Quantitative determination and pharmacokinetic study of the novel anti-Parkinson's disease candidate drug FLZ in rat brain by high performance liquid chromatography–tandem mass spectrometry

FLZ (N-[2-(4-hydroxy-phenyl)-ethyl]-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxyphenyl)-acrylamide) is a novel anti-Parkinson's disease candidate drug. A sensitive and specific high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated for the quantification of FLZ in rat brain. Carbamazepine was selected as the internal standard. Sample preparation involved double liquid–liquid extraction by n-hexane and ethyl acetate with high extraction efficiency. The chromatographic separation was achieved on a Zorbax SB-C18 column (100 mm × 2.1 mm, 3.5 μm) with an isocratic elution system comprised of acetonitrile and 0.3% aqueous acetic acid at a flow rate of 0.3 ml/min. The elutes were detected under positive electrospray ionization (ESI) and the target analytes were quantified by multiple reaction monitoring (MRM) mode. The method was sensitive with the lowest limit of quantification (LLOQ) at 1.0 ng/g brain tissue. Good linearity (r > 0.99) was obtained over the range of 1.0–400 ng/g. The intra- and inter-day precision ranged from 0.68% to 12%, while the accuracy between 92.7% and 111%. In addition, the stability, recovery and matrix effect involved in this method were also validated. The method was used to investigate the pharmacokinetics of FLZ in rat brain successfully after intravenous administration. The brain distribution studies showed that the brain distribution of FLZ was limited with the penetration ratio less than 0.1 in rats, with no target effect in the seven collected regions. Inhibition of P-glycoprotein (P-gp) by zosuquidar·3HCl ((2R)-1-{4-[(1aR,10bS)-1,1-difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c][7]annulen-6-yl]-1-piperazinyl}-3-(5-quinolinyloxy)-2-propanol trihydrochloride) resulted in a significant increase in brain-to-plasma ratio, while no significant increase by inhibition of breast cancer resistance protein (BCRP) by ko143 (2-methyl-2-propanyl 3-[(3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-3-yl]propanoate). The results indicated that FLZ had poor penetration to the brain due to the P-gp transport system.

► We developed a new sensitive, rapid and reliable HPLC–MS/MS method to determine FLZ concentration in rat brain.
► The double liquid–liquid extraction method we used could avoid matrix effect and contamination.
► The method was used to investigate pharmacokinetics of FLZ in rat brain successfully.
► The regional distribution of FLZ to rat brain showed no target effect.
► Inhibition of P-gp resulted in a significant increase in brain-to-plasma ratio of FLZ.