Reversed-phase high-performance liquid chromatography (RP-HPLC) of human
globin chains is an important tool for detecting
thalassemias and
hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of
globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the
globin chains (pre-β, β, δ, α, (G)γ and (A)γ) were denatured and separated from the
heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P<0.05) among three groups (normal, Hb H and β
thalassemia) were found in the area ratio of α/pre-β+β applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α
thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/β and α/pre-β+β area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating β
thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of
hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of δ/β for β
thalassemia carriers and 0.626 of α/pre-β+β for Hb H disease. In addition, abnormal
hemoglobin hemoglobin E (Hb E) and
Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human
globin chains could be of use for similar work.