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


CONCLUSIONS
This HPLC method was comparable to IEC and could represent an alternative for amino acid analysis.
Schwarz
Elisabeth L
EL
ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratory, Salt Lake City, Utah 84108, USA. schwarl@aruplab.com
Roberts
William L
WL
Pasquali
Marzia
M
D000596:Amino Acids / Q000032:analysis* / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D005452:Fluoresceins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013997:Time Factors
D000596:Amino Acids; D005452:Fluoresceins