Glycosylated haemoglobin: high-performance liquid chromatographic determination of 5-(hydroxymethyl)-2-furfuraldehyde after haemoglobin hydrolysis.
A specific and accurate method for the quantitation of the azomethine linkage present in non-enzymatically glycosylated haemoglobin is described. This protein is hydrolysed for 5 h in 1 M oxalic acid at 100 degrees C to yield 5-(hydroxymethyl)-2-furfur-aldehyde (5-HMF), known as a specific degradation product of hexoses linked to the protein. 5-HMF is then purified through a Sep-Pak C18 cartridge and measured by its absorption at 280 nm after separation on a C18 reversed-phase silica column. Quantitation is made accurate by using 1-methylxanthine as internal standard throughout the whole procedure. The identity and the purity of the 5-HMF chromatographic peak was ascertained by UV spectroscopy, gas chromatography on a glass capillary column and mass spectrometry. The method has been successfully used for 5-HMF determinations in monitoring diabetes mellitus patients. The mean values, expressed as nmol of 5-HMF per mg of haemoglobin were 0.64 +/- 0.13 (S.D.) for 27 controls and 1.32 +/- 0.39 for 78 diabetic patients. Unlike the usually employed thiobarbituric acid assay, the present procedure is truly specific for the 5-HMF determination.
D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005662:Furaldehyde / Q000031:analogs & derivatives* / Q000032:analysis; D006442:Glycated Hemoglobin / Q000032:analysis*; D006461:Hemolysis; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D013056:Spectrophotometry, Ultraviolet
D005662:Furaldehyde; D006442:Glycated Hemoglobin; C008046:5-hydroxymethylfurfural