Analytical Method for Gradient Separation of Monoamine, Adenine Nucleotide, and β‐Endorphin Neurotransmitters Using Amperometric and Spectrometric Detection

Baranyi, Mária and Karácsony, Orsolya and Sperlágh, Beáta (2025) Analytical Method for Gradient Separation of Monoamine, Adenine Nucleotide, and β‐Endorphin Neurotransmitters Using Amperometric and Spectrometric Detection. SEPARATION SCIENCE PLUS, 8 (10). ISSN 2573-1815

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Abstract

A novel analytical method combining gradient separation with amperometric/spectrometric detection is presented for the determination of monoamines: dopamine (DA), norepinephrine (NE), serotonin (5‐HT), nucleotides: adenosine triphosphate (ATP) and its metabolites, adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (Ado), and the opioid peptide β‐endorphin (BE). This method is capable of simultaneous detection of eight neurotransmitters with different chemical properties, thus significantly different from specific protocols focusing on only a few analytes. Chromatographic online enrichment was performed using a core–shell structure phenyl–hexyl (75 × 2.1 mm 2 , 5 µm) column followed by separation on an Ultra Core Super C‐18 (150 × 2.1 mm 2 , 5 µm) column. The mobile phases were based on an aqueous potassium phosphate buffer solution with controlled pH, chelating and ion‐pairing components, and buffer B supplemented with the organic modifier (methanol–acetonitrile) for gradient separation. With constant composition parameters, the separation was quasi‐isocratic; the organic component resulted in baseline drift, but no additional time was required to re‐equilibrate the column between runs. The experimental parameters used for separation and detection were tested for linearity, ( R 2 ) 0.9959 and 0.9994; repeatability, accuracy, and intra‐ and inter‐day coefficients of variation 99.44, 0.24–9.36, and 0.85%–9.67%, respectively; and sensitivity, with detection ranges of 18.2–79.7 pg/mL and quantification ranges of 55.3–241.5 pg/mL. The applicability of the method was demonstrated by analyzing plasma and brain tissue samples from a mouse model of chronic fatigue syndrome and calculating the intra‐laboratory variability from those data. This validated analytical method, with its ability to detect concentrations in the nanogram‐range in the measurement of ATP, ADP, AMP, Ado, DA, NE, 5‐HT, and BE therefore opens the way for potential applications.

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