Rapid analysis of hemoglobin variants by cation-exchange HPLC

Abstract

We investigated the use of a 3.5 × 0.46 cm HPLC column packed with 5-μm particles of porous (100 nm) silica coated with polyaspartic acid for hemoglobin analysis. A 13-min gradient was produced between two mobile phases. The method is capable of separating more than 35 commonly encountered hemoglobin variants within 12 min. Hemoglobin variants identified include Bart's, acetyl F, H, A1c, F, Camden, N-Baltimore, J-Baltimore, N-Seattle, Grady, Fannin-Lubbock, A, G-Georgia, Lepore-Baltimore, P-Galveston, G-Coushatta, Lepore-Boston, E, Osu Christiansborg, A, G-Philadelphia, Korle Bu, Russ, Richmond, D-Los Angeles, Deer Lodge, Montgomery, S, Q-Thailand, G-San Jose, A2′, Hasharon, Q-India, Tampa, GS hybrid, C-Harlem, O-Arab, British Columbia, and C. Between-run precision of an in-house pooled hemoglobin control material, AFSCA2 gave CVs of 2-5% for the A, F, S, and C and 8% for the A2 over a 6-month period. The simplicity of sample preparation, high resolution of the system, and high accuracy of the method, combined with complete automation, make this an ideal methodology for the routine diagnosis of hemoglobin disorders in a clinical laboratory.