Conclu sions

1. Simultaneous quantification of biogenic monoamines by liquid chromatography with fluorescence detection Maria Ilieş1, Alina Uifălean1, SimonaCodruţa Hegheş1, Cristina Iuga1*, Felicia Loghin2 1Department of Pharmaceutical Analysis, Faculty of Pharmacy, “Iuliu Hațieganu” University of Medicine and Pharmacy Cluj-Napoca, Romania. 2 Department of Toxicology, Faculty of Pharmacy, “Iuliu Hațieganu” University of Medicine and Pharmacy Cluj-Napoca, Romania. *Corresponding author: iugac@umfcluj.ro Introduction: Serotonin (ST), noradrenaline (NA), adrenaline (AD) and dopamine (DA) could be used as indicators of neurological functions. Since they have the potential to be exploited as theranostic biomarkers, the development of a validated analytical instrument for their quantification is extremely useful in clinical practice (1-3). The aim of this study was the optimization of simultaneous derivatization of biogenic monoamines using a mixture of reagents and the subsequent separation and quantification of fluorescent derivatives in a single run analysis by high performance liquid chromatography (HPLC) with fluorescence detection (FLD). Materials and Methods: Solvents: MQ wather (Millipore MilliQ50), methanol, acetonitrile (Sigma-Aldrich), acetic acid 96%, clorhidric acid 37% (HCl) (Merck). Chemicals: 3-cyclohexylamino-1-propanesulfonic acid (CAPS), benzylamine hydrochloride (BA), 1,2-diphenylethylenediamine (DPE), glycine (Gly) (Sigma-Aldrich), potassium hexacianoferrate(III) (K3[Fe(CN)6]), sodium acetate, heptane-1-sulfonic acid sodium salt (Merck). Solutions: BA 0.3M, CAPS 0.03M pH=10.00 in apăMQ:metanol (10:90 v/v), K3[Fe(CN)6] 20mM in MQwater:methanol (50:50 v/v), DPE 0.05M inmethanol:MQwater:HCl 3M, Gly 0.3M in MQwater. Derivatizationreagents: Reagent I: BA 0.3M:CAPS 0.03M: K3[Fe(CN)6] 0.02M: methanol 2:6:3:24 (v/v); ReagentII: DPE 0.05M:Gly 0.3M 1:1 (v/v). Standards: adrenaline, noradrenaline, dopamine (Fluka), serotonine, (−)-3,4-Dihydroxynorephedrine (internal standard) (DHN) (Sigma-Aldrich), Stock solutions (1mg/mL) were prepared in HCl 0.1M, kept at -20°C and further diluted with water to desired concentrations before use. • Derivatization procedure: • Chromatographic conditions: • Chromatogphy was performed with aWaters 2695 Alliance HPLC system with a Waters 2475 Multi λ Fluorescence Detector, which was operated at λex=345 nm/ λem=480 nm. The separation was achieved using a Waters XBridge C18 column(4.6x150mm, 3.5μm)with a XBridge C18 precolumn(4.6 x 20mm, 3.5μm), at 30°C column temperature. The mobile phase consisted ofacetonitrile and acetate buffer (10mM, pH=5.30, 1.00mM heptane-1-sulfonic acid sodium salt) 33:67 (v/v), 1mL/min flow rate. Results: • Fig1. Optimization of the derivatization conditions. Effects on the fluorescence intensity: • Fig2. The chromatogram of the fluorescent derivatives: The solvent for RD I DPEconcentration DPE solubilization solvent K3[Fe(CN)6] concentration The biogenic amines were completely separated at: NA tR=5.14min, ST tR=6.43min, DHN tR= 7.53min, AD tR=12.91min, DA tR=19.48min. Reactiontime Conclusions Optimum conditions for derivatization were: methanol as solvent for reagent I, K3[Fe(CN)6] 0.02M, DPE 0.05M, methanol: HCl: water 14:1:15 as solvent for DPE and 20 min reaction time. This method allows the simultaneous analysis of biogenic amines after derivatization with two complementary reagents, benzylamine and 1,2-diphenylethylenediamine. This method will undergo to bioanalyticalmethod validation and will be applied for quantification of ST, NA, AD and DA in biofluids. Acknowledgment:This paper was published under the frame of European Social Found, Human Resources Development OperationlProgramme 2007-2013, project no. POSDRU/159/1.5/136893. References: 1. Marc DT, Ailts JW, Campeau DC, Bull MJ, Olson KL. Neurotransmitters excreted in the urine as biomarkers of nervous system activity: validity and clinical applicability. Neuroscience and biobehavioral reviews. 2011;35(3):635-44. 2.Yoshitake T, Kehr J, Todoroki K, Nohta H, Yamaguchi M. Derivatization chemistries for determination of serotonin, norepinephrine and dopamine in brain microdialysis samples by liquid chromatography with fluorescence detection. Biomed Chromatogr. 2006;20(3):267-81. 3. Liu L, Li Q, Li N, Ling J, Liu R, Wang Y, et al. Simultaneous determination of catecholamines and their metabolites related to Alzheimer's disease in human urine. Journal of separation science. 2011;34(10):1198-204.