Chemiluminescence methods (present and future)

Chemiluminescence Me hods (P esen and Fu u e) By A.M. Jimenez* and M.J. Na as Depa amen o de Química Analí ica. Facul ad de Fa macia. Uni e sidad de Se illa. c/P o . Ga cía González, s/n. 41012-Se illa (España) e-mail: jimenez@ a a .us.es CONTENTS 1. In oduc ion 2. Chemiluminescence eac ions 2.1. Chemiluminescence eac ion wi h luminol 2.2. Chemiluminescence eac ion wi h Ce(IV) 2.3. Chemiluminescence eac ion wi h KMnO4 2.4. Chemiluminescence eac ion wi h is (2,2’-bipy idyl) u henium (III) 3. An ioxidan ac i i y and adical sca enging 4. Chemiluminescence and oli e oil 5. Analy e seawa e de e mina ion by chemiluminescence 6. Chemiluminescence in pha maceu ical samples 7. Conclusions Re e ences RESUMEN Mé odos de quimiluminiscencia (p esen e y u u o). Es e a ículo da una isión gene al de los mé odos de quimi- luminiscencia en algunas de las aplicaciones más ecien es en análisis de d ogas, análisis del agua ma ina o la ac i idad an ioxi- dan e de p oduc os na u ales y de sín esis (incluyendo el acei e de oli a). Las conside aciones p ác icas no es án incluidas ya que el p incipal in e és es es ablece , a a és de las aplicaciones mencionadas, que la quimiluminiscencia ha sido, es y se á en los p óximos años una he amien a e sá il de la Química Analí ica. PALABRAS-CLAVE: Ac i idad an ioxidan e - Análisis de agua ma ina - Análisis de d ogas - Quimiluminiscencia. SUMMARY Chemiluminescence me hods (p esen and u u e). This a icle p o ides a gene al e iew o chemiluminescen me hods in some o hei ecen applica ions in d ug analysis, sea wa e analysis o an ioxidan ac i i y o na u al and syn he ic p oduc s (including oli e oil). P ac ical conside a ions a e no included in he e iew since he main in e es is o s a e, h ough he a o emen ioned applica ions, ha chemiluminescence has been, is, and will be a e sa ile ool o Analy ical Chemis y in u u e yea s. KEY-WORDS: An ioxidan ac i i y - Chemiluminescence - D ug analysis - Radical sca enging - Seawa e analysis. 1. INTRODUCTION We can de ine he e m chemiluminescence (CL) as he emission o ul a iole , isible o in a- ed adia ion om a molecule o a om as he esul o he ansi ion o an elec onically exci ed s a e, ha ing been p oduced as a consequence o a chemical eac ion. This chemical eac ion p oduces ene gy in su icien amoun o induce he ansi ion o an elec on om i s g ound s a e o an exci ed elec onic s a e. This elec onic ansi ion is o en accompanied by ib a ional and o a ional changes in he molecule. In o ganic molecules, ansi ions om a π bonding o a π * an i-bonding o bi al (π → π*) o om a non-bonding o an an i-bonding o bi al (n → π*) a e mos equen ly encoun e ed. (Dodeigne e al., 2000). When he eac ion occu s in a li ing sys em o i is de i ed om one, he p ocess is called bioluminescence (BL). Luminescen eac ions has been obse ed since ancien ime, luminous animals a e known in he G eek ci ilisa ion, howe e he i s epo o a i icial chemiluminescence occu ed in 1669. The Ge man physician Henning B and isola ed om u ine a subs ance ha glowed con inuously in he da k. He called he subs ance “phospho us mi abilis”, and i is be e known oday as whi e phospho us (Ba ne and Lewis, 1996). In he 19 h cen u y i was ound ha a he simple o ganic compounds could also gi e ise o chemiluminescence. Radziszewski obse ed he g een ligh emission when oxygen was bubbled in o an alkaline e hanolic solu ion o 2,4,5- iphenylimidazole (lophine) (Isacsson and We e ma k, 1974). This disco e y was published in he yea 1877. The e m chemiluminescence was i s coined in 1888 by Eilha d Weidemann, as a pa o his classi ica ion o “cold ligh ” (luminescence). Fo y yea s la e , Alb ech in 1928 epo ed he luminescen p ope ies o 5-amino-2,3-dihyd oph alazine-1,4-dione (luminol). Ea ly esea ch on CL was mainly ocused on he obse a ion o a eac ion and in es iga ion o he mechanism and he analy ical applica ions o he phenomenon appea ed in he li e a u e in 1960s (Palilis and Caloke inos, 2000). Nowadays, a lo o ino ganic and o ganic CL eac ion a e known. A ypical CL eac ion would be (Townshend, 1990): whe e (*) indica es an elec onically exci ed s a e. Some imes, The exci ed p oduc (C*) is an ine ec i e emi e , bu i can ans e he exci a ion ene gy o an e icien luo opho e (F) added o he sys em: G asas y Acei es 64 Vol. 53. Fasc. 1 (2002), 64-75 A + B C* + D C + Ligh Now, he emission is iden ical wi h he luo escence o F and we can classi y ha as indi ec , sensi ised, o ene gy ans e chemiluminescence. The ligh emission gene a ed om a chemical eac ion equi es no ligh sou ce o exci a ion, he analy ical signal appea s ou o an essen ially black backg ound, and he only backg ound signal is ha o he pho omul iplie ube’s da k cu en . Analy ically, he CL eac ions a e a ac i e due o: (a) Excellen sensibili y and excellen de ec ion limi s because he e is absence o sou ce noise and sca e . (b) Some imes high selec i i y due o he limi ed numbe o a ailable eac ions. (c) Simple, obus and inexpensi e ins umen a ion (Schmid , 1999) sui able o bo h ba ch and low analy ical echniques. Fu he mo e, he in oduc ion o low injec ion analysis has made CL me hods e en mo e a ac i e because i is possible o mix sample and eagen apidly wi h high ep oducibili y. Chemiluminogenic eac ions mainly occu in solu ion and in he gas phase. The mos common o well known solu ion phase sys ems in ol e luminol (o i s de i a i es), oxala e es e s, lucigenin (N,N‘-dime hyl-9-9‘-diac idinium ni a e) o i s de i a i es, u henium is-bipy idine and luci e in. Gas phase examples include he ozone- and luo ine- induced, sodium apo , and chlo ine dioxide chemiluminescence de ec o s o gas ch oma og aphy (Van Flee -S alde and Chas een). The CL echniques ha e been applied o a g ea a ie y o analy es and samples. In li e a u e we can ind se e al dedica ed e iews which show he impo ance o his echniques in some in e es ing ields as clinical (K icka, 1994, Roda e al., 2000), ood (Na as and Jiménez, 1996), en i onmen al (Jiménez and Na as, 1997; Na as e al., 1997) o analy ical chemis y (Huang and Fang, 2000; Jiménez and Na as, 1999; Palilis and Caloke inos, 2000). In he igu e 1 we ha e summa ised he mos impo an applica ions o CL me hods in se e al ields. Cu en ly, i is possible o conside he CL me hods as a powe ul ools in Analy ical Chemis y. Today, he g ea a ie y o CL applica ions ha ha e appea ed in li e a u e makes an exhaus i e e ision o hem e y di icul . In his e iew we ha e selec ed and summa ised some in e es ing applica ions o CL echniques such as d ug analysis, sea wa e analysis o an ioxidan ac i i y o na u al and syn he ic p oduc s, wi h he aim o gi ing an o e iew o he possibili ies o hese me hods in some aspec s o he en i onmen al, clinical and ood ields as oli e oil. The selec ed bibliog aphy is a small sample o he nume ous epo s appea ed in he ecen yea s since we ha e only ied o e lec he cu en endency owa ds he CL echniques in hese ields. P e iously, he eagen s and eac ions which a e he basis o he CL me hods applied in he mos desc ibed cases a e b ie ly summa ised. 2. CHEMILUMINESCENCE REACTIONS 2.1. Chemiluminescence eac ion wi h luminol In aqueous solu ions, he mos commonly used chemiluminescen species is luminol. The chemiluminescence o luminol (5-amino-2,3- dihyd oph halazine-1,4-dione) was i s desc ibed by Alb ech in 1928. This compound eac s wi h a po en oxidizing agen (e.g. H2O2) in he p esence o a ca alys (gene ally a me al o me al-con aining compound o a enzyme) in alkaline solu ion o yield 3-aminoph hala e in an exci ed elec onic s a e which e u ns o g ound s a e wi h he p oduc ion o ligh . The mos ob ious use o he eac ion has been o de e mine oxidan s o compounds which in e ac wi h oxidan , bu i is possible o de e mine me al ions by hei ca alysis o he CL eac ion o p oduc s o enzyme-ca alyzed eac ions. 2.2. Chemiluminescence eac ion wi h Ce(IV) Acco ding o he in es iga ion o CL p ope ies o he luo opho e-sensi ized Ce(IV) eac ion sys em by Zhang e al. (1995), a possible CL mechanism o he eac ion may be a ibu ed o he ollowing eac ions: Ce(IV) + analy e ( ed) → Ce(III)* + analy e (ox) Ce(III)* → Ce(III) + ligh and/o Ce(III)-analy e complex* → Ce(III) + analy e + ligh Figu e 1 Some possible applica ions o CL me hods. $QDO WLFDO &KHPLVWU High sensi i e ace analysis De ec o o HPLC, Capilla y elec opho esis, Supe c i ical luids, ... &OLQLFDO &KHPLVWU D ug analysis Diagnos ic ool in medicine Biomedical esea ch Radical sca enging and an ioxidan e ec in oxida i e s ess... (QYLURQPHQWDO &KHPLVWU Ag icul u al analysis Ai analysis Wa e analysis Soil analysis... )RRG &KHPLVWU Quali y con ol Oxida ion and de e io a ion Oli e oil Residue de e mina ion... &+(0,/80,1(6&(1&( $3/,&$7,216 C* + F C + F* F + Ligh Vol. 53. Fasc. 1 (2002) 65 In he p esence o a luo opho e, Ce(III) ions o he Ce(III)-analy e complex ans e he excess ene gy o he luo opho e which in u n gene a es CL emission: Ce(III)* + luo opho e → Ce(III) + luo opho e* and/o Ce(III)-analy e complex* + luo opho e → Ce(III) + analy e + luo opho e* luo opho e* → luo opho e + ligh 2.3. Chemiluminescence eac ion wi h KMnO4 A possible CL mechanism p oposed by Aly e al. (1998) may be a ibu ed o he ollowing eac ions: analy e + MnO4- + 8 H+ → Oxidized analy e* + Mn++ + 4 H2O In he p esence o a luo opho e, he ene gy esul ing om he edox eac ion can be e ec i ely ans e ed o quinine which in u n gene a es CL emission. Oxidized analy e* + luo opho e → Oxidized analy e + luo opho e* luo opho e* → luo opho e + ligh 2.4. Chemiluminescence eac ion wi h is (2,2‘-bipy idyl) u henium (III) Since he ini ial disco e y o Ru(bipy)32+ chemiluminescence, i s u ili y has been applied o he p oduc ion o eac i e oxidan , Ru(bipy)33+, ollowed by educ ion, by an analy e species, o p oduce an emission o ligh (Aly e al., 2000 b): Ge a di e al., 1999 summa ises some mechanis ic conside a ions o he CL eac ion p oposed by some au ho s. 3. ANTIOXIDANT ACTIVITY AND RADICAL SCAVENGING The in e es in scien i ic li e a u e owa ds p oduc s which exhibi ing a p o ec i e unc ion in he oxida i e changes which ake place in oods (mainly, in ela ion o he p oduc ion o eac i e oxygen species) has been inc easing in ecen yea s, as e lec ed in he nume ous e iews ocussing on his opic (Roba ds and An olo ich, 1997, Roba ds e al., 1999, P io and Guohua, 2000). Oxida i e changes in oods a e impo an in e ms o nu i ional quali y, la o , odo , spoilage, and po en ial oxici y esul ing om inges ion o oxida ion eac ion p oduc s (Yasaei e al., 1996). The e m an ioxidan s in ood is applied o hose compounds which ac in e up ing he chain o ee adicals (p ima y an ioxidan s), consuming oxygen o discomposing hyd ope oxides in o s able p oduc s (seconda y an ioxidan s), (Kochlah and Rosell ci ed by Galeano Diaz, 1996), o anyway ac as enzyma ic an ioxidan s o chela ing agen . On he o he hand, oxida i e s ess is inc easingly becoming an impo an hypo hesis o explain he genesis o se e al pa hologies, including cance , a he oscle osis, aging (Blache e al. 1999) o Alzheime ’s disease (McIn osh e al. 1997, Smi h e al. 2000). Die a y ac o s (among o he s) a e known o be in ol ed in he p oduc ion o eac i e oxygen species which play impo an oles in oxida i e s ess, bu on he o he hand, p o ec i e unc ions o se e al componen s o oods and na u al p oduc s (Phenolic compounds, i amins...), in he a o emen ioned pa hologies, seem o be la gely due o hei abili y o sca enging eac i e oxygen species. In li ing sys ems, die a y an ioxidan s (α- ocophe ol, β-ca o ene, asco bic acid) and endogenous enzymes p o ec agains oxida i e damage (Mou e e al., 2001). Enzymes such as supe oxide dismu ase (ca alyze he dismu a ion o he supe oxide adical o H2O2 and molecula oxygen) o ca alase (ca alyzes he b eakdown o oxic H2O2 o wa e , p e en ing he seconda y gene a ion o hyd oxyl adical) a e in ol ed in hese de ensi e p ocesses. The e o e, an ioxidan s a e species which could play an impo an ole in he p o ec ion om damage p oduced by oxida i e p ocesses ha ake place in ou body. In his sec ion we ha e ied o compile some o he mos ecen epo s on he possibili ies o chemiluminescence me hods as a eliable ool o de ec ion and e alua ion o he an ioxida i e ac i i y o a g ea deal o na u al and syn he ic p oduc s, which wo k by mechanisms men ioned abo e. The g owing in e es in s udying he an ioxidan p ope ies o some componen s o na u al p oduc s is di ec ly ela ed o hei p o ec i e unc ion in oods and in human heal h, he e o e pape s e iewed he e a e ocused in bo h di ec ions. A he same ime, he summa ized li e a u e shows how CL me hods ha e been widely applied as sensi i e assay o de ec ion and measu emen o eac i e oxygen species in ol ed in he oxida i e p ocesses. Oxygen-de i ed ee adicals such as single molecula oxygen (SMO) (1O2), supe oxide anion adical (SAR) (O2-) and hyd oxyl adical (HR) (.OH) a e a e y high eac i e species. The iden i ica ion o adical species is no easy due o hei sho li e imes and di e en chemical p ope ies. Thei di ec Ru(bipy)32+ Ru(bipy)33+ Ru(bipy)32+* Ru(bipy)32+ + ligh oxida ion educ ion analy e 66 G asas y Acei es de ec ion and e alua ion is di icul , elec on spin esonance echniques usually being applied (Kole a e al. 2000). Me hods o de ec ion o moni o ing hese adicals, o compounds which p esen sca enge e ec s o e hese adicals, include CL me hods based on eac ions such as luminol, pi ogallol o Cyp idina luci e in. Suzuki e al. (1998) epo me hods o de ec ing ac i e oxygen species (single oxygen and supe oxide anion) using chemiluminescence phenomena. Fo single oxygen de ec ion, i s ligh emission in he nea -in a ed (1268 nm) is u ilized. The au ho s make an applica ion o measu ing eac ion a e cons an o some chemiluminescen compounds and supe oxide dismu ase wi h 1O2 . The a e cons an s we e measu ed by quenching he 1268 nm emission. They ha e also de eloped a me hod o measu ing eac ion a e cons an o an ioxidan s wi h O2- , using quenching expe imen o CL o Cyp idina luci e in analogues and supe oxide by supe oxide dismu ase. On he o he hand, chemiluminescence om Cyp idina luci e in has been used (K uk e al., 2000) o de e mining he e ec s o hymoquinone (TQ), hymol (TOH) and di hymoquinone (TQ2) [cons i uen s o he ola ile oil o black seed ( Nigella Sa i a Linn) ] on eac ions gene a ing eac i e oxygen species. The e ec o TOH, TQ and TQ2 on a ligh emission a ising om he enzyma ic (xan ine-oxidase-hypoxan ine-bo ine albumin) and nonenzyma ic sys em o gene a ing O2.- we e ollowed by a chemiluminescence me hod. Luminol can emi luminescence when supe oxide anion adical is p esen . This Cl eac ion can se e as use ul de ec ion me hod o adicals o an ioxidan . Dec ease o chemiluminescence in ensi y de i ed om luminol and supe oxide anion adical, gene a ed om he enzyme eac ion o xan hine oxidase (XO) wi h hypoxan ine, has been in es iga ed as a sc eening me hod o he de e mina ion o an ioxidan s in eal samples. The LC-CL me hod (Ogawa e al., 1999) has been applied o ex ac s o g een ea lea es ( Thea sinensis L.). The mos po en an ioxidan in g een ea ex ac s was (-)-epica echin, de ec ed in he ex ac s dilu ed 2000 imes. The au ho s conclude ha , hough he an ioxidan migh inhibi he CL by inhibi ing XO, i p e en s oxida ion o luminol, and quench exci ed aminoph hala e, as well as sca enging supe oxide adical anion. CL me hods based on luminol eac ion (in p esence and in he absence o SOD) ha e been employed (Kondo e al., 1999) o elucida e he sca enging mechanisms o speci ic ca echins, (-)-epigalloca echin galla e (EGCG) and (-)-epica echin galla e (ECG), on pe oxyl adicals. 2,2’-Azobis(2-aminop opane) hyd ochlo ide (AAPH), which gene a es pe oxyl adicals by i s eac ion wi h oxygen is used as an ini ia o o lipid pe oxida ion in he liposomal sys em and o adical oxida ion in he aqueous sys em. In EGC and EGCG he addi ion o SOD p oduced a ema kably inhibi o y e ec on CL, which allows he au ho s o sugges ha he py ogallol s uc u e in he B ing, gene a es supe oxide du ing he ac ion. Dapke icius e al., (1999) ha e employed he inhibi ion o luminol chemiluminescence o he on-line de ec ion o bo h, na u al and syn e hic an ioxidan s ( osma inic acid, ca nosic acid, α- ocophe ol, e -bu ylhyd oxi oluene, e - bu hylhyd oquinone, ca ac ol and hymol), sepa a ed by HPLC. De ec ion limi s (µg ml-1) epo ed by he au ho s we e in he ange 0.05 o 7.10. In a ecen publica ion (Dapke icius e al., 2001) hese au ho s make an e alua ion, op imiza ion and compa ison o wo echniques ( he on-line inhibi ion o luminol CL and he on-line 2,2’-diphenyl-1- pic yhyd azyl adical (DPPH.) bleaching assay) o he on-line de ec ion o analy es, which exhibi ing adical sca enging ac i i y, in HPLC elua es. Plan ex ac s o Thymus ulga is L. and s anda ds o some an ioxida i e compounds we e used o he pu pose o he in es iga ion. The au ho s conclude ha bo h me hods a e applicable o on-line sc eening o samples o na u al adical sca enge s, elucida ion o lipid pe oxida ion/an ioxida ion mechanisms and adical sca enging p ocesses, in gene al. Ne e heless, he DPPH. seems o be mo e obus . The o al eac i e an ioxidan po en ial (TRAP) o "Sang e de D ago", a ed iscous la ex ob ained om he ba k o C o on lechle i , has been measu ed by moni o ing he in ensi y o luminol-enhanced chemiluminescence (Desma chelie e al., 1997) using 2,2’-azo-bis(2-amidinop opane) as a pe oxyl adical sou ce. Resul s ob ained sugges o he au ho s ha Sang e de D ago is highly e ec i e in sca enging pe oxyl and hyd oxyl adicals a high concen a ions. Ne e heless, a lowe concen a ions i p esen ed p ooxidan ac i i y. An ioxidan ac i i y has been es ed using he hyd ope oxide ini ia ed chemiluminescence assay in a li e homogena es. The la ex was e ec i e in cap u ing pe oxyl and hyd oxyl adicals and in p e en ing oxida i e DNA damage in aqueous. A simila s udy has been ca ied ou by hese au ho s in aqueous and me hanolic ex ac s o se e al ees used in as an i-in lama o y in no heas e n B azil (Desma chelie e al., 1999). The chemiluminescence a ising om py ogallol oxida ion, is a phenomenon du ing which many di e en O2 species a e o med and each o hese species con ibu e o di e en mechanisms o he o e all phenomenon (Thanasoulias e al., 1999) . This ac has been used o s udy he in luence o compounds and enzymes ha ap o sca enge ce ain oxygen species (NaN3 o single oxygen, SOD o Vol. 53. Fasc. 1 (2002) 67 supe oxide anion, and ca alase and pe oxidase o H2O2 ) ha a e ac i e in oxida ion p ocesses. The compe i i e eac ion a e o a ious an ioxidan s, [bu yla ed hyd oxy anisole (BHA), bu yla ed hyd oxy oluene (BHT) and -bu yl hyd oquinone (TBHQ)] and lipids ha e been s udied o elucida e he oles and e ec i eness o an ioxidan in he p e en ion o ood oxida ion by single oxygen (Yasaei e al. 1996). Me hyl linolea e (as a model lipid) is oxidized by single oxygen gene a ed by i adia ing he solu ion wi h added ose bengal. The lipids and an ioxidan oxida ion p oduc s we e analyzed by HPLC wi h speci ics de ec ion o he hyd ope oxides by pos -column chemiluminescence and/o iodome ic de ec ion. The ob ained esul s allow o he au ho s conclude ha , mo e e icien single oxygen sca enge s ha can be added o oods may be equi ed o p o ec ion agains single oxygen oxida ion o lipids. The hyd oxyla ion o non chemiluminescen ph halic hyd azide by hyd oxyl adicals o gi e he s ongly chemiluminescen 3-hyd oxyph halic hyd azide supplies he basis o he CL me hod employed by Backa e al. (1997) o de ec ing hyd oxyl adicals. Selec i i y, sensi i i y o he me hod and he in luence o eac ion condi ions a e p esen ed. The au ho s e iew he applica ions ( ungal deg ada ion o ood, in he bleaching o pulp wi h ozone and oxygen) o he CL me hod in bo h, quan i a i e and quali a i e analyses o hyd oxyl adicals. Yildiz and Demi yüek (1998) ha e epo ed ha he hyd oxyl adical is gene a ed om he eac ion be ween e ous i on and molecula oxygen, and his eac ion induces luminol chemiluminescence. They ha e demons a ed ha CL signal is signi ican ly inhibi ed in a concen a ion-dependen manne by ei he supe oxide dismu ase (SOD) o ca alase, sugges ing ha supe oxide and H2O2 a e simul aneously gene a ed. A he same ime, speci ic hyd oxyl adical sca enge s (manni ol and dime hyl sul oxide) also p oduce a enua ion o e ous i on induced chemiluminescence. An ioxidan s, u a e, asco ba e and me hionine p oduced signi ican inhibi ions in his chemiluminescence. Kali chin e al (1997) ha e epo ed he an ioxidan ac i i y o ou choles e yl es e s o cinnamic acid de i a i esic acids (choles e yl 2" -hyd oxycinnama e, choles e yl 3 " -hyd oxy cinnama e, choles e yl 4 " -hyd oxycinnama e and choles e yl 3 " ,4 " -hyd oxycinnama e, as well as choles anyl 2 " -hyd oxycinnama e) by chemiluminescence. Te aline eac ion medium is chosen o his pu pose due o i s well known chemiluminescence and a e cons an in oxida ion adical chain eac ion. The oxida ion is ini ia ed by azo-bis- iso -bu y o ni ile. The au ho s epo ed ha hough all he choles e yl es e we e e icien adical accep o s, choles e yl 3 " ,4 " -hyd oxycinnama e esul ed pa icula ly e icien adical sca enge . Fu he mo e, he ac i i y o he inhibi o s s udied was compa able wi h ha o β-naph hol, eso cinol and hyd oquinone, de e mined in e aline a 353 K. In a la e epo (Kali chin e al. 1998), hese au ho s con inued making compa a i e s udies on he an ioxidan p ope ies o choles he yl es e s o subs i u ed cinnamic acids by chemiluminescence, in o de o e alua e he e ec o he me oxy g oup on hei an ioxidan p ope ies. Enhanced chemiluminescence and pho o- chemiluminescence ((luminol-p-indophenol pho o-induced chemiluminescence o luminol, espec i ely) me hods ha e been u ilized (Ama owicz and Raab, 1997) o es ablishing he e ec i eness o leguminous ex ac s (whi e bean, pea, len il, e e las ing pea, aba bean, and b oad bean) as na u al an ioxidan s. All ex ac s o leguminous seeds in es iga ed exhibi ed an ioxida i e p ope ies o g ea e o lesse deg ee. Red wine is a ich sou ce o la onoid an ioxidan s. On he o he hand, adical eac ions a e hough o play an impo an ole du ing bee oxida ion, and i has been demons a ed ha ee adicals a e p oduced in bee and educe quali y. The de e mina ion o an ioxidan capaci y o wine and he de e io a ion o bee quali y (due o oxida i e changes) by chemiluminescen me hods ha e been e iewed in a p eceden pape by us (Na as and Jiménez, 1999). A ho ough and e y in e es ing e iew abou lipid chemiluminescence as a book chap e (Whea ley, 1999) summa izes he scope o chemiluminescen me hods on his opic including sec ions dedica ed o chemiluminescen an ioxidan assays o lipid pe oxida ion de ec ion by Cl me hods based on di e en Cl eac ions (Luminol, pe oxala es...) and elec ogene a ed and ul aweak chemiluminescence. 4. CHEMILUMINESCENCE AND OLIVE OIL Fla onoids and o he plan -de i ed phenolic compounds a e known o exe s ong an i-oxida i e ac i i y. I is known ha he plan seeds, which a e he sou ce o edible oils, including oli e oil, a e abundan in hese an ioxidan compounds. These can help o p o ec agains degene a i e diso de s o he ca dio ascula sys em. Miyazawa e al. (1995) and Sugawa a e al. (1999) ha e analysed oils including oli e oil. Mono-, bis-, and is- hyd ope oxides o iacylglyce ols o med du ing au oxida ion and pho osensi ised oxida ion o oils we e de e mined by e e sed-phase high-pe o mance liquid ch oma og aphy in combina ion wi h chemiluminescence de ec ion. La e , his in es iga ion g oup (Sugawa a e al., 1999) s udied by he same echnique he de e mina ion o iglyce ide hyd ope oxides in oli e 68 G asas y Acei es oils. The au ho s concluded ha he CL-HPLC me hod, speci ic o he de ec ion o hyd ope oxides, should p o e use ul in s udies o iacylglyce ol oxida ion in ege able oils. The supp ession o he oxida ion o iglyce ide and me hyles e s o oli e oil by addi i es o na u al phenolic acids a 100oC was s udied by Kasaikina e al. (1997). The a e cons an s o he in e ac ion o hese acids wi h pe oxyl adicals in he oxida ion o cumene a 60oC we e de e mined by chemiluminescence me hod. The au ho s epo ed ha he ca eic acid is he mos e icien lipid an ioxidan , exceeding ionol and α- ocophe ol. ANALYTE DETERMINATION BY OBSERVATIONS CL REACTION DETECTION LIMIT OR MDC* REFERENCE Cobal (II) FIA - CL Column p econcen a ion using 8-quinolol immobilized on silica gel Gallic acid-hyd ogen pe oxide sys em 0,62 ng L-1 (3S) Hi a a e al. (1996) Sul u -con aining compounds GC wi h open ubula columns- CL de ec ion Speci ic sul u de ec o 0,1 ng L-1 Sa uk e al. (1995) A senic (V) Phospho us (V) FIA - CL P io sepa a ion by ion ch oma og aphy. Applied o analyses o a seaweed e e ence ma e ial CRM 9 Sa gasso Luminol in an NaOH medium. Acid solu ion o molybda e added o me a anada e ion 10 µg L-1 1 mg L-1 Fujiwa a e al. (1996) Ozone CL-ozone senso On line measu emen o he e ical u bulen ozone lux CL o an o ganic dye adso bed on silica gel in he eac ion wi h ozone < 0,1 ppb Güs en e al. (1997) Hyd ogen pe oxide FIA - CL The au ho s s udy he pho ogene a ion o H2O2 in ma ine wa e s In e compa ison wi h luo escence decay echnique Luminol-Co(II)-H2O2 in 0,1 M sodium ca bona e 10-me hyl-9-(p- o mylphenyl)-ac idinium ca boxyla e i luo ome hanesul ona e -hyd ogen pe oxide 10,6 nM H2O2 (signal- o-noise=3) 5 nM P ice e al. (1998) Coope e al. (2000) Coppe complexa ion FIA - CL Wi hou p econcen a ion and wi h minimal sample pe u ba ion Reac ion o coppe wi h 1,10-phenan oline- hyd ogen pe oxide 0,1 nM in undilu ed seawa e Zamzow e al. (1998) Manganese FIA - CL I on species emo ed h ough a 8-quinolinol chela e esin column Luminol-H2O20,029 nM (lowe ) 4 mM (uppe ) Okamu a e al. (1998) I on FIA - CL P io p econcen a ion using 8-hyd oxiquinoline On hyd ophilic inyl polyme using Toyopea l HW-40C esin Fe-ca alysed oxida ion o luminol by hyd ogen pe oxide and ye hilene e amine as sensi ize 0.021 nM (3S) (a e anging) Jong e al. (1998) Jong e al. (2000) To al dissol ed i on FIA - CL P io educ ion wi h sul i e and p econcen a ion on an 8-hyd oxyquinoline chela ing esin column Oxida ion o luminol ca alized by Fe ions, emi ing blue ligh (λmax 440 nm) 40 pM (3S) Bowie e al. (1998) I on (II) and To al i on FIA - CL p econcen a ion on an Ambe li e XAD-4 esin unc ionalized wi h N- hyd oxy e hyle hylenediamine B illian sul o la ine and H2O2 eagen sol. I on (II) 0,80 nmol L-1 in sea wa e samples usin g a concen a ion o 2 nmol-1 i on (II). Hi a a e al. (1999) Cobal (II) I on (II+III) FIA - CL P io p econcen a ion using 8-hyd oxyquinoline immobilized in Toyopea l 100 µM Na2SO3 as i on (III) educing agen Py ogallol-hyd ogen pe oxide-sodium hyd oxide Luminol-disol ed oxigen as he oxidan 5 pM (3S) 40 pM (3S) Cannizza o e al. (2000) Phospho us So p ion p econcen a ion o phospho us as a yellow anadomolybdophospho ic he e opoly acid (HPA) Alkaline-luminol eac ion and HPA as oxidan 0.02 µg o P L-1 (in he p esence o su ac an ) 0.1 µg o P L-1 (in he Zui and Bi ks (2000) Table I Analy e de e mina ion by chemiluminescence in seawa e Vol. 53. Fasc. 1 (2002) 69 Table II CL applica ions in pha maceu ical analysis ANALYTE CL METHOD OBSERVATION DETECTION LIMITS REFERENCE Asco bic acid Inhibi ion o i . C in he Cl eac ion be ween luminol and e ic y anide Luminol and e ic y anide immobilized on an anion-exchan g e esin column 5,5 x 10 -3 µ g ml -1 Zhan g and Qin., ( 1996 ) Vi amin B 12 Ca hal y ic e ec o cobal ( II ) on he CL eac ion be ween luminol and h y d o g en pe oxide Luminol immobilized elec os a icall y on an anion-exchan g e column, and h y d o g en pe oxide elec ochemicall y g ene a ed 3,5 x 10 -4 m g l -1 Qin e al. ( 1997 ) P y idoxine h y d ochlo ide Enhancin g e ec o anal y e on he CL g ene a ed b y he oxida ion o luminol wi h h y d o g en pe oxide in a q ueous po assium h y d oxide and sodium oxala e 6 µ g ml -1 Alwa han and Al y ( 1998 ) E ams y la e Quenchin g e ec o he anal y e in he luminol h y pochlo i e s y s em Elec o g ene a ed uns able ea g en in a low in j ec ion se up 6 x 10 -10 g ml -1 Zhan g e al. ( 1998a ) ca hecholamines ( dopamine, ad enaline and isop enaline ) Inhibi ion o he in ensi y o he CL om he luminol- h y pochlo i e s y s em The h y poclo i e was g ene a ed elec ochemicall y on-line Dopamine: 6 x 10 -10 g ml -1 Ad enaline: 8 x 10 -10 g ml -1 Isop enaline: 8 x 10 -10 g ml -1 Zhan g e al. ( 1998b ) Tannic acid Inhibi ion o he CL o he luminol-H 2 O 2 -Cu ++ s y s em b y annic acid 9 x 10 -9 mol l -1 Cui e al. ( 1998 ) β -lac am an ibio ics CL was di ec l y p oduced b y he eac ion o β -lac am an ibio ics wi h luminol in he p esence o a ca al y s in an alkaline solu ion wi hou H 2 O 2 hexac y ano e a e ( III ) and hexac y ano e a e ( II ) we e used as ca al y s Be ween 2 x 10 -1 o 20 µ g ml -1 Kubo e al. ( 1999 ) Isoniazid Enhancemen e ec o anal y e on he week emission o li g h p oduced b y CL eac ion be ween B O - and luminol The B O - was elec o g ene a ed on-line in KB solu ion 7 x 10 -9 g ml -1 Zhen g and Zhan g ( 1999 ) Lac a e Fe 2+ g ene a ed de ec ed b y he CL eac ion o luminol s y s em wi hou added oxidan Decomposi ion o lac a e in he p esence o UO 22+ and Fe 3+ , and p oduc ion o Fe 2+ 2 n g ml -1 Pé ez-Ruiz e al. ( 1999a ) Vi amin K 3 Moni o iza ion o H 2 O 2 p oduced b y CL eac ion wi h luminol ca al y sed b y hema in Pho ooxida ion o e hanol sensi ized b y i amin K 3 o y ield H 2 O 2 2,03 x 10 -9 mol l -1 Pé ez-Ruiz e al. ( 1999b ) Chlo amphenicol CL de ec ion o pho ol y ic a g men s whi luminol - Co ( II ) s y s em Pho ode g ada ion o ni o compounds 3 x 10 -9 mol l -1 Da id e al. ( 2000 ) Sulbac am sodium and cla ulanic acid Enhancin g e ec on he CL g ene a ed b y he oxida ion o luminol wi h H 2 O 2 in alkaline medium Sulbac am sodium: 0,05 µ g ml -1 . Cla ulanic acid: 0,01 µ g ml -1 Al y e al. ( 2000a ) A opine Anal y ical CL si g nal p oduced b y ce y l ime h y lammonium chlo ide – luminol – ion pai complex o e achlo oau a e ( III ) wi h a opinium On line ion-pai o ma ion and sol en ex ac ion me hod copupled wi h e e sed micella media ed CL Fu j iwa a e al. ( 2000 ) Penicillamine CL eac ion o hiol con ainin g d u g s wi h Ce ( IV ) in H 2 SO 4 medium Quinine as luo esce 15 pmol µ l -1 as hiol Zhan g e al. ( 1996 ) Tiop onin Oxida ion b y Ce ( IV ) in dilu e sul u ic medium Sensi ized b y q uinine 0,34 µ MZhao e al. ( 1997 ) Some pheno hiazines CL induced b y he oxida ion o d u g s wi h Ce ( IV ) in acid medium Rhodamine B as sensi ize Fluphenazine h y d ochlo ide: 0.01 µ g ml -1 Le omep omazine h y d ochlo ide and imep azine a a e: 0,1 µ g ml -1 Al y e al. ( 1998a ) Tiop onin CL eac ion o iop onin wi h Ce ( IV ) in sul u ic acid medium Rhodamine 6G and q uinine as luo opho s 3,6 x 10 -8 M Pé ez-Ruiz e al. ( 1998 ) H y d ochlo o hiazide CL eac ion o h y d ochlo o hiazide wi h Ce ( IV ) in sul u ic acid Sensi ized b y hodamine 6G 0,15 µ mol l -1 Ou y an g e al. ( 1998 ) Anal g in CL eac ion o anal g in wi h Ce ( IV ) sul a e in sul u ic acid Sensi ized b y Rhodamine 6G 0,02 m g ml -1 Huan g e al. ( 1999 ) H y d ochlo o hiazide and cap op il CL eac ion wi h Ce ( IV ) in sul u ic acid medium Sensi ized b y hodamine 6G H y d ochlo o hiazide: 0,2 µ mol l -1 Cap op il: 2,7 µ mol l -1 Ou y an g e al. ( 1999 ) Sodium 2- me cap oe hane sul ona e CL eac ion o hiol wi h Ce ( IV ) in sul u ic acid medium Quinine as sensi ize 1,38 µ g l -1 Capi án-Vall e y e al. ( 2000 ) Ce ad oxil monoh y d a e CL o anal y e wi h po assium pe man g ana e in sul u ic acid Sensi ized b y q uinine 0,05 µ g ml -1 Al y e al. ( 1998b ) Nal exone CL wi h po assium pe man g ana e in sul u ic acid medium wi hou FIA 25 n g ml -1 Campli g lio ( 1998 ) Pe phenazine CL eac ion wi h po assium pe man g ana e in sul u ic acid medium Me hod wi h low in j ec ion Sul an e al. ( 1999 ) Salbu amol and ani idine CL eac ion wi h acid po assium pe man g ana e in sodium pol y phospha e Weak CL om he eac ion be ween ani idine and KMnO 4 Salbu amol: 2,5 x 10 -8 M Rani idine: 1 x 10 -5 M Ba ne e al. ( 1999 ) Salic y lamide Oxida ion o he d u g b y po assium pe man g ana e in dilu e sul u ic acid A FI me hod o salic y lamide 30 n g ml -1 Fus e Mes e e al. ( 1999 ) P ocaine, benzocaine and e acaine Oxida ion o he anal y es b y pe man g ana e in sul u ic acid in he p esence o a ious CL enhance s De e mina ion b y se q uen ial in j ec ion anal y sis wi h CL de ec ion P ocaine h y d ochlo ide: and benzocaine: 0,3 µ g ml -1 Te acaine h y d ochlo ide: 0,1 µ g ml -1 Paseko á and Polásek ( 2000 ) Thio idazine h y d ochlo ide Oxida ion b y po assium pe man g ana e in acidic medium Flow-in j ec ion anal y sis 1,2 x 10 -6 MKo j lo e al. ( 2000 ) eac ion eac ion eac ion eac ion 70 G asas y Acei es De la Pue a e al. (1999) ha e employed he CL o s udy he e ec s o polyphenolic compounds om i gin oli e oils ( y osol, hyd oxy y osol, oleu opein and ca eic acid) in he inhibi ion o leukoci e 5-lipoxygenase by he quenching e ec p oduced on he CL signal due o eac i e oxygen species gene a ed by pho bol my is a e ace a e-s imula ed a leukocy es. This same in es iga ion g oup (Gu ie ez e al., 2001) ha e examined he e ec s o polyphenolic compounds om i gin oli e oils on he non-enzyma ic lipid pe oxida ion induced by asco ba e-Fe++ o a li e mic osomes by chemiluminescence. The ob ained esul showed ha he p incipal phenolics p esen wi hin he pola ac ion o Vi gin oli e oil possess an a ay o po en ially bene icial lipoxygenase-inhibi o y, p os aglandin-spa ing, and an ioxidan p ope ies. Sawa e al. (1998) and Kanazawa e al. (2000) ha e quan i ied he gene a ion o lipid pe oxyl adicals (LOO.) om oxidized oils and edible oils (including Ex a i gin and Vi gin oli e oils) by means o a luminol-enhanced chemiluminescence assay and hei implica ion in human heal h. The au ho s concluded ha a la ge p opo ion o an i-oxidan ac i i y, as well as adical sca enging o edible oils can be los by he con en ional p ocess o oil e ining and hey epo as Ex a i gin and Vi gin oli e oils a e ich in LOO. Sca enge s, so hey a e p e e ed no only o an an ica cinogenic po en ial, bu also o p e en ion o eac i e oxygen ela ed diseases. 5. ANALYTE SEAWATER DETERMINATION BY CHEMILUMINESCENCE Mos o he wa e o he Ea h’s su ace is in he ocean. Because o i s chemical and physical p ope ies, his wa e has had a g ea in luence on he con inuing biochemical e olu ion o his plane (Libes, 1992). Seawa e cons i u es a sou ce o a wide a ie y o chemical ions conside ed as majo cons i uen s (Mg2+, Cl-, Na+ , Ca2+ , SO42-...), ne e heless, he e a e a g ea numbe o o he elemen s o compounds which exis a ace le els and exhibi an impo an unc ion in ma ine geochemis y, o ins ance, i on and manganese a e known o be essen ial mic onu ien s o ma ine o ganisms. The analysis o ul a- ace elemen s in seawa e samples is one o he mos di icul analy ical asks in he ield o en i onmen al moni o ing as ex emely low de ec ion limi s o elemen s "bu ied" in a highly saline ma ix a e equi ed (Fe adello e al. 2001). One o he u ili ies o Chemiluminescen me hods in Analy ical Chemis y is he de e mina ion o ace me als due o he ac ha a g ea deal o CL eac ions equi e he p esence o me al jus as a ca alys o by i s edox p ope ies. The applica ion o CL me hods o seawa e analysis is compiled in Table I. 6. CHEMILUMINESCENCE IN PHARMACEUTICAL SAMPLES Table II (Con .) Codeine Ru ( bp y ) 3 2 + and codeine a e oxidized and upon eac in g g ene a e he exci ed p oduc * Rubp y 32+ Complex immobilized in a sin e ic ma ix 20 µ MMichel e al. ( 1999a ) Codeine Ru henium complex ( Ru ( bp y ) 32+ and Ru ( bp y ) 2 ( phen ) 2+ ) and codeine a e oxidized and upon eac in g g ene a e he exci ed p oduc Measu emen s ca ied ou in ba ch and FIA mode Ru ( bp y ) 32+ and ba ch mode: 0,5 µ M Ru ( bp y ) 2 ( phen ) 2+ and ba ch mode: 0,1 µ M Ru ( bp y ) 32+ and FIA mode: 100 µ M Ru ( bp y ) 2 ( phen ) 2+ and FIA mode: 50 µ M Michel e al. ( 1999b ) T ic y clic an idep essan s CL eac ion be ween Ru ( bp y ) 32+ and he e ia y amino g oups Ami ip y line: ml -1 Doxepin: 0 10 µ g ml -1 , No ip y line: 0,31 µ g ml -1 P omazine: 0,16 µ g ml -1 Chlo p omazine: 0,24 µ g ml -1 G eenwa y and Dolman ( 1999 ) µ g 0,09 Rani idine CL eac ion be ween Ru ( bp y ) 32+ and a e ia y and wo secunda y amine g oups Flow in j ec ion anal y sis wi h CL de ec ion 6 x 10 -7 MBa ne e al. ( 1999 ) Te ac y clines CL s y s em o Ru ( bp y ) 32+ oxidized b y acidic pe man g ana e in p esence o Mn ( II ) The li g h emission in ensi y is enhanced when he anal y e a e also p esen in he eac ion s y s em Te ac y cline: Chlo e ac y cline: Ox y e ac y cline: Han e al. ( 1999 ) , g ml -1 20 x 10 -8 , g ml -1 10 x 10 -8 , g ml -1 20 x 10 -8 Codeine Ru ( bp y ) 32+ CL eac ion Minia u e elec ochemiluminescence de ec o 100 µ ML´Hos is e al. ( 2000 ) Flu enamic and me enamic acids Ru ( bp y ) 32+ CL eac ion Chemical g ene a ion o Ru ( bp y ) 33+ b y mixin g wo s eams con ainin g solu ion o Ru ( bp y ) 32+ and acid Ce ( IV ) Flu enamic acid: 10 -9 M Me enamic acid: 2,1 x 10 -7 M Al y e al. ( 2000b ) 3,6 x Fluo o q uinolone de i a i es CL eac ion o anal y e wi h Ru ( bp y ) 32+ and Ce ( IV ) in sul u ic acid medium Ce ( IV ) used o p oduce Ru ( bp y ) 33+ wich is used o de e mina ion o anal y e 26 x 10 -8 M 2,6 x 10 -8 M O loxacin: Al y e al., ( 2001 ) No loxanin: pCi o loxacin: , 5,5 x 10 -9 M Vol. 53. Fasc. 1 (2002) 71 Accu a e de e mina ion o d ugs in pha maceu ical p epa a ions is e y impo an in he pha maceu ical indus ies. A a ie y o echniques ha e been used in he de e mina ion such as spec opho ome y (T a is e al., 1999; Sakia a e al, 1999, Al inoz and Du sun, 2000), spec o luo ime y (Ga i e al., 2000, Rizk e al., 2000), elec ochemical de ec ion (Yun e al., 1999, Wang, 2000), e c. The analy ical e alua ion o comme cially signi ican pha maceu ical d ugs using chemiluminescence de ec ion has a ac ed conside able a en ion in ecen yea s, due o hei highe sensi i i y and hei e y simple ins umen a ion (no monoch oma o equi ed). Mo eo e , he ep oducibili y and selec i i y can be excellen by combina ion wi h a low injec ion me hod. Table II summa ises he mos impo an CL applica ion o he de e mina ion o d ugs in pha maceu ical p epa a ion. The CL eac ion o luminol can se e as a basis o he de e mina ion o analy es which can enhance (py idoxine hyd ochlo ide, isoniazid, sulbac am sodium o cla ulanic acid) o inhibi (asco bic acid, e amsyla e, some ca hecholamines o annic acid) he chemiluminescence. Fu he mo e, o he analy es can be de e mined because hey di ec ly eac wi h luminol (β-lac am an ibio ics) o hey p oduce some compound ha would eac wi h luminol (lac a e o i amin K3). O he in e es ing pha maceu ical analy es can be oxidised by Ce(IV) in sul u ic acid medium and his CL eac ion can assis in hei quan i ica ion. The eac ion can be sensi ised by se e al luo opho s, such as quinine (penicillamine, iop onin, o sodium 2-me cap oe hane sul ona e) and hodamine 6G (some pheno hiazines, iop onin, hyd ochlo o hiazide, analgin o cap op il). In he same way, some d ugs can be oxidised and de e mined by po assium pe mangana e in a sul u ic acid medium. Usually, he me hod implies a FIA p ocedu e bu Campliglio (1998) p oposes he de e mina ion wi hou FIA. Nume ous analy ical applica ions o o ange emission o is(2,2’-bipy idiyl) u henium(II) in acid solu ion ha e appea ed in li e a u e. In he able we ha e summa ised some analy ical applica ions in pha maceu ical p epa a ion as de e mina ion o codeine, icyclic an idep essan s, ani idine, e acyclines , some luo oquinolone de i a i es o lu enamic and me enamic acids. 7. CONCLUSIONS Wi h he inc easing demand o highly sensi i e and selec i e analyses in many a eas o analy ical sciences, chemiluminescence echniques con inue o p o ide impo an and ascina ing ields o esea ch. In his pape we ha e selec ed and e iewed he mos ecen li e a u e in ela ion o h ee ele an ields o scien i ic, clinical, and en i onmen al in e es , in o de o show how chemiluminescence is a li ing echnique, wi h a ple ho a o eal possibili ies. Likewise, we ha e ied o show he ole ha he chemiluminescence plays in he de e mina ion o he an ioxidan ac i i y o na u al p oduc s, wi h special men ion o he oli e oil. Nowadays, echnological ad ances happen e y quickly, and his has enabled CL me hods o imp o e and has led o an inc ease in i s ields o applica ions. The awa eness o new CL eac ions, he coupling o hese new eac ions wi h o he s o analy ical in e es and, any case, he inc ease o new de ec ion sys ems ( eplacemen o he pho on coun ing pho omul iplie o he pho odiode) ha e con ibu ed o expanding he ange o he applica ions o CL echniques. We will conclude by saying ha CL is now and i will con inue being a echnique capable o sol ing analy ical p oblems o he new millennium. REFERENCES Alb ech , H.O. (1928). Chemiluminescence o aminoph halic hyd azide. Z. Phys. Chem. 136, 321. Al inoz, S. and Du sun, O.O. (2000). De e mina ion o nimesulide in pha maceu ical dosage o ms by second o de de i a i e UV spec opho ome y. J. Pha m. Biomed. Anal., 22, 175-182. Alwa han, A.A. and Aly, F.A. (1998). Chemiluminescen de e mina ion o py idoxine hyd ochlo ide in pha maceu ical samples using low injec ion. Talan a , 45, 1131-1138. Aly, F.A., Ala aj, N.A. and Alwa han, A.A. (1998a). 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