Method for the Analysis of Glycosaminoglycans, and Their Derivatives and Salts by Nuclear Magnetic Resonance

5) The method of any one of the above claims further comprising:

• a) comparing the intensities of said anomeric or target 1H chemical shift signals to the intensity of the reference 1H chemical shift signal;
• b) comparing the intensities of said anomeric or target 13C chemical shift signals to the intensity of the reference 13C chemical shift signal;
• or c) a combination of a) and b).

6) The method of any one of the above claims further comprising:

• a) obtaining the integrals for said anomeric or target 1H chemical shift signals and said reference 1H chemical shift signal;
• b) obtaining the integrals for said anomeric or target 13C chemical shift signals and said reference 13C chemical shift signal;
• or c) a combination of a) and b).

9) The method of any one of the above claims further comprising:

• a) correlating the relative proportions of said anomeric or target hydrogens with the relative proportions of corresponding saccharides present in said glycosaminoglycan;
• b) correlating the relative proportions of said anomeric or target carbons with the relative proportions of corresponding saccharides present in said glycosaminoglycan;
• or c) a combination of a) or b).

11) The method of any one of the above claims further comprising providing a reference 1H NMR spectrum and/or reference 13C NMR spectrum for each of said plural saccharides being quantified.

12) The method of any one of the above claims further comprising providing a reference 1H-NMR spectrum and/or reference 13C-NMR spectrum for said at least one glycosaminoglycan.

13) The method of any one of the above claims further comprising:

• a) determining the relative proportions of anomeric or target hydrogens in one or more reference glycosaminoglycans;
• b) determining the relative proportions of anomeric or target carbons in one or more reference glycosaminoglycans;
• or c) a combination of a) or b).

15) The method of any one of the above claims, wherein said concentration dependent intensity is substantially linear in the range of about 0.2 mM to about 2.5 mM of said at least one reference compound.

17) The method of any one of the above claims further comprising using the singlet 1H-NMR or 13C-NMR chemical shift of 3-(trimethylsilyl)-priopionic-D4 acid as a chemical shift reference for 0 ppm.

18) The method of any one of the above claims, wherein the two-dimensional 1H-13C-NMR analysis is heteronuclear single-quantum correlation (HSQC).

23) The invention of any one of the above claims, wherein said glycosaminoglycan is an oligosaccharide or polysaccharide.

24) The invention of any one of the above claims, wherein said deuterated solvent is selected from the group consisting of D2O, any solvent that will solubilize the at least one GAG and the at least one reference compound, and a combination of D2O and said any solvent.

25) The invention of any one of the above claims, wherein the at least one saccharide is selected from the group consisting of 4,5-unsaturated 2-O sulfo-uronic acid (ΔU2S), 4,5-unsaturated uronic acid (ΔU), 2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A), 2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M), 2-N-sulfo-6-O-sulfoglucosamine (ANS6S), 2,5-anhydro mannitol, N-sulfoglucosamine, glucuronic acid, N-sulfo-6-O-sulfoglucosamine, 2-O-sulfoiduronic acid, iduronic acid, N-sulfo-3-O-sulfoglucosamine, N-sulfo-3,6-di-O-sulfoglucosamine, galacturonic acid, Xylose, N-acetylglucosamine and N-acetyl-6-O-sulfoglucosamine.

26) The invention of any one of the above claims, wherein said at least one reference compound is present at a concentration in the range of about 0.2 mM to about 2.5 mM.

27) The invention of any one of the above claims, wherein said reference 1H-NMR chemical shift signal is outside the range of 3.2 to 6 ppm.

28) The invention of any one of the above claims, wherein said reference 13C-NMR chemical shift signal is outside the range of 55 to 115 ppm and outside the range of 22-24 ppm.

29) The invention of any one of the above claims, wherein said reference 1H-NMR chemical shift signal is a singlet within the range of about 1.2 ppm to about 1.6 ppm inclusive of the range limits.

30) The invention of any one of the above claims, wherein said reference 13C-NMR chemical shift signal is a singlet within the range of about 25 ppm to about 27 ppm inclusive of the range limits.

31) The invention of any one of the above claims, wherein said reference 1H-NMR chemical shift is relative to the singlet chemical shift of 3-(trimethylsilyl)-priopionic-D4 acid assigned as 0 ppm.

32) The invention of any one of the above claims, wherein said reference 13C-NMR chemical shift is relative to the singlet chemical shift of 3-(trimethylsilyl)-priopionic-D4 acid assigned as 0 ppm.

33) The invention of any one of the above claims, wherein said at least one reference compound is dimethylmalonic acid.

34) The invention according to any one of the above claims, wherein said at least one reference compound is present at a known or predetermined concentration or amount.

35) The invention according to any one of the above claims, wherein said at least one glycosaminoglycan is present at a known or predetermined concentration or amount or at a concentration in the range of about 0.02 to about 0.2 mg/μL.

36) The invention according to any one of the above claims, wherein said at least one glycosaminoglycan is selected from the group consisting of heparin, heparan, enoxaparin, bemiparin, dalteparin, tinzaparin, a salt of any of the preceding, a derivative of any of the preceding, a sulfated or non-sulfated form of any of the preceding, an ultra-low molecular weight form of any of the preceding, a low molecular weight form of any of the preceding, a high molecular weight form of any of the preceding, an unfractionated form of any of the preceding, a fractionated form of any of the preceding, and a combination of any thereof.