An integrated approach to identify rare hemoglobin variants using capillary electrophoresis, liquid chromatography...

Rare hemoglobin variants may produce atypical migration or retention patterns that require correlation across analytical techniques for accurate interpretation. Although capillary electrophoresis (CE) and cation exchange high-performance liquid chromatography (CX-HPLC) are routinely used for hemoglobin analysis, a structured integrated workflow for characterization of uncommon variants is not widely described. This study describes a combined analytical approach applied to four patients presenting with abnormal hemoglobin profiles. Complete blood count (CBC), CE, CX-HPLC, flow injection tandem quadrupole mass spectrometry (TQMS), and targeted MS/MS following tryptic digestion of globin chains were performed. Variants were identified using instrument integrated reference libraries and established hemoglobin variant databases to correlate electrophoretic zones, chromatographic retention windows, intact globin mass shifts, and diagnostic peptide fragment ions. CE and CX-HPLC demonstrated distinct variant fractions with characteristic migration zones, and/or retention times, and relative variant percentages. Correlation of electrophoretic, chromatographic, and intact globin mass data indicated β chain involvement in two cases and α chain involvement in two cases. Integrated analytical interpretation led to the identification of Hb D-Ouled Rabah (β + 14 Da) and a compound heterozygous Hb G-Coushatta (β - 58 Da) and Hb D-Punjab (β -1 Da), while the α chain variants corresponded to Hb O-Indonesia (α -1 Da) and Hb Chiapas (+59 Da). The β globin variant assignments were subsequently confirmed by molecular analysis using Sanger sequencing. Integration of electrophoretic, chromatographic, mass spectrometric, and reference database findings enabled characterization of these rare variants, demonstrating the value of coordinated analytical interpretation in routine laboratory practice.