Microcolumn separation of amine metabolites in the fruit fly

Abstract

Electrophoretic resolution of 14 biogenic amines and metabolites with similar mobilities is addressed by employing micellar electrokinetic capillary chromatography coupled to amperometric electrochemical detection. The present study describes the optimization of separation conditions to achieve resolution of analytes of biological significance within 20 min in a single separation. They include dopamine, epinephrine, norepinephrine, octopamine (OA), L-3, 4-dihydroxyphenylalanine, tyramine (TA), and serotonin as well as metabolites 5-hydroxyindolacetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine in addition to N-acetylated metabolites including N-acetyldopamine, N-acetyloctopamine (naOA), and N-acetylserotonin. The optimized conditions used result in excellent reproducibility and predictable peak shifting, thus enabling identification of several metabolites along with their biogenic amine precursors in biological samples, specifically from the fruit fly Drosophila melanogaster. The separation method is sensitive, selective, and quantitative as demonstrated by its capacity to detect changes in TA, OA, and naOA present in the head homogenates of the Canton-S and mutant inactive(1) Drosophila lines. Quantitative analysis of metabolites in conjunction with their biogenic amine precursors in a single separation offers tremendous potential to understand the physiological processes and underlying mechanisms mediated by various biogenic amines in Drosophila and other animals.

Figure 1

A single separation of 100 μM standards shown with the following peak identification: L-DOPA and naOA (1), naDA (2), na5-HT (3), HVA, 5-HIAA and DOPAC (4), NE (5), E (6), OA (7), DA (8), TA (9), 3-MT (10), 5-HT (11). Separation was performed in a 13-μm i.d. capillary with a separation potential of 550 V/cm. The working electrode was held at +750 mV vs. a Ag/AgCl reference electrode.

Figure 2

A single optimized separation of 100 μM standards (10 μM CAT, internal standard) shown with the following peak identification: DA (1), E (2), naOA (3), NE (4), OA (5), na5HT (6), naDA (7), 5-HIAA (8), L-DOPA (9), HVA (10), CAT (11), TA (12), 3-MT (13), 5-HT (14), DOPAC (15). Separations were performed in a 13-μm i.d. capillary with separation potentials of 333 V/cm. The working electrode was held at +750 mV vs. a Ag/AgCl reference electrode.

Figure 3

Three standard separations of 100 μM amines, metabolites and internal standard. The peak order is as in Figure 2. Separations were performed on a 13-μm i.d. capillary with separation potentials of 333 V/cm. Fly homogenate samples were run between standard separations. The working electrode was held at +750 mV vs. a Ag/AgCl reference electrode.

Figure 4

(A) Enlargement of 5.5 min to 9 min of a MEKC-EC separation of a Drosophila head homogenate highlighting DA (1), naOA (3), OA (5), na5HT (6), and naDA (7). (B) Enlargement of 9 min to 11 min of the separation emphasizing peaks for L-DOPA (9), and CAT (11). (C) Enlargement of 11 min to 15 min of the electropherogram showing TA (12). (D) Comparison of Canton-S (wt) and iav¹ Drosophila head homogenates highlighting the internal standard CAT (11) and TA (12). The numbers correspond to those in Figure 2. Field strength for the separation was 333 V/cm. The working electrode was held at +750 mV vs. Ag/AgCl.