Identification of Stimulant Drugs by Surface-Enhanced Raman Spectrometry on Colloidal Silver

Vibrational Spectroscopy, 2 (1991) 145-154 145 Elsevier Science Publishers B.V., Amsterdam Identification of stimulant drugs by surface-enhanced Raman spectrometry on colloidal silver A. RupCrez, R. Montes and J.J. Lasema * Department of Physical Chemistry and Department of Analytical Chemistry, Faculty of Sciences, Uniuersity of Ma’laga, 29071 Mrilaga (Spain) (Received 2nd February 1991) Abstract The surface-enhanced Raman (SER) spectra of stimulant drugs, including mefenorex, pentylenetetrazole, L- amphetamine and pemoline, were obtained on colloidal silver. Silver colloids are prepared in a single step, at room temperature, by chemical reduction of Ag+ with sodium tetrahydroborate. Spectra are recorded using drug concentrations at the pg ml-’ level. Individual drugs can be identified by characteristic vibrational bands. The SER spectrum of human urine and that of human urine spiked with mixtures of stimulant drugs are reported. Keywords: Raman spectrometry; Amphetamine; Mefenorex; Pemoline; Pentylenetetrazole; Pharmaceuticals; Sports doping; Stimulant drugs The increase over the last 30 years in the use In recent years, a number of papers have been of drugs in sport (doping) has led the Intema- published demonstrating the detectability of low tional Olympic Committee (IOC) to establish a molecule concentrations (< lop4 M) by adsorp- list of banned substances and to introduce formal tion on rough metal surfaces and subsequent drug testing. The doping classification includes Raman scattering [surface-enhanced Raman scat- stimulants as one of the five regulated categories. tering (SERS)]. Raman spectra of substances at Stimulants include various types of drugs which trace concentrations are readily obtained [6-l 11, increase alertness, competitiveness and hostility even in the presence of a strong luminescence and reduce fatigue. Stimulants also produce a background. Luminescence from adsorbed species false sense of ability and may cause a loss of is generally quenched by radiationless energy judgement, leading to accidents within sports. transfer to the metal surface [12-141. The first These include irregular heart rate, which may enhancement of Raman signals by adsorption on lead to cardiac arrest, the development of para- a rough metal surface was observed in 1974 [15], noid psychosis, a rise in body temperature, which the SER spectrum of pyridine on electrochemi- can induce dehydration in warm weather, and the tally roughened silver electrodes being obtained. masking of fatigue, which can lead to circulatory Since then, different metals have been reported collapse, heart exhaustion and stroke. to be SER-active, including silver, gold, copper The determination of stimulants in urine (the and, to a lesser extent, platinum, nickel, indium sample of choice in doping control) is accom- and others. Diverse roughening procedures have plished using a variety of techniques including gas been also investigated for the production of ac- chromatography [l], liquid chromatography [2,3] tive surfaces 116,171. Excitation with red wave- and gas chromatography mass spectrometry [4,5]. lengths is a requirement for large enhancement 0924-2031/91/$03.50 0 1991 - Elsevier Science Publishers B.V. All rights reserved 146 A.RUPkREZETAL. factors using gold and copper as substrates [18,191. mineralized water was used throughout. Mefeno- However, most visible wavelengths can be used rex, pentylenetetrazole, L-amphetamine and pe- with silver. For chemical characterization, col- moline were purchased from Sigma. Stock aque- loidal silver seems to be the most amenable sub- ous solutions (100 pg ml-‘) contained about 5% strate in terms of production, storage and sample methanol to help dissolve the drugs. Silver hydro- handling, although difficulties with the repro- sols were prepared at room temperature in test ducibility of SERS intensities have been reported tubes by adding 1 ml of 1.0 x 10e3 M AgNO, to La. 3 ml of 2.0 x 10e3 M NaBH,. Hydrosols so pre- A few papers [21,22] on the applicability of the pared showed an absorption maximum at 380 nm SERS technique to biological samples are avail- with no other spectral features in the long-wave- able. This whole demonstrates the analytical use- length side of this band. Each sample was pre- fulness of SERS for the identification of drugs in pared by adding 0.1 ml of the drug solution to 3 human urine samples using a silver hydrosol as a ml of the silver colloid at room temperature. substrate. The SER spectrum of urine and those After mixing, the resulting test solution was of urine spiked with four stimulants were ob- transferred into the liquid cell and placed in the tained. The procedure is easy to perform and the sample chamber of the spectrometer. The SER drugs can be readily identified by characteristic spectra of urine were obtained by spiking 0.1 ml bands in the SER spectrum of spiked urine. of unfiltered urine with 0.1 ml of drug solution and adding the resulting sample to 3 ml of silver colloid at room temperature. Urine samples were EXPERIMENTAL taken from healthy human subjects. Instrumentation The Raman system has been described in a RESULTS AND DISCUSSION previous paper [231. Basically, it consisted of a helium-neon laser (Siemens, Model LGK 76268) The SER spectra of mefenorex, pentylenete- tuned at 632.8 nm. The power at the sample was trazole, L-amphetamine and pemoline on silver typically 30 mW. Spectra were obtained with a colloid are shown in Fig. 1. Table 1 summarizes 0.22-m double-grating spectrometer (Spex Indus- the vibrational bands of these substances and tries, Model 1680B). The Raman scattering was shows selective spectral features corresponding to collected at right-angles and detected with a ther- the presence of different heteroatoms and sub- moelectrically cooled photomultiplier tube stituent groups on the ring systems. The peak (Hamamatsu, Model R928) and a photon count- positions of the conventional Raman (CR) spec- ing system (Stanford Research, Model SR 400). tra of the pure drugs (liquid state for L- Data acquisition, storage, processing and plotting amphetamine and solid state for the others) also were controlled by an IBM AT compatible micro- are listed in Table 1. computer. All spectra reported represent single The SER spectrum of mefenorex shows two scans and are provided with spectral smoothing medium bands at 990 and 651 cm-i and a weak of 11 or 17 points (Savitzky-Golay algorithm). mode at 1580 cm-‘, which appears in the CR The time constant of the detection electronics spectrum at 1598 cm-‘. The band at 651 cm-‘, was 0.3 s and each spectrum consisted of 968 data also appearing in the CR spectrum, can be at- points. Frequencies were accurate to within 3 tributed to the interaction of bending mode of cm-’ for strong, sharp bands. The spectrometer the carbon atoms in positions 1 and 4 with the resolution was generally set to 14 cm-‘. stretching of the bond connecting the substituent to the ring [24]. The C-X distance increases as Chemicals and procedure the distance between C-l and C-4 decreases. The All chemicals were of analytical-reagent grade band at 990 cm-’ corresponds to in-plane ring or equivalent, and were used as received. De- deformation. 147 SERS OF STIMULANT DRUGS - CH,CH(CH,)NH(CH,),CI / Q- a00 ,200 Raman shift (cm-‘) Raman shift (cm-‘) L-omphetamlne 1 I 800 lma Isc4 Roman shift (cm-‘) Fig. 1. Surface-enhanced Raman spectra of stimulant drugs on colloidal silver. Drug concentration, 25 fig ml-’ on the measured sample. 148 A. RUPl?REZ ET AL. The SER spectrum of pentylenetetrazole shows appears at 1580 cm-‘, probably due to the pres- a medium band at 1029 cm-’ and weak bands at ence of the C=N bond. Pemoline also exhibits a 674, 950, 1095, 1130, 1150 and 1584 cm-‘. Sev- medium band at 658 cm-’ and a strong band at eral other weak vibrational modes occur between 830 cm-‘. 1150 and 1550 cm-r and a medium band at 1640 The CR spectrum of mefenorex (Fig. 2) shows cm-‘. a strong band at 828 cm-’ which appear in the The SER spectrum of L-amphetamine shows SER spectrum as a weak band at 820 cm-‘. The the characteristic vibrational modes of the ben- medium band at 1379 cm- ’ could be due to zene ring at 990 and 1012 cm-’ and two modes at symmetric CH, deformation. Also, it exhibits 1112 and 1174 cm-‘, which may be due to the medium bands at 651, 1165 and 1316 cm-‘. Fig- primary amine group with a tertiary a-carbon ure 2c shows the CR spectrum of pentylenetetra- atom. A medium band at 1645 cm- ’ may be due zole, with the most prominent peaks at 1650 and to NH, bending. 661 cm-‘. Several weak bands occur in the spec- The SER spectrum of pemoline exhibits abun- tral zone examined. The weak SER band at 1385 dant spectral features. Medium bands at 1014 cm-’ appears in the CR spectrum at 1370 cm-‘. and 1047 cm-’ are observed. The medium band L-amphetamine (Fig. 2) shows a strong peak at at 1170 cm-’ and the very strong peak at 1640 990 cm-’ with a shoulder at 1012 cm-’ and weak cm-’ may be due to C-N stretching of the amine bands at 456, 493 and 821 cm-‘. These bands group [25] and NH, bending, respectively. The appear as weak vibrational modes in the SER weak band at 1745 cm-’ may be attributed to spectrum at similar Raman shifts. The other bands carbonyl group stretching. Another strong band in the CR spectrum also appear in the SER TABLE 1 Observed surface-enhanced Raman shifts (SERS, cm-‘) on colloidal silver, conventional Raman shifts (CRS, cm-‘) and relative peak intensities (in parentheses, on a semiquantitative scale from 1 to 10) of stimulant drugs L-Amphetamine

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