Ultra Micro Quantification of Dopamine and Homovanillic Acid in Human Brain Tissue: Quest for Higher Recovery and Sensitivity with CoulArray HPLC‐ECD System

Abstract

Dopamine (DA), a biogenic amine neurotransmitter is involved in regulating a number of functions of the central and peripheral nervous systems, including voluntary and involuntary motor functions and cognitive behaviors. Since the concentration of dopamine in different parts of the brain is extremely small and decreases further in neurodegenerative diseases, such as HIV‐1 infection, a highly sensitive and specific methodology is required to quantify DA and its metabolite, homovanillic acid (HVA) in small weights of tissues from different regions of the brain. The method presented in this report used a highly sensitive multielectrode CoulArray HPLC‐ECD system for quantification of <4 pg of DA and <10 pg of HVA in homogenates of human brain tissues.

A linear relationship in the calibration curves was observed between a wide range of concentration of standards of DA (50–500 pg/mL) and HVA (0.1–20.0 ng/mL) and the corresponding response factors. Separate aliquots from the same pool of brain tissue homogenate were used to extract DA and HVA by their specific extraction procedure and were quantified separately. Recovery of both DA and HVA from homogenate preparation and aqueous extraction were in the range of 88–98%. Stability of the system is evident from the consistency of retention times of analytes in different experiments. The reliability of the method is shown from the reproducibility of the values of DA and HVA obtained in different aliquots of the brain tissue homogenates.

Keywords:

• Dopamine
• HVA
• Brain tissues
• CoulArray HPLC‐ECD

Acknowledgments

This study was supported in part by the NIH grants NO: RO1 NS 43982, RO1 DA 13550, RO1 DA 12792, and RO1 NS 41205. The human brain tissue used for the study was supplied by the NIH supported National Neurological Tissue Consortium (NNTC).