Development and validation of a fluorometric method for the determination of hesperidin in human plasma and pharmaceutical forms

Abstract

A fluorometric method, based on the fluorescence properties of the aluminium(III)-hesperidin complex, 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 linearity range for pharmaceutical forms of hesperidin was 0.06-24.4 μg mL-1 with a limit of detection, LOD, of 0.016 μg mL-1 and a limit of quantification, LOQ, of 0.049 μg mL-1. 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 mL-1. The LOD was 0.032 μg mL-1 while LOQ was 0.096 μg mL-1. 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 mL-1. The LOD was 0.01 μg mL-1 and the LOQ was 0.03 μg mL-1. The linearity range for the determination of hesperidin in plasma was 0.02-10.00 μg mL-1 with an LOD 0.005 μg mL-1 and an LOQ of 0.015 μg mL-1. 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.

Razvijena je fluorimetrijska metoda za određivanje hesperidina u humanom serumu i farmaceutskim preparatima koja se zasniva na fluorescenciji kompleksa aluminijum(III)-hesperidin. Kompleks pokazuje intenzivnu fluorescenciju u prisustvu surfaktanta SB 12 na 476 nm prilikom ekscitacije na 390 nm. Linearna zavisnost intenziteta fluorescencije od koncentracije pri određivanju hesperidina u farmaceutskim preparatima dobijena je u koncentracionom opsegu 0,06-24,4 μg mL-1 sa granicom detekcije od 0,016 μg mL-1 i granicom kvantifikacije od 0,049 μg mL-1. Dobijene 'recovery' vrednosti u intervalu 99,3-99,7 % pokazuju veliku preciznost metode. Linearna zavisnost intenziteta fluorescencije kompeksa od koncentacije hesperidina dobijena je u koncentracionom opsegu 0,1-12,2 μg mL-1 sa granicom detekcije od 0,032 μg mL-1 i granicom kvantifikacije od 0,096 μg mL-1. 'Recovery' vrednosti su dobijene u opsegu 98,4 do 99,8 %. Pouzdanost metode proverena je LC-MS/MS metodom za određivanje hesperidina u serumu, a HPLC/UV metodom proverena je pouzdanost prilikom određivanja hesperidina u farmaceutskim preparatima. Linearna zavisnost pri određivanju hesperidina u farmaceutskim preparatima dobijena je u intervalu 0,05-10,00 μg mL-1. Granica detekcije je iznosila 0,01, a granica kvantifikacije je 0,03 μg mL-1. Linearna zavisnost pri određivanju hesperidina u humanom serumu je dobijena u intervalu 0,02-10,00 μg mL-1 sa granicom detekcije od 0,005 i granicom kvantifikacije od 0,015 μg mL-1. Dobro slaganje između ove dve metode pokazuje primenljivost fluorimetrijske 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.