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Clin Chim Acta. 2005 Apr;354(1-2):83-90. Epub 2004 Dec 25.

Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection.

Author information

1
ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratory, Salt Lake City, Utah 84108, USA. schwarl@aruplab.com

Abstract

BACKGROUND:

Plasma amino acids are usually analyzed by ion-exchange chromatography (IEC), a reproducible but time consuming method. Here, we test whether plasma amino acids can be analyzed using reverse-phase high performance liquid chromatography (HPLC).

METHODS:

Filtered plasma, with S-carboxymethyl-l-cysteine as the internal standard, was derivatized and analyzed by an Agilent 1100 HPLC system. Primary amino acids were derivatized with o-phthalaldehyde 3-mercaptopropionic acid (OPA) and detected by a diode array detector. Secondary amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC) and detected fluorometrically. Chromatographic separation is achieved by two gradient elutions (two injections per sample), starting at different pHs, on a reverse phase Agilent Zorbax Eclipse C(18) column AAA (4.6 x 150 mm).

RESULTS:

The HPLC method evaluated correlated well with IEC (0.89</=r</=1.00) with linearity up to 2500 mumol/l. The between- and within-run CVs were <6.0%. In addition, this method is able to separate argininosuccinic acid, homocystine and allo-isoleucine, rare but clinically significant amino acids.

CONCLUSION:

This HPLC method was comparable to IEC and could represent an alternative for amino acid analysis.

PMID:
15748603
DOI:
10.1016/j.cccn.2004.11.016
[Indexed for MEDLINE]

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