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

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 220nm) 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 220nm than at 280nm. 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.