Automatic semi-microcolumn liquid chromatographic determination of catecholamines in rat plasma utilizing peroxyoxalate chemiluminescence reaction

Abstract

A fully automated and highly sensitive method with a semi-microcolumn liquid chromatography system for the determination of rat plasma catecholamines (CAs) was developed. Automated on-line extraction of CAs in diluted plasma using a precolumn packed with strong acidic cation exchange resin was coupled with separation of CAs on a semi-microcolumn (250 x 1.5 mm id). fluorogenic derivatization with ethylenediamine and finally postcolumn peroxyoxalate chemiluminescence detection utilizing bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate (TDPO) and hydrogen peroxide. The detection limits were 0.91, 0.36 and 1.1 fmol for norepinephrine (noradrenaline), epinephrine (adrenaline) and dopamine, respectively, at a signal-to-noise ratio of 3. A good linearity of the calibration curve for each CA was observed in the range of 5.0 to 500 fmol for each CA using N-methyldopamine (N-MeDA) as an internal standard. The RSD for the proposed method (n = 5) were 3.7-9.5% for the intra-day assay and 6.6-10.0% for the inter-day assay. The volume of rat plasma required for the determination of CAs was 10 microliters.

Determination of catecholamines and their 3‐O‐methyl metabolites in mouse plasma

“…The basal CAs and their 3-Omethyl metabolite concentrations in the mouse plasma are summarized in Table 1. Plasma CAs and their 3-O-methyl metabolite concentrations of the mouse plasma were similar to or slightly higher than those of rat (Lenders et al, 1993;Prados et al, 1994;Tsunoda et al, 1999;Takezawa et al, 2000).…”

Chemiluminescence detection in liquid chromatography: Applications to clinical, pharmaceutical, environmental and food analysis—A review

“…Imai's group has extensively studied the determination of CAs by HPLC-CL by means of the PO reaction. They proposed a fully automated method with a semi-microcolumn HPLC system for the determination of CAs in rat plasma [42]. The method used a precolumn packed with strong acidic cation exchange resin coupled with separation on a semi-microcolumn, followed by fluorogenic derivatization with ethylenediamine (ED) in a reaction coil immersed in a thermostatically controlled bath.…”

Analysis of plasma catecholamines by liquid chromatography with amperometric detection using a novel SPE ion‐exchange procedure

“…Fluorimetric detection after derivatisation has been also reported [15]. An automated HPLC determination of CAs in rat plasma using the peroxyoxalate chemiluminescence reaction has also been reported [16], as well as chemiluminescence determination of CAs in human blood plasma using 1,2-bis(3-chlorophenyl)ethylenediamine as a derivatisation reagent [17]. Recently, we developed an HPLC method with coulometric detection with a careful SPE procedure for the extraction of CAs from plasma, based on their complexation with diphenylborate [18].…”