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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Hematology

Background:

• Reversed-phase high-performance liquid chromatography (RP-HPLC) is crucial for identifying thalassemias and hemoglobin variants.
• Current RP-HPLC methods face limitations in clinical application due to long analysis times and complex sample preparation.

Purpose of the Study:

• To develop a simple, rapid, and high-resolution RP-HPLC method for human globin chain separation.
• To improve the clinical applicability of globin chain analysis for hemoglobinopathies.

Main Methods:

• Human red blood cells were directly injected onto a C18 reversed-phase column.
• Globin chains were separated and denatured from heme groups within 12 minutes using optimized HPLC conditions.
• UV detection at 280 nm was employed for quantification.

Main Results:

• The method achieved high resolution with low coefficient of variation (CV) for retention time (0.11–1.29%) and peak area (0.32–4.86%).
• Significant differences in globin chain area ratios (α/pre-β+β) were observed between normal, Hb H, and β thalassemia groups (P<0.05).
• Receiver operating characteristic (ROC) curve analysis demonstrated 100% sensitivity and specificity for detecting β thalassemia carriers and high accuracy for predicting Hb H disease.

Conclusions:

• The developed rapid RP-HPLC method is effective for the fast and accurate separation of human globin chains.
• This method shows high diagnostic potential for identifying β thalassemia carriers and predicting Hb H disease.
• The technique successfully identified abnormal hemoglobins like Hb E and Hb WS, offering a valuable tool for clinical diagnostics.