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have developed a method for the analysis of all major primary amino acids in plasma, using automated pre- column derivatization with OPA in combination with.
CUN.

CHEM. 40/2, 245-249

(1994)

#{149} Automation and

Analytical

Techniques

Plasma Amino Acids Determined by Liquid Chromatography Within 17 Minutes Tom Teerlink,1’3

Paul A. M. van Leeuwen,2

and Alexander

We present an HPLC method for the determination of amino acids in plasma. The method is based on automated precolumn derivatization of amino acids with o-phthalaldehyde, separation of the derivatives by reversed-phase chromatography, and quantification by fluorescence detection. Complete separation was achieved within 12 mm. Total analysis time, includingderivatization, chromatography, and reequilibration of the column, was 17 mm. The assay was linear from 5 to 800 mol/L for all amino acids. Recovery of amino acids added to plasma samples was 96-106%, except for tryptophan (89%). Within-run precision (CV) was 1.8-6.4%, and betweenrun precision was 2.1-7.2%. The method can be used for determining primary amino acids in plasma and cerebrospinal fluid. The simple sample preparation and short analysis time make the method particularly suitable for routine analysis of large series of samples. IndexIng

Terms: chromatography, reversed-phase/fluorometr,’

Determination of plasma increasingly important in

requirements

amino

become the nutritional

acids

has

evaluating of patients. This has resulted

in supplementation of nutrition with amino acids such as glutamine, arginune, and branched-chain amino acids (1-3). The beneficial effects of these feedings on the nutritional status of patients has stimulated clinicians and researchers to study amino acid metabolism in different disease states (4-6). These developments have led to an increase in the number of plasma amino acid determinations and the need for a rapid determination method. Physiological amino acids are traditionally determined by ion-exchange chromatography in combination with postcolumn ninhydrin detection. These analyses, usually performed on dedicated instruments, are somewhat laborious, costly, and time-consuming. Alternatively, amino acids can be analyzed by reversed-phase HPLC after precolumn derivatisation. Various reagents for the precolumn derivatization of amino acids can be used, e.g., dabsylchloride (7), phenylisothiocyanate (8-11), naphthylisocyanate (12), 9-fluorenylmethyl chioroformate (13, 14), and o-phthalaldehyde (OPA) (15-23). Of these reagents, OPA is most widely used. In the presence of a thiol compound, OPA reacts Departments

sity Hospital, lands.

of ‘Clinical Chemistry and 2Surgery,

P.O. Box

7057,

1007

Free UniverMB, Amsterdam, The Nether-

3Mdress correspondence to this author. Fax +31 205487673. ‘Nonstandard abbreviations: OPA, o-phthaladehyde; MPA, 3-mercaptopropionic acid; and SSA, 5-sulfosalicylic acid. Received August 16, 1993; accepted October 11, 1993.

Houdi.jk2 readily with primary amino acids, forming highly fluorescent derivatives. Although the isoindole derivatives formed are not very stable (24), accurate results can be obtained by automation of the derivatization reaction. Stabifity of the derivatives depends on the choice of the sulfhydryl reagent added to the derivatization mixture (24). 3-Mercaptopropionic acid (MPA) reportedly results in more stable products than does 2-mercaptoethanol (18, 23). Many of the published methods that include OPA derivati.zation require analysis times close to 1 h, limiting the number of samples that can be analyzed in a single run. Furthermore, most reports indicate that not all amino acids in plasma are adequately separated. We have developed a method for the analysis of all major primary amino acids in plasma, using automated precolumn derivatization with OPA in combination with MPA. Separation is achieved in 0.999). Within-run precision was determined by replicate analysis of an amino acid standard solution (n = 33) and a plasma sample (n = 28). For the standard solution the CV was 1.0-1.8%. For the plasma sample the CV was 1.8-4.9% with the exception of aspartic acid (6.4%). For quality-control purposes a plasma pool was stored at - 70#{176}C in small aliquots; this sample was analyzed in each run. Between-run CV, assessed from the results from 24 consecutive runs performed over 4 months, was