A novel UPLC-ECD method for simultaneous quantification of levodopa, benserazide, and their major metabolites in human plasma and cerebrospinal fluid.

Levodopa (LD) combined with peripheral dopa decarboxylase inhibitors (DDCIs) remains the cornerstone therapy for Parkinson's disease (PD). Benserazide (BZ), a widely used DDCI, is rapidly converted in vivo to 2,3,4-trihydroxybenzylhydrazine (THBH), an active metabolite with comparable inhibitory activity. Although BZ and THBH are generally considered to exhibit negligible penetration across the blood-brain barrier, evaluation of their potential central exposure requires highly sensitive quantification in cerebrospinal fluid (CSF). However, no validated method is currently available for simultaneous determination of LD, dopamine (DA), BZ, and THBH in paired human plasma and CSF. Here, we developed and validated a rapid ultra-performance liquid chromatography-electrochemical detection (UPLC-ECD) method enabling concurrent quantification of all four analytes in both matrices. The assay provides baseline separation with minimal endogenous interference, excellent linearity across clinically relevant ranges, and low limits of quantification. Precision, accuracy, recovery, and stability met current bioanalytical validation criteria. This method offers a robust analytical platform for clinical and translational studies of LD-BZ therapy.