Adsorption of Beta-Adrenergic Agonists Used in Sport Doping on

Aug 30, 2010 - Instituto de Estructura de la Materia, CSIC, Serrano, 121, 28006-Madrid, Spain. Received June 26, 2010. Revised Manuscript Received Jul...
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Adsorption of Beta-Adrenergic Agonists Used in Sport Doping on Metal Nanoparticles: A Detection Study Based on Surface-Enhanced Raman Scattering I. Izquierdo-Lorenzo, S. Sanchez-Cortes,* and J. V. Garcia-Ramos Instituto de Estructura de la Materia, CSIC, Serrano, 121, 28006-Madrid, Spain Received June 26, 2010. Revised Manuscript Received July 28, 2010 The adsorption of beta2-adrenergic agonist (βAA) drugs clenbuterol, salbutamol, and terbutaline on metal surfaces has been investigated in this work by means of surface-enhanced Raman scattering (SERS). To assist in this investigation, a previous vibrational (IR and normal Raman) characterization of these drugs was performed, supported by ab initio density functional theory calculations. The application of SERS was aimed to apply this highly sensitive technique, based on localized surface plasmon resonance, in the detection of βAA at trace concentrations and as a possible alternative method which can be postulated in routine antidoping analysis. The adsorption of these drugs was studied in depth at different experimental conditions: on Au and Ag, at different pHs, and with varying adsorbate concentration. Moreover, plasmon resonance spectroscopy was employed to investigate the adsorption of these drugs on the metal nanoparticles as well as their aggregation. It was found that the adsorption of these molecules is more effective on gold nanoparticles and at acidic pH, based on the direct interaction of the aromatic or aliphatic moieties through ionic or coordination bonds with the metal. These drugs followed a Langmuir adsorption model from which the adsorption constant and the limit of detection can be determined.

Introduction Clenbuterol (CB), salbutamol (SB), and terbutaline (TB) (Scheme 1) are phenylethanolamine drugs which act as activators of β2-adrenergic receptors causing a beta2-adrenergic agonist (βAA) effect.1 These receptors are distributed throughout the body and their activation provokes responses such as bronchodilation, increased heart rate, and vasodilatation. Effects seen in the skeletal muscle include increased growth and speed of contraction.2 They are therapeutically employed for the treatment of asthma in some countries because of their bronchodilator activity. Also, these drugs can be illicitly employed by sportsmen to improve performance or for their anabolic effects.3-7 Thus, its consumption is regulated or even prohibited, in the case of clenbuterol, by the World Anti-Doping Agency.8 In order to control illegal use of βAA drugs, various methods of determination have been developed. Up to now, the most commonly used are based in LC-MS and capillary electrophoresis (CE).9-15 (1) Davis, E.; Loiacono, R.; Summers, R. J. Br. J. Pharmacol. 2008, 154, 584. (2) Bowman, W.; Anden, N. In Adrenergic Activators and Inhibitors; Szekeres, L., Ed.; Springer-Verlag: Berlin, 1981; pp 47-128. (3) Caruso, J. F.; Signorile, J. F.; Perry, J. F. Med. Sci. Sports Exercice 1995, 27, 1471. (4) Moore, N. G.; Pegg, G. G.; Sillence, M. N. Am. J. Physiol. 1994, 267, E475. (5) van Baak, M. A.; de Hon, O. M.; Hartgens, F.; Kuipers, H. Inhaled salbutamol and endurance cycling performance in non-asthmatic-athletes. Int. J. Sports Med. 2004, 25 (7), 533-538. (6) van Baak, M. A.; Mayer, L. H.; Kempinski, R. E. Med. Sci. Sports Exercice 2000, 32, 1300. (7) Yang, Y. T.; McElligott, M. A. Multiple actions of beta-adrenergic agonists on skeletal muscle and adipose tissue. Biochem. J. 1989, 261 (1), 1-10. (8) Agency, T. W. A.-D. The 2010 Prohibited List - International Standard, 2010. (9) Brambilla, G.; di Bez, S.; Pietraforte, D.; Minetti, M.; Campanella, L.; Loizzo, A. Ex vivo formation of gastric metabolites of clenbuterol: Preliminary characterisation of their chemical structure. Anal. Chim. Acta 2007, 586, 426-431. (10) Brunelli, C.; Bicchi, C.; Di Stilo, A.; Salomone, A.; Vincenti, M. High-speed gas chromatography in doping control: Fast-GC and fast-GC/MS determination of -adrenoceptor ligands and diuretics J. Sep. Sci. 2006, 29 (18), 2765-2771. (11) Huang, J. F.; Zhang, H. J.; Lin, B.; Yu, Q. W.; Feng, Y. Q. Multiresidue analysis of beta-agonists in pork by coupling polymer monolith microextraction to electrospray quadrupole time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21 (17), 2895-2904.

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However, LC-MS requires preconcentration and purification treatments due to the low concentrations of analytes and the complexity of the matrixes. On the other hand, CE lacks any molecular information about the structure of the studied compound. Surface-enhanced raman scattering (SERS) is a highly sensitive analytical tool that allows trace level detection in combination with detailed qualitative information, giving characteristic responses for each molecule that are true fingerprints of a certain compound. This technique has attracted a great deal of interest in recent years mainly due to single molecule detection and the preparation of new active surfaces of improved sensitivity, specificity, and reproducibility.16-18 At a more practical level, SERS-based sensors have been developed for the detection of diverse molecules of environmental or biological interest.19,20 It is the purpose of (12) Huang, L.; Lin, J. M.; Yu, L. S.; Xu, L. J.; Chen, G. N. Improved simultaneous enantioseparation of beta-agonists in CE using beta-CD and ionic liquids. Electrophoresis 2009, 30 (6), 1030-1036. (13) Kong, J.; Jiang, L.; Su, X. O.; Qin, J. H.; Du, Y. G.; Lin, B. C. Integrated microfluidic immunoassay for the rapid determination of clenbuterol. Lab Chip 2009, 9 (11), 1541-1547. (14) Li, S. T.; Wang, J. S.; Zhao, S. L. Determination of terbutaline sulfate by capillary electrophoresis with chemiluminescence detection. J. Chromatogr., B 2009, 877 (3), 155-158. (15) Luo, W.; Zhu, L.; Deng, J.; Liu, A.; Guo, B.; Tan, W.; Dai, R. Simultaneous analysis of bambuterol and its active metabolite terbutaline enantiomers in rat plasma by chiral liquid chromatography-tandem mass spectrometry. J. Pharm. Biomed. Anal. 2010, 52, 227-231. (16) Aroca, R. Surface-enhanced Vibrational Spectroscopy; John Wiley & Sons: Chichester, 2006. (17) Haynes, C. L.; McFarland, A. D.; Van Duyne, R. P. Surface-Enhanced Raman Spectroscopy. Anal. Chem. 2005, 77 (17), 338A-346A. (18) Guerrini, L.; Garcia-Ramos, J. V.; Domingo, C.; Sanchez-Cortes, S. Functionalization of Ag nanoparticles with dithiocarbamate calix[4]arene as an effective supramolecular host for the surface-enhanced Raman scattering detection of polycyclic aromatic hydrocarbons. Langmuir 2006, 22 (26), 10924-10926. (19) Guerrini, L.; Garcia-Ramos, J. V.; Domingo, C.; Sanchez-Cortes, S. Sensing Polycyclic Aromatic Hydrocarbons with Dithiocarbamate-Functionalized Ag Nanoparticles by Surface-Enhanced Raman Scattering. Anal. Chem. 2009, 81 (3), 953-960. (20) Zhang, X.; Shah, N. C.; Van Duyne, R. P., Sensitive and selective chem/bio sensing based on surface-enhanced Raman spectroscopy (SERS). Vib. Spactrosc. 2006, 42 (1), 2-8.

Published on Web 08/30/2010

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Scheme 1. Structural Formulas of Clenbuterol (a), Salbutamol (SB), and Terbutaline (TB)

this paper to employ SERS for the univocal detection of doping drugs at trace concentrations, with the final goal being the development of an analytical method to detect these drugs by SERS. The analytical ability of SERS applied to the detection of sport doping drugs has already been demonstrated by previous SERS studies.21,22 Since in practice the use of SERS implies the adsorption of the studied molecule on a metallic surface, the present work intends to analyze in a deeper way the adsorption of CB, SB, and TB drugs, aimed to implement a method for the molecular detection of these doping drugs by SERS spectroscopy at trace concentrations. This study is important to find the optimal conditions for the SERS detection of these drugs. The interpretation of SERS spectra needs a previous vibrational characterization of the studied βΑΑ drugs. Hemisulfate SB and TB were recently studied by vibrational spectroscopy by Ali et al.23,24 However, for CB, no previous vibrational study has been reported so far. In this paper, we have performed a previous vibrational analysis of CB and SB by infrared and Raman spectroscopy, with the latter under the neutral form. Thus, this study is reported for the first time for CB, while the vibrational treatment was carried out for the neutral SB and compared to the previous vibrational study performed for the corresponding hemisulphate form by Ali et al.23 The SERS experiments were carried out on Au and Ag nanoparticles (NPs) because of their stronger SERS efficiency.25-27 A study of the adsorption of the drugs at different experimental conditions (metal, pH, excitation wavelength, drug concentration) was performed on these substrates in order to find the optimal conditions for their adsorption and detection. Furthermore, the effect of adsorption on the plasmon resonance of the metal NPs was carried out at different adsorbate concentrations, and this was correlated to the SERS intensities.

Experimental Section HAuCl4 (99.5%) and KNO3 (99%) were purchased from Merck. Clenbuterol hydrochloride (95%), salbutamol, and terbutaline (21) Alvarez-Puebla, R. A.; Bravo-Vasquez, J. P.; Cui, B.; Veres, T.; Fenniri, H. SERS classification of highly related performance enhancers. ChemMedChem 2007, 2 (8), 1165-1167. (22) Trachta, G.; Schwarze, B.; Sagmuller, B.; Brehm, G.; Schneider, S. Combination of high-performance liquid chromatography and SERS detection applied to the analysis of drugs in human blood and urine. J. Mol. Struct. 2004, 693 (1-3), 175-185. (23) Ali, H. R. H.; Edwards, H. G. M.; Kendrick, J.; Scowen, I. J. Vibrational spectroscopic study of salbutamol hemisulphate. Drug Test. Anal. 2009, 1 (1-2), 51-56. (24) Ali, H. R. H.; Edwards, H. G. M.; Kendrick, J.; Scowen, I. J. Vibrational spectroscopic study of terbutaline hemisulphate. Spectrochim. Acta, Part A 2009, 72 (4), 715-719. (25) Anderson, D. J.; Moskovits, M. A SERS-Active System Based on Silver Nanoparticles Tethered to a Deposited Silver Film. J. Phys. Chem. B 2006, 110 (28), 13722-13727. (26) Leyton, P.; Sanchez-Cortes, S.; Campos-Vallette, M.; Domingo, C.; Saitz, C.; Garcia-Ramos, J. V. Surface-Enhanced Micro-Raman Detection and Characterization of Calix[4]Arene-Polycyclic Aromatic Hydrocarbon Host-Guest Complexes Appl. Spectrosc. 2005, 59 (8), 1009-1015. (27) Vlckova, B.; Moskovits, M.; Pavel, I.; Siskova, K.; Sladkova, M.; Slouf, M. Single-molecule surface-enhanced Raman spectroscopy from a molecularlybridged silver nanoparticle dimer. Chem. Phys. Lett. 2008, 455 (4), 131-134.

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hemisulfate were purchased from Sigma. Stock solutions of CB, SB, and TB were prepared in water or ethanol at a concentration of 10-2 M. All aqueous solutions were prepared using Milli-Q water. Absolute ethanol was of analytical grade, 99.5% purity, and was purchased from Merck. The gold colloid was prepared using the method described by Sutherland and Winefordner.28 Briefly, 0.1 mL of an aqueous solution of 0.118 M HAuCl4 was diluted in 40 mL of water, under intense stirring. A total of 1 mL of sodium citrate solution at 1% in volume was added dropwise. The yellow solution was refluxed for 5 min, resulting in a red colloidal suspension. The resulting colloid is formed by spherical Au NPs showing a mean diameter of 15 nm. Besides, this method leads to metal NPs with a net negative charge due to the anionic species adsorbed on the surface, that is, citrate and the oxidation products of this anion. All samples for SERS measurements were prepared as follows. A total of 1 ml of the gold colloid was activated by the addition of KNO3 up to 0.03 M. This activation consisted of the induced aggregation of Au NPs. An aliquot of an aqueous solution of the βΑΑ drugs at a certain concentration was then added to the sample. SERS samples obtained at different pH were prepared by adding nitric acid or NaOH. Each SERS experiment in the analytical study was carried out on three samples at each concentration. The SERS intensities registered for the quantitative study at different drug concentrations were acquired three times at a certain concentration, and then referred to the intensity of the AgNO3- band (at 250 cm-1) obtained at low pH values in the presence of each drug. As every sample contained the same amount of KNO3, the SERS intensity of this band could be considered invariable for all the studied systems and used as a reference. From this method, an average value and the corresponding standard deviations (SD) were calculated and plotted against the adsorbate concentration. On the other hand, the limits of detection (LOD) were calculated for a signal-to-noise ratio of 3. SERS experiments were carried out by focusing the laser beam inside a cuvette (1  1 cm) that contains the colloidal suspension. At these conditions, the registered SERS spectra are an average of all those signals emitted by the diverse nanoparticles that cross the beam focus during the time the experiment lasts. This sort of protocol leads to reproducible results because of their statistical nature. IR spectra were measured in transmission mode (KBr pellet) using a Bruker IFS66 instrument equipped with a DTGS detector operating at room temperature. Raman spectra of the pure solids were registered using a Bruker RFS/100S FT-Raman instrument, equipped with a Nd:YAG laser beam at 1064 nm and a germanium detector cooled with liquid nitrogen. SERS spectra were registered with a Renishaw Raman RM2000 instrument, equipped with an electrically cooled CCD camera, employing as excitation lines both the He/Ne laser at 632.8 nm and the diode laser at 785 nm. The best SERS results were obtained by employing the excitation line at 785 nm. Each SERS spectrum was registered by using a time of 10 s. Plasmon absorption spectra were registered employing an UV-visible-NIR Shimadzu 3600 spectrometer equipped with a PMT for light detection in the UV-visible range and an InGaAs detector for the NIR. The colloids were diluted at 20% in water for these measurements. Theoretical calculations were made using the Gaussian 0329 program. Geometry optimization was initially performed using a Hartree-Fock approximation and further refined using the BLYP functional, choosing a 6-311G basis set with the d polarization function for clenbuterol, and 6-311þG for salbutamol. Vibrational frequencies were computed by determining the second derivatives of the energy with respect to the Cartesian nuclear coordinates. (28) Sutherland, W. S.; Winefordner, J. D. Colloid filtration: A novel substrate preparation method for surface-enhanced Raman spectroscopy J. Colloid Interface Sci. 1992, 148 (1), 129-141.

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Figure 1. Vibrational spectra of clenbuterol hydrochloride: (a) Infrared spectrum of the solid in KBr pellet, (b) Raman spectrum of the solid state, and (c) Raman spectrum of 0.1 M aqueous solution. Raman excitation line: 1064 nm.

Figure 2. Vibrational spectra of salbutamol: (a) Infrared spectrum of the solid in KBr pellet and (b) Raman spectrum of the solid state registered with λexc = 1064 nm.

Results and Discussion

Table 1. Experimental and Calculated Vibrational Wavenumbers (cm-1) of Clenbuterol

Vibrational Characterization of Drugs. A previous step in the application of SERS to the detection of CB, SB, and TB drugs is their vibrational characterization by means of IR and Raman spectroscopy. Figures 1 and 2 show the vibrational spectra of these compounds along with the assignment of the main features. Tables 1 and 2 show the main wavenumbers and their assignments for CB and SB. This assignment was accomplished by carrying out a density functional theoretical study of the vibrational modes, which was further completed with the help of previous works on vibrational characterization of other structurally related βΑΑ drugs24,30 and beta blockers.31 The vibrational spectra of these molecules show various common features, which are due to the common molecular groups existing in their structure. In particular, two main parts can be distinguished in these molecules corresponding to the aromatic and the aliphatic moieties. The characteristic bands due to the first moiety are those corresponding to the aromatic stretching vibrations, anilinic NH2 group vibrations, C-H deformations, and C-Cl or C-OH stretchings. On the other hand, the modes corresponding to the aliphatic moiety correspond to the vibrations of tert-butyl group and the aliphatic OH and NH groups, essentially different from the aromatic ones also existing in the aromatic ring. In general, the most polar vibrations appear most intensified in the IR spectra than in the Raman one. (29) Frisch, M. J.; G. W. T., Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; M. A. Al-Laham, Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc: Wallingford, CT, 2004. (30) Socrates, G. Infrared and Raman Characteristic Group Frequiencies, 3rd ed.; John Wiley & Sons, Ltd.: New York, 1994. (31) Ruperez, A.; Laserna, J. J. Surface-enhanced Raman spectrometry of chiral b-blocker drugs on colloidal silver. Anal. Chim. Acta 1996, 87.

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Raman

IR

calculated wavenumbers

assignmenta

νas(NH2) ν(OH) 3244 3248 νs(NH2) 3204 ν(NH) 3065 νar(CH) 3032 νas(CH3) 2979 2974 νas(CH3) 2922 νs(CH3) 2800 νs(CH3) 1622 δ(NH2) 1597 ν(CdC)/δ(NH2) 1581 ν(CdC) 1482 1485 νar/νar(C-N) 1445 1454 δ(CH3)/δ(CH2)/δ(CH) 1425 δ(CH3) 1365 1377 1344 ω(CH2)/δal(CH) 1327 1323 t(CH2)/δ(OH)/δ(CH) 1287 1283 1297 νar/νar(C-N) 1237 1241 1237 ν(CC3)/t(CH2)/δ(OH) 1211 1210 1203 t(CH2)/δ(OH)/δ(CH)/νal(C-N) 1104 1083 ν(C-O)/νal(C-N) 1080 1080 1061 F(NH2)/νal(C-N) 1053 1053 1036 F(CH3) 1003 1003 992 F(CH3)/ν(CC) 924 922 899 γar(CH) 874 885 849 γar(CH)/F(CH3) 851 849 γar(CH) 784 753 νas(C-Cl)/νar 747 744 ν(C-Cl) 648 603 τal 378 357 δip(C-C-Cl) a Symbols: ν, stretching; δ, bending; γ, out-of-plane-bending; τ, torsion; F, rocking; ω wagging; t, twisting; s, symmetric; as, asymmetric; ip, in-plane; op, out-of-plane; ar, aromatic; al, aliphatic. 3351

3579 3486 3479 3387 3126 3088 3020 3004 2944 1643 1590 1541 1471 1416

The IR and Raman spectra of CB are shown in Figure 1. In the region corresponding to higher wavenumbers (3500-3000 cm-1) of the IR spectrum, the most intense bands are those assigned to NH and OH stretching vibrations, which are stronger than those corresponding to C-H stretching bands appearing at 2800-3000 cm-1. On the other hand, the bands seen below 2800 cm-1 can be attributed to the existence of H-bonds in the solid structure. DOI: 10.1021/la102590f

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Table 2. Experimental and Calculated Vibrational Wavenumbers (cm-1) of Salbutamol Raman

IR

calculated wavenumbers

assignmenta

3434 ν(NH) 3420 ν(OH) 3055 3118 νar(CH) 2980 3041 νas(CH3)/ν(CH) 2967 3041 νas(CH3) 2930 2977 νs(CH) 2883 2940 νs(CH2) 2879 2929 ν(CH) 1611 1610 1626 νar(CdC) 1506 1530 δ(NH) 1486 1517 δ(CH3) 1466 1456 1498 δ(CH2)/δ(CH3)/δ(CH) 1394 1445 δ(CH3) νar 1366 1380 ω(CH2)/δal(CH) 1354 1343 1358 δ(CH)/t(CH2)/δ(OH) 1271 1270 1303 t(CH2)/δal(CH) 1236 1235 1260 ν(CC3)/νar(CO)/δal(CH) 1219 1236 ν(CC3)/νal(CN)/δ(OH) 1189 1189 t(CH2)/δ(CH) 1106 1106 1150 δar(CH)/δ(OH) 1082 1081 1102 ν(CO)/δar(CH)/νar 1040 1051 F(CH3)/ν(CC3) 1023 1025 1042 F(CH3)/ν(CC3) 966 999 νal(CO) 962 953 969 γar(CH)/F(CH2) 922 902 928 F(CH2)/F(CH3) 883 906 νal(CC) 852 875 νal(CC) 814 822 818 γar(CH) 775 779 γar(CH) 723 722 755 τal 613 615 617 τal a Symbols: ν, stretching; δ, bending; γ, out-of-plane-bending; τ, torsion; F, rocking; ω wagging; t, twisting; s, symmetric; as, asymmetric; ip, in-plane; op, out-of-plane; ar, aromatic; al, aliphatic.

Figure 3. (a-g) SERS spectra of clenbuterol at a fixed concentration of 10-5 M and pH values of (a) 3, (b) 4, (c) 5, (d) 7, (e) 8, (f) 9.5, (g) 11, and (h) Raman spectrum of clenbuterol. Inset: Scheme of the proposed adsorption on changing the pH. Excitation line at 785 nm.

However, the NH and OH stretching vibrations are very weak in Raman spectra. At lower wavenumbers ( TB > SB. Also, the LOD of each molecule obtained by SER spectroscopy showed this leaning, and the best results were obtained for CB. Although the SERS intensification is due to both electromagnetic and chemical effects, it is commonly accepted that the main SERS enhancement is produced via the electromagnetic mechanism.16,34 Since the Raman cross section of all three molecules is similar, the differences in the enhancement factors leading to different SERS intensities should be due to the strength of the interaction of each molecule with the surface, which determines in turn the number of molecules adsorbed onto the surface. The higher sensitivity observed for CB is related to the strong interaction of the anilinic moiety in this drug with the Au surface, which seems to interact more strongly with the surface than the aliphatic amino groups and the resorcinol moiety. The LOD obtained in this work are lower than those found in the literature for CE coupled with UV-visible detection.35 However, (34) Moskovits, M. Surface-enhanced spectroscopy. Rev. Mod. Phys. 1985, 57 (3), 783-826. (35) Sirichai, S.; Khanatharana, P. Rapid analysis of clenbuterol, salbutamol, procaterol, and fenoterol in pharmaceuticals and human urine by capillary electrophoresis. Talanta 2008, 76 (5), 1194-1198.

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Figure 7. (a) Absorption spectrum of clenbuterol in aqueous solution. (b) UV-visible absorption spectra of the gold colloid in the presence of 0.03 M KNO3, (c) with 10-5 M clenbuterol, (d) with 10-5 M terbutaline, and (e) with 10-5 M salbutamol.

better LOD have been reported employing methods including indirect detection by labeling the molecules with a luminescent dye14 or preconcentration steps required to improve the signal when making use of amperometric detection.36 Furthermore, other techniques providing both qualitative and quantitative information of the compounds (by MS detection) require demanding sample pretreatment that should not be necessary for SERS detection, and their LOD are comparable to those presented in this paper.10,11 Resonance Plasmon Spectra. Figure 7 shows the plasmon absorption spectra of the Au colloid in the absence (Figure 7b) and in the presence of CB, SB, and TB at a constant concentration (10-5 M), along with the absorption spectrum of CB in aqueous solution (Figure 7a). The red Au colloid shows a characteristic absorption band at 518 nm.28 When the studied molecules are added to the colloid, a color change to blue takes place, and a wide band appearing at longer wavelengths appears, due aggregation of the colloid37 induced by the adsorption of the molecules on the nanoparticle surface. The relative absorption intensity of this second band is directly related to the relative amount of drug adsorbed onto the metal surface. As can be seen, these intensities correlate quite well to the adsorption parameters deduced from the above quantitative SERS analysis. The lower intensity measured for SB indicates again the lower affinity of this drug toward the Au surface. The intensity of the aggregation band varies with the concentration of βΑΑ drug in the suspension. Figure 8 shows the plasmon resonance spectra measured for SB at different drug concentrations. This method could be use to determine the concentration of these drugs by plasmon resonance. The inset plot displays the intensity of this aggregation band against the concentrations of SB and CB. As can be seen, this method also shows a clear difference between these two compounds, as the sensitivity is higher for the case of CB, since it is more strongly attached to the metal surface. This technique, however, does not reach the level of sensitivity achieved with SERS, and it does not allow a proper differentiation between the molecular components (36) Zheng, L. H.; Tong, P.; Zheng, X. Y.; Chi, Y. W.; Chen, G. N.; Zhang, L. Ellectrostacking online sample pre-concentration capillary electrophoresis with amperometric detection for beta(2)-agonists in human urine. J. Sep. Sci. 2008, 31 (20), 3556-3564. (37) Nordlander, P.; Oubre, C.; Prodan, E.; Li, K.; Stockman, M. I. Plasmon hybridizaton in nanoparticle dimers. Nano Lett. 2004, 4 (5), 899-903.

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Figure 8. UV-visible absorption spectra of the gold colloid in the

presence of (a) 0.03 M KNO3 and with (b) 10-6 M, (c) 10-5 M, and (d)10-4 M salbutamol. The inset represents the variation of the intensity of the band of the aggregates when increasing concentration of salbutamol and clenbuterol.

in a multicomponent mixture, giving rise to very similar responses for both CB and TB. An analysis by SERS, in contrast, gives exclusive fingerprint spectra of each one.

Conclusions SERS spectroscopy was proven to be a suitable technique for trace analysis of beta2-adrenergic agonist (βAA) drugs clenbuterol, salbutamol, and terbutaline using metal nanostructures. These compounds presented substrate selectivity, as they were SERS-active

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only on gold colloid. The analysis of the corresponding Raman spectra was done at different pHs, revealing the existence of two main interaction points to the surface: the aromatic moiety and the N atom placed in the aliphatic chain. The basicity of the aromatic part determines the affinity of these compounds to the metal surface at low pH. Therefore, this affinity varies in the following order: CB > TB > SB, as corresponds to the stronger basicity of aniline and resorcinol with respect to the phenol in SB. At alkaline pH, the interaction takes place through a direct interaction of the aliphatic secondary amine with the metal. The adsorption analysis derived from the SERS study revealed that the adsorption of βAA drugs follows a Langmuir model. The adsorption constant deduced from this analysis correlates very well the different affinity deduced from the direct study of SERS spectra. Finally, the LOD deduced was lower than that obtained for other techniques in the direct, low concentration level detection of these compounds. Plasmon resonance analysis correlates quite well with the SERS experiments and may also be used as an analytical technique for detection of any of the studied drugs, although it lacks the sensitivity and the molecular information given by SERS. This work is a first step toward real sample analysis by SERS as a promising technique to be applied in the field of sport doping detection. Acknowledgment. This work has been supported by the Spanish Ministerio de Ciencia e Innovaci on (Grant FIS2007-63065) and Comunidad de Madrid through the MICROSERES II network (Grant S2009/TIC-1476). I.I.-L. also acknowledges a Ph.D. scholarship from CSIC.

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