Problems of Forensic Sciences 2009 Vol. 79 (LXXIX) 314-326
DETERMINATION OF GLYCATED HAEMOGLOBIN HbA1c IN AUTOPSY MATERIAL USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONISATION TANDEM MASS SPECTROMETRY (HPLC-ESI-MS)
Krzysztof TUTAJ, Krzysztof BAŃKA, Grzegorz BUSZEWICZ, Grzegorz TERESIŃSKI, Roman MĄDRO
Chair and Department of Forensic Medicine, Medical University, Lublin, Poland
Streszczenie
Glycated haemoglobin (HbA1c) contains the stable adduct of glucose with the N-terminal group of the chain. To date, attempts to use HbA1c determinations for autopsy diagnostics of intravital metabolic abnormalities have been based on blood tests designed for clinical purposes, which neglected validation of post-mortem haemolysis and background effects. The aim of the present study was thus to design such a procedure of HbA1c determinations using HPLC-ESI-MS, which could be useful for autopsy diagnostics and to compare the results obtained using this method and other immunological techniques. For this purpose, the HPLC-ESI-MS method designed and approved by the International Federation of Clinical Chemistry (IFCC) was modified in such a way that the erythrocyte centrifugation phase was omitted (the haemolysate of whole blood was digested with endoproteinase). The hexapeptide standards were replaced with glycated haemoglobin standards exposed to digestion. Moreover, the HPLC conditions were changed – the steep gradient was replaced with a linear one, in order to obtain better separation of nonglycated hexapeptide from glycated hexapeptide and from the background. No essential changes in ESI-MS conditions were necessary. Complete validation of the modified method showed its high specificity, accuracy, precision. Furthermore, a linear relation between HbA1c concentration and signals from ions 429 and 857 m/z (mass-to-charge-ratio) was demonstrated. A comparison of HbA1c concentrations (in autopsy blood samples) determined using the modified IFCC reference method and two other immunological techniques (i.e. Konelab and Cobas) did not reveal statistically significant differences. It follows that each of the compared methods may be used for post-mortem diagnosis of diabetes, but due to the high specificity of detection of glycated hexapeptides by the HPLC-ESI-MS method, it should be considered as the reference one.
Słowa kluczowe
Glycated haemoglobin; HPLC-ESI-MS; Post-mortem diagnosis of diabetes.