Simultaneous determination of glycated haemoglobin, a long term biomarker of diabetes mellitus, and total haemoglobin by isotope dilution and HPLC-ICP-MS

Abstract

The measurement of glycated haemoglobin (particularly HbA_1c) serves as a powerful indicator for the evaluation and treatment of patients with diabetes mellitus. The separation of the glycated form of this protein (present about 3–4% in non-diabetic patients) is achieved by cation exchange chromatography, employing a Mono S column. The detection is carried out by measuring the Fe contained in the haemo-group of the protein, using an inductively coupled plasma mass spectrometer (ICP-MS) as the Fe selective detector. Using this experimental set-up, speciation of glycated and non-glycated haemoglobin has been investigated and detection limits around 1 μg mL⁻¹ of protein have been obtained. Furthermore, accurate determination of the Fe naturally present in glycated and non-glycated haemoglobin peaks can be conducted by post-column addition of a solution containing isotopically labeled iron (⁵⁷Fe, 92.4%) and applying isotope dilution calculations. The proposed method has been validated by determining glycated and non-glycated Hb (physiological levels about 10–13 g dL⁻¹) in reference standards commonly used to calibrate clinical analyzers and also in a reference material (RM 405). Additionally, the ICP-MS based quantification method has been applied to the analysis of 11 blood specimens of patients diagnosed with diabetes. The relative results for HbA_1c are compared to those obtained for the same samples by an external clinical laboratory. A good agreement in all cases was found. Finally, the potential of the complementary use of electrospray mass spectrometry (ESI-MS) is shown to study peak purity, identity and purity of the HPLC separated fractions before on-line ICP-MS detection.