Reverse Phase HPLC Method for Detection and Quantification of Lupin Seed γ-Conglutin

Abstract

γ-Conglutin is a bioactive protein from lupin seeds with demonstrated antidiabetic properties, yet routine quantification of this protein from lupin seed extracts lacked a validated analytical method. A simple, selective, and accurate reverse phase high-performance liquid chromatography (RP-HPLC) method was developed for the detection and quantitation of γ-conglutin from lupin seed extract.

The method employs a linear gradient of water and acetonitrile containing trifluoroacetic acid (TFA) as a mobile phase modifier on a reverse phase C-18 column (Agilent Zorbax 300SB C-18) at a flow rate of 0.8 mL/min, producing a sharp symmetric peak at a retention time of 29.16 min. The identity of γ-conglutin was confirmed by mass spectrometry (MS/MS) and SDS-PAGE analysis. The method demonstrated a detection limit of 2.68 μg/mL and a quantitation limit of 8.12 μg/mL. Intra-day and inter-day precision values were less than 0.5% with recovery greater than 97%, confirming high reproducibility. The method exhibited high selectivity with no matrix interference from other lupin seed proteins. Comprehensive validation studies demonstrated that the method is suitable for routine analysis of γ-conglutin from lupin seed extract, supporting research and quality control applications in nutraceutical development.

@article{mane2017reverse,
  title         = {Reverse Phase HPLC Method for Detection
                  and Quantification of Lupin Seed {\ensuremath{\gamma}}-Conglutin},
  author        = {Mane, Sharmilee and Bringans, Scott and Johnson, Stuart and Pareek, Vishnu
                  and Utikar, Ranjeet},
  year          = 2017,
  journal       = {Journal of Chromatography B},
  publisher     = {Elsevier},
  volume        = 1063,
  pages         = {123--129},
  doi           = {10.1016/j.jchromb.2017.08.025}
}