Development and validation of a simple and sensitive method for quantification of levodopa and carbidopa in rat and monkey plasma using derivatization and UPLC–MS/MS

• Derivatization of levodopa and carbidopa with fluorescamine.
• Derivatization improved chromatographic retention and sensitivity.
• The method was validated over the concentration range of 5/3–5000/3000 ng/mL for levodopa/carbidopa.
• This method was successfully employed in the analysis of rat and monkey plasma study samples.

A simple, selective, and sensitive quantitative method has been developed for the simultaneous determination of levodopa and carbidopa in rat and monkey plasma by protein precipitation using acetonitrile containing the derivatizing reagent, flourescamine. Derivatized products of levodopa and carbidopa were separated on a BEH C18 column (2.1 mm × 50 mm; 1.7 μm particle size) using ultra high performance liquid chromatography (UHPLC) without any further purification. Tandem mass spectrometry (MS/MS) was used for detection. The method was validated over the concentration range of 5–5000 ng/mL and 3–3000 ng/mL for levodopa and carbidopa, respectively in rat and monkey plasma. Due to the poor stability of the investigated analytes in biological matrices, a mixture of sodium metabisulfite and hydrazine dihydrochloride was used as a stabilizer. This method was successfully utilized to support pharmacokinetic studies in both species. The results from assay validations and incurred samples re-analysis show that the method is selective, sensitive and robust. To our knowledge, this is the first UHPLC–MS/MS based method that utilizes derivatization with fluorescamine and provides adequate sensitivity for both levodopa and carbidopa with 50 μL of sample and a run time of 3.5 min.