Micellar Electrokinetic Capillary Chromatography- Electrochemical Detection for Analysis of Biogenic Amines in Drosophila melanogaster

Abstract

Micellar electrokinetic chromatography coupled to amperometric electrochemical detection was used to investigate the chemical environment of the fruit fly, Drosophila melanogaster. Preliminary studies focused on the employment and optimization of the system to separate electroactive amine-containing molecules present in the head and body of male and female flies. Ultimately, biogenic amines significant to the fly including L-3,4-dihydroxyphenylalanine, dopamine, tyramine, and serotonin were identified and their relative abundance quantified. Transgenic Drosophila with functionally ablated dopamine and serotonin neurons were analyzed to demonstrate the sensitivity of the technique. The separation method developed in this study should offer an advantage in elucidating the critical role that electroactive biogenic amines play in complex physiological processes correlated with Drosophila behavior.

Analysis of Biogenic Amine Variability among Individual Fly Heads with Micellar Electrokinetic Capillary Chromatography−Electrochemical Detection

“…Nine different analytes have been reproducibly identified in a single Drosophila head homogenate without sample cleanup, which was previously adopted in the studies of Drosophila population homogenates. 24,25 The identified peaks shown in Figure 4 are consistent with previous population studies on Drosophila. [19][20][21][22][23][24][25] The presence of L-DOPA is expected as it is a synthetic precursor to DA.…”

“…This contrasts with our previous work where 30-50 heads were homogenized together in volumes of 1 μL/head and OA is not detectable. 25 It is notable that the current results, in general, agree with other published reports, 20,24 and it is not entirely clear why we do not observe OA under the previously described conditions. One possibility is that OA is below the detection limit for a real sample under those conditions and the better detection limits gained with the lower volume and better homogenization described here allow its detection.…”

“…The separation profile of a single Drosophila head in a 1-μL homogenization volume very closely matches that of previous population studies. 25 This result clearly demonstrates the utility of the miniature tissue homogenizer for homogenizing a single fly head. Subsequently, addition of homogenization solution in 750-, 500-, 250-, and 100-nL quantities to a single head was performed (Figure 3b-e).…”

Utility of separation science in metabolomic studies

“…These compounds are involved in a variety of physiological processes including emotion, adaptive behaviors such as learning, memory, and drug addiction, and stress-induced responses [54,55]. The amines and their metabolites that were quantitatively assayed included dopamine, epinephrine, norepinephrine (NE), octopamine (OA), L-3, 4-dihydroxyphenylalanine, tyramine (TA), serotonin, 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.…”

Recent advances in micellar electrokinetic chromatography

“…Amperometric detection has been applied to biogenic amines in rice spirits [126], pesticides [127], and homogenized fruit fly (heads or bodies) [128]. For these reports, detection limits were submicromolar [126,127].…”