Box-Behnken Design Assisted Ultra-Performance Liquid Chromatography-Photodiode Array Detection Method Development, Validation and Quantification of Lunasin Peptide from Nutraceutical Formulation

Abstract

A simple, specific, accurate, precise and robust ultra-performance liquid chromatography-photodiode array detection method for quantifying lunasin peptide in pure form or in plant based vegan supplements was developed and validated. Chromatographic separation was achieved on Accucore C18 column (50 mm×4.6 mm, 2.6 µm) with 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) delivered isocratically (50:50 v/v) at a flow rate of 0.4 ml/min for 10 min and the detection was monitored at 216 nm. Lunasin was eluted at a retention time of 4.94 min with a total runtime of less than 6 min. The chromatographic parameters were optimized using the Box-Behnken design with a sample set of 30 experimental runs. The developed ultraperformance liquid chromatography-photodiode array detection method was validated in accordance with the International Council for Harmonisation Q2(R1) guideline and evaluated the parameters such as system suitability, linearity, sensitivity, accuracy, precision, specificity and robustness. The method was found to be linear within the concentration range of 0.50-10.0 µg/ml. The developed method could be a viable candidate for routine quantification of lunasin peptide in nutraceutical formulations.