Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.

Abstract

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.

• Authors: Jun-Hui Wan, Pei-Ling Tian, Wei-Hao Luo, Bing-Yi Wu, Fu Xiong, Wan-Jun Zhou, Xiang-Cai Wei, Xiang-Min Xu
• Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (J Chromatogr B Analyt Technol Biomed Life Sci) Vol. 901 Pg. 53-8 (Jul 15 2012) ISSN: 1873-376X [Electronic] Netherlands
• PMID: 22727753 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier B.V. All rights reserved.

Chemical References

• Hemoglobin Subunits
• Hemoglobins, Abnormal
• Heme

Topics

• Adult
• Analysis of Variance
• Case-Control Studies
• Chromatography, High Pressure Liquid (methods)
• Chromatography, Reverse-Phase (methods)
• Heme (chemistry)
• Hemoglobin Subunits (analysis, chemistry, classification)
• Hemoglobins, Abnormal (analysis, chemistry, classification)
• Humans
• Hydrogen-Ion Concentration
• Infant, Newborn
• ROC Curve
• Reproducibility of Results
• Sensitivity and Specificity
• Thalassemia (blood)
• alpha-Thalassemia (blood)