Abstract— The aim of the study was to determine if fluo- rescence chromatography can be used to measure modified beta-2-microgobulin (B2M) from the spent dialysate. Amyloid B2M is the main pathogenic component of dialysis-related amyloidosis. This component is in our sphere of interest being one of the fluorescent advanced glycation end products (AGE). AGEs are potential uremic toxins that can cause amyloidosis and cardiovascular problems in chronic kidney failure pa- tients. Two haemodialysis patients with high levels of B2M were selected for this study. Their spent dialysate samples were collected 10 minutes after the start of the dialysis process and less hydrophilic compounds were concentrated using solid phase extraction (SPE) column. Sediment from the concentrate and spent dialysate were analysed with electrospray ionisation mass spectrometer (ESI-MS) MicrOTOF-Q II coupled to high pressure liquid chromatography (HPLC) Dionex UltiMate 3000 RS. The sediment was analysed with Poroshell 120 EC- C18 column and spent dialysate with Kinetex C18 100A col- umn. MagTran was used to interpret mass spectra. Brown coloured fluorescent sediment of the concentrate was identified as amyloid B2M on the basis of MS and fluores- cence spectra. AGE modified B2M was also found from spent dialysate. However the fluorescence intensity was very low compared to overall fluorescence of spent dialysate. In summary, the study revealed that the fluorescence of AGE modified B2M is possible to detect in spent dialysate. However, the measuring system needs high selectivity and sensitivity for detection due to low contribution of AGE modi- fied B2M to overall fluorescence. Keywords— Advanced glycation, beta-2-microgobulin, fluores- cence, mass spectrum, dialysate I. INTRODUCTION Advanced glycation end products (AGEs) are a hetero- geneous group of molecules that accumulate in uremic pa- tients’ plasma [1]. AGEs are formed when a carbonyl of a reducing sugar condenses with a reactive amino group in target protein [2]. Free AGEs probably originate mainly from the glycation of proteins and from food. AGEs accu- mulate as the result of decreased excretion and increased formation from oxidative stress. Accumulation of AGEs may be linked to the increased risk of cardiovascular dis- ease in patients with renal failure [3]. Also, AGEs can pre- dict diabetic microvascular complications [4]. To date, various AGEs have been identified e.g. N(6)- carboxymethyl lysine – the most used marker for AGEs in food analysis [5]. Our interest has captured beta-2- microglobulin (B2M) which can be advanced glycated. B2M is a polypeptide with a molecular weight of 11.8 kDa. It is present on the surface of most nucleated cells and in biological fluids [6]. The normal serum B2M concentration is 1.5–3 mg/l, and its average normal production rate has been estimated to be 2.4 mg/kg/day [7]. In patients with chronic kidney failure, B2M concentration increases as the glomerular filtration rate decreases. The accumulation of AGE modified B2M can lead to different health complica- tions like dialysis-related amyloidosis [2,8]. AGEs are classified as being potential uremic toxins [9], therefore removal of AGEs can be used as an indicator of the dialysis therapy. B2M could be a marker to describe the elimination of middle molecular weight solutes [2] because levels of B2M are not affected by food intake [2]. As a group, AGEs are difficult to measure, but individual com- pounds have been analyzed. There is no universal method for AGE measurements. B2M is mostly measured via B2M human enzyme-linked immunosorbent assay (ELISA) kit or high pressure liquid chromatography (HPLC). In a health care setting these methods are expensive and the results cannot be used to modify an ongoing dialysis process. An online measuring method would be advantageous. Fluores- cence detector is one device that could be used in online measurements. The aim of the study was to determine if fluorescence chromatography can be used to measure AGE modified B2M from spent dialysate. II. MATERIALS AND METHOD This study was performed after approval of the protocol by the Regional Ethical Review Board, Estonia. An in- formed consent was obtained from all participating patients. Two haemodialysis patients with high levels of B2M were selected for this study. Their spent dialysate samples were collected from the drain tube of the dialysis machine 10 minutes after the start of the dialysis session. Less hy- drophilic compounds were concentrated from spent dialy- sate using C18 solid phase extraction (SPE) column. The concentration consisted of 5 phases: (1) column was washed Fluorescence of Beta-2-microglobulin in the Spent Dialysate S. Kalle, H. Kressa, R. Tanner, J. Holmar, and I. Fridolin Department of Biomedical Engineering, Technomedicum, Tallinn University of Technology, 19086, Ehitajate tee 5, Tallinn, Estonia © Springer International Publishing Switzerland 2015 H. Mindedal and M. Persson (eds.), 16th Nordic-Baltic Conference on Biomedical Engineering, 59 IFMBE Proceedings 48, DOI: 10.1007/978-3-319-12967-9_16