Method development and validation for the quantitative determination of total flavonoids through the complexation of iron (III) and its application in real sample.

ABSTRACT

BACKGROUND: The determination of flavonoids in real sample using UV-Vis spectrophotometer commonly uses quercetin and catechin with Al+3 complexing agent as reference materials for the calibration of the instrument. However, getting these standard materials is challenging due to its expense and unavailability in the chemical reserve of the country. Moreover, the Al+3 - quercetin complexation standard method demands high amount of quercetin in spite of its high cost. Hence, developing alternative method that can solve this problem is crucial for the determination of flavonoids in the real sample. RESULTS: An iron-based complexation method for the determination of flavonoids in the real sample was developed that reduces the amount of quercetin by 200 times (1 mg/mL to 0.005 mg/mL) during the calibration of UV-Vis spectroscopy as an alternative method. The reaction parameters (incubation time, pH, and concentration of quercetin) were optimized using software Design Expert 11 and confirmed by the practical experiments. The kinetics of reaction between iron and quercetin was found to be pseudo first order with rate constant of kobs at 340 and 510 nm. The analysis window for the flavonoid complex was achieved with the kinetic discrimination of the interferences at its optimized time of complexation 20 min and absorbance maxima of 510 nm. The developed method was validated by evaluating its precision, accuracy, recovery test (84-117%), detection limit and quantification limit following the standard protocols. The calibration of the instrument has been developed for the new method and the linear regression coefficient (R2) of 0.998 was obtained. SIGNIFICANCE: Applying the developed standard material (Fe3+ - quercetin complex) gives freedom for the analytical chemists to find the standard materials that is accessible and cheaper than the existing one (Al3+-quercetin complex). The developed method can also be easily applied for determination of flavonoid in the real samples without potential interferences coming from sample matrix.