Spektrofluorimetrijsko ispitivanje kompleksnih jedinjenja morina, hesperidina i kvarcetina sa aluminijumom

Abstract

Morin, hesperidin i kvercetin pripadaju flavonoidima, koji imaju značajnubiološku i fiziološku aktivnost, zbog čega je bilo potrebno razviti jednostavne, preciznei tačne metode za njihovo određivanje u različitim uzorcima.Predložena je spektrofluorimetijska metoda za određivanje morina u 70 v/v %etanolu i u humanom serumu, zasnovana na fluorescenciji kompleksa aluminijum(III)–morin. Ovaj kompleks ima sastav aluminijum(III) : morin = 1 : 2. Formirani kompleks jestabilan u pH oblasti od 3,0 do 6,0 i pokazuje intenzivnu fluorescenciju na lem = 500 nmpri eksitaciji na lex = 410 nm. Intenzitet fluorescencije nastalog kompleksa zavisi od pHvrednosti rastvora, i najveći je na pH = 4,40, a vrednost konstante stabilnosti na ovojvrednosti pH iznosi log β2 = 16,96. Dobijena je linearna zavisnost intenziteta emitovanogzračenja od koncentracije morina u oblasti od 1,5 do 30,5 ng cm-3. Granica detekcije jeiznosila 0,02 ng cm-3 a granica kvantifikacije 0,06 ng cm-3. Pouzdanost predloženemetode proverena je referentnom RP-HPLC/UV metodom.Razvijena je spektrofluorimetijska metoda za određivanje hesperidina u humanojplazmi i farmaceutskim preparatima koja se zasniva na fluorescenciji kompleksaaluminijum(III)–hesperidin, čiji je sastav aluminijum(III) : hesperidin = 1 : 1. Komplekspokazuje intenzivnu fluorescenciju u prisustvu surfaktanta SB 12 na lem = 476 nm priekscitaciji na lex =390 nm i stabilan je u pH opsegu od 3,0 do 7,0. Konstanta stabilnostiovog kompleksa na pH = 4,50 je log β1 = 9,20. Linearna zavisnost intenzitetafluorescencije od koncentracije, pri određivanju hesperidina u farmaceutskimpreparatima dobijena je u koncentracionom opsegu 0,06–24,4 μg cm-3sa granicom detekcije od 0,016 μg cm-3 i granicom kvantifikacije od 0,049 μg cm-3.Dobijene „recovery“ vrednosti u intervalu od 99,3–99,7 % pokazuju veliku tačnostmetode. Linearna zavisnost intenziteta fluorescencije kompeksa od koncentacijehesperidina pri određivanju u uzorcima humane plazme dobijena je u koncentracionomopsegu od 0,1–12,2 μg cm-3 sa granicom detekcije od 0,032 μg cm-3 i granicomkvantifikacije od 0,096 μg cm-3. „Recovery“ vrednosti su dobijene u opsegu 98,4 do 99,8%. Pouzdanost metode proverena je LC–MS/MS metodom za određivanje hesperidina uhumanoj plazmi, a HPLC/UV metodom prilikom određivanja hesperidina ufarmaceutskim preparatima. Linearna zavisnost pri određivanju hesperidina ufarmaceutskim preparatima dobijena je u intervalu od 0,05–10,00 μg cm-3. Granicadetekcije, LOD je iznosila 0,01 μg cm-3, granica kvantifikacije, LOQ 0,03 μg cm-3.Linearna zavisnost pri određivanju hesperidina u humanoj plazmi je dobijena u intervalu0,02–10,00 μg cm-3 sa granicom detekcije od 0,005 μg cm-3 i granicom kvantifikacije od0,015 μg cm-3. Dobro slaganje između ove dve metode pokazuje primenljivostspektrofluorimetrijske metode u kliničkim laboratorijama i laboratorijama za kontrolukvaliteta. Predložena fluorimetrijska metoda je jednostavna, pouzdana i precizna zaodređivanje hesperidina u humanom serumu i farmaceutskim preparatima.Predložena je spektrofluorimetrijska metoda za određivanje sadržaja hesperidina usokovima od pomorandže zasnovana na fluorescenciji kompleksa aluminijum–hesperidin. Linearna zavisnost za određivanje hesperidina u rastvorima metanol-vodadobijena je u koncentracionom intervalu od 0,08 do 18,0 μg cm-3, pri čemu je granicadetekcije iznosila LOD = 0,023 μg cm-3, a granica kvantifikacije LOQ = 0,070 μg cm-3.Predložena metoda je pojednostavljena izostavljanjem površinski aktivnih materija kojese koriste u sličnim procedurama i uspešno je primenjena za određivanje sadržajahesperidina u sokovima od pomorandže prisutnim na tržištu Srbije.Predložena je spektrofluorimetijska metoda za određivanje kvercetina ufarmaceutskim preparatima, koja je zasnovana na fluorescenciji kompleksaaluminijum(III)–kvercetin, sastava aluminijum(III) : kvercetin = 2 : 1. Kompleksaluminijum(III)–kvercetin pokazuje intenzivnu fluorescenciju na lem = 480 nm prieksitaciji na lex = 420 nm, i stabilan je u pH oblasti od 2,0 do 5,5. Intenzitet fluorescencijenastalog kompleksa zavisi od pH vrednosti rastvora, i najveći je na pH = 3,33. Konstantastabilnosti ovog kompleksa na pH = 3,20 iznosi log β = 27,79. Linearnost je dobijena ukoncentracionom opsegu od 1,5 do 60,5 ng cm-3 kvercetina. Granica detekcije je iznosilaLOD = 0,09 ng cm-3, a granica kvantifikacije LOQ = 0,27 ng cm-3. Pouzdanost predloženemetode proverena je referentnom RP-HPLC/UV metodom. Linearnost je dobijena ukoncentracionom opsegu od 1,0 do 200,0 μg cm-3 Granica detekcije kvercetina RPHPLC/UV metodom je iznosila 0,066 μg cm-3 a granica kvantifikacije 0,200 μg cm-3kvrercetina.Predložena je spektrofluorimetrijska metoda za određivanje ukupnog sadržajapolifenola u soku od jabuka pri čemu je sadržaj ukupnih polifenola izražen u odnosuekvivalent kvercetina (QE), koji je određivan spektrofluorimetrijski.

Morin, hesperidin and quercetin belong to flavonoids, a large class of compounds.Flavonoids have important biological and physiological activity. Thus, it is of interest todevelop simple, accurate and precise method for the determination of morin, hesperidinand quercetin in different samples.Morin is a flavonol antioxidant. In ethanol–water mixtures (70 v/v % of ethanol)it reacts with aluminium (III) ion to give Al(Mor)2 in the pH range 3–6. The complexshows strong fluorescence emission at 500 nm upon excitation at 410 nm. Thefluorescence intensity is pH dependent with maximum emission at pH 4,40. Theconditional stability constant of this complex at 298 K was found to be log β2 = 16,96 atpH 4,40. Since the complexation reaction enhances the fluorescence of morin, thisproperty was used for the determination of morin in human serum. A linear dependenceof the intensity of fluorescence of the complex on the concentration of morin was obtainedin morin concentration range from 1,5–30,5 ng cm-3. The limit of detection, LOD was0,02 ng cm-3 while limit of quantification, LOQ was 1,0 ng cm-3. Serum concentration ofmorin was also determined using RP-HPLC as a reference method.A spectrofluorometric method, based on the fluorescence properties of thealuminium(III)–hesperidin complex, AlHesp2+, for the determination of hesperidin inhuman plasma and pharmaceutical forms has been developed and validated. The complexshows a strong emission in the presence of the surfactant betain sulphonate SB 12 at 476nm with excitation at 390 nm. The conditional stability constant of this complex at 298 Kwas found to be log β1 = 9,20 at pH 4,50. The linearity range for pharmaceutical forms ofhesperidin was 0,06–24,4 μg cm-3 with a limit of detection, LOD, of 0,016 μg cm-3 anda limit of quantification, LOQ, of 0,049 μg cm-3. Recovery values in the range 99,3–99,7% indicate good accuracy of the method. A linear dependence of the intensity offluorescence of the complex on the concentration of hesperidin in plasma was obtainedin concentration range from 0,1–12,2 μg cm-3. The LOD was 0,032 μg cm-3 while LOQwas 0,096 μg cm-3. Recovery values were in the range 98,4–99,8 %. The reliability of themethod was checked by an LC–MS/MS method for plasma samples and an HPLC/UVmethod for tablets with direct determination of hesperidin after separation. Linearityrange in determination of hesperidin in pharmaceutical forms was obtained in the rangefrom 0,05 to 10,00 μg cm-3. The LOD was 0,01 μg cm-3 and the LOQ was 0,03 μg cm-3.The linearity range for the determination of hesperidin in human plasma was 0,02–10,00μg cm-3 with an LOD 0,005 μg cm-3 and an LOQ of 0,015 μg cm-3. The good agreementbetween the two methods indicates the usability of the proposed fluorometric method forthe simple, precise and accurate determination of hesperidin in clinical and quality controllaboratories.The spectrofluorometric method based on fluorescence ability of aluminium –hesperidin complex for the determination of hesperidin in orange juice is proposed. Thelinearity range of hesperidin in methanolic-aqueous solution was 0,08 – 18,0 μg cm-3 withLOD and LOQ values as 0,023 μg cm-3 and 0,070 μg cm-3, respectively. Method wassimplified by omitting any of surphactant agents usually applied in similar procedures,and successfully applied for the determination of hesperidin in orange juicescommercially available on the Serbian market.Quercetin is a flavonol antioxidant. IIn methanol – water mixtures (70 v/v % ofethanol) it reacts with aluminium (III) ion to give Al2Querc+4 in the pH range 2,0–5,5.The complex shows strong fluorescence emission at 480 nm upon excitation at 420 nm.The fluorescence intensity is pH dependent with maximum emission at pH 3,33. Theconditional stability constant of this complex at 298 K was found to be log β = 27,79 atpH 3,20. Since the complexation reaction enhances the fluorescence of quercetin, thisproperty was used for the determination of quercetin in dosage forms. A lineardependence of the intensity of fluorescence of the complex on the concentration ofquercetin was obtained in quercetin concentration range from 1,5 to 60,5 ng cm-3. TheLOD was 0,09 ng cm-3 while LOQ was 0,27 ng cm-3. Concentration of quercetin was alsodetermined using RP-HPLC as a reference method.Quercetin and aluminium(III)-ion form a stable complex and the resulted emissionat 480 nm can be used for the determination of the total phenols concentration expressedin terms of “quercetin equivalent” (QE).