JPH01213258A - Purification of carnitine and carnitine amide - Google Patents

Abstract

PURPOSE:To conduct the isolation and purification of carnitine and carnitine amide by treating a carnitine aqueous solution containing carnitine amide with a cation-exchange resin. CONSTITUTION:A carnitine aqueous solution containing carnitine amide is brought into contact with a cation exchange resin equilibrated with an aqueous solution of 1-5pH whereby carnitine and carnitine amide are adsorbed to the resin. Then, the resin is treated with aqueous ammonia, a variety of ammonium salt and amine solutions to elute carnitine preferentially. Carnitine can be isolated by allowing carnitine amide to remain in the resin. Carnitine has L- and D-optical isomers, and L-carnitine acts as a carrier of fatty acids passing through the mitochondria membrane. Thus, the compound is used as a therapeutic agent for diseases in the cardiovascular system and nephrectopathy.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a method for separating and purifying carnitinamide from an aqueous carnitine solution using a cation exchange resin.

Carnitine (-monohydroxy-γ-trimethylaminobutyrofi) exists as tiL and D optical isomers.

-It has been revealed that monocarnitine normally exists in living organisms and acts as a carrier when fatty acids pass through the mitochondrial membrane.

Conventionally, the racemic form of carnitine has been used as an appetite stimulant, but in recent years, it has been used as an appetite stimulant. P
It has been found that the use of only 5 forms is effective in the fields of diseases related to the kidney system and diseases of the kidney system, and interest in the 'L form is increasing.

←) Conventional technology: There are many known methods for producing carnitine. Among them, there is a method in which L-carnitine is obtained by asymmetrically hydrolyzing DL-carnitinamide with an enzyme produced by microorganisms (patent application). In Sho 6l-198173), it is necessary to completely remove D-carnitinamide remaining in the L-carnitine water bath solution obtained. Until now, there is no known method for separating and purifying carnitine and carnitinamide from a mixed aqueous solution.

(c) Problems to be solved by the invention and means to solve the problems A representative example of the known purification method for carnitine is to adsorb carnitine on an H+ type cation exchange resin, and then use ammonia There is a method to elute and purify carnitine by embedding it in water or dilute hydrochloric acid. However, when carnitinamide is brought into contact with a cation exchange resin in the H+ form, most of the carnitinamide decomposes into carnitine, so this method cannot be used to separate L-carnitine and D-carnitinamide from a mixed aqueous solution. Cation exchange resins in ionic form other than H+ form, Na+ form, X+ form, and Mu
Carnitinamide is adsorbed on cation exchange resins such as - type, but carnitine is hardly adsorbed. Although it is possible to utilize this fact to separate carnitine and carnitinamide from a mixed aqueous solution and produce #I,
This method is not industrially satisfactory, as it requires a purification process for non-adsorbed carnitine.

Furthermore, carnitinamide adsorbed to the resin cannot be eluted even when treated with concentrated ammonia water, and when treated with dilute hydrochloric acid or dilute sulfuric acid, carnitinamide decomposes into carnitine during elution depending on their concentration. - There were also difficulties in purifying tinamide.

The present inventors conducted intensive research on a method for efficiently separating and purifying carnitine and carnitinamide, and as a result, p
By using a cation exchange resin equilibrated with an aqueous solution of salts of H1-5, we have found a method for efficiently separating and purifying both.

According to the present invention, carnitinamide is adsorbed on a cation exchange resin without being decomposed into carnitine, and is eluted without being decomposed into carnitine.
When separating L-carnitine and D-carnitinamide from a mixed aqueous solution, it is possible to prevent a decrease in the optical purity of L-carnitine due to contamination with D-carnitine produced by decomposition of D-carnitinamide.

The cation exchange resin used in the present invention can be equilibrated with an aqueous solution of pH 1 to 5 such as ammonium acetate-acetic acid or ammonium sulfate-sulfuric acid after normal pretreatment or regeneration treatment, but the pH of the equilibrated aqueous solution is 1~
For a period of 50 minutes, carnitine and carnitinamide can be combined with the resin by using aqueous solutions of ammonium phosphate, ammonium chloride, sodium acetate, sodium phosphate, potassium acetate, potassium phosphate, and other organic acid salts and free salts other than these aqueous solutions. can be adsorbed to.

When a resin adsorbed with carnitine and carnitinamide is treated with aqueous ammonia, only carnitine can be specifically eluted while carnitine remains in the resin. Furthermore, cartin can be preferentially eluted using aqueous solutions of various ammonium salts or amines.

In this case, in order to efficiently recover carnitine, an aqueous solution with a relatively low concentration that does not allow elution of carnitinamide is used. Although this concentration varies depending on the type of ammonium salt or amine, a concentration of 1M or less is usually suitable.

After the elution of carnitine is completed, the carnitinamide remaining in the resin can be removed from the resin without being decomposed into cycarnitine by increasing the concentration of various ammonium salts or amines or aqueous solutions of amine salts. It can be eluted and recovered quantitatively.

As the amine, various amines such as trimethylamine, triethylamine, dimethylamine, diethylamine, monomethylamine, and monoethylamine can be used.

Further, carnitine and carnitinamide can be similarly separated using a macroporous resin in addition to a gel type resin as the cation exchange resin.

Cycarnitine inner salt can be obtained by concentrating and drying the carnitine fraction eluted from the resin.

In addition, when obtaining carnitine in the form of various salts such as hydrochloride, the carnitine fraction eluted from the resin is concentrated until no ammonia is present, then equimolar amounts of various acids are added, reconcentrated, and ethanol is added. / Acetone mixture may be added, cooled, and crystallized.

On the other hand, carnitinamide can be prepared from the eluted carnitinamide fraction according to the method described in Japanese Patent Publication No. 3B-25TLC.

Next, examples of the present invention will be shown.

Example 1 1t of carnitine hydrochloride and 1f of carnitinamide hydrochloride were dissolved in 10mM ammonium acetate-acetic acid (pH 50), and after correcting the pH to 50, 1011M ammonium acetate-acetic acid was dissolved.
It was passed through a glass column (φ12 mm, D) packed with 50 m of strongly acidic cation exchange fat-resistant (Diaion BK IB) equilibrated with acetic acid (pH 50).Then, the column was washed with water. After that, only carnitine was eluted by passing 2 drops of ammonia water through the resin. (Recovery rate: 100) Furthermore, carnitinamide remaining in the resin was eluted and recovered by passing 8 drops of ammonium acetate aqueous solution. (
Recovery rate 80%) Implementation f12 Carnitine hydrochloride 1f and carnitinamide hydrochloride 1ft
-tjl dissolved in 350mM ammonium sulfate-sulfuric acid (pH 2-o), corrected to pBt-2,0, and equilibrated with 501EIM ammonium sulfate-sulfuric acid (pH 2,0)
i Acidic cation exchange resin (Diaion 811B)
A glass column (φ12■1.D,
) was passed. Next, after washing the column with water, aqueous ammonia was passed through the column, and only carnitine was eluted. (
Further, carnitinamide remaining in the resin was eluted and recovered by passing an 8% ammonium acetate aqueous solution through the resin. (
Example 3 A mixture containing 18 ft of each of carnitine hydrochloride and carnitinamide hydrochloride was passed through a column containing a resin equilibrated to 1PH 2.0 in the same manner as in Example 2, and the resin was washed with water. , only carnitine was specifically eluted using aqueous ammonia.

(Recovery rate: 100%) Next, after washing the resin with water, cycarnitinamide was eluted from the resin by passing a 50% ammonium sulfate aqueous solution through the resin.

(Recovery rate 10096) Calcium hydroxide was added to this fraction and left at 4°C. After separating the resulting precipitate, the F solution was concentrated, and an ethanol aqueous solution was added to the residue. When cooled, 2.5 tt of carnitinamide sulfate crystals were obtained. -I got it. (Recovery rate 50%
) Run 4 In the same manner as in Run 911, a mixed aqueous solution of carnitine and carnitinamide was passed through the column, the column was washed with water, and then only cartin was eluted from the resin by passing a 1% aqueous trimethylamine solution through the column. (Recovery rate 1oo%) Next, carnitinamide t-3% trimethylamine salt I remaining in the resin! i! (pH 9,0) was eluted by passing through the solution. (Recovery rate 93%) Example row 5 A mixed solution of carnitine and carnitinamide similar to that in Example 1 was mixed with 10 mM #, eIR ammonium-vinegar #1. Cp
Passed through a glass column packed with a 50-strong acidic cation exchange resin (Diaion 8K IB) equilibrated with H50). After washing the column with the same aqueous solution,
) Tacarnithine was eluted from the resin by passing an aqueous ammonium acetate solution through the resin. (Recovery rate 95%) Next, cycarnitinamide was eluted by passing an 8% aqueous ammonium acetate solution through the solution. (Recovery rate 79%) Practical row 6 DOWEX HOR-W as a strong acidic cation exchange resin
2 and the same operation as in Example 5 was carried out to separate and elute carnitine and carnitinamide. The recovery rate was 95% for carnitine and 74% for carnitinamide.

Implementation row 7 Using a macroporous type (Dowex 88) as a strongly acidic cation exchange resin, cartin and carnitinamide were separated and eluted in the same manner as in the actual m915. The recovery rate was 96% for carnitine and 84% for carnitinamide.

Example 8 When a carnitinamide degrading enzyme-producing bacterium was applied to 501 phosphate buffer (pH 7.0) containing 20% (W/V) DL-carnitinamide hydrochloride, L-carnitine and D-
Carnitinamide was obtained. This reaction product supernatant was treated with acid to remove the resulting precipitate, and then 10mM ammonium acetate-vinegar 1! The solution was passed through a strongly acidic positive exchange resin (Diaion 8K I P ) 1001 equilibrated with ff1 (pH 1 No.). After washing the resin with water, only L-carnitine could be eluted from the resin by passing 2 qb aqueous ammonia through the resin. This fraction was concentrated to dryness and further dried in a vacuum dryer to obtain 9L-carnitine inner salt 12k.
I got Ii.

(Recovery rate: 90 people) [“l”n = 2 a 6 (a = 10 e Ht
O) m, p, = 195-205 (dec) Carnitine purity = 100% (apbc) 5-body ratio =
D-carnitinamide remaining in the 92% (HPL()Y11 fat) was eluted from the resin by passing an 8% ammonium acetate aqueous solution through it. This fraction was condensed to remove the precipitated salt, and then diluted with dilute hydrochloric acid. was added to form carnitinamide hydrochloride and condensed again. When ethanol was added to the residue, crude crystals of carnitinamide hydrochloride were precipitated. By recrystallizing this from dilute ethanol, fiD-carnitinamide hydrochloride crystal 2 was obtained. .45 kg was obtained.

(Recovery rate 82%) Ctx3 back = +1! >9 (0=10, 1!, O)
m, p, = 255-240 (dec) Carnitinamide purity = 94% (TlPLC) D-isomer ratio
=981 (HPLO) Carnitine contamination rate αo
1% (HPIIO) L-carcine contamination rate = 100
9% (DTNB Method) (e) Effects of the Invention As described above, the present invention has made it possible to efficiently separate and purify carnitine and carnitinamide from an aqueous solution containing both compounds.

Beka Nakayama Kiyoshi

Claims (1)

[Claims] 1. A carnitine aqueous solution containing carnitinamide is
After contacting with a cation exchange resin equilibrated with an aqueous solution of salts of H1 to 5 to adsorb carnitine and carnitinamide onto the resin, the resin is treated with an aqueous solution of ammonia water, an amine or an ammonium salt. 1. A method for separating and purifying carnitine and carnitinamide, which comprises separating carnitine and carnitinamide by eluting only carnitine and leaving carnitinamide in an adsorbed state on a resin. 2. Separation and purification of carnitinamide, characterized in that carnitinamide is eluted and recovered by treating a cation exchange resin adsorbing carnitinamide with an aqueous solution of ammonium salt, amine, or amine salt. Law.