Development of molecularly imprinted chromatographic column material for glycosaminoglycan analysis

Proteoglycans which are among the most abundant molecules of the inter-cellular structure contain one or more glycosaminoglycan (GAG) chains that are covalently attached to the core protein. The levels of GAG excreted via urine is used as a marker to monitor and detect lysosomal storage diseases, especially mucopolysaccharidoses. Recently, studies that report GAG as a useful biomarker in different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes etc.) have been published. However, GAG analysis is not simple due to the high polarity and microheterogenity of GAG structure. Therefore there is a need to develop new, specific and easily applicable methods for GAG analysis. In this study, determination of biologically important species of glycosaminoglycans, heparan sulfate, chondroitin sulfate and dermatan sulfate determination was carried out by an improved material for chromatographic column separation. First of all, these three glycosaminoglycans were chemically broken into fragments, afterwards these fragments were characterized by mass spectrometry, which were Multiple Reaction Monitoring (MRM) and Surface Induced Reaction (SIR) were evaluated, specifically, then a library was created from obtained fragments. Polymers based on these measurements have been characterized. Molecularly imprinted polymer material can only exhibiting selectivity against these three molecules to glycosaminoglycans negatively charged sulfate groups of those showing non-ionic interest was derived from acrylamide monomer. The polymers prepared by the precipitation polymerization technique as a molecular imprinting technique obtained by thermal polymerization. By using this technique, the resulting polymers were filled to steel HPLC column, after quenched washing, and after the removal of glycosaminoglycans from these imprinted polymers, the sample application were characterized based on the retention capacity. According to different retention times, the characterization was carried out by considering these results. Standard graphics creation was carried out in the serial dilution at 1000ng / ml the highest level. Glycosaminoglycan detection in urine was also carried out and to increase the accuracy 500 ng/mL GAGs were added to each urine samples. In studies with non-imprinted polymers, glycosaminoglycan selectivity was not shown. By performing this assay development to design a chromatographic column solid state material on the selection of discharged GAGs in urine was performed more accurate, faster and more economical. This new methodological approach prepared by selective polymers for GAGs, GAG molecules in urine samples analyzed more accurately and faster and they will be used in chromatographic separation in an economical manner. Thus, we developed a new and easy method of urine GAG analysis and monitoring of of these diseases associated with GAG determination by polymers overcoming limitations/shortcomings and they could be used in routine chromatographic column packing material.