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

ABSTRACT 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.