Use of commercially available hemoglobin standards to quantitatively calibrate a high performance liquid chromatography method

High performance liquid chromatography (HPLC) has proven to be an extremely useful analytical technique to separate and identify different types of hemoglobins, particularly A, C, F and S in blood samples, and compute their relative percentages. Such data provide useful information in the diagnosis of hemoglobinopathies including sickle cell anemia, beta-thalassemia, hemoglobin C disease,etc. In the present investigation, we have explored the determination of absolute concentrations of individual hemoglobins in g/dL and recommend it as an additional parameter which could be included as part of clinical data. Possible correlations between g/dL hemoglobin and the severity of a specific hemoglobinopathy could be established and aid in the diagnosis and/or treatment of such diseases. Several commercially available hemoglobin standards were analyzed and evaluated for use in creating g/dL calibration graphs for the HPLC. Appropriate calibration procedures have been developed and are presented. Also, linear regression graphs and sample chromatograms are included. Preliminary results obtained demonstrate the feasibility of using commercially available hemoglobin standards to calibrate an HPLC method for the estimation of absolute hemoglobin concentrations.