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 bioanalytical method validation and will be applied for quantification of ST, NA, AD and DA in biofluids.

Results:

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.

Maria Ilieş1, Alina Uifălean1, Simona Codruţa Hegheş1, Cristina Iuga1*, Felicia Loghin2

1 Department 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

Simultaneous quantification of biogenic monoamines by liquid chromatography with fluorescence detection

Fig1. Optimization of the derivatization conditions. Effects on the fluorescence intensity:

DPE concentration

Fig2. The chromatogram of the fluorescent derivatives:

Acknowledgment: This paper was published under the frame of European Social Found, Human Resources Development Operationl Programme 2007-2013, project no. POSDRU/159/1.5/136893.

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.

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 in methanol:MQwater:HCl 3M, Gly 0.3M in MQwater. Derivatization reagents: Reagent I: BA 0.3M: CAPS 0.03M: K3[Fe(CN)6] 0.02M: methanol 2:6:3:24 (v/v); Reagent II: 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.

Chromatographic conditions: Chromatogphy was performed with a Waters 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 of acetonitrile and acetate buffer (10mM, pH=5.30, 1.00mM heptane-1-sulfonic acid sodium salt) 33:67 (v/v), 1mL/min flow rate.

Derivatization procedure:

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).

NH

HO

NH2

Serotonine

CH2NH2O

N

NH

NH2

Benzylamine

Fluorescent derivative

NH2

NH2

1,2-Diphenylethylenediamine

O

N

N

R1

R2

HO

HONH

R2

R1

Catecholamine

Fluorescent derivative

RD I 2 minroom T0C

RD II 20 min 500C

CH2NH2

Benzylamine

Chromatographic analysis

Icecooling 20 min

followed by

The solvent for RD I

NA DHN AD DA0E+002E+064E+066E+068E+06

MetOH ACN

K3[Fe(CN)6] concentration

NA DHN AD DA0E+002E+064E+066E+068E+06

0.02M 0.10M

Reaction time

NA ST DHN AD DA0E+001E+052E+053E+054E+05

20 min 40 min 60 min

NA DHN AD DA0E+002E+064E+066E+068E+06

RD II DPE 0.10M:Gly 0.3M 1:1 (v/v) RD II DPE 0.05M:Gly 0.3M 1:1 (v/v)

DPE solubilization solvent

NA ST DHN AD DA0E+002E+064E+066E+068E+061E+07

RD II in MetOH:HCl 30:1 (v/v) RD II in MetOH:HCl:H20 14:1:15 (v/v)