Crystalline binary sodium salts of 5-methyl-(6S)-tetrahydrofolic acid with organic bases

Abstract

The present invention is directed to crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid comprising (i) 5-methyl-(6S)-tetrahydrofolic acid, (ii) sodium and (iii) an organic base having a pKa value from 6 to 11; wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the sodium is from 1:0.5 to 1:1.5 and the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the organic base is 1:0.5 to 1:1.5 and/or hydrates and/or solvates thereof, as well as, a processes of obtaining the same.

Claims

1. A crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid, comprising (i) 5-methyl-(6S)-tetrahydrofolic acid, (ii) sodium and (iii) an organic base having a pKa value of 6 to 11; wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the sodium is 1:0.5 to 1:1.5 and the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the organic base is 1:0.5 to 1:1.5; or a hydrate or solvate thereof.

2. The crystalline salt of claim 1, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the sodium is 1:0.75 to 1:1.25 and the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the organic base is 1:0.75 to 1:1.25.

3. The crystalline salt of claim 1, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the sodium is approximately 1:1 and the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the organic base is approximately 1:1.

4. A crystalline salt of claim 1, wherein the organic base having a pKa value of 6 to 11 is 4-(2-hydroxyethyl)-morpholine, 1-(2-hydroxyethyl)-pyrrolidine, imidazole, 2-dimethylaminoethanol, or tert-butylamine; or is a mixture thereof.

5. The crystalline salt of claim 1, wherein the organic base is 4-(2-hydroxyethyl)-morpholine and which crystalline salt has a PXRD pattern with at least one characteristic peak, which is expressed in 2θ±0.2° 2θ with CuKα radiation, at 14.1, 15.8, 16.2, 16.6, 18.2, 19.9, 21.8, and/or 25.0.

6. The crystalline salt of claim 1, wherein the organic base is 4-(2-hydroxyethyl)-morpholine and which crystalline salt has a PXRD pattern with at least one characteristic peak, which is expressed in 2θ±0.2° 2θ with CuKα radiation, at 8.3, 13.9, 14.1, 14.2, 15.5, 15.8, 16.2, 16.6, 18.2, 19.4, 19.6, 19.9, 20.1, 20.8, 21.8, 23.6, 23.9, 25.0, 25.9, 28.1, 28.5, and/or 29.6.

7. The crystalline salt of claim 1, wherein the organic base is 4-(2-hydroxyethyl)-morpholine and which crystalline salt has a PXRD pattern substantially as shown in FIG. 1.

8. The crystalline salt of claim 1, wherein the organic base is 1-(2-hydroxyethyl)-pyrrolidine and which crystalline salt has a PXRD pattern with at least one characteristic peak, which is expressed in 2θ±0.2° 2θ with CuKα radiation, at 8.3, 14.3, 14.4, 15.6, 16.0, 16.7, 18.5, 20.0, 21.8, and/or 25.2.

9. The crystalline salt of claim 8, wherein the organic base is 1-(2-hydroxyethyl)-pyrrolidine and which crystalline salt has a PXRD pattern with at least one characteristic peak, which is expressed in 2θ±0.2° 2θ with CuKα radiation, at 4.8, 8.3, 12.5, 13.6, 14.3, 14.4, 15.6, 16.0, 16.1, 16.7, 18.3, 18.5, 19.6, 20.0, 20.7, 21.8, 22.3, 22.7, 23.8, 24.0, and/or 25.2.

10. The crystalline salt of claim 8, wherein the organic base is 1-(2-hydroxyethyl)-pyrrolidine and which crystalline salt has a PXRD pattern substantially as shown in FIG. 6.

11. The crystalline salt of claim 1, wherein the organic base is 2-dimethylaminoethanol and which crystalline salt has a PXRD pattern with at least one characteristic peak, which is expressed in 2θ±0.2° 2θ with CuKα radiation, at 8.3, 13.6, 14.2, 14.4, 15.8, 16.7, 18.5, 19.8, 19.9, 24.0, 25.1, and/or 30.2.

12. The crystalline salt of claim 11, wherein the organic base is 2-dimethylaminoethanol and which crystalline salt has a PXRD pattern with at least one characteristic peak, which is expressed in 2θ±0.2° 2θ with CuKα radiation, at 8.3, 13.6, 14.2, 14.4, 15.7, 15.8, 16.0, 16.7, 17.1, 18.5, 19.8, 19.9, 21.9, 22.1, 22.7, 24.0, 25.1, 25.5, 25.9, 27.8, 28.5 and/or 30.2.

13. The crystalline salt of claim 11, wherein the organic base is 2-dimethylaminoethanol and which crystalline salt has a PXRD pattern substantially as shown in FIG. 8.

14. The crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid according to claim 1, having at least 99% or more chemical and stereoisomerical purity.

15. A process for obtaining the crystalline sodium salt according to claim 1, comprising the steps of: a) providing 5-methyl-(6S)-tetrahydrofolic acid, optionally in a solvent or a mixture of solvents; b) adding sodium hydroxide; c) adding an organic base having a pKa value of 6 to 11; wherein the order of steps b) and c) may be interchanged; d) optionally adding a solvent or a mixture of solvents to the resultant composition of step b) or step c); e) crystallizing; f) optionally adding more solvent or mixture of solvents; and g) isolating obtained solid.

16. The process of claim 15, wherein the 5-methyl-(6S)-tetrahydrofolic acid and sodium hydroxide are present at a molar ratio in step b) in the range of 1:0.5 to 1:1.5.

17. The process of claim 15, wherein the organic base of step c) is 4-(2-hydroxyethyl)-morpholine, 1-(2-hydroxyethyl)-pyrrolidine, imidazole, 2-dimethylaminoethanol, or tert-butylamine; or is a mixture thereof.

18. A pharmaceutical composition, food additive, vitamin and/or other preparation comprising the crystalline salt of 5-methyl-(6S)-tetrahydrofolic according to claim 1 and one or more acceptable excipients suitable for forming said pharmaceutical composition, food additive, vitamin and/or other preparation.

19. The pharmaceutical composition according to claim 18, which is in the form of a tablet, capsule, oral liquid preparation, powder, lyophilisate, granule, lozenge, reconstitutable powder, injectable or infusable solution or suspension, or suppository.

20. A method for the treatment in homocysteine-lowering, of anemia, neural tube defects, cardiovascular diseases, depression, cognitive impairment, Alzheimer's disease, osteoporosis and/or dietary management of low plasma and/or low red blood cell and/or low cerebrospinal fluid and/or low peripheral or central nervous system folate, comprising administering to a subject in need thereof a crystalline salt of claim 1.

21. The process of claim 15, wherein the 5-methyl-(6S)-tetrahydrofolic acid and organic base are present at a molar ratio in step c) in the range of 1:0.5 to 1:3.

22. The process of claim 15, wherein the solvent and/or mixture of solvents according to step a), d) and/or f) is selected from the group consisting of water, water-soluble alcohols, methanol, ethanol, isopropanol, n-propanol, acetonitrile, tetrahydrofuran, acetone, methyl ethyl ketone, methyl isobutyl ketone, benzylalcohol, and mixtures thereof.

23. The process of claim 15, wherein steps b) and c) are interchanged.

24. The process of claim 15, wherein in step c), d) and/or e) the temperature is less than 60° C.

25. The process of claim 15, wherein in step a), b), c), d) and/or e) seed crystals are added.

26. The pharmaceutical composition according to claim 18, further comprising at least one additional therapeutic agent.

27. The pharmaceutical composition according to claim 18, which is a pharmaceutical composition for oral, parenteral, intramuscular, intraspinal, intrathecal, peridontal, topical or rectal administration.

28. The crystalline salt of claim 1, wherein the organic base is 4-(2-hydroxyethyl)-morpholine and which crystalline salt has a PXRD pattern with characteristic peaks, which are expressed in 2θ±0.2° 2θ with CuKα radiation, at 14.1, 15.8, 16.2, 16.6, 18.2, 19.9, 21.8, and 25.0.

29. A crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid, comprising (i) 5-methyl-(6S)-tetrahydrofolic acid, (ii) sodium and (iii) an organic base having a pKa value of 6 to 11; wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the sodium is 1:0.5 to 1:1.5 and the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the organic base is 1:0.5 to 1:1.5.

Description

EXPERIMENTAL

(1) Powder X-Ray Diffraction:

(2) Stoe Stadi P equipped with a Mythen1K Detector; Cu-Kα1 radiation; standard measurement conditions: transmission; 40 kV and 40 mA tube power; curved Ge monochromator; 0.02°2θ step size, 48 s step time, 1.5-50.5°2θ scanning range; detector mode: step scan; 1°2θ detector step; standard sample preparation: 10 to 20 mg sample was placed between two acetate foils; sample holder: Stoe transmission sample holder; the sample was rotated during the measurement. All sample preparation and measurement was done in an ambient air atmosphere.

(3) Microscopy:

(4) Light microscopy was performed on a Leitz Orthoplan polarized microscope, generally a 10×10 magnification was applied.

(5) TG-FTIR:

(6) Thermogravimetric measurements were carried out with a Netzsch Thermo-Microbalance TG 209 coupled to a Bruker FTIR Spectrometer Vector 22 (sample pans with a pinhole, N.sub.2 atmosphere, heating rate 10 K/min).

(7) Raman Spectroscopy:

(8) FT-Raman spectra were recorded on a Bruker MultiRAM FT-Raman or a Bruker RFS 100 FT-Raman system with a near infrared Nd:YAG laser operating at 1064 nm and a liquid nitrogen-cooled germanium detector. 64 scans with a resolution of 2 cm.sup.−1 were accumulated in the range from 3500 to −50 cm.sup.−1; however, only data above 100 cm.sup.−1 are evaluated due to filter cutoff effects. Nominal laser powers are typically 100 or 300 mW.

Example 1: Preparation of the Crystalline Sodium Salt of 5-methyl-(6S)-tetrahydrofolic Acid and Comprising 4-(2-hydroxy-ethyl)-morpholine

(9) To 468 mg of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 5-methyltetrahydrofolic acid 97.65% w/w) were weighed into a glass vial equipped with a magnetic stirrer bar. 1.0 mL of sodium hydroxide standard solution 1.00 mol/L and 140 μl of 4-(2-hydroxy-ethyl)-morpholine were added and the mixture was sonicated to obtain an essentially clear solution. At ambient temperature, 1.0 mL ethanol was added followed by seeding with about three mg of crystalline monosodium salt. Then 2.0 mL ethanol was added and the seeding step was repeated. Upon seeding, a thick suspension was obtained that was diluted with 1.0 mL of an ethanol-water 2:1 mixture. After stirring at r.t. for about one hour the solid product was separated by filtration and after drying in air at r.t., characterized by powder X-ray diffraction (FIG. 1, Table 2), .sup.1H-NMR, light microscopy and TG-FTIR. The yield was about 129 mg. The sample was further examined by HPLC, Raman spectroscopy, and IC-OES for determination of the sodium content. .sup.1H-NMR shows that when the integral for the two protons of 5-methyl-(6S)-tetrahydrofolic acid near 7.6 is normalized to 2.0 the resultant integral for the six protons of the 4-(2-hydroxy-ethyl)-morpholine that appear near 3.7 is 5.96. This shows that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to 4-(2-hydroxy-ethyl)-morpholine is essentially 1:1. Further analysis by TG-FTIR shows a water content of about 14 wt-%. The sodium content was determined by ICP-OES (inductively coupled plasma atomic emission spectroscopy) and a content of 3.2 wt-% sodium was found. This shows that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to sodium is essentially 1:1. The product was further investigated by Raman spectroscopy FIG. 2, Table 1.

(10) TABLE-US-00001 TABLE 1 Raman peaktable for the crystalline sodium salt of 5-methyl- (6S)-tetrahydrofolic acid and 4-(2-hydroxyethyl)-morpholine. intensity wavenumber (cm.sup.−1) (arbitrary units) 2954 0.34 2875 0.23 1610 1.74 1582 0.40 1547 0.35 1482 0.23 1463 0.28 1419 0.19 1340 0.48 1293 0.50 1217 0.14 1184 0.31 1154 0.16 1069 0.12 1023 0.14 946 0.14 892 0.15 861 0.25 836 0.13 778 0.17 649 0.56 638 0.35 624 0.18 479 0.20 415 0.12 374 0.15

(11) TABLE-US-00002 TABLE 2 PXRD peaktable the crystalline sodium salt of 5-methyl- (6S)-tetrahydrofolic acid and 4-(2-hydroxyethyl)-morpholine. intensity °2-theta d-spacings [Å] (qualitative) 4.9 18.0 w 6.1 14.5 vw 8.3 10.7 m 9.8 9.0 w 10.7 8.3 vw 11.9 7.4 w 12.4 7.1 w 13.3 6.6 w 13.9 6.3 s 14.1 6.3 vs 14.2 6.2 vs 14.8 5.99 w 15.5 5.72 s 15.8 5.59 vs 16.2 5.47 vs 16.6 5.34 vs 17.0 5.21 m 17.5 5.05 w 18.2 4.86 vs 19.4 4.58 m 19.6 4.52 s 19.9 4.45 vs 20.1 4.41 m 20.8 4.27 s 21.0 4.23 w 21.4 4.15 vw 21.6 4.10 w 21.8 4.07 vs 22.1 4.02 m 22.4 3.97 m 22.8 3.90 w 23.6 3.76 s 23.9 3.72 s 24.3 3.66 vw 24.5 3.63 w 24.8 3.59 m 25.0 3.56 vs 25.2 3.53 m 25.5 3.49 w 25.9 3.43 s 26.6 3.35 w 26.8 3.33 m 27.3 3.26 m 27.8 3.21 w 28.1 3.17 s 28.5 3.13 s 28.6 3.11 w 28.9 3.08 w 29.6 3.02 s 30.8 2.90 m 33.5 2.67 m

Example 2: Kinetic Solubility of 5-methyl-(6S)-tetrahydrofolic Acid Sodium Salt Comprising 4-(2-hydroxyethyl)-morpholine

(12) 27.1 mg of crystalline 5-methyl-(6S)-tetrahydrofolic acid sodium salt containing 4-(2-hydroxy-ethyl)-morpholine (according to Example 1) with a stoichiometric ratio of 1:1:1 are weighed into a 4 ml glass vial with screw cap. 0.5 ml of purified/de-ionized water (for instance water for chromatography) is added. The mixture is vigorously agitated at room temperature and briefly sonicated and a clear slightly yellow solution is readily obtained (within a few seconds). Thus the solubility is greater than 54 mg per 1 ml of water. The solution remains clear for several hours at r.t.

Reference Example 3: Kinetic Solubility of the Calcium Salt of 5-methyl-(6S)-tetrahydrofolic Acid

(13) 27.9 mg of crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid (containing about 11% of water, thus corresponding to a dry weight of about 25 mg) are weighed into a 4 ml glass vial with screw cap. 2.535 ml of purified/de-ionized water (for instance water for chromatography) is added to the solid using an adjustable volumetric pipette. The mixture is vigorously agitated at room temperature and briefly sonicated. No clear solution can be obtained and a fairly concentrated suspension persists. Thus the kinetic solubility measured as described here is smaller than 10 mg per 1 ml of water.

Example 4: Microscopy of 5-methyl-(6S)-tetrahydrofolic Acid Sodium Salt Comprising 4-(2-hydroxyethyl)-morpholine

(14) A small aliquot (a few microliters) of the suspension obtained upon crystallization of the sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 4-(2-hydroxyethyl)-morpholine is transferred on a microscopy glass slide and examined with a light microscope from on Leitz Orthoplan under polarized light. A 10×10 magnification was applied. Microscopy shows rod shaped particles with lengths of up to about 100 μm and a thickness of about 10 to 20β. Such particles are easy to handle and have favorable filtration and drying properties. An image is shown in FIG. 4.

Reference Example 5: Microscopy of the Calcium Salt of 5-methyl-(6S)-tetrahydrofolic Acid

(15) A small aliquot (a few micrograms) of crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid is transferred on a microscopy glass slide and examined as dry powder with a light microscope under polarized light. A 10×10 magnification was applied. Microscopy shows large agglomerates that consist of a multitude of very small particles. Such particles are difficult to handle. An image is shown in FIG. 5.

Example 6: Preparation of a Crystalline Sodium Salt of 5-methyl-(6S)-tetrahydrofolic Acid Comprising 1-(2-hydroxyethyl)-pyrrolidine

(16) To 467 mg of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 5-methyltetrahydrofolic acid 97.65% w/w) were weighed into a glass vial equipped with a magnetic stirrer bar. 1.0 mL of sodium hydroxide standard solution 1.00 mol/L, then 126 μl of 1-(2-hydroxyethyl)-pyrrolidine and 4.0 mL ethanol were added. The resultant mixture was sonicated and stirred at ambient temperature for two hours. A thick suspension was obtained that was diluted with 2.0 mL of an ethanol-water mixture 4:1 v/v and stirring was continued for one hour before the solid product was separated by filtration. After drying in air at ambient temperature for half an hour the product was characterized by powder X-ray diffraction (FIG. 6, Table 3), H-NMR, and TG-FTIR. The yield was about 350 mg. The sample was further examined by HPLC and showed an HPLC purity of 99.16 area-%. H-NMR spectroscopy shows that when the integral for the two protons of 5-methyl-(6S)-tetrahydrofolic acid near 7.6 is normalized to 2.0, the resultant integral for the four methylene protons in the five-ring near 1.7 ppm of 1-(2-hydroxyethyl)-pyrrolidine is 4.2. This suggests that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to 1-(2-hydroxyethyl)-pyrrolidine is essentially 1:1. Further analysis by TG-FTIR suggests a water content of about 9%. The sodium content was determined by ICP-OES (inductively coupled plasma atomic emission spectroscopy) and a content of 3.5 wt-% sodium was found. This shows that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to sodium is essentially 1:1. The product was further investigated by Raman spectroscopy FIG. 7, Table 4.

(17) TABLE-US-00003 TABLE 3 PXRD peaklist for sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 1-(2-hydroxyethyl)-pyrrolidine with 2-theta angles, d-spacing values in Ångstrom and qualitative intensity values as follows: vs = very strong, s = strong, m = medium, w = weak and vw = very weak. intensity °2-theta d-spacings [Å] (qualitative) 4.8 18.2 m 8.3 10.6 s 11.9 7.5 w 12.5 7.1 m 13.6 6.5 m 14.3 6.2 vs 14.4 6.1 vs 15.6 5.68 m 16.0 5.54 s 16.1 5.51 s 16.7 5.29 s 17.0 5.22 w 18.3 4.84 m 18.5 4.79 vs 19.6 4.53 m 20.0 4.44 s 20.7 4.29 m 21.0 4.22 w 21.8 4.08 s 22.3 3.99 w 22.7 3.92 w 23.8 3.73 w 24.0 3.70 m 24.9 3.58 m 25.2 3.53 vs 25.5 3.49 m 25.8 3.45 m 26.1 3.41 w 26.7 3.33 w 27.1 3.29 w 27.8 3.21 w 28.4 3.14 m 29.7 3.01 w 30.1 2.96 m

(18) TABLE-US-00004 TABLE 4 Raman data for the sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 1-(2-hydroxyethyl)-pyrrolidine with wavenumber in cm.sup.−1 and intensity values. It should be noted that the intensities vary with Laser power, sample amount and other factors. wavenumber (cm.sup.−1) intensity (arbitrary units) 3058 0.17 2954 0.46 2875 0.27 1611 2.10 1583 0.44 1548 0.47 1529 0.42 1480 0.28 1464 0.37 1419 0.26 1341 0.61 1295 0.60 1272 0.45 1216 0.16 1183 0.41 1155 0.19 1068 0.15 947 0.18 890 0.21 861 0.27 836 0.16 779 0.15 648 0.71 637 0.44 624 0.22 480 0.24 416 0.15 375 0.18

Example 7: Kinetic Solubility of the Sodium Salt of 5-methyl-(6S)-tetrahydrofolic Acid Comprising 1-(2-hydroxyethyl)-pyrrolidine

(19) 78.5 mg of crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 1-(2-hydroxyethyl)-pyrrolidine is weighed into a 4 ml glass vial with screw cap. Then 0.785 ml of purified/de-ionized water is added. The mixture is agitated at room temperature and a clear solution is readily obtained (within a few seconds). Thus the solubility is greater than 100 mg per 1 ml of water. The solution remains clear for several hours at r.t.

Example 8: Preparation of a Crystalline Sodium Salt of 5-methyl-(6S)-tetrahydrofolic Acid Comprising 2-dimethylaminoethanol

(20) 467 mg of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 5-methyltetrahydrofolic acid 97.65% w/w) were weighed into a glass vial equipped with a magnetic stirrer bar. The added 1.0 mL of sodium hydroxide standard solution 1.00 mol/L, then added 110 μl of 2-dimethylaminoethanol (˜1.1 equivalents). To the essentially clear solution 2.0 mL of ethanol was added. The resultant mixture was sonicated, seeded with crystalline salt, and stirred at ambient temperature for one hour. A thick suspension was obtained that was diluted with 2.0 mL of an ethanol-water mixture 4:1 v/v and stirring was continued for one hour before the solid product was separated by filtration and after drying in air at r.t. The crystalline product was characterized by H-NMR, Raman spectroscopy, PXRD, and TG-FTIR. Powder X-ray diffraction shows that the sample is clearly crystalline in nature (FIG. 8, Table 5). H-NMR spectroscopy shows that when the integral for the two protons of 5-methyl-(6S)-tetrahydrofolic acid near 7.6 is normalized to 2.0, the resultant integral for the six protons of the two methyl groups 2-dimethylaminoethanol near 2.1 ppm of is 6.6. This suggests that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to 2-dimethylaminoethanol is essentially 1:1. The sample was further examined by Raman spectroscopy and it shows a Raman spectrum as described in Table 6 and shown in FIG. 9. The sodium content was determined by ICP-OES (inductively coupled plasma atomic emission spectroscopy) and a content of 3.2 wt-% sodium was found. Further analysis by TG-FTIR suggests a water content of about 10%. Based on the two results, the product has a 1:1 molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to sodium.

(21) TABLE-US-00005 TABLE 5 PXRD peaklist for sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 2-dimethylaminoethanol with 2-theta angles, d-spacing values in Ångstrom and qualitative intensity values as follows: vs = very strong, s = strong, m = medium, 32 weak and vw = very weak. °2-theta d-spacings [A] intensity (qualitative) 4.8 18.4 w 8.3 10.6 s 11.8 7.5 w 12.5 7.1 w 13.6 6.5 s 14.2 6.2 vs 14.4 6.1 s 15.7 5.66 m 15.8 5.59 s 16.0 5.52 m 16.7 5.30 s 17.1 5.19 m 17.3 5.12 w 18.5 4.80 vs 19.8 4.49 s 19.9 4.45 s 20.8 4.27 w 21.0 4.22 w 21.9 4.06 m 22.1 4.02 w 22.7 3.92 m 23.7 3.75 w 24.0 3.70 s 25.1 3.54 s 25.5 3.49 m 25.9 3.44 m 26.8 3.33 w 26.9 3.31 w 27.2 3.28 w 27.8 3.20 m 28.5 3.13 m 29.6 3.02 w 29.9 2.98 w 30.2 2.96 s

(22) TABLE-US-00006 TABLE 6 Raman data for the sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 2-dimethylaminoethanol with wavenumber in cm.sup.−1 and intensity values. It should be noted that the intensities vary with Laser power, sample amount and other factors. wavenumber (cm.sup.−1) intensity (arbitrary units) 3054 0.08 2955 0.16 2871 0.10 1609 0.91 1580 0.27 1552 0.27 1463 0.20 1420 0.14 1345 0.33 1307 0.34 1272 0.27 1183 0.21 1059 0.08 1023 0.07 948 0.08 890 0.10 864 0.15 836 0.09 777 0.10 649 0.30 637 0.20 480 0.11 414 0.08 372 0.09

Example 9: Kinetic Solubility of the Sodium Salt of 5-methyl-(6S)-tetrahydrofolic Acid Comprising 2-dimethylaminoethanol

(23) 55 mg of crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 2-dimethylaminoethanol is weighed into a 4 ml glass vial with screw cap. Then 1.0 ml of purified/de-ionized water is added. The mixture is agitated at room temperature and a clear solution is readily obtained (within a few seconds). Thus the solubility is greater than 55 mg per 1 ml of water. The solution remains clear for several hours at r.t.

Example 10: Preparation of the Crystalline 1:1:1 Salt of 5-methyl-(6S)-tetrahydrofolic Acid, Sodium and 4-(2-hydroxyethyl)-morpholine Starting from 5-methyl-(6S)-tetrahydrofolic Acid

(24) To a mixture of 40 g 5-methyl-(6S)-tetrahydrofolic acid (assay 5-methyltetrahydrofolic acid 95.42% w/w) and 86 g water 3.46 g solid sodium hydroxide and 21.79 g 4-(2-hydroxyethyl) morpholine were added at room temperature under a nitrogen atmosphere while stirring. The mixture was added within 5.5 hours to a mixture of 472 g ethanol with 5% v/v 2-propanol and 0.4 g crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 23° C. while stirring. 3 g water were added and the mixture was stirred for an additional hour at 23° C. The solids were separated by filtration, washed three times with each 133 mL ethanol 5% 2-propanol/water 1:7 v/v and dried in vacuum at 40° C. for 16.5 hours to give 46.07 g of an off-white powder corresponding to 85% yield (assay 5-methyltetrahydrofolic acid 70.6% w/w). PXRD confirmed the crystalline nature of the sample. .sup.1H-NMR shows that when the integral for the two protons of 5-methyl-(6S)-tetrahydrofolic acid near 7.6 is normalized to 2.0 the resultant integral for the six protons of the 4-(2-hydroxy-ethyl)-morpholine that appear near 3.7 is 5.96. This shows that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to 4-(2-hydroxy-ethyl)-morpholine is essentially 1:1. Further investigation by TGA (Thermogravimetric Analysis) revealed a mass loss of about 5.58%. The sodium content found by IC (Ion chromatography) was 3.5%. This shows that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to sodium is essentially 1:1. HPLC showed a purity of 98.2%.

BRIEF DESCRIPTION OF THE DRAWINGS

(25) FIG. 1: PXRD of the crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 4-(2-hydroxyethyl)-morpholine according to Example 1.

(26) FIG. 2: Raman spectrum of the crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 4-(2-hydroxyethyl)-morpholine according to Example 1.

(27) FIG. 3: Microscopy image of sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 4-(2-hydroxyethyl)-morpholine according to Example 1.

(28) FIG. 4: Microscopy image of calcium salt of 5-methyl-(6S)-tetrahydrofolic acid (dry powder sample).

(29) FIG. 5: Microscopy image of calcium salt of 5-methyl-(6S)-tetrahydrofolic acid (dispersed in paraffin oil).

(30) FIG. 6: PXRD pattern of the crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 1-(2-hydroxyethyl)-pyrrolidine according to Example 6

(31) FIG. 7: Raman spectrum of the crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 1-(2-hydroxyethyl)-pyrrolidine according to Example 6

(32) FIG. 8: PXRD pattern of the crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 2-dimethylaminoethanol according to Example 8

(33) FIG. 9: Raman spectrum of the crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid comprising 2-dimethylaminoethanol according to Example 8