hydrochloric acid and the two eluates are combined and made up to a final volume of 10 ml with distilled water. This solution is centrifuged at high speed and the clear supernatant used for injection on to the reversed-phase column for HPLC determination.The test sample results were calculated using the internal standard results obtained by subtracting the peak areas of the Test assays from those of the Test + internal standard assays. The use of HPLC permits the identification and determination of catecholamines in tumour and urine extracts. Reversed-phase techniques ensure the prior identification of noradrena- line, adrenaline and dopamine even when the patient is on drug therapy such as methyldopa. The facility for identifying and measuring adrenaline, noradrenaline and dopamine has great advantages over the conventional chemical methods of analysis.Tumours of neurological origin, particularly neuroblastomas, often do not produce large amounts of vanillylmandelic acid (VMA) [4-hydroxy-3-methoxymandelic acid (HMMA)] and therefore are not detected by the laboratories that only perform this test. HPLC analysis enables the biochemist to separ- ate, identify and quantitate the excretion of dopamine, thus establishing the diagnosis. Fig. 2 shows results for the measurement of dopamine in tumour extracts. The analysis of tumour extracts is of considerable value in establishing the question of malignancy. Immediately on removzl from the patient a Fortion of the tumour is placed in 5% hydrochloric acid and forwarded to the laboratory.The tissue is homogenised in the acid and the filtered extract is diluted and injected on to the column. The differentiation between the malignant phaeochromocytoma is shcwn (Fig. 3) by the increased dopamine content, the benign tumour largely secreting noradrenaline and the malignant tumour secreting dopamine. Typical results are shown in Fig. 1. Conclusion Separation of the catecholamines by reversed-phase HPLC and quantitation by measure- ment of the native fluorescence of catecholamines has several advantages over the establishedApril, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 185 D C 6 I I L I I I 1 1 1 1 21 18 15 12 9 6 3 0 Timeimin Fig. 1. Catecholamine standards showing repeatability after continuous use of the column for 1 h.Peaks: A, a-methyldopa (200 ng); B, metadrenaline (20 ng) ; C, dopamine (20 ng) ; D, normetadrenaline (20 ng) ; E, adrenaline (20 ng) ; and F, noradrenaline (20 ng). Eluent, 0.17 M acetic acid; flow-rate, 1 ml min-'. Sample volume, 20 p1; temperature, 22 "C; pressure, 1580 lb in-2. Other conditions as in text. fluorimetric trihydroxyindole methods, which are interference prone4 and do not measure dopamine, the determination of which is essential in order to determine the presence of malig- L C Fig. 2. Measurement of dopamine in tumour extracts. A, Child, 8 yrs, neuroblastoma, VMA + +, HVA + + ; B, baby, 1 yr, neuroblastoma, VMA normal, HVA + + ; C, baby, 1 yr, normal; and D, control. Peaks: 1, dopamine ; and 2, noradrenaline.186 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC -2 B -1 C Fig.3. Differentiation between benign and malignant tumours. A, Benign tumour; B, malignant tumour, phaeochro- mocytoma; and C, controls. Peaks: 1, dopamine; 2, nor- adrenaline ; 3, adrenaline; and 4, a-methyldopa. Anal. Proc. nancy. The method is more precise and reliable, and the rapid analysis time of less than 10 min after alumina extraction and the stability and repeatability of the system enable the bio- chemist to establish a routine bench procedure suitable for busy hospital clinical biochemistry departments. References 1. 2. 3. 4. Minao, A. M., and Bennett, W. A., N . Engl. J . Med., 1954, 251, 959. Barbeau, A., Union Med. Can., 1957, 86, 1045. Mell, L. D., and Gustafson, A. R., Clzn. Chem., 1977, 23, 473. Crout, J. R., Clin.Chem., 1961, 3, 62. Analysis of Clinically Important Bile Pigments by High-performance Liquid Chromatography Rosalind V. A. Bull and C. K. Lim Division of Clinical Chemistry, Clinical Research Centre, Harrow, Middlesex, H A 1 3 U J The naturally occurring bile pigments are linear tetrapyrroles, end-products of the metabolism of the haem of haem protein ; biliverdin IXa [Fig. 1 (a)] is first formed, is converted into bilirubin [Fig. l ( b ) ] , which is conjugated in the liver mostly with glucuronic acid, and reaches the gut via the bile duct. In the gut bilirubin is hydrogenated by intestinal flora to “urobilinogens,” which are the colourless chromogens of “urobilins,” orange - red pigments including l-stercobilin [Fig. 1 (c)], half-stercobilin [Fig. 1 ( d ) ] and i- and d-urobilins [Fig.1 (e)]. The urobilinogens are excreted in the faeces, and are usually completely oxidised either before excretion in the faeces or during their extraction. Daily excretion of faecal bile pigments may be measured spectrophotometrically with Ehrlich’s reagent after their complete reduction with iron(I1) by hydroxide to urobilinogens. This has been used as an estimate of the severity of haemolysis, especially in diseases in which there is an abnormally high turnover of haem as in haemolytic anaemias such as sickle cell anaemia. However, “faecal urobilinogens” are now rarely determined because of their com- plexity and the instability of some of the urobilins and, as their molar absorptivities differ from one another, separation of these compounds before determination is essential.Bilirubin, either conjugated or unconjugated, or both, can accumulate in the blood under various pathological conditions to cause jaundice. They are mainly determined by the directApril, 1982 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC 187 and indirect diazo reaction1v2 under specific conditions, although solvent partition3 and direct spectrophotometry have also been used. These methods are non-specific and ina~curate,~ and a specific and reliable method is therefore needed. This paper describes high-performance liquid chromatographic (HPLC) methods for the analysis of faecal urobilinoids (“urobilins”) and for the determination of conjugated and unconjugated bilirubin in serum. Experimental Materials and Reagents Acetonitrile was of HPLC grade from Rathburn Chemicals (Walkerburn) ; tetraethylene- pentamine (TEPA) was of technical grade; dimethyl sulphoxide (DMSO), heptane, methyl acetate, diethylamine (DEA) , boron trifluoride in methanol (14% m/V) and ethylenediamine- tetraacetic acid (EDTA) were of analytical-reagent grade ; methanol, glacial acetic acid, hydrochloric acid (12 M), diethyl ether, chloroform, light petroleum (boiling range 60-80 “C), sodium acetate, sodium hydrogen carbonate, L-ascorbic acid, potassium hydroxide and glycine were of AnalaR grade from BDH Chemicals (Poole).Methods Urobilinoids The urobilinoids were extracted from a 6-day collection of faeces from a patient with haemo- lytic anaemia by Watson’s m e t h ~ d , ~ but the final extract that was precipitated as the hydro- chloride from light petroleum was not crystallised.A portion of the product was dissolved in methanol (3 ml), boron trifluoride in methanol (1 ml) was added and the mixture was refluxed for 10 min. After dilution with water the esterified pigment was extracted into chloroform, washed once with dilute sodium hydrogen carbonate solution, once with water and dried. Portions of the “free” and esterified urobilinoids were dissolved in chloroform for analysis by HPLC. For conditions see Table I. TABLE I k’ VALUES OF “FREE” AND ESTERIFIED UROBILINOIDS Column: Hypersil (250 x 5 mm i.d.). Mobile phase: 1, acetonitrile - water - TEPA (85 + 15 + 0.05); 2, heptane - methyl acetate - 1% DEA in methanol (75 + 25 + 2). Detection: 450 nm. Injection volume: 20 pl.Flow-rate: 1 ml min-l. Compound Mobile phase . . . . .. .. 1 . . . . . . .. 1 . . . . .. .. 1 Stercobilin .. . . Half-stercobilin . . . . Urobilin . . . . . . Stercobilin dimethyl ester (RR SS) . . .. .. 2 Half-stercobilin dimethyl ester (RR S S ) . . . . 2 Urobilin dimethyl ester (RR S S ) . . . . . . 2 Half-stercobilin dimethyl ester (RS SR) . . . . 2 Urobilin dimethyl ester (RS SR) . . . . .. 2 k’ 4.56 5.15 5.74 2.23 2.97 3.97 4.73 6.07 Bilirubin and its conjugates Bilirubin and its mono- and diconjugates were extracted by Blanckaert et al.’s method6 from 200 p1 of serum from a patient with conjugated hyperbilirubinaemia; the conjugates were con- verted into the corresponding methyl esters and extracted together with unconjugated bilirubin into chloroform.The dried extract was taken up in DMSO (100 pl) before injection on to the HPLC column. For conditions see Fig. 2. Results and Discussion Table I shows the chromatographic conditions and capacity ratios (k’ values) of “free” and esterified urobilinoids. Stercobilin, half-stercobilin and urobilin were eluted as a single peak when reversed-phase and reversed-phase ion-pair chromatography on Hypersil-ODS were attempted. These pigments are best resolved on a Hypersil column with acetonitrile - water - TEPA (85 + 15 + 0.05) as the eluent. Although similar systems have been used for the188 CLINICAL AND BIOLOGICAL DEVELOPMENTS I N HPLC Me V Me P P Me Me V H H H 1 2 0 2 4 6 810 Anal. Proc. 2 Time/min HPLC separation of bilirubin and its mono- and dimethyl esters from serum of a patient with conjugated hyper- bilirubinaemia.Column, Hypersil-SAS (100 X 5 mm) ; mobile phase, acetonitrile - dimethyl sulphoxide - water (34 + 34 4- 32) ; flow-rate, 1 ml min-1; detection, 450 nm; injection volume, 20 pl. Peaks: 1, bilirubin monomethyl ester ; 2, bilirubin : and 3, bilirubin dimethyl ester. Fig. 2. Fig. 1. Structures of bile pigments. (a) Biliverdin IXcc; ( b ) bilirubin IXa; (c) stercobilin ; ( d ) half-sterco- bilin; and (e) urobilin. Me = methyl; V = vinyl; P = propionic acid; and Et = ethyl. separation of oligosaccharides and shown to be operating by the normal-phase rnechani~m,~ ion-exchange and ion-pair mechanisms cannot be excluded because the bile pigments are ionis- able compounds. The separation of the urobilinoids by this system could improve quantitation and might be of clinical value in permitting a re-evaluation of the importance of the determin- ation of faecal bile pigments in clinical work.As the or-carbon atoms of the end rings are asymmetric [Fig. l ( c ) , (d) and ( e ) ] , the urobilin- oids can exist as RR SS and RS SR diastereoisomers, as indicated in Table I. The diastereo- isomers can be resolved using heptane - methyl acetate - 1% DEA in methanol (75 + 25 + 2) a s the mobile phase on a Hypersil column (Table I). Natural stercobilin was entirely in the RR SS forms, in contrast to the findings of Petryka.8 The detailed analysis of these faecal bile pigments might lead to a better understanding of haem catabolism in man. Unconjugated bilirubin in serum can be analysed directly by HPLC,9 but the simultaneous separation of unconjugated and conjugated bilirubin requires alkaline methanolysis (trans- esterification)6 to release the tightly bound conjugates from proteins.The reaction converts bilirubin mono- and diconjugates into bilirubin mono- and dimethyl esters, respectively, whereas unconjugated bilirubin is unaffected. Fig. 2 shows the separation of bilirubins in serum (after transesterification) of a patient with conjugated hyperbilirubinaemia using this method. The column was Hypersil-SAS eluted with acetonitrile - DMSO - water (34 + 34 + 32). DMSO eliminated peak tailing due to the insolubility of bilirubin in acetonitrile and water. This HPLC method is simple, fast and specific. It is ideal for both routine and research laboratories where bilirubin analysis is required and should be important in the diagnosis and treatment of neonatal jaundice. R. V. A. Bull was supported by a grant from the Medical Research Council to Professor C. H. Gray.April, 1982 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS References 189 1 . 2. 3. 4. 5. 6. 7. 8. 9 Billing, B. H., Haslam, R., and Wald, N., A n n . Clin. Biochem., 1971, 8, 21. Heirwegh, K. P. M., van Hees, G. P., Leroy, P., van Roy, F. P., and Jansen, F. H., Biochem. J., Weber, A. P., and Schalm, L., Clin. Chim. Acta, 1962, 7, 805. Ostrow, J . D., and Boonyapisit, S. T., Biochem. J . , 1978, 173, 263. Watson, C. J., J , Biol. Chem., 1934, 105, 469. Blanckaert, N., Kabra, P. M., Farma, F. A., Stafford, B. E., Martan, L. J., and Schmid, R., J . Lab. Wheals, B. B., and White, P. C . , J . Chromatogr., 1981, 204, 219. Petryka, 2. J., in Derk, P. D., and Berlin, N. I., Editors, “Chemistry and Physiology of Bile Pig- ments,” Fogarty International Centre Proceedings, No. 35, National Institutes of Health, USA, Lim, C . K., Bull, R. V. A., and Rideout, J . M., J . Chromatogr., 1981, 204, 219. 1970, 120, 877. Clin. Med., 1980, 96, 198. 1977, pp. 499-508.

ISSN:0144-557X    

DOI:10.1039/AP9821900180

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

April, 1982 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS 189 Official Methods of Analysis and Stability Testing of New Drugs The following are summaries of seven of the papers presented at a Joint Meeting of the East Anglia Region and the Chromatography and Electrophoresis and Joint Pharmaceutical Analysis Groups held on March 25th--26th, 1981, at the University of Cambridge. The last three summaries were presented at a full-day session on “The Work of the Analytical Methods Committee.” A summary of one paper in this session, by D. R. Williams, entitled “Develop- ment and Application of New Analytical Methods-the Work of the Analytical Methods Committee,” was published in connection with another meeting in the November 1981 issue of Analytical Proceedings (p. 480). Use of Chromatography Through the Development Phase of the Bulk Drug F.Bailey I c I Pharmaceuticals Division, Hurdsfield Industrial Estate, Macclesjield, Cheshire The development phase of a new chemical entity from the first biological screen that demon- strates interesting pharmacological activity to the sale of a cream/ointment, liquid, tablet/ capsule is a long and expensive process covering the whole scientific spectrum, chemical, pharmaceutical analytical, biological, pharmacological, toxicological and clinical, spanning a time typically of 6-10 years for the UK and Europe and 10-12 years for the USA and Japan and costing E10-15 million. Any inroads that can be made in reducing the time a compound spends in development and SO bring it to the market place quicker means a longer patent life and a bigger realisation on the research capital invested, and for a research-based industry this is of prime importance.Let us consider the bulk drug from three aspects: ’* Pre-development this order because 1 and 2 cover stability 2. Stability 3. Bulk drug control. Pre-development The sample submitted will be of R and D origin, of limited amount (50-100 mg) and the work will usually be confined exclusively to the stability unit-at this stage little information will be available (melting-point, route of synthesis, biological data). The objective of this phase is by some rapid tests (maybe less precise) to give an early warning to process development chemists, and the formulator, of potential problems. For this phase of the work we frequently use tritiated material.This work covers both solid-phase and solution studies. This covers 6-8 weeks.190 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS Solid Phase The questions asked are as follows: 1. Is it stable to light? 2. Is it stable to heat 3. Is it hygroscopic? These are studied by chromatographic methods. (90 "C, 100 "C)? Anal. Proc. Drug - excipient interaction (1% solid sample of tritiated drug in excipient) incompatability and recommend to the formulator the excipients to avoid. scanning calorimetry is not reliable. equipment and has very little advantage over solution spectroscopy. is not sensitive enough, even for C=:O-containing compounds. Method Tritiated drug is intimately mixed with unlabelled drug by dissolving both in the minimum amount of solvent; the aim is to produce a count of 10 x 103-20 x lo3 d.p.m.when mixed with excipient at the 1% level. This sample is then stored under varying conditions of temperature (60 "C for 2 weeks, 70 "C for 1 week) and relative humidity (RH) over potassium iodide in a Kilner jar (60% RH). Samples of drug and drug plus excipient are also stored at 4 "C for reference. After storage of the sample a portion is transferred to a TLC plate, chromatographed in the normal manner, dried and then scanned using a Berthold scanner with a 27r detection head. Degradation and incompatabilities may be assessed by comparing the chromatographic results for the stored sample with those for the reference. From the information generated in this test the analyst is able to advise the formulator on excipients that appear acceptable and those to be avoided.Solution tests carried out at this stage usually cover the following range : 1. Samples stored under nitrogen or oxygen. 2. Effect of pH, i.e., pH 2-10 profile. 3. Effect of anions (phosphate, acetate, citrate, hydrogen carbonate, borate), usually 0.2 M. 4. Effect of cations (iron, copper). 5. Effect of temperature: McIlvaine's buffer at pH 8 stored a t 100,80 and 70 "C, examined a t 2, 4, 6, 8, 10 and 20 days. At the end of this pre-development phase we are attempting to achieve a portfolio of information that can be of value to all individuals subsequently working on this development. Any samples throughout this pre-development phase that contain degradation products are subjected to some separation technique in order to isolate and identify them.Currently we would prefer to use HPLC and would scale up from the normal analytical 2-in column to a $- or &in column. Bulk Drug Stability Longer term stability is normally set down as soon as possible after the chemical production process has been worked out and taken through a scale of procedure to semi-plant scale; 10-g samples are used in the longer term stability tests stored under a variety of conditions. Room temperature dark (RTD), room temperature light (RTL), 37 "C and 50 "C hygro- scopicity are checked by exposing the bulk drug to a humid environment at various temperatures: 50% RH at room temperature, 78% RH at room temperature and 78% RH at 37 "C. The samples are examined after various periods (more frequently initially), 1 and 2 weeks, 1, 3, 6, 9, 12 and 18 months, 2, 3, 4 and 5 years, for physical change, strength by a stability-indicating method (this is checked by degrading a sample), TLC properties and moisture.The stability-indicating methods are usually some form of chromatographic separation. From the results of these tests we are able to predict the shelf-life of the bulk drug. Many physical techniques have been used in the past to try to assess this interaction, predict Differential Diffuse reflectance spectroscopy involves expensive Infrared spectroscopy Bulk Drug Evaluation Through the development phase we check the following criteria in an attempt to characterise If related substances are the compound: proof of structure; purity; and related substances.present we need to isolate and identify them and check their toxicity if necessary.AfiriL, 1982 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS 191 As the process development chemist scales up we now check, under good manufacturing practice, raw materials, isolated key intermediates and the finished product. We check the related substance profile to ensure that on scale-up the identity and quality of material that went into the toxicity testing are maintained into the early clinical work. Any route change carried out that alters any of the information generated by this testing schedule has to be treated virtually as a new chemical through the analytical development and stability phase. This work leads to the production of a standard testing schedule (standard official method) and a tentative specification.This is a compromise between the toxicologist (100.0% material) , the process chemists (realistic strength depending on his process, cost, yield) and the analyst (reasonably tight specification factors of primary interest in a specifica- tion or pharmacopoeia monograph) to help us to judge the quality, as follows. Physical state This can be judged by the purchaser without recourse to laboratory facilities. Purity an assessment of general purity obtained. Physical criteria capable of accurate measurement can be compared with standard data and Identity This should be sufficient to confirm identity when evaluated collectively. Active agent techniques. individual impurities is effected by titration, colorimetry, spectroscopy, polarimetry, etc.Related substances and degradation products Few registration authorities will accept a compound in the absence of requirements restricting related sub- stances and degradation products. Authorities demand data regarding products of degradation and selective methods for their determination. I t is easier to register a product with a marked degree of instability than one which is stable in accelerated stability tests. These are a selection of the factors to be considered when establishing a testing schedule and specification for a new pharmaceutical. The analyst must retain flexibility in both of the above areas, as each compound is examined in the light of the best information available at a particular time and knowing the purpose for which the new compound is to be used.Direct and selective determination of the active constituent is carried out by chromatographic Non-selective determination of the active constituent with separate determination of Related substances. These have grown to a position of prime importance. Degradation products. Chromatography in the Testing of New Drug Formulations J. Tillman Fisons Limited, Pharmaceutical Division, Research and Development Laboratories, Bakewell Road, Lough- borough, Leicestershire The development of new drug formulations is an expensive and time-consuming process. The pharmaceutical analyst is involved in all stages of formulation development and chroma- tographic techniques play an important role in the service he is required to provide.I t is important, however, that each problem is considered on its own merits and that the analyst uses his experience to employ the most appropriate technique. Analytical involvement in new drug development can be summarised as follows: (1) proof of purity of raw drug (specification); (2) preliminary stability studies on raw drug.; (3) pre- formulation studies on raw drug; and (4) formulation studies. Thus, before formulation studies can start, it is essential to show that the drug substance is of a known purity, reasonably192 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS Ana2. PYOC. stable and, in the pre-formulation studies, to investigate those properties which may affect formulation development (eg., crystal form, hygroscopicity, solubility).The object of formulation studies is to produce a final product that is capable of being manufactured reproducibly, has a useful shelf life (ie., physically stable, minimum drug degradation or interaction with components of the formulation and pack, and safe and effective in use by the patient). Analytical Involvement in Formulation Studies Pre-formulation Compatibility Studies These are early studies designed to investigate the possibility of chemical interaction between the drug substance and potential formulation ingredients or excipients. They involve the preparation of mixtures of the drug and each ingredient, either in the solid state or in solution, and storage of these mixtures at elevated temperatures (say 50-100 "C). These mixtures are then examined for reaction products.It should be made clear that many new compounds are nominated for development, but that most will not reach the market place. Failure to do so may be due to reasons such as lack of efficacy, insufficient safety or stability problems. For this reason, it is desirable t o utilise relatively simple techniques at this stage of development and then to look in greater depth as the potential product looks increasingly viable. Thin-layer chromato- graphy (TLC) generally meets these requirements and has many advantages as an indicator of possible interactions. It is simple and cheap and all components on the plate may be rendered visible (from base-line to front-running spots). The major disadvantage with high-performance liquid chromatography (HPLC), a t this stage of product development, is that components retained on the column are not detected unless time-consuming gradient elution is employed.Further, TLC systems are easily developed on a small scale and once optimised ten or more samples may be examined simultaneously on the same plate. Reactions may be followed, and by use of low loadings of standards, degradation products may be determined semi-quantitatively down to about 0.5% with a precision that is sufficient for these initial studies. The usefulness of TLC has been greatly enhanced over recent years by the introduction of HPTLC plates, concentration zone plates and reversed-phase plates. Mixing Studies Progress towards a suitable formulation involves mixing studies on a gradually increasing scale.These require numerous analyses of samples taken from bulk blends, various parts of the mixing equipment and unit dose assays. Stability is not usually a problem and a rapid, non-specific assay is generally appropriate. Ultraviolet spectrophotometry is often applicable and has the advantage of being relatively simple and readily automated. Automated HPLC also finds a use at this stage, but is generally not so rapid a technique as ultraviolet spectro- photometry. Feasibility Stability Studies These are generally of short duration (up to 1 year) and at temperatures up to 45 "C. They may involve several alternative formulations and often include pack feasibility tests. The objective of these studies is to assist the choice of the final formulation and to obtain estimates of potential shelf-life. Again, simple TLC procedures are preferred, but it is often more appropriate to utilise a more specific and precise technique.The analytical problems now become more difficult as it is desired to determine the intact drug molecule not only in the presence of its own degrada- tion products, but also in the presence of other formulation ingredients and their degradation products. Formal Stability Studies They are longer term storage tests, carried out to confirm the predicted shelf-life of the product under marketing conditions. Storage will normally be for up to 5 years at different conditions of temperature and humidity, usually on three batches of product in the final marketing pack(s). The next stage of product development is to perform feasibility storage tests. Formal stability tests are, as their name suggests, a mere formality.April, 1982 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS 193 In feasibility stability testing, degradation is often measured in a semi-quantitative manner.In formal stability studies it is essential to assay, as accurately and precisely as possible, the amount of intact drug molecule in the presence of its degradation products and formulation ingredients. 1. Measure the intact drug molecule. If a specific method is available, then separation from degradation products may not be necessary. A non-specific method may be used if the intact drug molecule can be separated from interfering degradation products. A disadvantage of this approach is that analytical variation may mask the presence of a small amount of degradation.2. Measure a specific degradation product (with or without separation from the intact molecule). This is the preferred approach if a suitable analytical procedure is available so that small amounts can be determined with precision, but it is suitable only if the degradation product is stable itself. 3. Measure both the intact molecule and a specific degradation product. This is the ideal approach, but the requirements of the first two approaches must be met. I t is in this area of formal stability testing that chromatographic techniques come into their own. Until recently, quantitative TLC was used extensively as a stability indicating assay. Its main drawback was that it was often time consuming and required considerable manipulative skills.HPLC has now proved to be generally invaluable, often enabling the “ideal” approach to be adopted. In many instances it is possible to assay both the intact molecule and its degradation products in the same solution, by varying the amount injected or monitoring at different wavelengths or different detector sensitivities. Occasionally a different mobile phase or alternative column may be necessary. With autosampling facilities and electronic data reduction a large number of samples can be processed with ease. The problem can be approached in three basic ways: Conclusions Product development is seen to be a continual build-up of information concerning the ability to manufacture reproducibly, ensuring compatibility with formulation ingredients and pack and obtaining a stable product. Obvious degradation products will be separated, characterised, synthesised and toxicity tested to enable shelf-life limits to be set.Chromatographic techniques play an important part during the whole of this development process, but it is stressed that results are usually required in the shortest possible time or a t the minimum of cost. It is the responsibility of the analyst to choose that technique which will conform to these requirements and still give meaningful results. Some Applications of Liquid Chromatography to the Analysis of Medicinal Products R. G. Hopkins Medicinal Products Sub-division, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SE1 9NQ The Laboratory of the Govenment Chemist provides an analytical service to Government Departments and under certain circumstances to other bodies.Frequently, the Medicinal Products Sub-division is asked to examine medicinal products to see if these comply with pharmaceutical standards of quality, but other information may be required. Recourse to Pharmacopoeia1 Monographs and methods of analysis may not be appropriate where there is insufficient sample, the sample contains components that interfere with the methods of analy- sis, the customer requirzs other information, or simply because there is no monograph relevant to the problem. The applicability of high-performance liquid chromatography (HPLC) to a wide range of medicinal product analyses can be demonstrated by the following eight cases.194 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS Anal.PYOC. Conjugated Oestrogens For tariff purposes it was necessary to identify and quantify each of the steroids in a sample of conjugated oestrogens. The existing pharmacopoeial methods could be used to assay several of the components, such as oestrone and equilin, but not the oestradiols. An HPLC method was developed that was used to resolve and quantify each of the oestrogens of interest, including both 17a- and 17p-oestradiol. Ampicillin Syrup It was not possible to assess the storage stability of a sample of strong ampicillin syrup using the method of the BPC 1973 monograph, because flavouring and colouring materials in the syrup interfered in the official colorimetric assay. A reversed-phase HPLC system was devel- oped which resolved ampicillin from all interferences in the syrup and permitted assessment of compliance with the storage stability requirements.Anaesthetic Injection No monograph procedure was available for the analysis of a steroid anaesthetic injection. The active ingredients, alphaxalone and alphadalone acetate, are closely related in structure but differ in terms of anaesthetic activity. Although they are both poorly absorbing at accessible ultraviolet wavelengths, an HPLC system with UV detection was devised to resolve and quantify the steroid ingredients of the injection. Racestyptine Cord Racestyptine cord is a product used in dentistry. The cord is impregnated with local anaesthetic, lignocaine base in this instance, and inorganic salts.HPLC was used to obtain a rapid, clean and accurate determination of the degree of lignocaine impregnation. Sample preparation was limited to extracting known lengths of cord with methanol and injecting the extract directly on to the chromatograph. The system was reproducible and the lignocaine assay was consistent with that of the other ingredients of the impregnation, which were deter- mined independently. Single Tablet Assays Two samples received for examination consisted of a single tablet. In both instances monograph procedures were not appropriate. The first sample was of a proprietary deconges- tant containing paracetamol, guaiaphenesin and phenylpropanolamine hydrochloride. The relative concentrations of the components and their relative molar absorbances represented a range of three orders of magnitude of detectability.A reversed-phase HPLC system was developed that resolved and permitted quantification of each of the active ingredients. By the same system it was possible to check for degradation by assaying the content of 4-amino- phenol. The contents were found to comply with the label claim; 4-aminophenol was not detected at a limit of detection of 0.004y0 and the product had the same chromatographic profile as an authentic specimen. For a sample restricted to a single tablet the assays and other information would have been difficult to achieve by application of classical methods. The second sample was of a single tablet alleged to contain phenylbutazone and thought to be several years old.The Laboratory was asked to determine the content of any phenylbuta- zone and to report any degradation products. The active ingredient was confirmed to be phenylbutazone by spectrophotometric techniques. The two reported major degradation products of phenylbutazone were synthesised and a normal-phase HPLC system developed that resolved these from phenylbutazone. This HPLC system was used to determine the phenylbutazone content of the sample and to demonstrate that no degradation products were present to a limit of O.Olyo. The chromatographic data demonstrated that the tablet was in remarkably good condition. As with the decongestant, it would have been difficult to obtain these data from a single tablet by the application of the classical pharmacopoeial titrimetric method.April, 1982 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS Ergometrine Maleate 195 The Laboratory examined a wide range of ergometrine maleate injections because some preparations had been alleged to be ineffective.It was not possible to use the methods of analysis defined in the BP monograph for two reasons : additives in the injections interfered with the TLC limit test for related substances and high proportions of related substances were found to react with the complexing reagent of the colorimetric assay, thus apparently assaying as ergometrine. A reversed-phase HPLC system was developed that was used to resolve ergo- metrine from both related substances and additives. This permitted the determination of both the ergometrine maleate and the additives.In addition, this system also permitted some long-term indication of the development of degradation products. Amy gdalin A reversed-phase system was used to quantify amygdalin, amygdalinamide and amygdalinic acid in a sample and two comparison materials. HPLC was used because amygdalin is not defined in any monograph and also, because of instability, it must be assayed under relatively mild condi- tions. Semi-preparative HPLC was used to collect fractions for identification by 13C NMR spectroscopy. By modification of the solvent system, making it less polar, it was possible to resolve the R- and S-epimers of amygdalin and amygdalinamide. An HPLC system was developed for the analysis of amygdalin injection. Conclusion The eight examples cited demonstrate some applications of HPLC to difficult analytical problems.The technique possesses the further significant advantage that sample preparation, with its inherent errors, may be minimised. Tablets generally only require extraction and centrifuging and injections may be introduced directly from the ampoule or after the addition of internal standard. The data obtained by HPLC on the samples discussed would have been difficult to obtain by other means. The Author thanks the Government Chemist for permission to publish this paper. Recommended Methods of Analysis in the Water Industry and Their Use of Chromatography B. T. Croll Co-ordinator of Scienti,fic Research and Laboratories, Anglian Water Authority, Diploma House, Grammar School Walk, Huntingdon, Cambridgeshire, PE18 6NZ Recommended methods of analysis in the water industry date back to the Royal Commission on Sewage Pollution of 1904.Part V of the 4th report of the Commission was entitled “Methods of Chemical Analysis as Applied to Sewage and Sewage Effluents.” This report was updated in 1929 and 1956. In 1958 a joint committee of the Association of British Chemical Manufacturers and the Society for Analytical Chemistry published a book entitled ‘Recommended Methods of Analysis for Trade Effluents,” complementing the 1956 revision of the government publication. The Ministry of Housing and Local Government (1964) ap- pointed a committee to revise and combine the 1956 and 1958 publications, resulting in the book “Analysis of Raw, Potable and Waste Waters” in 1972. The present organisation SUC- ceeded the 1964 committee and was revised early in its life, on the reorganisation of the Water industry in 1974, to form the joint DOEINational Water Council Standing Committee of Analysts. The work of the committee is to review and revise methods of analysis used in all parts of the water cycle and, unlike earlier documents, its publications include methods for sediments, sludge, biota, etc.Membership is drawn from all interested bodies, including government departments, Water Authorities, industry, consultants and the academic world.196 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS Committee Structure Anal. Proc. The field of work is large and, in order to obtain a maximum degree of expert input to the individual analytical methods, a sub-structure of permanent Working Groups covering nar- rower fields was created, as follows: WG.1 . . .. .. . . General Principles of Sampling and Results WG. 2 .. .. .. . . Instrumentation and On-line Analysis WG. 3 . . .. . . . . Empirical and Physical Methods WG. 4 . . . . . . . . Metals and Metalloids WG. 5 . . . . . I . . General and Non-metallic Substances WG. 6 . . . . .. . . Organic Impurities WG. 7 . . .. .. . . Biological Methods WG. 8 . . .. . . . . Sludge and Other Solids WG. 9 .. .. * . . . Radiochemical Methods The Working Groups are further sub-divided into panels who write the methods and arrange Panels are formed for a specific purpose and are disbanded when their performance testing. task is completed. Format of Publication In order to avoid the inevitable delays in the production of a book, where the last contribu- tion dictates the timing, and minor revisions are not possible, it was decided to publish a series of pamphlets containing individual or related groups of methods.Each method would be well defined, unambiguous and thoroughly performance tested. Methods where perform- ance data from only one source are available are labelled “Tentative.” Methods are written in a format originally devised at the Central Electricity Research Laboratory and broadly follow IS0 guidelines. The first page summarises the method performance characteristics, the experimental instructions are given in a two-column action and note form. Such a format is clear and quickly enables the analyst to assess whether the method suits his needs.Measure- ment tolerances are given in all instances and in general care is taken to provide unambiguous instructions. Progress The work of the committees effectively began in 1975, following the reorganisation of the water industry, and the first methods were produced in 1976 and published in 1977. The major delays in production of methods have been in securing the necessary time and effort to produce performance characteristics in a period of continually increasing economic pressure. To date 26 booklets have been published, 31 are being edited or awaiting printing and 40 are in various stages of draft circulation. The total number of indvidual methods written is ap- proximately 250, and these have been combined into the number of booklets indicated above.Most of the work of the committee in revising, re-writing and adding to earlier publications will be completed by the end of 1981. Its role will then be to introduce new methodology where it will show advantages over existing published methods and methods for new determin- ands as circumstances dictate. EEC leglisation may be a major input on this latter point. Chromatographic Methods Only three chromatographic methods have been published, all using gas chromatography : The Determination of Volatile Fatty Acids in Sewage Sludge ; The Analysis of Sludge Digestor Gas; Organochlorine Insecticides and Polychlorinated Biphenyls in Waters. Methods have been written, and are at various stages of development, for several other methods, namely, the gas-chromatographic analysis of oils, various pesticides, methanol, phenols and trihalogenated methanes ; thin-layer chromatographic analysis of polynuclear aromatic hydrocarbons, oils and non-ionic detergents ; and the high-performance liquid chromatographic analysis of polynuclear aromatic hydrocarbons.There were originally some doubts as to whether chromatographic methods could be written to the format adopted by the committee; these have proved unfounded, but some difficultiesApril, 1982 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS 197 have arisen. Chromatographic methods are more dependent on instrument than spectro- photometric methods and the committee decision not to name manufacturers has caused some problems. In some instances naming has been unavoidable, for instance where a reagent is manufactured by only one company, and the clause “or equivalent’’ has been inserted after the reagent name in order not to prejudice future commercial developments.Specification of gas-chromatographic columns has proved difficult, as each manufacturer uses different lengths and diameters, and recommendations with respect to items such as sup- port particle size vary. Additionally, many analyses may be performed on a variety of sta- tionary phases with equallygood results. The approach in these instanceshas been to specify the desired separation characteristics (e.g., theoretical platage) required and to quote some columns that have proved satisfactory. These are often defined as the peak-to-peak base-line noise multiplied by a constant.Such a system takes no account of interfering peaks in a sample and the policy adopted has been to measure replicates of blanks and to calculate limits of detection based on a significant difference of samples from the blank a t the 950/;, confidence level. The system works well until one encounters a method where the base line is absolutely flat under the chromatographic conditions employed for the working range of the method, and obviously nothing can be calculated from a series of zero values. Two approaches are possible in order to obtain real values: firstly to increase the amplifier gain until noise is encountered or secondly to measure the scatter of low-level samples. Both approaches will be found in the methods. Wherever possible a larger column selection is tabulated.Calculation of limits of detection has caused some problems. Fingerprinting by Programmed-temperature Gas Chromatography Using Packed Columns A. M. Humphrey Bush Boake A l l a n Ltd., Blackhorse Road, London, El7 5QP Essential oils are particularly suited to examination by gas chromatography, and the intro- duction of temperature programming has allowed the separation of most of the major com- ponents normally found. Under conditions of temperature programming, essential oils give a gas chromatographic “fingerprint” that can be used to identify the oil and its geographic origin and possibly detect the presence of adulteration or other irregularities. The advantages of being able to produce reproducible fingerprints are obvious. Results obtained in different laboratories or in the same laboratory with a time interval could be reliably compared, but attempts to achieve this in the past have failed.This was largely due to the inability to prepare reproducible column packings and hence to reproduce chromatographic performance. Close specifications of the operating conditions of the gas chromatograph were not sufficient in the absence of standardised column packings and the AMC Essential Oils Sub-committee has sought ways of achieving and specifying such columns. In retrospect, they have relied heavily on the work of Van den Do01 and Kratz on this subject. Background The approach of Van den Do01 and Kratz was to define the polarity interactions of a packed column with six test compounds by applying a series of calculations to the experi- mentally determined relative retention indices.The procedure finally produces a single figure described as the G-pack value for the column and is a measure of its polarity. This figure can then be used to specify the performance of a column in respect of its polarity and the choice of the test compounds is made such that the resolving powers of columns prepared with specific stationary phases can also be determined and hence specified. The nature of the six test compounds is related to essential oil analyses and they are limonene, linalol, linalyl acetate, naphthalene, acetophenone and cinnamic alcohol. When using polyethylene glycol 20M columns, linalol and linalyl acetate are closely eluted and their separation can be used to measure chromatographic efficiency.With non-polar columns, such as methyl polysiloxane, the pair limonene and acetophenone can be similarly used. In addition, the presence of linalyl acetate in the mixture can be used to detect any catalytic decompositionAnal. Proc. caused by the columns. Linalyl acetate is particularly sensitive to this type of decomposition and the appearance of spurious peaks and a reduction in the expected peak height for linalyl acetate serves to indicate catalytic activity. The Sub-committee adopted the method of Van den Do01 and Kratz to define column performance and detect ageing or other changes in column performance indicated by polarity or resolving power changes. Having established confidence in the value of the G-pack determinations, the Sub-committee proceeded to investigate the effects of changes in operating parameters in order to establish the levels of variations that could be tolerated whilst still achieving reproducible results.The first parameter to be investigated in detail was the stationary phase loading. Operating Parameters When using the slurry coating method of preparing packings, the exact stationary phase loading is not known accurately and so an alternative procedure was adopted in which the stationary phase was dissolved in the minimum volume of solvent and the entire solution absorbed on to the support. Under these conditions, the stationary phase and its solvent are rapidly distributed throughout the support, which remains externally dry and free flowing. After storage in a closed flask for 24 h, a perfectly even distribution is obtained and the solvent is allowed to evaporate, leaving an accurately known level of stationary phase on the support.The "absorption"-coated packings were found to be superior to "slurry"-coated packings in several respects. They are very reproducible in both methods of preparation and performance and in addition have a significantly lower bleed at elevated temperatures. They have flatter Van Deemter curves, which in practice means that their optimum resolving power is less affected by changes in carrier gas flow-rate and they do not require extensive conditioning prior to use. Each of these advantages is attributed to a better and more even coating of the stationary phase on the support. Using absorption-coated supports with polyethylene glycol 20M, it was established that changes in stationary phase loading had very little effect on the relative retention indices, although there was a small but measurable decrease in the polarity of the columns as the stationary phase loading was decreased.However, the effect on retention temperatures showed a sinusoidal relationship with the point of minimum inflection at 15% loading on Chromosorb W HP. Similar curves were obtained when using Gas-Chrom Q but the point of minimum inflection occurred at 12yo and the change was attributed to the lower specific surface area of the Gas-Chrom Q. These findings are used in support of the final recommenda- tion. The effect of changes in the temperature programming rate were also examined with a view to determining the optimum rate required to achieve maximum resolution with the lowest elution temperatures consistent with economic analysis times.Graphs of programming rates against relative retention indices showed that they were unaffected by the programming rate but that elution temperatures were, as expected, markedly affected. The actual figures showed that a programming rate of 2 "C min-l allowed a convenient elution of the C,, alkane at 225 "C when using polyethylene glycol 20M. I t had been previously established by a gravimetric examination of an absorption-coated packing that an upper temperature limit of 225 "C could be applied almost indefinitely without damaging a column prepared with such a packing. In order to achieve reproducible fingerprints it is necessary to achieve reproducible elution temperatures for the test compounds and the n-alkanes used as markers, because of the variable dependence of relative retention indices on temperature.The effect is more marked in general for aromatic than non-aromatic compounds. In addition, the relative retention indices are markedly affected by the polarity of the column and hence the necessity of defining the G-pack values of the columns used within close limits. In practice, the elution of the C,, alkane at 225 "C may be adjusted by varying the carrier gas flow-rate, taking advantage of the minimal effect that this has on the efficiency of absorption-coated packings. Conclusions The final proposals made by the Sub-committee for the reproducible fingerprinting of essential oils by programmed-temperature gas chromatography on packed columns using polyethylene glycol 20M were based on collaborative studies, and after acceptance by the 198 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGSApril, 1982 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS 199 AMC, were published in The Analyst, 1980, 105, 262.A second proposal on the use of non- polar stationary phases of the methyl polysiloxane type has also been prepared, accepted and published (An,alyst, 1981, 106, 448). Development of Official Methods for the Analysis of Medicinal Additives in Animal Feeds Ronald S. Hatfull County Chemical Laboratory, Staffordshire County Council, Martin Street, Staffoord The need for analytical procedures of proven validity for inclusion in the Statutory Instruments required for enforcement of the Medicines Act, as it applied to animal feeding stuffs, has been the driving force behind the establishment of specialist Sub-committees under the Analytical Methods Committee of the RSC Analytical Division.The history and formation of these Sub-committees was briefly surveyed and an outline was given of the approach of the Sub-committee to this work. Typical examples of this work were described, and it was shown how this work over the past 20 years had reflected progress in analytical chemistry, in particular with instrumental methods. Chromatography in its various applications had been a major analytical tool with a gradual transition to an increasing application of high-performance liquid chromatography.The Analytical Methods Committee-The Work of the New Animal Feedingstuffs Sub-committee G. H. Smith Pauls & Whites Foods Ltd., Ipszvich The review of the work on the Analytical Methods Committee (AMC), some 2 years ago, led to the proposal to form several new Sub-Committees, of which the Animal Feeds Sub-committee was one. Following discussions with the Ministry of Agriculture, Fisheries and Food (MAFF), the following Terms of Reference of the Sub- committee were agreed : 1. To arrange such collaborative studies as may be considered necessary with the agreement of the Government Chemist acting for MAFF on proposals for statutory methods of analysis for animal feedingstuffs, including pet foods. These would essentially appear under Part IV of the Agriculture Act 1970.2. To discuss the results of these studies and advise MAFF for possible incorporation, after formal consultation with interests, into Regulations. 3. To publish new and agreed methods in The Analyst. A grant for some expenses was to be made by MAFF. The AMC has always been concerned with quality, as the recent review by Eganl has shown. Consumer protection, in terms of analytical methods, to form the basis of official standards, was an early feature of the AMC in the 1920s. In fact, a group was working on a fibre method for feeding-stuffs as early as 1926, so there was an early precedent for the type of work covered by the present Committee. The essentials are, in my view, to subject products to statutory control where, in the broadest sense, it is in the customers’ interest so to do, and if this means analytical control of stated content, or freedom from undesirable substances, then clearly analytical methods must be capable of accurate use in quality control by the manufacturer without rendering the process of production uneconomic.As compound animal feeds contain such a wide range of naturaI products, it is becoming clear that there will always be a measure of risk of not conforming to one or more of the many, and increasing, statutory controls. The first meeting was held in May 1980.200 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS Membership Anal. Proc. Membership was initially proposed to represent various interests related to animal feeds : (a) TJze Feed Trade-three members, including the Chairman and one from the specialist area of petfoods.(b) The Agricultural Development and Advisory Service (ADAS)-two members. This Government service includes nutrition chemists, responsible for advice to farmers, and analytical chemists in 13 laboratories in the UK. (c). Oficial Agriculturai Analysts-two members from areas that are largely agricultural. One is in practice as a consultant with the Agricultural Analyst appointment, and the second is from a County Analyst’s Department. (d) TJze Laboratory of the Government Chemist-the Agricultural Materials Division is repre- sented by two members. Proposals for methods to be considered came from this Department, which has a referee function in cases of dispute arising from analysis of Trading Standards Departments’ samples.(e) A statistician-for consultation both in presentation of results and in the early stages of planning collaborative services. I t does appear from papers prepared by Steiner2 on this subject and his short address recently to the AMC, that restriction of the number of laboratories participating is perhaps one of the main difficulties. (f) Others-we recognised that there would be specialists organisations who might make valuable contributions, according to the subject under consideration. (g) Secretary of the Analytical Methods Committee. May supply methods into Trade Committees. Subjects for Consideration Initially, the Laboratory of the Government Chemist proposed consideration of analysis for the following parameters: (1) vitamin A; (2) oils and fats; (3) x-tocopherols (vitamin E); (4) vitamin D ; (5) carotenoids; (6) nicotinic acid; (7) choline; (8) amino acids; (9) vinyl- oxazolidinethione (VOT) ; (10) volatile mustard oil (isothiocyanates, ITC) ; (11) theobromine; (12) hydrocyanic acid.Topics 9-12 relate to substances deleterious to the recipient animal, e.g., VOT and volatile mustard oil occur in rapeseed and are toxic principles leading to goitrogenic effects. Volatile mustard oil is a general term for allyl, but-3-enyl and pent-3-enyl isothiocyanates. Fortunately, rapeseed varieties are now being bred in which these toxins are at a very low level. Theobromine occurs in cocoa products sometimes used in feeds. Hydrocyanic acid can occur in manioc (cassava or tapioca), a rich source of starch that has become increasingly considered alongside traditional cereals in this country.Some of the substances listed for our attention are already the subject of statutory control under the Agriculture Act 1970, Part IV. As members of EEC, the UK must recognise methods of analysis that include those which were already embodied in EEC legislation 10 years ago. Several methods were then unfamiliar to UK analysts and the process of harmon- isation is long and often difficult. Subjects 1-8 are related to substances of nutritional importance. EEC Directives EEC Directives from 1971 to 1978 give detailed methods of analysis, some of which had been laid down when the UK joined the Community. The first methods, published in 1971, we would hardly regard as being of first priority in the UK and certainly not the simplest to agree upon, although a simple ashing method appears.In the UK 1973 Regulations under Part IV of the Agriculture Act 1970 there were at that time a total of 13 statutory methods of analysis. Methods published in Directives were discussed in the presentation and amount to 38 methods for nutritional parameters or deleterious substances with a further 16 methods relating to medicines. By 1976 UK Regulations had incorporated methods for 22 parameters plus five alternative methods. In 1981 new Regulations will list at least 33 methods, some of which are currently little used in UK laboratories and have not been tested here by collaborative work. Pet foods will be embodied in the Regulations with the attendant difficulties of applying methods developed for relatively dry materials to high moisture canned products.April, 1982 OFFICIAL METHODS OF ANALYSIS OF NEW DRUGS 20 1 The relationship of this Sub-committee in the development of Statutory EEC methods is shown in Fig.1. ANALYTICAL METHODS COMMITTEE The Analyst -ms 1x14- 17 1 !::heodd 47 UK Representative EEC COMMITTEE OF EXPERTS Interests: UKASTNFEFAC Consultation, PROPOSED METHOD I EEC COUNCIL APPROVAL I Fig. 1. Official methods in the EEC. Present Work of the Committee Oil in Oilseeds and Their By-products The UK method has traditionally been extraction, using boiling light petroleum (boiling range 40”-60 “C) in a Soxhlet or straight-through extractor for about 4 h, evaporation of the solvent and weighing the oil residue.I t is an empirical standard but works well in commercial practice. I t has a number of pitfalls, for example, the definition of the solvent is imprecise and some batches may leave a residue; some natural products such as milk powders do not yield their oil content to this extraction without other treatment, so in applying this method to such materials a separate technique is given in our UK Regulations. For some years the UK has been trying to achieve a change from diethyl ether, used in the EEC method, because the use of this solvent tends to extract a wider range of lipids, particu- larly waxy substances of little use to the animal, and is, of course, more hazardous in use. The EEC method appeared about 10 years ago and is now under review. Results from our col- laborative exercises are being used by the UK delegates to the Committee of Experts in Brussels in support of a change.Vitamin A We are seeing at the present time the rapid adoption of HPLC for many applications in feed particularly for medicinal additives and it is this technique which has been proposed as th? basis for the new method for determining vitamin A. The existing method in the 1976 Regulations in a sample of feed (a) separates the unsaponi- fiable matter, (b) separates the vitamin A by column chromatography and (c) finally measures the colour of this extract after reaction with Carr - Price reagent. I t was stated not to be suitable for highly pigmented feeds below 10 units g1 or below 4 units g-l in other feeds. No- one knows exactly what is meant by “highly pigmented.” It is common practice to add between 5 and 10 units g-l of vitamin A to poultry layer foods, so it followed that in many instances the method was not suitable.The present statutory method is unsatisfactory at the column chromatographic stage in that there is poor definition of the fluorescent zone of vitamin A on the aluminium oxide column-Anal. R o c . this leads to low results from being too careful and collecting a small fraction or problems aris- ing from collecting too great a fraction which includes interfering pigments. Further, it is clear that sample size and preparation are important, as the gelatine beadlets each contain about 4 units of vitamin A so that in the 30-g sample of feed, when the final product should contain 10 units g-l, there can be an average prediction of 75 beadlets. Break- ing beadlets in sample grinding can lead to loss, so there is a built-in tendency for low results to be obtained. In the present situation, Denmark has proposed a method in which an extract of unsaponi- fiable matter is subjected to HPLC using a Kieselgel column and gradient elution. The LGC method, which is at present under trial, favours reversed-phase columns; it also recognises the relatively high cost of gradient elution apparatus. The aim of the method is to separate the vitamin A isomers (together if necessary) from interfering substances. 202 THERMAL ANALYSIS VOT and ITC The toxic principles in rapeseed, vinyloxazolidinethione (VOT) and volatile mustard oil, or isothiocyanates (ITC) , have been examined in a preliminary manner. ITCs are determined as allyl, but-3-enyl and pent-4-enyl isocyanates, but “volatile mustard oil” is expressed as allyl isocyanate derived from the sum of these ITCs. Treatment of the rapeseed with thioglucosidase prepared from freshly ground mustard seed (Sinapsis alba) results in the liberation of the ITCs, which are determined by GLC using an integral standard of butyl isothiocyanate. Two methods for the determination of VOT were attempted-GLC and HPLC. The latter technique was favoured by most members, but it is too early to say that the method as it stands is satisfactory. It is becoming clear from the vitamin A work and the VOT method that it is difficult to lay down precise HPLC conditions. Standardisation of a regulatory method in this new field does present apparent problems, although practical results from the technique are obtained so much faster that HPLC is clearly the most important technique in the field of feed analysis since atomic-absorption spectrophotometry became widely adopted in the trade nearly 20 years ago. Finally, it should not be forgotten that in any collaborative work sample presentation and sample preparation are vital. Simple addition of a component to one raw material or a mixture of feed ingredients may be necessary during method development, but finally the method must be valid on a mixed feed as sold and interaction of feed ingredients losses in the extrusion process for pellets, etc., must not be ignored. Preliminary results were encouraging. References 1. 2. Egan, H., Chem. Brit., 1980, 16, 642. Youden, W. J., and Steiner, E. H., “Statistical Manual of the Association of Official Analytical Chemists,” AOAC, Washington, D.C., 1975.

ISSN:0144-557X    

DOI:10.1039/AP9821900189

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

202 THERMAL ANALYSIS Anal. R o c . Thermal Analysis The following is a summary of the paper presented at a Joint Meeting of the Scottish Region with the South West Scotland Section of the RSC and the Andersonian Society held on November 20th, 1980, at the University of Strathclyde,, Glasgow. Thermal Analysis and Some Analytical Applications R. C. Mackenzie The Macaulay Institute for Soil Research, Craigiebuckler, A berdeen, A B9 2Q J According to current definition and usage,l thermal analysis covers “a group of techniques in which a physical property of a substance is measured as a function of temperature whilst ihe substance is subjected to a controlled temperature programme.” This gives no indica-April, 1982 THERMAL ANALYSIS 203 tion of the criteria employed in the dictionary definition of “analysis,” which, in this context, is therefore a misnomer : yet many of the techniques included have analytical applications. As it is impossible in brief compass to cover all of the methods available,l only the more common, namely thermogravimetry (TG), derivative thermogravimetry (DTG), differential thermal analysis (DTA) , differential scanning calorimetry (DSC) and evolved gas analysis (EGA), will be referred to and only a few relevant applications can be mentioned in illustra- tion.Standard definitions of the various techniques have been given elsewherel and equipment will not be discussed here, as many commercial instruments are now on the market and criteria for assessment of the best for a specific application can be found in standard text^.^-^ Many of the illustrations below are taken from these volumes. These may, however, suffice to indicate the type of information that can be gained. Thermogravimetry and Derivative Thermogravimetry A typical curve obtained for sample mass against temperature consists of a series of plateaux connected by more or less steep sloping sections or steps : on the DTG curve, which is the first derivative with respect to either time or temperature, the steps appear as peaks and small changes of slope are much more readily observed than on the integral curve.The classical use of TG in analytical chemistry was that of Duval,6 who successfully employed it to assess the most likely precipitates for gravimetric analysis, using as a criterion the temperature interval of mass constancy and rejecting materials that gave no reasonable mass-constant segment on the curve.However, even this criterion must be applied with care, as the curves are obtained under a dynamic temperature programme, whereas precipi- tates are ignited at a constant or near constant temperature. Consequently, each likely precipitate must be tested under normal gravimetric conditions before its suitability can be correctly assessed. Another obvious application of TG or DTG in analytical chemistry is in the quantitative determination of volatile materials or of the compounds that produce them, the height of the step in TG, or the area of the peak in DTG, giving accurate mass-change values. Again, however, care must be exercised, particularly with heating in air, as any readily oxidisable ion in a decomposing substance can be oxidised during decomposition, thus giving a mass change that is the result of the mass loss due to decomposition and the mass gain due to oxidation.This difficulty can be overcome by careful selection of the furnace atmosphere. Similar care is necessary with other classes of compounds such as carbonates: for example, the mineral ankerite (Ca,Fe,Mg)CO,, when heated in air may show only one decomposition step, but in a carbon dioxide atmosphere three steps (owing to the decomposition of three components) become readily visible. Differential Thermal Analysis and Differential Scanning Calorimetry The distinction between these two techniques has already been considered’ and need not be elaborated, except to note that, although the two grade into each other, DSC is the better quantitative technique for detecting enthalpy change.As with TG, great care must be exercised in atmosphere selection but, unlike TG, neither has much direct analytical chemical application. One possible direct application of DTA to chemical analysis arises from the observation that the DTA curves of an ion-exchange resin saturated with a specific cation are unique for that cation.s However, the curves are so complex that, should several cations be present, it is unlikely that the mixture could be disentangled. Although the occasional possibility of distinguishing several polymers in admixtureg might also be regarded as a direct analytical application, the main uses of both DTA and DSC in chemical analysis are undoubtedly indirect.Thus, it is frequently of little use to know the chemical composition of a substance without having any information as to how the elements present are combined into compounds. Although X-ray diffraction is usually the best technique for solving such problems, DTA can add its own quota of information. It has indeed been used in clay mineralogy for such purposes and, even where the information it yields is not completely diagnostic, sometimes a simple chemical pre-treatment can render it so.l0 In clay mineralogy, too, DTA is particu-204 THERMAL ANALYSIS Anal. Proc. larly sensitive to such accessory minerals as carbonates and sesquioxide-hydroxides and can reveal their presence in amounts that might be missed by X-ray diffraction.3 In the recent past, there have been several tragedies through the sudden collapse of buildings constructed with high-alumina cement concerete and simple methods for assessing the “degree of conversion’’ of this material have been in demand.DTA has proved to be one such method and is now a standard method of assessment.ll The application of DTA and DSC to organic compounds has increased tremendously over the past decade: this has been particularly so in the fields of polymers and pharmaceuticals, where these methods have been applied to detect glass transitions, degree of cure, impurities, polymorphism, etc. Moreover, natural products can be readily distinguished from substi- t u t e ~ , ~ proteins can be distinguished in aqueous solution12 and DSC can be employed for the quantative determination of the purity of highly purified samples (greater than 99%) of organic c o r n p o ~ n d s .~ ~ , ~ ~ Therefore, these techniques can provide very valuable information to the analyst. Evolved Gas Analysis Evolved gas analysis is usually employed along with TG and/or DTA, the volatile sub- stances produced on heating being passed through a suitable detecting system by a stream of carrier gas. Two types of detector can be used: where the gases likely to be evolved are known, detectors specific for one gas can be used either alone or in seriesl5 or, where this information is not available, a universal system, such as a mass spectrometer,16 can be employed. The second system is certainly the more versatile and ensures that no informa- tion is likely to be lost, but care must be taken with the interface to ensure no volatile product is preferentially enhanced or depleted.Such a system can readily, for example, indicate whether a mass or enthalpy change is due to loss of carbon dioxide, sulphur dioxide, water, etc., and thus reveal the nature of the material decomposing15 as well as the amount of volatile material evolved: it can also be used to elucidate the mechanism of decompo~itionl~ and even to enable design improvements to be made to electronic components, such as semiconductor contacts.ls Conclusions It will be evident that thermal analysis covers very few truly analytical techniques but that it can yield information that is of immense value to the analyst, although whether this justifies use of the term “analysis” must remain in dispute.Information on other analytical uses of the above techniques, and of several techniques not mentioned, will be found in Thermal Analysis Abstracts, published every two months by Heyden and Son, London, for the International Confederation for Thermal Analysis. 1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. References Mackenzie, R. C., Thermochim. Acta, 1979, 28, 1. Lodding, W., Editor, “Gas Effluent Analysis,” Marcel Dekker, New York, 1967. Mackenzie, R. C., Editor, “Differential Thermal Analysis,” Academic Press, London, 1970 and Wendlandt, W. W., “Thermal Methods of Analysis,” Second Edition, John Wiley, New York, 1974. Keattch, C. J., and Dollimore, D., “Introduction to Thermogravimetry,” Heyden, London, 1975.Duval, C., “Inorganic Thermogravimetric Analysis,” Second Edition, Elsevier, Amsterdam, 1963. Mackenzie, R. C., Anal. Proc., 1980, 17, 217. Garcia-Ramos, L. A., Montagut, M., and Vinyes-Miralpeix, J . M., J . Therm. Anal., 1970, 2, 429. Chiu, J., Du Pont Thermogram, 1965, 2, 9. Mackenzie, R. C., and Robertson, R. H. S., Acta Univ. Carol. Geol., Suppl. 1, 1961, 139. Midgley, H. G., J . Therm. Anal., 1978, 13, 515. Steim, J . M., Arch. Biochem. Biophys., 1965, 112, 599. Plato, C., and Glasgow, A. R., Anal. Chem., 1969, 41, 330. DeAngelis, M. J., and Papariello, G. J., J . Pharm. Sci., 1968, 57, 1868. Morgan, D. J . , J . Therm. Anal., 1977, 12. 245. Chiu, J., and Beattie, A. J., Thermochim. Acta, 1980, 40, 251. Price, D., Dollimore, D., Fatemi, N.S . , and Whitehead, R., Thermochim. A d a , 1980, 40, 323. G.allagher, P. K., in Wiedemann, H. G., Editor, “Proceedings of the Sixth International Conference on Thermal Analysis, Bayreuth, 1980,” Volume 1, Birkhauser Verlag, Bade, 1980, p. 13. 1972 (2 volumes).204 THERMAL ANALYSIS Anal. Proc. larly sensitive to such accessory minerals as carbonates and sesquioxide-hydroxides and can reveal their presence in amounts that might be missed by X-ray diffraction.3 In the recent past, there have been several tragedies through the sudden collapse of buildings constructed with high-alumina cement concerete and simple methods for assessing the “degree of conversion’’ of this material have been in demand. DTA has proved to be one such method and is now a standard method of assessment.ll The application of DTA and DSC to organic compounds has increased tremendously over the past decade: this has been particularly so in the fields of polymers and pharmaceuticals, where these methods have been applied to detect glass transitions, degree of cure, impurities, polymorphism, etc.Moreover, natural products can be readily distinguished from substi- t u t e ~ , ~ proteins can be distinguished in aqueous solution12 and DSC can be employed for the quantative determination of the purity of highly purified samples (greater than 99%) of organic c o r n p o ~ n d s . ~ ~ , ~ ~ Therefore, these techniques can provide very valuable information to the analyst. Evolved Gas Analysis Evolved gas analysis is usually employed along with TG and/or DTA, the volatile sub- stances produced on heating being passed through a suitable detecting system by a stream of carrier gas.Two types of detector can be used: where the gases likely to be evolved are known, detectors specific for one gas can be used either alone or in seriesl5 or, where this information is not available, a universal system, such as a mass spectrometer,16 can be employed. The second system is certainly the more versatile and ensures that no informa- tion is likely to be lost, but care must be taken with the interface to ensure no volatile product is preferentially enhanced or depleted. Such a system can readily, for example, indicate whether a mass or enthalpy change is due to loss of carbon dioxide, sulphur dioxide, water, etc., and thus reveal the nature of the material decomposing15 as well as the amount of volatile material evolved: it can also be used to elucidate the mechanism of decompo~itionl~ and even to enable design improvements to be made to electronic components, such as semiconductor contacts.ls Conclusions It will be evident that thermal analysis covers very few truly analytical techniques but that it can yield information that is of immense value to the analyst, although whether this justifies use of the term “analysis” must remain in dispute.Information on other analytical uses of the above techniques, and of several techniques not mentioned, will be found in Thermal Analysis Abstracts, published every two months by Heyden and Son, London, for the International Confederation for Thermal Analysis.1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. References Mackenzie, R. C., Thermochim. Acta, 1979, 28, 1. Lodding, W., Editor, “Gas Effluent Analysis,” Marcel Dekker, New York, 1967. Mackenzie, R. C., Editor, “Differential Thermal Analysis,” Academic Press, London, 1970 and Wendlandt, W. W., “Thermal Methods of Analysis,” Second Edition, John Wiley, New York, 1974. Keattch, C. J., and Dollimore, D., “Introduction to Thermogravimetry,” Heyden, London, 1975. Duval, C., “Inorganic Thermogravimetric Analysis,” Second Edition, Elsevier, Amsterdam, 1963. Mackenzie, R. C., Anal. Proc., 1980, 17, 217. Garcia-Ramos, L. A., Montagut, M., and Vinyes-Miralpeix, J . M., J . Therm. Anal., 1970, 2, 429. Chiu, J., Du Pont Thermogram, 1965, 2, 9. Mackenzie, R. C., and Robertson, R. H. S., Acta Univ. Carol. Geol., Suppl. 1, 1961, 139. Midgley, H. G., J . Therm. Anal., 1978, 13, 515. Steim, J . M., Arch. Biochem. Biophys., 1965, 112, 599. Plato, C., and Glasgow, A. R., Anal. Chem., 1969, 41, 330. DeAngelis, M. J., and Papariello, G. J., J . Pharm. Sci., 1968, 57, 1868. Morgan, D. J . , J . Therm. Anal., 1977, 12. 245. Chiu, J., and Beattie, A. J., Thermochim. Acta, 1980, 40, 251. Price, D., Dollimore, D., Fatemi, N. S . , and Whitehead, R., Thermochim. A d a , 1980, 40, 323. G.allagher, P. K., in Wiedemann, H. G., Editor, “Proceedings of the Sixth International Conference on Thermal Analysis, Bayreuth, 1980,” Volume 1, Birkhauser Verlag, Bade, 1980, p. 13. 1972 (2 volumes).

ISSN:0144-557X    

DOI:10.1039/AP9821900202

出版商:RSC    

年代:1982

数据来源: RSC