Basic Information
Poster Number: 860-3    
Author Name: Neil D Danielson Affiliation: Miami University
Session Title: Pharmaceutical LC, HILIC and GPC I
Event Type: Poster
Event Title: Spectroscopic Visualization Approaches for Chromatographic Detection of Glycoaminoglycans
Presider(s):   Start Time: ( Slot # 3 )
Date: Monday, March 12th, 2012 Location: Red Area on Exposition Floor, Aisles 1300-1500
Keywords: Flow Injection Analysis, HPLC Detection, Spectrophotometry

Co-Authors
NameAffiliation
Loegel, Thomas NMiami University
Santiago, StevenMiami University

Abstract Content
Glycoaminoglycans (GAGs) are long, unbranched molecules containing repeating sulfonated disaccharide units; heparin used pharmaceutically as an anticoagulant is one of the most important. In the last several years, GAGs have become of analytical interest due to the problems associated with contaminated batches of imported heparin. Due to the lack of an easily detectable chromophore in the GAG structure, current HPLC detection approaches are based on either low UV wavelength or electrochemical detection. We have explored the use of dyes such as methylene blue (MB) and acridine for respectively selective colorimetric and fluorometric visualization of heparin. The response of acridine and related dyes such as lucigenin for heparin is not favorable, showing modest quenching from 0 – 10 uM. In contrast, we have confirmed that MB shows a strong decrease in absorbance at 660 nm for the same heparin molarity range and also an increase in absorbance at 560 nm before leveling out. Analogous absorbance changes at the same wavelengths for dermatan sulfate are also found but the sensitivity is less. To understand the compatibility of this detection scheme for HPLC, the stability and retention of the MB-heparin and MB-dermatan sulfate complexes on a reversed phase HPLC column are studied. Using 50 uM MB, linearity in the decrease in peak area response of both complexes from 0 – 7 uM at 660 nm on a PRP-X100 polymeric column using a 70% methanol-30% water mobile phase is linear. Unexpectedly, a positive linear response from 7- 10 uM is noted before the response levels out at higher concentrations. Linearity can be extended to about 40 uM heparin using a 250 uM MB solution. A shift to longer retention time as expected is noted for the MB-heparin complex peak using a 50% methanol-50% water mobile phase. Optimization of the mobile phase by use of a gradient is in progress to gain separation of different GAGs.