摘要:

168N Analyst, November 1996, Vol. 121 Technical Abbreviations and Acronyms The presence of an abbreviation or acronym in this list should NOT be read as a recommendation for its use. However, those defined here need not be defined in the text of your manuscript. AAS ac A P ADC ANOVA AOAC ASTM bP BSA BSI CEN CPm CMOS c.m.c. CRM CVAAS CW CZE dc DRIFT DELFIA DNA EDTA ELISA emf ETAAS EXAFS EPA FAAS FAB dPm FAO-WHO FIR FT FPLC FPD GC GLC HGAAS HPLC ICP id INAA 1R ISFET iv im IGFET ISE LC LED LOD LOQ atomic absorption spectrometry alternating current analogue-to-digital analogue-to-digital converter analysis of variance Association of Official Analytical Chemists American Society for Testing and Materials boiling point bovine berum albumin British Standards Institution European Committee for Standardization counts per minute complementary metal oxide silicon critical micellization concentration certified reference material cold vapour atomic absorption spectrometry continuous wave capillary zone electrophoresis direct current disintegrations per minute diffuse reflectance infrared Fourier transform spectroscopy dissociation enhanced lanthanide fluorescence immunoassay deoxyribonucleic acid ethylenediaminetetraacetic acid enzyme linked immunosorbent assay electromotive force electrothermal atomic absorption spectrometry extended X-ray absorption fine structure spectroscopy Environmental Protection Agency flame atomic absorption spectrometry fast atom bombardment Food and Agriculture Organization, far-infrared Fourier transform fast protein liquid chromatography flame photometric detector gas chromatography gas-liquid chromatography hydride generation atomic absorption high-performance liquid inductively coupled plasma internal diameter instrumental neutron activation infrared ion-selective effect transistor intravenous intramuscular insulated gate field effect transistor ion-selective electrode liquid chromatography light emitting diode limit determination limit of quantification World Health Organization spectroscopy chromatography analysis mP MRL mRNA MS NIR NMR NIST od OES PBS PCB PAH PGE PIXE PPt PPb PPm PTFE PVC PDVB QC QA REE rf RIMS rms rpm RNA SCE SE SEM SIMS SIMCA S/N SRM STM STP TIMS TLC TOF TGA TMS tris TRIS uv UV/VIS VDU XRD XRF YAG melting point maximum residue limit messenger ribonucleic acid mass spectrometry near-infrared nuclear magnetic resonance National Institute of Standards and Technology outer diameter optical emission spectrometry phosphate buffered saline polychlorinated biphenyl polycyclic aromatic hydrocarbon platinum group element particle/proton-induced X-ray parts per trillion (1012; pg g-J) parts per billion (1 09; ng g-1) parts per million (106; pg g-1) poly( tetrafluoroethylene) pol y (v in y 1 chloride) poly(diviny1 benzene) quality control quality assurance rare earth element radio frequency resonance ionization mass spectrometry root mean square revolutions per minute ribonucleic acid saturated calomel (reference) electrode standard error scanning/surfxe (reflection) electron microscopy secondary-ion mass spectrometry soft independent modelling of class signal-to-noise ratio Standard Reference Material scanning tunnelling (electron) standard temperature and pressure thermal ionization mass spectrometry thin-layer chromatography time-of-flight themogravimetric analysis trimethylsilane 2-amino-2-( hydroxymethy1)- propane-l,3-diol (ligand) 2-amino-2-(hydroxymethyl)- propane- 1,3-diol (reagent) ultraviolet ultraviolet-visible visual display unit X-ray diffraction X-ray fluorescence yttrium aluminium garnet emission analogy microscopy Commonly Used Symbols M molecular mass Mr relative molecular mass r correlation coefficient s standard deviation U atomic mass

ISSN:0003-2654    

DOI:10.1039/AN996210168N

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, November 1996, VoI. 121 1525 Foreword Vllth International Symposium on Luminescence Spectrometry in Biomedical Analysis. Detection Techniques and Applications in Chromatography and Capillary Electrophoresis The simposium was held in Sophia Antipolis, Nice, France, on April 17-19, 1996. The chairman, Professor Willy R. G. Baeyens, from the Faculty of Pharmaceutical Sciences at the University of Gent, joined with his colleagues Kazuhiro Imai (University of Tokyo), Benito Del Castillo (Complutense University of Madrid), Michel Rouillard and Roland Fellous (both from the University of Nice) to arrange this unique common event. The scientific programme featured seven invited lectures and three selected oral communications as well as poster presentations. Twelve contributions are published in this issue of The Analyst.G. Rao (University of Maryland, Baltimore, USA) empha- sized the most recent advances in fluorescence-based sensors and instrumentation. Traditional techniques of analyte concen- tration measurements relying on fluorescence intensity are now superseded by time-resolved techniques using phase-modula- tion fluorimetry. The topic of time-resolved luminescence of lanthanides as detection mode in HPLC was presented by A. Rieutord (Universitk de Paris-Sud, France). The probe can be added directly to the mobile phase or as post-column reagent. N. Dovichi (University of Alberta, Edmonton, Canada) reported on his studies on single alkaline phosphatase molecules captured in a capillary filled with fluorogenic substrate, Single enzyme molecules show a distribution of activity, while the activation energy of the reaction catalysed by a single molecule can be determined with high precision. Furthermore, the activity is measured after partial heat denaturation. J.-J.Aaron (Universitk Denis Diderot Paris 7, France) described how enhanced fluorescence detection techniques in flow injection and liquid chromatography can effectively serve in environmental monitoring and biomedical analysis. The lecture presented an overview of principles, recent instrumental aspects, performances and applications of these methods. H. Lingeman (Free University of Amsterdam, The Nether- lands) showed effectively how diode-laser induced fluores- cence detection can overcome limitations with regard to concentration sensitivity in capillary electrophoresis.Labelling procedures for thiols, maines, aldehydes, carboxylic acids and alcohols were also presented. The paper by D. L. Massart (Free University of Brussels, Belgium) gave an introduction to some of the chemometrical concepts as applied to spectral data (multivariate calibration methods, pattern recognition, evolving methods). W. Adam used the firefly to demonstrate a highly efficient light-producing system based on decomposition of dioxetanes. From here, he developed chemical models for bioanalytical applications, in particular chemiluminescent immunoassay probes. Novel developements in capillary electrophoresis were depticted in the contribution by N. Guzman (Princeton Biochemicals, USA), in which he particularly expanded on online derivatization of a peptide mixture by immobilized fluorescein isothiocyanate and peptide mapping.D. W. Fink (Merck Research Laboratories, West Point, USA) described how dehydrative aromatization provided a selective fluorogenic derivatization of the non-fluorescent antiparasitic agent ivermectin. The review chronicled the evolution of this reaction in bioanalysis with continuous improvements in sensitivity. A systematic approach to chemiluminescence reactions was developed by A. C. Calokerinos (University of Athens, Greece). He discussed the scepticism behind the investigation of novel chemiluminescence reactions and explained analytically useful methodologies emanating from intensive research. C. Poupon-Fleuret (Eduard Herriot Hospital, Lyon, France) presented in her paper the optimization of a method for the detection of metalloporphyrins by luminol chemiluminescence.The effect of hydrogen peroxide and of the composition of the reaction mixture was examined, while insight into the mecha- nism allowed the application of effective enhancers. The bright Mediterranean sun invited participants to stroll along the famous ‘Promenade des Anglais’, but it turned out that the motivated group of participants, coming from some 22 countries, was not ready to be tempted, obviously as a result of the high quality of the scientific programme. W. R. G. Baeyens D. De Keukeleire Faculty qf Pharmaceutical Sciences University of Gent, BelgiumCSI XXX PRE-SYMPOSIUM The Third International Conference on SPECIATION OF ELEMENTS IN BIOLOGICAL, ENVIRONMENTAL AND TOXICOLOGICAL SCIENCES The Torresian Resort Port Douglas, Queensland, Australia, September 15-1 9, 1997 INVITATION AND CALL FOR PAPERS The Organising Committee extends an invitation to all individuals involved in element research or its applications.A major goal of the symposium is to facilitate interdisciplinary and inter-sector discussion about all aspects of elements requiring an understanding of speciation, the five main themes of this symposium being : A, Speciation of Elements in Biology, Toxicology and Medicine; B, Speciation of Elements in Nutrition; C, Speciation of Elements in Environmental Toxicology; D, Surface and Particle Characterisation; and E, New Developments in Methods/Techniques of Species Determination.A small number of travel scholarships will be provided to encourage overseas graduate students to attend and participate. The symposium programme will comprise four days of oral presentations, posters and discussion. All presenters will be asked to focus on new developments in research. Oral presentations (invited or submitted) will be 20 or 30 mins in duration. As at previous symposia (Loen, Norway, 199 1 and 1994) posters will play a central role, after formal viewing each poster presenter will be given five minutes to present the salient features of their work to a discussion group to encourage in-depth feedback. The venue for the symposium, is The Torresian Resort of Port Douglas, Australia. This tropical Queensland location is situated near Cairns, between the Great Barrier Reef and the Daintree Rainforest.A Symposium Package rate has been arranged: AUD $155(per person, per night, twin share) and AUD $225 (single occupancy) and includes accommodation (Garden View Room) all meals and morning and afternoon teas. A limited amount of less expensive accommodation (room and board) will be available. This conference (as a pre-symposium to CSI XXX) is scheduled to allow the participants to join the XXX Colloquium Spectroscopicum Internationale (21-26 September) in Melbourne. As with previous Speciation Symposia (see The Analyst 117; 549-691 and 120; 29-30N and 583-763) all papers presented as posters or lectures may be submitted as full papers for publication in a special issue of The Analyst, subject to the normal review procedure of this journal.SOCIAL PROGRAMME All participants and accompanying persons are invited to the symposium reception on Monday evening, September 15, and the dinner on Friday evening, September 19. Because of the numerous attractions available (e.g., swimming, all other watersports, cruises, canoeing, hiking, horse riding etc.) no other formal social events are planned. However, please note that for each full day of scientific sessions, the period 15.30 onwards will be set aside for the enjoyment of the mentioned activities by all. Port Douglas has a comfortable, year round, tropical climate. Day tours to the outer Barrier Reef are available. The registration fee per delegate is AUD $480 (AUD $150 for students)and includes the cost of the symposium dinner. SECRETARIAT Local (Registration) THE SCIENTIFIC PROGRAMME SYMPOSIUM LOCATION AND DETAILS CONFERENCE PROCEEDINGS REGISTRATION FEE Third Speciation Symposium c/o Dr J. P. Matousek, Department of Analytical Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia Tel: + 61 2 3854713 or + 61 2 4512322 (home) Fax : + 61 2 3856141 E- m a i I : M atou se k@un sw . edu. au The University of New South Wales (Sydney, Australia) The National Institute Of Occupational Health (Oslo, Norway) The Institute of Environment and Health (Universities of Toronto and McMaster, Canada) MAFF CSL Food Science Laboratory (Norwich, UK) ORGAN IZING COMMITTEES Local Programme THE SYMPOSNJM IS ORGANISED BY : Graeme Batley (CSIRO, Lucas Heights) R. (Dick) Finlayson (Sydney, NSW) D. Brynn Hibbert (Sydney, NSW) Jarda P. Matousek (Sydney, NSW) Helen Crews (MAFF CSL, UK) Jarda P. Matousek (Sydney, NSW) Evert Nieboer (Hamilton, Canada) Yngvar Thomassen (Oslo, Norway)

ISSN:0003-2654    

DOI:10.1039/AN9962101525

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, November 1996, Vol. 121 (1527-1531) 1527 From the Firefly Bioluminescence to the Dioxetane-based (AMPPD) C hemil umi nescence lmmunoassay : a Retroanalysis* Waldemar Adam, Dirk Reinhardt and Chantu R. Saha-Moiler Institute of Organic Chemistry, University of Wurzburg, Am Huhland, 0-97074 Wiirzburg, Germany. E-mail: Adam@chemie.uni-wuerzburg.de Chemiluminescent dioxetanes, which are enzymically triggeiable, have proved to be a valuable tool in diagnostic clinical applications. In this context, the phenomenon of firefly bioluminescence (the most efficient light-producing system so far) has served as an excellent example to develop new chemiluminescent probes for bioanalysis, in particular chemiluminescence immunoassays. From the recognition of the molecular details of the firefly bioluminescence, a chemical equivalent has been conceived which possesses the essential features for efficient generation of light, i.e., thermal persistence through spiroadamantyl substitution, spontaneous chemically initiated electron exchange luminescence (C1EEL)-active decomposition after enzymic triggering, efficient light emission as a lasting glow and convenient synthesis by photooxygenation.This has led to a rationally designed dioxetane, namely the 3-(2’-spiroadamantyl)-4-rnethoxy-4-(3’’-phosphoryloxy)- phenyl-1,2-dioxetane (AMPPD) derivative, which serves as a substrate for alkaline phosphatase and, in terms of sensitivity, has surpassed the hazardous radioactive immunoassay probes. This paper gives a historical account in the form of a retroanalysis of the success story behind the rational design of the first enzymically triggerable, CIEEL-active AMPPD chemiluminescent probe.Keywords: Dioxetanes; a-peroxy lactones; firefly luciferin; bioluminescence; chemiluminescence; immunoassay The phenomenon of luminescence is the emission of light in the visible region (400-700 nm), derived either from living organisms (bioluminescence) or from chemical processes (chemiluminescence), and has become an important analytical tool in recent years, in particular for biomedical applications. In this context, the intriguing natural phenomenon of biolu- minescence has attracted the attention of scientists over many centuries and has been intensively investigated in recent Of the many known cases in nature, firefly luminescence represents the most thoroughly studied and best understood type of bioluminescence. The molecular mechanism of light emission by the firefly, elucidated in the 1960s,4 was proposed to involve a four- membered cyclic peroxide, the so-called dioxetanone or a- peroxy lactone, formed by luciferase-catalysed enzymic oxida- tion of firefly luciferin with molecular oxygen (Fig.1). The a-peroxy lactone belongs to the family of 1,2-dioxetanes, a class of high-energy molecules, which were postulated as * Presented at the VIIth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17-1 9, 1996. reaction intermediates at the beginning of this century.536 However, only in 1969 was the first persistent dioxetane, namely the trimethyl derivative (Fig.2), synthesized by Kopecky and Mumford.7 Shortly thereafter, the preparation of the first persistent a-peroxy lactone (Fig. 2), which serves as the energy source in the firefly luminescence, was reported by our group.* Because of the unique property of dioxetanes to generate electronically excited carbonyl products on thermolysis and thereby emit light, they have received a great deal of attention during the last two decades and developed from once elusive molecules to commercial products for biochemical analysis. In this paper, we illuminate the secret behind the successful development of dioxetanes as commercial probes for chem ilu- minescence immunoassays.9-1* The first question that should be raised is why do dioxrtanes emit light.? One of the most important criteria for the emission of light is the existence of sufficient energy to generate electronically excited molecules, i.e., an exergonic chemical process is necessary.Thus, the formation of excited carbonyl compounds on thermal decomposition of dioxetanes must be favoured energetically to emit light. Since the energy of the lowest excited states, namely the n,n* states, of simple carbonyl compounds such as ketones lies in the range 75-85 kcal mol-1 for singlet and 60-78 kcal mol-l for triplet excitation, the energy sufficiency criterion demands that at least 70-85 kcal mol-1 of energy must be made available in the transition state during the decomposition of the dioxetane to generate one of the carbonyl fragments in its n,Jc*-excited state. For tetramethyldioxetane, as an example (Fig.3), an activation LUCIFERIN 1 OXYLUCIFERIN 1 coz 1 J Fig. 1 Molecular mechanism of firefly luciferin bioluminescence. 0-0 0-0 I I I I I I I 0 H3C - C - C - H Bur- C - C,\ H3C CH3 H Fig. 2 derivatives. Structures of the first isolated dioxetane and a-peroxy lactone1528 Analyst, November 1996, Vol. 121 energy of 25 kcal mol-1 has been measured13 and a reaction enthalpy of 60 kcal mol-1 calculated.13 Thus, sufficient energy is being released in the thermolysis of the dioxetane to form singlet and/or triplet carbonyl products, of which the former subsequently emits fluorescence. Now that it has been established that dioxetanes fulfil the energy requirements to emit light, the next question to be addressed is how efficiently do dioxetanes produce light? Dioxetanes and a-peroxy lactones, upon direct thermal decom- position, produce mainly triplet-excited ketones (Figs.4 and 5), which exhibit only weak if any light emi~si0n.l~ For the observation of efficient luminescence, the dioxetane decom- position products should be formed in singlet-excited states. The luminescence of dioxetanes or a-peroxy lactones can be enhanced by adding an appropriate energy acceptor (fluor- escer), which after energy transfer fluoresces more efficiently than the dioxetane decomposition products. 13 0 *s [*I .... _ _ _ _ _ _ _ _ _ _ _ ~ E,- 25 I 0-0 'CH, Fig. 3 Energy sufficiency for the generation of electronically n,n*-excited singlet and triplet acetone in the thermolysis of tetramethyldioxetane.@*/& - 300 Fig. 4 in the direct thermal decomposition of tetramethyldioxetane. Excited-state efficiencies (W for singlet and @T for triplet states) - 15 Fig. 5 Excited-state efficiencies in the direct thermal decomposition of dimethyl or-peroxy lactone. To develop an efficient luminescent dioxetane one must ask, how does the firefly dioxetane produce light so efficiently? The firefly luciferin decomposes to oxyluciferin with efficient emission of greenish yellow light (Q> = 90%); thus, with at least 90 out of 100 oxyluciferin molecules singlet-excited, the firefly represents the most efficient bioluminescent system known so far (Fig. 6). It is significant that when the phenolic hydroxy group is methylated, although the luciferase oxidizes this protected luciferin equally well to the corresponding o x y l ~ c i f e r i n , ~ ~ - ~ ~ the light emission is as feeble as for the simple dioxetanes in Figs.4 and 5. Why is this so? Unquestionably, the phenolate functionality offers an im- portant clue to the high efficiency in this bioluminescence. In the 1970s, Schuster and co-workers established that the formation of electronically excited fluorophores can be induced by chemical electron transfer processes. I 7 7 l 8 This phenomenon is known as chemically initiated electron exchange lumines- cence (CIEEL), a fundamental process of general scope. This mechanism was first proposed for organic peroxides, l7,Ip, but soon applied to the firefly luciferin bioluminescence.19 As shown in Fig.6, the free phenolate ion of the firefly dioxetane acts as an intramolecular electron donor and transfers an electron to the peroxide moiety with cleavage of the 0-0 bond. Subsequently, the carbon dioxide radical anion is released and immediate back-transfer of the electron to the phenoxyl radical leads preferentially to singlet-excited states of the oxyluciferin anion, which efficiently fluoresces. The over-all chemilumines- cent process possesses a quantum yield of about 90% and, as already stated, it constitutes so far the most efficient bio- luminescent system, which is unquestionably much more efficient than any chemiluminescent process known to date. Now that the secret behind the molecular mechanism of light production by the firefly has been unravelled, the next question one might ask is how can the natural phenomenon of firefly bioluminescence serve as model system to develop chem- iluminescent probes for biomedical applications? This is important in the sense that although during the last two decades numerous chemiluminescent systems have been developed (original work, refs.20-22; reviews, refs. 23-25), there is still great demand for efficient chemiluminescent probes for analyt- ical applications in medicine and biology. A still closer look at the firefly bioluminescent system (Fig. 7) reveals that it consists of three essential molecular components, namely (i) the a- Fig. 6 The mechanism for chemically induced electron exchange luminescence (CIEEL) of the firefly bioluminescence.Analyst, November 1996, Vol.121 1529 peroxy lactone as energy source, (ii) the conjugated benzthiazo- line moiety as both activator and fluorophore and (iii) the easily deprotonated hydroxy group as a trigger for CIEEL. With this fundamental recognition of the molecular details of firefly bioluminescence, a chemical equivalent can be con- ceived that possesses the essential features for the efficient generation of light (Fig. 8). In order to develop a dioxetane- based chemical equivalent from the firefly bioluminescence system, one must first ask the question, what features are essential in designing dioxetanes as chemiluminescent probes for analytical applications? The requirements for an effective chemiluminescent probe are (i) thermal persistence, (ii) sponta- neous decomposition after triggering, (iii) efficient light emission and (iv) convenient synthesis.It is known that dioxetanes are usually fairly labile com- pounds and tend to decompose thermally even at moderate temperatures. However, for bioanalytical applications, ther- mally pFrsistent dioxetanes are required. How can thermal persistence be achieved? In the early 1970s, the adamanty- lideneadamantane dioxetane was reported by our group,26 which possesses remarkable thermal stability, manifested by a melting point of 174-176 "C without decomposition (beyond 180 "C with a light flash!), an activation energy of 34.6 kcal mol-1 and a half-life of about 10000 years at 25 "C. Detailed studies on the thermal stability of dioxetanes revealed that the bulky and rigid spiroadamantane moiety exerts a tremendous stabilizing effect on dioxetanes and permits the isolation and purification of these otherwise thermally labile compounds.27 Therefore, spiroadamantane substitution is an effective tool for the stabilization of thermally labile dioxetanes (Fig.9). As already pointed out, a triggerable activator initiates the decomposition of the firefly bioluminescent system and a high yield of singlet-excited states is generated by the CIEEL ENERGY SOURCE c - - - - - -7' and FIXJOROPHORE Fig. 7 The three essential molecular components, energy source, activa- tor/fluorophore and trigger, for efficient light production in firefly bioluminescence. ED-X ENERGY ACTIVATOR TRIGGER SOURCE and FLUOROPHORE Fig. 8 The chemical equivalent of the firefly system. ED = electron donor. E,: 34.6 25.8 < 20 kcal mol-' STABLE STABILIZED LABILE (PERSISTENT) Fig.9 labile dioxetanes. E, = activation energy. Spiroadamantane substitution for the stabilization of thermally process. An effective chemical equivalent of the firefly system will also require a CIEEL-prone activator, but which chemical activator can facilitate CIEEL-type decomposition of thermally persistent dioxetanes? In the firefly system (Fig. 6) a protected phenolate serves as an activator. As soon as the free phenolate is released (by deprotonation of the phenol group), the a-peroxy lactone moiety decomposes by the CIEEL mechanism. Conse- quently, an efficient chemiluminescence probe should possess a phenolate functionality as activator. Since phenols deprotonate readily even under very mild conditions (uncontrollable pH change), more suitable protective groups, which may be removed selectively and conveniently, are required for analyt- ical applications. Among others, acetates and silyl ethers may be used as chemically28 and phosphates and glycosides as enzymicallyg removable protective groups (Fig. 10).Another important requirement for efficient light emission is a high fluorescence quantum yield of the light-emitting species formed during the triggered dioxetane decomposition and we ask, which triggerable fZuorophores sene as efficient emitters? Examples of suitable triggerable and CIEEL-active arene chromophores are shown in Fig. 11. For the proper choice of a suitable fluorophore the following requisite must be fulfilled: what structural features in the emitter are necessary to produce a lasting glow required for bioanalytical purposes rather than a brief flash? Again, we consult the firefly model system, which produces short flashes, to learn how to avoid the latter in order to generate a persistent glow.Inspection of the oxyluciferin structure (Fig. 6) reveals that the phenolate ion is conjugated with the carbonyl group; thus, such extended conjugation is propitious for flashing rather than glowing. In this context, Edwards et al.29 provided a clue, reporting that singlet efficiencies in the enzyme-triggered decomposition of dioxetanes depended on the substitution pattern of the aromatic fluorophore. Analogous to the firefly oxyluciferin, extended conjugated substitution led to a flash, accompanied by low chemiexcitation efficiencies, while the cross-conjugated regioisomers exhibited a steady-state glow over several minutes with higher excitation yields.Molecular phosphatase 0 - p032- galactosidase { M} 0 - sugar Fig. 10 Acetates and silyl ethers for chemically and phosphates and glycosides for enzymically triggered activators in luminescent applica- tions. I____________ X = RCO, R3Si T R I G G E R S{ ;--%%-!-; X = Poi2, sugar Fig. 11 iluminescence. Triggerable, CIEEL-active fluorophores for dioxetane chem-1530 Analyst, November 1996, Vol. 121 orbital calculations29 and a qualitative pictorial description30 confirmed that charge transfer from the donor (ED) to the acceptor (C02R) occurs more effectively when the two groups are cross-conjugated (Fig.12). Such charge transfer enhances excited state formation, ensures high chemiexcitation effi- ciencies and provides a persistent glow through stabilization of the incipient excited state. In contrast, extended conjugation stabilizes the ground state through dipolar resonance, which results in demotion of the excited state and, consequently, low efficiencies and short flashes. A suitable fluorophore that meets these requirements reasonably well is methyl m-hydrox- ybenzoate (Fig. 1 1 ; R = Me), which exhibits good fluorescence quantum yields, especially in the polar, aprotic organic solvents acetonitrile and dimethyl sulfoxide (Qfl = 3 1 %). To fulfil the above-mentioned molecular requirements for an efficient luminescent dioxetane as a chemical equivalent of the firefly system, the structure shown in Fig.13 serves the purpose well. .The question which still remains open is, how can this rationally designed dioxetane be synthesized? Dioxetanes are conveniently accessible by photooxygenation of electron-rich alkenes. One of the first examples mentioned in literature is the [2 + 21 cycloaddition of singlet oxygen to dimethoxystilbene to afford the corresponding dioxetane.3 1 Fortunately, the photo- oxygenation of the methoxy-substituted adamantylidene leads to the desired dioxetane in excellent yield (Fig. 14). Now that the rationally designed dioxetane has become accessible, let us examine its triggering process to generate chemiluminescence. As shown in Fig. 15, the silyloxy- substituted and the phosphorylated spiroadamantyl-substituted dioxetanes can be readily deprotected with an appropriate trigger (chemical or an enzyme).In the latter enzymically triggered case, the 3-(2’-spiroadamanty1)-4-methoxy-4-(3’’- It, n* EXCITED STATE (Charge ’Ransfer) GROUND STATE (Dipolar Resonance) Fig. 12 Cross-conjugated (metu substitution) and extended-conjugated @am substitution) fluorophores to provide glowing versus flashing triggerable CIEEL-active chemiluminescent systems. ENERGY SOURCE - STABILIZER ACTIVATOR and FLUOROPHORE Fig. 13 dioxetane based on the firefly bioluminescent system. The rationally designed persistent, triggerable and CIEEL-active I OX ox Fig. 14 genation of the enol ether. Synthesis of the rationally designed dioxetane by photooxy- phosphory1oxy)phenyl- 1,2-dioxetane (AMPPD) derivative serves as a substrate for the alkaline phosphatase.32.33 The free phenolate dioxetane decomposes through the CIEEL process (Fig.15) with efficient light emission (@ = 25%).28 What does this mean in terms of sensitivity as a chemilu- minescent probe for bioanalytical applications? The answer is startling: less than zeptomole amounts of analyte (fewer than 1000 molecules) can be detected (Fig. 16), which places the sensitivity level beyond the number of photons which can be perceived by the human eye, and there is still room for improvement. The question that remains, especially in view of sensitivity, is, what advances have dioxetanes promoted in biomedical analysis? Manifold applications in chemilumines- cence immunoassays emphasize the importance of this method and show clearly the unrivalled dominance with regard to sensitivity and selectivity and also environmental acceptance compared with radioactive, colorimetric and fluorescence detection systems.Table 1 gives a brief overview of some analytical applications of chemiluminescence for a variety of systems, of which the most frequently used are luciferin/ luciferase, luminol, acridinium ester, oxalate ester and, of 0SiR3 0- 1 Electron Transfer Back Electron Transfer r------_------------ cH30%1 *- j L - - - - - - - - - - - - - - _ _ _ - - , hv (a up to 25 %) 1 0- Fig. 15 CIEEL mechanism for the generation of the electronically excited m-carbomethoxyphenolate from the chemically or enzymically deprotected spiroadamantyl-substituted dioxetane.Fig. 16 Sensitivity level of the dioxetane-based chemiluminescent probes.Analyst, November 1996, Vol. 121 1531 course, dioxetanes, which are increasingly replacing more risky radioactive assays. What have we learnedfronz our retroanalysis of the firefly bioluminescent system in the rational design of triggerable, CIEEL-active dioxetanes .for hioanalytical applications of chemiluminescence? The salient features which the firefly has taught us are: (i) as a triggerable, CIEEL-active component, a protected phenol moiety serves the purpose, which can be triggered either chemically (by hydrolysis or desilylation) or enzymically (by phosphatase or galactosidase); (ii) a phenolate functionality constitutes a suitable activa- tor; (iii) a dioxetane provides the essential chemical energy source; (iv) an intense, lasting glow is achieved by selecting fluorophores in which the released phenolate is cross-conju- gated with the incipient carbonyl functionality.Unquestionably, firefly bioluminescence has been indis- pensable as model role in the rational design of triggerable, CIEEL-active dioxetanes as chemiluminescent probes. How- ever, organic chemistry has also paid its tribute in this success story, in that thermal persistence for ease of handling and long shelf-life of such dioxetanes have been achieved through spiroadamantyl substitution, while photooxygenation has pro- vided a convenient and efficient synthetic entry. It is truly remarkable to realize that a once curiosity to unravel the secret behind nature’s shining wonder, namely the firefly, and the recognition through fundamental research that the high-energy dioxetanes are involved, permitted the commercialization of this phenomenon in less than 20 years.Thus, the commercial dioxetane AMPPD and a newer compound, 3-(2’-spiro-S’- chloroadamantane)-4-methoxy-4-( 3”-phosphory1oxy)phenyl- 1,2, dioxetane (CSPD),34 nowadays represent valuable com- modities through their bioanalytical applications in medicine and biology.2G25 Of course, the intensive interdisciplinary efforts of scientists in the fields of biology, chemistry, medicine and physics were essential for the success, and bespeaks once more of the necessity for fundamental research for innovation in the service of mankind. Financial support by the Deutsche Forschungsgemeinschaft (SFB I 72: Molekulare Mechanismen kanzerogener Primar- veranderungen) and the Fonds der Chemischen Industrie is gratefully acknowledged.Special appreciation is due to Dr. 1. Bronstein and her staff of Tropix (Bedford, MA, USA) for stimulating this historical account of the rational design of the commercial dioxetane product AMPPD for chemiluminescence immunoassay applications and their constructive and critical Table 1 Chemiluminescence applied in chemical, biochemical and molecular bioiogical analysis Chemistry Biochemistry Molecular biology Toxic trace elements Toxic arenes Amino acids Nitrogen oxides (NO,) Sulfur oxides (SO,) Food assays Textile assays Immunoassays DNA hybridization assays Ligand-binding DNA sequencing Cell metabolism PCR products Lipid peroxidation Mutagenicity assays discussions.W.A. dedicates this paper to his Junior High School chemistry teacher, Helmut Linge, on the occasion of his 85th birthday in appreciation for having encouraged him even as a young student to dare to ask. References 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Adam, W., Chem. Unserer Zeit, 1973, 7, 182. Matern, U., Bid. Unserer Zeit, 1984, 14, 140. Albrecht, S., Brandl, H., and Adam, W., Cheni. Unserer Zeit, 1990, 24, 227. McElroy, W. D., Seliger, H. H., and White, E. H., Photochem. Photohiol., 1969, 10, 153. Staudinger, H., Chem. Ber., 1925, 58, 1075. Staudinger, H., Dyckerhoff, K., Klever, H. W., and Ruzicka, L., Chem. Bpi.., 1925, 58, 1079. Kopecky, K.R., and Mumford, C., Cun. J . Chem., 1969, 47, 709. Adam, W., and Liu, J.-C., J . Am. Chem. Soc., 1972, 94, 2894. Bronstein, I., Edwards, B., and Voyta, J. C., .I. Biolumin. Chem- ilumin., 1989, 4, 99. Bronstein, I., Voyta, J. C., Thorpe, G. H. G., Kricka, L. J., and Armstrong, G., Clin. Chem., 1989, 35, 144 1. Thorpe, G. H. G., Bronstein, I., Kricka, L. J., Edwards, B., and Voyta. J. C., Clin. Chenr., 1989, 35, 2319. Bronstein, I., and Sparks, A., in Immunochemical Assuys and Biosensor Technology,fbr the 1990s; ed. Nakamura, R. M., Kasahara, Y . , and Rechnitz, G. A., American Society for Microbiology, Washington, DC, 1991, pp. 229-250. Adam, W., and Yany, F., in Small Ring Heterocycles, ed. Hassner, A., Wiley, New York, 1985, vol. 42, part 3, ch. IV. White, E.H., Wiirther, H., Field, G. F., and McElroy, W. D., 1. Org. Chem., 1965.30, 2344. White, E. H., and Worther, H., .I. Org. Chem., 1966, 31, 1484. White, E. H., Worther, H., Seliger, H. H., and McElroy, W. D., .I. Am. Chenz. Soc., 1966, 88, 2015. Koo, J.-Y., and Schuster, G. B., J . Am. Chem. Soc., 1977, 99, 6107. Schuster, G. B., AK. Chem. Res., 1979, 12, 366. Koo, J.-Y., Schmidt, S. P., and Schuster, G. B., Proc. Nutl. A c d . Sci. USA, 1978,75, 30. Bronstein, I., Voyta, J. C., and Edwards, B., Anal. Biochem., 1989, 180, 95. Bronstein, I., and Voyta, J . C., Clin. Chem., 1989, 35, 1856. Tizard, R., Cate, R. L., Ramachandran, K. L., Wysk, M., Voyta, J. C., Murphy, 0. J., and Bronstein, I., Proc. Nutl. Acad. Sci. USA, 1990,87, 45 14. Beck, S., and Koster, H., Anal. Chem., 1990, 62, 2258. Albrecht, S., Brandl, H., Schonfels, C., and Adam, W., Chem. Unserer Zeit, 1992, 26, 63. Mayer, A., and Neuenhofer, S . , Angew. Chem., 1994, 106, 1097; Angew. Chem., Int. Ed. Engl., 1994, 33, 1044. Wieringa, J. H., Strating, J., Wynberg, H.. and Adam, W., Tetrahedron Lett., 1972, 169. Adam, W., Encarnacion, L. A. A., and Zinner, K., Chern. Ber., 1983, 116, 839. Schaap, A. P., Chen, T.-S., Handley, R. S., DeSilva, R., and Giri, B. P., Tetrahedron Lett., 1987, 28, 1155. Edwards, B., Sparks, A., Voyta, J. C., and Bronstein, I., J. Biolumin. Clzemilumin., 1990. 5 , 1. McCapra, F., Tetrahedron Lett., 1993, 34, 6941. Rio, G., and Berthelot, J., Bull. Soc. Chinz. Fr., 1971, 10, 3555. Bronstein, I., PCT Int. Appl., WO 88100695, 1988; Chem. Ahstr., 1989, 110, P72157u. Edwards, B., and Bronstein, I., PCT Int. Appl., WO 89106226, 1989; Chem. Abstr., 1990. 112, P77166x. Bronstein, I., Juo, R. K., Voyta, J. C., and Edwards, B., in Bioluminescence and Chemiluminescence: Current Status, ed. Stan- ley, P. E., and Kricka, L. J., Wiley, Chichester, 1991, pp. 73-82. Enzyme catalysis Reporter gene assays Small biomolecules Virus detection Forensic medicine Microbe detection Paper 610.3281 H Received May 10, 1996 Accepted June 24,1996

ISSN:0003-2654    

DOI:10.1039/AN9962101527

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, November 1996, Vol. 121 (1533-1537) 1533 Evolution of a Specific Fluorogenic Derivatization of lvermectin for Bioanalytical Applications* A Review David W. Fink, Pierre deMontigny and Jung-Sook Kim Shim Merck Research Laboratories, P.O. Box 4, West Point, PA 19486, USA The evolution of the fluorogenic derivatization of ivermectin is traced through a series of continual modifications that have resulted in improvements in speed and sensitivity. Since the original development of this selective analytical technique, the reaction time has been shortened from 24 h at 100 "C to <30 s at room temperature and, through modifications of the derivatization reagent and catalyst, the sensitivity has also been increased 50-fold to 20 pg of analyte with no significant decrease in precision.A procedure is reported, based on the use of fluorescence derivatization, which eliminates the use of solid-phase columns for sample preparation and fluorophore isolation, and is faster and less cumbersome than previous methods. The method was evaluated with cattle and canine plasma samples over the concentration range 1.0-40 ng ml-l of ivermectin. It has an accuracy of 1.9% (mean relative error) over this concentration range and a precision of 5.6% (RSD) at the 1 ng ml-I ivermectin concentration level in a 1 ml plasma sample. Keywords: Ivermectin determination; derivatization; fluorescence detection; hioanalytical; review not require the separation of excess fluorescent reagent or of reaction by-products from the analytical fluorophore. The reaction was designed specifically based on consideration of the structural features of ivermectin, and not to produce fluorescent derivatives of unidentified endogenous plasma components.The two hydroxyls having protons bonded trans to each on the dihydroxycyclohexene ring of this drug (11) are sterically positioned to allow for the facile elimination of water to produce a delocalized electronic system, i.e., heating with acetic anhydride in pyridine causes the elimination of 2 mol of water, producing a fluorophore consisting of an aromatic ring2 which is in conjugation with a diene system (111: A,,,: 37.5 nm; kern: 47.5 nm): J P (1) 6 1 OO'C, 24 hr ' I -2H20 ' CH, i '.. CH3 H 'H HS) Introduction Dehydrative aromatization using non-fluorescent reagents pro- vides a selective fluorogenic derivatization of the non-fluores- cent antiparasitic agent ivermectin (I) because this reaction does OCH3 c H , A 0 * 0 A CH3/)J OH I * Presented at the VIIth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17-19, 1996.I1 111 The mechanism of this analytical reaction involves acetyla- tion of the hydroxyl groups in advance of dehydration; the original development of this derivatization required a reaction time of 24 h.3 The evolution of this method has resulted in continual improvements in speed and sensitivity. Connors and co-workers435 identified several nucleophilic catalysts for acylation by acetic anhydride which are superior to pyridine, including 4-dimethylaminopyridine and some N-alkylated imi- dazoles.Use of the more powerful nucleophilic l-methyli- midazole as an acetylation catalyst instead of pyridine for this derivatization shortened the reaction time to 1 h.6 The method was later improved by the use of solid-phase extraction from the plasma sample to replace liquid-liquid partiti~ning.~ Further, introducing the better leaving group trifluoroacetyl by replacing acetic anhydride with trifluoroacetic anhydride as the acetyla- tion reagent further shortened the reaction time to <30 s and detection limits to about 20 pg (S/N = 2).8 The detection limit was later lowered even further by the use of laser-induced fluorescence detection9 This review chronicles the evolution of this analytical derivatization reaction and its numerous bioanalytical applica- tions.In our most recent iteration, we have simplified the method further by eliminating the chromatographic isolation step through the use of automated derivatization (as described by Rabel et al.9). This maintains an accuracy of 2% (mean1534 Analyst, November 1996, Vol. 121 relative error) and a precision of 5% (RSD) at the 1 ng measurement level in an even shorter and less cumbersome analytical procedure. Experimental Principle Ivermectin is extracted from a 1 ml plasma sample into acetonitrile and is derivatized with trifluoroacetic anhydride and N-methylimidazole in acetonitrile solution in an autoinjector to produce a fluorescent derivative. The derivative is determined using HPLC on a Zorbax Cx column at 30 "C with a mobile phase of acetonitrile-tetrahydrofuran-water (6 + 2 + 1) and fluorescence detection at 475 nm with 365 nm excitation.Abamectin is used as an internal standard. This method is applicable to the determination of ivermectin in cat, dog and cattle plasma over the concentration range 1-40 ng ml- 1 . Reagents Acetonitrile and tetrahydrofuran (THF), Optima, HPLC grade, and water, HPLC grade, were obtained from Fisher (Pittsburgh, PA, USA) and N-methylimidazole and trifluoroacetic anhy- dride, both 99 + %, from Aldrich (Milwaukee, WI, USA). Internal standard solution. Transfer about the equivalent of 50 mg of abamectin standard into a 50 ml calibrated flask and dissolve in acetonitrile, dilute to volume and mix well. Dilute 1.0 ml to 100 ml with acetonitrile (10 pg ml-I).Further dilute 2.0 ml to 100 ml with acetonitrile (200 ng ml-1). Finally, dilute 10.0 ml of the last solution to 200 ml with acetonitrile (10 ng ml- '). This is the working internal standard solution. Ivei-vzec*tin standard solution. Accurately weigh about the equivalent of 50 mg of ivermectin standard of known HZBl, purity into a 50 ml calibrated flask, dissolve in acetonitrile, dilute to volume and mix well (approximately 1 mg ml-l). Dilute 1.0 ml to 100 ml with acetonitrile (approximately 10 pg ml-1). Further dilute 1.0 ml to 100 ml with acetonitrile (approximately 100 ng ml-1). Finally, dilute 20 ml of the last solution to 200 ml with acetonitrile (approximately 10 ng ml- l ) . This is the working ivennectin standard solution. Apparatus The following were used: an analytical balance, capable of weighing to 1.00 mg accuracy; a 15 ml centrifuge tube, polypropylene, disposable; 1, 2, 10 and 20 ml volumetric pipettes; 50-250 and 200-1000 p1 Eppendorf or equivalent pipettes; a Vortex mixer; a centrifuge; and an N-Evaporator or equivalent. The HPLC system consisted of an LC pump [Shimadzu (Kyoto, Japan) LC-6001, a fluorescence HPLC monitor (Shimadzu RF-535) an autoinjector (Shimadzu SIL9A) and an integrator [Spectra-Physics (San Jose, CA, USA) SP42901.Chromatographic Conditions The following conditions were adopted: column: Zorbax Cg, 250 X 4.6 mm id, operated at 30 "C; mobile phase, acetonitrile- THF-Water (6 + 2 + I); flow rate, 1.0 ml min-l; and fluorescence detection with excitation wavelength 365 nm and emission wavelength 475 nm.Under these conditions, the retention times of ivermectin and the internal standard were approximately 18 and 12 min, respectively. Sample Preparation Transfer 1 .00 ml (V,) of well mixed plasma sample and 1 .OO ml of the working internal standard solution (10 ng of abamectin in 1 .O ml of CH3CN) into a 15 ml centrifuge tube, vortex mix, add 4.0 ml of acetonitrile, vortex mix for 1 min to denature and promote protein precipitation and centrifuge for 12 min to obtain a clear supernatant. Standard Preparation Mix 1.0 ml (V,) of the working ivermectin standard solution (approximately 10 ng ml-1 in CH3CN), 1 .O ml of the working internal standard solution (10 ng of abamectin in 1.0 ml CH3CN) and 4.0 ml of acetonitrile in a 15 ml centrifuge tube.Follow the same procedure for the sample and standard as follows: transfer carefully 1.0 ml of the clear extract of the sample and the standard into separate 100 X 13 mm id disposable culture tubes, evaporate to dryness with a stream of nitrogen at < 40 "C and dissolve the residue in 110 pl of N- methylimidazole-CH3CN ( 1 + 1). Transfer 100 pl of the solution obtained into an autosampler vial and cap it. Pro- gramme the rapid addition of 150 pl of trifluoroacetic anhydride-CH3CN (1 + 2) to the sample vial with N- methylimidazole and proceed with the automated mixing by the autosampler. Within 2 min after mixing, inject 100 pl into the HPLC system. Measure the peak heights of ivennectin (H2BIa) and internal standard (abamectin, B1,) for the calculation. Calculations PHR, VS PHR, VY X C, X- = ng ml-1 ivermectin (H2B1,) where peak height of ivennectin (HZB,,) in sample peak height of internal standard (B1,) in sample peak height of ivermectin (H2BIa) in standard peak height of internal standard (Bla) in standard ' C, = concentration of standard in ng ml-1 = M X P X 1/5 [M = mass of standard in mg; P = purity of ivermectin (H2B1,) standard in as a decimal]; PHR, = ' PHR, = V , = Volume of standard in ml; V , = Volume of sample in ml.Results and Discussion The evolution of this HPLC-fluorescence method is outlined in Table I , which shows the experimental conditions (reagent, catalyst, reaction temperature) as they were changed, and the resulting figures of merit of the method (reaction time, LOD and precision). The reaction time has been decreased from 24 h to <30 s (at room temperature) and the limit of detection has concomitantly been reduced from 1 ng to < 10 pg.The bar chart in Fig. 1 illustrates the continual decrease in reaction time and limit of detection with the modifications of the method. It can be seen that the exceptional increase in sensitivity is not accompanied by a loss of precision at the lower concentrations, as would be expected. Indeed, following the initial decrease in the RSD from 8% to 4% with the change of catalyst, it still remains in the range 4-6%, without a significant increase at even lower LODs. In the original version of the method, the RSD was 8% because a derivatization time of 24 h was required for maximum chemical yield, after which reaction time the HPLC-fluorescence intensity decreased, probably reflecting the formation of other by-products under these reaction conditions.3 In addition, 4"-acetylation also occurs r a ~ i d l y , ~ forming a labile ester which could hydrolyse during derivatiza- tion; accordingly, the careful timing required of the derivatiza- tion reaction under these original conditions was a component of variance contributing to the 8% RSD (at the 200 ng measurement level). Under these conditions, the optimized chemical yield of the fluorescent derivative was 60%; theAnalyst, November 1996, Vol.121 1535 precision was later improved as derivatization efficiency was increased to 100% quantitative yield with the introduction of 1 -methylimidazole as the improved catalyst.The initial acetylation step in the mechanism of this dehydration is catalysed by the nucleophilic base catalyst pyridine. In a mixed mechanism, this base can form the pyridinium cation in this organic (CH3CN) reaction medium, in addition to some possible acetylpyridinium cation; the resulting analyte alkoxide ion is then available for nucleophilic attack on the carbonyl of the acetic anhydride derivatization reagent. exhibiting an emission maximum at 480 nm, providing the sensitivity for this HPLC method. As shown in Fig. 2, the excitation spectrum of this fluorophore has two excitation peaks at energies that correspond to the new absorption h,,, values created by the derivatization reaction. With current methods, trifluoroacetylation of the 4"-hydroxyl of ivermectin, a position remote to the aromatization site, still leads to the formation of a labile ester, therefore still requiring careful timing of the derivatization (if no internal standard is used).Hydrolysis of the trifluoroacetylated derivative leads to 9- + 2 : N t T W OH 0- 0- N-Methylimidazole has been shown to be a much more efficient catalyst for acylations of hydroxy groups in both aqueous and non-aqueous media.5 It is a stronger base,10 (pK, = 7.1) than pyridinelo (pK, = 5.2) as its conjugate imidazoli- nium cation and also the possible accompanying acylimidazol- inium cation can be stabilized by electron delocalization.5 the formation of a more polar product which can compromise the sensitivity of the reversed-phase HPLC conditions. To obviate these complications, an automated derivatization of ivermectin has now been devised to provide a rapid and reproducible method for the determination of the drug at low concentrations with minimal sample preparation.The solid- OH 0- + 2H+N6N-CH3 - HNANtCH3 L/ u + 2 : N ~ N - C H ~ L/ (3) OH 0- For this reason, N-methylimidazole was used to replace pyridine in version I1 of this method, which effected a decrease in derivatization time from 24 h to 1 h at 95 "C. Following acetylation, the analytical derivatization then results from the loss of acetic acid via elimination of the acyl substituents and the protons trans to these. Because the trifluoracetyl moiety is a better leaving group than the original unsubstituted acyl functional group, this substitution was then introduced in version 111 of the method, which accomplished a further reduction of the analytical derivatization time to < 30 s.Derivatization Ivermectin has a single absorption band at A,,, 244 nm and does not absorb at h > 270 nm.I1 The drug is not intrinsically fluorescent. With aromatization [reaction (l)], two new low- energy absorption bands appear at A,,, 280 and 360 nm (Fig. 2), which are transitions on the extended aromatic-diene conju- gated system which is created by this analytical derivatization reaction. These electronic absorption transitions are accom- panied by the appearance of an intense visible fluorescence Fig. 1 Evolution of the analytical method. Changes in reaction time, LOD and precision with method revisions. Method versions I, 111, IV and V are in chronological order (Table 1).Table 1 Evolution of the analytical method Version Year Reagent Catalyst Time TemperaturePC Column I 1980 (CH3C0)20 Pyridine 24 h 100 Florisil and silica gel I1 198 1 (CH3C0)20 N-Me-imidazole l h 95 -a 111 1987 (CH3C0)20 N-Me-imidazole I h 95 Solid-phase extraction IV 1990 (CF3C0)20 N-Me-imidazole < 30 s 25 Sep-Pak V 1993 (CF3C0)20 N-Me-imidazole < 30 s Room temp. Sep-Pak RSD LOD (%) Comments 1 ng 8 60% yield -a -11 Quantitative yield 0.5 ng 4 - <20pg 5 - <lOpg 6 Laser excitation Q This method applied to tissue samples; all others refer to plasma analyses.1536 Analyst, November 1996, Vol. 121 phase extraction that was used to prepare the sample prior to derivatization has been eliminated to simplify further the sample preparation and to minimize drug loss.The present simplified method requires no solid-phase extraction prior to pre-column on-line derivatization for the determination of ivermectin in plasma at concentrations reaching 1 ng ml-I. In this method, the analyte is extracted into CH3CN and an aliquot of the extract is evaporated to dryness. To the dry residue obtained following extraction, 1 10 pl of N-methylimidazole catalyst in acetonitrile (1 + 1 v/v) is added. This solution is then transferred into a 1.1 ml sample vial. The autosampler (Shimadzu LC-9A) is programmed to add 150 pl of the trifluoracetic anhydride reagent in acetonitrile (1 + 2 v/v) automatically. After mixing, 100 pl of the analyte solution are injected directly into the liquid chromatograph for analysis.Aqcuracy and Precision of the Analytical Method To determine the accuracy and precision of this procedure, a series of 1 ml samples of drug-free plasma collected from cattle and from dogs was supplemented with known amounts of ivermectin to cover the concentration range 1-40 ng ml-* in the plasma. Each of these samples was then analyzed by this revised analytical procedure, and the results are presented in Table 2. All of these analyses gave results within k0.4 ng of the true ivermectin content, and the mean relative error was 1.9% over this concentration range. There is no statistically significant difference (with a high level of significance; P > 0.8) between the results obtained in cattle or canine plasma, indicating that this method is equally applicable to both species.A linear least- squares standard calibration line of HPLC-fluorescence peak height as a function of known ivermectin concentration was linear with a correlation coefficient r2 > 0.999 over the concentration range 0-160 ng ml-1 ivermectin in 12 plasma samples. Table 2 includes eight replicate analyses of 1 ml 0.6 3 0.4 E $ 9 0.2 200 250 1 I I 300 . 400 460 . 560 Wavelength/nm Fig. 2 Spectra associated with the derivatization. Top: absorption spectra of (A) ivermectin and (B) fluorescent derivative. Bottom: (C) excitation spectrum and (D) fluorescence spectrum of the derivative. plasma samples containing 1 ng of the analyte. At this concentration, the method has an accuracy of 2.1% (mean relative error) and a precision of 5.6% (RSD).Comparison With Other Analytical Methods The results obtained by the present procedure were verified by direct comparison with two other methods. To obviate the derivatization, an alternative quantitative procedure had been developed previously using HPLC with direct UV absorp- tiometric detection.12 This measurement, however, which can only be applied to plasma samples containing 2 2 ng ml-1 of ivermectin, requires two consecutive solid-phase extraction steps to remove unidentified endogenous UV-absorbing plasma interferences in order to prepare a sample sufficiently purified for the use of this non-specific detection mode. Table 3 shows the results of the direct comparison of the present HPLC- fluorescence procedure with the HPLC procedure using direct Table 2 Analyses of plasma supplemented with known concentrations of ivermectin Ivermectin Cattle plasma Canine plasma added to 1 ml of Ivennectin Relative Ivennectin Relative plasma/ng found& error (96) found/ng error (%) 0.00 0.96 4.80 9.59 14.4 19.2 24.0 28.8 33.6 38.4 0.00 0.00 0.93 0.86 0.95 0.99 4.60 9.58 14.4 19.3 24.2 29.0 33.7 38.5 0.0 0.0 3.1 10.4 1 .0 3.1 4.2 0.1 0.0 0.5 0.8 0.7 0.3 0.3 0.00 0.00 0.99 0.92 0.87 0.99 4.80 9.21 14.I 19.4 24.0 28.9 33.5 38.5 0.0 0.0 3.1 4.2 9.4 3.1 0.0 4.0 2.1 I .0 0.0 0.3 0.3 0.3 Mean 1.8 2.0 Table 3 Comparison of results of HPLC-fluorescence method with UV absorption detection’ Ivermectin concentration/ng ml- I Plasma sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 HPLC- fluorescence detection 2.0 3.0 2.2 4.1 5.3 5.3 4.8 6.5 8.0 11.8 13.6 17.0 19.4 20.1 21.3 23.7 27.5 30.8 uv absorption detection Difference 2 0 3 0 3 0.8 4 0.1 5 0.3 5 0.3 5 0.2 6 0.5 8 0.0 11 0.8 13 0.6 17 0.0 18 1.4 19 1.1 20 1.3 23 0.7 26 1.5 30 0.8 Av.: 0.6Analyst, November 1996, Vol.121 1537 absorption detection. The 18 plasma samples in this table were actual naturally incurred ivermectin plasma samples that were collected from dosed animals, rather than drug-free plasma that had been supplemented with the drug for the accuracy measurements (Table 2). The mean difference between the methods was 0.6 ng over the concentration range 2-30 ng ml- l , Table 4 Comparison of results of HPLC-fluorescence detection with previous version of HPLC method8 Ivermectin concentratiodng ml- Plasma sample 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 HPLC- fluorescence detection 0.24 0.25 0.24 0.53 0.67 0.67 0.93 1.05 1.06 I .04 1.17 1.22 1.13 I .80 1.93 2.09 1.96 2.54 4.12 4.39 4.64 6.68 7.77 10.4 41.9 Previous method8 0.20 0.2 1 0.23 0.43 0.56 0.63 0.73 0.92 0.93 0.95 1 .00 1 .OO 1.06 1.60 1.80 1.83 1.84 2.16 3.73 3.92 4.61 6.67 7.09 9.40 38.6 Difference 0.04 0.04 0.0 1 0.10 0.11 0.04 0.20 0.13 0.13 0.09 0.17 0.22 0.07 0.20 0.13 0.26 0.12 0.38 0.39 0.47 0.03 0.01 0.68 1 .0 3.3 Av.: 0.3 demonstrating close agreement.The largest difference found, 1.5 ng (plasma sample 17), is only 5.5% of the drug concentration, which is within the 5.6% RSD of the method. Table 4 shows a comparison of analytical results between the present method and the previous more complicated version using fluorescence detection.8 Over the concentration range 0.20-38.6 ng ml-1 including 17 of these 25 samples at concentrations 1 2 ng ml-1, the results of these methods are in close agreement, with the difference between the two averaging 0.3 ng ml-1.Plasma samples 1-3 in Table 4 represent the measurement of a total 0.3 pmol of the analyte in a 1.0 ml plasma sample, to illustrate the sensitivity of this fluorescence derivatization HPLC procedure. References 1 2 3 4 5 6 7 8 9 10 11 12 Fink, D. W., Trends Anal. Chem., 1982, 1, 254. Mrozik, H., Eskola, P., Fisher, M. H., Egerton, J. R., Cifelli, S., and Ostlind, D. A., J. Med. Chem., 1982, 25, 658. Tolan, J. W., Eskola, P., Fink, D. W., Mrozik, H., and Zimmerman, L. A., J . Chromatogr., 1980, 190, 367. Connors, K. A., and Albert, K. S., J . Phurm. Sci., 1973, 62, 845. Pandit, N. K., and Connors, K. A., J. Phurm. Sci., 1982, 71, 485. Tway, P. C., Wood, J. S., and Downing, G. V., J . Agric. Food Chem., 1981,29, 1059. Kojima, K., Yamamoto, K., and Nakanishi, Y., J . Chromatogr., Biorned. Appl., 1987, 413, 326. deMontigny, P., Shim, J.-S. K., and Pivnichny, J. V., J. Pharm.. Biomed. Anal., 1990, 8, 507. Rabel, S. R., Stobaugh, J. F., Heinig, R., and Bostick, J. M., J. Chromatogr., Biomed. Appl., 1993, 617, 79. Sill&, L. G., and Martell, A. E., Stability Constants of Metal-Ion Complexes, Special Publication No. 17, Chemical Society, London, 1994. Fink, D. W., in Anulytrcd Profiles of Drug Suhstunceh, ed. Florey, K., Academic Press, San Diego, 1988, vol. 17, pp. 156-84. Pivnichny, J. V., Shim, J.-S. K., and Zimmerman, L. A., .I. Pharm. Sci., 1983, 72, 1447. Paper 6/02 7841 Received April 22, 1996 Accepted June 24, I996

ISSN:0003-2654    

DOI:10.1039/AN9962101533

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, Novenzber 1996, Vol. 121 (1539-1543) 1539 Lum inol C hem i I um i nescence-based Porp hyri n Assays Without Hydrogen Peroxide: a Spectral Study of Mechanism and Enhancement* Carole Poupon-Fleuret", Jean-Paul Steghens" and Jean-Claude Bernengob a Lahoratoire de Biochimie C, HGpital Edouard Herriot, 69437 Lyon Cedex 3, France h Unit6 INSERM I2 I , I8 Avenue du Doyen Lkpine, 69500 Bron, France In ordFr to use metalloporphyrins as labels in immunoassays or in nucleic probes, a detection method based on luminol chemiluminescence (CL) at alkaline pH was developed. It was found that omitting H202 improves the light yield, and that a dramatic enhancement of CL is achieved by the association of nicotinamide adenine dinucleotide (NAD) and flavins [flavin mononucleotide (FMN) or riboflavin].To explain the mechanism of the catalytic cycle in the absence of H202, electron spin resonance (ESR) measurements were performed. Using 5,5'-dimethyl-l-pyrroline N-oxide as a spin trap agent, ESR showed the production of OH* and 02*- radicals. This free radical production may explain the luminol oxidation by metalloporphyrins. The role of 0 2 ' - was confirmed by almost complete inhibition of light emission when superoxide dismutase was added to the CL reaction. The contribution of oxygen was also confirmed by the large decrease in the CL emission when deaerated solutions were used. A reaction scheme is proposed in which a redox cycle between Fe" and FeIII porphyrins is coupled to the production of oxy radicals. Owing to their redox properties, FMN and NAD enhancers could act at this level through an increase of the exchange rate between FeI1 and Fell'.Moreover, in the presence of FMN, a significant red shift and shape change of the luminol emission spectrum is observed, which arises from an energy transfer phenomenon in the final luminescent step of the reaction. On the other hand, neither a spectral shape change nor a shift was observed in the presence of NAD. These data were collected by using a high-performance laboratory-made spectroluminometer. Keywords: Porphyrins; luminol; chemiluminescence; .flavins; free radicals Introduction Metalloporphyrins represent the active site of many enzymes (cytochromes, catalases, peroxidases, etc.) and are therefore widely used as a model in biomimetic chemistry (models for oxygen transport, cytochrome P450, ligninase, etc.).l-3 Horse- radish peroxidase (HRP) includes an Fe"'-protoporphyrin IX bound to a protein by an imidazole axial ligand. The peroxidase activity of haematin (natural porphyrin) and synthetic porphyr- ins has been used for hydrogen peroxide (H202) determination.4 The chemiluminescence (CL) reaction of luminol (3-amino- phthalhydrazide) with HRP in the presence of an oxidant such as hydrogen peroxide in alkaline aqueous solution is well * Presented at the VIIth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17-19, 1996. known. Some metal complexes, metal-containing compounds and metalloporphyrins have also been reported to cause CL from luminol in aqueous solution, even in the absence of H202.5-9 Classical enhancers of the luminol CL with HRP are phenol derivativeslo or more recently arylboronic acids; 1 * unsaturated long-chain fatty acids and surface-active com- pounds were found to be enhancers in the case of metal- loporphyrins.7~8 The HRP catalytic cycle is well described] but the mechanism of metalloporphyrin catalysis remains un- explained.The low relative molecular mass (around 1000) of these molecules facilitates their coupling to antibodies and haptens. Moreover, it is known that porphyrins have an affinity to nucleic a~ids.12-1~ Therefore, the fact that luminol CL detects them suggests the use of metalloporphyrins as labels in immunoassays or in nucleic probes. For this purpose, we first optimized a method for metallo- porphyrin detection by luminol CL and secondly tried to explain the catalytic mechanism by performing electron spin resonance (ESR) and spectroluminescence studies.Experimental Reagents All solutions were prepared with ultra-pure water (resistivity > 18 M a cm) (Elga, Villeurbanne, France) and stored in dark bottles. meso-Tetraphenylporphyrins were either commercially available from Porphyrins Products (Logan, UT, USA) and Aldrich (L'Isle d'Abeau, France) or synthesized by P. Battioni (URA 400, CNRS, Paris, France). The porphyrins were dissolved in water or, for water-insoluble species, in dichloro- methane-methanol (1 + 1 v/v), to prepare 2 X 10-8 rnol 1-1 solutions. Traces of free metal were removed by addition of an iminodiacetic resin.Luminol (Aldrich) was purified by recrystallization from 1 moll- NaOH as described by Stott and Kricka15 and dissolved in dimethyl sulfoxide (DMSO) (Sigma, L'Isle d' Abeau, France) to obtain a 5 X 10-2 mol 1-1 luminol solution, used daily to prepare a more dilute solution (1 X rnol 1-1 NaOH. Luminol in DMSO is stable for at least 3 months. Nicotinamide adenine dinucleotide (NAD) and the reduced form (NADH) (Boehringer Mannheim) were used at 6 X 10-3 mol 1-1 in water unless specified otherwise. Flavins [flavin mononucleotide (FMN), (Boehringer Mann- heim), flavin adenine dinucleotide (FAD) (Fluka, L'Isle d' Abeau, France) and riboflavins (Sigma)] were dissolved in 5 X 10-2 moll-1 NaOH to give 1 X 10-3 rnol 1-1 solutions. mol 1-1) was prepared by dissolving commercial 30% v/v H202 solution (Merck, Nogent- sur-Marne, France) in 1 X 10-3 rnol 1-I nitric acid.moll-I) in 5 X Hydrogen peroxide (2 X1540 Analyst, Novevriher 1996, Vol. 121 5,s'-Dimethyl- 1 -pyrroline N-oxide (DMPO) was purchased from Sigma at a concentration of 9 x 10-3 mol 1-1. Bovine superoxide dismutase (SOD) (Sigma) was dissolved in TRIS-cacodylic acid buffer (pH 8) to obtain a final concentration of 100 mg I--'. The activity of the solution, as measured by pyrogallol autoxidation, was 650 U ml- I . Apparatus Luminescence was measured on an Optocomp 1 luminometer, (MGM Instruments, Hamden, CT, USA) in which polystyrene tests-tubes were introduced. Photons were counted for 30 s. Results are given in relative light units (RLU); 1 RLU =: 10 counts.Luminescence spectra were obtained from a low-noise laboratory-made spectroluminometer with a 5 nm bandwidth and a spectral range of 380-700 nm. The photomultiplier was a Hamamatsu R634-0 1. ESR measurements were carried out on an ER lOOD spectrometer (Bruker, Wissembourg, France) operating at 9.5 GHz. Continuous-wave X-band ESR spectra were recorded at room temperature. Influence of the Reaction Medium on Light Emission A 5 pl volume of 2 X mol 1 - I iron meso-tetrakis- (4-sulfonatopheny1)porphyrin [Fe"'(TS03NaPP] solution were added to 200 pl of 1 X 10-5 moll-' luminol solution in various solvents: ethanol, TRIS-5 x mol I-' HCI buffer (pH 8.5) or 5 X 10-3, 10 X 10-3 and 50 X 10-3 mol 1 - 1 NaOH. After the addition of the catalyst (assay measurement), each sample tube was inserted in the luminometer and, in some experiments, 100 pl of 2 X 10-2 mol 1-1 H202 were injected automatically. The blank was measured by replacing the catalyst with water.The net signal was calculated as RLU,,,,, - RLUblank. All subsequent experiments were performed in the absence of H202. Porphyrin Structure-Luminescence Relationship Seventeen meso-tetraphenylporphyrins differing in the substi- tuents on the phenyl ring or the chelated metal (Fe, Mn or Co) were tested. Nine of them were water soluble. The metal content was determined by ETAAS. The percentage of metallation was calculated as (measured metal content/theoretical metal con- tent) ~ 1 0 0 . Enhancement Conditions NAD and NADH were tested as enhancers. Volumes of 10 pl of a solution containing different proportions of NAD and NADH (final concentration 3 x 10-4 mol I- l) were added to 200 pl of reagent ( I X 10 -5 moll-1 luminol in 5 X mol 1 - I NaOH) before the addition of the porphyrin. In a further experiment, various concentrations of NAD (final concentration 2.5 X 10-6-6 X 10-4 mol 1- 1 ) were used in the absence or presence of 1 x 10-5 inol 1-1 flavins (FMN or riboflavin) (final concentration in the reagent). The optimum incubation time of NAD in the reagent was also examined.Mechanism Luminol, FMN and NAD were used at the-same concentration as described previously. Fe"' (TS03NaPP) was used at 2 X moll-I for ESR studies. DMPO (0.45 mol 1-l) was used as the spin trap agent. The reaction solution was prepared and transferred to an ESR-calibrated capillary tube.For the luminescence spectroscopic study, Fe"' (TS03NaPP) was used at 2 X 10-7 mol 1-1. Effect of superoxide disniutuse Volumes of 10 p1 of serial dilutions of the 100 mg I-' solution were added to the FMN-containing luminol-NaOH reagent to obtain final activities of 32.5-32.5 X U mi-'. NAD and porphyrin were subsequently added before CL measurement. Role (v oxygen The reagent (luminol-NaOH-FMN) and porphyrin solutions were divided into two parts. One part was deaerated by bubbling overnight with nitrogen and the other part was stored without treatment. After deaeration, the CL reaction was conducted under anaerobic conditions. The deaerated solutions were then exposed to ambient air and the CL reaction was measured after 3 and 6 h.For comparison, the CL was also measured using the untreated solutions (no deaerated reagents). Results and Discussion Optimization of Porphyrin Detection The best medium for the CL of luminol catalysed by Fe"' (TS03NaPP) was found to be 5 X 10-2 moll-' NaOH (results not shown) . Omitting H202 causes a drastic decrease in the blank and a slightly higher light yield (15%); the net signal is 16 times higher than that with H202 [Fig. l(a)]. This clearly demon- strates that the porphyrin-catalysed CL differs from that of peroxidase, which needs the presence of a peroxide, generally H202. Fig. 2 compares the activity (net signals) and the percentage of metallation of the 17 rneso-tetraphenylporphyrins tested. The 250 000 , i A 200 000 - 150 000 -- 2 100 000 -- 50 000 -_.0 Blank Assay Difference 1 400 000 I 1 1 2 200 000 800 600 400 200 000 000 000 000 000 000 0 Blank Assay Fig. 1 Light emission of luminol CL catalysed by Fe"'(TS03NaPP) in either the presence (0) or absence (H) of hydrogen peroxide without enhancers (A) and/or enhanced by FMN and NAD in absence of hydrogen peroxide (0, no enhancer; a, FMN; M, NAD; 8, FMN -t NAD) (B). Blank values were obtained by replacing Fe"' (TSO?NaPPj with water.Analyst, November 1996, Vol. 121 1541 results indicate that water-soluble porphyrins are generally better catalysts and that F P (TSO3NaPP) (compound 15) displays the best activity. Unlike Motsenbocker et al.,7 we found that, with the same structure and a similar percentage of metallation, the iron porphyrin is a better catalyst than the manganese porphyrin (compounds 15 and 16, respectively).It has already been shown that porphyrins having no central metal atom have no CL a ~ t i v i t y . ~ The differences in metal content could explain the disagreement between our results and those of Motsenbocker et al. Moreover, the porphyrins with a low metal content could be very efficient if they were fully metallized (compounds 10, 12 and 13 in Fig. 2). Finally, we observe that Fe"' (TCOOHPP) (compound 9) is fairly active and, since it can be easily bound to amine functions as a label, it could be potentially used in further coupling studies. A preliminary study using 1 1 different fluorochromes had shown that FMN was the most efficient enhancer. In alkaline medium, flavins are unstable16 and are converted into lumifla- vin,17 .suggesting that, in our system, lumiflavin could be the actual enhancer.Therefore, FMN and other flavins (FAD, riboflavin) can be used equally well (results not shown). In order to compare the redox properties of different compounds, the NAD-NADH couple was also tested: NAD is a more efficient enhancer than NADH (Table 1). The optimum concentration of NAD is 3 X 10-4 mol 1 - 1 ; it needs 1 h of incubation in the reagent for the best results. The study of the reaction, either without enhancer or with FMN alone, NAD alone or with FMN + NAD, shows that the maximum signal is obtained with FMN-NAD [Fig. l(h)j. The kinetic study of this optimized method indicates that the light emission is stable from 5 to 20 min after the start and then slowly decreases (less than 1% min-1) (Fig.3). Study of the Mechanism When Fe"I(TSO3NaPP) is dissolved in 5 X lo-* moll-' NaOH, a characteristic ESR spectrum of a spin adduct of DMPO with OH. radicals (DMPO-OH) is observed [Fig. 4(h)]. The luminol addition causes the disappearance of the ESR signal [Fig. 4(c)], indicating that luminol competes with DMPO to trap the radicals formed. Knowing that the DMPO-trapped superoxide radical (DMPO-OOH) is unstable in alkaline medium, it appears that DMPO-OH can be also formed from DMPO- OOH.18,i9 This means we cannot know if the ESR signal observed is due to OH- or/and 02*- formation. 1 000 000 800 000 g 600 000 2 200 000 0 To prove 0 2 ' - formation during the course of the reaction, the effect of SOD on the CL signal was studied (Fig. 5).A 50% inhibition of the light emission is obtained with the lowest SOD concentration (32.5 X 10-3 U ml-1). Inhibition is dose dependent and almost total (99%) with the highest SOD concentration tested (32.5 U ml-I). It can be concluded that OH. and 0 2 . - radicals are produced by the reaction between Fe"' (TS03NaPP) and NaOH; these radicals could be produced in a redox cycle of the porphyrin in which reduction of Fe"' to Fe" results in OH. formation from OH-, and reoxidation of Fe" by O2 generates 02.- (Fig. 6). Redox potentials calculated for the reaction in Fig. 6 lead to a minimum value of 0.7 V for the reduction of Fell' to Fell which agrees with the literature data.20.2' This radical production can explain the luminol oxidation by metalloporphyrins, since luminol is known to Table 1 Effects of different mixtures of NAD-NADH on the luminol CL NAD NADH Net signal 0 0 46 000 I00 0 I 600000 50 50 1300000 0 100 2 000 ("/c) (RLU) 1 400 000 1 200 000 1 000 000 800 000 600 000 400 000 200 000 0 2 0 10 20 30 40 50 60 Tirne/min Fig.3 both NAD and FMN as enhancers. A, Blank; and B, assay. Time course of the optimized porphyrin detection reaction, using - 80 - 60 - 40 - 20 1- 0 1 2 3 4 5 6 7 8 91011121314151617 Fig. 2 Relationship between structure atid luminescence for 17 porphyrins. Bars represent the activity (= net signal = URL;,,,,, - URLhltlnk) and black points represent the percentage of metallation calculated as (measured metal content/theorical metal content) X 100. Porphyrins (U = URA 400 CNRS; A = Aldrich; P = Poiphyrins Products): 1, Fe meso-tetraphenylporphyrin (U); 2, Fe nzeso-tetrakis(pentafluoropheny1)porphyrin (U); 3, Fe ~rzeso-tetrakis(2,6- dichloropheny1)porphyrin (U); 4, Mn meso-tetraphenylporphyrin (U); 5 , Mn meso-tetratoluylporphyrin (U); 6, Mn meso-tetrakis(2,6-dichloro- pheny1)porphyrin (U); 7, Fe meso-tetrakis(pentafluoropheny1)porphyrin (A); 8, Fe nzeso-tetrakis(4-methoxypheny1)porphyrin (A); 9, Fe meso-tetrdkis(4- carboxypheny1)porphyrin (U); 10, Fe nzrso-tetrakis(2,6-dichloro-_?-sulfonatophenyl)porphyrin (U); 1 1.Fe meso-tetrakis(4-methylpyridy1)porphyrin (U); 12, Mn meso-tetrakis(4-methylpyridy1)porphyrin (U); 13, Mn nzeso-tetrakis(2-methylpyridy1)porphyrin (U); 14, Fe n~eso-tetrakis(2,3,5,6-tetrafluoro- 4-aminoethyltrimethylamnioniutn)porphyrin (U); 15, Fe meso-tetrakis(4-su1fonatophenyl)porphyrin (P); 16, Mn meso-tetrakis(4-sulfonatopheny1)porphyrIn (P); and 17, Co 1neso-tetrakis(4-sulfonatophenyl)porphyrin (P).1542 Analyst, November 1996, Vol.121 I I 1 1 - I generate light in the presence of free radicals.22 Therefore, the mechanism of our system obviously differs from the HRP catalytic cycle described in the presence of H202.l The kinetics of the reactions conducted with deaerated or partially reareated reagents and non-deareated reagents are shown in Fig. 7. No significant luminescence could be obtained with the deareated reagents, demonstrating that this reaction requires 0 2 . The initial luminescence signal is progressively restored by reoxygenation. After partial reoxygenation, the CL decreases with time, suggesting a rapid consumption of oxygen.t I I - 50 3474 G + 50 Fig. 4 ESK spectra obtained with NaOH + DMPO (a), NaOH + Fe"l (TS03NaPP) + DMPO (b) and NaOH + Fdtk (TS03NaPP) + luminol + DMPO (c). Scan range = 100 G. 2.5 1 I 2 h a 1.5 ?5. = J 1 J [I 0.5 This result favours the above-described mechanism based on the reoxidation of Fe" by 0 2 . According to this reaction scheme, FMN and NAD enhancers could act at this level, owing to their redox properties. On the other hand, an energy transfer could also arise in the final luminescent step of the reaction chain. We tested this latter hypothesis using a laboratory made spectroluminometer, vali- dated with the well known shift of light emission when fluorescein is added to lumino1.2"24 Luminescent spectra obtained with luminol alone or associated with NAD or FMN are shown in Fig.8. In the presence of FMN, we observed a significant red shift ( 12 nm) of the luminol-porphyrin emission 7000 - - * - ~ 90 000 D __.---- 80 000 - 70 000 5000 - 4000 - 3000 - 2000 - 60 000 - 50 000 40 000 . 30 000 20 000 A 1 - 1 - 1 - 1 - I 1 1 I A i~ 10 000 1000 - ,". . . * . . * I 0 I 5 10 O I 0 Time/min Fig. 7 Role of oxygen: light emission (RLU) measured with deaerated solutions (luminol-NaOH-FMN; porphyrin and water) by bubbling overnight with nitrogen (A) and then exposed to ambient air for 3 and 6 h (B and C, respectively). Control of the emitted light with the same solutions but not deaerated is represented in D. A, R and C refer to the left-hand scale and D to the right-hand scale.400 Fig. 5 Inhibition effect of superoxide dismutase. Volumes of 10 vl of serial dilutions of SOD solutions were added to the FMN-containing lumi- nol-NaOH reagent. NAD and porphyrin were added before CL measure- ment. Final concentrations in SOD were 0.032,0.325,3.25 and 32.5 U ml-1 (log scale); 0 represents the control without SOD. 700 Wavelengthhm Fig. 8 Luminescent spectra obtained for luminol alone (a) and in presence of NAD (b), FMN (c) and NAD + FMN (d). The scale of each spectrum was adjusted for comparison. FeII(porphyrin) Fig. 6 Reaction scheme: electron transfer process between Fe"' and Fe" porphyrin and oxy radicals.Analyst, November 1996, Vol. 121 I543 Table 2 Shape functions parameters obtained from mathematical Rix and MalengC25 treatment of luminescent spectra, taking luminol alone as reference.The first line (luminol versus luminol) represents the maximum intrinsic variability for the parameters. A, Slope (widening factor); B, intercept (translation factor); s, standard deviation from the best square regression line (shape factor) System A B s Luminol versus luminol 0.998 2 0.03 Luminol versus luminol + FMN 0.948 19 0.14 Luminol versus luminol + NAD 0.988 6 0.09 Luminol versus luminol + FMN + NAD 0.969 12 0.13 Luminol versus luminol + fluorescein 0.804 56 0.27 spectrum, which suggests that an energy transfer related to the final radiative step is involved (unlikely to be photon coupled). However, no spectral shape change or shift was observed in the presence of NAD, indicating that NAD is involved in the former steps of the catalytic cycle.The spectra were compared on a shape factor basis, using a mathematical treatment proposed by Rix and MalengC,2' which involves normalized spectral repartition functions (Table 2). The aim of this comparison was to look for a possible photon- coupled emission process between luminol and FMN. This showed that interactions between NAD and luminol did not shift or enlarge the spectrum, while the action of FMN consisted in significant translation and widening, together with a shape factor change. A mathematical adjustment has shown that the luminol + FMN + NAD spectrum can be obtained through a linear combination of luminol + FMN and luminol + NAD, which is suggestive of two independent emission mechanisms. A careful examination of luminol and luminol + FMN spectral shapes suggest a chemically induced electron exchange lumi- nescence energy transfer.26 The authors are grateful to J.Michon (LEDSS, Grenoble, France) for the ESR measurements, P. Battioni (URA 400, CNRS, Paris, France) for the synthesis of the metalloporphyrins and J. Poupon (Laboratoire de Biochimie, H6pital Lariboisikre, Paris, France) for the AAS measurements. Financial support for this study was provided by the RCgion Rh6ne-Alpes (grant No. M01443 92 to J.-P.S. 1992). References 1 Xie, L. Y., and Dolphin, D., in Handbook of Metal-Ligand Interactions in Biological Fluids, ed. Berthon, G., Marcel Dekker, New York, 1995, pp. 339-351. 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 Mansuy, D., Battioni, P., and Battioni, J.P., Eur. J . Biochem., 1989, 184, 267. Labat, G., Seris, J. L., and Meunier, B., Angew. Chem., Int. Ed. Engl., 1990,29, 1471. Genfa, Z., and Dasgupta, P. K., Anal. Chem., 1992, 64, 5 17. Klopf, L. L., and Nieman, T. A., Anal. Chem., 1983, 55, 1080. Ojima, H., and Nonoyama, K., in Dioxygen Activation and Homoge- neous Catalytic Oxidation, ed. Simandi, L. I., Elsevier, Amsterdam, 1991, pp. 417-427. Motsenbocker, M., Ichimori, Y., and Kondo, K., Anal. Chem., 1993, 65, 397. Motsenbocker, M., Oda, K., and Ichimori, Y., J . Biolumin. Chem- ilumin., 1994, 9, 7 . Green, M. D., Mount, D. L., Todd, G. D., and Capomacchia, A. C., J . Chromutogr. A , 1995, 695, 237. Thorpe, G. H. G., Kricka, L. J., Moseley, S.B., and Whitehead, T. P., Clin. Chem., 1985, 31, 1335. Kricka, L. J., and Ji, X., Clin. Chem., 1994, 40, 1828. Sehlstedt, U., Kim, S. K., Carter, P., Goodisman, J., Vollano, J. F., Norden, B., and Dabrowiak, J. C., Biochemistry, 1994, 33, 417. Nicoreta, T. M., Munson, B. R., and Fiel, R. J., Photochem. Photobiol., 1994, 60, 295. Bustamante, C., Gurrieri, S., Pastemack, R. F., Purrello, R., and Rizzarelli, E., Biopolymers, 1994, 34, 1099. Stott, R., and Kricka, L. J., in Bioluminescence and Chemilu- minescence. New Perspectives, ed. Scholmerisch, J., Andreesen, R., Kapp, A., Emst, M., and Woods, W. G., Wiley, Chichester, 1987, Beaucamp, K., Bergmeyer, H. U., and Beutler, H. O., in Methods of Enzymatic Analysis, ed. Bergmeyer, H. U., Verlag Chemie, Wein- heim and Academic Press, New York, 2nd Engl. edn., 1974, vol. 1, pp. 532-533. Beinert, H., in The Enzymes, ed. Boyer, P. D., Lardy, H., and Myrbkk, K., Academic Press, New York, 2nd edn., 1960, vol. 2, pp. 339416. Gaudu, P., Touati, D., Niviere, V., and Fontecave, M., J . Biol. Chem., 1994,269, 8182. Weiner, L. W., Methods Enzymol., 1994, 233, 92. Koppenol, W. H., in Membrane Lipid Oxidation, ed. Vigo-Pelfrey, C., CRC Press, Boca Raton, FL, 1990, vol. I, pp. 6-7. Kochi, J. K., in Free Radicals, ed. Kochi, J. K., Wiley, New York, 1973, vol. I, pp. 628-629. Merenyi, G., Lind, J., and Ericksen, T. E., J . Biolumin. Chemilumin., 1990, 5, 53. Chemiluminescence, ed. Campbell, A. K., VCH Publishers, Cambridge, 1988, p. 480. Patel, A., and Campbell, A. K., Clin. Chem., 1983, 29, 1604. Rix, H., and MalengC, J. P., IEEE Trans. Syst. Man Cybern., 1980,10, 90. Chemiluminescence, ed. Campbell, A. K., VCH Publishers, Cambridge, 1988, pp. 510-516. pp. 237-240. Paper 6102789J Received April 22, 1996 Accepted June 24,1996

ISSN:0003-2654    

DOI:10.1039/AN9962101539

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, November 1996, Vol. 121 (1545-1549) 1545 Photochemical-Spectrof luorimetric Determination of Two Pyrethroid Insecticides Using an Anionic Micellar Medium* Jean- Jacques Aaron and Atanasse Coly' Institut de Topologie et de Dynamique des Syst2mes de I' Universitk Denis Diderot-Paris 7, Laboratoire Associk au CNRS, URA 34, 1 Rue Guy de la Brosse, 75005 Paris, France The effect of sodium dodecyl sulfate (SDS) micelles on the photochemically induced fluorimetric (PIF) method was investigated in the determination of fenvalerate and deltamethrin insecticides. Physical and chemical variables affecting the sensitivity of the method were optimized. Linear calibration curves were established over more than two orders of magnitude. The limits of detection were 2.2-2.5 times lower in SDS micelles than in common organic solvents.The RSDs were between 3.7 and 5.2%. Application of the method to the analysis of commercial technical formulations and spiked water samples was evaluated. Satisfactory recoveries were found, ranging from 88.4 to 112.6%. The possible origins of the observed PIF enhancement effect in micellar solution are discussed. Keywords: Photochemically induced fluorescence; pyrethroid insecticides; anionic micellar medium; technical formulations; water analysis Introduction Organized media, viz., cyclodextrins,1.2 micellesl.3-7 and lipo- somic vesicles,8 are known for their ability to solubilize selectively, by forming aggregates in solution, a variety of hydrophobic fluorophores, resulting in an increase in lumines- cence intensity. Since the microenvironment of the analytes associated with these aggregates is very different from that in a bulk solution, differences are often found in quantum yields,4 lifetimes of the excited singlet4 or triplet9-12 states of the probe, excitation and emission spectra6 and quenching or deactivation processes.4~10 These differences frequently induce an enhance- ment of the luminescence intensities and decrease the inter- ference from impurities.From an analytical point of view, these phenomena are extremely valuable. Nowadays, organized media used for analytical purposes involve micelles rather than cyclodextrins or vesicles. The former systems are more versatile and generally lead to enhanced intensities and selectivities. Although micelles are not size-selective like cyclodextrins, 1 they offer charge selection through the choice of anionic, cationic, neutral or zwitterionic surfactants3 and polarity selection through the use of normal (aqueous) micelles4~s or reversed (apolar organic) micelles.Therefore, micellar fluorimetry is emerging as a competitive alternative to fibre-optic and laser-based fluorimetric tech- niques, which require more sophisticated and expensive instrumentation. 13 Other interesting features include the speed of analysis of complex mixtures7 and the increase in the solubility of organic analytes in aqueous solutions. In spite of * Presented at the VIIth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17-19, 1996.t On leave from the Facult6 des Sciences et Techniques, Departement de Chimie, UniversitC Cheikh Anta DlOP, Dakar. SCnCgal. this obvious analytical interest, micellar fluorimetry has rarely been applied to the determination of pesticides.'4 In this paper, we describe the effect of sodium dodecyl sulfate (SDS) micellar solutions on the photochemically induced fluorimetric (PIF) analysis of two pyrethroid insecticides, fenvalerate and deltamethrin. Application to their determination in technical formulations and tap water samples was evaluated. The influence of the immediate fluorophore microenvironment in terms of solvent polarity and viscosity and the presence of molecular oxygen on the change in molar absorptivity and PIF quantum efficiency is discussed. The aim of this work was to demonstrate that the use of UV radiation to form fluorescent photoproducts by direct irradiation of the pesticide solubilized in the micelle core is a valuable method for the rapid screening of non-fluorescent pesticides in environmental analysis.Experimental Reagents Fenvalerate and deltamethrin (HPLC grade, 99% m/m) were purchased from Riedel-de Haen (Hannover, Germany) and used without further purification. Technical formulations of these insecticides were obtained from the Office of Plant Protection (DPV, Dakar, Shkgal). Two types of formulations, ultra-low-volume solutions (ULV) and emulsifiable concentrate (EC), were utilized. SDS of analytical-reagent grade was purchased from Aldrich (Milwaukee, WT, USA) and used as received. Solvents used were methanol (spectroscopic grade, Merck, Darmstadt, Germany) acetonitrile (spectroscopic grade, Aldrich) and de-ionized water.Apparatus Fluorescence measurements were performed on a Kontron SFM-25 spectrofluorimeter interfaced with a Geocom micro- computer. Fluorescence spectra were recorded and memorized using a Kontron SFM-25 data control and acquisition program. An Osram 200 W HBO high-pressure mercury lamp with an Oriel Model 8500 power supply was utilized for the photolysis reactions. The photochemical set-up included a light-box, consisting of a fan, the mercury lamp and a convenient quartz lens, and a 1 cm quartz cuvette placed on an optical bench 45 cm from the mercury lamp. Procedure Solutions Stock standard solutions of the pesticides (10-3 mol 1- I ) were freshly prepared by dissolving the compounds in acetonitrile for fenvalerate and methanol for deltamethrin. Serial dilutions were performed to obtain working standard solutions.A solution of the optimized surfactant (SDS) concentration (0.04 mol 1- I )1546 Analyst, November 1996, Vol. 121 was obtained from a 0.1 rnol 1-1 stock solution. Micellar solutions of the pesticides were prepared by transferring 10-100 pl of the pesticide working standard solution into a 10 ml flask, containing 4 ml of the surfactant stock solution, and adding the requisite amount of de-ionized water. The solutions were then stirred ultrasonically for 10 rnin before irradiation. The amount of organic solvent remaining in the micelles is known to decrease their chemical potential and to diminish the c.m.c.15 Therefore, we maintained a constant concentration of about 1 % v/v of the organic solvent used for all calibration plots instead of evaporating it.4 Photolysis reactions and Calibration curves Photolysis reactions were performed by irradiating the pesticide micellar solutions with UV radiation for a fixed time.All photolysis studies were carried out at room temperature. Curves of fluorescence intensity versus UV irradiation time were constructed at constant excitation (he-) and emission (hem) analytical wavelengths of the pesticide photoproducts at 5 min intervals. Linear calibration curves were obtained at the same pesticide photoproduct hex and hem values by measuring the PIF signal corresponding to the optimum irradiation time.Write Plus, version 3.10, application software was used for statistical treatment of the data. Treatment of Technical Formulations Commercial technical formulations, i.e., sumicombi EC (a binary mixture containing 250 g 1- of fenitrothion and 50 g 1- of fenvalerate) and deltamethrin ULV (12.5 g I-l), were either extracted with methanol, utilizing the previously described extraction procedure,lG and then irradiated without any clean-up procedure, or used as received. Recoveries from the treated and untreated technical formulations were evaluated by means of the standard additions method. Results and Discussion Photochemical Reactivity Both pyrethroids were naturally non-fluorescent, whereas an intense fluorescence band appeared upon UV irradiation. The mechanism of deltamethrin photodecomposition upon UV irradiation was established under various experimental con- ditions.17 It was found that this insecticide can produce 25 photoproducts via four main photodegradation pathways.A photochemical study of the solar irradiation of fenvaleratel8 showed that the predominant reactions were decarboxylation and cleavage of the ester linkage in water. Under our experimental conditions (see Experimental), the photolysis of fenvalerate and deltamethrin (irradiated in an organic solvent for 15 and 30 min, respectively) led to the formation of five and three photoproducts, respectively, which were determined by GC-MS. We identified the main fluores- cent degradation photoproduct of fenvalerate as Z-oxo(3- phenoxypheny1)acetonitrile and that of deltamethrin as 3-phe- noxymethyl benzoate.PIF Spectral Properties in Micellar Medium We investigated the micelle effect on the photochemical fluorescence spectral properties of fenvalerate and deltameth- rin. A surfactant (SDS) concentration of 0.04 mol 1-1, corresponding to about 4.6 times the c.m.c. (8.6 X 10-3 mol 1-1),3,4 was used in order to ensure micelle formation. The PIF emission spectra of both pesticides were recorded in micellar and non-organized media, using irradiation times of 6 rnin for fenvalerate (hex = 293 nm) and 25 rnin for deltamethrin ~ _ _ _ _ _ (hex = 291 nm) (Figs. 1 and 2). It can be seen that for the deltamethrin photoproduct, the emission maximum wavelength (Lem = 3 19 nm) is the same in micelle solution and in methanol, whereas a 5 nm redshift of the emission wavelength is observed for the fenvalerate photoproduct on going from acetonitrile (kern = 333 nm) to the SDS solution (hem = 338 nm).In pure water, the PIF emission spectra present a very unusual shape with a minimum in the 350-380 nm region, probably due to light diffusion by the pesticide molecule aggregates formed in aqueous suspension (Figs. 1 and 2). Fenvalerate and deltameth- rin are particularly hydrophobic and poorly soluble in water (<0.4 pg ml-I). At the SDS concentration used (0.04 moll- l), an important enhancement of the PIF signal occurs in micellar medium resulting from the solubilization of substantial amounts of 200 i\ t \ Wavelengthtnm Fig. 1 PIF emission spectra of fenvalerate (1 0-5 mol 1- I ) in water (curve l ) , acetonitrile (curve 2) and 0.04 moll-' SDS aqueous solution (curve 5).Curves 3 and 4 were obtained using fenvalerate concentrations of 2 X 10-6 and 6 X mol I-l, respectively, in 0.04 mol 1-' SDS solution. All spectra were recorded at he, = 293 nm with an irradiation time of 6 min. 200 150 x v) c a, c a, +-' .- - .- g 100 8 2 ii v) 0 3 50 r\\ 1 350 400 450 ! I0 Wave1 en g t h/nm Fig. 2 PIF emission spectra of deltamethrin (10-5 moll-I) in water (curve I), methanol (curve 2) and 0.04 mol I-' SDS aqueous solution (curve 5). Curves 3 and 4 were obtained using deltamethrin concentrations of 2 X 10-6 and 6 X 10-6 mol l-l, respectively, in 0.04 moll-' SDS solution. All spectra were recorded at he, = 291 nm with an irradiation time of 25 min.Analyst, November 1996, Vol.121 1547 pesticide in the micellar aggregates formed. Indeed, the observed PIF intensities of fenvalerate and deltamethrin in aqueous SDS solution are enhanced about 3.2 times relative to acetonitrile and 2.9 times relative to methanol respectively, (Figs. 1 and 2). This PIF micellar enhancement factor may be attributed to the increase in the rigidity of the pesticide photoproduct molecules when they are adsorbed at the micelle surface.6 This clearly indicates that the presence of micelles is required for observing PIF emission from the excited singlet state of both pyrethroid photoproducts in aqueous solution. Effect of the SDS Concentration As is generally o b s e r ~ e d , ~ ? ~ an increase in surfactant (SDS) concentration has a marked influence on the PIF intensity of analytes.Plots of pesticide photoproduct fluorescence intensity versus the logarithm of surfactant concentration are shown in Fig. 3. The general shape of these curves is in agreement with those found for other aromatic compounds in the presence of SDS.4,6 At surfactant concentrations far below the c.m.c., SDS is dispersed mostly as monomers, although dimers, trimers, etc., can exist. Therefore, the resulting PIF intensity is weak as virtually all pesticide molecules are dispersed in a disordered suspension of monomer. When the surfactant concentration is increased, the PIF signal remains unaffected until the c.m.c. value is reached. Close to the c.m.c. value, monomers assemble progressively in aggregates to form micelles roughly spherical in shape and consisting on an average of 62 monomers per aggregate unit.3.4 Therefore, the PIF signal of the pesticide increases proportionally to the number of micelles formed.As the concentration of surfactant is increased above the c.m.c. value, more micellar assemblies are formed, while the amount 1 60 - 0 A 20 - b 80 - 0 0 A A 08 A 0 0 of free monomer remains approximately constant and equal to the c.m.c.;3 as a consequence, the PIF intensity continues to increase progressively. As the surfactant concentration in- creases further, the number and size of micelles also increase and a dynamic equilibrium exists between the pesticide molecules adsorbed at the micelle surface and those in the bulk solution. The ratio of bound to free molecules becomes larger until saturation is reached.For both pyrethroids, the selected SDS concentration was 0.04 mol 1- I . Optimum Irradiation Time The curves of fluorescence intensity of the pesticide photopro- ducts versus UV irradiation time (Fig. 4) show an initial increase in the fluorescence signal, which reaches a maximum value, then a marked plateau, and finally a decrease. The general shape of these curves indicates a two-step photolysis mecha- nism.16 For fenvalerate, the optimum irradiation time (defined as the irradiation time corresponding to the maximum fluores- cence intensity) is slightly longer (12 min) in micellar medium than in acetonitrile (10 min) (Table 1), whereas for deltameth- rin, it is substantially increased, from 23 to 35 min, on going from methanol to SDS.Therefore, these irradiation times were chosen for analytical purposes, except for deltamethrin, for which 25 min was adopted. Analytical Figures of Merit A series of standard solutions of fenvalerate and deltamethrin solubilized in micelles were irradiated in triplicate ([SDS] = 0.04 mol 1-*), in order to test the linearity and the reproducibility of the log-log calibration plots. Satisfactory linearity was obtained, as indicated by the values of the correlation coefficients ( r ) , which are close to unit (Table 1). 240 > cn C a, c. .- 180 .- 0 0 c a, 3 120 s? ii 0 60 0 7 14 21 18 35 Time/min Log( [SDS]/mol I -' ) Fig. 3 Influence of SDS concentration of the PIF intensity of fenvalerate (0). concentration = 5 X 10-6 mol I-' (irradiation time 12 min) and deltamethrin (A), concentration = mol 1-I (irradiation time 25 min) .Fig. 4 Influence of the UV irradiation time on the PIF intensity of fenvalerate in different solvents. Concentrations: A, 10-5 mol 1-1 in acetonitrile and B, 2 X 10-6, C, 6 X 10-6 and D, 10-5 mol 1-1 in 0.04 moll-' SDS aqueous solution. Table 1 Analytical figures of merit for the PIF determination of pyrethroids Concentration LOD' RSD (Yo) Compound Solvent range/ng ml-1 Slope" r ng ml-1 &J/min (n = 3) Fenvalerate 0.04 moll-' SDS 10-2520 1.05 0.998 7 12 3.7 MeCN 42-2520 1.00 0.991 16 10 2.5 Deltamethrin 0.04 moll-' SDS 20-5040 0.98 0.995 11 35 5.2 MeOH 50-5040 0.85 0.994 27 23 2.9 * Slope of the log-log calibration curves. 7 Defined as the amount of analyte giving a signal-to-noise ratio of 3.* Optimum irradiation time, corresponding to the maximum PIF intensity. The irradiation time selected for deltamethrin in micellar solution was 25 min instead of 35 min.1548 Analyst, November 1996, Vol. 121 The limits of detection (LODs) are 2.2-2.5 times lower in micellar solutions than in common organic solvents. The linear concentration ranges are also larger in micelles. The reproduci- bility is satisfactory in micelle solutions with RSDs ranging from 3.7 to 5.2%. Our LOD values are significantly lower than those reported for the determination of the same insecticides by other methods, e.g., the LODs obtained by reversed-phase HPLC with UV detection were 40-70 ml-1 for fenvalerate and deltamethrin. 19 Analytical Applications The proposed method was applied to the determination of fenvalerate and deltamethrin in treated and untreated com- mercial technical formulations and in spiked water.Recovery studies were performed by adding known amounts of the pesticide to real samples and determining the concentration by u'sing the standard additions method. In most instances, the accuracy and recovery from real samples were satisfactory. Treated commercial technical formulations Fenvalerate and deltamethrin were determined in technical formulations according to the extraction procedure described previously.16 Table 2 gives the results of three replicate analyses. The recoveries obtained ranged from about 93 to 109%. Untreated commercial technical formulations Technical formulations of the two pesticides were solubilized in micellar solution as received, and the recoveries obtained were between 88 and 113% (Table 2).Table 2 Determination of pyrethroids in technical formulations and spiked water Concentration/ng ml- I Compound Treated formulutions- Fenvalerate Deltamethrin Untreuted formulutions- Fenvalerate Deltamethrin Spiked wuter- Fenvalerate Deltamethrin Added 867 1245 2085 2505 379 546 1359 2883 872 1124 1350 2090 704 87 1 2192 3208 5.58 I524 I944 2364 184 657 1673 2689 Found* 832 f 8 1209 f 3 1960f 18 414 f 6 579 f 9 1340 f 6 3021 k 6 859 k 5 1021 + 3 1156 f 3 1848 k 8 775 f 25 981 + 9 2175 k 104 3064 f 74 528 f 5 1586 f 3 2002 k 8 2301 f 15 180 f 6 678 f 6 1617 k 25 2665 f 18 2324 k a Mean recovery1 (%) 96.0 97.1 94.0 92.8 109.2 106.0 98.6 104.8 98.5 90.8 92.5 88.4 110.1 112.6 99.2 95 .5 94.6 104.0 102.9 97.3 97.8 103.2 96.6 99.1 * Mean & standard deviation of three replicates.+ Values measured using standard additions procedure. Spiked wuter analvsis Table 2 summarizes the results obtained with the proposed method for the analysis of tap water samples spiked with different amounts of fenvalerate and deltamethrin. Satisfactory recoveries were obtained, ranging from 95 to 104%. Origins of the PIF Enhancement in Micellar Solution The quantitative luminescence relationship indicates that the observed enhancement of the PIF signal, for a constant pesticide concentration, in micellar medium compared with that in bulk organic solvent or water and under identical instrumental conditions (I0 and I constant), results from an increase in the quantum yield, @f and/or molar absorptivity, E.The absorption spectra of the photoproducts formed in micellar medium and organic solvents showed no significant difference in the absorbance intensity of the studied spectra in both media. Therefore, the PIF enhancement in the micellar systems can be attributed to an increase in the quantum yield of both pesticide photoproducts, implying a significant decrease in the rate of the competitive radiationless processes. The factors that are able to induce the increase of quantum yield in micellar medium are numerous, including changes in micropolarity and in viscosity of the medium and effective shielding of the excited singlet state from quenchers such as molecular oxygen.4.5 Table 3 summarizes the influence of these parameters on the PIF of the pesticide photoproducts under study.Influence of micropolarity Table 3 shows that ethylene glycol is less polar than SDS. Nevertheless, the PIF intensity of both pesticides is larger in the former solvent. Therefore, we can conclude that the polarity of the microenvironment has only a limited effect on the observed micellar PIF enhancement of both pesticides. Znjluence of molecular oxygen Molecular oxygen is known to be an efficient quencher of excited singlet states. As expected, the PIF intensities of the pesticides measured after deoxygenating the organic solution for 30 min are larger than those determined in the corresponding aerated solvent. Consequently, the observed micellar PIF enhancement can be ascribed in part to the probable micelle Table 3 Study of the various factors influencing the enhancement of the PIF signal of both lo-' mol I-' fenvalerate and deltamethrin in miccllar media.Viscosityi/ Compound Solvent &* CP I f + f,,Jmin Fenvalerate SDS (0.04 moll-') Ethylene glycol Deoxygenated MeCN MeCNs Deltamethrin SDS (0.04 mol 1-I) Ethylene glycol Deoxy genated MeOH MeOH3 40-55 37.7 37.5 37.5 40-5 5 37.7 32.63 32.63 28-39 19.9 0.345 0.345 28-39 19.9 0.547 0.547 3.3 12 5.4 15 1.5 10 1.0 10 3.7 3s 5.8 20 2.4 23 1.0 23 E = Relative permittivity: for micelles, see ref. 4. For micelles, see ref. 4. Zf = relative PIF intensity, corrected for the solvent signal and normalized to the lowest PIF intensity, for each compound. 4 Deoxygen- ation time 30 min.Analyst, November 1996, Vol.121 1549 protection of the pesticide molecules from the different quenchers present in solution. As a result, the local concentra- tion of molecular oxygen would decrease in the micellar phase. Influence (f niic.i-ovisc.osit~ Very large PIF signals were observed for both pesticide photoproducts when using high-viscosity media such as SDS and ethylene glycol. We can assume that the microenvironment viscosity is involved in the micellar PIF enhancement. The larger PIF intensities obtained in ethylene glycol may be due to the intervention of hydrogen bonds, which exert a significant effect in the excited singlet states. This study has demonstrated that a combination of the various factors investigated is accountable for the PIF enhance- ment in micelles.Indeed, micellar media provide a favourable microenvjronment for both pesticide photoproducts so that their excited singlet state is stabilized and the efficiency of PIF is improved. Conclusion We have shown the usefulness of the effect of micelles for improving the sensitivity, selectivity and simplicity of the PIF method for the determination of aromatic pesticides. Moreover, its application has revealed that nanogram levels of the two pyrethroids can be detected under better conditions using SDS micellar media rather than organic solutions. For real environ- mental samples, purification and/or preconcentration steps may be needed. In addition, for environmental analysis purposes, simultaneous PIF determination of both pyrethroid insecticides may be performed by selecting a different UV irradiation time for each pesticide, or by applying specific techniques such as derivative spectroscopy or partial least-squares multivariate calibration. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Cline Love, L.J., Graycski, M. L., and Noroski, J., Anal. Chim. Acfa, 1985, 170 3. Marquez, J. C., Hernandez, M., and Sanchez, F. G., Analyst, 1990, 115, 1003. Cline Love, L. J., Habarta, J. G. and Dorsey, J. G., Anal. Chem., 1984, 56, 1132A. Singh, H., and Hinze, W. L., Anal. Lett., 1982, 15, 221. Armstrong, D. W., Hinze, W. L., Bui, K. H., and Singh, H. N., And. Lett., 1981, 14, 1659. Kessler, M. A., and Wolfbeis, 0. S., Ber. Bunsenges. Phys. Chem., 1989, 93, 927. Berthod, A., Asensio, J. M., and Lascma, J. J., ,I. Liy. Chromatogr., 1989,12, 2621. Gonzalez Alvarez, M. J., Diaz Garcia, M. E., and Sanz-Medel, A., Anal. Chim. Acta, 1990, 234, 181. Cline Love, L. J., Skrilec, M., and Habarta, J. G., Anal. Chrm., 1980, 52, 754. Alingren, M., Grieser, F., and Thomas, J. K., J . Am Chem. Sor.., 1979,101, 279. Mwalupindi, A. G., Blyshak, L. A., Ndou, T. T., and Warner, 1. M., Anal. Chern., 1991, 63, 1328. Blyshak, L. A., Rollie-Taylor, M., Sylvester, D. W., Underwood, A. L., Patonay, G., and Warner, 1. M., J . Colloid Interface Sci., 1990, 136, 509. Warner, I. M., and McGown, L. B., Anal. Chem., 1992, 64, 343R. De la Guardia, M., Hernandez, M. L., Sancenon, S., and Carrion, J. L., Colloids Surf., 1990, 48, 57. Hayase, K., and Hayano, S., Bull. Cliem. SOC. Jp., 1978, 51, 933. Coly, A., and Aaron, J. J., Analyst, 1994, 119, 1205. Ruzo, L. O., Holmstead, R. L., and Casida, J. E., J . Agt-ic. Food Chem., 1977, 25, 1385. Mikami, N., Takahashi, N., Hayashi, K., and Miyamoto, J., Pesric. Sci., 1980, 5 , 225. Haddad, P. R., Brayan, J. G., Sharp, G. J., Dilli, S . , and Desmarchelier, J. M., J . Clzromatogr., 1989, 461, 337. Paper 6/02 788A Received April 22, 1996 Accepted June 13, 1996

ISSN:0003-2654    

DOI:10.1039/AN9962101545

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, November 1996, Vol. 121 (1551-1556) 1551 Effects of pH and Solvent on the Fluorescence Properties of Biomedically Important Benzam ides. Application to Determination in Drugs and in Human Urine* Mihaela Bunaa, Jean-Jacques Aarona,? Patrice Prognonb and Georges Mahuzierb a Institut de Topologie et de Dynamique des Systdmes de I'Universite' Paris 7-Denis Diderot, associk au CNRS (URA 34) 1, rue Guy de la Brosse, 75005 Paris, France b Lahoratoire de Chimie Analytique II-Bioanalyse, Faculte' de Pharmacie, Universite' Paris-Sud, 5, rue J . B. Cle'ment, 92290 Chatenay-Malahry, France The fluorescence properties of five substituted benzamides, including alizapride, metoclopramide, sulpiride, sultopride and tiapride, were investigated at several pH values and in various solvents (dimethyl sulfoxide, ethanol, ethylene glycol, methanol, propan-2-01, tetrahydrofuran and water).Except for alizapride, the fluorescence intensities were found to be higher at acidic (1-6) than at alkaline (8-12) pH values. Using the optimum solvent (aqueous solutions) and appropriate pH conditions, linear spectrofluorimetric calibration curves were established over a concentration range of about two orders of magnitude, with correlation coefficients larger than 0.996. Limits of detection were between 1 and 13 ng ml-1, depending on the compound. The method was applied to the determination of benzamides in pharmaceutical preparations and in human urine, with recoveries ranging from 94 to 108% and from 93 to 104 %, respectively. Keywords: Benzamides; spectrojhorimetry; pH and solvent efseects; drugs; urine analysis Introduction Substituted benzamides, e.g, alizapride (1), metoclopramide (2), sulpiride (3), sultopride (4) and tiapride (5) (Fig.l), are a homogeneous class of chemicals which, depending on their structure, exhibit antipsychotic properties (e.g., 3 and 5) and can prevent nausea and vomiting (e.g., 1 and 2). Originally derived from procainamide, benzamides are antagonists of the dopamine D2 receptors, which distinguishes these compounds from other antipsychotic agents. This singular feature may explain the very low incidence of side effects on the extrapyramidal system. In addition to their antipsychotic action, substituted benzamides present anti-emetic (1-3) antidyskinetic (5) and antihypertensive (4) action.l.2 In fact, metoclopramide (2) is used for the treatment of various gastro-intestinal disorders and alizapride (1) in the prophylaxis of nausea and vomiting induced by chemotherapy.The therapeutic importance of these compounds required the development of selective, rapid and accessible methods for their assay in industrial quality control and clinical monitoring. For this reason, most studies concerning these substituted benza- mides have mainly practical and analytical purposes, and only * Presented at the VIIth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17-19. 1996. t To whom correspondence should be addressed. seldom a theoretical purpose. With the aim of finding a correlation between the chemical properties of substituted benzamides and their pharmacological activity, Van Damme and co-workers3.4 determined the ionization constants of sulpiride and investigated the mass, UV, IR and NMR spectra of several benzamides.Most analytical methods employed for the determination of benzamides are chromatographic,s--21 includ- 0 U Alizapride CONHCH~CH~N(C~H~)P CI @OCH3 NH2 Metoclopramide Sulpiride n 0 Sultropide ,C2H5 CONHCH2-N, H3C-S OJ&OCH3 I I C2H5 I I 0 Tiapride Fig. 1 Structures of substituted benzamides.1552 Analyst, November 1996, Vol. 121 ing HPLC,s-7>10-21 GC8 and GC-MS.9 Although the use of fluorescence as an HPLC detection mode for substituted benzamides in biological media has been relatively widely reported,5--7,21 very few direct spectrofluorimetric techniques have been implemented.'1-22 In fact, a sole fluorescence study concerning metoclopramide has been reported.22 In this context, the first aim of this work was to assess the general fluorescence properties of the five benzamides studied and their change with pH and solvent, and to investigate their photolysis properties, which are of interest for the evaluation of their photostability in dosage forms.Second, taking into account this spectroscopic study, we propose a simple and rapid procedure for the determination of benzamides in pharmaceuti- cal preparations and in biological matrices such as human urine. Experimental Apparatus Perkin-Elmer (Norwalk, CT, USA) LS-S and LS-50 spectro- fluorimeters were used for fluorescence studies.All measure- ments were performed in quartz cuvettes (10 mm optical path) at 22 k 2 "C. The excitation and emission slit widths were 5 nm. Electronic absorption spectra were measured on a Varian (Palo Alto, CA, USA) Cary-2 10 spectrophotometer. An Osram (Berlin, Germany) 200 W mercury arc lamp with an Oriel (Les Vlis, France) Model 8500 power supply was used for the photolysis study. Reagents Benzamides were a kind gift from Laboratoires Delagrange (Chilly-Mazarin, France). No further purification was perfor- med. Stock standard solutions (10-7 mol I-') of the substituted benzamides were prepared in de-ionized water and in other solvents from the corresponding chlorohydrate (1,2,4 and 5) or base compound (3). Plitican (alizapride), Primperan (metoclo- pramide), Dogmatil (sulpiride), Barnetil (sultopride) and Tia- pridal (tiapride) formulations (injection ampoules) were also provided by Laboratoires Delagrange.Solvents [ethanol, propan-2-01, methanol, diethyl ether, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acet- onitrile, tetrahydrofuran (THF) and ethylene glycol] were of spectrometric grade and purchased from Aldrich (Milwaukee, WI, USA), Janssen (Beerse, Belgium) or Merck (Darmstadt, Germany). Chloroform used for extraction from urine was of analytical-reagent grade (Prolabo, Paris, France). Buffer solu- tions of different pH were obtained from Fluka (Buchs, Switzerland) or Merck. Commercial 36% hydrochloric acid and aqueous solutions of sodium hydroxide were also used. For spectroscopic studies, the concentration ranges of the dilute solutions of the benzamides were between 10-7 and 2 X 10-5 mol 1- I , depending on the molecule.For urine samples, standard solutions of different concen- trations (5.2-1 1.8 yg ml-l) were prepared in de-ionized water for each series of analyses and each compound. Procedure Photolytic study An aliquot of each benzamide aqueous solution (concentrations between and 2 X 10-5 moll-1) was introduced into a 10 mm quartz cuvette, then irradiated at room temperature with a mercury arc lamp for short periods of time, specific for each molecule. The photolysis kinetics were monitored spectro- fluorimetrically . Fluorescence quantum yield measurements Fluorescence quantum yields (GF) of the benzamides were determined in water at 22.0 k 2.0 "C by the comparative method, using quinine in 0.05 mol I-' H2S04 as a standard.Emission spectra were corrected by the Perkin-Elmer LS-SO FLDM program. Quantum yields were calculated using the equation where @pf = quinine quantum yield (0.58),23 IF and ZEef are the corrected emission spectra areas of the benzamide and quinine, respectively, E and &Ref are the corresponding molar absorption coefficients and C and CRef are the corresponding concentra- tions. Solvent e#ect Fluorescence spectra of benzamide solutions in de-ionized water, ethanol, propan-2-01, methanol, ethyl ether, DMF, DMSO, acetonitrile, THF and ethylene glycol were recorded at 22 (+ 2) "C. Working conditions were identical for each compound. pH effect Fluorescence spectra of the aqueous solutions of each com- pound, containing a 20% volume of buffer solutions of different pH (ranging from 1.8 to 1 l.S), were recorded under the same working conditions, specific to each molecule under study.Measurements at extreme pH values (i.e., 1 and 12) were also performed in non-buffered solution (0.1 mol 1-1 HCl and 0.01 mol 1-1 NaOH, respectively). Assays In pharmaceutical preparations Vials of the five benzamides were assayed. For each prepara- tion, a constant volume was collected from the contents of one vial and diluted with de-ionized water to a concentration of 0.175-0.300 yg ml-1, depending on the compound. Quantifi- cation was performed according to the standard additions method. In urine The extraction procedure is an adaptation of that of Kleimola et al.24 A known volume of standard solution was introduced into a screw-capped centrifugation tube.A 1 ml volume of human urine was added, then the mixture was adjusted to an alkaline pH value, specific for each compound, with NaOH solution. To this mixture chloroform was added ( 5 ml), then the tube was shaken mechanically for 3 min. After centrifugation for 5 min, the upper aqueous layer was discarded and a 3 or 4 ml (depending on the compound) sample of the organic layer was transferred into another similar tube containing 4 ml of dilute HCl so that the resulting pH was 1.8. The mixture was shaken for 3 min and centrifuged for S min, then the fluorescence of the upper aqueous layer was measured. Results and Discussion Electronic Absorption and Fluorescence Characteristics The electronic absorption spectral properties of the substituted benzamides are presented in Table 1.It can be seen that benzamides have two main absorption bands in the ranges 2 10-225 and 270-3 10 nm. Only metoclopramide shows threeAnalyst, November 1996, Vol. 121 1553 absorption bands. Compounds 3-5, with similar structures, have very close absorption characteristics with a shoulder at about 236-240 nm. It can be assumed that all the electronic transitions are of a n-n* type, since the molar absorption coefficients are high (103-104 1 mol-1 cm-I), those correspond- ing to the shorter wavelengths being larger than those corresponding to longer wavelengths. All the benzamides displayed native fluorescence, as ex- pected from their structure. Fluorescence characteristics are given in Table 1.The quantum yields are between 0.025 and 0.35. It has been shown that for orthomethoxybenzamides, a planar conformation is favoured by an intramolecular six- membered ring hydrogen bond between the amide N-H and the oxygen of the methoxy group.25 Hence this common feature of the studied compounds with increasing coplanarity of the benzenic moiety is favourable with respect to the fluorescence properties. Alizapride, which has the most extensively conju- gated system, exhibits logically the highest fluorescence quantum yield (Table 1). As shown in Table 1, most compounds exhibit very close fluorescence emission wavelengths, from which it can be deduced that the substituent of the amide group has virtually no effect on the fluorescence spectra.Photolysis Studies In order to evaluate whether it was possible to determine the studied compounds by the photochemical-fluorimetric method,26 photolysis was performed under the conditions described under Experimental. When the benzamides were submitted to UV irradiation, a similar behaviour was observed for all the compounds, that is, a rapid decrease of the fluorescence intensity. The half-lives depended on the molecule and ranged from 45 to 9200 s. No shift of the excitation or emission spectra occurred upon irradiation (Table 2). No formation of fluorescent photoproducts was observed. To establish the photolysis kinetics log(l/lo) versus time curves were plotted using the fluorescence intensities measured after each irradiation interval.Linear relationships were obtained for all compounds (Fig. 2). Therefore, all photolysis kinetics obeyed a first-order law. The observed rate constants were between 3 X and 4.1 X s-l. It should be pointed out that these photochemical results are of importance from a pharmaceutical point of view i.e., benzamide dosage forms should be strictly protected from light to ensure good preservation. Under the conditions used for our fluorimetric assays (5 nm excitation slit-width and pulsed low-power excitation lamp), the benzamides were photostable and no change of fluorescence intensity was observed during the measurement time. Solvent Effect Table 3 shows the solvent effect on the fluorescence properties of the substituted benzamides. Slight emission spectral shifts (AA < 15 nm) were noted.In contrast, important variations in the fluorescence intensity occurred on changing the solvent. A strong enhancement of the fluorescence signal was observed for most compounds in water, which appears to be the most convenient solvent from the analytical standpoint. For all the other protic solvents tested, except ethylene glycol, a much weaker emission signal was generally recorded. It seems that the high viscosity of ethylene glycol relative to that of the other alcohols under study may cause, by reduction of the non- radiative deactivation process, an increase in fluorescence signal. Concerning the other protic solvents, it can be suggested that the intramolecular hydrogen bonds, characteristic of the benzamide structure in pure water, could be weakened and thus the planarity of the benzamide system altered, producing a decrease in the emission intensity.Furthermore, certain mole- cules were insoluble or very weakly soluble in solvents such as Table 2 Experimental conditions and results of the photolysis study of substituted benzamides. All photolysis measurements were performed in aqueous solution at room temperature Concen- tration/ he,,/ A,,,,/ Compound No. mol 1-1 nm nm tIj2* s k,' q-1 Alizapride 1 10-7 286 366 540 1.4X lop7 Metoclopramide 2 10-5 272 356 45 4.1 X Sulpiride 3 10-5 294 344 9200 1.3 X Tiapride 5 2 X 240 335 3210 3.0X * Half-life corresponding to the irradiation time at which the initial fluorescence intensity of the benzamide has decreased by 50%. + Observed first-order rate constant.Sultopride 4 1 0 237 338 19x0 3 . 5 ~ 1 0 - 4 / 1.30 I 1 0.26 0.52 L L 4 I / u 0 8 16 24 32 40 Ti me/s Fig. 2 water. Photolysis kinetic plot for metoclopramide (lo-' mol 1-l) in Table 1 Electronic absorption and fluorescence and spectral characteristics of the substituted benzamides in water Fluorescence Quantum Compound* Absorption: h/nm (log E)+ h,,/nm* h,,/nm* yields Alizapride 223 (4.56); 296 (3.82) - 286 375 0.35 Metoclopramide 2 12 (4.39); 272 (4.1); 307 (4.02) 272; 3 1 I 356 0.10 Sulpiride 212 (4.18); 236; 291 (2.95) - 239; 294 342 0.02s Sultopride 212 (4.55); 240; 288 (3.02) - 237; 289 338 0.072 Tiapride 212 (4.39); 240; 280 (3.12) - 240; 296 339 0.056 * The concentrations of the benzamides were between 10-6 and mol I-', depending on the compound.1 Absorption maxima. Wavelength precision, f l nm. Wavelength values in italics correspond to the shoulders. The logarithm of the molar absorption coefficient E (1 mol-1 cm-1) is given in parentheses. * Excitation (hex) and emission (he,,) maxima. Wavelength precision, +I nm. Secondary excitation peaks are not underlined. 9 Quantum yields ($F) measured at he, = 250 nm (except for 2: A,, = 260 nm) relative to a 0.05 mol 1-1 H2S04 quinine hydrogensulfate aqueous solution ($F = 0.55).*'1554 Analyst, November 1996, Vol. 121 acetonitrile and THF. For these reasons, water was finally selected as the working solvent for further experiments. pH Effect As depicted in Fig. 3, the compounds studied can be divided in two groups: the benzamides exhibiting a fluorescence signal as a function of pH similar to that of a titration curve (metoclopra- mide, sultopride and tiapride) [Fig.3(a)] and those which present two fluorescence maxima depending on the pH (sulpiride and alizapride) [Fig. 3(b)]. For the first group, the pH corresponding to the mid-point of the curve inflection (9.06, 8.9 and 8.9 for metoclopramide, sultopride and tiapride, respectively) might reflect the pK, values of these compounds. Hence it seems that an acidic pH stabilizes the intramolecular bond between the N-H amide group and the ortho OCH3 substituent. Conversely, a higher pH medium acts as an unfavourable environment, as shown by the drastic decrease in the fluorescence emission under these conditions. For the second group, the reason for the existence of two fluorescence maxima remains unclear. For example, sulpiride displayed two optimum pH values of comparable fluorescence intensities, 6.1 and 1.2 [Fig.3(h)]. Since the blank signal for the latter was relatively more important, we considered only the former for analytical purposes. Its value is virtually identical with that of the aqueous solution containing no buffer. Table 3 Solvent effects on the fluorescence properties of substituted benzamides Compound* Alizapride Metoclopramide Sulpiride Sultopride Tiapride Solvent DMSO Ethanol Ethylene glycol Methanol Propan-2-01 THF Water DMSO Ethanol Ethylene glycol Methanol Propan-2-01 THF Water DMSO Ethanol Methanol Propan-2-01 THF Water DMSO Ethanol Ethylene glycol Methanol Propan-2-olt THF) Water DMSO Ethanol Ethylene glycol Methanol Propan-2-01 THF Water L l nm 298 298 300 298 29 1 NFI 287 NF 272 279 272 272 NF 272 NF NF 294 NF NF 294 NF NF 24 1 230 NF 237 NF 246 246 246 246 NF 246 - Llnl nm 359 375 365 366 377 NF 366 NF 346 361 355 346 NF 356 NF NF 344 NF NF 344 NF NF 335 336 NF 333 NF 344 334 334 348 NF 336 - Fluorescence intensity 1176 784 6740 1940 1515 8960 640 1075 1840 300 1680 - - - - - 145 - - 920 - - 204 1 1567 - - 2649 629 2330 1747 47 8 3600 - - * Concentrations were between and mol I-', depending on the compound.1 NF = compound not fluorescent in this solvent. 4 Opalescent solution. g Compound not soluble in this solvent. Analytical Figures of Merit The analytical figures of merit for the spectrofluorimetric determination of the five benzamides under the optimum conditions are given in Table 4.These figures were obtained 1600 (4 1280 ~ 960 - x 640 6 320 c. .- cn + 0 2.6 5.2 7.8 10.4 13.0 PH Fig. 3 sulpiride. Effect of pH on the fluorescence intensity of ((I) tiapride and (h) Table 4 Analytical figures of merit for the spectrofluorimetric determina- tion of substituted benzamides in aqueous solution Concen- tration range/ Analyte pg ml - I Alizapride 0.08-1.5 Metoclopramide 0.05-0.6 Sulpiride 0.07-0.7 Sultopride 0.2-1 .0 Tiapride 0.2-1 .o Regression equation" Correlation LODtI RSD A B coefficient ng ml-' (%) 535 0.3 0.999 1.0 3.4: 13.0 2.45 443 1.2 0.996 183 0.7 0.998 2.0 1.67 384 0.7 0.999 4.0 3.611 264 6.6 0.998 2.0 2.8** * IF = Ac + B , where IF = relative fluorescence intensity and c = analyte concentration (pg ml-I).t LOD = limit of detection defined as the concentration of analyte giving a signal-to-noise ratio of 3. RSD for alizapride concentration of 0.15 pg ml- I (n = 6). 3 RSD for a metoclopra- mide concentration of 0.18 bg ml- (n = 6). 7 RSD for a sulpiride concentration of 0.24 pg ml- (n = 6). 11 RSD for a sultopride concentration of 1.50 pg ml-' (n = 6). ** RSD for a tiapride concentration of0.24 pg ml - - I ( n = 6). Table 5 Fluorimetric determination of benzamides in pharmaceutical formulations Analyte Regression equation* A B coefficient (%) Correlation Recovery; Alizapride (Plitican) 533 143 0.999 94-1 00 Metoclopramide (Primperan) 438 52 0.999 99- 1 07 Sulpiride (Dogtamil) 182 36 0.999 103- 1 06 Sultopride (Bametil) 315 62 0.999 95-101 Tiapride (Tiapridal) 268 94 0.998 101-108 * I F = A c + B , where Zr; = relative fluorescence intensity and c = analyte concentration (pg ml- I ) .+ Measured using the standard additions procedure.Analyst, November 1996, Vol. 121 1555 Table 6 Fluorimetric determination of benzamides in urine samples Regression Concentra- equation’ tion range/ Correlation LOD*/ Recoveryo Analyte yg ml- I pH* A B coefficient ngml-I RSD (9%) (9%) Metoclopramide 0.1-1.3 I .7 405 3.0 0.992 7 0.7; 3.211 93-101 (428) (148) (260) (343) Sulpiride 0.2-1.5 1.5 108 8.0 0.999 45 2.0; 5.011 96-104 S ultopri de 0.2-2.2 I .8 253 23 0.999 19 2.1; 4.3** 99-101 Tiapride 0.1-2.1 1.9 342 10 0.999 10 0.6; 1.2?-’ 94-102 * pH of the acid aqueous layer in which the analytical measurements were performed (see extraction procedure).I F = Ac + B, where IF = relative fluorescence intensity and c’ = analyte concentration (pg ml-I). The slope ( A ) of the calibration curve at the same pH is given in parentheses. 4: LOD = limit of detection defined as the concentration of analyte giving a signal-to-noise ratio of 3. Measured using the standard additions procedure. 1 RSD for metoclopramide concentrations of 1.3 and 0.26 pg ml-I, respectively ( n = 6). 11 RSD for sulpiride concentrations of 1.25 and 0.35 pg ml-I, respectively ( n = 6). -’-’ RSD for sultopride concentrations of 1 .OO and 0.32 pg ml-1, respectively (n = 6). t+ RSD for tiapride concentrations of 1.60 and 0.325 pg ml- I, rcspectively (n = 6). from measurements performed in triplicate at 22 5- 2 “C, for at least five concentrations of each compound.Linear calibration plots were established over a concentration range of about two orders of magnitude. The correlation coefficients were very close to unity, indicating a satisfactory precision for the analytical curves. The limits of detection (LOD) were fairly low, ranging from 1 ng ml-1 for alizapride to 13 ng ml-l for metoclopramide. The RSDs were small, between I .6 and 3.4%, demonstrating good reproducibility of spectro- fluorimetric measurements of benzamides. Analytical Applications In order to evaluate the analytical usefulness of the spectroflu- orimetric method, several benzamides were determined in pharmaceutical preparations and in real samples (human urine), using the standard additions procedure (Tables 5 and 6).Good linearity was obtained for the standard addition plots with both pharmaceuticals and urine samples and, except for sulpiride, the slopes were close to those measured for standard calibration curves obtained at the same pH (Table 6). This indicates the absence of significant interferences from matrix effects for pharmaceuticals or from compounds possibly present in human urine. Satisfactory recoveries were found for all the benzamides under study, ranging from 94 to 108% for pharmaceutical preparations and from 93 to 104% for urine samples. The LODs for the assay of benzamides in urine were satisfactory, ranging from 7 to 45 ng ml-’ according to the analyte (Table 6). They are comparable to those obtained previously by HPLC for the determination of benzamides in biological fluids. For instance, LODs of 5-8 ng ml-I in serum”’* and 100 ng ml-1 in urine21 for metoclopramide, 5-10 ng ml-1 in serum6 and 200 ng ml-1 in urine5 for sulpiride and 15 ng ml-1 in plasma for sultopride14 have been reported.A recent study on benzamide determination in urine of treated patients showed that the lowest urine benzamide concentration levels ranged between about 100 and 200 ng ml-1.27 Conse- quently, our proposed method appears to be suitable for the determination of benzamide in pharmacokinetic studies. Conclusion The usefulness of the proposed spectrofluorimetric method for the assay of substituted benzamides in pharmaceutical prepara- tions and urine with good sensitivity, rapidity and precision has been demonstrated.Moreover, recent studies have demon- strated that metabolism appears to be of limited importance in the urine excretion of some benzamides.gJ0 As a consequence, the direct and rapid spectrofluorimetric method reported here appears convenient for therapeutic drug monitoring in urine. In addition, the combination of this spectrofluorimetric technique with flow injection, requiring smaller volumes of sample, may be valuable for routine drug screening of patients under treatment. The authors gratefully acknowledge Laboratoires Delagrange (Chilly-Mazarin, France) for their kind gift of pure alizapride, metoclopramide, sulpiride, sultopride and tiapride. References 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Spano, R. F., Trabucchi, M., Corsini, G. U., and Gessa, G. L., in Sulpiride and Other Benxmides, Experimental and Clinical Phar-ma- c~Aogy, Raven Press, New York, 1979.Lecuyer, M., Sem. Hop., 1979, 60, 1872. Van Dainne, M., Hanocq, M., Topart, J., and Molle, L., Annlusis, 1976, 4, 299. Topart, J., Hanocq, M., Van Damme, M., and Molle, L., Phnrm. Acta Hel\-., 1976, 51, I I. Alfredsson, G., Sedvall, G., and Wiesel, F. A., J . Chromatogr., 1979, 164, 177. Nicolas, P.. Fauvelle, F., Ennachachibi. A., Merdjan, H., and Petitjean, O., .I. Chromatop-., 1986, 381, 393. Coulais, Y., Campistron, G., Caillard, C., and Houin, G., J . Chroma- togr., 1986, 374, 425. Tam, Y. K., Axelson, J. E., and Ongley, R., .I. Pharm. Sci., 1979,68, 1254. Wayne Riggs, K.. Szeitz, A., Rurak, D. W., Mutlib, A. E., Abbott, F. S., and Axelson, J. E., J . Chromatogr., 1994, 660, 315. Bishop-Freudling, B., and Vergin, H., J . Chromutogr.. 1983, 273, 453. Bryson, S. M.. McGovern, E. M., and Gilbert, M., Clin. Hosp. Phar-m., 1984, 9, 263. Cohen, J. L., Hisayasu, G. H., McDermed, J. E., and Strum, S. B., Pharm. Res., 1984, 1, 43. Bron, J., Wittebrod, A. J., de Jong, ,4. P., and du Chstinier, W. M., .I. Pharm. Belg., 1987, 42, 1 and 47. Bressolle, F., and Bress, J., .I. Chromutogr., 1985, 341, 391. Bressolle, F., Bress, J., and Snoussi, M., J . Chromatogr., 1985, 343, 443. Kuss, H. J., and Nathmann, M., Arzneim.-Forsch. Drug. Res., 1978, 28, 1301. Rey d’Athis, P., Richard, M. O., Delauture, D., and Olive, G., Inr. .I. Clin. Pharnzacol. Ther. Toxicol., 1982, 20, 62. Norman, T. R., James, R. H., and Gregory, M. S., .I. Chromatogr., 1986, 375, 197. Houin, G., Bree, F., Lerumeur, N., and Tillement, J. P., J . Pharm. Sci., 1983, 72, 7 1.1556 Analyst, Nol7ernher 1996, Vol. 121 20 Kuo, B. S., Poole, J. C., Hwang, K. K., and Cheng, H., J . Phurm. Sci., 1993, 82, 694. 21 Nygard, G., Lovett, L. J., and Wahba Khalil, S. K., .I. Liq. Chromutogr., 1986, 9, 157. 22 Baeyens. W., and De Moerloore, P., AnulyJt, 1978, 103, 359. 23 Eartman, J. W., Photochem. Photohiol., 1967, 6, 55. 24 Kleimola, T., Leppanen, O., Kanto, J., Mantyla, R., and Syvalahti, E., Ann. Clin. Res., 1976, 8, 104. 25 Clark, C. R., Wells, M. J. M., Sansom, R. T., Humerick, J. l., Brown, W. B., and Commander, B. J., J . Chromutogr. Sci., 1984, 22, 75. 26 Aaron, J. J., in Molecular Luminescenc e Specti-oscopy: Methods and Applications, ed. Schulman, S. G., Wiley, New York, 1993, pt. 3, Ascalone, V., Ripamonti, M., and Malavasi, B., J . Chromuto<gr. R , 1996, 676, 95. p. 85. 27 Paper 6104002K Received June 7, 1996 Accepted July 26, I996

ISSN:0003-2654    

DOI:10.1039/AN9962101551

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, November 1996, Vol. 121 (1557-1560) 1557 Analytical Applications of Ret i no id-Cyclodext r i n I ncl us ion Com plexes* Part 2.t Luminescence Properties at Room Temperature S. Munoz Botella", D. A. Lernerh, B. del Castillo" and M. A. Martha Analitica, Facwltad de Furmacia, Uniilersidad Complutense, 28040 Madrid, Laboratorio de Te'cnicas Instrumentales, Secci6n Departamental de Quimica Spain Noi-male, 34053 Montpellier Cede-- 2, France Lahoizrtoire de Chimie Physique et Informatiyue, E.N.S.C.M Retinoids are polyenic molecules of biological importance. They are generally very hydrophobic. This paper describes the luminescence behaviour of inclusion complexes between all-trans-retinal and its geometric isomers, 9 4 s and 13-cis-retinal, retinoic acid, retinol and retinyl acetate and different cyclodextrins (CDs), viz., a-, P and y-CD, as well as the modified dimethyl-P-CD, trimethyl-P-CD and hydroxypropyl-P-CD.These complexes were found to emit room-temperature luminescence. The position and intensity of the observed emission maxima depend on the CD and on the guest retinoid. The formation of these inclusion complexes was also followed by UV-visible spectrophotometry. The results obtained allow the quantitative and selective determination of retinoids in aqueous solution owing to the solubility of these inclusion complexes in water. Keywords: Retinoid; cydodextrin; inclusion complex; luminescence Introduction Cyclodextrins (CDs) have been widely used in analytical luminescence techniques because the formation of inclusion complexes of hydrophobic luminophores increases their appar- ent solubility and therefore improves the sensitivity of the detection.Very often, this complexation is associated with an enhancement of the luminescence quantum yield as a con- sequence of the protection of the excited state. Thus, a notable increase in fluorescent emission is observed for the inclusion complexes1 of pyrene,* narcotics3 and hallucinogenic drugs.4 Retinoids play a fundamental role in various fields such as cell growth and differentiation (retinoic acid), photochemistry of the vision process (retinal) and nutrition (retinol and its esters). It is well known that retinol and its esters exhibit room- temperature luminescence.~--7 However, retinal shows a very weak luminescence only in thoroughly de-gassed media.X Retinal and retinoic acid emit at 77 K in dry hydrocarbon mediaq In this context, we described preliminary results on the luminescence properties of the retinal-CD inclusion com- plexes'() and we have recently characterized the isolated solid retinal-P-CD complex by various instrumental techniques.' Recently, Guo et al.I obtained the association constants of the vitamin A-P-CD complexes by absorption spectrophotometry. * Presenkd at the VIlth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17-19, 1996. I For Part I see ref. 1 . 8 rue de I'Ecole The present paper describes the luminescence properties of a variety of retinoid-CD inclusion complexes in aqueous solu- tions and evaluates their potential for detection from an analytical point of view in comparison with UV-visible spec trophotometry .Experimental Apparatus and Reagents UV-visible absorption spectra were obtained with a Kontron (Zurich, Switzerland) Uvikon 8 10 double- beam spectropho- tometer. Uncorrected fluorescence excitation and emission spectra were recorded with a Perkin-Elmer (Norwalk, CT, USA) Model MPF-2A and/or Model LS-3 spectrofluorimeter. A thermostated multimagnetic stirrer was used to prepare the complexes, All reagents and solvents employed were of analytical-reagent or spectroscopic grade. Water was de-ionized and doubly distilled prior to use. All samples were prepared and handled in a dark room. All-trans, 9-cis and 13-cis-retinal were purchased from Sigma (St.Louis, MO, USA) and all-trans- retinol, all-trans-retinyl acetate and all-trans-retinoic acid were products from Fluka (Buchs, Switzerland). The cyclodextrins a-CD and y-CD were purchased from Merck (Darmstadt, Germany). p-CD and HP-(3-CD (hydroxypropyl-(3-cyclodex- trin) were a generous gift from RhBne-Poulenc (Courbevoie, France). DM-P-CD (2,6-di-U-methyl-~-cyclodextrin) and TM- (3-CD (2,3,6-tri-U-methyl-P-cyclodextrin) were purchased from Sigma. Procedures Several procedures were followed to obtain the inclusion complexes. Procedure 1 Stock solutions of retinoids (in ethanol for retinoic acid and in hexane for the other retinoids) were freshly prepared. Aliquots of 10 or 20 pl of these solutions were placed in a round- bottomed flask and hexane ( 5 ml) was added.The solvent was then evaporated under reduced pressure while the solution was stirred mechanically so that a thin film of retinoid was left on the wall of the flask. To this film were added 10 ml of an aqueous solution of the required CD ( 1 .O X moll- l, prepared 24 h before use). The retinoid-CD mixture was stirred magnetically for 24 or 48 h in order to obtain the complexes. The concentration of the retinoids in these solutions was I .O X 10-5 mol 1 - 1 .1558 Analyst, November 1996, Vol. 121 Procedure 2 An appropriate amount of retinoid and CD were precisely weighed to obtain concentrations of 1.OX 10-5 and 1.0 X 10-2 mol 1-I, respectively, in the final solution. The solid products were gently ground in order to obtain a fine powder.The mixture was then dissolved in water and stirred magnetically for 24 h. The solutions obtained have the same composition as in procedure 1. Procedure 3 Ternary inclusion complexes of retinoid-CD and tert-butanol were prepared according to the procedures described in the literature. '2713 The retinoid-CD inclusion complexes were first prepared following procedure 1. After stirring magnetically for 24 h, an aliquot of tert-butanol was added to produce a final concentration of 10% v/v in tert-butanol and magnetic stirring was again applied for a further 24 h. Results and Discussion Retinoids are polyenic compounds with a marked hydrophobic character and they should consequently form annular inclusion complexes with CDs of the correct size. Retinol, retinal and retinoic acid in organic solvents all exhibit an intense UV absorption band between 300 and 400 nm.Considering the aqueous insolubility of these compounds, the appearance of such a chwacteristic absorption band in aqueous solutions of CDs indicates the solubilization of retinoids and the formation of an inclusion complex. Fig. I shows the UV-visible absorption spectra of retinal in organic solvents and in the presence of different CDs. The absorption band obtained for the retinal complexes appears slightly red-shifted (10-15 nm) with respect to the maximum wavelength observed in hexane and is very close to the values observed in ethanol = 385 nm). Hence, the average polarity of the cavity of CDs is similar to that of hydroxylic solvents. l 4 However, with these complexes the molar absorptivity is lower than in organic solvents.The absorption spectra result from a satisfactory solubilization of the retinoids following procedures 1, 2 and 3. The fluorescence spectrum was more resolved if procedure 1 was followed (Fig. 2). This procedure is also quantitatively more accurate 0'5 It li 0.4 200 300 400 500 Wnm Fig. 1 UV-visible absorption spectra of retinal (the full-scale absorbance is given in parentheses): I , retinal in hexane (2); 2, retinal in ethanol (0.5); 3, retinal-P-CD (2); 4, retinal-HP-fi-CD (0.5); 5, retinal-TM-fi-CD (0.5); and 6, retinal-a-CD (0.5). because there is no loss of powder as in procedure 2 and no possible side effects associated with the heat generated by even a soft grinding process. It has been shown previously that addition of tert-butanol in procedure 3 results in a stabilization of the inclusion ~omplexes.13,1~.~~ However, under our experi- mental conditions the presence of tert-butanol did not increase the stability of these complexes.Therefore, procedure 3 was not used further. The contact time necessary to obtain a notable fluorescence signal from the complexes was also tested. The fluorescence signal increased with the stirring time. After 48 h, a slight increase in fluorescence intensity is still observed, but such time delays are so long that they decrease the usefulness of the method. The long-term stability of the complexes was also verified. The decrease in the fluorescence intensity after 10 or 20 d in the dark at 20 "C is in the worst case 15%; hence it can be concluded that the guest molecule is not altered when the inclusion complexes are formed.This is particularly important with retinoids, which are labile molecules in solution. Evolution of complex formation as a function of stirring time and stabilization is similar for all the CDs studied and was also checked by UV-visible spectrophotometry. For retinoic acid, which possesses an ionizable group, complexation was also studied in acidic (pH = 2.0) and alkaline media (pH = 10.0). Ionization in an alkaline medium results in a larger global solubilization and hence in an increased absorption due to free and complexed molecules. However, the fluorescence that is emitted only by included molecules is greater than in water and acidic media and in the absence of further information this can be attributed to a larger quantum yield for the complex with the ionized species.The low luminescence yields measured in homogeneous solutions of retinoids are explained on the basis of photoisomerization processes which compete with radiative emission. It should be noted that there are some doubts as to the nature of the emitting state of these polyenes.7.17 The most significant result of the present work is that a room temperature emission is obtained for retinoids even in aqueous media. Table 1 summarizes the absorption and fluorescence maxima for the different inclusion complexes of retinoids. It appears that the forces acting to hold the CDs and the guest molecule together hamper molecular movements and so diminish the efficiency of the photoisomerization processes for retinoids.The wave- lengths of the emission maxima for the different all-trans- retinal-CD complexes are very similar, although their absolute emission intensities differ notably under the same conditions. Thus, the most intense luminescence is produced with P-CD and HP-P-CD. The excitation and emission spectra are also better resolved in the complexes with (3-CD and this can be attributed 70 1 1 6o J 200 300 400 500 600 Nnm Fig. 2 Excitation and fluorescence spectra of p-CD complexes of retinal: 1, retinal-b-CD, soluble complex (hex = 355 nm, he,, = 430 nm); 2, retinal-O-CD, solid complex (Acx = 355 nm, he, = 430 nm).Analyst, November 1996, Vol. 121 1559 20.0 7 17.5 - ., 15.0 - & 12.5 C c .- cn c..- $ 10.0 g 7.5 : K a, 2 0 2 5.0 : LL 2.5 - to the fact that the cavity of 13-CD accommodates and fits retinal efficiently. As retinal is too large to penetrate into the cavity of a-CD totally, this results in a weak complex and poor luminescence intensity. For y-CD, the cavity is too large to fit retinal tightly. The behaviour of the complexes with 13-cis- retinal is similar to that of all-trans-retinal. Therefore, we can propose for the structure of their complexes with the different b- CDs a geometry in which the trimethylcyclohexenyl ring penetrates first into the cavity. The polyenic side chain is less hydrophobic and too long to be totally included in the cavity. The behaviour of 9-cis-retinal is peculiar as the fluorescence intensity and absorbance were very low for all its complexes with the different (3-CDs.Furthermore, the formation of the inclusion complexes was very slow as it was necessary to wait 48 h to obtain a weak luminescence. As HP-P-CD has long chains of hydroxypropyl substituents, the bent geometry of the 9-cis isomer guest probably hinders the formation of the complex. These geometrical constraints explain why the 9-cis- retinal-a-CD and 9-c-is-retinal-TM-6-CD complexes exhibit absorbance and fluorescence intensities, respectively, similar to those of the 9-cis-retinal-P-CD and 9-cis-retinal-HP-P-CD complexes: the trimethylcyclohexenyl ring cannot enter the cavity completely as the bent part of the side chain leans against the rim of the CD. The most relevant analytical consequence of these phenomena is the selectivity presented by the inclusion complexes of CDs, which allows a distinction among geome- trical isomers in the same way that the changes in the fluorescence lifetime after inclusion in P-CD differentiate the diastereoisomers' 8 of (-)-a-( 1 -naphthyl)ethylamine and (+)-a- ( 1 -naphthyl)ethylamine or 5-methoxypsoralene from 8-me- thoxypsoralene.19 In its complexes with (3-CD and HP-P-CD, retinyl acetate shows spectra with a better resolution than in ethanol or hexane (Fig. 3). The emission intensity is higher than, or similar to, that observed in ethanolic solutions but lower than that obtained in hexane. This confirms the existence of the inclusion complexes and results in an enhanced sensitivity for detection. Although retinol and retinyl acetate are compounds with similar chemical and spectroscopic properties, they react in a very different way to form complexes.Many difficulties were encountered in reproducing the absorbances and fluorescence intensities of the inclusion complexes of retinol (this explains why results for retinol do not appear in Table 2). The Table 1 Room-temperature fluorescence data for the complexes of different retinoids with a set of CDs HP- DM- TM- fi-CD (3-CD p-CD (3-CD a-CD y-CD All-tr~ns- 355* 335* 335@ 335" Retinal 430i 4307 430t 430' 601: 14* 13* 9 7 13-cis-Retinal 335* 340* 335* 335* 420' 420t 420t 420' 28* 364 24* 16; 9-ci~-Retinal 340* 335* 335* 335* 430' 420t 420t 420+ 24* 29* 2 2 3 20t Retinol 330* 360* 340* 9 470t 5101 4651 b 181 171: 3t 4 Retinyl acetate 335* 37W 350* 350* 430t 5051 475' 430t Retinoic acid 335' 355* 355* 355* 43.5' 430t 430t 4301 1st 17* 4.51- 2.5* 55* 3.2' 3.21 494 335* 4307 335* 4207 48* 335* 420 1- 23t 350% 450: 355* 430t § D 9 4.9z 3.5* 7 s 335* 430t 1 0* 335* 420t 26* 3 3 s 4201- 28* 9 § § 355* 470' 68t § 9: a * Excitation wavelength (nm).t Emission wavelength (nm). * Emis- sion intensity in arbitrary units. 5 The data did not allow the values to be calculated. fluorescence intensity of its complexes was lower than in organic solvents, possibly as a result of hydrogen bonding of retinol to the CD. The shapes of the emission spectra for retinoic acid and retinal are almost identical. For the former, a normally non-luminescent compound, room-temperature luminescence was also detected. Nowadays, the use of a reversed phase in HPLC is very common because it has a number of advantages compared with normal-phase chromatography. However, the use of mixtures of organic and aqueous solvents decreases the sensitivity of the analytical detection when molecules are highly hydrophobic. The introduction of CDs into the mobile phase enhances both the analytical sensitivity and selectivity because CDs are water- soluble, stable, do not absorb in the UV-visible region and lead to reversible and selective complexation processes.20,21 The most popular mode of detection for retinoids is still by UV- visible absorption.Considering the high absorbance values obtained for the different retinoid-CD inclusion complexes, it was decided to study the quantitative aspects of this complexa- tion with a view to the determination of the various retinoids.In Table 2 are given the correlation coefficients and linear ranges obtained for some CDs. These results show that the quantifi- cation of retinoids in aqueous solution is possible. In all these experiments procedure 1 was followed to prepare the inclusion complexes with the following modification: stock solutions of the retinoids with concentrations from 5.0 X 10-3 to 5.0 X 10-2 moll-1 were used. In order to test the advantage of the method, reference solutions of the retinoids in ethanol-water (0.1 + 9.9) were prepared. The retinoids were first completely dissolved in ethanol and then the solutions were diluted with water. The correlation coefficients obtained under these conditions were 0.0 1 200 Alnm Fig.3 Excitation and fluorescence spectra of retinyl acetate under various conditions: 1, retinyl acetate-b-CD; 2, retinyl acetate-HP-b-CD; and 3 , retinyl acetate in ethanol. Table 2 Linear regression parameters obtained for the determination of retinoids in aqueous solution through inclusion complex formation (data were obtained in triplicate) m h r Retinal 13.9% 0.0040* 0.9659* 29.2' 0.0 I491 0.9989' Retinyl acetate* 10.2* 0.0061 * 0.8655* 12.71 0.0184t 0.9965t Retinoic acid 17.4* 0.0022* 0.798W 35.1 ' -0.0205t 0.9953' * Ethanol-water (0.1 + 9.9). Linear range 1-5.5 pmol I-'. 1 Aqueous Linear range 5-40 inclusion complexes with (3-CD or HP-P-CD. pmol I- 1 .1560 Analyst, November 1996, Vol. 121 not satisfactory owing to the insolubility of these compounds in water.It was shown above that the correlation coefficients obtained for the inclusion complexes in the same range of concentrations are good. Therefore, the inclusion complexes with CDs not only allow a differentiation of the geometrical isomers but also facilitate the quantification of hydrophobic compounds such as retinoids in aqueous solution. These results provide a basis for the sensitive detection of these compounds by reversed-phase HPLC. The authors thank UCM for a Research Fellowship for S. Mufioz Botella and MEC (Ministerio de Educacion y Ciencia, Spain) for support for a sabbatical leave for D. A. Lerner. References M u i i o ~ Botella, S., Martin, M. A., del Castillo, B., Menendez, J. C., Vazquez, L., and Lemer, D. A,, J . Phurm. Bionzed.Anal., 1996, 14, 909. Hashimoto, S., and Thomas, J. K., J . Am. Chem. S o c . , 1985, 107, 4655. Cline Love, L. J., Grayeski, M. L., Noroski, J., and Weinberger, R., And. Chim. Acta, 1985, 170, 3. Jules, O., Scypinski, S., and Cline Love, L. J., And. Chim. Actu, 1985, 169, 3. Lerner, D. A., C.K. Acad. Scz Paris, 1969, 268, 1740. Thomson, A. J.. J.Chem. Phys., 1969, 51, 4106. Becker, R. S., Photoc hem. Photobiol., 1988, 48, 369. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Lerner, D. A., Mani, J. C., and Mousseron-Canet, M., Bull. Soc. Chim. Fr., 1970, 1968. Takemura, T., Chihara, K., Becker, R. S., Das, P. K., and Hug, G. L., J . Am. Chem. Soc., 1980,102,2604. Lerner, D. A., del Castillo, B., and Muiioz Botella, S., Anal. Chim. Actu, 1989, 227, 297. Guo, Q. X., Ren, T., Fang, Y. P., and Liu, Y. C., J. Incl. Phenom. Mol Recognit. Chem., 1995, 22, 251. Nelson, G., Patonay, G . , and Warner, I. M., J . Incl. Phenom., 1988,6, 277. Muiioz de la Peiia, A., Ndou, T. T., Anigbogu, V. C., and Warner, I. M., Anal. Chem., 1991, 63, 1018. Frankewich, R. P., Thimmaiah, K. N., and Hinze, W. L., Anal. Chem., 1991,63,2924. Mufioz de la Peiia, A., Ndou, T. T., Zurg, J. B., Greene, K. L., Live, D. H., and Warner, I. M., J . Am. Chem. Soc., 1991,113, 1573. Guilleux, J. C., Barnouin, K., Ricchierro, F., and Lerner, D. A., J . Liq. Chromatogr., 1994, 17, 282 I . Becker, R. S., Hug, G., Das, P. K., Schaffer, A. M., Takemura, T., Yamamato, N., and Waddell, W., J . Phys. Chem., 1976, 80, 2265. Tran, C. D., and Fendler, J. H., J . Phys. Chem., 1984, 88, 2167. Blais, J., Prognon, P., Mahuzier, G., and Vigny, P., J . Photochem. Photobiol. B, 1988, 2, 455. Burkert, W. G., Owensby, C. N., and Hinze, W. L., J . Liy. Chromatogr., 198 1, 4, 1065. Cserhti, T., Bojarski, J., Fenyveski, E., and Szejtli, J., J . Chromutugr., 1986,351, 356. Paper 6/02 791 A Received April 16, 1996 Accepted July 16, 1996

ISSN:0003-2654    

DOI:10.1039/AN9962101557

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, November 1996, Vol. 121 (1561-1.564) 1561 Stoichiometry and Association Constants of the Inclusion Complexes of Ellipticine With Modified P-Cyclodextrin* M. Sbaia, S. Ait Lyazidib, D. A. Lernerc, B. del Castillo" and M. A. Martha a Laboratorio dc Tkcnicas Instrumentales, Seccidn Departamental de Quimica Analitica, Facultad de Famacia, Universidad Complutense, 28040 Madrid, Spain h Dkpurtement de Physique, Facultk des Sciences, Univei-site' Mouluy Ismail, Me kn 2s , Mo K ~ K O Normule, 34053 Montpellier Cedex 2 , France Lahoratoire de Chimie Physique et Informatiyue, E.N.S.C.M. 8 rue de I'Ecole Ellipticine is the most representative compound of the small family of pyrido[4,3-b]carbazole alkaloids which exhibit antitumour activity. However, its use is plagued by problems associated with its insolubility in water and with its detection in clinical analysis.As the use of cyclodextrins (CDs) may alleviate these problems, the inclusion complexes obtained from ellipticine and y-CD as well as a set of complexes with p-CD or modified p-CDs were characterized by means of their stoichiometry and association constants. The stoichoimetry was 1 : 1 (ellipticine : CD) for complexes with p-CD and modified p-CDs and 1 : 2 for the ellipticine-y-CD complex. Association constants were calculated using spectrofluorimetric data. As they depend on the pH of the medium, they were calculated for different pH values (l.O? 9.1 and 13.0). Modified p-CDs together with y-CD gave the highest association constants. The association constants obtained for cationic and neutral ellipticine with the different p-CDs did not differ significantly. These results might lead to an improvement in the chromatographic analysis and clinical use of ellipticine.Keywords: Ellipticine; cyclodextrin; modified /3-cyclodextrin; JZuorescence; association constant; stoichiometry Introduction Ellipticine, 9-methoxyellipticine lactate and 2-methyl-9-hy- droxyellipticinium acetate exhibit a significant biological activity. These pyridocarbazoles are active in the treatment of solid tumours due to their interaction with DNA.' Ellipticine free base shows a very slight solubility in water whereas cationic species such as ellipticinium are water-soluble. These cationic species of ellipticine and its derivatives are formed at pH values below 7.42 as a consequence of the protonation of their pyridine-like nitrogen.One of the main problems of ellipticine as a drug resides in its low solubility in water. Inclusion complexes with cyclodextrins (CDs j should increase the solubility of the neutral species in water and its bioavailability . Ellipticine exhibits a marked native fluorescence and this property has been used in fluorimetric a s ~ a y s . ~ - ~ HPLC with fluorescence detection has been successfully applied to the separation and determination of ellipticine and related com- pounds.6.7 However, resolution under acidic conditions is not as * Presented at the VIIth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17-19, 1996. efficient because the cations of ellipticine and its derivatives are not separated.6 It is well known that micellar and CD solutions increase the selectivity of chromatographic separations and the sensitivity of fluorimetric detection in HPLC.8-11 With ellipti- cine, the presence of micellar medial2 or CD complexesI3 increases the emission intensity and alters the acid-base equilibria of the analyte.The present paper describes the preparation and characteristics of different inclusion complexes between ellipticine and CDs as well as the effect of pH on the complexation processes of the different species of ellipticine. The results show that the association constants for neutral ellipticine are higher and depend on the type of CD and this should, therefore, influence the chromatographic behaviour of ellipticine.Experimental Apparatus and Reagents The stoichiometry and association constants of the different CD complexes were derived from spectrofluorimetric data. Un- corrected fluorescence spectra were obtained with a Perkin- Elmer (Norwalk, CT, USAj MPF-2A fluorimeter. All reagents employed were of analytical-reagent grade. p-CD and y-CD were obtained from Merck (Darmstadt, Germany). (2,6-Di-0- methyl)-13-cyclodextrin (DM-(3-CD), and (2,3,6-tri-O-methylj- P-cyclodextrin (TM-P-CD) were purchased from Sigma (St. Louis, MO, USA) and HP-p-CD (hydroxypropyl-(3-cyclodex- trinj was a generous gift from Rhone-Poulenc (Coubervoie, France j. Doubly distilled, and de-ionized water (Millipore, Milford, MA, USAj was used in all experiments.Procedures Inclusion complex formation Aliquots of a stock solution of ellipticine (1.0 X 10-3 moll-' in ethanol) were placed in a round-bottomed flask and the solvent was evaporated in vacuo at room temperature. A 5 or 10 ml volume of an aqueous CD solution was then added to the resulting thin film of ellipticine. In the solutions thus obtained, the concentrations were 1 .O X 10-6 mol 1-1 for ellipticine and 1.0 X mol 1-l for the CD. All solutions were protected from light and stirred magnetically (1 8 hj in a thermostated (20 k 2 "C) water-bath before use. Stoichiometry The same general procedure as above was followed to prepare sets of samples such as [ellipticine] + [CD] = 1.0 x 10-21562 Analyst, November 1996, Vol. 121 - mol 1-1 to apply the method of continuous variation of concentrations.14 These solutions were stirred magnetically for 48 h.They were diluted 100 times for the spectrofluorimetric measurements in order to avoid an inner-filter effect and their spectra were recorded. The solutions were then filtered (0.22 pm Millipore filters) and the filtrates were used to acquire a second set of spectrofluorimetric data. The filtered solids were then dissolved in water and the fluorescence spectra of the resulting solutions were also recorded. Association constants The general procedure for preparing the inclusion complexes was followed. The ellipticine concentration was fixed at 1.0 X 10-6 mol 1-1 for all samples. CD concentrations were varied from 1.0 X 10-4 to 1.0 X lo-' rnol 1-1. The association constants were evaluated at different pH values to take into account the presence of the different forms of ellipticine.At pH 9.2, the solutions of the CDs were prepared in buffered aqueous solutions (0.2 rnol 1-1 KH2P04-0.2 rnol 1-1 NaOH up to a pH value of 9.2, i.e., Britton-Welford buffer solution). In order to obtain the association constants in acidic or alkaline media, 1 ml of 10 moll-1 NaOH or 10 moll-' HC1 was added to the film obtained from ellipticine after ethanol evaporation. CD solu- tions of the chosen concentration were then added to obtain a final volume of 10 ml. Results and Discussion Ellipticine and its derivatives have been extensively investi- gated for their interesting pharmacological properties. Never- theless, many aspects related to the spectrofluorimetric behav- iour of ellipticine remain unexplored. Its acid-base behaviour can be expected to be similar to that of carbazole. However, ellipticine also possesses a pyridinic nitrogen and consequently could be related to a (S-carboline moiety.Fig. 1 shows the emission spectrum of ellipticine in buffered aqueous solutions and in aqueous alkaline and acidic media. In acidic media the emission at 520 nm (Aex = 302 nm) corresponds to the cationic species of ellipticine with a protonated pyridine nitrogen. In alkaline media, the emission band corresponding to the neutral or anionic form appears at 430 nm with an excitation maximum at 291 nm. In buffered aqueous solutions (pH 7.83), a strong band appears at 340 nm (Aex = 291 nm) together with the emission band at 520 nm, the latter being of very weak intensity.When the pH is increased to 9.1-9.5, the emission intensities at 340 and 520 nm both decrease with a new band appearing at 430 nm. This behaviour can be explained if we consider that ellipticine at concentrations below 1 X 10-5 mol 1-l is solubilized in water as a cationic (Aenl = 520 nm) or a zwitterionic species emitting at 340 nm. The latter co-exists in equilibrium with the neutral and cationic species as represented in Scheme 1. Its acid-base characteristics are similar to those ascribed to (S-carboline alkaloids.15 The neutral form emits at 430 nm and exists in pure polar aprotic solvents (dimethyl sulfoxide4), polar protic solvents (Fig. 2) or in micellar (Brij-35) aqueous solutions. l2 The emission band at 340 nm, which corresponds to the other neutral species, the zwitterionic CH, Scheme 1 Equilibria of the different species (neutral and ionic forms) of ellipticine.2ol l5 t 5 0 200 300 400 500 600 700 Alnm Fig. 2 Fluorescence emission spectra of ellipticine (c = 1 pmol 1-1) in: 0- in ethanol (Aex = 294 nm), -- in ethanol and NaOH (Aex = 294 nmj, ~ in ethanol and HC1 (Aex = 307 nmj. Fluorescence intensity for - is nine times and for - ~ three times lower than for the ethanolic solution.Analyst, November 1996, Vol. 121 1563 species, does not appear under these various conditions. Addition of HCI to ethanolic solutions results in an emission at 520 nm (Aex = 307 nm). Addition of NaOH increases the emission intensity at 430 nm. Consequently, the latter emission band could be attributed to the neutral as well as to the anionic species because solubilization of ellipticine in 10 moll-’ NaOH solution produced only the emission at 430 nm, but its intensity was higher than in buffered aqueous solution (pH 13).Inclusion of ellipticine in y-CD2 or in modified (3-CDl” increases the solubility in water and hence the over-all fluorescence intensity due to ellipticine. Inclusion of cationic species which predominate in aqueous solutions is questioned because these species are water-soluble. However, as the concentration of CDs is increased (0, I .O X 10-4 to 9.0 X 10-3 mol 1-1) in a solution containing such a cationic species, a notable increase in the emission intensity of the cationic band is observed. The fluorescence is ten times more intense in a 9.0 X 1.0-3 mol I-’ solution of any CD than in a solution without that CD.The increase in the emission intensity of the zwitterionic band under the same conditions is much weaker. This behaviour is common to all the ellipticine-CD complexes studied and has been described for other complexes of charged species.16.17 An analogous increase in the fluorescence intensity with increasing concentration of CDs was observed for complexes prepared in alkaline media (pH = 13) where the emission band corresponds to the neutral or anionic form (A,,, = 430 nm). Although the amplification factor for fluorescence intensity depends on the nature of the CD, this effect is seen for all the CDs and substantiates the formation of inclusion complexes.These spectroscopic changes also allow an estimate of the stoichio- metry and of the apparent association constants of the complexes (Table 1). The complexation of ellipticine at pH 9.2 was also tested. At this pH, the complexation of the neutral form of ellipticine should be favoured because this pH value is above 7.42 (pK, of ellipticine). However, CD inclusion can modify the apparent pK, value of the included molecules and, with ellipticine, it fdcilitates or hampers ionization in the acid-base equilibrium depending on the nature of the CD.13 In Fig. 3 it is seen that the fluorescence maxima and the shape of the emission band change according to the nature of the CD. Thus, for 0-CD, the emission band is structured, probably as a result of the strong interaction between ellipticine and (S-CD.For y-CD, the emission maximum appears at 480 nm, in agreement with the spectrum described by El Hage Chahine et af.2 Fig. 4 shows the stoichiometry plots based on the spectroflu- orimetric data acquired for the different CDs in aqueous solutions. The data were processed by subtracting the signal of the fluorescent emission due to free ellipticine. For p-CD and modified (S-CD, a value of 0.5 is obtained for the molar ratio (ellipticine) : [(ellipticine) + (CD)], which implies a 1 : 1 ellipticine : CD stoichiometry. These results are in agreement with those deduced from the association constant calculations. However, it is necessary to consider the different range of CD concentrations employed in the stoichiometry and association constant experiments.In spite of the extensive dilution of the solutions to avoid the inner-filter effect in the fluorescence measurements, a large fraction of the ellipticine or of the ellipticine-CD complexes is not solubilized. For this reason these solutions were filtered. The solid precipitate was then dissolved in water. Fig. 5 shows the stoichiometry plots for the unfiltered solutions, the filtrates and the solid precipitate. A maximum at a molar ratio of [ellipticine]:([CD] + [ellipticine]) = 0.5 is observed for the unfiltered solutions and filtrates. However, a high fluorescence intensity is observed for a molar ratio of [ellipticine] : ([CD] + [ellipticine]) of 0.7 and, for the solid precipitate, the maximum is obtained at this molar ratio. This can be explained on the basis of the co-existence of two types of complex each with a different stoichiometry, viz., I : 1 and 2 : 1 (ellipticine : CD).The formation of the latter is less probable in view of the steric hindrance resulting from the size of the cavity of the (3-CDs. L 200 300 400 500 600 700 Wnm Fig. 3 Fluorescence emission spectra of neutral ellipticine ( c = 1 pmol I-’) in aqueous solutions of CDs: ~ p-CD (Aex = 292 nm), -__ HP-P-CD (hex = 294 nm), - - - - DM-P-CD (hex = 294 nm), - TM-P-CD (Aex = 296 nm), - - y-CD (Acx = 282 nm). Fluorescence intensity for 0-CD, HP-(3-CD, TM-P-CD is three times lower and for y-CD nine times lower than in DM-P-CD. Table 1 Association constants and stoichiometry (in parentheses) of ellipticine-CD complexes (cationic, neutral and anionic ellipticine species) Log (KJI mol-1) for cationic ellipticine (pH = 1) Log (KJl mol-1) for neutral ellipticine (pH = 9.IS) Log (K$1 mol - 1 ) for anionic ellipticine (pH = 13) CD Ellipticine : CD 1/F* 1/(F - FJt 1 / F 1/(F - Fo)-t 1/F* l/(F - F J f p-CD (1: 1) 1. * $ 2.65 1.87 DM-fi-CD (1 : 1 ) 2.90 2.43 3.20 2.95 3.1 1 2.52 y-CD (1:2) + 6.58 6.43 $ I HP-P-CD ( 1 : 1) 2.80 2.55 2.96 2.47 2.93 1.91 2.95 2.66 2.87 2.50 2.68 1.87 TM-6-CD (1 1) * The variable chosen for the double reciprocal plot was the fluorescence intensity (arbitrary units) F at h = 520 nm at pH = 1, h = 430 nm at pH =: 13 and h = 410 nm for (3-CD, 440 nm for HP-P-CD and DM-P-CD, 430 nm for TM-(3-CD and 480 nm for y-CD at pH L- 9.15. + The variable chosen for the double reciprocal plot was the fluorescence intensity (arbitrary units), in aqueous solution, Fo, and after addition of CD, F .F , Fo at h = 520 nm at pH = 1, h = 430 nm at pH = 13 and h = 410 nm for O-CD, 440 nm for HP-0-CD and DM-0-CD, 430 nm for TM-(3-CD and 480 nm for y-CD at pH = 9.15. $ The data did not allow a satisfactory computation of the values.1564 Analyst, November 1996, Vol. 121 Table 1 lists the values of the apparent association constants (K,) deduced from the double reciprocal plots at different pH values. A linear plot is expected from this treatment, which should allow an estimate of the association constant from the slope and intercept. IX,Ic) The values of the association constants were obtained from the total fluorescence, F , as well as from the ratio l/(F - Fo), where the signal due to free solubilized ellipticine, Fo, was subtracted from the total fluorescence, F.We consider the latter procedure to be more accurate. However, at low CD concentration (< 1.0 X mol 1-I), problems arise because F =: Fo. Bright et a1.20 also consider that this procedure introduces significant errors if the spectral overlap of the complex and the substrate is strong. The overlap of the emission signal due to free ellipticine at pH 1.0 also gives noisier signals and contributes in part to overestimated values of K,. The values obtained by employing the latter treatment for the association constant of the ellipticine-y-CD complex are in agreement with those reported by El Hage Chahine et a1.2 for neutral ellipticine, because for these complexes the double reciprocal plots are a function of 1/(CD).2 This implies the formation of inclusion complexes with a stoichiometry of 1 : 2 (ellipticine : CD).This happens because only y-CD has a cavity that can include ellipticine through its dimethylisoquinoline moiety. The association constant obtained for this complex under our experimental conditions at pH 9.15 (Table 1) was very similar to the global constant deduced from the dissocia- tion constants calculated by El Hage Chahine et a1.2 for neutral ellipticine (Ed, = 9.80 X 10-5, Kd2 = 1.85 X 10-3; therefore, KD = Kdl X Kd2 = 1.81 X and log K, = 6.74). Here, Kdl and Kd2 are dissociation constants and K, is the global association constant. The existence of this type of complex also explains the effective protection against fluorescence quench- ing by bromide ion with ellipticine-y-CD compared with O1 0.2 0.4 0.6 0.8 1-.0 [El I i]/( [ El I i]+[C D]) Fig.4 Determination of the stoichiometry of the complexes by the continuous variation method, from fluorescence data for ellipticine-CD complexes at 360 nm: 1 , CJ-CD; 2, HP-fi-CD; 3 , DM-@-CD; 4, TM-P-CD; and 5, y-CD. Fluorescence intensity for curves 1 and 4 was multiplied by a factor of 3 to make the graph morc readable. [Ellipticine] + [CD] = 1 .O X lop2 mol 1- I . [ Elli]/( [ EIIi]+[CD]) Fig. 5 Determination of the stoichiometry of the complex by the continuous variation method, from fluorescence data for the ellipticine- DM-fi-CD complex at 360 nm: 1, complex in solution; 2, solution after filtration; and 3.complex precipitated and dissolved in water. [Ellipticine] + [CD] = 1.0 X lo-’ mol I-’. ellipticine-(S-CD and ellipticine-modified p-CD. 13 For the above-mentioned p-CD complexes, the association constants (Table 1) obtained for the complexes with 1 : 1 stoichiometry are in agreement with those reported by Orstan and Ross21 for the (S-CD-indole complex. Therefore, we can propose the inclusion of ellipticine in modified p-CD by its indole- containing end with the isoquinoline moiety protruding from the CD. This mode of inclusion can also explain the acid-base behaviour of ellipticine in the presence of modificd P-CD as well as the complexation of the cationic form of ellipticine, because the hydrophobic part of the molecule penetrates inside the CDs. HP-P-CD and DM-P-CD, which have longer cavities, exhibit values for the association constants greater than that for TM-6-CD.For TM-p-CD, the presence of the two methyl substituents on the secondary hydroxyl groups causes steric hindrance and a destabilization of the strength of the com- plex. In conclusion, evidence has been provided for the complex- ation of neutral species of ellipticine with various CDs. The latter may then be used as vectors to increase the solubility of neutral ellipticine in aqueous medium. Furthermore, since the acid form of ellipticine can also be complexed by various CDs, it is expected that the use of these CDs will result in better chromatographic separations of ellipticine and its analogues. The authors thank ICMA (Instituto de Cooperacion con el Mundo Arabe, Mediterrhneo y Paises en Desarrollo, Spain) for a Research Fellowship for M.Sbai and MEC (Ministerio de Educacion y Ciencia, Spain) for support for a sabbatical leave for D. A. Lerner (BOE 11/01/96). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1s 16 17 18 19 20 21 Gribble, G. W., in The Alkaloids Chentistry and Phurmacology, ed. Brossi, A., Academic Press, San Diego, CA, 1990, vol. 39, ch. 7. El Hage Chahine, J. M., Bertigny, J. P., and Schwaller, M. A,, .I. Chem. Soc., Perkin Trans. I / , 1989, 629. Sureau, F., Morean, F., Millot, J. M., Manfait, M., Allard, B., Aubard, J., and Schwaller, M. A., Biophys. .I., 1993, 65, 1767. Montagu, M., Levillain, P., Chenieux, J. C., and Rideau, M., J . Clzromatogr., 1987, 409, 426. Dufour, E.: Roger, P., and Haertle. T., J . Protein Chem., 1992, 11, 645. Muzard, G., and Le Pecq, J. B., .I. Chromutogr., 1979, 169, 446. Bykadi, G., Flora, K. P., Cradoc, J. C., and Poochikian, G. K., J . Chromatogr., 1982, 231, 137. Singh, H. N., and Hinze, W. L., Analyst, 1982, 107, 1073. Armstrong, D. W., and Henry, S. J., J . Liq. Chronzutcgr., 1980, 3, 657. Armstrong, D. W., Hinze, W. L., Rui, K. H., and Singh, H. N., Anal. Left., 1981, 14, 1659. Kasturi, A., and Gilpin, R. K., .I. Chromatogr. Sci., 1987, 25, 29. Sbai, M., Ait-Lyazidi, S., Lemer, D. A., del Castillo, R.. and Martin, M. A., J . Pharm. Biomed. Anal., 1996, 14, 959. Sbai, M., Ait-Lyazidi, S., Lerner, D. A., del Castillo, €3.. and Martin, M. A., Anal. Cliim. Acta, 1995, 303, 47. Vosburgh, W. C., and Cooper, G. R., J . Am. Chcm. Soc., 1941. 63, 437. Balon, M., Hidalgo, J., Guardado, P., Mufioz, M. A., and Carmona, C., J . Chmz. Soc., Perkin Trans. I l , 1993, 99. Connors, K. A., and Pendergast, D. D., .J. An?. Ciiem. Sot.., 1984, 106, 7607. Buvari, A., and Barcza, L., .I. Chem. Sot., Perkin Trans. I/, 1988, 543. Harada, A. . Furue, M., and NoLakur, S., Macromolec~iiles, 1977, 10, 676. Kusumoto, Y., Chenz. Phys. Lett., 1987, 136, 535. Bright, F. V., Keimig. T. L., and McGown, L. B.. Anal Chim. Acra, 1985, 175, 189. Orstan, A., and Ross, J. B. A., J . Phys Ciiem., 1987, 91, 2739. Paper 6/02 792J Received April 22, I996 Accepted July 16, 1996

ISSN:0003-2654    

DOI:10.1039/AN9962101561

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, November 1996, Vol. I21 (1 565-1 568) 1565 Precipitation Flow Injection lmmunoassay for Human Immunoglobulin G* Andrea Hackera, Gerald Giibitza?* Juan-Manuel Fernandez-Romerob, Maria D. Luque de Castroh and Miguel Valcarcelh A-801 0 Graz, Austria h Depui-tment of Analytic-al Chemistry, Faculty cv Science, University of Cordoba, E- I4004 Coi-doha, Spain Institute of Pharmac.eutica1 Chemistry, Karl-Franzens-Uni-ciersity of Graz, The development of a precipitation flow injection immunoassay is described. This approach is based on the 'immunoprecipitin reaction, whereby the precipitate formed by binding of the sample antigen (HIgG) to fluorescein-labelled antibodies is retained on a filter built in on-line in a flow-injection system. After dissolving the precipitate with sodium hydroxide solution the liberation of fluorescein-labelled antibodies results in a fluorescence signal that is directly proportional to the concentration of HIgG.The instrumental set-up is very simple and fully automated. One assay cycle takes about 10 min. The RSD ranged between 1 and 3%, depending on concentration. The detection limit was about 140 fmol. The recovery from spiked serum samples was between 96 and 102%. Keywords: Human ininiunoglobulin G; immunoprecipitin reaction ; pi.ec*ipitation flow injection immunoassay Introduction Immunoassay methods are becoming increasingly important in biomedical and environmental analysis because of their high selectivity and sensitivity. Classical immunoassay techniques are usually time consuming, handling is difficult and automa- tion requires high instrumental complexity.Flow injection immunoassays (FIIA), which have attracted great interest in recent years,' have advantages over conventional immu- noassay techniques with regard to speed, precision, sensitivity and simplicity of automation. Regarding sample throughput, FIIA techniques cannot yet compete with automated im- munoassay analysers. However, the advantage is the tlexibility if many different types of assays have to be carried out and rapid results from a single assay are required. Precipitation flow injection analysis was introduced recently.4.5 A precipitate formed in situ by a reaction is retained on a filter which is arranged on-line in the FI system. After dissolution of the precipitate, the liberated component causes a signal at the detector.This approach has been successfully applied to the determination of both inorganic and organic corn pound^.^-^ We have adapted this principle to the development of a precipitation flow injection immunoassay making use of the well known fact that precipitation occurs during the immuno binding reaction, which can be accelerated by the addition of polyethylene glycol. The basic principle of the so-called immunoprecipitin reaction has been used in classical immunoassays7 and subsequently in continuous flow and F1 systems with nephelometricg-9 and turbidimetric l o detection. respectively. Since these detection methods are not very sensitive, we aimed to improve the * Presented at the VlIth Inkmational Symposiurn on Luminescence Spectrometry in Biomedical Analysis.Sophia Antipolis, Nice, France. April 17-1 9, 1996. To whom correspondence should be addresscd. sensitivity by developing an immunoassay in combination with sensitive fluorescence detection. A very rapid homogeneous enzymatic immunoassay for human immunoglobulin G (HIgG) with fluorescence detection using horseradish peroxidase- labelled antibodies together with leuco-diacetyldichlorofluor- escein was published by Kelly and Christian. I 1 This paper describes the development of a simple precipita- tion flow injection immunoassay with fluorescence detection for HIgG using fluorescein-labelled antibodies in the form of a non-compe ti tive assay. Experiment a1 Materials Human IgG and anti-human IgG (Fab specific) fluorescein isothiocyanate (FITC) conjugate were purchased from Sigma (Deisenhofen, Germany).Polyethylene glycol (PEG) 6000 was obtained from Fluka (Buchs, Switzerland) and disodium hydrogenphosphate dodecahydrate, sodium chloride and so- dium hydroxide from Merck (Vienna, Austria). All other buffer components were of analytical-reagent grade. Preparation of Human IgG Samples Different concentrations of samples were prepared by diluting a stock standard solution ( 5 mg ml- 1) with dilution buffer (SO mmol l-1 Na2HP04, pH 7.0, containing 0.9% NaCl). The stock standard solution was stored at -18 "C and the dilutions used were freshly prepared. The highest concentration was 68.3 pmol ml- l . Preparation of Labelled Anti-human IgG Stock standard solution (4.3 mg ml- l ) was diluted with dilution buffer to give a 10-fold molar excess of the highest concentra- tion of antigens.Determination of Human IgG in Serum Samples Serum samples were diluted 1 : 10000 with dilution buffer. The IgG content was calculated from the relative peak area using known amounts of authentic IgG as a standard. For recovery studies, serum samples with known IgG contents were spiked with two different amounts of IgG (680 and 340 nmol ml-1). Determination of Detection Limit A solution of 4.6 pmol ml-1 was prepared and treated with a 10-fold molar excess of anti-human IgG to this concentration. Apparatus A schematic diagram of the instrumental set-up is shown in Fig. 1. It consists of a solvent pump [Minipuls 3 (Gilson,1566 Analyst, November 1996, Vol.121 Villiers-le-Bel, France)], a Gilson liquid handler 221 XL, an automated Rheodyne (Cotati, CA, USA) injection valve, a switching valve controlled by the autosampler, a PTFE reaction coil (2 m X 0.76 mm id) immersed in a water-bath (37 "C), a metallic filter ( 5 pm pore size) and a fluorescence detector [Perkin-Elmer (Norwalk, CT, USA) LS-SB]. An Axxiom (Moorpark, CA, USA) 737 system was used for data acquisi- tion. Once a week the filter should be cleaned in an ultrasonic bath to avoid clogging. Assay Procedure Before starting, the system was equilibrated with carrier buffer ( S O mmol 1-1 Na2HP04, pH 7 , containing 6% PEG 6000 and 0.9% NaCl) at 0.5 ml min-'. The wavelengths were set to he, 485 nm (slit IS) and he, 521 nm (slit 20). The first sample was pre-mixed by the autosampler.The sequences of events are given in Table 1 . After measurement, the system should be washed with doubly distilled water. Results and Discussion Although heterogeneous FIIAs3 are very sensitive and precise, the need to prepare an immunoreactor and its limited stability are certain drawbacks. a .i C U b d Fig. 1 Instrumental set-up. a, Autosampler with an automated injection valve; b, pump; c, switching valve, controlled by the autosampler; d, water- bath (37 "C) with the reaction coil; e, metallic filter; f, fluorimeter; g, personal computer for data aquisition; I. carrier buffer; 11, dissolution reagent. Carrier buffer c 0.5 mi min-' + 0 0 Dissolution buffer d t = 7 Filter arranged on-line Premixing 0 0 Homogeneous FIIAs, although less sensitive, are easier to handle and more flexible.A serious problem with homogeneous FIIAs is detection, as a basic requirement is a change in the detection properties of the immuno-complex compared with the free antigens and antibodies. This can be solved, for example, by using an exitation energy-transfer principle in fluorescence detection, l 2 which, however, requires a complicated labelling technique. Simple detection techniques, which are based on the immunoprecipitin reaction, are nephelometric8.9 and turbidi- metric1() detection, but they are not very sensitive. To enhance the sensitivity, we applied the principle of the immunoprecipitin reaction for the development of an FIIA using a precipitation FI system in combination with a fluores- cence detector (Fig.1). HIgG and fluorescein-labelled antibodies are pre-mixed by an autosampler and injected automatically. The carrier buffer containing PEG, which is known to enhance the immunopre- cipitin reaction,"' 3 transports the mixture through a reaction coil to a metallic filter arranged on-line in the FI system. The precipitate formed is retained on the filter and excess labelled antibodies are washed through by the carrier buffer. The immunoprecipitate is dissolved by switching to 1 mol 1-1 Table 1 Sequence of events of the assay Step Event* Flow ratel Time/ ml min--I min 1 2 The first sample or standard is pre- mixed by the autosampler Inject 75.4 pl of a mixture of 130 1.11 of standard or sample and 195 1.11 of labelled anti-human HIgG 0.5 3 Excess of labelled anti-human IgG reaches the detector; change flow rate 0.7 next solution to inject (1 mol 1-I NaOH) 0.7 7 Change flow rate 0.5 4 5 Change to dissolution reagent 6 Change to carrier buffer 8 Next injection Autosampler starts to pre-mix the * All steps are automated.0 0 2 5 7 8 9.5 10 0.5 ml min-' / t = O &O &O Carrier buffer 0.5 ml min-' New injection 0 --- 0 t = 8 t = 9.5 t=10 0.7 ml min-' t = 2 + Human immunoglobulin G (sample) FlTC labelled anti-human immunoglobulin G 0' Fig. 2. Scheme of the assay procedure. 0Analyst, November 1996, Vol. 121 I567 Incubation time/s Fig. 3. Influence of the incubation time: stopped-flow experiments with nephelornetric detection at a flow rate of 5.22 U 1 - I (0.5 ml min-l). Flow is stopped for various intervals after the immuno-complex reaches the detection cell.Fig. 4 Influence of flow rate; continous flow experiments with nephelo- metric detection at different flow rates were made using a PTFE reaction coil of 2 m X 0.76 mm id. 2.06 pmol c 11111 0 s 10 1.44 pmol k 1.03 pmol NaOH, resulting in a fluorescence signal caused by the liberated fluorescein-labelled antibodies (Fig. 2). Kinetic studies were performed to determine the optimum reaction conditions. Stopped-flow and continuous flow experiments with nephe- lometric detection showed that an incubation time of 90 s is sufficient for quantitative reaction (Fig. 3). A flow rate of 0.5 nil min-1 and a reaction coil of 2 m X 0.76 mm id were found to meet these requirements. The dependence of the signal intensity on the flow rate is shown in Fig.4. During the dissolution process with NaOH the flow rate was increased to 0.7 ml min-1. Comparison of the peak area of the signal for the excess antibodies and the signal for the dissolved antibodies showed an approximately 95% degree of reaction. The influence of PEG concentration on the reaction rate was also studied. Increasing amounts of PEG increased the reaction rate; however, clogging was a major problem. Hence a concentration of 6% PEG in the carrier buffer was used. 3 6 12 15 10 0 5 Molar excess of labelled antibodies Fig. 5 antibodies; continuous flow experiments with fluorimetric detection. Dependence of the reaction yield on the excess of labelled ----- 0 5 10 0 5 I0 0 5 100 5 100 5 10 Timdmin Signals obtained for a decreasing concentration of HIgG.Fig. 61568 Analyst, November 1996, Vol. 121 WE 100- $ 80- 3 60- 40 - 20 - Table 2 Intra- and inter-assay reproducibility for HIgG during 3 d using FITC-labelled anti-human IgG in a 10-fold molar excess over the highest concentration of HIgG Results (day 1) Results (day 2) Results (day 3) Results over 3 d x* A* x * x* ( n = 7) (T* Orcl ( n = 7) (T* or,1 ( n = 7) (T* B,,l ( n = 21) (T* 0x1 Blank 0.14 0.001 0.80 0.14 0.001 0.80 0.14 0.001 0.80 0.14 0.001 0.80 206.5 fmol per injection 0.29 0.006 1.99 0.27 0.008 3.02 0.30 0.008 2.54 0.29 0.014 4.96 413.0 fmol per injection 0.50 0.011 2.24 0.47 0.013 2.68 0.48 0.010 2.01 0.49 0.014 2.96 I .03 pmol per injection 0.89 0.014 1.56 0.87 0.011 1.23 0.88 0.013 1.47 0.88 0.014 1.55 I .44 pmol per injection 0.98 0.012 1.18 0.96 0.010 1.01 0.98 0.011 1.10 0.97 0.012 1.28 2.06 pmol per injection 1.39 0.017 1.21 1.39 0.018 1.27 1.4 0.014 1.01 1.39 0.016 1.16 * Peak area in cm2.1607 120 01 1 I 0 500 1 000 1 so0 2000 2500 Hurnan IgG/fniol per injection (75.4 pl) Fig. 7 Calibration curve for HIgG. Phosphate buffer of pH 7 was found to be optimum as the carrier buffer. For the dissolution of the precipitate, HCl and NaOH were tested. NaOH was chosen because of the higher fluorescence yield of the fluorescein label at high pH. Another parameter influencing the immunoreaction is tem- perature. The best results were obtained under physiological conditions. Therefore, the reaction coil was maintained at 37 "C. In addition, the degree of conversion was found to be dependent on the excess of labelled antibodies (Fig.5). As the blank signal increased with increasing antibody excess, proba- bly caused by adsorption on the metallic filter, a 10-fold molar excess of labelled antibodies was used as a compromise. Several types of filters were checked. Since filters with pore siLes > 0.5 pm did not quantitatively retain the precipitate, the choice was limited. A serious problem was clogging of the filters by the fine amorphous precipitate. A basic requirement was stability at high pH, as 1 mol 1-I NaOH is used for the dissolution of the precipitate. Among the materials tested, only the metallic filter met these requirements. A disadvantage of the metallic filters, however, is the adsorption of a certain amount of labelled antibodies, resulting in a blank signal, which limits the sensitivity.Fig. 6 shows typical signals for various concentrations of HIgG. The first peak corresponds to the excess labelled antibodies and the second to the liberated antibodies; the latter is directly proportional to the concentration of HIgG. The resulting calibration curve is shown in Fig. 7. There is a linear range of more than one order of magnitude with a typical correlation coefficient of 0.997. The detection limit, defined as a signal that is three standard deviations of the blank signal, was found to be 140 fmol per injection (4.6 pmol ml-1 sample). The standard deviation of the blank signal is given in Table 2. The precision of the assay was checked both within one assay series on one day and in the course of several days (Table 2).The intra-assay reproducibility was 1-3% and the inter-assay reproducibility was 1-5% (RSD). Preliminary experiments with serum samples showed that there are no interferences and no sample pretreatment is necessary. The recoveries from serum samples spiked with 340 and 680 nmol ml-' were 102 and 96.2%, respectively, with a reproducibility of about 2% (RSD). Conclusion Precipitation flow injection immunoassay has been shown to be a simple and sensitive approach for the analysis of biological samples. The instrumental set-up is very simple and easily automatable. No stopped-flow is necessary. One assay cycle takes about 10 min. This time could be shortened considerably by using a multiple reaction system. An array of three reaction coils and filters connected in parallel may shorten the time for one assay to about 3.5 min.Further, this approach would allow successive assays for different analytes within one series. The method shows excellent precision, ranging between 1 and 3% for series measured on a single day and between 1 and 5% for continuous measurements over 3 d. The detection limit for HIgG was 140 fmol per injection or 4.6 pmol ml-I of sample. Detection limits in this range are certainly not relevant for IgG analysis; however, this model assay was developed with a view towards future applications to several other analytes in trace amounts. The recovery from serum samples was between 96 and 102%. References 1 2 3 4 5 6 7 8 9 10 11 12 13 Puchades, R., Maguieira, A., Atienza, J., and Montoya, A., Crit. Rev. Anal. Chem., 1992, 23, 301. Pollema, C. H., Ruzicka, J., Lernmark, A., and Christian, G. D., Microchern. J., 1992, 45, 121. Gubitz, G. and Shellurn, C., Anal. Chini. Acta, 1993, 283, 421. Valcarcel, M., and Gallego, M., Trends Anal. Chem., 1989, 8, 34. Martinez, P., Gallego, M., and Valcarcel, M., Anal. Chem., 1987, 59, 69. Kuban, V., FreJenius' J . Anal. Chem., 1993, 346, 873. Marrack, J. R., and Richards, C. R., Immunology, 1971, 20, 1019. Killingsworth, L. M., Britain, C. E., and Woodard, L. L., Clin. Chem., 1975,21, 1465. Sternberg, J. C., Int. Clin. Prod. Rev., 1984, 3, 16. Worsfold, P. J., Hughes, A., and Mowthorpe, D. J., Analy~t, 1985, 110, 1303. Kelly, T. A., and Christian, G. D., Talanta, 1982, 29, 1109. Lim, C. S., Miller, J. M., and Bridges, J. W., Anal. Chzm. Acta, 1980, 114, 183. Creighton, W. D., Lambert, P. H., and Miescher, P. A., J. fmmunol., 1973, 111, 1219. Paper 61031 48J Received May 7, 1996 Accepted July 4, 1996

ISSN:0003-2654    

DOI:10.1039/AN9962101565

出版商:RSC    

年代:1996

数据来源: RSC