Design of Experiments Approach for the Development of a Validated UPLC‐Q‐ToF/MS Method to Quantitate Soy‐Derived Bioactive Peptide Lunasin in Rabbit Plasma: Application to a Pharmacokinetic Study

A novel ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometric (UPLC-Q-ToF/MS) method for quantifying soy-derived bioactive peptide lunasin in rabbit plasma was developed using design of experiments (DoE) methodology. Lunasin and Neuropeptide Y as an internal standard (IS) were separated on a BEH-C18 column (50 mm × 2.1 mm, 1.7 μm particle size) using 0.1% formic acid solution and acetonitrile as mobile phase delivered for 9 min at a constant flow rate of 0.4 mL/min in gradient mode. The Q-ToF mass spectrometer, equipped with electrospray combined ionization (ESCi) interface, was operated in positive ion mode, and the quantifier ions of lunasin and IS were at m/z 838 and m/z 602, respectively. The lower limit of quantification (LLOQ) was 34.6 ng/mL, and the assay was linear over the concentration range 35-10,000 ng/mL. The accuracy was within a range from 86.7% to 88.9% in terms of mean recovery (R), and the intraday and interday precisions in terms of relative standard deviation (RSD) were < 2.65% and < 6.22%, respectively. The method was successfully applied to a pharmacokinetic study involving oral administration of lunasin-rich processed soybeans (6.56 and 19.1 g/kg) to rabbits.