Total and Non-Protein-bound Fractions of 3,4-Dihydroxyphenylalanine

¹ Laboratorio di Analisi Chimico Cliniche, Ospedale di Circolo, Università di Pavia, 21100 Varese, Italy,
² Servizio di Analisi Chimico Cliniche, IRCCS S. Matteo, Università di Pavia, 27100 Pavia, Italy,
³ Unità di Farmacologia Clinica, Università di Pavia, 27100 Pavia, Italy
^a Address correspondence to this author at: Piazza Berengario 5, 27100 Pavia, Italy. Fax 0332 260017.

To the Editor:

Several analytical methods for the determination of plasma 3,4-dihydroxyphenylalanine (L-DOPA) by HPLC have been reported. Many of these methods require preliminary purification of the plasma sample. Such purification procedures include adsorption on alumina (1) and solid-phase (2) or solvent extraction (3). The direct injection of deproteinized plasma supernatant (4) and the determination of the non-protein-bound fraction by ultrafiltration (5) offer simplified alternatives for rapid assay.

Dethy et al. (6), propose microdialysis-HPLC to determine L-DOPA and its metabolites (3,4-dihydroxyphenylacetic acid, dopamine, and homovanillic acid) in the plasma of patients with advanced Parkinson's disease. We would like to make a number of comments concerning this attractive new method. First, it should be noted that microdialysis excludes large molecules (the authors do not specify the molecular weight excluded by the membrane) and thus separates protein-bound L-DOPA that is not dialyzed from unbound L-DOPA that passes through the membrane. Although L-DOPA binds to plasma proteins only to a minor extent, the unbound fraction is influenced by the total concentration in the sample. In fact, it increases from an average of ~70% at 100 µg/L L-DOPA to ~90% at 1000 µg/L (7). Although for therapeutic drug monitoring purposes unbound plasma concentrations are considered to more accurately reflect drug concentrations at the site of action, variability in protein binding implies that results in plasma dialysates and whole plasma may not be comparable.

Another important issue is the poor stability of L-DOPA, particularly when it is separated from its plasma matrix. Addition of antioxidants like ascorbic acid (5 mM) or sodium metabisulfite (5 mM) does not markedly improve the stability of an aqueous solution of L-DOPA, whose concentration, even in the presence of the above antioxidants, decreases by 60% after 3 hours at room temperature (8). Moreover, antioxidants may increase the background current of the electrochemical detector and may give rise to additional unidentified peaks (8). It would, therefore, be important to determine the stability of the drug in plasma and dialysates using the antioxidant solution proposed by the authors.

Finally, with respect to the chromatographic separation, the peaks of L-DOPA, 3,4-dihydroxyphenylacetic acid, and DA are not well separated from each other, from the solvent front, or from two unidentified peaks. For these reasons, it would be useful to know the procedures used to confirm the identity of peaks and to exclude possible coelution with unidentified substances.

References

1. Premet-Cabit A, Allain P. Simultaneous liquid chromatographic determination of norepinephrine, 3,4-dihydroxyphenylethyleneglycol, 3,4-dihydroxyphenylalanine and 3,4-dihydroxyphenylacetic acid in human plasma. J Chromatogr 1988;434:187-190.[Medline] [Order article via Infotrieve]
2. Notha H, Yamaguchi E, Ohkura Y. Measurement of catecholamines, their precursor and metabolites in human urine and plasma by solid-phase extraction followed by high-performance liquid chromatography with fluorescence derivatization. J Chromatogr 1989;493:15-26.[Web of Science][Medline] [Order article via Infotrieve]
3. Tsuchiya H. Determination of L-3,4-dihydroxyphenylalanine in blood by high-performance liquid chromatography after solvent extraction. J Chromatogr 1989;491:291-298.[Medline] [Order article via Infotrieve]
4. Zurcher G. Da Prada M. Simple automated high-performance liquid chromatographic column-switching technique for the measurement of dopa and 3-O-methyldopa in plasma. J Chromatogr 1990;530:253-262.[Medline] [Order article via Infotrieve]
5. . Melzi d'Eril GV, Rizzo V. Determination of free DOPA and 3-O-methyl-DOPA in human plasma by high-performance liquid chromatography with electrochemical detection. J Chromatogr 1991;553:265-269.[Medline] [Order article via Infotrieve]
6. Dethy S, Laute MA, Van Blercom N, Damhaut P, Goldman S, Hildebrand J. Microdialysis-HPLC for plasma levodopa and metabolites monitoring in parkinsonian patients. Clin Chem 1997;43:740-744.[Abstract/Free Full Text]
7. Rizzo V, Memmi M, Moratti R, Melzi d'Eril GV, Perucca E. Concentrations of L-dopa in plasma and plasma ultrafiltrates. J Pharm Biomed Anal 1996;14:1043-1046.[Web of Science][Medline] [Order article via Infotrieve]
8. Rizzo V, Pastore R, Pankopf S, Melzi d'Eril GV, Moratti R. Determination of total and non-protein-bound fractions of L-DOPA in blood plasma by liquid chromatography and electrochemistry. Biog Amines 1996;12:1-7.