Further advances in identification of pentosan polysulfate monosaccharide composition by NMR.

Several publications have recently proposed NMR spectroscopy to evaluate the critical quality attributes (CQA) of pentosan polysulfate sodium (PPS), the active ingredient of Elmiron™ approved to treat interstitial cystitis. PPS is a polymer of sulfated ß(1-4)-d-xylopyranose residues randomly substituted by 4-O-methyl-glucopyranosyluronic acid, containing, beyond the main xylose-2,3-O-disulfate repetitive unit, some minor residues that can be marker of both the starting material and preparation process. In the present study we assigned some previously unknown cross-peaks in 1H-13C HSQC NMR of PPS related to its minor sequences adding additional details to its CQA. Four anomeric cross-peaks related to glucuronate-branched xylose and different sulfation pattern as well as the preceding xyloses were identified. Two minor process-related signals of monosulfated xyloses (unsubstituted in position 2 or 3) were also assigned. The isolation of a disaccharide fraction allowed the assignment of the reducing end xylose-α/ß as well as the preceding xylose residues to be corrected. Additionally, the oversulfation of PPS allowed detection of the reducing end xylose-tri-1,2,3-O-sulfate. The newly identified cross-peaks were integrated into an updated quantitative NMR method. Finally, we demonstrated that an in-depth PPS analysis can be obtained using NMR instruments at medium magnetic fields (500 MHz/600 MHz), commonly available in pharmaceutical industries.

Quantitative 2D 1H, 13C HSQC NMR Spectroscopy for the Determination of Chondroitin Sulfate and Dermatan Sulfate Content in Danaparoid Sodium.

OBJECTIVE: Danaparoid sodium is a biopolymeric complex drug composed of the most abundant heparan sulfate (HS) followed in descending order by dermatan sulfate (DS) and chondroitin sulfate (CS). This composite nature explains its peculiar antithrombotic and anticoagulant properties and make it particularly advantageous when the risk of heparin-induced thrombocytopenia occurs. A specific control of the danaparoid composition is required by the Ph. Eur. The monograph includes the CS and DS limit contents and describes the method for their quantification through selective enzymatic degradations. MATERIALS AND METHODS: In this study, a quantitative two-dimensional nuclear magnetic resonance (NMR) method is proposed as a new method suitable for CS and DS quantification. Statistical comparison of the results provided by the analysis of a series of danaparoid samples with both NMR and enzymatic methods highlights a small systematic difference, likely derived from lyase-resistant sequences bearing oxidized terminals. Some modified structures, whose survival to the enzymatic action was confirmed by mass spectrometry, can be detected and quantified by NMR. CONCLUSION AND RESULTS: The proposed NMR method can serve for the determination of DS and CS contents, is an easy-to-apply method with no dependence from enzymes and standards, and provides extensive structural information on the overall glycosaminoglycans mixture.

Saturated tetrasaccharide profile of enoxaparin. An additional piece to the heparin biosynthesis puzzle.

Enoxaparin, widely used antithrombotic drug, is a polydisperse glycosaminoglycan with highly microheterogeneous structure dictated by both parent heparin heterogeneity and depolymerization conditions. While the process-related modifications of internal and terminal sequences of enoxaparin have been extensively studied, very little is known about the authentic non-reducing ends (NRE). In the present study a multi-step isolation and thorough structural elucidation by NMR and LC/MS allowed to identify 16 saturated tetramers along with 23 unsaturated ones in the complex enoxaparin tetrasaccharide fraction. Altogether the elucidated structures represent a unique enoxaparin signature, whereas the composition of saturated tetramers provides a structural readout strictly related to the biosynthesis of parent heparin NRE. In particular, both glucuronic and iduronic acids were detected at the NRE of macromolecular heparin. The tetrasaccharides bearing glucosamine at the NRE are most likely associated with the heparanase hydrolytic action. High sulfation degree and 3-O-sulfation are characteristic for both types of NRE.

Characterization of Danaparoid Complex Extractive Drug by an Orthogonal Analytical Approach.

Danaparoid sodium salt, is the active component of ORGARAN, an anticoagulant and antithrombotic drug constituted of three glycosaminoglycans (GAGs) obtained from porcine intestinal mucosa extracts. Heparan sulfate is the major component, dermatan sulfate and chondroitin sulfate being the minor ones. Currently dermatan sulfate and chondroitin sulfate are quantified by UV detection of their unsaturated disaccharides obtained by enzymatic depolymerization. Due to the complexity of danaparoid biopolymers and the presence of shared components, an orthogonal approach has been applied using more advanced tools and methods. To integrate the analytical profile, 2D heteronuclear single quantum coherence (HSQC) NMR spectroscopy was applied and found effective to identify and quantify GAG component signals as well as those of some process signatures of danaparoid active pharmaceutical ingredient (API) batches. Analyses of components of both API samples and size separated fractions proceeded through the determination and distribution of the molecular weight (Mw) by high performance size exclusion chromatographic triple detector array (HP-SEC-TDA), chain mapping by LC/MS, and mono- (¹H and 13C) and bi-dimensional (HSQC) NMR spectroscopy. Finally, large scale chromatographic isolation and depolymerization of each GAG followed by LC/MS and 2D-NMR analysis, allowed the sequences to be defined and components to be evaluated of each GAG including oxidized residues of hexosamines and uronic acids at the reducing ends.

Structural peculiarity and antithrombin binding region profile of mucosal bovine and porcine heparins.

The major compositional differences between bovine mucosal heparin (BMH) and the currently employed porcine mucosal heparin (PMH) have been reported to essentially consist of reduced 6-O-sulfation of the glucosamine residues in BMH and somewhat lower 2-O-sulfation of the iduronate residues in PMH. The present work is based on direct comparison of several BMH and PMH commercial preparations. A combined study by 2D (heteronuclear single quantum coherence, HSQC) NMR and ion-pair reversed-phase high performance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) on the heparins, extended to the analysis of their heparinases digests and fractions separated by affinity chromatography on antithrombin (AT), confirmed the previously reported lower degree of 6-O-sulfation and showed lower 3-O-sulfated glucosamine content in BMH. More detailed studies allowed the identification of structural variants of AT-binding region (ATBR) structural variants, showing higher content of the N-sulfated components in BMH than in PMH.

Reversed-phase ion-pair ultra-high-performance-liquid chromatography-mass spectrometry for fingerprinting low-molecular-weight heparins.

Heparin is a complex mixture of sulfated linear carbohydrate polymers. It is widely used as an antithrombotic drug, though it has been shown to have a myriad of additional biological activities. Heparin is often partially depolymerized in order to decrease the average molecular weight, as it has been shown that low molecular weight heparins (LMWH) possess more desirable pharmacokinetic and pharmacodynamic properties than unfractionated heparin (UFH). Due to the prevalence of LMWHs in the market and the emerging availability of generic LMWH products, it is important that analytical methods be developed to ensure the drug quality. This work explores the use of tributylamine (TrBA), dibutylamine (DBA), and pentylamine (PTA) as ion-pairing reagents in conjunction with acetonitrile and methanol modified mobile phases for reversed-phase ion-pairing ultraperformance liquid chromatography coupled to mass spectrometry (RPIP-UPLC-MS) for fingerprint analysis of LMWH preparations. RPIP-UPLC-MS fingerprints are presented and compared for tinzaparinand enoxaparin.

How to find a needle (or anything else) in a haystack: two-dimensional correlation spectroscopy-filtering with iterative random sampling applied to pharmaceutical heparin.

Risks of contamination of the major clinical anticoagulant heparin can arise from deliberate adulteration with unnatural or natural polysaccharides, including heparin from other animal sources, other natural products, or artifacts of manufacture, and these can escape detection by conventional means. Currently, there is no generally applicable, objective test recommended by regulators that can detect these in pharmaceutical heparin, and this continues to leave heparin exposed to contamination risks. Two-dimensional correlation spectroscopic-filtering with iterative random sampling (2D-COS-firs) is reported. It employs a difference covariance matrix with iterative random sampling, and is capable of revealing contamination in pharmaceutical heparin to a high level of sensitivity irrespective of the nature of those features. The technique is suitable to any situation in which a comparison of a single entity to a family of heterogeneous entities, particularly natural products and biosimilars, needs to be made, and will find application in pharmaceutical monitoring, manufacturing quality control, materials science, biotechnology, and metabolomic investigations.