Optimization and development of a green high performance liquid chromatography method for determination of 8 anti-epileptic drugs and 2 active metabolites in human serum assessed using AGREE, AGREEprep, GAPI and BAGI.

Pharmacological treatment with antiepileptic drugs (AEDs) remains the primary choice for most patients with epilepsy, while therapeutic drug monitoring is an important strategy to ensure the efficacy and safety of AEDs. The aim of this study was to optimize and develop a green high-performance liquid chromatography (HPLC) method suitable for simultaneous detection of 8 AEDs and 2 active metabolites including primidone (PRM), lamotrigine (LTG), carbamazepine (CBZ) and its active metabolite 10,11-epoxide (CBZE), oxcarbazepine (OXC) and its active metabolite 10,11-dihydro-10-hydroxyl carbamazepine (MHD), phenobarbital (PHB), eslicarbazepine acetate (ESLA), clonazepam (CZP) and diazepam (DZP) in human serum. The results showed that using chlorzoxazone as the internal standard, ethanol as the protein precipitant of serum, a ZORBAX Eclipse Plus-C18 (150 × 4.6 mm, 3.5 μm) column as the stationary phase, and gradient ethanol and 15 mM potassium dihydrogen phosphate water solution as the green alternative mobile phase, good chromatographic separation for 10 target analytes could be obtained. The calibration curves of 10 analytes demonstrated good linearity within the test range (r > 0.996). The intra- and inter-batch precision and accuracy were all less than 15%, while extraction recoveries were in the range of 74.57-90.89% with relative standard deviation values less than 15%. The results of greenness assessment showed that the proposed method was ecofriendly. Finally, the validated method was successfully applied in the analysis of real serum samples.