Utilizing of square wave voltammetry to detect flavonoids in the presence of human urine

Senso s 2007, 7, 2402-2418 senso s ISSN 1424-8220 © 2007 by MDPI www.mdpi.o g/senso s Full Resea ch Pape U ilizing o Squa e Wa e Vol amme y o De ec Fla onoids in he P esence o Human U ine Voj ech Adam 1,2, Radka Mikelo a 3, Ja omi Hubalek 4, Pa el Hanus iak 5, Mi osla a Beklo a 5, Pe Hodek 6, Ales Ho na 7, Libuse T nko a 4, Ma ie S ibo o a 6, Ladisla Zeman 2 and Rene Kizek 1,* 1 Depa men o Chemis y and Biochemis y, Mendel Uni e si y o Ag icul u e and Fo es y, Zemedelska 1, CZ-613 00 B no, Czech Republic 2 Depa men o Animal Nu i ion and Fo age P oduc ion, Facul y o Ag onomy, Mendel Uni e si y o Ag icul u e and Fo es y, Zemedelska 1, CZ-613 00 B no, Czech Republic 3 Depa men o Chemis y, Facul y o Science, Masa yk Uni e si y, Ko la ska 37, CZ-611 37 B no, Czech Republic 4 Depa men o Mic oelec onics, Facul y o Elec ical Enginee ing and Communica ion, B no Uni e si y o Technology, Udolni 53, CZ-602 00 B no, Czech Republic 5 Depa men o Ve e ina y Ecology and En i onmen al P o ec ion, Facul y o Ve e ina y Hygiene and Ecology, Uni e si y o Ve e ina y and Pha maceu ical Sciences, Palackeho 1-3, CZ-612 42 B no, Czech Republic 6 Depa men o Biochemis y, Facul y o Science, Cha les Uni e si y, Albe o 2030, CZ-128 40 P ague, Czech Republic 7 Depa men o Food Enginee ing, Facul y o Technology, Tomas Ba a Uni e si y, CZ-762 72 Zlin, Czech Republic * Au ho o whom co espondence should be add essed. E-mail: [email p o ec ed] Recei ed: 10 Sep embe 2007 / Accep ed: 17 Oc obe 2007 / Published: 19 Oc obe 2007 Abs ac : Abou biological a ec ing o la onoids on animal o ganisms is known less, hus we selec ed la onoids, la anones and la ones, and hei glycosides, which we e examined as po en ial induce s o cy och ome(s) P450 when adminis a ed by ga ages in o expe imen al male a s. The s udy was ocused on induc ion o CYP1A1, he majo cy och ome P450 in ol ed in ca cinogen ac i a ion. The da a ob ained demons a e he necessi y o aking in o accoun no only abili y o la onoids o bind o Ah ecep o (induc ion ac o ) bu also o concen a e on hei dis ibu ion and me abolism (including colon mic o lo a) in he body. A e ha we examined ce ain la onoids as po en ial Senso s 2007, 7 2403 induce s o cy och ome P450, we wan ed o sugges and op imize sui able elec ochemical echnique o de e mina ion o selec ed la onoids (que ce in, que ci in, u in, ch ysin and diosmin) in body liquids. Fo hese pu poses, we selec ed squa e wa e ol anne y using ca bon pas e elec ode. P ima ily we aimed on in es iga ion o hei basic elec ochemical beha iou . A e ha we ha e op imized equency, s ep po en ial and suppo ing elec oly e. Based on he esul s ob ained, we selec ed he mos sui able condi ions o de e mina ion o he la onoids as ollows: equency 180 Hz, s ep po en ial 1.95 mV/s and phospha e bu e o pH 7 as suppo ing elec oly e. De ec ion limi s (3 S/N) o he la onoids we e om uni s o ens o nM excep diosmin, whe e he limi we e highe han μM. In addi ion, we a emp ed o sugges a senso o analysis o la onoids in u ine. I clea ly ollows om he esul s ob ained ha la onoids can be analysed in he p esence o animal u ine, because u ine did no in luence much he signals o la onoids ( eco e ies o he signals we e abou 90 %). Keywo ds: la onoids, an ioxidan , ca bon pas e elec ode, squa e wa e ol amme y, wes e n blo analysis, cy och ome P450, cance 1. In oduc ion Fla onoids a e a g oup o polyphenolic compounds widely dis ibu ed h oughou he plan kingdom. They a e cha ac e ized by he p esence o wo benzene ings ha a e linked by a 3-ca bon b idge ( o o m chalcones) o by a py ane o py one ing (Figu e 1a). On he basis o he posi ion o and he modi ica ions o hese ings, mo e han 4,000 la onoids disco e ed o da e can be classi ied in o se e al classes, including chalcones, la andiols, la ones, la onols, la anones, an hocyanins, ca echins, and au ones [1-3]. Fla onoids mainly occu as aglycones (consis ing o a benzene ing condensed wi h a six membe ing which possesses a phenyl ing a he 2 posi ion), glycosides ( ha ca y one o mo e suga esidues on he ing) o me hyla ed de i a i es [4]. Exis ence o ee aglycones is a e, bu i was obse ed ha concen a ion o ee aglycones could inc ease unde speci ic condi ions (du ing echnological p ocessing a high empe a u es and also in acidic medium) when hyd olysis o glycosides may p oceed [3,5]. Fla onoids ha e ecei ed conside able a en ion because o hei bene icial e ec s as an ioxidan s in he p e en ion o human diseases such as cance and ca dio ascula diseases, and some pa hological diso de s o gas ic and duodenal ulce s, alle gies, ascula agili y, and i al and bac e ial in ec ions [6]. They con ibu e o he an ioxidan p ope ies o g een ege ables, ui s, oli e and soybean oils, ed wine, chocola e, and eas [7]. Some la onoids ha e been epo ed o possess a a ie y o biological ac i i ies, including an ialle gic, an iin lamma o y, an i i al, an ip oli e a i e, and an ica cinogenic ac i i ies, in addi ion o ha ing e ec s on mammalian me abolism [6-8]. Se e al mechanisms by which la onoids play an impo an ole in cy o oxici y ha e been iden i ied. An i- umou ac i i y o se e al la onoids (pinos obin, que ce in, my ice in, mo in) is a ibu ed o hei e iciencies o inhibi opoisome ase I and II [9,10]. Fla onoids migh slow down cell p oli e a ion as a consequence o hei binding o es ogen ecep o [11]. On he o he hand, ce ain la onoids like some Senso s 2007, 7 2404 o he xenobio ics, including 2,3,7,8- e achlo odibenzo-p-dioxin, can induce ac i i y o cy och ome P450s [12], which a e esponsible o ac i a ion o ca cinogens such as benzo[a]py ene, 7,12- dime hylbenz[a]an h acene, a la oxin B1 and mea -de i ed he e ocyclic a oma ic amines [13]. The e ec o di e en la onoids on cy och ome P450s has been e iewed in [14]. A numbe o spec opho ome ic me hods o quan i ica ion o la onoids in ma e ials o di e en kind ha e been de eloped. These assays a e based on di e en p inciples and a e used o de e mine a ious s uc u al g oups p esen in he compounds o in e es [15]. Elec opho e ic and high pe o mance liquid ch oma og aphic echniques a e used widely o bo h sepa a ion and quan i ica ion o hem [16-22]. Ne e heless, elec ochemical de ec ion is an a ac i e al e na i e me hod o elec oac i e species de ec ion, because o i s inhe en ad an ages o simplici y, ease o minia u iza ion, high sensi i i y and ela i ely low cos [23-45]. Thus, elec ochemical me hods such as di e en ial pulse, cyclic and squa e wa e ol amme y ha e been in ensi ely u ilizing o analysis o ce ain la onoids [46-60]. Due o high abundance o la onoids in human die , we decided no only o op imize he squa e wa e ol amme y o he de ec ion o que ce in, que ci in, u in, ch ysin and diosmin a nM le el bu also o examine hem as po en ial induce s o cy och ome(s) a male a s. 2. Expe imen al Sec ion 2.1 Chemicals β-naph ho la one, diosmin, ch ysin, que ce in dehyd a e, la one, na ingin, na ingenin, hespe e in, hespe idin, baicalin, F eund’s adju an s, polye hylene glycol 6000, abbi an i-chicken IgY-alkaline phospha ase conjuga e, 5-b omo-4-chlo o-3-indolylphospha e/ni oblue e azolium (BCIP/NBT) and NaH2PO4 we e pu chased om Sigma-Ald ich Chemical Co p. (S . Louis, MO, USA). Ru in ihyd a e and que ci in dihyd a e we e om Ro h GmbH (Ka ls uhe, Ge many); PVDF memb ane om Millipo e (Bed o d, MA, USA), bicinchoninic acid om Pie ce (Rock o d, IL, USA) and Na2HPO4 om Me ck (Da ms ad , Ge many). O he chemicals used we e pu chased om Sigma-Ald ich unless no ed o he wise. S ock s anda d solu ions o he la onoids we e p epa ed by ACS wa e (Sigma- Ald ich, USA) and s o ed in he da k a he empe a u e o -20 °C. Wo king s anda d solu ions we e p epa ed daily by dilu ion o he s ock solu ions. The pH alue was measu ed using pH me e WTW inoLab (Weilheim, Ge many). The pH-elec ode (SenTix- H, pH 0–14/3M KCl) was egula ly calib a ed by se o WTW bu e s (Weilheim, Ge many). 2.2 Elec ochemical measu emen s Elec ochemical measu emen s we e pe o med wi h AUTOLAB Analyse (EcoChemie, Ne he lands) connec ed o VA-S and 663 (Me ohm, Swi ze land), using a s anda d cell wi h h ee elec odes. The wo king elec ode was a ca bon pas e elec ode, he e e ence elec ode an Ag/AgCl/3M KCl elec ode and he auxilia y elec ode a g aphi e elec ode. Phospha e bu e (0.1 M NaH2PO4 + 0.1 M Na2HPO4, pH 7.0) was used as suppo ing elec oly e. Fo smoo hing and baseline co ec ion he so wa e GPES 4.9 supplied by EcoChemie was employed. All expe imen s we e ca ied ou a oom empe a u e. Squa e wa e ol amme ic pa ame e s we e as ollows: ini ial Senso s 2007, 7 2405 po en ial 0.0 V, end po en ial 1.2 V, pulse ampli ude 49.85 mV. S ep po en ial and equency ha e been op imized, o mo e de ails see “Resul s and Discussion” sec ion. 2.3 P epa a ion o ca bon pas e elec ode The ca bon pas e (abou 0.5 g) was made o 70 % g aphi e powde (Sigma-Ald ich) and 30 % mine al oil (Sigma-Ald ich; ee o DNase, RNase, and p o ease). This pas e was housed in a Te lon body ha ing a 2.5-mm-diame e disk su ace. P io o measu emen s, an elec ode su ace was enewed by polishing wi h a so il e pape [26,61]. 2.4 P epa a ion o human u ine Human u ine (ob ained om heal hy labo a o y s a ) was il e ed h ough a Te lon disc il e (0.45 µm and 13 mm diame e , All ech Associa es, Dee ield, Il, USA) and 1,000 imes dilu ed wi h phospha e bu e be o e measu emen s. We added que ce in, que ci in, u in and diosmin a 5, 7.5 and 10 μM o 1,000 imes dilu e solu ion o human u ine. As o diosmin, we used he ollowed concen a ions: 300, 400 and 500 μM. 2.5 Animal ea men and p epa a ion o mic osomes Ra males di ided in o g oups (3 animals) we e ea ed by ga ages wi h es ed la onoids (60 mg/kg) o sun lowe oil ( ehiculum) o 3 consecu i e days. F om li e s and colons mic osomal ac ions we e p epa ed by di e en ial cen i uga ion as desc ibed elsewhe e [62]. P o ein concen a ions we e assessed using he bicinchoninic acid p o ein assay [63]. The concen a ion o CYP was es ima ed acco ding o Omu a and Sa o [64]. All p epa a ions we e s o ed in small aliquo s in liquid ni ogen p io o use. 2.6 P epa a ion o an i- a CYP1A1 Legho n chickens we e immunized subcu aneously ones a week o h ee consecu i e weeks by a ecombinan CYP1A1 (0.1 mg/animal) emulsi ied in comple e F eund’s adju an o he i s injec ion and in incomple e one o boos e s. Immunoglobulin ac ion was isola ed om pooled egg yolks as desc ibed by Polson e al. [65,66] using ac iona ion by polye hylene glycol 6000, and an ibody was pa ially pu i ied by an a ini y ch oma og aphy [67]. 2.7 Wes e n blo analysis o hepa ic and colon mic osomes Using Wes e n blo ing wi h speci ic an i-CYP1A1 an ibodies induc ion o espec i e cy och ome(s) P450 was de e mined in mic osomal samples o he li e and colon issues p epa ed o a exposed o es ed la onoids [68]. Molecula weigh s anda ds (SigmaMa ke ), li e and colon (7.5 μg/well) mic osomal p o eins we e elec opho e ically sepa a ed on SDS-PAGE using 7.5% sepa a ion gel and hen elec o- ans e ed om he gel o a PVDF memb ane on a semi-d y blo appa a us (Biome a, Ge many) using 0.8 mA/cm2 o 10 min. and 2.0 mA/cm2 o 45 min. Memb ane was hen incuba ed a 4 °C o e nigh wi h 3% skim milk solu ion in PBS con aining 0.3% T i on X100 (PBSM) o block unoccupied memb ane binding si es. A e wa ds, he memb ane was cu in o wo pa s and he Senso s 2007, 7 2406 i s one incuba ed o 2 h s unde shaking wi h he p eimmune an ibody, he second one wi h he speci ic an ibody. Fo de ec ion o cy och ome(s) P4501A chicken an ibodies we e dilu ed o 20 μg/ml o PBSM. A e washing memb anes we e indi idually incuba ed (1 h ) wi h he seconda y an ibody, abbi an i-chicken IgY-alkaline phospha ase conjuga e, dilu ed 2000 imes wi h PBSM. A e washing wi h PBSM and PBS, he memb anes we e de eloped wi h BCIP/NBT subs a e. 2.8 S a is ical analysis STATGRAPHICS® (S a is ical G aphics Co p®, USA) was used o s a is ical analyses. Resul s a e exp essed as mean ± S.D. unless no ed o he wise. Di e ences wi h p < 0.05 we e conside ed signi ican . 3. Resul s 3.1 Biological a ec ing o ce ain la onoids on a s Abou biological a ec ing o la onoids on animal o ganisms is known less, hus we selec ed la onoids, la anones and la ones, and hei glycosides, which we e examined as po en ial induce s o cy och ome(s) P450 (CYP) when adminis a ed by ga ages in o expe imen al male a s. The s udy was ocused on induc ion o CYP1A1, he majo cy och ome P450 in ol ed in ca cinogen ac i a ion. Enhanced exp ession o his enzyme in colon issue migh be esponsible o inc easing incidence o colo ec al ca cinoma ound in humans. The e is a plen y o expe imen al da a a ailable in he li e a u e dealing wi h he induc ion e ec o la onoids in a ious a i icial sys ems (e.g. cell cul u es), howe e , esul s a e equen ly con adic o y possible because o he inadequa e sys em used. The goal o he p esen wo k is a di ec e idence o la onoid induc ion o CYP1A1 in colon a e la onoid in ake which is iden ical o he human exposu e. Wes e n blo ing o colon and li e mic osomal samples o β-naph ho la one (β -NF) ea ed a s shows ha β -NF, known CYP1A1 (used as a posi i e con ol), is e ec i e induce o bo h issues e en when adminis e ed by ga ages (see Figu e 1). Simila ly, na u al aglycons, ch ysine, and que ce in and u inoside, diosmin, caused in bo h issues CYP1A1 induc ion. As glycosides a e belie ed no o be abso bed om he colon mic obial glycosidases a e p obable in ol ed diosme in (aglycone) o ma ion. When compa ing CYP1A1 induc ion in li e and colon issues by o he la onoids shown in Figu e 1A,B, i is clea ha none o la anones used (hespe e in, hespe idin) as well as la one, baicalin (glucu onide) e ec ed CYP1A1 exp ession. O ypical ci us la onoids, na ingin and na ingenin (aglycone), he only induc ion was de e mined in he case o na ingenin (aglycone) in he colon. Su p isingly, la one ( la onoid lacking hyd oxyl o me hoxyl g oups) was no e icien induce in colon while he inc eased CYP1A1 exp ession was de ec ed in li e . This obse a ion is in acco dance wi h he assump ion ha less pola la onoids a e eely passing memb ane ba ie and hus hey a e abso bed om colon o blood ci cula ion. A e me abolic con e sion o la one in li e (mul iple hyd oxyla ion) i migh se e as CYP1A1 induce like ch ysin (5,7-dihyd oxy la one). These da a demons a e he necessi y o aking in o accoun no only abili y o la onoids o bind o Ah ecep o (induc ion ac o ) bu also o concen a e on hei dis ibu ion and me abolism (including colon mic o lo a) in he body. Senso s 2007, 7 2407 Figu e 1. Immunode ec ion o CYP1A1 in colon (A) and li e (B) mic osomes om la onoid ea ed a s. Elec opho esed mic osomal p o eins (7.5 µg/well) we e ans e ed o Immobilon-P memb ane and p obed wi h an ibody agains CYP1A1. 3.2 Elec oanaly ical analysis o la onoids A e ha we examined ce ain la onoids as po en ial induce s o cy och ome P450, we wan ed o sugges and op imize sui able elec ochemical echnique o de e mina ion o selec ed la onoids (que ce in, que ci in, u in, ch ysin and diosmin) in body liquids. I is common knowledge ha phenolic compounds dissol e poo ly in pola sol en s (wa e ) in compa ison wi h non-pola ones (me hanol), whe e hese compounds ha e se e al imes highe solubili y. Thus, we wan ed o selec a sui able sol en whe e he solubili y o compounds o in e es would be highes . Me hanol was selec ed as he mos sui able sol en o hese pu poses. Recen ly we ha e ound ou ha p esence o an o ganic sol en ma kedly dec eased heigh o cu en esponses o compounds o in e es [69-71]. Thus, we sugges ed ha con en o o ganic sol en in he suppo ing elec oly e (2 ml) did no exceed 0.1 % ( / ), which did no in luence elec ochemical de e mina ion o compounds o in e es . Recen ly, se e al pape s, whe e he au ho s u ilized glassy ca bon elec ode o de e mina ion o phenolic compounds, we e published [46-48,58]. He e, we u ilized squa e wa e ol amme y o de e mina ion o que ce in, que ci in, u in, ch ysin and diosmin using ca bon pas e elec ode due o easie enewing o he elec ode su ace by polishing wi h a so il e pape in compa ison wi h glassy ca bon elec odes (Figu e 2). Senso s 2007, 7 2408 Figu e 2. Fla onoid s uc u e made o wo benzene ings linked by he e ocyclic py ane (a). Squa e– wa e ol ammog ams o he la onoids (10 μM) and hei s uc u es – que ci in (b), u in (c), que ce in (d), ch ysin (e) and 100 μM o diosmin ( ). Squa e wa e ol amme ic pa ame e s we e as ollows: ini ial po en ial 0.0 V, end po en ial 1.2 V, pulse ampli ude 49.85 mV, s ep po en ial 1.5 mV, and equency 100 Hz. Phospha e bu e (0.1 M NaH2PO4 + 0.1 M Na2HPO4, pH 7.0) was used as a suppo ing elec oly e. Fo o he de ails see Ma e ials and Me hods sec ion. 3.2.1 Basic elec ochemical beha iou o la onoids measu ed on he su ace o ca bon pas e elec ode Squa e wa e ol ammog ams o he la onoids measu ed in he p esence o 0.1 M phospha e bu e a e shown in Figs. 2b- . Que ce in, que ci in and diosmin ga e h ee oxida ion signals, bu u in and ch ysin only wo. B e desc ibed ha peaks 1 and 2 associa es wi h oxida ion o oxida ion o hyd oxyl g oup in posi ion 7 o la onoids s uc u e [46]. Besides ha , we obse ed wo o he peaks (peaks 3 and 4, Figs. 2b,d,e, ), which could co esponds o oxida ion o hyd oxyl g oup in posi ion 7 o la onoids s uc u e [50]. In spi e o ha he analyzed la onoids gi e signals co esponding o oxida ion o he same g oups in hei s uc u e, hei po en ials di e ma kedly [48,59,60]. We obse ed ha peak 1 o que ce in (0.25 V) had po en ial shi ed abou 100 mV in compa ison wi h peak 1 belonging o que ci in (0.35 V). This shi p obably ela es wi h p esence o saccha ide g oup O O OH OH O O OH OH O OH OH OCH 2 OH OH OH O O CH3 O H O H OH O O OH OH O OH OH O CH3 OH OH OH O O O OH O OH CH3 OCH2 OH OH OH O O CH3 OH OH OH 50 μA Que ci in peak 1 peak 2 b 50 μA Ru in c 5 μA Ch ysin Po en ial (V) 5 μA Diosmin Po en ial (V) Po en ial (V) 0.25 0.50 1.000.75 1.25 0.0 0.25 0.50 1.000.75 1.25 0.0 0.25 0.50 1.000.75 1.25 0.0 0.25 0.50 1.000.75 1.25 0.0 Po en ial (V) 0.25 0.50 1.000.75 1.25 0.0 0.25 0.50 1.000.75 1.25 0.0 0.25 0.50 1.000.75 1.25 0.0 0.25 0.50 1.000.75 1.25 0.0 peak 2 peak 1 e peak 2 peak 4 peak 3 ScanScan ScanScan ScanScan ScanScan O O OH OH OH O H OH 50 μA Que ce in peak 2 peak 3 d 0.25 0.50 1.000.75 1.25 0.0 peak 1 ScanScan Po en ial (V) Elec oly e Elec oly e Elec oly e Elec oly e Elec oly e a O O 8 7 6 54 12 3 6 / 5 / 4 / 3 / 2 / 1 / O O 8 7 6 54 12 3 6 / 5 / 4 / 3 / 2 / 1 / Basic la onoids s uc u e 500 nA peak 2 peak 3 0.51 V 0.78 V 500 nA peak 2 peak 3 0.51 V 0.78 V peak 3 peak 4 1 μA peak 2 peak 4 0.80 V 0.96 V 1 μA peak 2 peak 4 0.80 V 0.96 V peak 2 200 nA peak 3 peak 2 peak 4 0.51 V 0.77 V 0.95 V 200 nA peak 3 peak 2 peak 4 0.51 V 0.77 V 0.95 V Senso s 2007, 7 2409 in he s uc u e o que ci in in compa ison wi h “non-saccha ide” s uc u e o que ce in. Peak 2 desc ibed a bo h ch ysin and diosmin is hough o be he same, bu he po en ials di e again. This shi could be also associa ed wi h p esence o disaccha ide g oup in he s uc u e o diosmin (Figu e 2). Figu e 3. In luence o di e en equencies (A and inse ) and s ep po en ials (B and inse ) on peak heigh s and po en ials o he la onoids. Fo o he de ails see Figu e 2. 3.2.2 In luence o equency, s ep po en ial and suppo ing elec oly e on SWV de e mina ion o la onoids As soon as we desc ibed basic elec ochemical beha iou o he analyzed la onoids measu ed on he su ace o ca bon pas e elec ode by SWV, we decided o op imize pa ame e s ( equency and s ep po en ial) o he ol amme ic me hod used. Fo e alua ion o esul s ob ained he signals o single la onoids ha e been selec ed as ollows: peak 1 o u in, que ce in and que ci in, and peak 2 o ch ysin and diosmin. In luence o equency has been s udied wi hin he ange om 20 o 220 Hz (Figu e 3A). The signals o la onoids inc eased wi h inc easing equency up o 180 – 190 Hz, and hen sha p dec ease o heigh o diosmin and u in peaks ollowed. This ma ked change could be 0 25 50 75 100 0 25 50 75 100 0.1 0.5 0.9 0 50 100 150 200 250 F equency (Hz) Que ce in Que ci in Diosmin Que ce in Que ci in Diosmin Peak heigh (%) 0 25 50 75 100 0 50 100 150 200 250 F equency (Hz) Peak po en ial (V) 0.1 0.5 0.9 0 50 100 150 200 250 F equency (Hz) Peak Po en ial (V) Ru in Ch ysin Ru in Ch ysin Peak heigh (%) 0 25 50 75 100 01234 S ep po en ial (mV) 0 0.2 0.4 0.6 0.8 01234 S ep po en ial (mV) Peak po en ial (V) 0.1 0.5 0.9 01234 S ep po en ial (mV) Peak Po en ial (V) Que ce in Que ci in Diosmin Que ce in Que ci in Diosmin Ru in Ch ysin Ru in Ch ysin AB Senso s 2007, 7 2410 associa ed wi h s uc u al o mulas o u in and diosmin, because bo h o hem ha e disaccha ide g oups in hei s uc u e, whe e highe equency could dec ease elec oac i i y o posi ions need o oxidize. In addi ion, heigh o peaks o u in, que ce in and que ci in dec eased mo e sligh ly wi h equency highe han 190 Hz, whe eas hei po en ials did no change much wi hin whole s udied ange. Ch ysin and diosmin peaks si h ed o mo e posi i e po en ials wi h inc easing equency (inse s in Figu e 3A).We used equency o 150 Hz o ollowing analysis, because mos o all compounds o in e es ga e he highes esponses he e. Mo eo e , we we e in e es ed in he issue how do hese compounds beha e wi h inc easing s ep po en ial om 0.15 o 4 mV (pe 0.5 mV). The inc easing s ep po en ial caused dec ease in peaks heigh o que ce in and que ci in, whe eas u in ones did no change much (Figu e 3B). Ch ysin and diosmin beha e e y simila again, whe eas heigh o hei peaks inc eased wi h inc easing s ep po en ial ma kedly. Dependences o po en ials o he s udied la onoids on s ep po en ial was simila shape as he equency dependences (inse s in Figu e 3B). Based on he esul s ob ained, s ep po en ial o 1.95 mV was he mos sui able o ollowing pu poses. The op imiza ion o la onoids de e mina ion p oceeded wi h selec ion o suppo ing elec oly e and i s pH. We wan ed o s udy a b oad ange o pH om acidic o basic, hus, we used B i on- Robinson bu e wi hin he ange om pH 2 o 8 (Figu e 4A). The highes peaks o que ce in and diosmin we e obse ed a pH 4, and hen he signals ma kedly dec eased up o pH 8, whe e he heigh o diosmin peak came up o uni s o pe cen s in compa ison wi h he highes ones and peak o que ce in did no de ec able. In addi ion, u in and que ci in ga e he highes signal a pH 2 and hen dec eased wi hin he whole ange o highe pH alues. The elec ochemical beha iou o ch ysin di e ed ma kedly om o he ones. The cu en esponse o he ch ysin peak inc eased wi h inc easing pH up o 7 and hen dec eased sligh ly (Figu e 4A). This phenomenon p obably ela es wi h basici y o his la onoid [48,58,72]. Peaks o he la onoids shi ed o mo e nega i e po en ials wi h inc easing pH (inse in Figu e 4A). I clea ly ollows om he esul s ob ained ha acidic pHs is sui able o analysis o que ce in, que ci in, u in and ch ysin, bu he basic pHs o diosmin analysis. In addi ion, we we e in e es ed in he issue how can di e en bu e s in luence he analysis o he la onoids. Thus we used ace a e (pH 4) and phospha e (pHs 6, 7 and 8) bu e s. The esul s ob ained ha e been compa ed wi h analysis in he p esence o B i on-Robinson bu e (Figu e 4B). I clea ly ollows om he esul s ob ained ha ace a e bu e is no sui able o de e mina ion o compounds o in e es . I clea ly ollows o he esul s ob ained ha u in, que ce in, que ci in and ch ysin ga e he highes signals in he p esence o phospha e bu e and diosmin in he p esence o B i on-Robinson bu e . In spi e o low cu en esponse o diosmin peaks in he p esence o phospha e bu e , we decide o use i o ollowing analysis o he la onoids. Thus, mo e comp ehensi e in es iga ion o beha iou o hem in he p esence o his bu e had o be done. We choose pH ange om 5.5 o 8 and obse ed he changes in heigh s and po en ials o la onoids’ peaks (Figu e 4C). The highes peaks o u in and que ce in we e ob ained a pH 6 and hen he peaks dec eased sligh ly (up o 25 % in compa ison wi h he highes ones). The heigh o que ce in peak did no change much wi h inc easing pH (abou 10 %). In addi ion, diosmin peak dec eased sligh ly up o pH 7 and hen ma kedly ( he signal measu ed a pH 8 dec eased o mo e han 60 % in compa ison wi h he highes signal ob ained a pH 5). In he case o ch ysin, we obse ed e y di e en beha iou in compa ison wi h o he s udied la onoids again. I s signal inc eased up o pH 7.5 and hen dec eased abou 20 %. Peaks o all compounds o in e es Senso s 2007, 7 2417 54. Timbola, A.K.; de Souza, C.D.; Giacomelli, C.; Spinelli, A. Elec ochemical oxida ion o que ce in in hyd o-alcoholic solu ion. J. B az. Chem. Soc. 2006, 17, 139-148. 55. 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