Neurochemical Changes Following Botulinum Toxin Type A in Chronic Migraine: An LC–MS/MS and HPLC Evaluation of Plasma and Urinary Biomarkers

<b>Background:</b> Botulinum toxin type A (BoNT-A) is an established preventive therapy for chronic migraine (CM), yet the accompanying neurochemical changes remain incompletely characterized. <b>Objective:</b> To evaluate the effects of BoNT-A on plasma substance P (SP), γ-aminobutyric acid (GABA), glutamate, glutamine, and 5-hydroxytryptamine (5-HT), and on urinary 5-HT, and to explore relationships with clinical outcomes. <b>Methods:</b> In this prospective study, plasma neurotransmitters were analyzed in CM patients (n = 31) at baseline and one month after BoNT-A (155 U; PREEMPT protocol) and in healthy controls (n = 30). Plasma SP was measured using enzyme-linked immunosorbent assay (ELISA); plasma GABA, glutamate, and glutamine were quantified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) with isotopically labeled internal standards; plasma and urinary 5-HT were determined by high-performance liquid chromatography (HPLC). Clinical outcomes included monthly headache frequency, Visual Analog Scale (VAS), and Migraine Disability Assessment (MIDAS). Statistical analyses applied appropriate parametric or non-parametric tests with <i>p</i> < 0.05 considered significant. <b>Results:</b> One month post-BoNT-A, headache frequency, MIDAS, and VAS were significantly reduced (all <i>p</i> < 0.001). SP levels were significantly higher after BoNT-A than at baseline and versus controls. Plasma 5-HT increased post-BoNT-A, while urinary 5-HT decreased. Plasma GABA was elevated in patients versus controls without statistical significance. Glutamine was significantly higher before treatment, whereas the Glu/Gln ratio increased after BoNT-A. Correlations revealed that higher GABA was associated with lower VAS and attack frequency post-treatment. <b>Conclusions:</b> BoNT-A provided short-term clinical improvement with distinct neurochemical changes, including increased plasma SP and 5-HT, decreased urinary 5-HT, reduced glutamine, and a higher Glu/Gln ratio. These biomarkers, particularly Glu/Gln, may serve as indicators of cortical excitability and therapeutic response in CM.