Validation procedure of 96-well plate spectrophotometric method for the quantification of flavonoids and total phenolic compounds

Abstract

Validation procedure for determination of flavonoids and total phenolic compounds (TPC) was prepared for Biosan HiPo MPP-96 Microplate Photometer (Latvia). The advantage of new assay based on 96-well plate procedure is simple, fast and inexpensive measurement decreasing total assay solution volume up to 4 times in comparison with the previously optimized non-automated spectrophotometric assay using manual pipetting and spectrophotometer (Spectronic, Milton Roy, USA) [1, 2]. Calibration standards of rutin solution were prepared within the range of 0.01–1.00 mg/ml. For determination of flavonoids absorbance was measured at 407 nm (Milton Roy) and 405 nm (Biosan) and for determination of TPC was measured at 760 nm (Milton Roy) and 694 nm (Biosan). Calibration curve was built of 5 concentrations. To evaluate the linearity within described concentration range and determination coeficient R2, accuracy, precision, detection limit and quantification limit new assay was validated according to the recommendations of ICH Guideline Q2(R1) [3]. Accuracy was evaluated using known intermediate rutin solutions. The interday precision was evaluated following the same procedure for different days. The precision of the measurements was reported as the relative standard deviation (RSD%). Detection limit and quantification limit were determined based on the standard deviation of the response and on the slope of the calibration curve [3].