Method for the analysis of glycosaminoglycans, and their derivatives and salts by nuclear magnetic resonance

Abstract

An analytical method employing nuclear magnetic resonance of glycosaminoglycans in general, and of heparins and low molecular weight heparins and their derivatives in particular, is provided. The method is used for identification and the relative quantification of characteristic signals by .sup.1H-NMR and/or .sup.1H-.sup.13C HSQC.

Claims

1. A composition comprising: at least one glycosaminoglycan comprising at least one saccharide comprising anomeric or target hydrogen exhibiting respective anomeric or target .sup.1H chemical shift signal in the range of about 3.2 ppm to about 6 ppm when analyzed by NMR; at least one reference compound comprising at least one reference hydrogen atom having a NMR signal t1 longitudinal relaxation time of about 1 s or less, wherein the at least one reference hydrogen exhibits a reference .sup.1H NMR chemical shift signal separated from said anomeric or target .sup.1H chemical shift signals, and said reference .sup.1H chemical shift signal exhibits a concentration dependent signal intensity.

2. The composition of claim 1, wherein said reference .sup.1H NMR chemical shift signal is in the range of about 1.2 to about 1.6 ppm.

3. The composition of claim 1, wherein said glycosaminoglycan is an oligosaccharide or polysaccharide.

4. The composition of claim 1, wherein the at least one saccharide is selected from the group consisting of 4,5-unsaturated 2-O sulfo-uronic acid (U2S), 4,5-unsaturated uronic acid (U), 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.

5. The composition of claim 1, 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.

6. The composition of claim 1, wherein said reference .sup.1H-NMR chemical shift signal is outside the range of 3.2 to 6 ppm.

7. The composition of claim 1, wherein said reference .sup.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.

8. The composition of claim 1, wherein said reference .sup.1H-NMR chemical shift is relative to the singlet chemical shift of 3-(trimethylsilyl)-priopionic-D4 acid assigned as 0 ppm.

9. The composition of claim 1, wherein said at least one reference compound is dimethylmalonic acid.

10. The composition of claim 1, wherein said at least one reference compound is present at a known or predetermined concentration or amount.

11. The composition of claim 1, 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/L.

12. The composition of claim 1, 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.

13. The composition of claim 1 further comprising a chemical shift reference standard defining 0 ppm.

14. The composition of claim 13 further comprising at least one deuterated solvent for said at least one glycosaminoglycan, said at least one reference compound, and said chemical shift reference standard.

15. The composition of claim 14, wherein said deuterated solvent is selected from the group consisting of D.sub.2O, any solvent that will solubilize the at least one GAG and the at least one reference compound, and a combination of D.sub.2O and said any solvent.

16. A composition comprising: at least one glycosaminoglycan comprising at least one saccharide comprising anomeric or target carbon exhibiting respective anomeric or target .sup.13C chemical shift signal in the range of about 55 ppm to about 115 ppm when analyzed by NMR; at least one reference compound comprising at least one reference carbon atom that exhibits a reference .sup.13C NMR chemical shift signal separated from said anomeric or target .sup.13C chemical shift signal, and said reference .sup.13C chemical shift signal exhibits a concentration dependent signal intensity.

17. The composition of claim 16, wherein said reference .sup.13C NMR chemical shift signal is in the range of about 25-27 ppm.

18. The composition of claim 16, wherein said reference .sup.13C-NMR chemical shift signal is outside the range of 55 to 115 ppm and outside the range of 22-24 ppm.

19. The composition of claim 16, wherein said reference .sup.13C-NMR chemical shift signal is a singlet within the range of about 25 ppm to about 27 ppm inclusive of the range limits.

20. The composition of claim 16, wherein said reference .sup.13C-NMR chemical shift is relative to the singlet chemical shift of 3-(trimethylsilyl)-priopionic-D4 acid assigned as 0 ppm.

21. The composition of claim 16 further comprising a chemical shift reference standard defining 0 ppm.

22. The composition of claim 21 further comprising at least one deuterated solvent for said at least one glycosaminoglycan, said at least one reference compound, and said chemical shift reference standard.

23. The composition of claim 22, wherein said deuterated solvent is selected from the group consisting of D.sub.2O, any solvent that will solubilize the at least one GAG and the at least one reference compound, and a combination of D.sub.2O and said any solvent.

24. A composition comprising at least one glycosaminoglycan comprising at least one saccharide comprising a) anomeric or target hydrogen exhibiting respective anomeric or target .sup.1H chemical shift signal in the range of about 3.2 ppm to about 6 ppm when analyzed by NMR; or b) anomeric or target carbon exhibiting respective anomeric or target .sup.13C chemical shift signal in the range of about 55 ppm to about 115 ppm when analyzed by NMR; wherein said glycosaminoglycan is an oligosaccharide or polysaccharide; and dimethylmalonic acid.

25. The composition of claim 24 further comprising a chemical shift reference standard defining 0 ppm.

26. The composition of claim 25 further comprising at least one deuterated solvent for said at least one glycosaminoglycan, said at least one reference compound, and said chemical shift reference standard.

27. The composition of claim 24, wherein said at least one saccharide is selected from the group consisting of 4,5-unsaturated 2-O sulfo-uronic acid (U2S), 4,5-unsaturated uronic acid (U), 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.

28. The composition of claim 24, 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; and said at least one saccharide is selected from the group consisting of 4,5-unsaturated 2-O sulfo-uronic acid (U2S), 4,5-unsaturated uronic acid (U), 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; wherein said glycosaminoglycan is an oligosaccharide or polysaccharide; and said composition further comprises: dimethylmalonic acid; 3-(trimethylsilyl)-priopionic-D4 acid; and at least one deuterated solvent for said at least one glycosaminoglycan, dimethylmalonic acid, and 3-(trimethylsilyl)-priopionic-D4 acid.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Diagram of the biosynthetic process of heparin.

(2) FIG. 2: Specificity. .sup.1H-NMR spectra: a) SEL-I-D2O: spectrum of D.sub.2O solvent; b) SEL-II-D2O-TSP: spectrum of combination of D.sub.2O solvent and TSP; c) SEL-III-DMMA-D2O: spectrum of combination of D.sub.2O solvent and DMMA; d) SEL-IV-ENOX-DMMA-D2O: spectrum of combination of enoxaparin, D.sub.2O solvent and DMMA.

(3) FIG. 3: Specificity. .sup.1H-.sup.13C HSQC spectra of SEL-IV-ENOX-DMMA-D2O. The spectrum was obtained under the following conditions: Temperature: 298.0 K; number of scans: 12; receiver gain: 2050.0; relaxation delay: 1.8; pulse width: 11.11; acquisition time: 0.1068 s; JCH: 170; spectrometer frequency: 800.13, 201.49; spectral width (ppm): 4795.4, 24154.6; lowest frequency: 447.9, 575.7.

(4) FIG. 4: .sup.1H-NMR linearity. Graph depicting the linearity of concentration (mM) of DMMA (variable X) versus the integral (variable Y) of its .sup.1H chemical shift signal. Under the conditions of the assay employed, the linearity is defined by the following equation: y=1,136,561.5669x42,152.0764 with an R.sup.2 value of 0.9985.

(5) FIG. 5: .sup.1H-.sup.13C HSQC linearity. Graph depicting the linearity of concentration (mM) of DMMA (variable X) versus the integral (variable Y) of its .sup.1H chemical shift signal. Under the conditions of the assay employed, the linearity is defined by the following equation: y=59,069,197.0868x4,422,535.0841, with an R.sup.2 value of 0.9966

(6) FIG. 6: .sup.1H-NMR spectrum of enoxaparin sodium. The spectrum was obtained under the following conditions: Temperature: 298.0 K; number of scans: 12; receiver gain: 2.0; relaxation delay: 13.5; pulse width: 10.69; acquisition time: 6.8157 s; JCH: 1; spectrometer frequency: 800.13; spectral width (ppm): 12.0.

(7) FIG. 7: .sup.1H-.sup.13C HSQC spectrum of enoxaparin sodium. The spectrum was obtained under the following conditions: Temperature: 298.0 K; number of scans: 12; receiver gain: 2050.0; relaxation delay: 1.8; pulse width: 10.69; acquisition time: 0.1068 s; JCH: 170; spectrometer frequency: 800.13, 201.19; spectral width: 4795.4, 24154.6; lowest frequency: 447.9, 575.7.

ABBREVIATIONS AND ACRONYMS

(8) The following abbreviations and acronyms are used in the present specification: NMR: Nuclear magnetic resonance HSQC: Heteronuclear Single-Quantum Correlation GAG: Glycosaminoglycan UFH: Unfractionated heparin LMWH: Low Molecular Weight Heparin ULMWH: Ultra Low Molecular Weight Heparin Da: Dalton U2S: 4,5-unsaturated 2-O sulfo uronic acid U: 4,5-unsaturated uronic acid 1,6-an. A: 2-N-sulfo-1,6-anhydroglucosamine 1,6-an.M: 2-N-sulfo-1,6-anhydro-mannosamine TOCSY: TOtal Correlation SpectroscopY TSP: Sodium salt of 3-(Trimethylsilyl)-Propionic-D4 acid DMMA: Dimethylmalonic acid, derivative thereof, and or salt thereof MHz: Megahertz ppm: parts per million : chemical shift SW: Spectral width TD: Time domain T1: Longitudinal relaxation time ANS: N-sulfoglucosamine G: Glucuronic acid ANS6S: N-sulfo-6-O-sulfoglucosamine I2S: 2-O-sulfoiduronic acid I: duronic acid ANS3S: N-sulfo-3-O-sulfoglucosamine Gal: Galacturonic acid Xyl: Xylose ANAc: N-acetylglucosamine A6S: 6-O-sulfoglucosamine A6OH: Glucosamine G2S: Sulfoglucuronic 2-O acid M: Mannosamine MNS6S: N-sulfo-6-O-sulfomannosamine Epox: Epoxide red: anomer red: anomer.

DETAILED DESCRIPTION OF THE INVENTION

(9) As used herein, monosaccharide residue present in heparin chains refers to all monosaccharide residues or components that are typically present in LMWH/UFH/GAG chains. These residues are generally selected from the group consisting of 4,5-unsaturated 2-O sulfo uronic acid (U2S), 4,5-unsaturated uronic acid (U), 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.

(10) The invention provides a composition for NMR analysis of GAG's. The composition comprises at least one GAG (comprising plural saccharide residues having respective anomeric or target hydrogens) and at least one internal reference compound having a T1 for hydrogen of about the T1 for the anomeric or target hydrogen of at least one said plural saccharide residues. The composition can further comprise a deuterated solvent in which said at least one GAG is at least partially soluble or is fully soluble and in which said at least one internal reference compound is at least partially soluble or is fully soluble.

(11) In some embodiments, the composition comprises at least one GAG, DMMA, and at least one deuterated solvent. The content of said components in the composition can be as described herein. The composition can be a sample composition.

(12) A GAG comprises plural saccharide residues or at least two different saccharide residues. A saccharide residue will usually comprise an anomeric and/or target hydrogen atom(s). Anomeric or target hydrogens typically exhibit a T1 of about 1 sec or 10.2 sec or about 1 sec or less, of about 1 sec. Anomeric hydrogens typically exhibit a chemical shift signal of about 4.6-6 ppm for .sup.1H-NMR. At least hydrogen atom(s) of the internal reference standard will exhibit a singlet chemical shift reference signal outside the range of about 4.6-6 ppm, and the intensity of said reference signal will be concentration dependent. At least hydrogen atom(s) of the internal reference standard can exhibit a singlet chemical shift reference signal in the range of about 1.2 to about 1.4 ppm for .sup.1H-NMR analysis and a singlet chemical shift signal within the range of about 26 ppm to about 27 ppm .sup.13C-NMR. The concentration versus intensity typically exhibits an approximately linear relationship when said reference standard is present in the composition at least in the concentration range of about 0.2 mM to about 2.5 mM, and the linearity of said relationship can also exist outside said concentration range. The linearity can have a correlation coefficient of 0.90, 0.95, 0.98, or 0.99. 3-(trimethylsilyl)-priopionic-D4 acid (deuterated TSP) can be used as an internal chemical shift signal reference for 0 ppm in .sup.1H-NMR. The concentration of total amount (concentration) of GAG, or at least one of its saccharide residues, is typically in the range of about 15 to about 700 mM, about 25 to about 600 mM, about 30 to about 500 mM, about 30 to about 400 mM, about 33 to about 333 mM, about 0.005 to about 1 mg/L, about 0.01 mg/L to about 0.5 mg/L, or about 0.02 to about 0.2 mg/L. At least one hydrogen of the internal reference standard will exhibit a T1 that approximates, is equal to, or is less than the T1 of the anomeric or target hydrogen of said saccharide residue(s), e.g. a T1 of about 1 sec or 10.2 sec or about 1 sec or less, of about 1 sec. Combinations of two or more deuterated solvents can be used. An aqueous deuterated solvent can be used, e.g. D.sub.2O or D.sub.2O in combination with one or more deuterated organic solvents. The solvent or combination of solvents used will dissolve at least a portion of the GAG or will dissolve all of the GAG in the composition. The free acid or salt form of DMMA can be used.

(13) The present invention provides a NMR method of determining the identity and relative content of individual saccharide residues forming a respective GAG. A NMR method of the invention is generally performed by: a) providing a sample composition comprising at least one GAG (comprising plural saccharide residues, each comprising at least one respective hydrogen and at least one respective carbon), at least one internal reference compound (as described herein for concentration standard), TSP (internal standard defining 0 ppm), and at least one deuterated solvent; b) conducting a 1D or 2D NMR analysis on said sample composition to obtain at least one reference chemical shift signal (for at least one hydrogen of said at least one internal reference and/or for at least one carbon of said at least one internal reference) and to obtain plural saccharide residue chemical shift signals (for said at least one respective hydrogens and/or for said at least one respective carbon of said respective said plural saccharide residue); c) normalizing said plural saccharide residue chemical shift signals relative to (with respect to) said at least one reference chemical shift signal(s); d) determining the relative percentage of individual ones of said plural saccharide residues in said at least one GAG; and e) correlating said plural saccharide residue chemical shift signals to known chemical shift signals for saccharide residue standards to determine the identity of individual ones of said plural saccharide residue. The above steps after b) need not be conducted sequentially, meaning steps c) through e) can be conducted in any order.

(14) The relative percentage of individual saccharide residues in a GAG can be determined according to the following formula:

(15) $\%\mathrm{signal}X=\frac{\mathrm{normalized}\mathrm{value}\mathrm{for}\mathrm{signal}X}{\mathrm{normalized}\mathrm{value}\mathrm{for}\mathrm{all}\mathrm{the}\mathrm{signals}}100.$
wherein:
X is the target hydrogen.
normalized value for signal X is the chemical shift signal intensity for X after said signal has been normalized relative to the reference chemical shift signal, and normalized value for all the signals is the sum total of all the normalized values.

(16) A chemical shift signal pattern (or NMR fingerprint), as used herein, refers to the set of the signals corresponding to the peaks found in a determined NMR spectrum, whether one-dimensional .sup.1H-NMR and/or two-dimensional .sup.1H-.sup.13C HSQC, or the absence thereof, in the relative proportions of its normalized integrals indicated by the parameter relative proportion (%). The chemical shift signal pattern can be a graphical spectrum (visual linear graph) or a numerical spectrum (group of values in a data set). The absence of particular chemical shift signals corresponds to the absence of respective saccharide residues and the presence of particular chemical shift signals corresponds to the presence of respective saccharide residues. The chemical shift signal pattern is used to identify, characterize, specify the saccharide composition of a corresponding GAG.

(17) The method and composition of the invention can be practiced with any GAG known to date and any as yet unknown GAG, meaning any GAG discovered or first prepared after the date of the present invention.

(18) Experimental Assays

(19) The quantitative NMR assays have been performed using a Bruker AVIII-600 o AVIII-800 nuclear magnetic resonance (NMR) spectrometer. Reagents used were deuterium oxide (D.sub.2O) 99.9%, sodium salt of 3-(Trimethylsilyl)-Propionic-D4 acid (TSP) and Dimethylmalonic acid (DMMA, standard for quantitative NMR, TraceCERT grade) as internal standard. a) Equipment Conditions Frequency: 1H: 600/800 MHz, 13C: 150,9/201.2 MHz Temperature: 298 K b) Acquisition Parameters (quantitative .sup.1H NMR) 90 pulse: it is determined from a qualitative 1H spectrum Acquisition window: SW=10-12 ppm/TD=64-128 k Inter scans delay d1 must fulfil the condition d1+AQ20 s No. of scans: 12 c) Acquisition Parameters (HSQC) 90 pulse: it is determined from a qualitative .sup.1H spectrum Acquisition window: SW2 (.sup.1H)=6 ppm/TD(F2)=1 k SW1 (.sup.13C)=120 ppm/TD(F1)=256384 Time between pulses d1=1.82 s No. of scans: 12 d) Processing Parameters (.sup.1H) Processing window: SI=64256 K Window function: None Phase adjustment: manual Baseline adjustment: automatic (abs) e) Processing Parameters (HSQC) Processing window: SI(F2)=2 k Processing function: QSINE, SSB=2 Phase adjustment: manual Baseline adjustment: automatic f) Preparation of the Sample: the Following Solutions were Prepared: Solution A (TSP) 1 mg/mL Solution B (D.sub.2O-TSP) 0.002 mg/mL: 40 L A (TSP)+19.96 mL D.sub.2O, Total volume=20 mL Solution C (DMMA) 1.2 mg dimethylmalonic/mL of D.sub.2O Test sample: 50 mg of product to study in 500 L of the solution B (D.sub.2O-TSP) and add 100 L of solution C (DMMA) and place in 5 mm-diameter NMR tube.

(20) The NMR tube containing the sample is introduced in the spectrometer. Then, the homogeneity of the magnetic field is adjusted and the harmony of the wave is optimized for the .sup.1H and .sup.13C nuclei. A qualitative .sup.1H spectrum is then performed, with parameters similar to the aforementioned, except the following: Time between pulses d1=12 s; No. of scans: 1-4.

(21) Then, the value of the 90 pulse is determined with automatic pulse program (TOPSPIN). Next, the quantitative .sup.1H spectrum is performed, with the parameters indicated in the analytical method and the 90 pulse value (P1) previously determined. After the HSQC spectrum is obtained with the aforementioned parameters. The spectra obtained are then processed according to the aforementioned parameters, taking as chemical shift reference, the TSP-d4 signal at 0 ppm.

(22) The dimethylmalonic acid reference chemical shift signals appear at the following approximate chemical shifts: .sup.1H NMR: singlet that appears at about 1.2 to about 1.5 ppm, about 1.3 to about 1.5 ppm, about 1.4 to about 1.5 ppm, about 1.3 ppm, and about 1.4 ppm; HSQC: signal at about 1.2 to about 1.5 ppm, about 1.3 to about 1.5 ppm, about 1.4 to about 1.5 ppm, about 1.3 ppm, and about 1.4 ppm (.sup.1H) and about 25 to about 27 ppm, about 26 ppm to about 27 ppm (.sup.13C).

(23) It should be understood that all chemical shift signals described herein are approximate and can vary slightly according to experimental conditions; however, said signals are obtained within the specified ranges when corresponding NMR analyses are conducted as described herein. Moreover, all ranges specified herein are inclusive of the range limits and all integer and fractional values therein especially as defined by the definition of the term about as used herein, and each said chemical shift is relative to TSP as the internal reference defining 0 ppm.

(24) The parameters assessed to determine validation of the method for quantitative NMR have been the following:

(25) Specificity

(26) This aspect of the method determines the capacity of the analytical method for measuring and/or identifying, simultaneously or separately, the analytes of interest unequivocally in presence of other chemical substances that may be present in the sample.

(27) The data obtained in the .sup.1H NMR analyses were as follows:

(28) TABLE-US-00002 Chemical shift, Sample Composition ppm I, solvent D.sub.2O 4.79 II, chemical shift reference D.sub.2O-TSP 0.00 III, internal standard D.sub.2O-DMMA 1.42 IV, GAG sample D.sub.2O-TSP- 1.8-8.0 DMMA-GAG

(29) The data obtained in the .sup.1H .sup.13C-HSQC analyses were as follows:

(30) TABLE-US-00003 Chemical shift Chemical shift Sample Composition .sup.1H, ppm .sup.13C, ppm III, internal standard D.sub.2O-DMMA 1.42 25.42 IV, sample: GAG D.sub.2O-TSP- 1.8-8.0 24-112 DMMA-GAG

(31) We found no interference between chemical shift signal for the DMMA and GAG in the .sup.1H NMR or .sup.1H .sup.13C-HSQC spectra (FIGS. 2 and 3). The data demonstrate that the method is capable of discriminating, without interference or undue chemical shift overlap, the chemical shift signals of the GAG from those of other products present in the sample such as the solvent (deuterium oxide, D.sub.2O), the internal standard (DMMA) and the chemical shift reference (TSP-d4).

(32) Limit of Quantification and Linearity

(33) Under these parameters, on the one hand, the minimum quantity of analyte that may be suitably quantified precisely and accurately is determined and, on the other hand, the capacity of the method to obtain results directly (by means of mathematical transformations) proportional to the concentration of the analyte in the sample, within an established interval.

(34) To assess the limit of quantification and the linearity (of chemical shift signal intensity dependence upon concentration of sample) of the integrals (for said chemical shift signal) of the DMMA, corresponding chemical shift signals were quantified. Solution comprising enoxaparin sodium (fixed concentration) and DMMA (seven different concentrations of DMMA) were prepared as follows: 0.2 mM of DMMA: 13.5% of the working concentration 0.3 Mm of DMMA: 20.3% of the working concentration 0.76 mM of DMMA: 50% of the working concentration 1.2 mM of DMMA: 80.1% of the working concentration 1.5 mM of DMMA: 100% of the working concentration 1.8 mM of DMMA: 120% of the working concentration 2.27 mM of DMMA: 150.2% of the working concentration.

(35) The acceptance criteria established to fulfil this linearity criterion is that in the line obtained the correlation coefficient for both experiments is 0.99. In FIGS. 4 and 5 depicts a graph of the DMMA signal integral values vs. DMMA concentration (mM), for the .sup.1H NMR and .sup.1H .sup.13C-HSQC spectra. The acceptance criterion is easily fulfilled both for one and the other.

(36) The lower limit of quantification for a DMMA is a concentration of about 0.20 mM or 20 mM. Thus, the signals of the samples studied with intensity less than the intensity of the DMMA signal corresponding to this concentration, cannot be suitably quantified and, therefore, they cannot be taken to determine the relative proportion of the residues present in the molecule. This means that the GAG, or at least one of its corresponding saccharide residues, should be present in the sample composition at a concentration of at least about 0.2 mM or 20 mM.

(37) Accuracy

(38) This aspect of the methods determines the proximity between the value which is accepted conventionally as true or reference value and the experimental value found. To calculate the experimental value of the concentration of the samples in analyte solution, you correlate the signal integral of the analyte to the signal integral of the DMMA, for example the equation defining the linearity (described above) of signal intensity versus concentration for DMMA can be used to determine the corresponding concentration of analyte in the sample.

(39) The accuracy is expressed as recovery percentage in the value of a known quantity of internal standard:

(40) $\mathrm{Recovery}\mathrm{percentage}\left(R\right)=\frac{\mathrm{Xm}}{}100$
Where: Xm is mean value found, and is the value accepted as true.

(41) The acceptance criteria established is that the recovery values are between 70.0-130.0% for the concentration corresponding to the concentration limit and 80.0-120.0% for the other levels.

(42) The data obtained for the .sup.1H NMR and HSQC experiments were the following: a) .sup.1H NMR

(43) TABLE-US-00004 Concentration, mM Conc. Calculated, mM Recovery, .sup.1H NMR 0.204 0.228 111.77 0.307 0.330 107.31 0.758 0.737 97.26 1.212 1.175 96.90 1.514 1.488 98.29 1.817 1.810 99.65 2.274 2.318 101.94 b) HSQC

(44) TABLE-US-00005 Concentration, mM Conc. Calculated, mM Recovery, HSQC 0.204 0.243 119.17 0.307 0.337 109.81 0.758 0.727 95.96 1.212 1.140 94.03 1.514 1.488 98.29 1.817 1.823 100.32 2.274 2.328 102.37

(45) Both the .sup.1H NMR and HSQC methods provide accuracy in compliance with the acceptance criteria for the accuracy parameters for those signals corresponding to the sample, with intensity higher than that of the limit of quantification.

(46) PrecisionRepeatabilityReproducibility

(47) The intra-sample variability of the method is studied by performing a series of analyses on the same sample in the same operating conditions in a same laboratory and in a short period of time.

(48) To do this, three consecutive analyses were performed for each concentration. The repeatability of a method is expressed as the coefficient of variation (CV) of a series of measurements and is mathematically calculated as follows:

(49) $\mathrm{CV}\left(\%\right)=\frac{s}{X}100,$
where: s is standard deviation, and X is arithmetic means of the results.

(50) The acceptance criterion established to fulfill these accuracy criteria is a coefficient of variation for all levels of 7%.

(51) The data obtained for both experiments was the following:

(52) TABLE-US-00006 Concentration, mM CV, % .sup.1H NMR CV, % HSQC 0.204 5.55 3.40 0.307 0.09 2.29 0.758 1.62 1.45 1.212 1.74 2.52 1.514 1.41 1.01 1.817 2.16 3.99 2.274 2.30 2.73

(53) Both the .sup.1H NMR and HSQC methods provide reproducibility in compliance with the acceptance criterion for the accuracy parameters for those signals corresponding to the sample, with intensity higher than that of the limit of quantification.

EXAMPLES

(54) The following specific examples provided below serve to illustrate the nature of the present invention. These examples are included only for illustration purposes and are not to be deemed to limit the invention to just said exemplary embodiments. The invention is defined by the claims, drawings, abstract and entire specification.

Example 1

.SUP.1.H NMR of Enoxaparin Sodium

(55) Enoxaparin sodium (50 mg) are dissolved in 500 L of a D.sub.2O-TSP (solution B) solution. Then 100 L of DMMA solution (solution C) are added. The resulting solution is introduced in a 5 mm diameter tube.

(56) The resulting DMMA concentration in the solution is 1.5 mM. The experiments are performed on a Bruker AVIII-800 nuclear magnetic resonance spectrometer. The main signals identified are as follows:

(57) TABLE-US-00007 Signal Chemical shift, ppm H4 U2S 5.992 H4 U2 5.825 H1 1,6-AnA 5.616 H1 ANS(-G) 5.585 H1 1,6-AnM 5.569 H1 U2S 5.509 H1 ANS6S 5.405 H1 I2S 5.228 H1 I 5.012 H5 I2S 4.836 H1 G 4.628 H6 ANS6S 4.344 H6 ANS6S 4.210 H3 ANS 3.670 H2 ANS3S 3.395 H2 ANS 3.293 NAc 2.047 DMMA 1.320 TSP 0.069

(58) Once the values of the integrals of the signals both of DMMA and the rest of the residues have been obtained, the normalized values are obtained of said residues dividing the value of its integrals by the value of the integral corresponding to the DMMA signal. This normalization can be performed, because the concentration of the internal standard is kept constant with respect to the saccharide residue concentration for all experiments, thus avoiding the inter-experimental variability that may arise in the analysis of a series of several product batches.

(59) Once the normalized values of the residues have been obtained, the relative percentage of each one of them is calculated in accordance with the following formula:

(60) $\%\mathrm{signal}X=\frac{\mathrm{normalized}\mathrm{value}\mathrm{signal}X}{\mathrm{normalized}\mathrm{value}\mathrm{all}\mathrm{the}\mathrm{signals}}100$

(61) To clarify the steps performed, the results obtained are shown for a series of four samples M1, M2, M3 and M4 of enoxaparin sodium. The following integral values of each one of the chemical shift signals selected were obtained.

(62) TABLE-US-00008 Integral Signal M 1 M 2 M 3 M 4 H4 U2S 2767732.83 2988384.02 3075705.31 2332763.55 H4 U 114294.94 135658.19 143037.09 94201.86 H1 1,6-an.A 404060.73 472871.53 509930.86 351978.17 H1 1,6-an.M 2535227.20 2700027.94 2844751.53 2160178.72 H1 U2S 3541377.06 3818336.16 3951288.34 2999870.34 H1 ANS6S 10350026.69 10716882.48 10780945.33 8442504.14 H1 I2S 8922576.12 9202191.38 9486264.66 7346708.47 H5 I2S 8924825.12 9540022.53 10041127.64 7751650.03 H2 ANS 16351247.22 16802874.61 17051664.14 13189968.22 NAc 8054931.36 7973188.48 8124533.56 6388424.34 DMMA 1464255.27 1415020.17 1485242.95 1279612.94
where M corresponds to sample.

(63) To obtain the normalized values of the integrals of the chemical shift signals, their respective integrals are divided by the integral of the DMMA (present at a known or determined concentration):

(64) TABLE-US-00009 Normalized integral Signal M 1 M 2 M 3 M 4 H4 U2S 1.890 2.112 2.071 1.823 H4 U 0.078 0.096 0.096 0.074 H1 1,6-an.A 0.276 0.334 0.343 0.275 H1 1,6-an.M 1.731 1.908 1.915 1.688 H1 U2S 2.419 2.698 2.660 2.344 H1 ANS6S 7.068 7.574 7.259 6.598 H1 I2S 6.094 6.503 6.387 5.741 H5 I2S 6.095 6.742 6.761 6.058 H2 ANS 11.167 11.875 11.481 10.308 NAc 5.501 5.635 5.470 4.992 DMMA 1.000 1.000 1.000 1.000

(65) From these normalized values, the relative proportion of each one of the chemical shift signals is calculated with respect to the sum of all the normalized chemical shift signals.

(66) TABLE-US-00010 Relative proportion, % Signal M 1 M 2 M 3 M 4 H4 U2S 4.47 4.64 4.66 4.57 H4 U 0.18 0.21 0.22 0.18 H1 1,6-an.A 0.65 0.73 0.77 0.69 H1 1,6-an.M 4.09 4.20 4.31 4.23 H1 U2S 5.72 5.93 5.99 5.88 H1 ANS6S 16.70 16.65 16.33 16.54 H1 I2S 14.40 14.30 14.37 14.39 H5 I2S 14.40 14.83 15.21 15.18 H2 ANS 26.39 26.11 25.83 25.83 NAc 13.00 12.39 12.31 12.51

(67) The quantification of the characteristic and well-differentiated signals of enoxaparin sodium (generally those corresponding to the anomeric or target protons, H1) are shown in the following table with the observed relative proportion values:

(68) TABLE-US-00011 Signal Chemical shift, ppm Relative proportion, % H4 U2S 5.99 4.3-4.7 H4 U 5.82 0.2 H1 1,6-an.A 5.62 0.7-0.9 H1 1,6-an.M 5.57 4.1-4.4 H1 U2S 5.51 5.7-6.0 H1 ANS6S 5.40 16.1-16.7 H1 I2S 5.23 13.1-14.4 H5 I2S 4.84 14.3-16.3 H2 ANS 3.29 24.3-26.6 NAc 2.05 12.0-15.3

(69) The set of the signals corresponding to the peaks found in a determined NMR spectrum, whether one-dimensional .sup.1H-NMR and/or two-dimensional .sup.1H-.sup.13C HSQC, or the absence thereof, in the relative proportions of its normalized integrals indicated by the parameter relative proportion (%) is what in the present specification is called signal pattern or simply pattern.

Example 2

(70) The same solution used in Example 1 is used to perform the study by .sup.1H-.sup.13C HSQC. The main signals identified are as follows:

(71) TABLE-US-00012 Signal ppm .sup.13C, ppm .sup.1H, ppm C4-H4 U 110.71 5.82 C4-H4 U2S 108.97 5.99 C1-H1 G // Gal 106.62 4.66 C1-H1 Xyl 105.79 4.45 C1-H1 G(-ANAc) 105.13 4.50 C1-H1 I(-A6S) 104.94 5.01 C1-H1 G(-ANS) 104.77 4.60 C1-H1 I(-A6OH) 104.67 4.94 C1-H1 Gal 104.30 4.54 C1-H1 1,6-an.A 104.22 5.61 C1-H1 G(-ANS3S) 103.91 4.61 C1-H1 1,6-an.M 103.91 5.57 C1-H1 U 103.88 5.16 C1-H1 G2S 102.99 4.75 C1-H1_I2S 102.09 5.22 C1-H1 I2S(-1,6-an.M) 101.59 5.36 C1-H1 ANS(-G) 100.50 5.58 C1-H1 ANAc 100.23 5.31 C1-H1 U2S 100.18 5.51 C1-H1 ANS(-I2S) 99.78 5.40 C1-H1 ANS6S 99.43 5.43 C1-H1 ANS,3S 99.06 5.51 C1-H1 ANS red 98.73 4.71 C1-H1 ANS(-I) 98.42 5.34 C1-H1 M red 95.74 5.39 C1-H1 I2S red 95.70 5.42 C1-H1 I2S red 94.76 4.97 C1-H1 ANS red// 93.97 5.45 ANS6S red C3-H3 Gal 85.45 3.78 C3-H3 Gal 84.85 3.83 C4-H4 ANS6S(-G)// 80.94 3.84 ANS6S red C2-H2 I2S 78.53 4.34 C3-H3 Xyl 77.82 3.72 C2-H2 U2S 77.42 4.62 C2-H2 G(-AN6S) 75.70 3.40 C3-H3 ANS6S (-G) 72.47 3.66 C3-H3 ANS6S red 72.29 3.77 C5-H5 I2S 72.01 4.83 C3-H3 I2S 71.87 4.21 C5-H5 ANS6S(-G) 71.72 4.09 C5-H5 MNS6S red 70.98 4.15 C5-H5 ANS6S red 70.64 4.12 C6-H6 1,6-an.A// 67.53 3.77 1,6-an.M C5-H5 Xyl 65.89 4.12 C5-H5 Xyl 65.86 3.40 C3-H3 U2S 65.75 4.32 C6-H6 Gal 63.90 3.74 C2-H2 ANS6S red// 60.82 3.28 ANS(-I2S) C2-H2 ANS6S(-G) 60.52 3.29 C2-H2 MNS6S red 60.38 3.60 C2-H2 1,6-an.A 58.50 3.21 C2-H2 ANAc 56.68 3.92 C2-H2 1,6-an.M 55.09 3.47 DMMA 26.73 1.32 NAc 24.87 2.05

(72) These signals are then correlated with monosaccharide components of the molecule, so that their quantification provides a chemical shift signal pattern representative of the monosaccharide content of the GAG.

(73) The integral for each one of these .sup.13C chemical shift signals was normalized from the value set for the integral of the reference .sup.13C chemical shift signal of DMMA, using the same process (calculations) described for the .sup.1H NMR experiments. The quantification of the characteristic signals of enoxaparin sodium is shown in the following table:

(74) TABLE-US-00013 Signal Relative proportion, % C1-H1 ANS-I2S 25.6-26.9 C1-H1 ANS-I 2.6-3.0 C1-H1 ANS-G 5.1-5.5 C1-H1 ANS.3S 1.5-1.7 C1-H1 ANAc 2.7-3.5 C1-H1 ANAc-red <LC C1-H1 ANS-red 3.8-4.9 C1-H1 1,6-an.A 1.2-1.5 C1-H1 1,6-an.M 1.6-1.9 C1-H1 MNS-red 1.0-1.3 C1-H1 I2S 24.5-27.5 C1-H1 I-A6S 2.4-2.7 C1-H1 I-A6OH 0.3-0.4 C1-H1 G-ANS.3S 1.4-1.6 C1-H1 G-ANS 4.2-4.4 C1-H1 G-ANAc 1.9-2.6 C1-H1 G2S 1.1-1.6 C1-H1 U2S 11.5-12.4 C1-H1 U 0.3-0.5 C1-H1 I2S-red 1.0-1.4 C5-H5 Gal-A <LC-0.5 Epox <LC-0.4

(75) These experiments demonstrate that, using the experimental conditions described above, it is possible to obtain an analysis method by nuclear magnetic resonance (.sup.1H-NMR and .sup.1H-.sup.13C HSQC) of glycosaminoglycans in general, and of heparins and low molecular weight heparins and their derivatives in particular, which allows their quantitative analysis.

Example 3

Study by .SUP.1.H NMR of Bemiparin Sodium

(76) The main .sup.1H chemical shift signals identified were as follows:

(77) TABLE-US-00014 Chemical Signal shift, ppm H4 U2S 5.992 H4 U 5.825 H1 1,6-AnA 5.616 H1 ANS(-G) 5.585 H1 1,6-AnM 5.569 H1 U2S 5.509 H1 ANS6S 5.405 H1 I2S 5.228 H1 I 5.012 H5 I2S 4.836 H1 G 4.628 H6 ANS6S 4.344 H6 ANS6S 4.210 H3 ANS 3.670 H2 ANS3S 3.395 H2 ANS 3.293 NAc 2.047 DMMA 1.320 TSP 0.069

(78) The quantification of the characteristic and well-differentiated signals of bemiparin sodium (generally those corresponding to the anomeric or target protons, H1) are shown in the following table, with the values of relative proportions observed for a series of six samples.

(79) TABLE-US-00015 Signal Chemical shift, ppm Relative proportion, % H4 U2S 5.99 3.7-5.7 H4 U 5.82 0.2-2.5 H1 1,6-an.A 5.62 0.5-2.5 H1 1,6-an.M 5.57 2.5-6.0 H1 U2S 5.51 7.0-10.7 H1 ANS6S 5.40 19.0-21.3 H1 I2S 5.23 13.8-18.5 H2 ANS 3.29 18.7-26.3 NAc 2.05 9.4-14.4

Example 4

(80) The same solution used in example 3, is used to perform the study by .sup.1H-.sup.13C HSQC. The main .sup.1H and .sup.13C chemical shift signals identified were as follows:

(81) TABLE-US-00016 Signal ppm .sup.13C, ppm .sup.1H, ppm C4-H4 U 110.71 5.82 C4-H4 U2S 108.97 5.99 C1-H1 G // Gal 106.62 4.66 C1-H1 Xyl 105.79 4.45 C1-H1 G(-ANAc) 105.13 4.50 C1-H1 I(-A6S) 104.94 5.01 C1-H1 G(-ANS) 104.77 4.60 C1-H1 I(-A6OH) 104.67 4.94 C1-H1 Gal 104.30 4.54 C1-H1 1.6-an.A 104.22 5.61 C1-H1 G(-ANS3S) 103.91 4.61 C1-H1 1.6-an.M 103.91 5.57 C1-H1 U 103.88 5.16 C1-H1 G2S 102.99 4.75 C1-H1_I2S 102.09 5.22 C1-H1 I2S(-1,6-an.M) 101.59 5.36 C1-H1 ANS(-G) 100.50 5.58 C1-H1 ANAc 100.23 5.31 C1-H1 U2S 100.18 5.51 C1-H1 ANS(-I2S) 99.78 5.40 C1-H1 ANS6S 99.43 5.43 C1-H1 ANS,3S 99.06 5.51 C1-H1 ANS red 98.73 4.71 C1-H1 ANS(-I) 98.42 5.34 C1-H1 M red 95.74 5.39 C1-H1 I2S red 95.70 5.42 C1-H1 I2S red 94.76 4.97 C1-H1 ANS red// 93.97 5.45 ANS6S red C3-H3 Gal 85.45 3.78 C3-H3 Gal 84.85 3.83 C4-H4 ANS6S(-G)// 80.94 3.84 ANS6S red C2-H2 I2S 78.53 4.34 C3-H3 Xyl 77.82 3.72 C2-H2 U2S 77.42 4.62 C2-H2 G(-AN6S) 75.70 3.40 C3-H3 ANS6S (-G) 72.47 3.66 C3-H3 ANS6S red 72.29 3.77 C5-H5 I2S 72.01 4.83 C3-H3 I2S 71.87 4.21 C5-H5 ANS6S(-G) 71.72 4.09 C5-H5 MNS6S red 70.98 4.15 C5-H5 ANS6S red 70.64 4.12 C6-H6 1,6-an.A// 67.53 3.77 1,6-an.M C5-H5 Xyl 65.89 4.12 C5-H5 Xyl 65.86 3.40 C3-H3 U2S 65.75 4.32 C6-H6 Gal 63.90 3.74 C2-H2 ANS6S red// 60.82 3.28 ANS(-I2S) C2-H2 ANS6S(-G) 60.52 3.29 C2-H2 MNS6S red 60.38 3.60 C2-H2 1.6-an.A 58.50 3.21 C2-H2 ANAc 56.68 3.92 C2-H2 1.6-an.M 55.09 3.47 DMMA 26.73 1.32 NAc 24.87 2.05

(82) The signals were correlated with particular saccharides (based upon comparison of said signals to those of reference monosaccharides), and after normalization of the respective integrals of said signals with respect to the integral of the DMMA reference, and after determining the relative percentage of the individual signals, a quantitative chemical shift pattern for the GAG was obtained.

(83) Specifically, the integral of each one of the above signals was normalized with respect to the integral of DMMA, using the same procedure explained for the experiments .sup.1H MMR. Accordingly, the quantitative relative content (proportion) of each saccharide in bemiparin sodium is detailed in the following table.

(84) TABLE-US-00017 Signal Relative proportion, % C1-H1 ANS-I2S 26.5-30.6 C1-H1 ANS-I 1.7-5.3 C1-H1 ANS-G 2.1-3.8 C1-H1 ANS.3S 0.6-2.5 C1-H1 ANAc 1.7-3.0 C1-H1 ANAc-red <LC C1-H1 ANS-red 2.6-5.4 C1-H1 1,6-an.A <1.1 C1-H1 1,6-an.M <1.0 C1-H1 MNS-red 0.9-2.3 C1-H1 I2S 30.4-34.9 C1-H1 I-A6S 1.4-2.6 C1-H1 I-A6OH <0.2 C1-H1 G-ANS,3S <2.5 C1-H1 G-ANS 1.9-3.6 C1-H1 G-ANAc 0.4-1.4 C1-H1 G2S <0.5 C1-H1 U2S 10.9-14.9 C1-H1 U 0.6-1.6 C1-H1 I2S-red <0.5 C5-H5 Gal-A <0.3

Example 5

Study by .SUP.1.H NMR of Dalteparin Sodium

(85) The main .sup.1H chemical shift signals identified were as follows.

(86) TABLE-US-00018 Chemical Signal shift, ppm H1 ANS(-G) 5.585 H1 ANS6S 5.405 H1 I2S 5.228 H1 I2S-(AM.ol) 5.178 H1 I 5.012 H5 I2S 4.836 H1 G 4.628 H6 ANS6S 4.344 H6 ANS6S 4.210 H3 ANS 3.670 H2 ANS3S 3.395 H2 ANS 3.293 NAc 2.047 DMMA 1.320 TSP 0.069

(87) After normalization, correlation and relative content determination, the quantitative relative content (proportion) of each saccharide in dalteparin sodium was obtained and is detailed in the following table. The quantitation (based upon a series of six samples) is based upon the the characteristic and well-differentiated signals of dalteparin sodium (generally those corresponding to the anomeric or target protons, H1).

(88) TABLE-US-00019 Signal Chemical shift, ppm Relative proportion, % H1 ANS6S 5.40 25.5-25.8 H1 I2S 5.23 19.2-20.8 H1 I2S-(AM.ol) 5.18 9.5-9.8 H2 ANS 3.29 28.0-30.0 NAc 2.05 15.4-20.0

Example 6

(89) The same solution used in example 5 was used to perform the study by .sup.1H-.sup.13C HSQC. The main .sup.1H and .sup.13C chemical shift signals identified were as follows:

(90) TABLE-US-00020 Signal .sup.13C, ppm .sup.1H, ppm C1-H1 G // Gal 106.62 4.66 C1-H1 Xyl 105.79 4.45 C1-H1 G(-ANAc) 105.13 4.50 C1-H1 I(-A6S) 104.94 5.01 C1-H1 G(-ANS) 104.77 4.60 C1-H1 I(-A6OH) 104.67 4.94 C1-H1 G(-ANS3S) 103.91 4.61 C1-H1 G2S 102.99 4.75 C1-H1 I2S 102.09 5.22 C1-H1 ANS(-G) 100.50 5.58 C1-H1 ANAc 100.23 5.31 C1-H1 ANS(-I2S) 99.78 5.40 C1-H1 ANS6S 99.43 5.43 C1-H1 ANS,3S 99.06 5.51 C1-H1 ANS(-I) 98.42 5.34 C3-H3 Gal 85.45 3.78 C3-H3 Gal 84.85 3.83 C4-H4 ANS6S(-G) 80.94 3.84 C2-H2 I2S 78.53 4.34 C3-H3 Xyl 77.82 3.72 C2-H2 G(-AN6S) 75.70 3.40 C3-H3 ANS6S (-G) 72.47 3.66 C5-H5 I2S 72.01 4.83 C3-H3 I2S 71.87 4.21 C5-H5 ANS6S(-G) 71.72 4.09 C5-H5 Xyl 65.89 4.12 C5-H5 Xyl 65.86 3.40 C6-H6 Gal 63.90 3.74 AM.ol-6S 63.8/63.7 3.70/3.74 C2-H2 ANS(-I2S) 60.82 3.28 C2-H2 ANS6S(-G) 60.52 3.29 C2-H2 ANAc 56.68 3.92 DMMA 26.73 1.32 NAc 24.87 2.05

(91) These signals can be associated with the monosaccharide components of the molecule, so that their quantification allows for the determination of their monosaccharide composition.

(92) The integrals of each one of these signals were normalized starting from the value established for the integral of DMMA, using the same procedure explained for the experiments .sup.1H NMR. The quantification of the signals characteristic of dalteparin sodium are shown in the following table.

(93) TABLE-US-00021 Signal Relative proportion, % C1-H1 ANS-I2S 22.2-23.3 C1-H1 ANS-I 3.0-3.2 C1-H1 ANS-G 2.3-2.6 C1-H1 ANS,3S 2.1-2.9 C1-H1 ANAc 2.4-3.1 C1-H1 I2S 24.5-27.5 C1-H1 I-A6S 3.6-4.0 C1-H1 G-ANS,3S 1.8-2.3 C1-H1 G-ANS 2.5-3.5 C1-H1. C6-H6 AM.ol-6S 20.8-21.7

Example 7

Study by .SUP.1.H NMR of Tinzaparin Sodium

(94) The main .sup.1H chemical shift signals identified were as follows:

(95) TABLE-US-00022 Chemical Signal shift, ppm H4 U2S 5.992 H4 U 5.825 H1 ANS(-G) 5.585 H1 U2S 5.509 H1 ANS6S 5.405 H1 I2S 5.228 H1 I 5.012 H5 I2S 4.836 H1 G 4.628 H6 ANS6S 4.344 H6 ANS6S 4.210 H3 ANS 3.670 H2 ANS3S 3.395 H2 ANS 3.293 NAc 2.047 DMMA 1.320 TSP 0.069

(96) The quantification of the characteristic and well-differentiated signals of tinzaparin sodium (generally those corresponding to the anomeric or target protons, H1) are shown in the following table, with the values of relative proportions observed (based upon a series of six samples).

(97) TABLE-US-00023 Signal Chemical shift, ppm Relative proportion, % H4 U2S 5.99 2.7 H1 U2S 5.51 5.3 H1 ANS6S 5.40 23.6 H1 I2S 5.23 21.0 H2 ANS 3.29 30.0 NAc 2.05 16.1

Example 8

(98) The same solution used in example 7 was used to perform the study by .sup.1H-.sup.13C HSQC. The main signals identified were as follows:

(99) TABLE-US-00024 Signal ppm .sup.13C, ppm .sup.1H, ppm C4-H4 U 110.71 5.82 C4-H4 U2S 108.97 5.99 C1-H1 G // Gal 106.62 4.66 C1-H1 Xyl 105.79 4.45 C1-H1 G(-ANAc) 105.13 4.50 C1-H1 I(-A6S) 104.94 5.01 C1-H1 G(-ANS) 104.77 4.60 C1-H1 I(-A6OH) 104.67 4.94 C1-H1 Gal 104.30 4.54 C1-H1 G(-ANS3S) 103.91 4.61 C1-H1 U 103.88 5.16 C1-H1 G2S 102.99 4.75 C1-H1 I2S 102.09 5.22 C1-H1 ANS(-G) 100.50 5.58 C1-H1 ANAc 100.23 5.31 C1-H1 U2S 100.18 5.51 C1-H1 ANS(-I2S) 99.78 5.40 C1-H1 ANS6S 99.43 5.43 C1-H1 ANS,3S 99.06 5.51 C1-H1 ANS red 98.73 4.71 C1-H1 ANS(-I) 98.42 5.34 C1-H1 I2S red 95.70 5.42 C1-H1 I2S red 94.76 4.97 C1-H1 ANS red// 93.97 5.45 ANS6S red C3-H3 Gal 85.45 3.78 C3-H3 Gal 84.85 3.83 C4-H4 ANS6S(-G)// 80.94 3.84 ANS6S red C2-H2 I2S 78.53 4.34 C3-H3 Xyl 77.82 3.72 C2-H2 U2S 77.42 4.62 C2-H2 G(-AN6S) 75.70 3.40 C3-H3 ANS6S (-G) 72.47 3.66 C3-H3 ANS6S red 72.29 3.77 C5-H5 I2S 72.01 4.83 C3-H3 I2S 71.87 4.21 C5-H5 ANS6S(-G) 71.72 4.09 C5-H5 ANS6S red 70.64 4.12 C5-H5 Xyl 65.89 4.12 C5-H5 Xyl 65.86 3.40 C3-H3 U2S 65.75 4.32 C6-H6 Gal 63.90 3.74 C2-H2 ANS6S red// 60.82 3.28 ANS(-I2S) C2-H2 ANS6S(-G) 60.52 3.29 C2-H2 MNS6S red 60.38 3.60 C2-H2 ANAc 56.68 3.92 DMMA 26.73 1.32 NAc 24.87 2.05

(100) These signals can be associated with the monosaccharide components of the molecule, so that their quantification allows the determination of their monosaccharide composition.

(101) The integrals of each one of these signals were normalized starting from the value established for the integral of DMMA, using the same procedure explained for the experiments .sup.1H RMN. The quantification of the signals characteristic of tinzaparin sodium are shown in the following table:

(102) TABLE-US-00025 Signal Relative proportion, % C1-H1 ANS-I2S 27.2 C1-H1 ANS-I 3.2 C1-H1 ANS-G 3.4 C1-H1 ANS.3S 1.2 C1-H1 ANAc 3.7 C1-H1 ANAc-red <LC C1-H1 ANS-red 6.5 C1-H1 I2S 35.1 C1-H1 I-A6S 3.1 C1-H1 I-A6OH 0.8 C1-H1 G-ANS,3S 1.5 C1-H1 G-ANS 3.4 C1-H1 G-ANAc 2.2 C1-H1 U2S 8.6 C1-H1 I2S-red <0.1

(103) These experiments show that, using the above-described experimental conditions, it is possible to obtain a method of analysis by nuclear magnetic resonance (.sup.1H-RMN y .sup.1H-.sup.13C HSQC) of glycosaminoglycans in general and of heparins and low molecular weight heparins and their derivatives in particular, which allows their quantitative analysis.

(104) Additional Disclosure

(105) The invention includes at least the following embodiments.

(106) A method for the analysis of a composition that contains monosaccharide residues present in heparin chains by means of .sup.1H-NMR one-dimensional nuclear magnetic resonance and/or .sup.1H-.sup.13C HSQC two-dimensional nuclear magnetic resonance comprising the steps of: providing a composition including at least one monosaccharide residue present in heparin chains and obtaining its spectrum of .sup.1H-NMR one-dimensional nuclear magnetic resonance and/or .sup.1H-.sup.13C HSQC two-dimensional nuclear magnetic resonance using dimethylmalonic acid (DMMA) as internal reference, and identifying in the NMR spectrum the presence or the absence of at least one signal of at least one residue selected from the group consisting of: 4,5-unsaturated 2-O sulfo uronic acid (U2S), 4,5-unsaturated uronic acid (U), 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, characterized in that the presence of said NMR signals in a determined relative proportion of its normalized integrals with respect to DMMA, or the absence thereof, forms a pattern which allows identifying the heparin which the monosaccharide residue comes from comparing the pattern obtained in the analysis with a standard pattern previously obtained for different heparins in the same conditions.

(107) The method according to any of the embodiments herein, wherein the following pattern is identified in the .sup.1H NMR spectrum:

(108) TABLE-US-00026 Signal Chemical shift, ppm Relative proportion, % H4 U2S 5.99 4.3-4.7 H4 U 5.82 0.2 Hl 1,6-an.A 5.62 0.7-0.9 Hl 1,6-an.M 5.57 4.1-4.4 Hl U2S 5.51 5.7-6.0 Hl ANS6S 5.40 16.1-16.7 Hl I2S 5.23 13.1-14.4 H5 I2S 4.84 14.3-16.3 H2 ANS 3.29 24.3-26.6 NAc 2.05 12.0-15.3
thereby determining the content of the monosaccharide residues in enoxaparin sodium.

(109) The method according to any of the embodiments herein, wherein the following pattern is identified in the .sup.1H-.sup.13C HSQC NMR spectrum:

(110) TABLE-US-00027 Signal Relative proportion, % C1-H1 ANS-I2S 25.6-26.9 C1-H1 ANS-I 2.6-3.0 C1-H1 ANS-G 5.1-5.5 C1-H1 ANS.3S 1.5-1.7 C1-H1 ANAc 2.7-3.5 C1-H1 ANAc-red <LC C1-H1 ANS-red 3.8-4.9 C1-H1 1,6-an.A 1.2-1.5 C1-H1 1,6-an.M 1.6-1.9 C1-H1 MNS-red 1.0-1.3 C1-H1 I2S 24.5-27.5 C1-H1 I-A6S 2.4-2.7 C1-H1 I-A6OH 0.3-0.4 C1-H1 G-ANS.3S 1.4-1.6 C1-H1 G-ANS 4.2-4.4 C1-H1 G-ANAc 1.9-2.6 C1-H1 G2S 1.1-1.6 C1-H1 U2S 11.5-12.4 C1-H1 U 0.3-0.5 C1-H1 I2S-red 1.0-1.4 C5-H5 Gal-A <LC-0.5 Epox <LC-0.4
where LC is limit of quantification,
thereby determining the content of the monosaccharide residues in enoxaparin sodium.

(111) The method according to any of the embodiments herein, wherein the following pattern is identified in the .sup.1H NMR spectrum:

(112) TABLE-US-00028 Signal Chemical shift, ppm Relative proportion, % H4 U2S 5.99 3.7-5.7 H4 U 5.82 0.2-2.5 H1 1,6-an.A 5.62 0.5-2.5 H1 1,6-an.M 5.57 2.5-6.0 H1 U2S 5.51 7.0-10.7 H1 ANS6S 5.40 19.0-21.3 H1 I2S 5.23 13.8-18.5 H2 ANS 3.29 18.7-26.3 NAc 2.05 9.4-14
thereby determining the content of the monosaccharide residues in bemiparin sodium.

(113) The method according to any of the embodiments herein, wherein the following pattern is identified in the .sup.1H-.sup.13C HSQC NMR spectrum:

(114) TABLE-US-00029 Signal Relative proportion, % C1-H1 ANS-I2S 26.5-30.6 C1-H1 ANS-I 1.7-5.3 C1-H1 ANS-G 2.1-3.8 C1-H1 ANS.3S 0.6-2.5 C1-H1 ANAc 1.7-3.0 C1-H1 ANAc-red <LC C1-H1 ANS-red 2.6-5.4 C1-H1 1,6-an.A <1.1 C1-H1 1,6-an.M <1.0 C1-H1 MNS-red 0.9-2.3 C1-H1 I2S 30.4-34.9 C1-H1 I-A6S 1.4-2.6 C1-H1 I-A6OH <0.2 C1-H1 G-ANS,3S <2.5 C1-H1 G-ANS 1.9-3.6 C1-H1 G-ANAc 0.4-1.4 C1-H1 G2S <0.5 C1-H1 U2S 10.9-14.9 C1-H1 U 0.6-1.6 C1-H1 I2S-red <0.5 C5-H5 Gal-A <0.3
thereby determining the content of the monosaccharide residues in bemiparin sodium.

(115) The method according to any of the embodiments herein, wherein the following pattern is identified in the .sup.1H NMR spectrum:

(116) TABLE-US-00030 Signal Chemical shift, ppm Relative proportion, % H1 ANS6S 5.40 25.5-25.8 H1 I2S 5.23 19.2-20.8 H1 I2S-(AM.ol) 5.18 9.5-9.8 H2 ANS 3.29 28.0-30.0 NAc 2.05 15.4-20.0
thereby determining the content of the monosaccharide residues in dalteparin sodium.

(117) The method according to any of the embodiments herein, wherein the following pattern is identified in the .sup.1H-.sup.13C HSQC NMR spectrum:

(118) TABLE-US-00031 Signal Relative proportion, % C1-H1 ANS-I2S 22.2-23.3 C1-H1 ANS-I 3.0-3.2 C1-H1 ANS-G 2.3-2.6 C1-H1 ANS,3S 2.1-2.9 C1-H1 ANAc 2.4-3.1 C1-H1 I2S 24.5-27.5 C1-H1 I-A6S 3.6-4.0 C1-H1 G-ANS.3S 1.8-2.3 C1-H1 G-ANS 2.5-3.5 C1-H1. C6-H6 AM.ol-6S 20.8-21.7
thereby determining the content of the monosaccharide residues in dalteparin sodium.

(119) The method according to any of the embodiments herein, wherein the following pattern is identified in the .sup.1H NMR spectrum:

(120) TABLE-US-00032 Signal Chemical shift, ppm Relative proportion, % H4 U2S 5.99 2.7 H1 U2S 5.51 5.3 H1 ANS6S 5.40 23.6 H1 I2S 5.23 21.0 H2 ANS 3.29 30.0 NAc 2.05 16.1
thereby determining the content of the monosaccharide residues in tinzaparin sodium.

(121) The method according to any of the embodiments herein, wherein the following pattern is identified in the .sup.1H-.sup.13C HSQC NMR spectrum:

(122) TABLE-US-00033 Signal Relative proportion, % C1-H1 ANS-I2S 27.2 C1-H1 ANS-I 3.2 C1-H1 ANS-G 3.4 C1-H1 ANS,3S 1.2 C1-H1 ANAc 3.7 C1-H1 ANAc-red <LC C1-H1 ANS-red 6.5 C1-H1 I2S 35.1 C1-H1 I-A6S 3.1 C1-H1 I-A6OH 0.8 C1-H1 G-ANS,3S 1.5 C1-H1 G-ANS 3.4 C1-H1 G-ANAc 2.2 C1-H1 U2S 8.6 C1-H1 I2S-red <0.1
thereby determining the content of the monosaccharide residues in tinzaparin sodium.

(123) The method according to any of the embodiments herein, wherein the signals corresponding to the N-acetyl groups appear in the region between 1.8 to 2.1 ppm in of .sup.1H-NMR spectroscopy.

(124) The method according to any of the embodiments herein, wherein the signals corresponding to the saccharide ring of said residues appear in the region between 2.8 to 6.0 ppm in .sup.1H-NMR spectroscopy.

(125) The method according to any of the embodiments herein, wherein the signals corresponding to the anomeric or target H1 protons, and that of the H4 protons of the non-reducing ends of one of said residues, appear in the region between 4.6 to 6.0 ppm in .sup.1H-NMR spectroscopy.

(126) The method according to any of the embodiments herein, wherein, in .sup.1H NMR spectroscopy, the 4,5-unsaturated 2-O-sulfo-uronic acid (U2S) signals appear at 5.99 and 5.51 ppm, for the H4 and anomeric protons respectively, the 4,5-unsaturated uronic acid (U) signal appears at 5.82 ppm for the H4 proton, the 2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A) signal appears at 5.62 ppm for the anomeric proton, the 2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M) signal appears at 5.57 ppm for the anomeric proton, and the 2-N-sulfo-6-O-sulfoglucosamine signals appear at 5.41 and 4.21-4.34 ppm for the H1 and H6 and H6 protons respectively.

(127) The method according to any of the embodiments herein, wherein, in .sup.1H-.sup.13C HSQC NMR spectroscopy, the 4,5-unsaturated 2-O sulfo uronic acid (U2S) signals appear at 6.0-109.0 ppm (H4-C4), 5.5-100.2 ppm (H1-C1), 4.6-77.4 ppm (H2-C2) or 4.3-66.8 ppm (H3-C3), the 4,5-unsaturated uronic acid (U) signals appear at 5.8-110.7 ppm (H4-C4) or 5.2-103.9 ppm (H1-C1), the 2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A) signals appear at 5.6-104.2 ppm (H1-C1), 3.2-58.5 ppm (H2-C2) or 3.8-67.5 ppm (H6-C6), the 2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M) signals appear at 5.6-103.9 ppm (H1-C1), 3.5-55.1 (H2-C2) or 3.8-67.5 ppm (H6-C6), and the 2-N-sulfo-6-O-sulfoglucosamine signals appear at 5.4-99.4 ppm (H1-C1), 3.8-80.9 ppm (H4-C4), 3.7 to 3.8-42.3 to 72.5 ppm (H3-C3), 4.1-70.6 to 71.7 ppm (H5-C5) or 3.3-60.5-60.8 ppm (H2-C2).

(128) The method according to any of the embodiments herein, wherein the pattern obtained in the analysis is such that it determines that the monosaccharide residues come from an unfractionated heparin.

(129) The method according to any of the embodiments herein, wherein the pattern obtained in the analysis is such that it determines that the monosaccharide residues come from a Low Molecular Weight Heparin (LMWH).

(130) The method according to any of the embodiments herein, wherein the pattern obtained in the analysis is such that it determines that the monosaccharide residues come from an Ultra Low Molecular Weight Heparin (ULMWH).

(131) The invention according to any of the embodiments herein, wherein the glycosaminoglycan is selected from the group consisting of enoxaparin, bemiparin, dalteparin, tinzaparin, a salt of any of the preceding, a derivative of any of the preceding, or a combination thereof.

(132) In view of the above description and the examples below, one of ordinary skill in the art will be able to practice the invention as claimed without undue experimentation. The foregoing will be better understood with reference to the following examples that detail certain procedures for the preparation and/or practice of embodiments of the present invention. All references made to these examples are for the purposes of illustration. The following examples should not be considered exhaustive, but merely illustrative of only a few of the many embodiments contemplated by the present invention.

(133) As used herein, the term about or approximately are taken to mean10%, 5%, 2.5% or 1% of a specified valued. As used herein, the term substantially is taken to mean to a large degree or at least a majority of or more than 50% of. Moreover, all ranges specified herein are inclusive of the range limits and all integer and fractional values therein especially as defined by the definition of the term about.

(134) As used herein a derivative is: a) a chemical substance that is related structurally to a first chemical substance and theoretically derivable from it; b) a compound that is formed from a similar first compound or a compound that can be imagined to arise from another first compound, if one atom of the first compound is replaced with another atom or group of atoms; c) a compound derived or obtained from a parent compound and containing essential elements of the parent compound; or d) a chemical compound that may be produced from first compound of similar structure in one or more steps. For example, a derivative may include a deuterated form, oxidized form, dehydrated, unsaturated, polymer conjugated or glycosilated form thereof or may include an ester, amide, lactone, homolog, ether, thioether, cyano, amino, alkylamino, sulfhydryl, heterocyclic, heterocyclic ring-fused, polymerized, pegylated, benzylidenyl, triazolyl, piperazinyl or deuterated form thereof.