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Standard and Specification > Natural Additives > Gibberellic Acid
54. Gibberellic Acid  
Chemical Formula C12H22O6 
Molecular Weight 346.37 
Definition Gibberella fujikuroi is cultured, which is then filtered. The filtrate is concentrated under a reduced pressure. The concentrate is extracted and crystallized. Gibberellic Acid is obtained by purifying the crystallized precipitates. 
[Compositional Specifications of Gibberellic Acid]
Content Gibberellic Acid should contain not less than 90.0% of gibberellic acid (C19H22O6 = 346.37). 
Description Gibberellic Acid is scentless white~pale yellow crystalline powder. 
Identification A solution of a few milligrams of Gibberellic Acid in 2 ml sulfuric acid is red in color with green. 
Purity (1) Specific Rotation : Approximately 5 g of Gibberellic Acid is precisely weighed and dissolved in alcohol to make 50 ml. (This solution should not be heated during preparation.) The polarity of this solution should be [α](20, D)= +75.0∼+90.0°.

(2) Arsenic : 0.25 g of Gibberellic Acid is placed in a platinum, quartz, or porcelain crucible. 10 ml of magnesium nitrate in ethyl alcohol (1→50) is added to the crucible and then ethanol is ignited. It is then reduced to ash by heating at 450∼550°. If carbonaceous substance persists, it is wetted with minute amount of nitric acid, which is further heat treated at 450∼550°. After cooling, 3 ml of hydrochloric acid is added to the residue, which is then dissolved by heating in a water bath. When test for arsenic is carried out with this test solution, it should not be more than 4ppm.

(3) Heavy Metals : 0.5 g of Gibberellic Acid are carbonized by heating mildly in a quartz or porcelain crucible. After cooling, add 2 ml of nitric acid and 5 drops of sulfuric acid, it is heated until white smoke disappears, which is then reduced to ash by further heating at 450∼550°. After cooling, 2 ml of hydrochloric acid is added, which is then evaporated to dryness in a water bath. 3 drops of hydrochloric acid and 10 ml of hot water are added to the resulting residue, which is then heated for 2 minutes. After cooling, 1 drop of phenolphthalein indicator solution is added, then ammonia solution is added until the color of the solution becomes pale red. The resulting solution is transferred into a Nestler cylinder by rinsing with water. 50 ml of test solution is prepared by adding 2 ml of dilute acetic acid (1→20) and water. When this solution tested for heavy metals, the content should not be more than 40ppm. Color standard solution is prepared by the following procedure. 2 ml of nitric acid, 5 drops of sulfuric acid, and 2 ml of hydrochloric acid are added and evaporated to dryness in a crucible that is made of the same material used for test solution preparation. 3 drops of hydrochloric acid are added to the residue, which is then transferred into another Nestler cylinder as described above. Finally, 2 ml of lead standard solution, 2 ml of dilute acetic acid (1→20), and water are added to bring the total volume to 50 ml.

(4) Lead : 0.8 g of Gibberellic Acid is slowly carbonized by heating, which is reduced ash by further heat treatment at a temperature below 500°. Carefully 20 ml of dilute nitric acid is added to the ash, which is then gently boiled for 5 minutes. It is then filtered (if necessary), the residue is washed with water, which is then added to the filtrate. Water is added so that total volume of this solution becomes 50 ml. This test solution is tested for lead. The detected amount of lead should not be more than 10ppm. 
Loss on Drying When Gibberellic Acid is dried for 7 hours at 100° under a reduced pressure of 20 mmHg, the loss should not be more than 3%. 
Assay Approximately 40 mg is precisely weighed and dissolved in methyl alcohol (total volume = 50 ml). 10 ml of this solution is diluted to 100 ml with methyl alcohol (Test Solution). Separately, 25 mg of gibberellic acid standard is precisely weighed and dissolved in methyl alcohol (total volume = 50 ml). 10 ml of this solution is diluted to 50 ml with methyl alcohol (Standard Solution). Each of 5 ml Test Solution, 4 ml, 5 ml Standard Solution is separately added into 3 test tubes. The test tubes are evaporated to dryness and further dried at 90° for 10 minutes. The tubes are cooled to normal temperature. The residue in each test tube is dissolved in 10 ml each of diluted sulfuric acid (8→10), which is then heated for 10 minutes in a water bath and cooled for 5 minutes in 10° water bath. Absorption of each solution is measured at 535 nm with 1 cm path length using dilute sulfuric acid as a reference. The content is measured using the following equation (standard absorption that is closer to the absorption of Test Solution is chosen from the absorption values measured by using 4 ml and 5 ml of Standard Solution).

                                                            V               Au                      100
       Content (%) = 500 × C × ━━━ × ━━━ × ━━━━━━━━━━
                                                            5               As          Weight of sample(g)
        
       C  : Concentration of Standard Solution (mg/ml)
       V  : Amount of Standard Solution
       Au : Absorption of Test Solution
       As : Absorption of Standard Solution 
Permitted Use Level of Gibberellic Acid Gibberellic acid and diluted additives  containing it should only be used for malting for fermented liquor and spirits.