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Amino Acids in Infant Formula and Adult/Pediatric Formulas
Ultra High Performance Liquid Chromatography
3
iii) L-Cysteic acid, 2.5mM:
Accurately weigh 23.40 mg of L-Cysteic acid monohydrate into a
50 ml standard volumetric flask. Dissolve and makeup to 50 ml with water. Store at
-20°C±2°C for 2 months.
iv) L-Methionine Sulfone, 2.5 mM:
Accurately weigh 22.65 mg of L-Methionine Sulfone into a
50 ml standard volumetric flask. Dissolve and makeup to 50 ml with water. Store at
-20°C±2°C for 2 months.
v) Tryptophan stock standard, 500 mg/L:
Accurately weigh 25.0 mg of tryptophan standard
into a 50 ml standard volumetric flask and dissolve in 0.1 M HCl and makeup to the mark.
vi) Norvaline, stock Internal Standard Preparation, 2.5 mM:
Accurately weigh 14.64 mg of
Norvaline into a 50 ml standard volumetric flask. Dissolve and makeup to 50 ml with
water. Store at -20 °C±2°C for 2 months.
(Note: Apply appropriate Purity, Salt and Moisture corrections to the stock standards
concentrations)
2) Preparation of Intermediate, working standards and derivatization procedure for Acid
Hydrolysis and Oxidative Acid Hydrolysis methods:
i) Table 2: Intermediate standards (IS) preparation:
Stock Std. Name
Stock
std.
Conc.
(mM)
Volume of
Stock std.
(ml)
Volume of
diluent
c
(ml)
Intemediate
std. Conc.
(mM)
Intermediate
std code
Amino acids,
Hydrolysate
2.5
0.40
0.20
1.0
IS Mix - 1
Taurine
2.5
0.40
L-Cysteic acid
2.5
0.40
0.20
1.0
IS Mix - 2
L-Methionine
Sulfone
2.5
0.40
Norvaline
2.5
0.10
0.90
0.25
--
Norvaline
0.25
0.20
0.80
0.05
--
c
Diluent
–
Laboratory water
Storage and Validity: -
20 ± 2˚C for 2 m
onths
ii) Table 3: Working standards preparation:
IS Mix std.
d
Conc. (mM)
Vol. of
working Std.
(mL)
Vol. of 2.5 mM
Norvaline (mL)
Vol. of
Diluent* (mL)
Final
Volume
(mL)
Working
std. Conc.
(mM)
1.00
0.50
0.10
0.40
1.00
0.50
1.00
0.25
0.10
0.65
1.00
0.25
1.00
0.10
0.10
0.80
1.00
0.10
0.50
0.10
0.10
0.80
1.00
0.05
0.10
0.10
0.10
0.80
1.00
0.01
d
Choose appropriate IS Mix standard based on the hydrolysis procedure.
iii) Preparation of Calibration standards by derivatization of working standards:
Quantitatively transfer 10 μL of each working standard into total recovery vials. Add 70 μL
of borate buffer to each vial and vortex for 30 seconds. Add 20 μL of reconstituted
AccQ.Tag Ultra reagent powder and vortex for 30 seconds. Let it stand for 1 minute at
Amino-03 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE