Abstract:
Disclosed are a crystal form of (6S)-5-methyltetrahydrofolate salt and a method for preparing the same. The crystal form is: Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 6.3±0.2 and 19.2±0.2; or the crystal form of (6S)-5-methyltetrahydrofolate strontium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 6.5±0.2 and 22.0±0.2. The crystal form of (6S)-5-methyltetrahydrofolate salt of the present invention has the advantages of excellent physicochemical properties, good stability, high purity, good reproducibility, and being more suitable for production on an industrial scale.

Description:
BACKGROUND 
       [0001]    1. Technical Field 
         [0002]    The present invention belongs to the field of crystal forms of compounds, and specifically relates to a crystal form of (6S)-5-methyltetrahydrofolate salt and methods for preparing the same and uses of the same. 
         [0003]    2. Related Art 
         [0004]    The crystal forms of active pharmaceutical ingredients are closely associated with the biological activity, bioavailability, dissolution, stability, and shelf life thereof. Therefore, during the research and development of new drugs, screening of crystal form is one of the most important tasks. Even if the drug has been on the market for many years, seeking more effective crystal forms of the drug is still the goal intensely pursued by pharmaceutical companies. 
         [0005]    5-methyltetrahydrofolic acid was first separated from a horse liver in the form of barium salt by Donaldson et al. in 1959 and was named as Prefolic-A, and was synthesized by a chemical method in 1961 (K. O. Donaldson et al., Fed. Proc, (1961), 20, 453). 
         [0006]    The 5-methyltetrahydrofolic acid molecule has two chiral carbon atoms, where the configuration of the chiral carbon atom at the glutamic acid site is certain, while the chiral carbon atom at Site 6 has two configurations R and S, and therefore, 5-methyltetrahydrofolic acid has been used in the form of a diastereomeric mixture. It is reported that, the two isomers have different effects with in vivo enzymes, where the compound with S configuration of the carbon atom at site 6 exhibits good efficacy, while the compound with R configuration of the carbon atom at site 6 almost has no efficacy in comparison. 
         [0007]    The chemical name of (6S)-5-methyltetrahydrofolic acid is (6S)—N[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-5-methyl-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid, referred to as (6S)-5-MTHF for short hereinafter. The structural formula is shown in Formula I: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0008]    (6S)-5-MTHF and salts thereof are very unstable and easily degraded, and especially are highly sensitive to oxygen and moisture (A. L. Fitzhugh, Pteridines 1993, 4(4), 187-191). In the air, (6S)-5-MTHF and salts thereof are easily oxidized into 5-methyldihydrofolic acid and/or folic acid and the like. Therefore, it is difficult to prepare high-purity and high-stability bulk pharmaceutical chemicals or food additives, to meet the quality standards. 
         [0009]    Due to the physical and chemical properties of (6S)-5-MTHF, it is difficult to prepare a stable crystal form of (6S)-5-MTHF by a conventional crystallization process. In the past few decades of production of (6S)-5-MTHF and preparation of formulations thereof, a reducing agent, for example, Vitamin C or 2-mercaptoethanol, is often added to achieve the purpose of anti-oxidation. 
         [0010]    Patent Document U.S. Pat. No. 5,223,500 reports a process for preparing a stable crystal form of (6S)-5-MTHF calcium salt. The process includes the following steps: first, preparing amorphous (6S)-5-MTHF calcium salt, then transferring into boiling water of 100° C. to form a solution, cooling and standing overnight at room temperature. The collected solid is called to be a stable crystal product. However, relevant crystal parameters are not reported in this patent. 
         [0011]    Patent Document U.S. Pat. No. 6,441,168 discloses a crystal form of (6S)-5-MTHF calcium salt with extremely high stability and a method for preparing the same. (6S)-5-MTHF sodium salt and calcium chloride are subjected to heat treatment in a polar solvent at about 90° C. to obtain four stable crystal forms of (6S)-5-MTHF calcium salt, which are respectively Form I having 2θ values of 6.3, 13.3, 16.8, and 20.1, Form II having 2θ values of 5.3, 6.9, 5.7, and 21.1, Form III having 2θ values of 6.8, 10.2, 15.4, and 22.5, and Form IV having 2θ values of 6.6, 15.9, 20.2, and 22.5. 
         [0012]    Patent Document WO2008144953 discloses a process for preparing a stable amorphous (6S)-5-MTHF calcium salt, in which a crystal form of (6S)-5-MTHF is used as a raw material, calcium chloride is added for slow crystallization. The whole crystallization process in this patent is very complex, and the crystallization time is 16 to 18 hours, thereby reducing the production capacity in the product procedure. 
       SUMMARY 
       [0013]    Surprisingly, it has been found now that by using ultrasonic waves to assist crystallization during the formation of a salt, a crystal form of (6S)-5-methyltetrahydrofolate salt with high stability and good chemical and optical purity can be obtained. 
         [0014]    In order to overcome the disadvantages in the prior art, an objective of the present invention is to provide a novel crystal form of (6S)-5-methyltetrahydrofolate salt with good stability, high purity, and good reproducibility. 
         [0015]    Preferably, the present invention sets forth Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 6.3±0.2 and 19.2±0.2. 
         [0016]    Preferably, the present invention sets forth a crystal form of (6S)-5-methyltetrahydrofolate strontium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 6.5±0.2 and 22.0±0.2. 
         [0017]    Preferably, the X-ray diffraction pattern of the Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt has diffraction peaks at the 2θ angles of 3.2±0.2, 6.3±0.2, 13.2±0.2, 14.6±0.2, 19.2±0.2, and 32.6±0.2. 
         [0018]    Preferably, the X-ray diffraction pattern of the crystal form of (6S)-5-methyltetrahydrofolate strontium salt has diffraction peaks at the 2θ angles of 6.5±0.2, 10.0±0.2, 13.7±0.2, 16.8±0.2, 17.1±0.2, 22.0±0.2, and 24.9±0.2. 
         [0019]    Preferably, the X-ray diffraction pattern of the Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt has diffraction peaks at the 2θ angles of 3.2±0.1, 6.3±0.1, 13.2±0.1, 14.6±0.1, 19.2±0.1, and 32.6±0.1; or, preferably, the X-ray diffraction pattern of the crystal form of (6S)-5-methyltetrahydrofolate strontium salt has diffraction peaks at the 2θ angles of 6.5±0.1, 10.0±0.1, 13.7±0.1, 16.8±0.1, 17.1±0.1, 22.0±0.1, and 24.9±0.1. 
         [0020]    Another objective of the present invention is to provide methods for preparing the crystal form of (6S)-5-methyltetrahydrofolate salt. 
         [0021]    The third objective of the present invention is to provide pharmaceutical compositions of the crystal form of (6S)-5-methyltetrahydrofolate salt. 
         [0022]    The fourth objective of the present invention is to provide uses of the crystal form of (6S)-5-methyltetrahydrofolate salt. 
         [0023]    The objectives of the present invention can be achieved according to the following ways: 
         [0024]    A crystal form of (6S)-5-methyltetrahydrofolate salt is provided, where the crystal form is Form C of the crystal form of (6S)-5-methyltetrahydrofolic acid calcium salt or a crystal form of (6S)-5-methyltetrahydrofolate strontium salt. 
         [0025]    In an aspect, the present invention provides Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt, where by using Cu-Ka radiation, the X-ray diffraction pattern has diffraction peaks at 2θ of 6.3±0.2 and 19.2±0.2 in degree, especially has one or more diffraction peaks at 2θ of 3.2±0.2, 6.3±0.2, 13.2±0.2, 14.6±0.2, 19.2±0.2, and 32.6±0.2, and preferably has one or more diffraction peaks at 2θ of 3.2±0.1, 6.3±0.1, 13.2±0.1, 14.6±0.1, 19.2±0.1, and 32.6±0.1. The X-ray diffraction pattern of the Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt exhibits strong diffraction peaks and low background spectrum, indicating high crystallinity. 
         [0026]    The further X-ray diffraction pattern of the Form C (6S)-5-methyltetrahydrofolic acid calcium salt is essentially shown in  FIG. 1 . The chemical purity of the Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt is further greater than 99.0%. 
         [0027]    In another aspect, the present invention provides a crystal form of (6S)-5-methyltetrahydrofolate strontium salt, where by using Cu-Ka radiation, the X-ray diffraction pattern has diffraction peaks at 2θ of 6.5±0.2 and 22.0±0.2 in degree, especially has one or more diffraction peaks at 2θ of 6.5±0.2, 10.0±0.2, 13.7±0.2, 16.8±0.2, 17.1±0.2, 22.0±0.2, and 24.9±0.2, and preferably has one or more diffraction peaks at 2θ of 6.5±0.1, 10.0±0.1, 13.7±0.1, 16.8±0.1, 17.1±0.1, 22.0±0.1, and 24.9±0.1. The X-ray diffraction pattern of the crystal form of (6S)-5-methyltetrahydrofolate strontium salt exhibits strong diffraction peaks and low background spectrum, indicating high crystallinity. 
         [0028]    The further X-ray diffraction pattern of the (6S)-5-methyltetrahydrofolate strontium salt is essentially shown in  FIG. 2 . 
         [0029]    In the present invention, the moisture content of the crystal form of (6S)-5-methyltetrahydrofolate salt is 10% to 18%, and further is 15% to 17%. 
         [0030]    In still another aspect, the present invention provides a method for preparing a (6S)-5-methyltetrahydrofolate salt, where the method includes crystallizing (6S)-5-methyltetrahydrofolate salt from a polar medium through ultrasonic assistance. 
         [0031]    In yet another aspect, the present invention provides a method for preparing a (6S)-5-methyltetrahydrofolate salt, specifically including the following steps: 
         [0032]    (1) Neutralization of (6S)-5-methyltetrahydrofolic acid with a base in a polar medium to full dissolution; the polar medium may be water, deionized water, or a solution formed by water and an organic solvent capable of being mixed uniformly with water, and may also be a salt; a preferred polar medium is water and deionized water. The base is an inorganic base or organic base capable of forming a salt with (6S)-5-methyltetrahydrofolic acid, the inorganic base is selected from alkali metal bases or alkaline earth metal bases, carbonates and bicarbonates; the organic base is selected from ammonia, amines, pyridines or piperazines, where potassium hydroxide, sodium hydroxide, calcium hydroxide, potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, ammonia, methylamine, 4-dimethyl-pyridine or piperazine are preferred. 
         [0033]    (2) Addition of an alkaline earth metal salt or an alkaline earth metal salt solution; the alkaline earth metal salt refers to an inorganic salt or organic salt that is soluble or partially solution in the polar medium, for example, calcium salt, magnesium salt, strontium salt, barium salt, and calcium chloride, hexahydrate calcium chloride and strontium chloride are preferred. 
         [0034]    (3) Heating to a temperature higher than 30° C., especially to a temperature of 30° C. to 60° C. or 60° C. to 100° C. 
         [0035]    (4) Introduction of ultrasonic waves and crystallization, and isolation of (6S)-5-methyltetrahydrofolate salt crystal. 
         [0036]    Ultrasonic technology is a simple, inexpensive technique, and is safe and convenient in use. On one hand, ultrasonic wave can strength the nucleation and growth of crystals. In the crystallization process, the introduction of ultrasonic waves may cause cavitation phenomenon, when cavitation bubbles burst, a certain micro-jet is produced, the micro-jet crushes crystal grains having a certain size, and a part of the crushed crystals serve as seed for crystal growth, thereby promoting the growth of crystals. On the other hand, in the crystallization process, ultrasonic waves are equivalent to a catalyst, and excite the molecular motion through translation, rotation and reversal, thereby increasing the crystallization rate of the entire system, and shortening the crystallization time. The ultrasonic waves can also improve the particle size distribution of the product, and with the increase of the ultrasonic power, the crystal particles show a tendency of decrease. Since no other reagents are added and no contaminants are introduced in the crystallization process, the ultrasonic waves can be used to prepare very pure crystalline materials, and this is very important for some materials with very strict requirements for purity, especially pharmaceutical products and foods. Compared with other crystallization starting methods such as stimulation crystallization starting method and seed charging crystallization starting method, the degree of supersaturation required by ultrasonic crystallization is low, the growth speed is fast, the resulting crystal is uniform, complete and clean, the crystal size distribution range is small, and the coefficient of variation is low. 
         [0037]    Ultrasonic waves have been used in crystalline area since 1927. In recent years, the utilization of ultrasonic waves in pharmaceutical and fine chemical industries is further promoted. Ishtiaq et al. reviewed the application of ultrasonic waves in the pharmaceutical field (World Applied sciences Journal (2009), 6(7), 856-893). However, so far, in the pharmaceutical industry, merely several papers and patents disclose and apply the ultrasonic crystallization technique, for example, paroxetine, aspartame, adipic acid, fenoterol hydrobromide (Organic Process Research &amp; Development 2005, 9, 923-932). 
         [0038]    The inventors apply ultrasonic waves in the field of crystallization of 5-methyltetrahydrofolic acid and salts thereof for the first time. We have found in experiments that when the ultrasonic power is 0.01 W/ml to 1.0 W/ml, the resulting crystal is uniform, complete, and clean, the crystal size distribution range is small, and the purity is high and is up to above 99.0%. Preferably, the ultrasonic power is 0.04 W/ml to 0.60 W/ml. 
         [0039]    Neutralization with a base in Step (1) generally refers to neutralization to a pH value of about 7.0, generally neutralization to a pH value of 6.5 to 8.5, preferably neutralization to a pH value of 7.0 to 7.5, and most preferably neutralization to a pH value of 7.0. The base may be directly added, and may also be added in the form of a solution (for example, aqueous solution). This method has not specific requirements on the amount of the polar medium, and an amount for a general reaction or a crystallization medium is suitable. 
         [0040]    When an alkaline earth metal salt solution is used in Step (2), generally a 5% to 50% aqueous solution of an alkaline earth metal salt, preferably a 25% to 50% aqueous solution of an alkaline earth metal salt, is used. 
         [0041]    The heating temperature in Step (3) is 30° C. to 60° C. or 60° C. to 100° C., preferably 40° C. to 80° C., and more preferably 65° C. to 70° C. 
         [0042]    In Step (4), after ultrasonic crystallization and isolation the crystal, a step of washing with water and drying (drying in vacuum at a temperature of 20° C. to 40° C.). 
         [0043]    Preparation of the crystal form of (6S)-5-methyltetrahydrofolate salt by using ultrasonic waves can be carried out naturally or be carried out by introducing a corresponding (6S)-5-methyltetrahydrofolate salt seed. 
         [0044]    In another aspect, the present invention relates to a composition containing at least one (6S)-5-methyltetrahydrofolate salt described above, since persons skilled in the art can understand based on the knowledge in the art that the composition of the present invention may further contain a pharmaceutically acceptable excipient or carrier. The carrier includes a diluent, a binder, a disintegrant, a lubricant, and an anti-oxidant, and these excipients are existing conventional excipients. The preparation form of the composition is an oral solid preparation or injection, for example, tablets, capsules, orally disintegrating tablets, lozenge, sustained-release preparations, injections, and lyophilized powder, prepared by adopting methods for corresponding formulations. 
         [0045]    A preparation is provided, which contains an effective amount of the crystal form of (6S)-5-methyltetrahydrofolate salt. 
         [0046]    In another aspect, the present invention discloses a use of the at least one (6S)-5-methyltetrahydrofolate salt and/or composition defined above in preparation of pharmaceuticals, food additives or nutritional supplements, where the pharmaceuticals, food additives, or nutritional supplements are used for preventing and/or treating defects or diseases positively impacted by administration of 5-methyltetrahydrofolate salt. 
         [0047]    Merely for example, the (6S)-5-methyltetrahydrofolate salt and/or composition of the present invention defined above may be used in preparation of pharmaceuticals, food additives, or nutritional supplements. The pharmaceuticals, food additives, or nutritional supplements are used for preventing and/or treating neurological diseases such as subacute encephali associated with dementia and vacuolar myelopathy; physiological and pathological vascular and cardiovascular disorders such as premature occlusive arterial diseases, severe vascular diseases in infancy and childhood, progressive arterial stenosis, intermittent claudication, renal vascular hypertension, ischemic cerebrovascular diseases, premature occlusive retinal artery and retinal vein, cerebral occlusive arterial diseases, occlusive peripheral arterial diseases, and premature death caused by thromboembolic diseases and/or ischemic heart diseases; autoimmune diseases such as psoriasis, celiac disease, arthritis and inflammatory conditions; megaloblastic anemia caused by folate inefficiency, intestinal malabsorption, used as antidote for folic acid antagonists (for example, methotrexate, pyrimethamine or trimethoprim); used for preventing serious toxic effects caused by methotrexate overdose or high-dose therapy, reducing risks of woman miscarriage and/or production of fetuses with neural tube defects, cleft defects and/or palate defects, keeping and/or normalizing homocysteine levels and/or metabolism; changes synthesis of and/or functions and/or variations in DNA and RNA and changes in synthesis of cells; and depression. 
         [0048]    The (6S)-5-methyltetrahydrofolate salt of the present invention exhibits long-term persistent chemical stability, and after long-term exposure in the air at a temperature of 40° C. and relative humidity of 60%, and the color of the crystal form has no significant changes, which is very important for application of the (6S)-5-methyltetrahydrofolate salt in pharmaceutical preparations. 
         [0049]    In addition to the rare high chemical stability, it may also be noted that, the (6S)-5-methyltetrahydrofolate salt of the present invention has good dissolution rate, and in water at a temperature of 25° C., the saturated state can be quickly reached within 1 min. The high dissolution rate can not only improve the producibility of preparations for parenteral administration such as injections, thereby facilitating industrial production, and can also be made into oral preparations, thereby having important biopharmaceutical advantages in oral administration of pharmaceuticals, because the high dissolution rate of the active pharmaceuticals improves the absorption rate of the active pharmaceuticals through the gastrointestinal wall. Additionally, the crystal form of the present invention further has the advantages of high crystallinity, uniform particle distribution, smooth surface, and high chemical purity of up to above 99.0%. 
         [0050]    The method for preparing the crystal form of (6S)-5-methyltetrahydrofolate salt of the present invention has the advantages that the reaction steps are simple, no pollution occurs, the obtained novel crystal form of (6S)-5-methyltetrahydrofolic acid calcium salt has high chemical stability, high purity, high dissolution rate, and high bioavailability, thereby providing a novel way for preparing (6S)-5-methyl-tetrahydrofolate crystalline salt. 
         [0051]    The crystal form of (6S)-5-methyltetrahydrofolate salt of the present invention has the advantages of excellent physicochemical properties, good stability, high purity, good reproducibility, and being more suitable for production on an industrial scale. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0052]    The disclosure will become more fully understood from the detailed description given herein below for illustration only, and thus are not limitative of the disclosure, and wherein: 
           [0053]      FIG. 1  shows an X-ray diffraction pattern of Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt; 
           [0054]      FIG. 2  shows an X-ray diffraction pattern of the crystal form of (6S)-5-methyltetrahydrofolate strontium salt; and 
           [0055]      FIG. 3  shows the particle diameter distribution of Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt obtained through the preparation method of the present invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0056]    Without further description, by means of the previous description, persons skilled in the art can implement the present invention to the maximum. The following preferred specific embodiments are just examples, and in no way limit the disclosure of the present invention. 
       Embodiment 1 
       [0057]    15.0 g (6S)-5-MTHF was added to 325 ml deionized water, and 10% NaOH solution was added with stirring for neutralization to a pH value of 7.8 till (6S)-5-MTHF was fully dissolved. Next, 37.5 g calcium chloride solution (containing 9.0 g calcium chloride) was added, the resulting reaction solution was transferred into an ultrasonic reactor having a power density of 0.04 W/ml at a temperature of 72° C., and after 40 min-ultrasonic reaction, the reaction solution was filtered, and washed with water, ethanol and acetone respectively. After drying in vacuum at 25° C., 13.5 g white Form C of (6S)-5-MTHF calcium salt was obtained. The chemical purity is 99.25% (detected by HPLC), and the moisture content is 10.67%. 
       Embodiment 2 
       [0058]    10.0 g (6S)-5-MTHF was added to 250 ml water, and 10% NaOH solution was added with stirring for neutralization to a pH value of 7.4 till (6S)-5-MTHF was fully dissolved. Next, 25 g calcium chloride solution (containing 6.0 g calcium chloride) was added, the resulting reaction solution was transferred into an ultrasonic reactor having a power density of 0.03 W/ml at a temperature of 60° C., and after 40 min-ultrasonic reaction, the reaction solution was filtered, and washed with water and acetone. After drying in vacuum at 30° C., 9.2 g white Form C of (6S)-5-MTHF calcium salt was obtained. The chemical purity is 99.01% (detected by HPLC), and the moisture content is 15.8%. 
       Embodiment 3 
       [0059]    10.0 g (6S)-5-MTHF was added to 150 ml water, and ammonia was added with stirring for neutralization to a pH value of 7.4 till (6S)-5-MTHF was fully dissolved. Next, 12 g calcium chloride solution (containing 3.0 g calcium chloride) was added, the resulting reaction solution was transferred into an ultrasonic reactor having a power density of 0.05 W/ml at a temperature of 40° C., and after 100 min-ultrasonic reaction, the reaction solution was filtered, and washed with water and acetone. After drying in vacuum at 25° C., 9.0 g white Form C of (6S)-5-MTHF calcium salt was obtained. The chemical purity is 99.60% (detected by HPLC), and the moisture content is 17.76%. 
       Embodiment 4 
       [0060]    40.0 g (6S)-5-MTHF was added to 1,000 ml water, and 10% NaOH solution was added with stirring for neutralization to a pH value of 7.8 till (6S)-5-MTHF was fully dissolved. Next, 96 g calcium chloride solution (containing 24 g calcium chloride) was added, the resulting reaction solution was transferred into an ultrasonic reactor having a power density of 0.56 W/ml at a temperature of 90° C., and after 30 min-ultrasonic reaction, the reaction solution was filtered, and washed with water and acetone. After drying in vacuum at 25° C., 36.0 g white Form C of (6S)-5-MTHF calcium salt was obtained. The chemical purity is 99.77% (detected by HPLC), and the moisture content is 16.39%. 
       Embodiment 5 
     Strontium Salt 
       [0061]    6.0 g (6S)-5-MTHF was added to 150 ml water, and 10% NaOH solution was added with stirring for neutralization to a pH value of 7.3 till (6S)-5-MTHF was fully dissolved. Next, 7.29 g strontium chloride solution (containing 1.8 g strontium chloride) was added, the resulting reaction solution was transferred into an ultrasonic reactor having a power density of 0.30 W/ml at a temperature of 70° C., and after 20 min-ultrasonic reaction, the reaction solution was filtered, and washed with water and acetone. After drying in vacuum at 25° C., 4.2 g white (6S)-5-MTHF strontium salt was obtained. The chemical purity is 97.57% (detected by HPLC), and the moisture content is 15.02%. 
       Embodiment 6 
       [0062]    9.0 g (6S)-5-MTHF was added to 225 ml water, and 10% NaOH solution was added with stirring for neutralization to a pH value of 7.1 till (6S)-5-MTHF was fully dissolved. Next, 10.2 g calcium chloride solution (containing 2.7 g calcium chloride) was added, the resulting reaction solution was transferred into an ultrasonic reactor having a power density of 0.20 W/ml at a temperature of 70° C., and after 20 min-ultrasonic reaction, the reaction solution was filtered, and washed with water and acetone. After drying in vacuum at 25° C., 6.1 g white Form C of (6S)-5-MTHF calcium salt was obtained. The chemical purity is 99.08% (detected by HPLC), and the moisture content is 15.20%. 
       Embodiment 7 
       [0063]    18.0 g (6S)-5-MTHF was added to 450 ml water, and 10% NaOH solution was added with stirring for neutralization to a pH value of 7.3 till (6S)-5-MTHF was fully dissolved. Next, 21.6 g calcium chloride solution (containing 5.4 g calcium chloride) was added, the resulting reaction solution was transferred into an ultrasonic reactor having a power density of 0.04 W/ml at a temperature of 70° C., and after 30 min-ultrasonic reaction, the reaction solution was filtered, and washed with water and acetone. After drying in vacuum at 25° C., 13.9 g white Form C of (6S)-5-MTHF calcium salt was obtained. The chemical purity is 99.53% (detected by HPLC), and the moisture content is 16.4%. 
       Embodiment 8 
       [0064]    2.0 g (6S)-5-MTHF was added to 50 ml water, and sodium hydroxide was added with stirring for neutralization to a pH value of 7.2 till (6S)-5-MTHF was fully dissolved. Next, 2 g calcium chloride solution (containing 0.5 g calcium chloride) was added, the resulting reaction solution was transferred into an ultrasonic reactor having a power density of 0.05 W/ml at a temperature of 50° C., and after 60 min-ultrasonic reaction, the reaction solution was filtered, and washed with water and acetone. After drying in vacuum at 40° C., 1.0 g white Form C of (6S)-5-MTHF calcium salt was obtained. The chemical purity is 99.01% (detected by HPLC), and the moisture content is 15.6%. 
         [0065]    Although the above specific embodiments merely disclose the preparation methods of the (6S)-5-methyltetrahydrofolic acid calcium salt crystal and the (6S)-5-methyltetrahydrofolate strontium salt crystal, persons skilled in the art can prepare other types of (6S)-5-methyltetrahydrofolate salt crystals according to the teaching of the preparation methods, particularly the (6S)-5-methyltetrahydrofolic acid alkaline earth metal salt crystals. 
       Embodiment 9 
     Stability Study 
       [0066]    In order to determine the stability of the novel crystal form of the Form C of (6S)-5-MTHF calcium salt, the crystal form was placed in the air at a temperature of 40° C. and a relative humidity of 60%, and the content of remained (6S)-5-MTHF calcium salt was periodically measured. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Crystal Form 
                 Storage Days 
                 Appearance 
                 Content 
               
               
                   
                   
               
             
             
               
                   
                 Form C 
                 0 
                 White crystal 
                 99.5% 
               
               
                   
                   
                 3 
                 White crystal 
                 99.1% 
               
               
                   
                   
                 6 
                 White crystal 
                 99.1% 
               
               
                   
                   
                 9 
                 White crystal 
                 98.4% 
               
               
                   
                   
               
             
          
         
       
     
         [0067]    The results show that the Form C of (6S)-5-MTHF calcium salt has good stability, which is beneficial to the production and storage of pharmaceutical preparations. 
       Embodiment 10 
     Particle Diameter Distribution of the Form C of the Crystal Form of (6S)-5-Methyltetrahydrofolate Calcium Salt 
       [0068]      FIG. 3  shows the particle diameter distribution of the Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt obtained through the preparation method of the present invention. It can be seen from  FIG. 3  that, the particle size is in normal distribution, indicating that the crystal treated by ultrasonic waves has a relatively uniform particle size. 
       Embodiment 11 
     Conditions and Data of the X-Ray Diffraction Pattern of the Crystal Form 
       [0069]    Instrument: Bruker DS advance XRD 
         [0070]    Diffraction ray: CuKα (40 kV, 40 mA) 
         [0071]    Scanning rate: 80°/min (2θ value) 
         [0072]    Scanning range: 2° to 45° (2θ value) 
         [0073]    Peak Search Report (37 Peaks, Max P/N=46.1) 
         [0074]    PEAK: 35-pts/Parabolic Filter, Threshold=3.0, Cutoff=0.1%, BG=3/1.0, Peak−Top=Sumnmit 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 # 
                 2-Theta 
                 d(A) 
                 BG 
                 Height 
                 I % 
                 Area 
                 I % 
                 FWHM 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 3.151 
                 28.0163 
                 612 
                 8740 
                 89.06 
                 165950 
                 100.00 
                 0.343 
               
               
                 2 
                 6.309 
                 13.9974 
                 689 
                 9814 
                 100.00 
                 155754 
                 93.86 
                 0.286 
               
               
                 3 
                 9.447 
                 9.3545 
                 681 
                 1601 
                 16.31 
                 16953 
                 10.22 
                 0.309 
               
               
                 4 
                 13.199 
                 6.7022 
                 913 
                 4338 
                 44.20 
                 46545 
                 28.05 
                 0.228 
               
               
                 5 
                 13.612 
                 6.4999 
                 1029 
                 2121 
                 21.61 
                 14775 
                 8.90 
                 0.227 
               
               
                 6 
                 14.166 
                 6.2469 
                 1072 
                 1514 
                 15.43 
                 12344 
                 7.44 
                 0.441 
               
               
                 7 
                 14.639 
                 6.0462 
                 1057 
                 4630 
                 47.18 
                 52370 
                 31.56 
                 0.246 
               
               
                 8 
                 15.329 
                 5.7755 
                 1055 
                 1310 
                 13.35 
                 2233 
                 1.35 
                 0.147 
               
               
                 9 
                 16.001 
                 5.5343 
                 1133 
                 2147 
                 21.88 
                 18187 
                 10.96 
                 0.301 
               
               
                 10 
                 16.534 
                 5.3572 
                 958 
                 1409 
                 14.36 
                 15394 
                 9.28 
                 0.539 
               
               
                 11 
                 17.046 
                 5.1973 
                 1089 
                 2406 
                 24.52 
                 14700 
                 8.86 
                 0.187 
               
               
                 12 
                 18.824 
                 4.7103 
                 1017 
                 3484 
                 35.50 
                 74762 
                 45.05 
                 0.479 
               
               
                 13 
                 19.158 
                 4.6288 
                 1118 
                 3998 
                 40.74 
                 84209 
                 50.74 
                 0.491 
               
               
                 14 
                 20.125 
                 4.4085 
                 1295 
                 3176 
                 32.36 
                 30820 
                 18.57 
                 0.275 
               
               
                 15 
                 20.976 
                 4.2316 
                 1169 
                 2397 
                 24.42 
                 22579 
                 13.61 
                 0.308 
               
               
                 16 
                 21.411 
                 4.1466 
                 1068 
                 1503 
                 15.31 
                 5525 
                 3.33 
                 0.213 
               
               
                 17 
                 22.614 
                 3.9287 
                 863 
                 1716 
                 17.49 
                 13799 
                 8.32 
                 0.271 
               
               
                 18 
                 24.073 
                 3.6937 
                 857 
                 1619 
                 16.50 
                 9785 
                 5.90 
                 0.215 
               
               
                 19 
                 24.785 
                 3.5892 
                 884 
                 1719 
                 17.52 
                 26584 
                 16.02 
                 0.503 
               
               
                 20 
                 25.022 
                 3.5558 
                 898 
                 1971 
                 20.08 
                 26647 
                 16.06 
                 0.417 
               
               
                 21 
                 25.914 
                 3.4354 
                 884 
                 1390 
                 14.16 
                 7075 
                 4.26 
                 0.235 
               
               
                 22 
                 26.858 
                 3.3168 
                 846 
                 1262 
                 12.86 
                 10476 
                 6.31 
                 0.423 
               
               
                 23 
                 27.334 
                 3.2601 
                 852 
                 1244 
                 12.68 
                 8923 
                 5.38 
                 0.359 
               
               
                 24 
                 27.674 
                 3.2207 
                 901 
                 1048 
                 10.68 
                 1050 
                 0.63 
                 0.113 
               
               
                 25 
                 28.358 
                 3.1446 
                 915 
                 1606 
                 16.36 
                 15814 
                 9.53 
                 0.384 
               
               
                 26 
                 28.908 
                 3.0860 
                 913 
                 1339 
                 13.64 
                 16265 
                 9.80 
                 0.641 
               
               
                 27 
                 29.444 
                 3.0310 
                 977 
                 1419 
                 14.46 
                 9424 
                 5.68 
                 0.358 
               
               
                 28 
                 30.251 
                 2.9520 
                 921 
                 1248 
                 12.72 
                 3742 
                 2.25 
                 0.192 
               
               
                 29 
                 30.769 
                 2.9035 
                 880 
                 1518 
                 15.47 
                 9728 
                 5.86 
                 0.256 
               
               
                 30 
                 31.537 
                 2.8345 
                 836 
                 1196 
                 12.19 
                 4349 
                 2.62 
                 0.203 
               
               
                 31 
                 32.602 
                 2.7443 
                 813 
                 1863 
                 18.98 
                 21721 
                 13.09 
                 0.347 
               
               
                 32 
                 35.722 
                 2.5115 
                 617 
                 929 
                 9.47 
                 4980 
                 3.00 
                 0.268 
               
               
                 33 
                 37.675 
                 2.3856 
                 659 
                 1229 
                 12.52 
                 10784 
                 6.50 
                 0.317 
               
               
                 34 
                 38.819 
                 2.3179 
                 633 
                 884 
                 9.01 
                 5939 
                 3.58 
                 0.397 
               
               
                 35 
                 40.263 
                 2.2381 
                 607 
                 776 
                 7.91 
                 4592 
                 2.77 
                 0.429 
               
               
                 36 
                 42.037 
                 2.1476 
                 580 
                 988 
                 10.07 
                 14181 
                 8.55 
                 0.583 
               
               
                 37 
                 43.615 
                 2.0735 
                 563 
                 841 
                 8.57 
                 6633 
                 4.00 
                 0.400 
               
               
                   
               
             
          
         
       
     
       Embodiment 12 
     Conditions and Data of the X-Ray Diffraction Pattern of the Crystal Form of Strontium Salt 
       [0075]    Instrument model: Bruker D8 advance XRD 
         [0076]    Diffraction ray: CuKα (40 kv, 40 mA) 
         [0077]    Scanning rate: 8°/min (2θ value) 
         [0078]    Scanning range: 5° to 45° (2θ value) 
         [0079]    Peak Search Report (36 Peaks, Max P/N=20.9) 
         [0080]    PEAK: 29-pts/Parabolic Filter, Threshold=3.0, Cutoff=0.1%, BG=3/1.0, Peak−Top=Summit 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 # 
                 2-Theta 
                 d(A) 
                 BG 
                 Height 
                 I % 
                 Area 
                 I % 
                 FWHM 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 6.54 
                 13.503 
                 581 
                 2213 
                 100 
                 46131 
                 100 
                 0.354 
               
               
                 2 
                 7.496 
                 11.7831 
                 565 
                 96 
                 4.3 
                 1563 
                 3.4 
                 0.277 
               
               
                 3 
                 8.38 
                 10.5426 
                 570 
                 136 
                 6.1 
                 2418 
                 5.2 
                 0.302 
               
               
                 4 
                 9.96 
                 8.8736 
                 610 
                 1379 
                 62.3 
                 35198 
                 76.3 
                 0.434 
               
               
                 5 
                 12.339 
                 7.1672 
                 647 
                 292 
                 13.2 
                 5831 
                 12.6 
                 0.339 
               
               
                 6 
                 13.66 
                 6.4772 
                 902 
                 604 
                 27.3 
                 20626 
                 44.7 
                 0.581 
               
               
                 7 
                 14.22 
                 6.2232 
                 826 
                 413 
                 18.7 
                 26525 
                 57.5 
                 1.092 
               
               
                 8 
                 14.741 
                 6.0044 
                 940 
                 355 
                 16 
                 5431 
                 11.8 
                 0.26 
               
               
                 9 
                 15.58 
                 5.6828 
                 842 
                 199 
                 9 
                 3770 
                 8.2 
                 0.322 
               
               
                 10 
                 16.279 
                 5.4403 
                 1049 
                 239 
                 10.8 
                 2625 
                 5.7 
                 0.187 
               
               
                 11 
                 16.8 
                 5.2729 
                 815 
                 533 
                 24.1 
                 28957 
                 62.8 
                 0.924 
               
               
                 12 
                 17.14 
                 5.169 
                 898 
                 672 
                 30.4 
                 16152 
                 35 
                 0.409 
               
               
                 13 
                 18.28 
                 4.8492 
                 796 
                 664 
                 30 
                 16290 
                 35.3 
                 0.417 
               
               
                 14 
                 19.54 
                 4.5393 
                 819 
                 598 
                 27 
                 15788 
                 34.2 
                 0.449 
               
               
                 15 
                 20.3 
                 4.371 
                 779 
                 328 
                 14.8 
                 5046 
                 10.9 
                 0.262 
               
               
                 16 
                 22.02 
                 4.0334 
                 699 
                 1281 
                 57.9 
                 31787 
                 68.9 
                 0.422 
               
               
                 17 
                 23.259 
                 3.8211 
                 764 
                 131 
                 5.9 
                 2094 
                 4.5 
                 0.272 
               
               
                 18 
                 24.399 
                 3.6451 
                 944 
                 589 
                 26.6 
                 22484 
                 48.7 
                 0.649 
               
               
                 19 
                 24.92 
                 3.5702 
                 911 
                 788 
                 35.6 
                 34957 
                 75.8 
                 0.754 
               
               
                 20 
                 26.42 
                 3.3707 
                 830 
                 673 
                 30.4 
                 12784 
                 27.7 
                 0.323 
               
               
                 21 
                 27.999 
                 3.1841 
                 838 
                 310 
                 14 
                 5810 
                 12.6 
                 0.319 
               
               
                 22 
                 28.961 
                 3.0805 
                 851 
                 249 
                 11.3 
                 4360 
                 9.5 
                 0.298 
               
               
                 23 
                 29.881 
                 2.9877 
                 723 
                 138 
                 6.2 
                 2168 
                 4.7 
                 0.267 
               
               
                 24 
                 30.84 
                 2.897 
                 679 
                 190 
                 8.6 
                 3775 
                 8.2 
                 0.338 
               
               
                 25 
                 32.04 
                 2.7912 
                 678 
                 220 
                 9.9 
                 7951 
                 17.2 
                 0.614 
               
               
                 26 
                 32.44 
                 2.7577 
                 683 
                 157 
                 7.1 
                 7115 
                 15.4 
                 0.77 
               
               
                 27 
                 33.12 
                 2.7026 
                 662 
                 190 
                 8.6 
                 2851 
                 6.2 
                 0.255 
               
               
                 28 
                 34.12 
                 2.6256 
                 655 
                 149 
                 6.7 
                 4534 
                 9.8 
                 0.517 
               
               
                 29 
                 35.041 
                 2.5587 
                 674 
                 88 
                 4 
                 2007 
                 4.4 
                 0.388 
               
               
                 30 
                 37.179 
                 2.4163 
                 663 
                 223 
                 10.1 
                 8948 
                 19.4 
                 0.682 
               
               
                 31 
                 37.723 
                 2.3827 
                 689 
                 142 
                 6.4 
                 7683 
                 16.7 
                 0.92 
               
               
                 32 
                 39.78 
                 2.2641 
                 592 
                 373 
                 16.9 
                 9421 
                 20.4 
                 0.429 
               
               
                 33 
                 41.019 
                 2.1985 
                 577 
                 122 
                 5.5 
                 3827 
                 8.3 
                 0.533 
               
               
                 34 
                 42.84 
                 2.1092 
                 648 
                 445 
                 20.1 
                 17304 
                 37.5 
                 0.661 
               
               
                 35 
                 43.298 
                 2.0879 
                 726 
                 211 
                 9.5 
                 10128 
                 22 
                 0.816 
               
               
                 36 
                 44.321 
                 2.0421 
                 683 
                 153 
                 6.9 
                 4026 
                 8.7 
                 0.447 
               
               
                   
               
             
          
         
       
     
         [0081]    Therefore, the present invention relates to the crystal form of (6S)-5-methyltetrahydrofolate salt prepared by the above method. 
         [0082]    The crystal form of (6S)-5-methyltetrahydrofolate salt is: 
         [0083]    (a) Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 6.3±0.2 and 19.2±0.2; or 
         [0084]    (b) Crystal form of (6S)-5-methyltetrahydrofolate strontium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 6.5±0.2 and 22.0±0.2. 
         [0085]    Preferably, the crystal form of (6S)-5-methyltetrahydrofolate salt is: 
         [0086]    (a) Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 3.2±0.2, 6.3±0.2, 13.2±0.2, 14.6±0.2, 19.2±0.2 and 32.6±0.2; or 
         [0087]    (b) the crystal form of (6S)-5-methyltetrahydrofolate strontium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 6.5±0.2, 10.0±0.2, 13.7±0.2, 16.8±0.2, 17.1±0.2, 22.0±0.2 and 24.9±0.2. 
         [0088]    Preferably, the crystal form of (6S)-5-methyltetrahydrofolate salt is: 
         [0089]    (a) Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 3.2±0.1, 6.3±0.1, 13.2±0.1, 14.6±0.1, 19.2±0.1 and 32.6±0.1; or 
         [0090]    (b) the crystal form of (6S)-5-methyltetrahydrofolate strontium salt, where the X-ray diffraction pattern has diffraction peaks at the 2θ angles of 6.5±0.1, 10.0±0.1, 13.7±0.1, 16.8±0.1, 17.1±0.1, 22.0±0.1 and 24.9±0.1. 
         [0091]    Specifically, the crystal form of (6S)-5-methyltetrahydrofolate salt is: 
         [0092]    (a) X-ray diffraction pattern of the Form C of the crystal form of (6S)-5-methyltetrahydrofolate calcium salt is essentially shown in  FIG. 1 ; or 
         [0093]    (b) X-ray diffraction pattern of the crystal form of (6S)-5-methyltetrahydrofolate strontium salt is essentially shown in  FIG. 2 . 
         [0094]    The preferred or specific embodiments of the present invention are described above in detail. It should be understood that persons skilled in the art can make various modifications and variations according to the design concept of the present invention without any creative work. Therefore, all technical solutions that can be obtained by persons skilled in the art through logical analysis, reasoning or limited experiments based on the prior art according to the design concept of the present invention shall fall within the scope of the present invention and/or the protection scope defined by the claims.