Electrochemical detection

Electrochemical detection is based on the principle that electroactive compounds are oxidized or reduced at a certain potential and the resulting flow of electrons creates a

measurable current. Catecholamines and metanephrines are easily oxidized to form quinones, as illustrated in Figure 7.3, and therefore are suitable for electrochemical detection. Because these biogenic amines are more easily oxidized than many other compounds, it is possible to obtain a certain degree of selectivity with electrochemical detection if the oxidizing potential is kept as low as possible. Two types of electrochemical detectors exist. The coulometric detector oxidizes 100% of the catecholamine molecules in the effluent and therefore creates a larger current than the amperometric detector, which oxidizes less than 10% of these molecules. It has been reported that the higher yield of the coulometric detector does not seem to increase its sensitivity compared with the ampero-metric detector because the background noise level increases in proportion to the increase in signal, so the two detectors have a similar signal-to-noise ratio.90 Nonetheless, the wide use of the coulometric detector for the trace analysis of free catecholamines and/or metane-phrines in urine reflects its high sensitivity in practical applications.62,74,91,92 This high sensitivity and selectivity of coulometric detection may be a result of the oxidation of the analytes in the guard cell followed by reduction at successively higher reduction potentials in the analytical cell.93 The response of the electrochemical detector is influenced by ionic strength and pH of the mobile phase, which must be taken into account when optimizing the HPLC assay. For all their simplicity, though, electrochemical detectors require considerable attention to troubleshooting and minimization of disturbances in order to obtain optimal

sensitivity.94

Electrochemical detection with ion-pairing adaptations of reversed-phase chromatogra-phy are the most common methodologies, and many techniques for measuring urinary catecholamines and metanephrines have been published.57,59,65,71,72,74,95-108

Ion-pairing with alkyl sulfonates or sulfates enhances the retention of cationic amine moieties on the lipophilic stationary phase. The effects of mobile phase composition (ion-pairing agents, ionic strength or organic components) on the chromatographic and electrochemical behavior of catecholamines and their metabolites has been reviewed previously.93109

The coupling of ion-exchange chromatography with electrochemical detection has been applied to the measurement of urinary catecholamines.62,110-113 Early applications of ionexchange chromatography were hampered by the lack of column reproducibility, resulting in the loss of catecholamines during the analytical process.110 Mefford reported that semiirreversible loading of C18 columns with lauroylsarcosine resulted in separations that are dependent on ion-exchange mechanisms.112 Subsequently, N-methyloleoyl taurate was applied for the ion-exchange chromatographic separation of catecholamines. This latter resin facilitated the elution of E prior to NE and, therefore, allowed enhanced sensitivity for the normally lower concentration of E.112 This enhanced sensitivity may be of particular value in detecting small tumors that preferentially secrete E. Sarzanini et al. reported an ion chromatographic procedure for the simultaneous purification and determination of urinary NE, E and DA.62

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