Kinetex 5 μm c18 100 250 4.6 mm

Plasma samples were thawed on ice and a spatula-tip (~5mg) of activated alumina powder (199966, Sigma) was added to each well of a 96-well AcroPrep filter plate with 0•2 µM wwPTFE membrane (514-1096, VWR). A100 µL of plasma sample, 1 µM DHBA (3,4-dihydroxybenzylamine hydrobromide, 858781, Sigma) as an internal standard, and 800 µL of TE buffer (2•5% EDTA; 1•5 M Tris/HCl pH 8•6) were added sequentially to the wells. Liquid was removed using a 96-well plate vacuum manifold and the alumina were washed twice with 800 µl of H2O. Catechols were eluted using 0•7% HClO4, which was incubated for 30 min at RT. Samples were injected in a HPLC-ED system (Ultimate 3000 SD HPLC system coupled to Ultimate 3000 ECD-3000RS electrochemical detector with a glassy carbon working electrode (DC amperometry at 800 mV), Thermo Scientific). Samples were analysed on a C18 column (Kinetex 5 μM, C18 100 Å, 250 × 4•6 mm, Phenomenex, Utrecht, The Netherlands) using a gradient of water/methanol with 0•1% formic acid (0-3 min, 99% H2O; 3-7 min, 99-30% H2O; 7-10 min 30-5% H2O; 10-11 min 5% H2O; 11-18 min, 99% H2O). Data recording and analysis were performed using Chromeleon software (version 6•8 SR13). Potential intake differences of levodopa were corrected by using carbidopa as an internal standard.