Gas chromatography mass spectrometry based pharmacometabolomic assessment of formulation-dependent metabolic responses of levetiracetam

• Gaz chromatography Mass spectrometry based pharmacometabolomics was applied to levetiracetam formulations. • Multivariate analysis showed formulation-specific metabolic clustering. • Energy and amino acid metabolism changed with systemic drug exposure. • Pharmacometabolomics revealed biochemical adaptation beyond pharmacokinetic profiles. . This study applied a GC–MS–based pharmacometabolomic approach to investigate formulation-dependent bioavailability and systemic metabolic responses following levetiracetam administration. Plasma samples from rabbits were analyzed after receiving three different formulations: a control formulation in which levetiracetam was mixed with deionized water, a multithick formulation prepared with an 8% modified maize starch thickener, and a thicken-up clear (TUC) formulation prepared with 3.6% xanthan gum thickener. Although dissolution profiles varied substantially among these formulations, Likewise, total levetiracetam exposure, represented by AUC, did not differ significantly among groups (p<0.05). Untargeted GC-MS analysis was annotated 97 metabolites and multivariate analysis revealed distinct metabolic clustering, with the TUC group showing the most pronounced separation. Correlation analysis identified five metabolites that were positively associated with pharmacokinetic parameters: pyrophosphate, glyceric acid, aconitic acid, methyl-2-butenedioic acid, and itaconic acid. Six amino acid–related metabolites showed negative associations, reflecting enhanced energy metabolism together with reduced excitatory neurotransmission, consistent with the known neuroprotective and antiepileptic actions of levetiracetam. Overall, these results demonstrate that formulation design influences not only dissolution behavior but also systemic metabolic responses. Pharmacometabolomics offers a quantitative framework for integrating chromatographic, pharmacokinetic and biochemical data to support rational formulation development and precision pharmacotherapy. .