Sulodexide Structure Analyzed Using MS-Based System

Soochow University scientists based in Jiangsu, China recently developed a new mass spectrometry (MS)-based system for better understanding the structure of Sulodexide, a drug meant for treating conditions like diabetic nephropathy. Their findings were published in the Journal of Chromatography A (1).

scientist holds an amber vial | Image Credit: © bigy9950 - stock.adobe.com

Cell-to-cell communication is when a cell gives and receives messages with itself and its environment (2). Cells survival depends on receiving and processing information from the outside environment. Cell-to-cell communication is vital for the development and repair in multicellular organisms. Glycosaminoglycan (GAG), a family of polysaccharides, are integral components of the extracellular matrix, plays a critical role in this process. GAGs, which include hyaluronic acid (HA), heparin (Hp), heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS) and keratan sulfate (KS), are highly negatively charged polysaccharides consisting of repeating disaccharide units of a hexuronic acid or a galactose residue linked to a hexosamine residue. They show many activities, including anti-inflammatory, anti-angiogenesis, anti-tumor and anti-tumor metastasis, with several important medicines being developed based on GAGs. One such medicine is Sulodexide, which can help treat diabetic nephropathy by reducing urinary albumin excretion rates in patients with type 1 and type 2 diabetes (3). Further, it is also used in clinic for prophylaxis and treating thromboembolic diseases.

Sulodexide’s structure is not well understood. It consists of polysaccharides characterized by differing sugar compositions, linkages, and sulfonation patterns; despite this, it shares common features, such as strong hydrophilicity, high native charges, and considerable polydispersity. This poses challenges when trying to characterize the drug using conventional chromatographic and online mass spectrometry (MS) characterization.

For this study, the scientists developed a novel analytical method, which combined multiple-heart cut two-dimensional liquid chromatography (2D-LC) and in-source acid-induced dissociation. With this approach, three polysaccharides in Sulodexide were separated via highly efficient strong-anion-exchange (SAX) chromatography, which is an FDA-authorized quality control method, typically used for detecting oversulfated chondroitin sulfate (OSCS) in pharmaceutical heparin since 2009. This was followed by desalting prior to MS with second-dimensional size-exclusion chromatography. Further, a multiple heart cut (MHC) 2D-LC system was employed alongside this system. Online analysis was conducted using a novel MS strategy that employed inAID techniques, which led to the initial identification of Sulodexide polysaccharide components.

With the system’s utilization of SAX enhanced with finely packed particles, a more convenient, robust, and specific alternative to previous electrophoresis methods was created. This allowed for deeper understanding of this complex medicine. When integrating on-line high-resolution MS with high energy inAID, direct analysis of the three polysaccharides in Sulodexide, HS-like, Hp-like, and DS, was enabled. The fragmentation patterns of commercial HS and Hp presented in inAID MS spectra are different based on the degree of sulfonation and N-acetylation. However, HS-like and Hp-like components from Sulodexide were indistinguishable from each other in their spectra. This was because the substance of HS-like has slightly higher degree of sulfonation and lower degree of N-acetylation than the commercial HS, and Hp-like has slightly lower degree of sulfonation and higher degree of N-acetylation than the commercial Hp. As for the DS component from Sulodexide, this was well-separated by SAX and characterized by online inAID MS.

With these findings, the scientists created a novel approach for analyzing heparinoid medicines like Sulodexide. This technique can allow a streamlined, automatic, and robust means for analyzing highly sulfated polysaccharides with enhanced resolution and specificity.

References

(1) Wei, Y.; Zhu, W.; Tian, H.; Liu, J.; et al. Structural Elucidation of Sulodexide with Multidimensional Chromatography and Online In-Source Acid-Induced Dissociation Mass Spectrometry. J. Chromatogr. A 2024, 1733, 465242. DOI: 10.1016/j.chroma.2024.465242

(2) Neitzel, J.; Rasband, M. Cell Communication. Nature Education 2014. https://www.nature.com/scitable/topic/cell-communication-14122659/ (accessed 2024-8-22)

(3) Sulodexide. Elsevier B.V. 2024. https://www.sciencedirect.com/topics/neuroscience/sulodexide (accessed 2024-8-22)