Development and Validation of a New LC-MS/MS Bioanalytical Method for the Simultaneous Determination of Levodopa, Levodopa Methyl Ester, and Carbidopa in Human Plasma Samples

Abstract

Levodopa (L-DOPA) treatment, combined with the administration of dopa-decarboxylase inhibitors (DDCIs), is still the most effective symptomatic treatment of Parkinson's disease (PD). Although its efficacy in the early stage of the disease has been confirmed, its complex pharmacokinetics (PK) increases the variability of the intra-individual motor response, thus amplifying the risk of motor/non-motor fluctuations and dyskinesia. Moreover, it has been demonstrated that L-DOPA PK is strongly influenced by several clinical, therapeutic, and lifestyle variables (e.g., dietary proteins). L-DOPA therapeutic monitoring is therefore crucial to provide personalized therapy, hence improving drug efficacy and safety. To this aim, we have developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify L-DOPA, levodopa methyl ester (LDME), and the DDCI carbidopa in human plasma. The compounds were extracted by protein precipitation and samples were analyzed with a triple quadrupole mass spectrometer. The method showed good selectivity and specificity for all compounds. No carryover was observed, and dilution integrity was demonstrated. No matrix effect could be retrieved; intra-day and inter-day precision and accuracy values met the acceptance criteria. Reinjection reproducibility was assessed. The described method was successfully applied to a 45-year-old male patient to compare the pharmacokinetic behavior of an L-DOPA-based medical treatment involving commercially available Mucuna pruriens extracts and an LDME/carbidopa (100/25 mg) formulation.

Keywords: Parkinson’s disease; analytical method; carbidopa; levodopa; levodopa methyl ester; liquid chromatography–mass spectrometry (LC–MS); therapeutic drug monitoring.

Figure 2

Representative chromatograms of blank plasma (1) compared to spiked plasma (2). In panel (A), L-DOPA (quan 198.09 → 152.13 m/z, RT: 0.89 min); in panel (B), LDME (quan 212.50 → 152.13 m/z, RT: 1.53 min); in panel (C), carbidopa (quan 227.07 → 181.11 m/z, RT: 1.44 min); in panel (D), internal standard L-DOPA-D3 (201.05 → 154.39 m/z, RT: 0.85 min). Qualifier ions are described in Table 3.

Figure 3

L-DOPA plasma concentrations (µg/L) and PK parameters at 0, 15, 30, 45, 60, 90, 120, 150, and 180 min after administration of two tablets of an LDME/carbidopa (100/25 mg) formulation (L-DOPA concentrations curve in green and carbidopa in red) (A) compared to plasma concentrations after the administration of 500 mg of L-DOPA from commercially available Mucuna pruriens extracts (Mucuna pruriens-derived L-DOPA concentrations curve in blue) (B).

Figure 1

Spectral data and fragment analysis of L-DOPA (panel (a)), LDME (panel (b)), and carbidopa (panel (c)). Panel (d) shows L-DOPA metabolism and enzymes involved in sequential transformation (catechol-O-methyltransferase COMT; monoamine oxidases (MAO).