Determination of dopamine, serotonin, biosynthesis precursors and metabolites in rat brain microdialysates by ultrasonic-assisted in situ derivatization–dispersive liquid–liquid microextraction coupled with UHPLC-MS/MS

• In situ UAD-DLLME was reported for multiple neurotransmitters for the first time.
• Lissamine rhodamine B sulfonyl chloride was firstly used as derivatization reagent.
• The method was simple, rapid, green, efficient, sensitive and low matrix effect.
• The method was successfully applied to brain microdialysates of normal and LID rats.

This paper, for the first time, reported a simple, rapid, sensitive and environmental friendly ultrasonic-assisted in situ derivatization-dispersive liquid–liquid microextraction (in situ UAD-DLLME) method followed by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of dopamine (DA), serotonin (5-HT) and their biosynthesis precursors and metabolites in rat brain microdialysates. In this work, a commercial reagent, Lissamine rhodamine B sulfonylchloride (LRSC), was proposed as a derivatization reagent. The ionization efficiency of neurotransmitters was greatly enhanced through the introduction of a permanent charged moiety of LRSC into their derivatives during electrospray ionization MS (ESI–MS) analysis. Parameters of in situ UAD-DLLME and UHPLC-MS/MS conditions were all optimized in detail. The optimum conditions of in situ UAD-DLLME were found to be as follows: a mixture of 150 μL of acetonitrile (dispersant) containing LRSC (derivatization reagents) and 50 μL of low toxic bromobenzene (extractant) was rapidly injected into an aqueous sample containing 30 μL of microdialysate and 800 μL of NaHCO3-Na2CO3 buffer solution (pH 10.5) at 37 °C. After ultrasonication for 3 min and centrifuging for 2 min, the sedimented phase was conveniently injected for UHPLC–MS/MS analysis. Under the optimized conditions, good linearity was observed with the limits of detection (LODs, S/N>3) and limits of quantification (LOQs, S/N>10) in the range of 0.002–0.008 and 0.015–0.040 nmol/L, respectively. Meanwhile, it also brought good results of precision (3.2–13.0%, peak area RSDs %), accuracy (86.4–112%), recovery (73.9–105%), matrix effect (86.2–105%), and stability (3.1–8.8%, peak area RSDs %). The developed method was successfully applied for the simultaneous determination of multiple neurotransmitters, their precursors and metabolites in brain microdialysates of normal and L-DOPA induced dyskinesias (LID) rats.

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