Bell, SEJ

Boyd, A

Dennis, AC

English

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a report by A  R  B o y d , S  E  J  B e l l  and A  C  D e n n i sAvalon Instruments LtdIn recent years, Raman spectroscopy has enjoyed astrong resurgence in popularity due to advances inthe technology, which gives higher sensitivity, easeof use and lower-cost instruments. In particular, ithas been finding application as a technique forroutine and non-routine analysis within a regulatedpharmaceutical environment. Many differenttechniques are already utilised by the pharmaceuticalindustry, including infrared (IR) spectroscopy,nuclear magnetic resonance (NMR), X-raydiffraction (XRD), high-performance liquidchromatography (HPLC) and thermal methods.Indeed, many of these techniques have beendeveloped in order to offer automated analyses.However, it is now evident that Raman spectroscopyis uniquely placed to support the growing demandsof the pharmaceutical industry, especially in relationto high-throughput screening (HTS), rapid analyses,non-invasive analyses (through sample vials andpackaging), no sample preparation and ease of use.The ‘Raman effect’ involves inelastic scattering oflight – the radiation scattered by molecules is shiftedto a different frequency (energy) than the incidentradiation. This inelastic scattering was first predictedin 1923 by A Smekal1, however it wasn’t until 1928that Sir C V Raman carried out the firstexperiments2, which confirmed the prediction andled to the award of his Nobel prize in 1930. Itreceived considerable attention as a method of non-destructive chemical analysis in the years following itsinitial discovery, however this interest eventuallywaned due to advances in IR spectroscopy. Thecurrent renaissance in Raman has been driven byadvances in the photonics sector, most notably by theintroduction of lasers in the 1960s. More recently,multichannel detectors (initially photodiode arrays(PDAs), but now charge-coupled detectors (CCDs)designed specifically for spectroscopy), moreadvanced laser sources and high-performance opticalfilters and spectrographs are all allowing thedevelopment of ever more sensitive and reliableRaman instruments. This process of innovation andimprovement has pushed backed the boundaries andallowed Raman spectroscopy to become a powerfulanalytical tool that can be accessed by specialists andnon-specialists alike. The technique has always hadthe potential to find wide application in thepharmaceutical industry but it is only now that theinstrumentation has evolved to the stage where itspotential can be realised. Wha t  i s  R aman ?Raman spectroscopy is based on detection of lightthat has been scattered inelastically by a sample (the‘Raman effect’). This article will concentrate on thevibrational data these measurements can give; theoverwhelming majority of studies and applications ofthe technique are vibrational Raman experiments. In general, when light interacts with a substance itdoes so in three main ways: the light may beabsorbed; it may be transmitted through the sampleunchanged; or it may be scattered. Figure 1 illustratesthe general principle of the measurement, which isthat the light from a powerful monochromatic lightsource (invariably a laser) is focused onto the sampleof interest and as many as possible of the photons thatscatter from the sample are collected and dispersed ina spectrometer. The photons that are scatteredelastically (i.e. with no change in their wavelength)comprise the Rayleigh scattering, which is intensebut carries no vibrational information and so isfiltered out of the signal. In addition to the Rayleighscattering, Raman scattering, in which the frequencyof the incident photons changes due to interactionwith the sample, can also be detected. Unfortunately,the Raman scattering comprises a very small fractionof the incident photons, typically 0.0001%, hence theneed for an intense source and sensitive detector. Not all Raman-scattered photons have the samechange in energy. Some photons lose part of theirenergy to give Stokes scattering, while others gainenergy and generate the anti-Stokes signal. Togenerate an anti-Stokes signal, photons mustencounter a vibrationally excited molecule in theRaman Spec t roscopy for Future Drug Di scover yB U S I N E S S  B R I E F I N G :  F U T U R E  D R U G  D I S C O V E R Y1Technology & Services1. A Smekal, Naturwissenschaften, 11, 1923, p. 873.2. C V Raman & K S Krishnan, Nature, 121, 1928, p. 501. sample; Stokes scattering has no such requirement soStokes signals tend to be stronger than anti-Stokes andspectra are normally recorded only on the Stokes(energy loss) side of the spectrum. Stokes photons loseenergy by depositing it into vibrational excitation ofthe sample so that the pattern of energy loss reflectsthe vibrational levels within the sample. In practice,plotting the intensity of Raman-scattered photonsagainst frequency difference between incident andscattered radiation maps the vibrational spectrum ofthe sample. Overall, Raman and IR signals measurethe same vibrational levels (with subtly differentselection rules), but the major difference is that thepresence of a vibrational band is detected in IRmeasurements directly by the absorption of an IRphoton, while in Raman experiments the samevibration causes the scattering of a photon, whichretains most of its energy but shows a small losscorresponding to the same energy absorption by thesample as occurs in IR spectroscopy.Every compound has its own unique Ramanspectrum that can be used for both sampleidentification and quantification. Raman and IRspectroscopy can be used as complementarytechniques because, due to differences in thespectroscopic selection rules, each is sensitive todifferent components of a given sample. Forexample, IR spectroscopy is generally more sensitiveto polar bonds such as O-H stretches, whereasRaman is much more sensitive to vibrations ofcarbon backbone structures and symmetrical bonds,such as C = C groups. Using both techniques tocharacterise a particular substance can provide twiceas much information on its chemical composition asthat obtained by using either technique on its own.Given that inexpensive Fourier-transform (FT) IRabsorption spectrometers are widely available, theadvantages of Raman spectroscopy would need to besignificant to justify changeover. There are indeedmany advantages, the most notable being:• no sample preparation is required – Nujol or KBrmatrices are not used; the laser is simply directedonto the sample; • wet samples or even aqueous solutions can beanalysed because water is a particularly poorRaman scatterer;• in situ analysis is straightforward with no samplepreparation required – Raman can even analysesamples through glass and plastic;• fibre optics up to hundreds of metres in length canbe used for remote analysis;• Raman bands are narrower than those typicallyobserved in mid-IR spectra and can be used morereadily for quantitative analysis;• the technique is suitable for use with both organicand inorganic materials;B U S I N E S S  B R I E F I N G :  F U T U R E  D R U G  D I S C O V E R Y2Technology & ServicesFigure 1Raman Spec t roscopy for Future Drug Di scover y• Raman spectroscopy can measure vibrations fromsymmetrical molecular modes, which arefrequently very weak in IR spectra; and• Raman bands can be related more easily tochemical structure due to the fact thatfundamental modes are measured.Most of the disadvantages of Raman methods arisedirectly from the fact that it is a weak effect, whichleads to the need for intense laser excitation sourcesand sensitive detectors. In the past, this, in turn, ledto relatively high costs for Raman instrumentation,which was one of the main obstacles to thewidespread application of Raman spectroscopy forroutine chemical and biological analyses. However,this situation is now changing, as laser and detectorcosts have fallen significantly, while theirperformance has improved steadily. There are numerous variations on generalexperimental Raman methodology but the mostcommonly encountered variants are dispersive andFT Raman methods. In general, Raman instruments,whether interferometric (FT) or dispersive areconstructed of three basic components: the laserexcitation source; the spectrometer (or energyanalyser); and the detector. FT-Raman typicallyemploys a 1,064nm excitation laser, an inferometer(which encodes the unique frequencies of theRaman spectrum into a single scan) and a single-channel near-infrared (NIR) detector. Incomparison, dispersive systems use visible or near-infrared excitation lasers (488, 532, 633 and ca.785nm are employed most commonly), a grating fordispersion and a multichannel CCD detector.Overall, the dispersive systems are much moresensitive than those based on 1,064nmlaser/inferometer combinations, primarily because ofthe exceptional quantum efficiency of modern CCDdetectors and the enhancement in Raman signal thatoccurs at shorter wavelengths. This increasedsensitivity allows data to be acquired over shortertime periods, which has significant implications forHTS applications. The dispersive instruments are alsoless expensive than FT instruments. A significant factor in considering any Ramanexperiment on unpurified samples is the possibilitythat the sample may give a broad optical emissionsignal. In Raman spectroscopy, such backgroundfluorescence is problematic because the Raman signalis relatively weak, so any other emission comingfrom the sample can drown it out. Problem levels ofbackground fluorescence can arise not only fromsamples that contain known flurorophores, but alsofrom adventitious fluorescent impurities that may bepresent at relatively low (sub-millimolar)concentrations. The problem of sampleluminescence has been recognised for a considerabletime and many different strategies have beenemployed, with reasonable success, to overcome it.These include quenching the luminescence by usingsurface-enhanced Raman spectroscopy (SERS) andB U S I N E S S  B R I E F I N G :  F U T U R E  D R U G  D I S C O V E R Y3Figure 2shifting the excitation wavelength to one that doesnot generate luminescence or gives luminescencethat lies at a different wavelength range to theRaman signal3. Until the 1990s, the only widelyavailable technique was FT Raman, which utilisedlong excitation sources and therefore did not excitethe fluorescence that was observed when usingvisible excitation. Recently, dispersive instruments,such as the example given in Figure 2, with longwavelength (NIR) excitation have begun to findfavour as a compromise, which reduces samplefluorescence in much the same way as FTinstruments, but gives the sensitivity advantagesinherent with multichannel CCD detection.Typically, these use ca. 785nm excitation and aclassical spectrograph, although the advantages ofusing an echelle spectrograph, which allows theentire useful spectral range to be recorded at highresolution with no moving parts, are now beingrecognised and exploited.The final major choice to be made in specifying aRaman spectrometer is whether to opt for amicroscope-based system or use ‘macroscopic’sampling. A wide range of commercial instrumentsbased on both sampling protocols is available and thechoice of a microscope-based system is easy ifsampling of very small (<10µm diameter) is required.If this is not the case, the choice is much morecomplex as it is necessary to balance the increased costand complexity of a microscope-based system (with itspotential for sample damage from the highly focusedlaser source) against typical macroscopic systems,which are simpler, more rugged and less costly andallow sampling of regions ca. 100–200µm diameter.This macroscopic sampling is clearly a technique thathas application for HTS, allowing the user to samplefrom a larger area and providing additional assurance,especially if the samples are inhomogeneous. Clearly,there is no single best option that would satisfy allusers; individual requirements are likely to determinethe optimum choice.R aman  S p e c t r o s c o p y  i n  t h eP h a rma c e u t i c a l  I n d u s t r yRaman spectroscopy has been applied successfully inthe study of various drugs, including pharmaceuticals4,narcotics5 and drug delivery devices6,12. Historically,visible Raman spectroscopy did not find applicationwithin the pharmaceutical industry due to theproblems associated with fluorescence andphotodegradation. FT-Raman yielded spectra outsidethe fluorescent region, although this techniquerequired high laser power, which often resulted insample decomposition. The use of dispersive Ramandid, however, offer an excellent compromise to theseproblems and has found increased utility in this area.The implementation of swift and safe drugdevelopment processes is clearly a priority for thepharmaceutical industry. With current technologyunable to match the pace of drug discovery anddesign, there is an obvious need to address thisbottleneck and provide innovative new technologyto improve and accelerate drug discovery. Already,the use of robotics, HTS and advanced software andcomputers has transformed the industry. However,there will always be a need to improve the efficiencyof such systems and drive costs downwards. Thedevelopment of analytical techniques that push backthe boundaries is therefore critical and should beimplemented to improve quality, offer additionalassurance, increase efficiency and reduce cost. Raman spectroscopy is uniquely placed to supportthe growing demands of the pharmaceutical industry,especially in relation to HTS, rapid analyses(sometimes less than one second per sample) andnon-invasive analyses (through sample vials andpackaging) – all without the need for samplepreparation. Many different techniques are alreadyutilised by the pharmaceutical industry, including IRspectroscopy, NMR, XRD, high-performanceliquid chromatography (HPLC) and thermalmethods. Indeed, many of these techniques havebeen developed in order to offer automated analyses.However, Raman spectroscopy offers a range ofsignificant advantages over these techniques and hasbeen applied in the investigation of pharmaceuticalsamples, including:• rapid identification of raw materials;• analysis of drug mixtures, APIs and excipients;• identification of contaminants in samples;• characterisation of formulated materials; and• process monitoring.In particular, Raman has found application in theanalysis of polymorphs (compounds with more thanone crystalline form)7. Compounds often exist asmore than one polymorph so the properties and,B U S I N E S S  B R I E F I N G :  F U T U R E  D R U G  D I S C O V E R Y4Technology & Services3. S E J Bell, E S O Bourguignon and A Dennis, Analyst, 123, 1998, pp. 1,729–1734.4. L S Taylor, American Pharmaceutical Review, 4, 2001, pp. 60–67.5. S E J Bell, D T Burns, A C Dennis and J S Speers, Analyst, 125, 2000, pp. 541–544.6. A C Dennis, J J McGarvey, D A Woolfson, A O’Grady and D F McCafferty, Proc. of the XVIIth Int. Conf. onRaman Spec., Beijieng, 2000, pp. 1,078–1,079.7. R Hilfiker, J Berghausen, C Marcolli, M Szelagiewicz and U Hofmeier, European Pharmaceutical Review, 2, 2002, 2, pp. 37–43.Raman Spec t roscopy for Future Drug Di scover yimportantly, the stability and reactivity of the drugwill be largely influenced by the existence of thesepolymorphs. If more than one polymorph exists, theRaman spectra will differ, usually quite significantly.In addition, Raman can also be used to distinguishbetween pseudo-polymorphs, where the degree ofhydration differs. Not only does Raman provide arapid method for the analysis of differentpolymorphs, it also allows the work to be carried outwithout additional sample preparation and is non-contact. IR spectroscopy usually requires grinding ofthe sample to form a mull or preparation of a KBrpellet, which may influence the crystalline form ofthe sample. Also, techniques such as XRD and DSCtake much longer when compared with Ramananalyses. Hence, Raman provides a unique platformto discriminate between different polymorphs,which is more rapid, flexible and robust thanexisting analytical techniques. Tetracaine gels are another example of the efficacy ofRaman spectroscopy for drug monitoring or analysis.Tetracaine, in an aqueous gel, provides an effectivepercutaneous anaesthesia of intact healthy skin and hasfound useful application in a bioadhesive transdermalpatch6. Its effectiveness is due to a low melting pointmetastable hydrate in aqueous media. Drug deliveryinvolves Tetracaine undergoing a phase change from asolid to an oily suspension at skin temperature12. Thefunction of Tetracaine is to diffuse through the outerlayers of the skin and desensitise the nociceptorswithin the epidermis. Figure 3(a) shows Raman spectrafor Tetracaine after various periods of exposure toskin. An appropriate internal standard (rutile-TiO2)was introduced into the sample in order to determinethe absolute concentration of the Tetracaine. Adecrease in the Tetracaine band intensity over timewith no change in TiO2 band intensity indicated adrop in the Tetracaine concentration as shown inFigure 3(b). The clinical applications of Tetracaineinclude painless venepuncture and miscellaneoustopical surgical procedures. Despite the success of Raman methods in analysingbulk materials, investigations involving very lowconcentrations were prohibited by the low sensitivity ofnormal Raman methods. However, the developmentof SERS, which involves the analysis of samples thathave been adsorbed to, or interact with, metal surfaces(typically a roughened metal electrode or a metalcolloid) has allowed trace analysis by Raman methodsto be carried out8,9. Under optimised conditions, SERScan provide spectral enhancements of 1014 or even 1015compared with normal Raman scattering10. ForB U S I N E S S  B R I E F I N G :  F U T U R E  D R U G  D I S C O V E R Y5Figures 3a and 3b8. A C Dennis, J J McGarvey and S E J Bell, Proc. of the XVIIth Int. Conf. on Raman Spec., Beijieng, 2000, pp. 682–683.9. Y Ye, J Hu, L He and Y Zeng, Vib. Spec., 20, 1999, pp. 1–4.10. A M Michaels, J Jiang and L Brus, Jn. Phys. Chem., B., 104, 2000, pp. 11,965–11,971.example, it can be used for HTS and detection of lowconcentrations of drug compounds11. These are only a few examples of how Ramanspectroscopy can be exploited for the analysis ofpharmaceuticals. However, what is clear from thiswork is that Raman has now become a veryimportant and powerful analytical tool. Its utilitywithin the pharmaceutical sector is now of particularsignificance, because it can provide selective, rapidHTS and highly sensitive methods of analysis, whichare low cost, non-contact and do not require anytime-consuming sample preparation of the type thatexists with many of the current analytical techniquesavailable. It has evolved in the last decade from acomplex research tool to a fairly user-friendlytechnique that is for everyday use.Raman spectroscopy has the potential to play a keyrole in the pharmaceutical industry and allow a morestreamlined approach to drug development.Undoubtedly, it will have a positive impact in thisarea in the coming years, with the capacity to be usedthroughout the drug development life-cycle. ■B U S I N E S S  B R I E F I N G :  F U T U R E  D R U G  D I S C O V E R Y6Technology & Services11. S E J Bell and S J Spence, Analyst, 126, 2001, pp. 1–3.12. A D Woolfson and D F McCafferty, Int. J. Pharm., 94, 1993, pp. 75–80.Contact InformationAvalon Instruments Ltd 97 Botanic Avenue, Belfast, BT7 1JN Northern Ireland, UKTel: +44 (0)28 9043 9555 Fax: +44 (0)28 9043 9646 e-Mail: a.boyd@avaloninst.com Web: http://www.avaloninst.com

Raman spectroscopy for future drug discovery

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Raman spectroscopy for future drug discovery

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