Research article

Received: 05 January 2011, Revised: 11 April 2011, Accepted: 18 May 2011, Published online in Wiley Online Library: 21 June 2011

(wileyonlinelibrary.com) DOI 10.1002/bio.1332

84

A terbium‐sensitized spectrofluorimetricmethod for determination of catecholaminesin a serum sample with micelle mediumMohammad Kamruzzamana, Al‐Mahmnur Alama, Sang Hak Leea,c*,Young Ho Kimb and Sung Hong Kimc

ABSTRACT: A terbium‐sensitized spectrofluorimetric method has been developed for determination of catecholamines such asnorepinephrine (NE), epinephrine (EP) and dopamine (DA), using sodium dodecyl benzene sulphonate (SDBS). Fluorescencesensitization of terbium ions (Tb3+) by complexation with catecholamines in the presence of SDBS was observed. The fluores-cence intensities of the Tb3+–catecholamine complexes were highly enhanced by introducing SDBS with an emission maxi-mum at 545nm after excitation at 290nm. The conditions for the complex formation of Tb3+–catecholamine wereinvestigated systematically and optimized to determine catecholamines in a serum sample. Under the optimum conditions,the fluorescence intensities of the Tb3+–catecholamine complexes were increased linearly with the concentration of NE, EPand DA over the ranges 2.5 × 10–10–1.0 × 10–8, 2.5 × 10–10–1.0 × 10–8 and 2.5 ×10–9–1.0 × 10–7 g/mL with correlation coefficientsof 0.999, 0.999 and 0.9996, respectively. The limits of detection (3δ) of NE, EP and DA were found to be 4.6 × 10–11, 7.8 × 10–11

and 8.38 × 10–10 g/mL, respectively. Precision of the method was tested at the concentration level of 1.2 × 10−7 g/mL for fivereplicate measurements of NE, EP and DA, giving relative standard deviations (RSDs) of 1.41%, 1.23% and 1.89%, respectively.The interaction mechanism of the Tb3+–catecholamine complexes system was investigated and presented with ultraviolet ab-sorption spectra. The proposed method has been applied for the quantitative determination of NE, EP and DA in aspiked serum sample and a pharmaceutical preparation sample. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: terbium(III); catecholamines; sodium dodecyl benzene sulphonate; fluorescence probe

* Correspondence to: S. H. Lee, Department of Chemistry, Kyungpook Na-tional University, Daegu 702–701, South Korea. E‐mail: shlee@knu.ac.kr

a Department of Chemistry, Kyungpook National University, Daegu, SouthKorea

b Research Institute of Advanced Energy Technology, Kyungpook NationalUniversity, Daegu, South Korea

c Korea Basic Science Institute Daegu Centre, Daegu, South Korea

IntroductionCatecholamines are sympathomimetic flight hormones releasedby the adrenal glands in response to stress. They are part ofthe sympathetic nervous system. Catecholamines are a class ofbiogenic amines that act as neurotransmitters, which play an im-portant role in the central nervous system. They affect the regu-lation of blood pressure and metabolic processes (1) thatpossess important intrinsic pharmacological properties and areused for the correction of haemodynamic disorders associatedwith shock episodes (2). They also play a crucial role in thepathomechanism of Parkinson’s disease, which results from re-duction of the efficiency of the extrapyramidal movement sys-tem (3). The most abundant catecholamines in the humanbody are epinephrine (EP; also called adrenaline), norepineph-rine (NE; also called noradrenaline) and dopamine (DA).

DA plays an important role in brain functions as a typical neuro-transmitter. Insufficient DA level due to the loss of DA‐producingcells may lead to Parkinson’s disease. The hydrochloride saltof DA is used in the treatment of shock caused by trauma,heart attack, open heart surgery, heart failure, kidney failureand severe bacterial infections of the blood (4,5). NE is themain neurotransmitter of the sympathetic nervous system andis used as an index for several diseases, such as hypertension,phaeochromocytoma and neuroblastoma septic shock, systemicinflammation or pharmacological vasodilatation. EP is the mainhormone of the adrenal medulla and has an important effecton blood pressure and cardiac output and exhibits vasoconstric-tion. Due to the significance of catecholamines in clinical

Luminescence 2012; 27: 84–90 Copyright © 2011 John

diagnosis and medical treatment, determination ofcatecholamines is important in the diagnosis of diseases andthe development of pharmaceuticals.

A variety of analytical methods have been reported to determinecatecholamines in pharmaceutical preparations andbiologicalfluids,such as spectrophotometry (6–9), fluorimetry (10,11), electro-chemistry (12), high‐performance liquid chromatography (HPLC)(13–15), chemiluminescence (CL) coupled with flow injection(16–21), capillary electrophoresis (CE) (22–24), non‐competitiveenzyme immunoassay (25) and modified electrodes (26,27).Among these methods, spectrofluorimetric methods that havehigh sensitivity and selectivity have been widely used to esti-mate catecholamines in pharmaceutical preparations and bio-logical fluids. Lanthanide ion complexes with a lanthanide ion,such as Tb(III) or Eu(III), have particular luminescencecharacteristics that arise from the transition between their f or-bital shielded by the 5 s2 and 5p6 subshell, and they show a largeStokes’ shift and narrow emission band fluorescence spectra

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Terbium‐sensitized spectrofluorimetric determination of catecholamines

with a long luminescence lifetime and strong interaction, espe-cially Tb3+ and Eu3+, because their energy levels overlap with ul-traviolet (UV) light. Based on the above characteristics, thecoordination complexes of the rare earth ions (especially Tb3+

and Eu3+) can be used as a fluorescence probe to determine var-ious pharmaceutical and biological substances, owing to thehigh fluorescence quantum efficiency of the Tb3+ chelates (28–31).

In this study, we report a terbium‐sensitized spectrofluorimetricmethod with a micelle medium to determine catecholamines. Wedevelop a simple and sensitive spectrofluorimetric method for thedetermination of catecholamines by improving terbium‐sensitizedfluorescence, using sodium dodecyl benzene sulphonate (SDBS).Under the optimum conditions, catecholamines concentrated attrace level in bio‐fluids and pharmaceutical samples were deter-mined by the terbium‐sensitized spectrofluorimetric method.

Experimental

Apparatus

Fluorescence intensity measurements of a sample were carried outwith a spectrofluorimeter (Model F‐4500, Hitachi, Japan) equippedwith a 150W xenon lamp. Emission lights of a sample solutionweremeasured and transduced to electric signals by a photomultipliertube (Model R 928, Hamamatsu, Japan). The high voltage for thephotomultiplier tube was set to 950V. A pH meter (Model Orion,520A, USA) was used for pH adjustment. Absorption spectra of asample were measured on a UV‐1800 spectrophotometer(Shimadzu, Japan) equipped with 10mm path length quartz cells.

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Reagents and solution

Catecholamines (NE, EP and DA), terbium(III) chloride hexahydrateand SDBS were purchased from Sigma‐Aldrich (USA). Stock stan-dard solutions of NE, EP and DA were prepared by dissolving indeionized (DI) water and preserved at 4°C. Stock solutions of Tb3+

(1 × 10–3mol/L) and SDBS (1 × 10–3mol/L) were prepared bydissolving 0.012 g and 0.017 g in a 50mL volumetric flask usingDI water and preserved at 4°C. Tris–HCl buffer was prepared bydissolving an appropriate amount of Tris(hydroxy methyl)aminomethane in 500mL DI water and pH was adjusted using0.1mol/L HCl. Working solutions were prepared daily from thestock solution by appropriate dilution immediately before use.Other chemicals were of analytical reagent grade. Double‐dis-tilled (DI) water was used throughout.

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Analytical procedure

The following solutions were added to a 10mL volumetric flask inorder: 1mL 1×10–3mol/L Tb3+ ion solution, 1mL 0.1mol/L Tris–HCl buffers, 1mL of a certain concentration of catecholaminesand 1mL 1× 10–3mol/L SDBS solution. The prepared mixturewas diluted up to the mark with DI water and mixed thoroughlyby shaking and then allowed to stand for 20min at room tem-perature. The fluorescence intensity of the system was measuredin a 1 cm quartz cell with excitation and emission wavelengths of290 and 545 nm, respectively. Both excitation and emission slitsfor all fluorescence measurements were 10 nm.

Excitation spectra (λem=545 nm)

Figure 1. Fluorescence emission (a) and excitation (b) spectra of the Tb3+–catecholamines–SDBS system. (1) Tb3+; (2) Tb3+–DA; (3) Tb3+–EP; (4) Tb3+–NE; (5)Tb3+–SDBS; (6) Tb3+–DA–SDBS; (7) Tb3+–EP–SDBS; (8) Tb3+–NE–SDBS. Conditions:NE, 1.0 × 10–8 g/mL; EP, 1.0 × 10–8 g/mL; DA, 1.0 × 10–7 g/mL; Tb3+, 8.5 × 10–6mol/L;SDBS, 5.5 × 10–4mol/L; Tris–HCl, 0.01mol/L; pH, 8.5. 85

Pharmaceutical sample preparation

The content of one ampoule of DA (declared content of DA in5mL solution was 4%) was transferred to a calibration flaskand diluted to 100mL with Tris–HCl buffer, pH 8.5. The contentof 10 ampoules of EP (declared content of EP in 1mL solution

Luminescence 2012; 27: 84–90 Copyright © 2011 John Wiley

was 0.1%) was transferred to a calibrated flask and diluted withTris–HCl buffer, pH 8.5, to 100mL. The content of one ampouleof NE (norepinephrine bitartrate, 1mg/mL, 4mL ampoule) waspowdered and transferred to a calibrated flask and dissolved in100mL Tris–HCl buffer, pH 8.5. Further dilutions were made toensure that the concentrations of the drug in the samplesolutions fall within the linear range of the method.

Serum sample preparation

The protein of a 1mL volume of serum sample was deproteinizedby adding 5mL 20% trichloroacetic acid (CCl3COOH) in a centri-fuge tube. The mixture was sonicated for 5min and centrifugedfor 15min at 8000 rpm. The supernatant was dissolved in 1mLDI water. The catecholamines were added to the protein‐free se-rum and diluted to 50mL with DI water in order to obtain a con-centration of catecholamines in the range of linearity.

Results and discussion

Characteristics of fluorescence spectra

Fluorescence emission and excitation spectra of Tb3+, Tb3+–NE,Tb3+–EP, Tb3+–DA, Tb3+–SDBS, Tb3+–SDBS–NE, Tb3+–SDBS–EPand Tb3+–SDBS–DA are shown in Fig. 1. Neither catecholamine

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Figure 2. Effect of pH on the FL intensity of Tb3+–catecholamines–SDBS system.Conditions: NE, 1.0 × 10–8 g/mL; EP, 1.0 × 10–8 g/mL; DA, 1.0 × 10–7 g/mL; Tb3+,8.5 × 10–6mol/L; SDBS, 5.5 × 10–4mol/L; Tris–HCl, 0.01mol/L.

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Figure 3. Effect of Tb3+ concentration on the FL intensity of Tb3+–catecholamines–SDBS system. Conditions: NE, 1.0 × 10–8 g/mL; EP, 1.0 × 10–8 g/mL;DA, 1.0 × 10–7 g/mL; SDBS, 5.5 × 10–4mol/L; Tris–HCl, 0.01mol/L; pH, 8.5.

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solutions nor Tb3+ solution (Fig. 1a, curve 1) exhibited an en-hanced fluorescence peak at 545 nm. The emission spectrum ofTb3+ was increased by using SDBS (Fig. 1a, curve 5), indicatingthat Tb3+ could interact with SDBS and resulted in energy trans-fer from the SDBS to Tb3+. When catecholamines were addedinto the Tb3+–SDBS system, the fluorescence intensities wereenhanced markedly (Fig. 1a, curves 6–8), which indicated the in-teraction of Tb3+ with catecholamines resulting in energy trans-fer from the catecholamines to Tb3+. The emission peaks wereobtained at 490 and 545 nm, which corresponded to theTb3+ transitions of 5D4 → 7 F5 and 5D4 → 7 F6, respectively.Among these emission spectra, the spectrum at 545 nmdisplayed the strongest signal. At the optimum concentra-tions of catecholamines, the fluorescence intensity of theTb3+–catecholamine–SDBS system could be enhanced moremarkedly than the Tb3+–catecholamine system (Fig. 1a, curves2–4). The excitation spectra of the Tb3+–catecholamine–SDBSsystem exhibited excitation peaks at about 273 nm (Fig. 1b).Then, considering the interference of multiple peaks, excitationand emission wavelengths were selected at 290 and 545 nm, re-spectively, in the study.

Effect of pH and buffer solution

The pH of a solution plays an important role in terms of obtainingmaximum FL intensity. The change in pH would influence thecompositions and stabilities of the fluorescent complexes and re-sult in changes in the fluorescence characters. The effect of pHvalues on the fluorescence intensity of the Tb3+–catecholamine–SDBS system is shown in Fig. 2. The fluorescence intensity

Table 1. Effect of buffer solution (pH 8.5) on fluorescence intensi

Buffers NH4Ac–HAc NH4Cl–NH3·H2O

FL intensity NE 20.7 2.8EP 19.6 2.1DA 18.2 3.2

Copyright © 201wileyonlinelibrary.com/journal/luminescence

increased with an increase in pH up to 8.5. The fluorescence in-tensity gradually decreased when the pH was > 8.5. In the pres-ent study, the fluorescence intensity exhibited a maximum atpH 8.5, which was chosen for this experiment.

The effect of the following buffer solutions on the fluorescenceintensity was studied: NH4Ac–HAc, NH4Cl–NH3·H2O, KH2PO4–NaOH, Tris–HCl and KH2PO4–Na2HPO4; the results are summa-rized in Table 1. The results indicated that the maximum fluores-cence intensities were obtained by using 0.01mol/L Tris–HClbuffer solutions. Tris–HCl buffer solution (0.01mol/L), pH 8.5,was selected for the study.

Effect of Tb(III) ion concentration

The effect of Tb3+ concentration on the fluorescence intensity wasinvestigated in the range 1.0× 10–6–1.6 × 10–5mol/L (Fig. 3). Thefluorescence intensity reached maximum at a Tb3+ concentra-tion of 8.5 × 10–6mol/L and the intensity gradually decreasedabove this concentration. Therefore, the Tb3+ concentration8.5 × 10–6mol/L was selected for further study.

Effect of surfactants

Spectrofluorimetric properties of catecholamines were investi-gated in a series of micellar media in the presence of variousconcentrations of surfactants, such as cetyl trimethyl ammoniumbromide (CTAB; cationic), Triton X‐100 (non‐ionic), Igepal (non‐ionic), sodium dodecyl sulphate (SDS, anionic) and SDBS (an-ionic). The results indicated that the fluorescence intensity wasincreased significantly in the presence of the anionic surfactantsSDS and SDBS, especially SDBS. The specific effect of anionicsurfactants on the fluorescence intensity can be associated with

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KH2PO4–NaOH Tris–HCl KH2PO4–Na2HPO4

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Figure 4. Effect of SDBS concentration on the FL intensity of Tb3+–catecholamines–SDBS system. Conditions: NE, 1.0 × 10–8 g/mL; EP, 1.0 × 10–8 g/mL;DA, 1.0 × 10–7 g/mL; Tb3+, 8.5 × 10–6mol/L; Tris–HCl, 0.01mol/L; pH, 8.5.

Terbium‐sensitized spectrofluorimetric determination of catecholamines

the ability of negatively charged micelles to concentrate Tb3+

ions at their surfaces by electrostatic interaction, while the solu-bilization of ligands is mainly due to the hydrophobic interactionof the hydrocarbon skeleton of the molecule with the hydrocar-bon radical of anionic surfactant ions. Therefore, anionicsurfactants (SDBS) are more effective for enhancing the fluores-cence intensity. The effect of SDBS concentration on the fluores-cence intensity of the system was investigated. The fluorescenceintensity of the system increased with increasing SDBS concen-tration up to 5.5 × 10–4mol/L (Fig. 4) and then decreased whenthe concentration of SDBS was > 5.5 × 10–4mol/L. For the presentstudy, 5.5 × 10–4mol/L SDBS was the chosen concentration.

Effect of reagent addition order and fluorescence stability

The effect of the order of addition of reagents on the fluores-cence intensity was investigated. The results showed that an ad-dition order of Tb3+, Tris–HCl, catecholamine and SDBS producedmaximum fluorescence intensity, which was chosen as optimumreagent addition order. The fluorescence intensity of the samplesolutions reached a maximum 20min after all the reagents hadbeen added and remained stable for 2 h.

Table 2. Tolerance limit of various foreign substances on the det

Interfering species

DA

Pb2+, Fe3+, Mn2+, Zn2+ 3000Na+, K+ 2000Cl−, SO4

2−, NO3− 1050

Sodium metabisulphite 1800Glucose, citric acid and resorcinol 700Glucose 180Lactose 150Ascorbic acid and quinol 120Sucrose and Fe2+ 80

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Effect of interfering species

The presence of interfering substances in a real sample mightsuppress or enhance a FL signal, although they have no signifi-cant effect on the intensity. The presence of interfering speciesin a sample was closely investigated by adding increasingamounts of an interfering species to a standard solution of1.0 × 10–7mol/L catecholamines. A foreign substance was con-sidered to interfere significantly when its presence produced avariation of > 5% in the fluorescence intensity of the analyte.The possible interfering species, such as various metal ions(Pb2+, Fe3+, Mn2+, Zn2+, Na+, K+), anions (Cl–, SO4

2–, NO3–) and

biochemical compounds (sodium metabisulphite, glucose, citricacid, resorcinol, lactose, etc.) that might influence the determi-nation of catecholamines in serum and pharmaceutical formulationswere examined by the proposed method. The experimental results ofthe tolerance level of various interferents are summarized in Table 2.The results showed that most of the excipient substances fre-quently found did not interfere in the determination ofcatecholamines. The proposed method can be used directly inthe determination of catecholamines.

Calibration curve and detection limit

A calibration curve of FL intensity versus NE, EP and DAconcentrations was obtained under the optimum conditions de-scribed above. Under the optimum conditions, the FL intensitywas increased linearly with concentrations of NE, EP and DA inthe range 2.5×10–10–1.0 × 10–8, 2.5 × 10–10–1.0 × 10–8 and2.5×10–9–1.0 × 10–7 g/mL, with correlation coefficients of 0.99903,0.999 and 0.99969, respectively (summarized in Table 3). The limitof detection (LOD) as defined by IUPAC, CLOD = 3 Sb/m [whereSb is the standard deviation (SD) of the blank signals and m isthe slope of the calibration curve], was found to be 4.6 × 10–11,7.8 × 10–11 and 8.38 × 10–10 g/mL for NE, EP and DA, respectively.The relative standard deviations (RSDs) were 1.41%, 1.23% and1.89% for five determinations of 1.2×10−7 g/mL NE, EP and DA,respectively. The reproducibility of the results of the proposedmethod showed that determination of a trace amount ofcatecholamines could be made using this method.

Pharmaceutical sample analysis

The proposed FL method was applied to determine cate-cholamines in real samples. The results obtained for the

ermination of catecholamines

Maximum tolerable concentration ratio

EP NE

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Table 3. Calibration curves for catecholamines

Catecholamines Linear range (g/mL) Regression equations (y= c+mx) Correlation coefficient (R2) LOD* (g/mL)

NE 2.5 × 10–10–1.0 × 10–8 y=201 + 2.53 × 10–10 CNE 0.9990 4.6 × 10–11

EP 2.5 × 10–10–1.0 × 10–8 y=140 + 1.5 × 10–10 CEP 0.9990 7.8 × 10–11

DA 2.5 × 10–9–1.0 × 10–7 y= 123+ 1.4 × 10–9 CDA 0.9996 8.38 × 10–10

*Limit of detection (3 Sb/m).

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determination of catecholamines are summarized in Table 4. Theresults obtained using the proposed method were in goodagreement with the label claims. The recoveries were in therange 97.5–107%. Hence, the method can be applied success-fully for the determination of catecholamines in commerciallyavailable pharmaceutical samples.

Determination of catecholamines in a serum sample

The proposed FL method was also applied for the determina-tion of catecholamines in a serum sample. The results areshown in Table 5. For this serum assay the freshly prepared sam-ple was diluted appropriately within the linear range of determi-nation. In order to compensate the effect of the biological matrixin the measurement, the standard addition method was appliedto the quantification of catecholamines in the serum sample. Re-coveries of catecholamine content in serum samples were 97–106.2%. The results showed that the proposed method can be

Table 4. Application of the proposed method for the determinat

Sample Amount (mg)

Claimed Obtained± RSD* Added

Dopaminum hydrochloricum 200 198.4 ± 1.03

Epinephrine hydrochloride 1 1.05 ± 0.83

Norepinephrine bitartrate 4 3.97 ± 0.81

*RSD for three replicate measurements.

Table 5. Determination of catecholamines in a serum sample

Sample Dopamine Epi

Added(µg/mL)

Found(µg/mL±RSD*)

Recovery (%) Added(µg/mL)

Fouµg/mL

Serum 0.2 0.194 ± 0.8 97.0 0.22 0.2180.4 0.403 ± 1.12 100.7 0.16 0.1590.6 0.597 ± 0.91 99.5 0.08 0.085

*RSD for three replicate measurements.

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applied to determine catecholamines in a serum sample withgood accuracy.

Possible interaction mechanism

The lanthanide complexes with Tb3+ or Eu3+ produce largeStokes’ shifts, narrow emission bandwidths and long emissionlifetimes. Due to the above characteristics, lanthanide complexesare broadly used in high‐sensitivity assay to analyse varioustypes of pharmaceuticals and biomolecules. Tb(III) ion is ahighly efficient luminescence centre and complexes of Tb3+ withstrong light‐absorbing ligands leads to energy transfer from theligand to the lanthanide ion. We showed that the fluorescenceintensity of the Tb3+–catecholamine complexes was enhancedmarkedly by adding SDBS. When the Tb3+–catecholaminecomplexes were dispersed and gathered together with themicelles, its microenvironment was changed to a great extent,which decreased the non‐radiative energy loss through

ion of catecholamines in pharmaceutical preparations

Standard addition method

(× 10–7), (mol/L) Found (× 10–7), (mol/L ± RSD*) Recovery (%)

1.0 1.03 ± 0.8 1032.0 2.04 ± 0.1 1023.0 2.96 ± 0.5 98.671.0 0.98 ± 1.12 982.0 2.05 ± 0.75 102.53.0 3.01 ± 0.95 100.331.0 1.07 ± 0.87 1072.0 1.95 ± 1.03 97.53.0 3.04 ± 1.13 101.33

nephrine Norepinephrine

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± 1.31 99.1 0.10 0.097 ± 0.65 97.0± 0.85 99.4 0.08 0.082 ± 1.03 102.5± 0.51 106.2 0.06 0.061 ± 0.96 101.7

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Figure 6. Absorption spectra of catecholamines in various systems. (1) Tb3+;(2) DA; (3) EP; (4) NE; (5) Tb3+–DA; (6) Tb3+–EP; (7) Tb3+–NE; (8) Tb3+–DA–SDBS;(9) Tb3+–EP–SDBS; (10) Tb3+–NE–SDBS. Conditions: NE, 5.0 × 10–7 g/mL; EP,5.0 × 10–7 g/mL; DA, 2.0 × 10–7 g/mL; Tb3+, 8.5 × 10–6mol/L; SDBS, 5.5 × 10–4mol/L;Tris–HCl, 0.01mol/L; pH, 8.5.

Terbium‐sensitized spectrofluorimetric determination of catecholamines

molecule collision and improved the quantum efficiency offluorescence. Catecholamines were excited to their excitedsinglet state after absorbing light energy and then changedto the triplet state via an inter‐system crossing. Moreover, thecoordination number of Tb3+ could not be contented after theformation of the Tb3+–catecholamine complex, so that Tb3+ inthe Tb3+–catecholamine complex could interact with SDBS byan ionic interaction. It was possible that the energy was trans-ferred not only from catecholamines to Tb3+ but also fromSDBS to Tb3+, which increased the fluorescence intensity of thesystem significantly. As shown in Fig. 5, the absorption intensityof the SDBS increased by introducing Tb3+ due to the interactionof SDBS with Tb3+, which resulted in the energy transfer fromSDBS to Tb3+.

The interaction of catecholamines, Tb3+ and SDBS was investi-gated by UV‐vis spectroscopy, as shown in Fig. 6. Cate-cholamines produced a absorption maximum wavelength atabout 280 nm (Fig. 6, curves 2–4) by increasing fluorescence in-tensity. However, the absorbance of catecholamines increasedsignificantly when both Tb3+ and SDBS solutions were added(Fig. 6, curves 8–10), which was in accordance with the fluores-cence enhancement of the fluorescence excitation spectrum ofthe Tb3+–catecholamines–SDBS system (see Fig. 1b), and themaximum absorption wavelength showed a red shift at about280–295 nm. Moreover, a multiple ionic associate was formedin the catecholamines–Tb3+–SDBS system to enhance the effec-tive absorption cross‐section of the complex. Thus, the fluores-cence intensity of the system was significantly increased withthe addition of SDBS. In addition, the optimum concentrationof SDBS in the presented study was approximated to the criticalmicelle concentration (CMC) of SDBS (CMC=0.63mmol/L) (32),and it can be concluded that the formation of micelles took partin the enhancement of the fluorescence intensity of the system.

ConclusionA sensitive spectrofluorimetric method with terbium(III) for thedetermination of catecholamines (NE, EP and DA) using SDBShas been described. The fluorescence intensity of the Tb3+–catecholamine complexes was enhanced markedly, proportionalto the concentration of catecholamines (NE, EP and DA) over theranges 2.5×10–10–1.0 × 10–8, 2.5×10–10–1.0× 10–8 and 2.5×10–9–

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Figure 5. Absorption spectra of SDBS in Tb3+ system. (1) Tb3+; (2) SDBS; (3) Tb3+–SDBS. Conditions: Tb3+, 8.5 × 10–6mol/L; SDBS, 5.5 × 10–4mol/L; Tris–HCl 0.01mol/L;pH, 8.5.

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1.0 × 10–7 g/mL, respectively. The limit of detection (3δ) for NE,EP and DA by the proposed method was found to be4.6 × 10–11, 7.8 × 10–11 and 8.38 × 10–10 g/mL, respectively. Inthe proposed interaction mechanism, the fluorescence inten-sity of the Tb3+–catecholamine complexes could be greatlyenhanced by introducing SDBS that provides a hydrophobicenvironment with relatively low polarity and high viscosity.This method has been applied to the determination ofcatecholamines in pharmaceutical preparations and serumsamples with a very low detection limit.

Acknowledgements

This research was supported by Kyungpook National UniversityResearch Fund, 2009.

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