Vitagen (3-Aminopropyl-L-ascorbylphosphate)

Vitagen is the diester of L-ascorbic acid and 3-aminopropanol phosphoric acid. When dried, it contains not less than 91.0 % of 3-aminopropyl-L-ascorbylphosphate (C₉H₁₆O₉NP : 313.20).

■ Manufacturing Method

The manufacturing step of Vitagen was obtained by reactions of Acid hydrolysis after which was reacted between 2-chlorotetrahydro-2H-1,3,2-oxazaphosphorine P-oxide and 5,6-isopropylidene- L-ascorbic. The total 5 step of its manufacturing method is as follows:

(1) reacting 3-amino-1-propanol with phosphorus oxychloride in an equivalent ratio of 1 : 1~1.3, in presence of an organic base, in an organic solvent, at a temperature of about 0~5°C for about 1~2 hours, to produce 2-chlorotetrahydro-2H-1,3,2-oxazaphosphorin P-oxide represented by following general formula (Ⅱ) :

(2) reacting said compound (Ⅱ) of said step (1) with 5,6-hydroxyl groups-protected L-ascorbic acid, in presence of an organic base, in an organic solvent, to produce L-ascorbic acid derivative represented by following general formula (Ⅲ) :

(3) hydrolyzing the L-ascorbic acid derivative (Ⅲ) of said step (2) by adding distilled water thereto, at a temperature of about 5~100°C for about 3 hours, to produce 2-(3-aminopropane phosphoric acid)-L-ascorbate ;

(4) re-crystallizing the product of said step (3) with a polar organic solvent ;

(5) optionally neutralizing the product of said step (4) with an alkali or a base, to produce a salt.

■ Reaction Scheme

■ Specification

Appearance : Vitagen is a white to pale yellow powder. It is odorless or has a slight odor.

Identification :

(1) Dissolve 0.1g of Vitagen in 5mL of water add 0.5mL of diluted nitric acid and heat for 5 minutes. After cooling, take 2mL of this solution and add iodine TS until the solution becomes slightly yellow in color. Then add 1 drop of a solution of cupric sulfate(1-1000) and 1 drop of pyrrole, and heat the mixture at 50° for 5 minutes: a bluish green or blue color develops.
(2) To 2mL of the decomposed acidic solution obtained in (1) add 2~3 drops of ammonium molybdate TS, and warm: a yellow color developed. Then heat for 5 minutes: the color of the solution changes to blue.

PH : 1.7 ~ 3.7 (1-100)

Purity

• (1) Clarity of solution Dissolve 1g of Vitagen in 10mL of water: the solution is colorless to pale yellow and clear.
  
• (2) Chloride Perform the test with 1.0g of Vitagen. Prepare the control solution with 0.3mL of 0.01N hydrochloric acid (not more than 0.011 %).
  
• (3) Heavy metals Proceed with 1.0g of Vitagen according to Method 2, and perform the test: the limit is not more than 20 ppm. Prepare the control solution with 2.0mL of Standard Lead Solution.
  
• (4) Arsenic Take 1.0g of Vitagen, prepare the test solution according to Method 3, and perform the test using Apparatus B with the test solution: the limit is not more than 2 ppm.
  
• (5) Free ascorbic acid Dissolve 1.0g of Vitagen in 50mL of a solution of metaphosphoric acid (1- 50), and add 1mL of starch TS and 2.3mL of 0.01N iodine TS: a blue color develops (not more than 0.2 %).
  
• (6) Free phosphoric acid Dissolve 0.4g of Vitagen in water and make exactly 100mL to use as the test solution. Take 10mL of this solution add 2.5mL of ammonium molybdate-sulfuric acid TS(Note 1) and 1mL of 1-amino-2-naphthol-4-sulfonate TS(Note 2), shake the mixture, add water to make 20mL, and allow to stand for 5 minutes: the solution has no more color than the following control solution: the limit is not more than 0.5%.
  Control solution : Proceed in the same manner as the test solution with 8mL of Standard Phosphoric Acid solution(Note 3).
  
• (7) Derivatives Dissolve 20mg of Vitagen in 5mL of water and use this solution as the test solution. Perform the test with 5 L of the test solution as directed under the High-performance Liquid Chromatography, according to the below conditions. By the automatic integration method, determine the total peak area A and the peak area B, which is other than the 3-aminoporpyl-L-ascorbylphosphate: the peak area of ascorbic acid derivatives other than the Vitagen is not more than 1.0%.
  Derivatives of ascorbic acid other than the Vitagen (%) = (B/A) 100
  Operating conditions:
  Detector : An ultraviolet absorption photometer (wavelength 254nm)
  Column : A stainless steel column about 4.6mm in inside diameter and 250mm in length, packed with octadecylsilanized silica gel (5 μm in particle diameter).
  Mobile phase : A buffer solution of 0.08M acetic acid-sodium acetate (pH 5.0) adjusted to contain 2.8mM of n-hexylamine, 0.1mM disodium edetate and 2% of ethanol.
  Flux : 1.0mL/min.

Water : Not more than 0.5 %

Assay : Weigh accurately about 0.1g of Vitagen, and dissolve in the phosphate buffer solution (pH 7.0) to make 100mL. To 10mL of this solution add the phosphate buffer solution (pH 7.0) to make 100mL. Read the absorbance A in a 10-mm cell at the maximum wavelength at about 259nm.