Development and validation of a spectrofluorimetric method for MHPG detection in plasma via rhodamine B fluorescence quenching: a Box-Behnken design approach with application to autism spectrum disorder

Abstract

This study presents the development and validation of a sensitive spectrofluorimetric method for detecting 3-methoxy-4-hydroxyphenylglycol (MHPG), a major metabolite of norepinephrine and proxy for central noradrenergic activity, in plasma based on Rhodamine B fluorescence quenching. The spectral characteristics of Rhodamine B and its ion pair complex formation with MHPG were investigated using UV–vis and fluorescence spectroscopy. Mechanistic studies employing Stern-Volmer kinetics, thermodynamic analysis, and Job's method of continuous variation revealed a static quenching mode with 1:1 complex formation stoichiometry. Critical method parameters such as pH, Rhodamine B volume, and incubation time were optimized using a Box-Behnken design of experiments, with desirability function analysis employed to maximize the quenching efficiency and sensitivity. The method was validated according to the ICH M10 guidelines and demonstrated excellent linearity (2–200 ng/mL), precision, accuracy and selectivity, with lower limits of detection and quantification of 0.655 and 1.966 ng/mL, respectively. Application to a pilot cohort of children with ASD and neurotypical controls revealed elevated plasma MHPG levels in the ASD group, although the difference did not reach statistical significance. Notably, age-related trends in MHPG levels differed markedly between groups, with a robust positive correlation observed in controls but absent in the ASD group. Sex-specific analyses further suggested more pronounced sex-related variations in noradrenergic function in ASD compared to controls. These preliminary results provide valuable insights into potential alterations of the noradrenergic system in ASD and highlight the utility of the developed spectrofluorimetric method, warranting further investigations in larger cohorts to elucidate the clinical relevance of disturbed noradrenergic function in ASD.