Analysis of pramipexole in human urine by CE-LIF after derivatisation with fluorescein isothiocyanate

Pramipexole (4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazolediamine) is a non-ergoline dopamine agonist, approved for the treatment of Parkinson’s disease as a monotherapy or in combination with levodopa in advanced disease patients with reduced response to levodopa. Pramipexole is orally administered as the dihydrochloride salt, at a starting dose of 0.375 mg/day, which is gradually increased up to 4.5 mg/day. Common side effects are orthostatic hypotension, dyskinesias, constipation, asthenia, insomnia hallucinations and others. Pramipexole undergoes a minimum hepatic metabolism, therefore 90% of the drug is eliminated unchanged in the urine, which represents the main elimination pathway. Pramipexole clearance can be reduced in several cases, such as in the elderly and in patients with renal insufficiency. Only a few HPLC methods, and no capillary electrophoretic method, can be found in the literature for the determination of Pramipexole. In order to accurately determine urinary Pramipexole levels in patients undergoing treatment, a method based on capillary electrophoresis with LIF (laser-induced fluorescence) detection has been developed. The method employs uncoated fused silica capillaries (75 µm internal diameter, 60 cm effective length) and a BGE composed of borate buffer containing tetrabutylammonium bromide and acetone. A complete removal of interference is obtained by means of a liquid/liquid extraction procedure with ethyl acetate, followed by derivatisation with fluorescein isothiocyanate at pH 9, which allows the detection by LIF (laser wavelength: 488 nm). A complete electrophoretic run lasts about 12 min when applying a 20 kV voltage, allowing the determination of Pramipexole in human urine. The method achieves satisfactory sensitivity, with LOD and LOQ values corresponding to 2 and 5 ng/mL, respectively. Preliminary results are very promising and the method is now undergoing validation.