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Applications of Raman spectroscopy in pharmaceutical analysis

T. Vankeirsbilck, A. Vercauteren, W. Baeyens*, G. Van der Weken Laboratory of Drug Quality Control, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium F. Verpoort Laboratory of Organometallics and Catalysis, Department of Inorganic and Physical Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281 (S3), B-9000 Ghent, Belgium G. Vergote, J.P. Remon Laboratory of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
As Raman spectroscopy enables rapid, non-destructive measurements, the technique appears a most promising tool for on-line process monitoring and analysis in the pharmaceutical industry. This article gives a short introduction to Raman spectroscopy and presents several applications in the pharmaceutical eld. # 2002 Published by Elsevier Science B.V. All rights reserved.
Keywords: Pharmaceutical analysis; Polymorphism; Quantitative Raman analysis; Raman spectroscopy

1. Introduction
The phenomenon of inelastic light scattering is known as Raman radiation and was rst documented by Raman and Krishnan in 1928 [1]. When a substance is irradiated with monochromatic light, most of the scattered energy comprises radiation of the incident frequency (Rayleigh scattering). In addition, a very small quantity (0.0001%) of photons with shifted frequency is observed. The fraction of photons scattered from molecular centres with less energy than they had before the interaction
*Corresponding author. Tel.: +32 9-264.80.97; Fax: +32 9-264.81.96. E-mail: willy.baeyens@rug.ac.be

is called Stokes scattering. The anti-Stokes photons have greater energy than those of the exciting radiation (Fig. 1). Both infrared (IR) and Raman spectra are concerned with measuring associated molecular vibration and rotational energy changes. However, the requirement for vibrational activity in Raman spectra is not a change in dipole moment, as it is in IR spectra, but a change in the polarizability of the molecule. (It is therefore possible to obtain spectral information from a homo-nuclear molecule by Raman spectroscopy.) The energy resulting from this shift is equal to the vibrational energy gap that is excited in IR spectroscopy. Two major technologies are used to collect the Raman spectra: dispersive Raman; and, Fourier transform Raman (FT-Raman) [2,3]. The differences between both technologies are the laser that is used and the way the Raman scattering is detected and analysed. Each technique has unique advantages and the method that best suits the sample should be preferred. The applications listed in Table 1 are limited to the analytical eld. Two related techniques are important in the pharmaceutical area. The rst is confocal Raman microscopy, which is a useful technique for non-destructively probing depths of the sample without cross-sectioning. Confocal
# 2002 Published by Elsevier Science B.V. All rights reserved.

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Raman microscopy can selectively probe any given XYZ-location in a sample with spatial resolution in the micron range. It is best done by dispersive Raman spectroscopy with short wavelengths. In confocal Raman microscopy, laser light from the probe-head is focused onto a diffraction-limited spot in the sample by the microscope objective. The backscattered Raman signal is refocused onto a small confocal aperture that acts as a spatial lter, passing the Raman signal excited at the beam waist, but eliminating Raman produced at other points above and

below the beam waist. The ltered Raman signal then returns to the spectrometer where it is dispersed onto a CCD (charge coupled device) camera to produce a spectrum. Another technique is surface-enhanced Raman spectroscopy (SERS). In SERS, the Raman scattering from a compound (or ion) adsorbed on a structured metal surface can be 103106 times greater than that in solution. Surface-enhanced Raman scattering is strongest on silver, but is observable on gold and copper as well. SERS arises from two mechanisms. The rst is an enhanced electromagnetic eld produced at the surface of the metal. The second is enhanced formation of a charge-transfer complex between the surface and the analyte molecule. Molecules with lone-pair electrons or pi clouds show the strongest surface-enhanced Raman scattering. Even singlemolecule detection by SERS has been reported [4].

2. Raman versus IR
Farquharson et al. [5] depicted Raman spectroscopy as moving out of the shadow of IR spectroscopy during the 1990s through the development of stable diode lasers, bre-optic

Fig. 1. Schematic representation of energy transitions in IR and Raman spectroscopy. Table 1 Comparison of dispersive and FT-Raman spectroscopy Dispersive Raman VIS : 785 nm, 633 nm, 532 nm, . . . Grating Silicon CCD detector

FT-Raman Laser NIR : 1064 nm Spectral analysis by Interferometer Detector - room temperature indium gallium arsenide - liquid nitrogen-cooled germanium Advantages - limited uorescence - maximal compatibility with libraries and analysis software Applications - in the pharmaceutical industry: - unknown identication - incoming raw material characterisation - nal product quality - quantitative analyses - investigating polymorphs - forensic analysis through sample containers or evidence bags

- higher sensitivity - higher spatial resolution for microscopy applications - lower laser power minor component analysis more sensitive for aqueous samples analysis of dark samples depth or cross-sectional information in samples

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sample probes, compact optical designs, highquantum-efciency detectors, and personal computers with fast electronics and associated data acquisition and analysis. These advantages provide for real-time, multi-component chemical analysis and suggest the use of Raman spectroscopy for process monitoring and control, according to these authors [5]. Single-ended ber-optic probes simplify coupling into process streams, allow the Raman instrument to be placed far from the sample point, and give Raman spectroscopy a decided advantage over IR spectroscopy for industrial liquid and solid process applications. They also proposed solutions to instrument limitations, such as uorescence interference, incomplete spectral coverage, wavelength reproducibility, and long-term instrument instability [5].

3.

4.

5. 6.

3. Major advantages of Raman spectroscopy

The following advantages of Raman spectroscopy are impressive and should be critically evaluated and considered for application by analytical chemists active in any scientic discipline.
1. Current procedures for the quality control of nal products frequently comprise an assay of the active ingredient. To carry out these assays, the product is often subjected to crushing (or grinding), dissolution, separation and nally determination of the active ingredient. The determination step is often automated, but the sampling procedures are time-consuming, labour-intensive and destructive. Sample preparations, such as grinding can also lead to changes in solid states (e.g. hydration state, polymorphism, hydrogen bonding), which sometimes have an impact on the nal detection method. Near-IR (NIR) spectroscopy is one of the methods for which very little sample preparation is required. However, NIR data are complicated, as non-parametric multivariate analysis or other pattern-recognition techniques are required to interpret the data. But Raman, too, requires virtually no sample preparation and that provides signicant cost savings. 2. Raman spectra can be obtained non-invasively, which means that bulk and nal products can be tested directly in their packaging, such as glass

7.

8.

bottles, plastics and blister packs. Methods can be applied to the on-line monitoring of production lines. Raman analysis can be used to study small particles within inhomogeneous sample matrices. This is important because, in the early stages of development, only a few mg of material are available. As many drugs are formulated as microcrystals in a solid matrix with an excipient ller material, these small particles can be studied in situ using Raman microscopy. Raman analysis time is short, thus enabling quick feedback from the quality-control department to the production-development team. Raman experiments can be carried out easily, so that the work can be done by minimally trained personnel. The good reproducibility of FT-Raman spectrometric experiments has been attributed to the large entrance aperture, which permits the laser to be focused into a relatively large sample volume. Since water is a weak Raman scatterer, Raman has minimal sensitivity towards interference by water. It is possible to analyse aqueous solutions. Sampling for Raman spectroscopy is further eased through the use of bre optics.

4. Disadvantages of Raman spectroscopy

It is clear that Raman spectroscopy also has drawbacks, which may be summarized as follows:
1. The cost of required equipment has been the main obstacle to the widespread adoption of Raman spectroscopy for routine analysis; 2. A major problem for Raman measurements lies in the high levels of uorescence (intrinsic or caused by impurities) overlaying the Raman bands. However, in most cases, this can be avoided by shifting the laser wavelength to the NIR spectral region; and, 3. If excitation intensities are too high, they may thermally decompose the sample.

5. Applications in pharmaceutical analysis

Pharmaceutical analysis may benet from the advantages offered by Raman spectroscopy in various ways:

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1. Identication of raw materials (building up an extensive library of Raman spectra); 2. Quantitative determination of active substances in different formulations; 3. Supporting polymorphic screenings (polymorphs have different solubility rates, thereby impacting the effective dosing); and, 4. Supporting chemical development process scale-up (as process steps are modied and rened to ascertain whether the same form of the same chemical is being produced or not).

5.1. Analysis of pharmaceutical formulations

Several pharmaceutical forms have already been studied by Raman spectroscopy. It is not the aim of the present review to discuss every single molecule, but to give a general view on the possibilities of this powerful spectroscopic technique. An overview of important pharmaceutical substances together with their corresponding references is presented in Table 2. One of the cited useful features of Raman spectroscopy is its ability to carry out direct measurements on solids. Among the parameters that inuence the intensity of the Raman signal detected from solids are particle size and packing density. Critical steps during sample preparation are grinding, mixing, the pressure applied and the period of time to prepare the discs. Precision is affected by not only changes in sample location, but also the degree of homogenization. A motorized positioner focuses the laser beam on the sample and a manual side-to-side adjuster allows the sample to be moved to maximise optical efciency. Rodriguez [45] describes various experimental FT-Raman imaging procedures and their ability to both obtain and spatially resolve chemical information in the analysis of formulated tablets of pharmaceutical interest. Experimental analytical procedures using the imaging techniques are outlined. In solid dispersions, the drug is suspended in the polymer carrier in an amorphous state. Recrystallization of the drug under stress conditions can inuence the therapeutic performance

of the carrier. Breitenbach et al. [35] used confocal Raman spectroscopy to examine solid dispersions of the anti-inammatory agent ibuprofen. The group investigated the physicochemical stability of the formulation under stress conditions (from 90  C to 90  C), together with the content and the homogeneity of the drug distribution in the formulation matrix. The method was found promising for monitoring the spatial distribution of drugs in solid dispersions and for detecting changes, such as recrystallization. It should be mentioned that this is only so if a detectable amount of another crystalline compound is observed as a shifted band. The authors state that confocal Raman spectroscopy can investigate different layers (e.g. coatings on a tablet), areas (e.g. phase separation) or simply the quality of mixing in a manufacturing process, which is of great industrial importance. Because of the wide range of formulation materials used, it is often impossible to identify and subsequently to visualize every ingredient within the formulation using a single spectroscopic technique. Clarke et al. [46] combined Raman and NIR spectroscopies in thoroughly describing heterogeneous mixtures of solid dosage forms. Where Raman directly probes the molecular structure of the compound, NIR displays only overtones and combinations of fundamental molecular vibrations. Raman spectroscopy is more useful in identifying different polymorphic forms, drug substances and inorganics, while NIR benets include detection of similar carbohydrate species, moisture and varying hydration states. In their new approach of chemical image fusion (CIF), Clarkes group acquired data from exactly the same area of the sample using both Raman and FT-NIR mapping. The best images for the components were then overlaid to produce a combined chemical image that visually describes the entire formulation. Niemczyk et al. [23,24] showed that Raman spectroscopy using NIR excitation has signicant potential as a rapid, non-destructive quality control method for pharmaceutical samples. They obtained useful spectral data directly from drug formulations in gel capsules

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and from gel capsules inside blister packs. The data obtained showed a decrease in intensity through reective losses as the excitation beam and the Raman scattering pass through the curved wall of the blister pack. For the multivariate analysis, cross-validation was employed. The largest source of error was the sample inhomogeneity, then noise caused by vibrations
Table 2 List of pharmaceuticals (and related) studied by Raman spectroscopy, with corresponding references Acebutolol Acetaminophen Alprenolol Amiloride Amoxycillin Amphetamine and/or related compounds Amphotericin A/B Arterenol Aspirin Atenolol Bucindolol Calcium carbonate and glycine Cephalotin Cimetidine Ciprooxacin Cocaine Diclofenac Ephedrine Flucloxacillin Fluconazole Fluocortolone Heroin Ibuprofen Indomethacin Isosorbide Lorazepam Lormetazepam Nicotinamide Nystatin Oxazepam Phenylephrine Phenylpropanolamine Piroxicam Promethazine Propanolol Spironolactone Strychnine Sulfamerazine Sulfadiazine Sulfamethazine Temazepam Theofylline Triamterene Triuoperazine [6] [7,8] [6] [9] [10] [1117] [18] [19] [2022] [6] [23,24] [25] [10] [26] [27] [12,17,2830] [31] [19] [10] [32] [33,34] [12,29] [35] [31] [36] [37] [37] [38] [18] [37] [19] [8] [39] [31] [6] [40] [41] [42] [42] [42] [37] [31] [43] [44]

from the stepper motor of the XY stage and the CCD camera, and laser instability. McCreery et al. [47] have reported on the use of NIR Raman spectroscopy in identifying pharmaceuticals inside amber USP (United States Pharmacopoeia) vials. Although the amber glass greatly attenuated the signal, adequate spectra were obtained for the determination of vial contents with 1-60 s integration. Identication was performed using a library of spectra and accuracy was in the range 88-96%. Most of the errors were caused by uorescent samples. This work clearly demonstrated the potential of Raman spectroscopy for on-line process monitoring. The activity, stability and texture of emulsions are strongly affected by their microstructure, so an effective method for chemically imaging these microstructures is needed. Andrew et al. [48] exploited Raman imaging in distinguishing complex multi-component, multi-phase emulsion systems of inherently low contrast. They described how a confocal Raman microscope with an automated stage can be used to produce high-resolution, three-dimensional maps of the chemical composition of heterogeneous and multi-phase materials. They have characterized several commercial product systems ranging from pharmaceutical and skin creams to toothpaste. Armstrong et al. [49] employed FT-Raman microscopy to construct a prole of estradiol distribution of a transdermal drug-delivery patch. Findlay and Bugay [50] described how variable temperature (VT)-Raman spectroscopy can be used to study the dynamics of crystallization of menthol from a solvent (ethanol). The authors stated that there are many potential future applications for VT-Raman spectroscopy. By changing the cooling rate of a supersaturated solution, it is possible to study the dynamics of crystallization of a polymorphic system, providing insight into why one polymorphic form is preferred over another under particular conditions. By increasing the temperature of a sample over another under ambient conditions, it is possible to study the effects of changing temperature on active components and excipients in

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drug preparations. The effects of changing temperature on the synthesis of drug substances can also be studied. Raman spectroscopy can also be applied as a quantitative technique, but some criteria must rst be considered critically, e.g. homogeneous sample mixing, particle size, and instrument variability and reproducibility [50]. The authors conclude that Raman spectroscopy can be used in the pharmaceutical analytical laboratory in a variety of ways. Traditional drug substance characterization is enhanced with the additional information provided by Raman spectral data, and quantitative polymorph assays can be developed. Raman spectroscopy can also be used qualitatively and semi-quantitatively

to support pharmaceutical development and clinical supply operations by performing identity testing and potency differentiation. The technique also allows differentiation between hydrated forms of molecules (Fig. 2). According to Langkilde et al. [51,52], differences can be seen between Raman spectra from different crystal forms of a compound, or between crystalline and amorphous forms (Fig. 3). These investigators showed that the possibility of minimal sample preparation and the sensitivity to polymorphism make Raman spectrometry ideal for the study of crystal forms of pharmaceutical compounds, as they observed different FT-Raman spectra from the two

Fig. 2. Differentiation between hydrated forms by FT-Raman.

Fig. 3. FT-Raman spectra of different samples of ketoprofen (amorphous state and three degrees of crystallinity).

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polymorphs A and B of a compound H. They identied a frequency shift that leads to well-resolved bonds and found that, for mixtures of A in B, the intensity of the two bonds was proportional to the amount of the A and B forms. Furthermore, FT-Raman spectra can be obtained from whole, undisturbed pharmaceutical tablets, they stated, and it is possible to study a pharmaceutical formulation directly whether the active ingredient has one crystal form or another, or is amorphous. Given the larger amount of information offered by vibrational spectra as compared to uorescence spectroscopic data, Mulvaney and Keating [53] expected ultra-sensitive SERS to become increasingly important in chemical sensing. Detection of single molecules has already been reported by several groups. Mulvaney and Keating [53] also mentioned that Raman spectroscopy is a valuable detection method in a variety of separation techniques, where it offers not only elution proles, but also structure-specic spectra of analytes. Raman spectroscopy can also be helpful in determining enantiomeric compositions. In 1994 and later, Spencer et al. [54,55] used scattered circularly polarized Raman optical activity (SCP ROA) to quantify the chiral purity of pharmaceuticals. SCP ROA utilizes incident linearly polarized light and measures changes in the scattered left and right circularly polarized light. Ruperez and Laserna [6] showed that SERS is better for showing differences between enantiomers than conventional Raman spectroscopy.

spectroscopy was achieved using either univariate or multivariate calibration and both methods were compared. Results were compared with the current USP 24 and National Formulary (NF 19). During quantitative analysis, normalised percent relative intensities or areas should be used to compensate for variations in excitation intensity, sample positioning and temperature uctuations, factors which strongly affect the precision and detection limit values of Raman spectroscopy. Normalisation results in increased stability of the calibration, compensating for changes in experimental parameters. It can be performed with using an internal or an external standard. An internal standard should not interact with the sample, or perturb or overlap the analyte spectrum. Internal standards should be strong emitters, so that a small quantity produces a signal of adequate intensity. As an external standard, the 802/cm band of cyclohexane was used. It was measured right after the acquisition of each sample using the same experimental parameters, such as laser power, sample holder position, resolution and number of scans. The use of an external standard compensates for not only excitation uctuations but also other parameters, such as changes in sample location.

7. Summary
Vibrational spectroscopy is an excellent method for identifying substances because it provides ngerprint spectra that are unique to each specic compound. Of the various vibrational spectroscopies available, Raman spectroscopy should be the method of rst choice because the spectra it produces are rich in information and because it needs virtually no sample preparation. This makes it ideal for the analysis of tablets, powders and liquids, thus avoiding mechanical changes during sample preparation, which could alter the physicochemical properties of the formulation. However, although Raman spectroscopy shows

6. Quantitative aspects
Skoulika and co-workers quantitatively determined methyl-parathion [56], fenthion [57] and diazinon [58] in pesticide formulations and ciprooxacin [27] in pharmaceutical solid dosage forms. They used FT-Raman spectroscopy based on band-intensity or band-area measurements. Band intensities and band areas were calculated employing a two-point baseline correction. Quantitative analysis by Raman

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advantages over the more traditional IR spectroscopic techniques, it should not be considered as the analytical technique to solve most problems, but as one more powerful, albeit expensive, technique that is part of a multidisciplinary approach to analysis.

References
[1] C.V. Raman, K.S. Krishnan, Nature 501 (1928) 3048. [2] D.P. Strommen, in: F.A. Settle (Editor), Handbook of Instrumental Techniques for Analytical Chemistry, Prentice Hall PTR, Upper Saddle River, NJ, USA, 1997, p. 285. [3] R.L. McCreery, Raman Spectroscopy for Chemical Analysis, Wiley-Interscience, Chichester, West Sussex, UK, 2000. [4] C.J.L. Constantino, T. Lemma, P.A. Antunes, R. Aroca, Anal. Chem. 73 (2001) 3674. [5] S. Farquharson, W.W. Smith, R.M. Carangelo, C.R. Brouillette, Proc. SPIE-Int. Soc. Opt. Eng. 3859 (1999) 14. [6] A. Ruperez, J.J. Laserna, Anal. Chim. Acta 335 (1996) 87. [7] J.P. Pestaner, F.G. Mullick, J.A. Centeno, J. Forensic Sci. 41 (1996) 1060. [8] T.H. King, C.K. Mann, T.J. Vickers, J. Pharm. Sci. 74 (1985) 443. [9] N. Calvo, R. Montes, J.J. Laserna, Anal. Chim. Acta 280 (1993) 263. [10] E.A. Cutmore, P.W. Skett, Spectrochim. Acta 49 (1993) 809. [11] S.E.J. Bell, D.T. Burns, A.C. Dennis, J.S. Speers, Analyst 125 (2000) 541. [12] A.G. Ryder, G.M. OConnor, T.J. Glynn, J. Forensic Sci. 44 (1999) 1013. [13] R.A. Sulk, R.C. Corcoran, K.T. Carron, Appl. Spectrosc. 53 (1999) 954. [14] H. Tsuchihashi, M. Katagi, M. Nishikawa, M. Tatsuno, H. Nishioka, A. Nara, E. Nishio, C. Petty, Appl. Spectrosc. 51 (1997) 1796. [15] B.A. Dawson, D.B. Black, T.D. Cyr, J.C. Ethier, A.W. By, G.A. Neville, H.F. Shurvell, Can. J. Anal. Sci. Spectrosc. 42 (1997) 84. [16] A. Ruperez, R. Montes, J. Laserna, J. Vib. Spectrosc. 43 (1991) 67. [17] C.M. Hodges, P.J. Hendra, H.A. Willis, T. Farley, J. Raman Spectrosc. 20 (1989) 745. [18] E.N. Lewis, V.F. Kalasinsky, I.W. Levin, Anal. Chem. 60 (1988) 2306. [19] A.M. Tudor, C.D. Melia, J.S. Binnis, P.J. Hendra, S. Church, M.C. Davies, J. Pharm. Biomed. Anal. 8 (1990) 717. [20] R.C. Schweitzer, A.S. Bangalore, P.J. Treado, Managing Mod. Lab. 5 (2000) 7. [21] C. Wang, T.J. Vickers, C.K. Mann, J. Pharm. Biomed. Anal. 16 (1997) 87. [22] C.G. Kontoyannis, M. Orkoula, Talanta 41 (1994) 1981.

[23] T.M. Niemczyk, M.M. Delgado-Lopez, F.S. Allen, Anal. Chem. 70 (1998) 2762. [24] T.M. Niemczyk, M.M. Delgado-Lopez, F.S. Allen, J.T. Clay, D.L. Arneberg, Appl. Spectrosc. 52 (1998) 513. [25] C.G. Kontoyannis, J. Pharm. Biomed. Anal. 13 (1995) 73. [26] G. Jalsovszky, O. Egyed, S. Holly, B. Hegedus, Appl. Spectrosc. 49 (1995) 1142. [27] S.G. Skoulika, C.A. Georgiou, Appl. Spectrosc. 55 (2001) 1259. [28] A.G. Ryder, G.M. Connor, T.J. Glynn, J. Raman Spectrosc. 31 (2000) 221. [29] S. Sands, I.P. Hayward, T.E. Kirkbride, R. Bennett, R.J. Lacey, D.N. Batchelder, J. Forensic Sci. 43 (1998) 509. [30] A.P. Gamot, G. Vergoten, G. Fleury, Talanta 32 (1985) 363. [31] M.C. Davies, J.S. Binns, C.D. Melia, P.J. Hendra, D. Bourgeois, S.P. Church, P.J. Stephenson, Int. J. Pharm. 66 (1990) 223. [32] T.D. Cyr, B.A. Dawson, G.A. Neville, H.F. Shurvell, J. Pharm. Biomed. Anal. 14 (1996) 247. [33] A.P. Gamot, G. Vergoten, M. Saudemon, G. Fleury, J. Barbillat, Talanta 33 (1986) 295. [34] A.P. Gamot, G. Vergoten, M. Saudemon, G. Fleury, Talanta 32 (1985) 373. [35] J. Breitenbach, W. Schrof, J. Neuman, J. Pharm. Res. 16 (1999) 1109. [36] I. Jedvert, M. Josefson, F. Langkilde, J. Near Infrared Spectrosc. 6 (1998) 279. [37] G.A. Neville, H.D. Beckstead, H.F. Shurvell, Can. J. Appl. Spectrosc. 37 (1992) 18. [38] T. Pal, V.A. Narayanan, D.L. Stokes, T. Vo-Dinh, Anal. Chim. Acta 368 (1998) 21. [39] A. Bertoluzza, P. Taddei, M. Zanol, P. Ventura, Spectrosc. Biol. Mol.: New Dir., 8th Eur. Conf.; Kluwer Academic Publishers, Dordrecht, The Netherlands, 1999, p. 605. [40] G.A. Neville, H.D. Beckstead, H.F. Shurvell, J. Pharm. Sci. 81 (1992) 114. [41] V.A. Narayanan, N.A. Stump, G.D. Del-Cul, T. Vo-Dinh, J. Raman Spectrosc. 30 (1999) 435. [42] W.S. Sutherland, J.J. Laserna, M.J. Angebranndt, J.D. Winefordner, Anal. Chem. 62 (1990) 689. [43] A. Ruperez, J.J. Laserna, Talanta 44 (1997) 213. [44] R. Perez, A. Ruperez, J.J. Laserna, N. Felidj, B. Laassis, J.J. Aaron, J. Aubard, Anal. Chim. Acta 369 (1998) 197. [45] C. Rodriguez, Proc. SPIE-Int. Soc. Opt. Eng. 3608 (1999) 37. [46] F.C. Clarke, M.J. Jamieson, D.A. Clark, S.V. Hammond, R.D. Jee, A.C. Moffat, Anal. Chem. 73 (2001) 2213. [47] R.L. McCreery, A.J. Horn, J. Spencer, E. Jefferson, J. Pharm. Sci. 87 (1998) 1. [48] J.J. Andrew, M.A. Browne, I.E. Clark, T.M. Hancewicz, A. Millichope, J. Appl. Spectrosc. 52 (1998) 790. [49] C.L. Armstrong, H.G. Edwards, D.W. Farwell, A.C. Williams, Vib. Spectrosc. 11 (1996) 105. [50] W.P. Findlay, D.E. Bugay, J. Pharm. Biomed. Anal. 16 (1998) 921. [51] F.W. Langkilde, J. Sjoblom, L. Tekenbergs-Hjelte, J. Mrak, J. Pharm. Biomed. Anal. 15 (1997) 687. [52] F.W. Langkilde, A.J. Svantesson, Pharm. Biomed. Spectrosc. 13 (1995) 409.

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[53] S.P. Mulvaney, C.D. Keating, Anal. Chem. 72 (2000) 145R. [54] K.M. Spencer, R.B. Edmonds, R.D. Rauh, M.M. Carrabba, Anal. Chem. 66 (1994) 1269. [55] K.M. Spencer, R.B. Edmonds, R.D. Rauh, Appl. Spectrosc. 50 (1996) 681.

[56] S.G. Skoulika, C.A. Georgiou, Appl. Spectrosc. 54 (2000) 747. [57] S.G. Skoulika, C.A. Georgiou, M.G. Polissiou, Appl. Spectrosc. 53 (1999) 1470. [58] S.G. Skoulika, C.A. Georgiou, M.G. Polissiou, Talanta 51 (2000) 599.