COMPOSITION COMPRISING (6S)-5-METHYL TETRAHYDROFOLIC ACID OR SALT THEREOF, AND PREPARATION AND USE THEREOF

Abstract

Disclosed are a composition including (6S)-5-methyl tetrahydrofolic acid or a salt thereof, and preparation and use thereof. In the composition, the content of (6S)-5-methyl tetrahydrofolic acid or the salt thereof is not less than 98.0%, the content of a related impurity JK12A is not greater than 0.1%, and 5-methyl tetrahydropterioic acid is not detectable.

Claims

1. A composition comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof, wherein the content of (6S)-5-methyl tetrahydrofolic acid or the salt thereof in the composition is not less than 98.0%, the content of a related impurity JK12A is not greater than 0.1%, and 5-methyl tetrahydropterioic acid is not detectable ##STR00007##

2. The composition comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof according to claim 1, wherein the content of (6S)-5-methyl tetrahydrofolic acid or the salt thereof is not less than 99.0%, the content of the related impurity JK12A is not greater than 0.1%, and 5-methyl tetrahydropterioic acid is not detectable.

3. The composition comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof according to claim 1, wherein the salt of (6S)-5-methyl tetrahydrofolic acid is selected from a potassium salt, a sodium salt, a calcium salt, a magnesium salt, a barium salt, a zinc salt, a D-glucosamine salt, a D-galactosamine salt, or an arginine salt.

4. A method for preparing a composition comprising high-purity (6S)-5-methyl tetrahydrofolic acid or a salt thereof according to claim 1, the method comprising a method A or a method B, wherein the method A comprises reacting a crude salt of (6S)-5-methyl tetrahydrofolic acid with a reverse reducing agent in water at pH 6-8 at a temperature of 50-90° C. directly, or reacting ultrasonically, to obtain a composition comprising the salt of (6S)-5-methyl tetrahydrofolic acid after reaction; and the method B comprises reacting crude (6S)-5-methyl tetrahydrofolic acid, a reverse reducing agent, and a corresponding salt in water at pH 6-8 at a temperature of 50-90° C., or reacting ultrasonically, to obtain a composition comprising a salt of (6S)-5-methyl tetrahydrofolic acid after reaction; wherein the reverse reducing agent is one or more selected from vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an ester of isovitamin C, a salt of isovitamin C, mercaptoethanol, cysteine, mercaptoethane sulfonic acid, dithiothreitol, reductive glutathione, or zinc sulfate.

5. The method for preparing a composition comprising high-purity (6S)-5-methyl tetrahydrofolic acid or a salt thereof according to claim 4, wherein the reverse reducing agent is one or more selected from vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an ester of isovitamin C, or a salt of isovitamin C and the corresponding salt is a chloride.

6. The method for preparing a composition comprising high-purity (6S)-5-methyl tetrahydrofolic acid or a salt thereof according to claim 4, wherein during the reaction of the method A or B, the reaction temperature is 60-80° C., and the pH is 7-7.5 and adjusted with sodium hydroxide; and the concentration of the reverse reducing agent in water is not less than 2%, by weight.

7. A solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof and a stabilizing agent, wherein the solid solution is composed essentially of (6S)-5-methyl tetrahydrofolic acid or a salt thereof and the stabilizing agent, and the total content of the stabilizing agent and (6S)-5-methyl tetrahydrofolic acid or the salt thereof in a liquid chromatogram of the solid solution is greater than 99.5%, wherein the stabilizing agent is one or more selected from vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an ester of isovitamin C, a salt of isovitamin C, dithiothreitol, reductive glutathione, or zinc sulfate.

8. The solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof and a stabilizing agent according to claim 7, wherein the total content of the stabilizing agent and (6S)-5-methyl tetrahydrofolic acid or the salt thereof in a liquid chromatogram of the solid solution is greater than 99.8%.

9. The solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof and a stabilizing agent according to claim 7, wherein the stabilizing agent is one or more selected from vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an ester of isovitamin C, or a salt of isovitamin C.

10. A method for preparing the solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof and a stabilizing agent according to claim 7, the method comprising a method C or a method D, wherein the method C comprises reacting crude (6S)-5-methyl tetrahydrofolic acid or a high-purity (6S)-5-methyl tetrahydrofolic acid with a stabilizing agent in water at pH 3-5 at a temperature of 50-90° C. directly, or reacting ultrasonically, and separating the solid that is crystallized out after reaction; and the method D comprises reacting crude (6S)-5-methyl tetrahydrofolic acid of or a high-purity (6S)-5-methyl tetrahydrofolic acid a stabilizing agent and a corresponding salt in water at pH 6-8 at a temperature of 50-90° C., or reacting ultrasonically, and separating the solid that is crystallized out after reaction; wherein the stabilizing agent is one or more selected from vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an ester of isovitamin C, a salt of isovitamin C, dithiothreitol, reductive glutathione or zinc sulfate; and the corresponding salt is a chloride and wherein the high-purity (6S)-5-methyl tetrahydrofolic acid is a composition comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof, wherein the content of (6S)-5-methyl tetrahydrofolic acid or the salt thereof in the composition is not less than 98.0%, the content of a related impurity JK12A is not greater than 0.1%, and 5-methyl tetrahydropterioic acid is not detectable ##STR00008##

11. The method for preparing a solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof and a stabilizing agent according to claim 10, wherein during the reaction of the method C, the reaction temperature is 60-80° C., and the pH is 3-4 and adjusted with hydrochloric acid; and during the reaction of the method D, the reaction temperature is 60-80° C., and the pH is 7-7.5 and adjusted with sodium hydroxide.

12. A pharmaceutical composition, comprising a) the composition comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof according to claim 1, or a solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof and a stabilizing agent, wherein the solid solution is composed essentially of (6S)-5-methyl tetrahydrofolic acid or a salt thereof and the stabilizing agent, and the total content of the stabilizing agent and (6S)-5-methyl tetrahydrofolic acid or the salt thereof in a liquid chromatogram of the solid solution is greater than 99.5%, Wherein the stabilizing agent is one or more selected from vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an ester isovitamin C, a salt of isovitamin C, dithiothreitol, reductive glutathione, or zinc sulfate; and b) a reducing agent, which is one or more selected from vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an ester of isovitamin C, a salt of isovitamin C, mercaptoethanol, cysteine, mercaptoethane sulfonic acid, dithiothreitol, reductive glutathione, or zinc sulfate.

13. The pharmaceutical composition according to claim 12, which is in the form of common tablets, sustained release tablets, controlled release tablets, effervescent tablets, granules, common capsules, sustained release capsules, controlled release capsules, dry suspensions, oral solutions, oral suspensions, injections, or freeze-dried powder injections, wherein the weight ratio of the reducing agent to the (6S)-5-methyl tetrahydrofolic acid or the salt thereof is 0.02-1000:1.

14. A method of treating or preventing diseases associated with deficiency of 5-methyl tetrahydrofolic acid or a salt thereof in mammals, comprising administering to a patient in need thereof a drug comprising (i) the composition comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof according to claim 1, or (ii) a solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof and a stabilizing agent, wherein the solid solution is composed essentially of (6S)-5-methyl tetrahydrofolic acid or a salt thereof and the stabilizing agent, and the total content of the stabilizing agent and (6S)-5-methyl tetrahydrofolic acid or the salt thereof in a liquid chromatogram of the solid solution is greater than 99.5%, wherein the stabilizing agent is one or more selected from vitamin C, an ester of vitamin C, a salt of vitamin C, isovitamin C, an ester of isovitamin C, a salt of isovitamin C, dithiothreitol, reductive glutathione, or zinc sulfate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0089] FIG. 1 is a partially enlarged view of a large number of vacuoles anatomically found after severe renal tubular necrosis of the mice 14 days after administration of JK1303.

DETAILED DESCRIPTION

[0090] For better understanding of the technical solutions of the present invention, the technical solutions of the present invention will be further described below with reference to specific embodiments; however the present invention is not limited thereto.

[0091] The conditions used for detection by HPLC in the present invention are as follows.

[0092] Chromatographic Conditions:

TABLE-US-00002 Chromatographic column C18-250 * 4.6 mm-5 μm Detection wavelength UV at 280 nm Flow rate 1.0 ml/min Injection volume 20 μl Column temperature 30° C.

[0093] Formulation of Mobile Phases

[0094] Mobile phase A: 7.80 g of NaH.sub.2PO.sub.4.2H.sub.2O was weighed, dissolved in 1000 ml of water, and adjusted to pH 6.5 with a 32% NaOH solution.

[0095] Mobile phase B: 5.07 g of NaH.sub.2PO.sub.4.2H.sub.2O was weighed, dissolved in 650 ml of water, added with 350 ml of methanol, and adjusted to pH 8.0 with a 32% NaOH solution.

[0096] Gradient Program:

TABLE-US-00003 T (min) A % B % 0 100 0 14 45 55 17 0 100 22 0 100 31 100 0

[0097] Preparation of Sample Solution:

[0098] A suitable amount of a sample was dissolved in cold water at 2-8° C. (which was prepared by adding sodium sulfite under nitrogen atmosphere, distilling, collecting, and storing, and cooled to 2-8° C.) under nitrogen atmosphere, to formulate a solution of about 0.5 mg/ml. The solution was prepared in situ when used.

[0099] In some example of the present invention, ultrasonic waves are used as an aid, which can obviously accelerate the reversion and crystallization.

[0100] For ease of description, the data detected in the examples is only with respect to 5-methyl tetrahydrofolic acid, salts thereof, and related substances.

Example 1. Preparation of JK12A

[0101] Under nitrogen atmosphere, 10 g of (6S)-5-methyl tetrahydrofolic acid was transferred to a reaction flask, and 80 g of water was added, and adjusted to pH 7.3 with a 10% sodium hydroxide solution with stirring. After the solid was completely dissolved, 5 g of activated carbon was added, and reacted overnight with stirring in open reactor. After the raw material was detected to be reacted completely by HPLC, the reaction solution was filtered, and the filtrate was adjusted to pH 4.0 with 50% acetic acid. A crystal was formed, and filtered. The filter cake was washed respectively with ethanol and acetone, and dried under vacuum, to obtain 6.0 g of JK12A as a yellow solid (chemical purity 99.42%).

Example 2. Preparation of 5-Methyl Tetrahydropterioic Acid

[0102] 61 g of (6S)-5-methyl tetrahydrofolic acid was slowly added to 600 ml of a 25% sodium hydroxide solution in water, heated to reflux, and reacted overnight. After the raw material was reacted completely, the reaction solution was cooled to room temperature, and the impurities were filtered off. The filtrate was adjusted to pH 9-10 with concentrated sulfuric acid, and the impurities were precipitated out. The reaction solution was cooled to 10-15° C., and filtered. The filtrate was further adjusted to pH 2 with concentrated sulfuric acid, and a solid was precipitated out, stirred for 0.5 hr at 60° C., and filtered while hot. The solid was washed with water, and dried, to obtain 44 g of a white solid (purity 96%).

Example 3. Preparation of JK1303

[0103] 5 g of (6S)-5-methyl tetrahydropterioic acid was fed to a three-neck flask, 50 g of water was added, stirred fully, and adjusted to pH 9.0 with a 10% sodium hydroxide solution. After the solid was completely dissolved, 3 g of activated carbon was added, the oxygen balloon was closed, and the stirring was continued overnight. The reaction solution was filtered, and the filter cake was washed with water. The filtrate was adjusted to pH 4 with a 50% acetic acid solution in water, and the crystallization was continued for 30 min with stirring. After filtering, the filter cake was washed twice with water, slurried twice in 30 ml acetone, and dried at 30° C. under vacuum, to obtain 3.04 g of a product (purity by HPLC: 94.3%).

Example 4. Reversion of JK12A

[0104] 10 g of water was added to 0.1 g of JK12A (purity by HPLC: 95.91%), and adjusted to pH 7.2 with 10% sodium hydroxide with stirring. After the solid was completely dissolved, 0.8 g of dithiothreitol was slowly added, and stirred for 1 hr. The reaction solution was sampled and detected to contain 97.53% of 5-methyl tetrahydrofolic acid and 0.72% of JK12A residue.

Example 5. Reversion of JK12A

[0105] 10 g of water was added to 0.1 g of JK12A (purity by HPLC: 95.91%), and adjusted to pH 7.2 with 10% sodium hydroxide with stirring. After the solid was completely dissolved, 0.5 g of cysteine was slowly added, and stirred for 1 hrs. The reaction solution was sampled and detected to contain 96.64% of 5-methyl tetrahydrofolic acid and 0.85% of JK12A residue.

Example 6. Reversion of JK12A

[0106] 0.1 g of JK12A (purity by HPLC: 74.04%) and 0.2 g of mercaptoethanol were added to 5 ml of water, and adjusted to pH 7.5 with 30% sodium hydroxide with stirring. After the solid was completely dissolved, stirring was further continued for 1 hr. Then the reaction solution was sampled and detected to contain 60.92% of 5-methyl tetrahydrofolic acid and 0.59% of JK12A residue.

Example 7. Reversion of JK12A

[0107] 0.1 g of JK12A (purity by HPLC: 74.04%) and 0.2 g of sodium mercaptoethane sulfonate were added to 5 ml of water, and adjusted to pH 7.5 with 30% sodium hydroxide with stirring. After the solid was completely dissolved, stirring was further continued for 1 hr. Then the reaction solution was sampled and detected to contain 72.85% of 5-methyl tetrahydrofolic acid and 0.61% of JK12A residue.

Example 8. Preparation and Conversion of Mixture Comprising (6S)-Mefox and JK12A

[0108] Under nitrogen atmosphere, 1 g of (6S)-5-methyl tetrahydrofolic acid was transferred to a reaction flask, and 8 g of water was added, stirred, and adjusted to pH 7.3 with a 10% sodium hydroxide solution. After the solid was completely dissolved, 3 g of activated carbon was added, and reacted for 10 hrs with stirring in open reactor. The reaction solution was detected by HPLC to contain 12.20% of 5-methyl tetrahydrofolic acid; 83.51% of JK12A, and 0.57% of (6S)-Mefox), and filtered. The filtrate was added with 10 g of vitamin C, and the pH was adjusted to and maintained at 7.0 with caustic soda liquid. After stirring for 3 hrs at 60° C., the reaction solution was sampled and detected to contain 96.49% of 5-methyl tetrahydrofolic acid; 0% of JK12A; and 0% of (6S)-Mefox

Example 9. Reversion of JK1303

[0109] 10 g of water was added to 0.1 g of JK1303 (purity by HPLC: 96.7%), and adjusted to pH 7.0 with 10% sodium hydroxide with stirring. After the solid was completely dissolved, 1 g of sodium vitamin C was slowly added, and stirred for 1 hr. The reaction solution was sampled and detected to contain 96.6% of 5-methyl tetrahydropterioic acid and 0% of JK1303 residue.

Example 10. Composition Comprising (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt

[0110] 50 ml of water and 50 g of vitamin C were neutralized to pH 7.0 with sodium hydroxide, and the solution became clear after the solid was completely dissolved. 1 g of crude (6S)-5-methyl tetrahydrofolic acid calcium salt was added batchwise, and ultrasonicated at 65° C. for 2 hrs. After filtering, washing, and drying under vacuum, 0.72 g of a composition comprising (6S)-5-methyl tetrahydrofolic acid calcium salt was obtained.

TABLE-US-00004 Crude (6S)-5- methyl tetrahydrofolic acid calcium Final Related substance salt/content product/content JK12A 0.49% — 5-methyl tetrahydrofolic acid 99.26% 99.9% 5-methyl tetrahydropterioic acid 0.14% — Other impurities 0.11% 0.01

[0111] The content is in percentages by weight, the same below.

Example 11. Composition Comprising (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt

[0112] 15 g of (6S)-5-methyl tetrahydrofolic acid and 150 g of vitamin C were added to 225 ml of water, and neutralized to pH 7.0 with a 30% sodium hydroxide solution in water. The solid was dissolved, and the solution was stirred for 1 hr. Then 15 g of a 40% calcium chloride solution in water was added and ultrasonicated at 70° C. for crystallization. After 3 hrs, white particles were crystallized out, filtered, and then washed with water. The filter cake was dried under vacuum, to obtain 10.1 g of a solid composition comprising (6S)-5-methyl tetrahydrofolic acid calcium salt.

TABLE-US-00005 Crude (6S)-5-methyl Final Related substance tetrahydrofolic acid/content product/content JK12A 0.22% 0.03% (6S)-Mefox 0.02% 0.01% 5-methyl tetrahydrofolic 99.40% 99.95% acid 5-methyl tetrahydropterioic 0.03% — acid Other impurities 0.33% 0.01%

Example 12. Composition Comprising (6S)-5-Methyl Tetrahydrofolate

[0113] 1 g of (6S)-5-methyl tetrahydrofolic acid, 1 g of vitamin C, and 0.5 g of NaCl were added to 10 ml of water, and neutralized to pH 7.0 with a 30% sodium hydroxide solution in water. The solid was dissolved, and the solution was stirred for 1 hr. Then 1 g of a 40% calcium chloride solution in water was added and ultrasonicated at 70° C. After 1 hr, a solid was crystallized out, filtered, and then washed. The filter cake was dried under vacuum, to obtain 0.52 g of a solid composition comprising (6S)-5-methyl tetrahydrofolic acid calcium salt.

TABLE-US-00006 Crude (6S)-5-methyl Final Related substance tetrahydrofolic acid/content product/content JK12A 0.22% 0.07% (6S)-Mefox 0.02% 0.01% 5-methyl tetrahydrofolic 99.40% 99.75% acid 5-methyl tetrahydropterioic 0.03% — acid Other impurities 0.33% 0.15%

Example 13. Composition Comprising (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt

[0114] 0.5 g of (6S)-5-methyl tetrahydrofolic acid, 10 mg of vitamin C, and 0.1 g of sodium chloride were added to 10 ml of water, and neutralized to pH 7.5 with a 30% sodium hydroxide solution in water. The solution became clear after the solid was completely dissolved. The reaction solution was stirred for 1 hr under nitrogen atmosphere. 1 g of a 20% calcium chloride solution in water was added, and ultrasonicated at 70° C. for crystallization. After 1.0 hr, white particles were crystallized out, filtered, and then washed. The filter cake was dried under vacuum, to obtain 0.38 g of a solid composition comprising (6S)-5-methyl tetrahydrofolic acid calcium salt.

TABLE-US-00007 Crude (6S)-5-methyl Final Related substance tetrahydrofolic acid/content product/content JK12A 0.22% 0.09% (6S)-Mefox 0.02% 5-methyl tetrahydrofolic 99.40% 99.63% acid 5-methyl tetrahydropterioic 0.03% — acid Other impurities 0.33% 0.27%

Example 14. Composition Comprising (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt

[0115] 1 g of (6S)-5-methyl tetrahydrofolic acid and 3 g of isovitamin C were added to 20 ml of water, and neutralized to pH 7.0 with a 30% sodium hydroxide solution in water. The solution became clear after the solid was completely dissolved. The reaction solution was stirred for 1 hr, and then 1 g of a 40% calcium chloride solution in water was added, and ultrasonicated at 60° C. After 1 hr, a solid were crystallized out, filtered, and then washed. The filter cake was dried under vacuum, to obtain 0.80 g of a composition comprising (6S)-5-methyl tetrahydrofolic acid calcium salt.

TABLE-US-00008 Crude (6S)-5-methyl Final Related substance tetrahydrofolic acid/content product/content JK12A 0.22% — (6S)-Mefox 0.02% 0.05% 5-methyl tetrahydrofolic 99.40% 99.75%  acid 5-methyl tetrahydropterioic 0.03% — acid Other impurities 0.33% 0.18%

Example 15. Composition Comprising (6S)-5-Methyl Tetrahydrofolic Acid

[0116] 0.5 g of the composition comprising calcium (6S)-5-methyl tetrahydrofolate prepared in Example 11 was heated to 40° C., and adjusted to pH 4.0 with a hydrochloric acid solution. The system was stirred for 1 hr for crystallization while the pH was maintained unchanged. After cooling to room temperature, filtering, washing, and drying at 25° C. under vacuum, 0.23 g of a composition comprising (6S)-5-methyl tetrahydrofolic acid was obtained, which contains 99.80% of (6S)-5-methyl tetrahydrofolic acid, 0% of JK12A, 0.07% of (6S)-Mefox, and 0% of 5-methyl tetrahydropterioic acid, as detected by HPLC.

TABLE-US-00009 Composition of (6S)-5- methyl tetrahydrofolic acid Final Related substance calcium salt/content product/content JK12A 0.03% — (6S)-Mefox 0.01% — 5-methyl tetrahydrofolic 99.95%  99.80% acid 5-methyl tetrahydropterioic — — acid Other impurities 0.01%  0.20%

Example 16. Composition Comprising 6R,S-5-Methyl Tetrahydrofolic Acid Calcium Salt

[0117] 1 g of 6R,S-5-methyl tetrahydrofolic acid and 10 g of vitamin C were added to 15 ml of water, and neutralized to pH 7.0 with a 30% sodium hydroxide solution in water. The solution became clear after the solid was completely dissolved. 1 g of a 40% calcium chloride solution in water was added, and ultrasonicated at 70° C. for crystallization. After filtering, washing with water, and drying under vacuum, 0.54 g of a composition comprising 6R,S-5-methyl tetrahydrofolate was obtained.

TABLE-US-00010 Crude 6R, S-5-methyl Final Related substance tetrahydrofolate/content product/content JK12A 0.21% — (6S)-Mefox — — 5-methyl tetrahydrofolic acid 99.15%  99.59%  5-methyl tetrahydropterioic 0.28% 0.08% acid Other impurities 0.36% 0.33%

Example 17. Composition Comprising Sodium 6R,S-5-Methyl Tetrahydrofolic Acid

[0118] 1 g of 6R,S-5-methyl tetrahydrofolic acid, and 5 g of vitamin C were added to 20 ml of water, and neutralized to pH 7.0 with a 30% sodium hydroxide solution in water. The solution became clear after the solid was completely dissolved. The reaction was continued for 2 hrs at 60° C. with oxygen being isolated. Then, the reaction solution was added dropwise to 200 ml of ethanol, to crystallize a sodium salt out, which was filtered, washed, and dried under vacuum, to obtain 0.52 g of a composition comprising sodium 6R,S-5-methyl tetrahydrofolate.

TABLE-US-00011 Crude 6R, S-5-methyl Final Related substance tetrahydrofolic acid/content product/content JK12A 0.21% — (6S)-Mefox — — 5-methyl tetrahydrofolic 99.15%  99.64%  acid 5-methyl tetrahydropterioic 0.28% 0.12% acid Other impurities 0.36% 0.34%

Example 18. Composition Comprising of (6S)-5-Methyl Tetrahydrofolic Acid Arginine Salt

[0119] 5 g of vitamin C was added to 5 ml of water, and neutralized to pH 7.0 with sodium hydroxide. The solution became clear after the solid was completely dissolved. 0.5 g of crude (6S)-5-methyl tetrahydrofolic acid arginine salt was added, dissolved completely, and ultrasonically stirred for 2 hrs at 60° C. The reaction solution was added dropwise to 50 ml of ethanol, and stirred to crystallize a solid out. The solid was filtered out and detected. 0.26 g of a composition comprising (6S)-5-methyl tetrahydrofolic acid arginine salt was obtained.

TABLE-US-00012 Crude 5-methyl tetrahydrofolic acid Final Related substance arginine salt/content product/content JK12A 0.76% — (6S)-Mefox 1.30% 0.27% 5-methyl tetrahydrofolic acid 97.69% 99.70%  5-methyl tetrahydropterioic acid 0.05% — Other impurities 0.20% 0.03%

Example 19. Composition Comprising (6S)-5-Methyl Tetrahydrofolic Acid D-Glucosamine Salt

[0120] 5 g of vitamin C was added to 10 ml of water, and neutralized to pH 7.0 with sodium hydroxide. The solution became clear after the solid was completely dissolved. 2.0 g of crude (6S)-5-methyl tetrahydrofolic acid D-glucosamine salt was added, dissolved completely, and ultrasonically stirred for 2 hrs at 60° C. Ethanol was slowly added dropwise, and a solid was crystallized out, which was filtered and recrystallized in ethanol, to obtain a composition comprising (6S)-5-methyl tetrahydrofolic acid D-glucosamine salt.

TABLE-US-00013 Crude 5-methyl tetrahydrofolic acid Final Related substance D-glucosamine salt/content product/content JK12A 0.56% — (6S)-Mefox 0.70% 0.14 5-methyl tetrahydrofolic 98.47% 99.81% acid D-glucosamine salt 5-methyl tetrahydropterioic 0.05% — acid Other impurities 0.22%  0.05%

Example 20. Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt and Vitamin C

[0121] 1 g of (6S)-5-methyl tetrahydrofolic acid and 2.0 g of vitamin C were added to 20 ml of water, and neutralized to pH 7.5 with a 30% sodium hydroxide solution in water. The solid was dissolved, and the solution was stirred for 1 hr. Then 1 g of a 40% calcium chloride solution in water was added and ultrasonicated at 70° C. for crystallization. After 1.0 hr, white particles were crystallized out, filtered, dried, and detected by HPLC to comprise 94.89% of (6S)-5-methyl tetrahydrofolic acid calcium salt, and 5.11% of vitamin C.

Example 21: Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt and Isovitamin C

[0122] 0.1 g of (6S)-5-methyl tetrahydrofolic acid and 0.3 g of isovitamin C were added to 1.5 ml of water, and neutralized to pH 7.5 with a 30% sodium hydroxide solution in water. The solid was dissolved, and the solution was stirred for 1 hr. Then 1 g of a 40% calcium chloride solution in water was added and ultrasonicated at 90° C. for crystallization. After 1.0 hr, white particles were crystallized out, which were filtered, dried, and detected by HPLC to comprise 29.29% of (6S)-5-methyl tetrahydrofolic acid calcium salt, and 70.66% of isovitamin C.

Example 22. Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt and Zinc Sulfate

[0123] 0.5 g of (6S)-5-methyl tetrahydrofolic acid and 1.0 g of zinc sulfate were added to 10 ml of water, and neutralized to pH 7.5 with a 30% sodium hydroxide solution in water. The solid was dissolved, and the solution was stirred for 1 hr. Then 1 g of a 20% calcium chloride solution in water was added and ultrasonicated at 70° C. for crystallization. After 1.0 hr, a solid solution was obtained, which was filtered, dried, and detected by HPLC (with an ELSD detector) to comprise 27.69% of (6S)-5-methyl tetrahydrofolic acid calcium salt, and 72.26% of zinc sulfate.

Example 23. Solid Solution Comprising 5-Methyl Tetrahydrofolic Acid Calcium Salt and Reductive Glutathione

[0124] 0.5 g of (6S)-5-methyl tetrahydrofolic acid and 1.5 g of reductive glutathione were added to 10 ml of water, and neutralized to pH 7.5 with a 30% sodium hydroxide solution in water. The solid was dissolved, and the solution was stirred for 1 hr. Then 1 g of a 20% calcium chloride solution in water was added and ultrasonicated at 70° C. for crystallization. After 1.0 hr, white particles were crystallized out, which were filtered, dried, and detected by HPLC (with an ELSD detector) to comprise 37.62% of (6S)-5-methyl tetrahydrofolic acid calcium salt, and 62.33% of reductive glutathione.

Example 24. Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic Acid and Vitamin C

[0125] 0.5 g of (6S)-5-methyl tetrahydrofolic acid and 3.0 g of vitamin C were added to 10 ml of water, and neutralized to pH 7.5 with a 30% sodium hydroxide solution in water. The solid was dissolved, and the solution was stirred for 1 hr. A suitable amount of 6N hydrochloric acid solution in water was added dropwise, to adjust the pH to 4.0, and stirred 50° C. for crystallization. After 1.0 hr, white particles were crystallized out, which were filtered, dried, and detected by HPLC to comprise 26.80% of (6S)-5-methyl tetrahydrofolic acid and 73.17% of vitamin C.

Example 25. Solid Solution Comprising (6S)-5-Methyl Tetrahydrofolic Acid Zinc Salt and Vitamin C

[0126] 0.5 g of (6S)-5-methyl tetrahydrofolic acid and 1.5 g of vitamin C were added to 10 ml of water, and neutralized to pH 7.5 with a 20% sodium hydroxide solution in water. The solution became clear after the solids were completely dissolved. 0.8 g of a 50% zinc chloride solution in water was added, crystallized for 2 hrs, and filtered under nitrogen atmosphere. The filter cake was dried under vacuum and detected by HPLC to comprise 88.7% of (6S)-5-methyl tetrahydrofolic acid zinc salt, and 11.0% of vitamin C.

Example 26: Solid Solution of (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt, and Magnesium Vitamin C Phosphate

[0127] 0.5 g of (6S)-5-methyl tetrahydrofolic acid, and 2.0 g of magnesium vitamin C phosphate were added to 10 ml of water, and neutralized to pH 7.5 with a 20% sodium hydroxide solution in water. The solution became clear after the solids were completely dissolved. 0.6 g of a 50% calcium chloride solution in water was added, and then reacted ultrasonically at 70° C. After 1.5 h, white particles were crystallized out, which were filtered under nitrogen atmosphere, dried and detected by HPLC to comprise 62.4% of (6S)-5-methyl tetrahydrofolic acid calcium salt, and 37.4% of vitamin C.

Example 27. Stability of Composition Comprising (6S)-5-Methyl Tetrahydrofolic Acid Calcium Salt Prepared Through METHOD JK and Vitamin C

[0128] 1 g of the composition of (6S)-5-methyl tetrahydrofolic acid calcium salt obtained in Example 11 was fully mixed by grinding with vitamin C in various proportions by weight, placed in an incubator at 25° C. and 40% humidity, and detected for (6S)-5-methyl tetrahydrofolic acid calcium salt and JK12A periodically.

TABLE-US-00014 Amount of VC 0 20% 100% (6S)-5-methyl (6S)-5-methyl (6S)-5-methyl tetrahydrofolic JK1 tetrahydrofolic JK1 tetrahydrofolic JK1 Days acid calcium salt 2A acid calcium salt 2A acid calcium salt 2A 0 99.95% 0.03% 99.95% 0.03% 99.95% 0.03% 7 99.90% 0.06% 99.94% 0.04% 99.94% 0.03% 15 99.85% 0.12% 99.89% 0.08% 99.90% 0.05% 30 99.63% 0.25% 99.71% 0.17% 99.90% 0.05% 60 99.40% 0.46% 99.50% 0.22% 99.86% 0.10% Amount of VC 500% 1000% 10000% (6S)-5-methyl (6S)-5-methyl (6S)-5-methyl tetrahydrofolic JK1 tetrahydrofolic JK1 tetrahydrofolic JK1 Days acid calcium salt 2A acid calcium salt 2A acid calcium salt 2A 0 99.95% 0.03% 99.95% 0.03% 99.95% 0.03% 7 100.00% — 100.00% — 100.00% — 15 100.00% — 100.00% — 100.00% — 30 99.98% — 100.00% — 100.00% — 60 99.96% — 100.00% — 100.00% —