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DETERMINATION OF AMINO ACIDS IN INFANT AND ADULT/PEDIATRIC
NUTRITIONAL FORMULA BY UHPLC/UV
Carmen Martín-Hernández* and Sabine Lahrichi.
Nestle Research Center. Nestec Ltd. Vers-chez-les Blanc. CH-1000 Lausanne, Switzerland.
*Corresponding author: Tel 41 21 7859353; E-mail:
carmen.martin-hernandez@rdls.nestle.com1
APPLICABILITY
This instruction describes the determination of total amino acids using AccQ•Tag derivatization
(Waters Corporation, Milford, MA, USA) in combination with UHPLC/UV detection. It allows the
determination in one single analysis of the following amino acids: aspartic acid (Asp), threonine (Thr),
serine (Ser), glutamic acid (Glu), proline (Pro), glycine (Gly), alanine (Ala), valine (Val), isoleucine (Ile),
leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys), histidine (His), arginine (Arg),
methionine (Met), (cystine (Cys2) + cysteine (Cys)) and taurine (Tau).
This method it is not suitable for the determination of tryptophan.
The method is applicable to infant and adult/pediatric nutritional formula and other matrices such as
infant cereals and pet foods.
Free amino amino acids can be determined using the same method but with a different sample
preparation.
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PRINCIPLE
Proteins are hydrolyzed with 6M HCL for 24 h at 110°C. Phenol (0.1 %) is added to prevent
halogenation of tyrosine. 3,3’-dithio-dipropionic acid (DDP) is added to convert cystine and cysteine
to S-2-carboxyethylthiocysteine as described by Barkholt & Jensen (1989).
After hydrolysis and neutralization, the amino acids and converted cystine are derivatized with 6-
aminoquinolyl-N-hydroxysuccinimidyl carbamate using the AccQ•Tag Ultra Derivatization Kit (Waters
Corporation, Milford, MA, USA). Amino acids are separated using reversed phase UHPLC with UV
detection at 260 nm.
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PERFORMANCE CHARACTERISTICS
3.1
Linearity
Linearity was assessed with at least six standard solutions ranging between 0.25 and 50 pmol/µl.
They were prepared from three independent stock solutions. Norvaline (Nva) was used as internal
standard.
Most amino acids studied follow a linear response within 0.25 to 50 pmol/µL, whereas the linearity
range for cystine is 0.5 to 10 pmol/µL and 0.25 to 30 pmol/µL for Tau, Pro and Tyr.
AMINO-01
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE