A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

Li Xie; Liqin Chen; Pan Gu; Lanlan Wei; Xuejun Kang

doi:10.3791/56445

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Method Article

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

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DOI:

10.3791/56445

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March 1st, 2018

Li Xie¹ , Liqin Chen³ , Pan Gu⁴ , Lanlan Wei¹ , Xuejun Kang²

¹School of Public Health of Southeast University, Laboratory of Environment and Biosafety Research Institute of Southeast University in Suzhou, ²Key Laboratory of Child Development and Learning Science (Ministry of Education), School of Biological Science & Medical Engineering, Southeast University, ³School of Public Health, Tianjin Medical University, ⁴British Columbia Academy, Nanjing Foreign Language School

Summary

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

Abstract

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets.

Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

Introduction

Catecholamine neurotransmitters and their metabolite contents in body fluids can affect neural function and affect the balance of response-to-stimulus states to a large extent¹. Abnormities may cause a variety of diseases, such as pheochromacytoma, ganglioneuroma, neuroblastoma, and neurological disorders¹^,². The extraction and determination of catecholamines in body fluids is meaningful to the diagnosis of the relevant diseases. However, catecholamines in biological samples exist in low concentrations and are easily oxidized. Furthermore, they are very difficult to elute because of the....