Sensing of Catecholamine in Human Urine Using a Simple Colorimetric Assay Based on Direct Melanochrome and Indolequinone Formation

Abstract

We used the first enzyme-free synthesis and stabilization of soluble melanochrome (MC) and 5,6-indolequinone (IQ) derived from levodopa (LD), dopamine (DA), and norepinephrine (NE) oxidation to develop a simple colorimetric assay for catecholamine detection in human urine, also elucidating the time-dependent formation and molecular weight of MC and IQ using UV-Vis spectroscopy and mass spectrometry. The quantitative detection of LD and DA was achieved in human urine using MC as a selective colorimetric reporter to demonstrate the potential assay applicability in a matrix of interest in therapeutic drug monitoring (TDM) and in clinical chemistry. The assay showed a linear dynamic range between 5.0 mg L^-1 and 50.0 mg L^-1, covering the concentration range of DA and LD found in urine samples from, e.g., Parkinson's patients undergoing LD-based pharmacological therapy. The data reproducibility in the real matrix was very good within this concentration range (RSD_av% 3.7% and 6.1% for DA and LD, respectively), also showing very good analytical performances with the limits of detection of 3.69 ± 0.17 mg L^-1 and 2.51 ± 0.08 mg L^-1 for DA and LD, respectively, thus paving the way for the effective and non-invasive monitoring of dopamine and levodopa in urine from patients during TDM in Parkinson's disease.

Keywords: Parkinson’s disease; catecholamines oxidation; colorimetric assay; dopamine; indolequinone; levodopa; melanochrome; norepinephrine; therapeutic drug monitoring; urine analysis.

Figure 1

Time-dependent oxidation of natural catecholamines 0.2 g L⁻¹ in DMSO:H₂O 1:1 (v/v) in the presence of 150 mM Mg(Ac)₂, 150 mM NH4Cl at pH 9.4 and 25 °C between 2 min and 144 min. UV–Vis spectra (upper panels) and absorbance values (lower panels) at 340 nm (orange circles/dashed line) and 590 nm (purple circles/dashed lines) for dopamine (DA), levodopa (LD), and norepinephrine (NE).

Figure 2

Catecholamines calibration curves in human urine spiked with levodopa (LD, purple circles), dopamine (DA, white circles), and norepinephrine (NE, orange circles), from 5.0 to 50.0 mg L⁻¹, and dilute 1:8 (v/v) with a proper buffer (150 mM Mg(Ac)₂, 150 mM NH₄Cl at pH 9.4 in DMSO:H₂O 1:1 (v/v)) [4]. Absorbance at 585 nm was acquired at 25 °C after 15 min of solution mixing. Each point represents the mean ± SD (n = 4).