Abstract:

The sensitive detection method of levodopa (L-DOPA) and dopamine (DA) in rat brain microdialysate of Parkinson's disease (PD) is an essential tool for the clinical study and attenuated synergistic drug screening for L-DOPA from traditional Chinese medicines. Using d₀/d₃-10-methyl-acridone-2-sulfonyl chloride (d₀/d₃-MASC) as stable isotope derivatization reagent, a novel ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for L-DOPA and DA by stable isotope-coded derivatization coupled with ultrasonic-assisted dispersive liquid-liquid microextraction (UA-DLLME). d₀-MASC (light) and d₃-MASC (heavy) were used as derivatization reagents for microdialysate samples and standards, respectively. Mixtures of the two solutions were prepared by UA-DLLME for UHPLC-MS/MS analysis with multiple reaction monitoring (MRM) mode. With d₃-MASC heavy derivatives as internal standards for corresponding light derivatives from samples, the stable isotope internal standard quantification for L-DOPA and DA was carried out. The stable derivatives were obtained in aqueous acetonitrile (pH 10.8 sodium carbonate-sodium bicarbonate buffer) at 37 ℃ for 3.0 min, and then were separated within 2.0 min using gradient elution. Linear range was 0.20-1500.0 nmol/L (R> 0.994). LODs were 0.005 and 0.009 nmol/L for DA and L-DOPA (S/N=3), respectively. This method was validated, and it showed obvious advantages in comparing with the reported methods in terms of sensitivity, analysis speed and anti-matrix interference. This method has been successfully applied to the study of effect of Shouwu Fang on L-DOPA and DA concentration fluctuations in PD rat brain microdialysate.

Key words: drug screening, mass spectrometry sensitization analysis, Parkinson's disease (PD), rat in vivo microdialysis, Showwu Fang, ultra high performance liquid chromatography (UHPLC)