L Histidine Monohydrochloride  
Monohydrate  
 
Specifications for feed additives  
Raw material for manufacturing  
Compositional specifications  
Content:  When this product is determined following a 3-hour drying at 105°C, it contains more 
than 9 8.0% of L Histidine Monohydrochloride  Monohydrate  (C6H9N3O2･HCl･H2O) 
Physical and chemical properties  
(1) It appears white crystals o r crystalline powder.  
(2) The pH of this solution or suspension (1 in 10) in water is between 3.5 and 4.5.  
Confirmation test:  
(1) 5mL of a solution of this (1 in 1,000) add into 5ml of Ninhydrin solution (1 in 1,000), and 
heat for 3 minutes: a purple color develops.  
   (2) A solution of this (1 in 10) reacts to qualitative test of Chloride.  
Purity test:  
(1) Specific rotation : Weigh about 5.5g of this to digit of 0.01g, and calculate the record 
converted as dry matter, and then dissolve it in 6mol/L Hydrochloric acid solution to make 
50mL, and filter if necessary: the Specific Rotation [ α]20D of this solution must be between 
+8.5° and +10.5 °. 
(2) Lead : Weigh 5.0g (4.95 to 5.04g) of this and  perform the test for lead as directed in the Lead 
Test ( Atomic Absorption Spectrophotometry Method 1); the amount of lead must be not more 
than 2 µg/g. Pipet 1.0mL of Standard Lead Solution and add nitric acid (1 in 15 0) to make 
exactly 10mL and  use this solution as the standard solution.  
(3) Arsenic : Place 1.0g (0.95 to 1.04g) of this in a decomposition flask, add 10 mL of Nitric acid 
and 5mL of Sulfuric acid, and heat gently. If the solution is brown in color, allow to cool, add 
1 to 2mL of Nitric acid, heat, and repeat this procedure until the solution is colorless to pale 
yellow. After cooling, add 0.5mL of Perchloric acid, and heat until white fumes are evolved.  
After cooling, add 15mL of a saturated solution of Ammonium oxalate, and heat until white 
fumes are ev olved again. After cooling, add water to make about 10mL, and use this solution 
as the sample solution. Perform the test with the sampl e solution according to the method with 
Apparatus A. Observe the color of the absorbing solution: the color produced must not be 
more intense than the standard color (not more than 2 µg/g). 
(4) Ammonium salt : The purity test iii. for the raw material for manufacturing  of aminoacetic acid 
is applied mutatis mutandis.  In this case, "0.1 g" shall be read as "0.05 g" and "1 mL" shall be 
read as "0.5 mL"(  not more than 0.04 %).  
Loss on drying:  ≤ 0.3 % (3 g, 105℃, 3 hours)  
Ignition residue : ≤ 0.1 % (1 g) Assay:  Dry this at 105°C for 3 hours and weigh about 0.5g of this to digit of 0.1mg, record its 
weigh, add water to dissolve and pour into 1,000mL measuring flask and add water to make 
1,000mL . Use this solution as the sample solution.  
 Perform the test with 5 µl of the sample solution as directed under Liquid Chromatography 
according to the following conditions. Determine the peak area of Histidine in the 
chromatogram, calculate the concentration of L -Histidine Monohydrochloride Monohydrate 
based on the calibration curve separately determined, and calculate the content.  
 Operating conditions  
 Detector: A ultraviolet absorptiometer  (mensural  waveleng th of 210 nm)  
 Column: A stainless s teel column 4.6mm in inside diameter  and 150mm in length, packed with 
Octadecylsilylated  silica gel for Liquid  Chromatography (3 µm in particle diameter).  
 Column temperature: 35 °C 
 Mobile phase: Dissolve 2.27g (2.265 to 2.274g) of Potassium dihydrogen phos phate and 1.08g 
(1.075 to 1.084g) of Sodium 1 -Octane sulfonate in 850mL of water. After adjusting the pH to 2.5 
with Phosphoric acid, add 100mL of Acetonitrile and mix and then add water to make 1,000mL 
of solution.  
 Flow rate: 1.0mL per minute  
 Creation of calibration curve  
 Weigh about 0.05g, 0.1g, 0.5g and 1g of L -Histidine chloride for assay to the digit of 0.1mg 
respectively and record their weight, add about 800mL of water to dissolve and pour into 
1,000mL measuring flask and add water to make 1,000mL . Use these solutions as the standard 
stock solution. (1mL of the solution contains 0.05mg, 0.1mg, 0.5mg or 1mg, respectively.) 
Perform the test with 5 µL each of the standard solution as directed under Liquid 
Chromatography in the same manner a s the sample solution. Determine the peak area of L -
Histidine in the Chromatogram and prepare a calibration curve.  
Standards for manufacturing methods : 
Culture Corynebacterium glutamicum  strain for Histidine production aerobically, filter the 
culture broth  to remove bacteria after completion of the culture, and isolate the crude L -Histidine 
crystal fraction. Furthermore, purity the crude crystals and dry the solid substances obtained.  
Standards for storage methods : 
Store in a well -closed container.  
 
Preparation  
Compositional specifications  
Apply the compositional specifications of L -Histidine raw material for manufacturing . 
Standards for storage methods  
Apply the standards for storage methods  of L-Histidine raw material for manufacturing .