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Amino Acids in Infant Formula and Adult/Pediatric Formulas
Ultra High Performance Liquid Chromatography
1
Applicable for the determination of amino acids (including Taurine) in all forms of infant, adult, and/or
pediatric formula (powders, ready to feed liquids and liquid concentrates). The LOQs for the method
ranged from 2.0 to 10000 mg/100g.
Caution:
Refer to Material Safety Data Sheet (MSDS) of chemicals prior to use, and follow the suggested
personal protective equipment.
A. Principle:
This procedure involves the conversion of proteins present in the foods to amino acids by
different hydrolysis methods (see table 1) based on the amino acids chemical nature. The resulting
amino acids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent
(except Tryptophan) to form stable urea derivatives of each amino acid. The derivatized forms of
amino acids are separated by UHPLC and quantitated by Diode Array Detector (Tryptophan by
Fluorescence detector) using Norvaline as internal standard.
Table 1: Different Hydrolysis Procedures and amino acids to be determined in each method
Procedure
Amino acids
Acid Hydrolysis
Histidine, Taurine, Serine, Arginine, Glycine, Aspartic acid (sum of
Aspartic acid and Asparagine), Glutamic acid (sum of Glutamic acid
and Glutamine), Threonine, Alanine, Proline, Lysine, Tyrosine,
Valine, Isoleucine, Leucine, Phenyl alanine.
Oxidative Acid Hydrolysis Cysteine
a
(sum of cysteine and cystine), Methionine
b
Alkaline Hydrolysis
Tryptophan
c
a, b
Cystine and Methionine are converted into Cysteic acid and Methionine sulfone by performic
acid oxidation prior to acid hydrolysis.
c
Determined by Fluorescence detector
B. Apparatus:
a) Liquid Chromatograph:
Agilent UHPLC Infinity series with Binary Solvent Manager capable of
1200 bar pressure.
b) Column:
AccQ.Tag
TM
Ultra, C18, 1.7 μm, 2.1 x 100 mm (Waters Part No. 205000343).
c) Detector:
Agilent Infinity series UV/DAD detector with 1 μL flow cell and Fluorescence detector.
d) Injector:
Agilent Infinity Series Auto sampler with column oven.
e) Data System:
Agilent Open lab CDS.
f) Centrifuge.
g) Hot air oven:
Capable of 110 ± 2°C
h)
Turbo-vap evaporator:
Concentration evaporator workstation (Biotage)
i)
pH meter.
j)
Digestion Vessels:
20 mL glass vials with relse cap (crimp caps and Teflon lined septas).
k)
Vortex mixer
l) Syringe filters:
0.22 μm, PTFE.
m) Total Recovery Vials: Waters (Part No.: 186000384C)
C. Reagents:
a) AccQ-Tag Eluent A concentrate:
Waters, Part No: 186003838
b) AccQ-Tag Ultra Derivatization kit:
Waters, Part No: 186003836. This kit contains 5 sets of vials
and each set contains 3 vials labeled with 1 (Borate Buffer), 2A (Reagent Powder), 2B (Reagent
Diluent).
c) De-ionized laboratory water -
≥ 18 Mohm/cm
d) Acetonitrile –
HPLC grade.
e) Methanol –
HPLC grade.
Amino-03 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE