Meeting Abstract - Poster Presentation

Developing RP-HPLC method for detection of peanut allergens
H. SINGH (1), P. Malave (1), G. B. Montes (1)
(1) California State University, Los Angeles, CA, U.S.A.
Cereal Foods World 56:A64

During this study various RP-HPLC conditions for detecting peanut allergens were compared. Three major peanut allergens Ara h1, Ara h2 and Ara h3 were targeted for identification using HPLC. Raw and unsalted U. S. Virginia peanuts were used for the preparation of crude peanut extract (CPE) using a published procedure. CPE, (0.0012 g/ml) was analyzed for allergens using a C18 column at various wavelengths (280 nm and 220 nm) and solvent conditions. Method 1 (Solvent A -0.05% TFA in HPLC water; B-linear gradient 0.05% TFA in methanol) and Method 2 (A - 0.1% TFA in HPLC water and B-linear gradient 0-100% acetonitrile) were used to run all the samples. CPE in distilled water (0.006 g/mL) was spiked with one of the three pure allergens obtained from USDA (New Orleans, LA). During spike test increase in peak height using pure allergens was used to identify allergen peaks in RP-HPLC chromatogram. HPLC profiles were compared for retention time of allergens, resolution (resolution from the allergen peak to the preceding protein peak) and peak heights. The best method was identified to be the one with lesser retention time, better resolution and more peak height. In general 220nm provided higher peak heights using method B. Under the best conditions Ara h 1 and Ara h 2 were individual peaks at 18.6 and 14.4 minutes, Ara h 3 eluted as a set of 3 peaks ranging from 19.2–21.2 minutes. To further confirm that the peaks are allergens, the fractions of corresponding allergens were collected, freeze-dried to run SDS-PAGE and immunoblotting tests.

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