Spektrofluorimetrijsko ispitivanje kompleksnih jedinjenja morina, hesperidina i kvarcetina sa aluminijumom

Abstract

Morin, hesperidin i kvercetin pripadaju flavonoidima, koji imaju značajnu biološku i fiziološku aktivnost, zbog čega je bilo potrebno razviti jednostavne, precizne i 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 je stabilan u pH oblasti od 3,0 do 6,0 i pokazuje intenzivnu fluorescenciju na lem = 500 nm pri eksitaciji na lex = 410 nm. Intenzitet fluorescencije nastalog kompleksa zavisi od pH vrednosti rastvora, i najveći je na pH = 4,40, a vrednost konstante stabilnosti na ovoj vrednosti pH iznosi log β2 = 16,96. Dobijena je linearna zavisnost intenziteta emitovanog zračenja od koncentracije morina u oblasti od 1,5 do 30,5 ng cm-3. Granica detekcije je iznosila 0,02 ng cm-3 a granica kvantifikacije 0,06 ng cm-3. Pouzdanost predložene metode proverena je referentnom RP-HPLC/UV metodom. Razvijena je spektrofluorimetijska metoda za određivanje hesperidina u humanoj plazmi i farmaceutskim preparatima koja se zasniva na fluorescenciji kompleksa aluminijum(III)–hesperidin, čiji je sastav aluminijum(III) : hesperidin = 1 : 1. Kompleks pokazuje intenzivnu fluorescenciju u prisustvu surfaktanta SB 12 na lem = 476 nm pri ekscitaciji na lex =390 nm i stabilan je u pH opsegu od 3,0 do 7,0. Konstanta stabilnosti ovog kompleksa na pH = 4,50 je log β1 = 9,20. Linearna zavisnost intenziteta fluorescencije od koncentracije, pri određivanju hesperidina u farmaceutskim preparatima dobijena je u koncentracionom opsegu 0,06–24,4 μg cm-3 sa 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 tačnost metode. Linearna zavisnost intenziteta fluorescencije kompeksa od koncentacije hesperidina pri određivanju u uzorcima humane plazme dobijena je u koncentracionom opsegu od 0,1–12,2 μg cm-3 sa granicom detekcije od 0,032 μg cm-3 i granicom kvantifikacije 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 u humanoj plazmi, a HPLC/UV metodom prilikom određivanja hesperidina u farmaceutskim preparatima. Linearna zavisnost pri određivanju hesperidina u farmaceutskim preparatima dobijena je u intervalu od 0,05–10,00 μg cm-3. Granica detekcije, 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 intervalu 0,02–10,00 μg cm-3 sa granicom detekcije od 0,005 μg cm-3 i granicom kvantifikacije od 0,015 μg cm-3. Dobro slaganje između ove dve metode pokazuje primenljivost spektrofluorimetrijske metode u kliničkim laboratorijama i laboratorijama za kontrolu kvaliteta. Predložena fluorimetrijska metoda je jednostavna, pouzdana i precizna za određivanje hesperidina u humanom serumu i farmaceutskim preparatima. Predložena je spektrofluorimetrijska metoda za određivanje sadržaja hesperidina u sokovima od pomorandže zasnovana na fluorescenciji kompleksa aluminijum– hesperidin. Linearna zavisnost za određivanje hesperidina u rastvorima metanol-voda dobijena je u koncentracionom intervalu od 0,08 do 18,0 μg cm-3, pri čemu je granica detekcije iznosila LOD = 0,023 μg cm-3, a granica kvantifikacije LOQ = 0,070 μg cm-3. Predložena metoda je pojednostavljena izostavljanjem površinski aktivnih materija koje se koriste u sličnim procedurama i uspešno je primenjena za određivanje sadržaja hesperidina u sokovima od pomorandže prisutnim na tržištu Srbije. Predložena je spektrofluorimetijska metoda za određivanje kvercetina u farmaceutskim preparatima, koja je zasnovana na fluorescenciji kompleksa aluminijum(III)–kvercetin, sastava aluminijum(III) : kvercetin = 2 : 1. Kompleks aluminijum(III)–kvercetin pokazuje intenzivnu fluorescenciju na lem = 480 nm pri eksitaciji na lex = 420 nm, i stabilan je u pH oblasti od 2,0 do 5,5. Intenzitet fluorescencije nastalog kompleksa zavisi od pH vrednosti rastvora, i najveći je na pH = 3,33. Konstanta stabilnosti ovog kompleksa na pH = 3,20 iznosi log β = 27,79. Linearnost je dobijena u koncentracionom opsegu od 1,5 do 60,5 ng cm-3 kvercetina. Granica detekcije je iznosila LOD = 0,09 ng cm-3, a granica kvantifikacije LOQ = 0,27 ng cm-3. Pouzdanost predložene metode proverena je referentnom RP-HPLC/UV metodom. Linearnost je dobijena u koncentracionom 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-3 kvrercetina. Predložena je spektrofluorimetrijska metoda za određivanje ukupnog sadržaja polifenola u soku od jabuka pri čemu je sadržaj ukupnih polifenola izražen u odnosu ekvivalent 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 to develop simple, accurate and precise method for the determination of morin, hesperidin and 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 complex shows strong fluorescence emission at 500 nm upon excitation at 410 nm. The fluorescence intensity is pH dependent with maximum emission at pH 4,40. The conditional stability constant of this complex at 298 K was found to be log β2 = 16,96 at pH 4,40. Since the complexation reaction enhances the fluorescence of morin, this property was used for the determination of morin in human serum. A linear dependence of the intensity of fluorescence of the complex on the concentration of morin was obtained in morin concentration range from 1,5–30,5 ng cm-3. The limit of detection, LOD was 0,02 ng cm-3 while limit of quantification, LOQ was 1,0 ng cm-3. Serum concentration of morin was also determined using RP-HPLC as a reference method. A spectrofluorometric method, based on the fluorescence properties of the aluminium(III)–hesperidin complex, AlHesp2+, for the determination of hesperidin in human plasma and pharmaceutical forms has been developed and validated. The complex shows a strong emission in the presence of the surfactant betain sulphonate SB 12 at 476 nm with excitation at 390 nm. The conditional stability constant of this complex at 298 K was found to be log β1 = 9,20 at pH 4,50. The linearity range for pharmaceutical forms of hesperidin was 0,06–24,4 μg cm-3 with a limit of detection, LOD, of 0,016 μg cm-3 and a 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 of fluorescence of the complex on the concentration of hesperidin in plasma was obtained in concentration range from 0,1–12,2 μg cm-3. The LOD was 0,032 μg cm-3 while LOQ was 0,096 μg cm-3. Recovery values were in the range 98,4–99,8 %. The reliability of the method was checked by an LC–MS/MS method for plasma samples and an HPLC/UV method for tablets with direct determination of hesperidin after separation. Linearity range in determination of hesperidin in pharmaceutical forms was obtained in the range from 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 0,02–10,00 μg cm-3 with an LOD 0,005 μg cm-3 and an LOQ of 0,015 μg cm-3. The good agreement between the two methods indicates the usability of the proposed fluorometric method for the simple, precise and accurate determination of hesperidin in clinical and quality control laboratories. The spectrofluorometric method based on fluorescence ability of aluminium – hesperidin complex for the determination of hesperidin in orange juice is proposed. The linearity range of hesperidin in methanolic-aqueous solution was 0,08 – 18,0 μg cm-3 with LOD and LOQ values as 0,023 μg cm-3 and 0,070 μg cm-3, respectively. Method was simplified by omitting any of surphactant agents usually applied in similar procedures, and successfully applied for the determination of hesperidin in orange juices commercially available on the Serbian market. Quercetin is a flavonol antioxidant. IIn methanol – water mixtures (70 v/v % of ethanol) 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. The conditional stability constant of this complex at 298 K was found to be log β = 27,79 at pH 3,20. Since the complexation reaction enhances the fluorescence of quercetin, this property was used for the determination of quercetin in dosage forms. A linear dependence of the intensity of fluorescence of the complex on the concentration of quercetin was obtained in quercetin concentration range from 1,5 to 60,5 ng cm-3. The LOD was 0,09 ng cm-3 while LOQ was 0,27 ng cm-3. Concentration of quercetin was also determined using RP-HPLC as a reference method. Quercetin and aluminium(III)-ion form a stable complex and the resulted emission at 480 nm can be used for the determination of the total phenols concentration expressed in terms of “quercetin equivalent” (QE).