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 HbA1c) 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 HbA1c 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.