Standardization of RP-HPLC methods for the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3

Authors

Harmit Singh, California State Polytechnic UniversityFollow
Mary Jo Cantoria, California State University
Poonam Malave, California State University
Denny Saputra, California State Polytechnic University
Soheila Maleki, United States Department of Agriculture, Southern Regional Research CenterFollow

Date of this Version

2016

Citation

Food Chemistry 194 (2016) 383–390

Comments

U.S. Government Work

Abstract

Crude peanut extract (CPE) was analyzed for three major allergens (Ara h 1, h 2, and h 3) using a C₁₂ and a C₁₈ column at two wavelengths (280 and 220 nm) and under different solvent conditions. HPLC profiles were compared for retention time, resolution, and peak heights. CPE samples were spiked with pure allergens to identify the peaks corresponding to allergens. The HPLC fractions of corresponding allergens were collected and freeze-dried in order to perform SDS–PAGE and immunoblotting tests. The best method was identified the one with a shorter retention time, better resolution, and greater peak height as compared with the other methods. In general, the peak heights were greater at 220 nm than at 280 nm. The major disadvantage of the C₁₂ column was the need for two sets of conditions to identify the allergens as compared to the C₁₈ column where all three allergens could be identified in one run.