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Amino Acids in Infant Formula and Adult/Pediatric Formulas
Ultra High Performance Liquid Chromatography
2
f) Hydrochloric acid, 35% -
AR grade
.
g) Ortho-Phosphoric acid, 85%-
AR grade.
h) Hydrogen peroxide,
30% v/v.
i) Formic acid, 99% -
AR & HPLC grade.
j) Ethanolamine –
HPLC grade.
k) 1,1,1-trichloro-2-methyl-2-propanol –
AR grade.
l) Phenol –
AR grade.
m) Sodium Hydroxide –
AR grade.
D. Standards:
a) Amino acids standard, Hydrolysate, 2.5 mM concentration of each amino acid except
Cystine(1.25mM) :
Waters, Part No.: WAT 088122, (contains Aspartic acid, Serine, Glutamic
acid, Glycine, Histidine, Arginine, Threonine, Alanine, Proline, Cystine, Tyrosine, Lysine, Valine,
Methionine, Isoleucine, Leucine and Phenyl Alanine).
b) Tryptophan
: Sigma, product No.: T0254 or equivalent).
c) Taurine
: Sigma, product No.: T0625 or equivalent).
d) Cysteic acid:
Sigma, product No.: 30170 or equivalent).
e) Methionone Sulfone
: Sigma, product No.: M0876 or equivalent).
f) Norvaline:
Sigma, product No.: N7627 or equivalent).
g) Standard Reference Material
–
NIST SRM 1849a.
E.
Solutions Preparation:
a) Hydrochloric acid, 6M :
Add 53 mL of Hydrochloric acid to 47 mL of water in a glass beaker.
Cover the beaker with a watch glass and boil for 2 minutes in a fume hood.
b) Hydrochloric acid, 20 mM:
Add 1.7 ml of 6M Hydrochloric acid to 500 mL laboratory water.
c) Mobile Phase A1:
Quantitatively transfer 25 ml of Eluent A concentrate into a 500 mL volumetric
flask, add approximately 200 mL laboratory water to dissolve and fill to volume with laboratory
water.
d) Mobile Phase B1
: Add 6.5(±0.1) mL Formic acid (HPLC grade) to 500 mL Acetonitrile and
dissolve.
e) Mobile Phase A2:
Dissolve 2.85 mL acetic acid in 900 ml laboratory water and add 50 mL
methanol containing 0.5g of 1,1,1-trichloro-2-methyl-2-propanol. Adjust to pH 5.0 with
Ethanolamine and makeup to 1000 ml.
f) Performic acid solution:
Dissolve 0.24g of phenol in 45 mL of formic acid. Add 5 mL of 30% v/v
Hydrogen peroxide and mix gently. Incubate the solution between 20 - 30 ºC for 1 hour to form
performic acid, then cool in an ice bath for 15 minutes before adding to the samples.
g) Reconstitution of Derivatizing agent from Derivatization kit:
Transfer 1 mL of diluent from vial 2B
to vial 2A. Cap the vial tightly and vortex for 2 minutes. Heat on a preheated water bath at 55°C
for 5 minutes.
h) Sodium Hydroxide solution, 4M:
Dissolve 16g sodium hydroxide pellets in 80 mL laboratory
water. Cool and dilute to 100 mL with laboratory water.
i) Orthophosphoric acid, 0.5M:
Add 3.4 mL of orthophosphoric acid to 80 mL laboratory water and
make upto 100 mL.
j) Hydrochloric acid, 1.0M:
Add 16.4 mL of hydrochloric acid (6M) to laboratory water and make
upto 100 mL with water.
k) Standards Preparation:
1) Preparation of Stock Standards:
i) Amino acids standard, Hydrolysate, 2.5mM:
Transfer the content from the ampoule into a
new HPLC vial, close tightly and store at -20°C±2°C for 3 months.
ii) Taurine Stock standard preparation, 2.5 mM:
Accurately weigh 15.64 mg of Taurine into a
50 mL standard volumetric flask. Dissolve and makeup to 50 mL with laboratory water.
Store at -20°C±2°C for 2 months.
Amino-03 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE