Journal of Chromatography B, 913–914 (2013) 41–47 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l ho me page: www.elsevier.com/locate/chromb A sensitive and selective quantification of catecholamine neurotransmitters in rat microdialysates by pre-column dansyl chloride derivatization using liquid chromatography–tandem mass spectrometry Ramakrishna Nirogi a,∗ , Prashanth Komarneni a , Vishwottam Kandikere a , Rajeshkumar Boggavarapu a , Gopinadh Bhyrapuneni a , Vijay Benade a , Srinivasarao Gorentla b a Biopharmaceutical Research, Drug Discovery, Suven Life Sciences Ltd., Serene Chambers, Road – 5, Avenue – 7, Banjara Hills, Hyderabad 500 034, India b Saastra College of Pharmaceutical Education and Research, Varigonda, Nellore 524 002, India a r t i c l e i n f o Article history: Received 5 April 2012 Accepted 23 September 2012 Available online 27 September 2012 Keywords: Dopamine Norepinephrine Dansyl chloride derivatization LC–MS/MS Microdialysates Rat prefrontal cortex a b s t r a c t A rapid and sensitive liquid chromatography tandem mass spectrometry method for simultaneous quan- tification of catecholamine neurotransmitters in microdialysates was developed. The catecholamine neurotransmitters dopamine (DA) and norepinephrine (NE) were pre-column derivatized with dansyl chloride and analyzed. A gradient elution method was used to separate the analytes from the inter- ferences on an Agilent Poroshell 120 EC-C18 outer porous micro particulate column. The method was robust and sensitive to determine with the lower limit of quantification value of 0.068 pmol/mL and 0.059 pmol/mL for DA and NE, respectively. It has acceptable precision and accuracy for concentrations over the standard curve range. The method was successfully applied for simultaneous quantitation of DA and NE in the prefrontal cortex (PFC) dialysates of rats obtained from a microdialysis study dosed with vehicle and atomoxetine through intra peritoneal (i.p.) route at a dose of 3 mg/kg to monitor the change in extracellular concentrations. Thus, accomplishment of this method would facilitate the neuro- chemical monitoring for discovery of new chemical entities targeted for the treatment of attention deficit hyperactivity disorder (ADHD). © 2012 Published by Elsevier B.V. 1. Introduction Dopamine (DA) and norepinephrine (NE) are two prominent catecholamine neurotransmitters in the extracellular fluid of the brain. They play a vital role in many functions of the brain and critical in attention and focus [1,2]. Attention-deficit/hyperactivity disorder (ADHD) is a psychiatric disorder that affects both children and adults. However, little is known about the mechanisms of drugs to treat the symptoms. Modulation of DA and NE in cortical (prefrontal cortex, PFC) and subcortical (nucleus accumbens, NACb) extracellular fluid is criti- cal in controlling the impulsive behavior associated in ADHD [1,2]. Adequate levels of DA and NE are vital in PFC function, and insuf- ficient levels can lead to PFC dysfunction and symptoms similar to ADHD [2–5]. Moreover, the extracellular concentrations of these neurotransmitters are significant for communication between neu- rons, thus monitoring of neurochemicals in extracellular fluids are essential in a variety of CNS disorders [3]. The in vivo brain microdialysis in preclinical species is the practical way to monitor ∗ Corresponding author. Tel.: +91 40 23556038x23541142; fax: +91 40 23541152. E-mail address: ramakrishna nirogi@yahoo.co.in (R. Nirogi). the modulation of catecholamines in PFC by new drugs designed to treat ADHD. It offers to measure the neurotransmitters from conscious and freely moving animal over the time [6–9]. How- ever, the samples from brain dialysis results small sample volumes (20–30 L) and pico molar range of dialysate concentrations of neurotransmitters necessitate the sensitive analytical methods. In the literature, various analytical methods are reported for the quantification of dialysates DA, NE and their metabolites. Among them, the most frequently used methods are high performance liquid chromatography (HPLC) coupled with electrochemical detection (HPLC–ECD) [10–15], HPLC coupled with fluorescence detection (HPLC–FLD) [16–20] and HPLC coupled with tandem mass spectrometry (HPLC–MS/MS) [7–9,21–26]. HPLC–ECD meth- ods have limitations like high background noise, low sensitivity and poor separation [10–15]. Similarly, the HPLC–FLD methods suf- fer with low detection limits, lengthy derivatization process and longer run times [16–20]. Although several HPLC–MS/MS methods are reported for neurochemical quantification, these methods have limitations like elution of analytes of interest in void time, noise interferences, peak resolution, lengthy derivatization processes and longer run times. Cai et al. [21] have reported a HPLC–MS/MS method for the quantification of dansylated monoamine, amino acid neurotransmitters and their metabolites in human plasma. In 1570-0232/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jchromb.2012.09.034