Discovery of a Rare Hemoglobin Variant Q‐India/D‐Punjab in Whole Blood From an Indian Patient Using Capillary Electrophoresis and Mass Spectrometry

ABSTRACT A definitive diagnosis of hemoglobinopathies requires a comprehensive evaluation encompassing clinical history, laboratory findings that include complete blood count, peripheral smear, chromatographic or electrophoretic techniques, mass spectrometry, and molecular studies. The clinically significant hemoglobin variants, such as S, C, E, D‐Punjab, and O‐Arab, are identified using the gold‐standard methods of cation exchange high‐performance liquid chromatography or capillary electrophoresis. The identification of compound heterozygous variants or the presence of two overlapping or rare variants in the same sample presents a significant diagnostic challenge. This study demonstrates the utility of combining migration time and hemoglobin fraction percentages from hemoglobin electrophoresis and glycated hemoglobin electrophoresis, and mass data from triple quadrupole MS to identify a compound heterozygous hemoglobin Q‐India and D‐Punjab variant in a patient sample. To our knowledge, this is the first report of a compound heterozygous Q‐India/D‐Punjab variant identified using an integrated approach involving capillary electrophoresis and mass spectrometry. The findings were further validated using HPLC, which effectively separates Q‐India, D‐Punjab, and a heterozygous hybrid Q‐India/D‐Punjab based on their distinct retention times on a cation exchange column.