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7.2. Infrared and Raman spectroscopy
7.2. Infrared and R a m a n Spectroscopy
Bd.la Heged{_.is
7.2.1. Introduction The last decades gave rapid development in the sensitivity and efficiency of chromatographic procedures as well as in such highly sophisticated structure elucidating techniques as NMR and MS. This development toppled the previous monopoly of vibrational and UV-VIS spectroscopy in organic chemical research. However, vibrational spectroscopy is regaining prestige due to its specific utility in the fast-spreading morphological research of bulk drugs: it is probably the most straightforward method that provides the kind of spectral fingerprint of the molecule that is sensitive to morphological differences stemming from differences in hydrogen bonds or the rotational hindrance of a group. There are of course exceptions where vibrational spectra show no difference between modifications. For example the polymorphism of enalapril maleate [1] comes from the different packing parameters of conformationally identical molecules. In such cases the application of other instrumental techniques like X-ray diffraction or ~3C-NMR solid state spectroscopy are recommended. Over the last few years FT-Raman spectroscopy has been rapidly gaining weight besides the long-established and universally employed FT-IR spectroscopy. Since FT-IR and FT-Raman spectroscopy are both vibrational methods, but utilise different selection rules, they are useful complementary tools as is reflected by their growing simultaneous application in morphological research [2-4].
7.2.2. FT-IR Measurements
7.2.2.1. Standard FT-IR Spectroscopy The principles and manual methods of the preparation of infrared spectra with proper spectral resolution and intensities using both the KBr and nujol technique are described in the European Pharmacopoeia 1997 [5]. Such spectra are needed for the examination of the morphological purity of bulk drugs. In
Estimation of Polymorphic Modifications as Impurities in Drugs
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this section we only touch upon those aspects of FT-IR where the application of the standard methodology can give misleading or false results and therefore a specialised sample preparation procedure must be employed. (a) The sample is hygroscopic. Bulk drug substances are often salts with a hygroscopic character. These materials are rather unfavourable in respect of controlling their morphological purity by infrared spectroscopy. First, the salt character of the sample generally broadens the infrared absorption bands. Secondly, the broad diffuse absorption band of protonated nitrogens covers the infrared spectrum in the region of 3000-2000 cm -1. In this case the pellet must be prepared under flushing dry nitrogen and measurement must commence as soon as possible. The application of nujol mull can be another means to obtain IR spectra of such materials, but in such cases the strong absorption bands of the suspending nujol will cover other parts of the spectrum in the 3000-2800 cm -1 and 1500-1300 cm -1 regions. (b) The morphological composition of the sample changes under mechanical treatment. The recrystallisation of metastable modifications into more stable forms provoked by mechanical influences is well known among bulk drug substances. For example, cefixamine trihydrate turns into a mixture of crystalline and amorphous forms on grinding [6]. Similarly, cyclophosphamide monohydrate looses its crystal water and changes its morphological composition on such mechanical treatments as grinding and tabletting [7]. To take an example from our own experience, modification C of mebendazole is a widely used drug in the veterinary practice. In the presence of traces of modification A, modification C tends to recrystallise into the more stable, but inefficient modification A. The latter reaches a well detectable level over a 1-2-min stirring period which is normally needed for the preparation of a homogeneous KBr pellet. If the usual amount of sample is increased 4-5fold and the homogenising procedure is reduced to a slight agitation with the tip of a spatula, any undesired changing of the sample can be avoided. The above examples involve interconversions between existing modifications, but it is important to note that mechanical treatment may also cause pseudo-polymorphism in materials which have secondary structures in the solid state, like proteins, hyaluronates, gelatines etc. [8]. As the destruction of the secondary structure is a random phenomenon, the same sample can give different spectra as a function of stirring time and local pressure values. These problems suggest the application of other methods instead of the usual infrared method. (c) The morphological features of the sample change without mechanical influences. Unstable samples may change morphologically with some statistical probability which depends on the light and heat influences reaching the sample [9]. Thus an undesired transition of the sample is more likely to occur as the time between sample preparation and measurement increases. Such a
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procedure was detected and followed by the combined use of IR and DSC in the case of six interconverting mexiletine hydrochloride modifications [10]. The time-dependent spontaneous recrystallisation of amorphous indomethacin was reported by Byrn et al. [6]. This uncertainty is one of the reasons behind the fast spread of in situ techniques (see NIR) which do not require any sample preparation. A rare phenomenon we had the fortune to encounter in our practice involves morphological change induced by the infrared radiation of the FTIR instrument [ 11]. A KBr pellet containing a relatively loose crystal-hydrate modification of cimetidine was left in the infrared beam of the instrument for some hours, while its spectrum was repeatedly recorded. As a result of this prolonged irradiation the monohydrate modification of cimetidine turned quantitatively into form A.
7.2.2.2. Other Techniques The rapid and diverse developments in instrumental analytical methods have also affected infrared spectroscopy, particularly in the spread of different reflection and combined methods. The basic principle of the ATR (attenuated total reflectance) technique, first applied by Fahrenfort in 1961 [12] relies on the phenomenon that in the course of total reflection the reflecting beam penetrates to some lxm depth into the material touching the outer surface of the reflecting plate. This coaction of the infrared radiation with the outer material results in the attenuation of light. In practice the measurement can be performed with the help of a special accessory, consisting of a quite large ZnSe (or other special material) crystal with a perfectly planparallel flat measuring surface and an inlet and an outlet plate of 45 ~ As the above depicted coaction attenuates the measuring beam to an extent that depends on the length of the coated measuring crystal, the light leaving the crystal shows a wavelength-intensity profile very similar (but not identical) to the usual infrared spectrum in the analytical region (4000-400 cm-1). The above-noted problem with the recrystallisation of metastable formulations during sample preparation can be easily circumvented in this way, as this examination requires no other sample preparation than the setting of the sample on the measuring crystal. The ATR method is a powerful tool for the examination of bulks, liquids and ointments. The examination of the morphological composition of ganciclovir bulk samples with the help of the ATR technique was reported by Salari et al. [13]. In a paper of Markovich et al. [9] the examination of the solid state amorphous to crystalline transition of an azetidinone derivative drug substance was described. The ATR method proved to be well applicable in monitoring the slow crystallisation of the active agent.
Estimation of Polymorphic Modifications as Impurities in Drugs
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The DRFTIS (diffuse reflectance Fourier transform infrared spectroscopy) technique is also applicable in the analytical infrared spectrum region. This method was first described by Fuller and Griffith in 1978 [ 14]. Its essence also lies in the attenuation of the infrared beam reflected from the sample. The DRFTIS method is also an easily performed technique, as the sample preparation only requires pouring the sample, or its powdered mixture with KBr, into the sample holder and flattening its surface. This flat surface of the sample has to be irradiated by the beam. The reflected radiation, containing the spectral information, is collected by a special optical arrangement and is worked up with the help of a standard software. This method can be a useful tool in the examination of the morphological composition of a drug substance, as demonstrated by Hartauer et al. [ 15] in the case of sulphamethoxazole. These authors report less than 4% relative differences between the measured and the theoretical values of the morphological composition of the samples. Its ease and rapidity makes the NIR (near infrared reflection) technique [16] an indispensable tool in the fast identification, and morphological purity control of bulk drugs. This method covers the 10 000-4000/cm region of the spectrum, which contains mainly overtones and combination bands. Nevertheless, owing to the computerised handling of the whole spectrum, NIR is a rather sensitive and reliable method. In the last few years combined measuring heads which are equipped with flexible fibre optics gave a further momentum to this technique. Norris et al. [17] report the application of a NIR equipment with an on-line fibre optic probe in the examination of the polymorphic conversion of trovafloxacin mesylate modifications in crystal slurry in organic solvents. The latest infrared microscopes, being the most multifunctional and most sensitive tools in morphological research, should also be mentioned [ 18]. They have a heatable stage and are equipped with a multichannel detector unit which facilitates the examination of the full spectra of separate spots of 20 • 20 txm as the function of temperature and time both in transmission and in reflection. The spread of these instruments in quality control laboratories is expected.
7.2.3. FT-Raman Measurements The principles of Raman spectroscopy [19], named after its Nobel awarded discoverer, Sir Chandrasekhara Venkata Raman, were first published in 1928. This technique which measures vibrational spectral transitions of the atoms and atomic groups of the molecules in the 4000-100 cm -1 spectral region, is an important complementary method to infrared spectroscopy. The widespread use of Raman spectroscopy had for a long time been hindered by various technical obstacles: at the beginning by the lack of sufficient light
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sources, later, when visible light lasers were used, frequently by strong fluorescence. It was the development of a new type of laser (see below), and its application in the well known Michaelson infrared interferometers that gave a real breakthrough in the utility of Raman spectroscopy after 1986. The Nd:YAG (an yttrium aluminium garnet crystal doped with triplyionised neodymium) laser produces a near infrared light beam of 9395 cm - 1 (1064 nm wavelength), and its output is up to 1000 mW power or more. The greatest advantage of this laser comes from its low photon energy, which makes it practically inactive in the field of fluorescence. As the latest instruments are equipped with efficient optical filters cutting off the 105-108 times more intense Rayleigh component from the reflected light, all the technical obstacles were removed, and an efficient method was established to perform measurements based on the well known Fourier transform method. On a modern FT-Raman equipment (very often combined with an FT-IR unit) Raman spectra can be recorded with arbitrary spectral resolution and a large number of scans. Details of the technical realisation and the measurement procedures are discussed in excellent handbooks [19,20]. Although the FT-Raman method has not yet been introduced in the field of quality control of bulk drugs by the pharmacopoeias, in the following some important practical aspects of this method will shortly be noted. Some advantageous features of Raman spectroscopy are as follows. 9 The greatest advantage of Raman spectroscopy lies in its high selectivity in respect of the polarity or polarisability of the given groups. This means that highly polarisable groups, including symmetrical vibrations which are inactive in the infrared spectra, are very good Raman scatterers, while the Raman spectrum is nearly blind in respect of highly polarised groups, like a loose O-H or a N+-H group, which are very intense in the IR spectra, sometimes covering the 3700-2000 cm -1 region. This characteristic gives a fairly flat baseline in the Raman spectrum, consequently baseline correction - in contrast to infrared spectra- is normally unnecessary. 9 Raman bandwidths are smaller than the corresponding infrared band widths. This results in reduced overlaps and more well-separated symmetrical bands, which can be used in the evaluation of the spectra. As a characteristic example the FT-Raman spectra of five polymorphic modifications and a monohydrate of cimetidine are presented in Fig. 7.2.A. 9 A further advantage of FT-Raman spectroscopy is its fast and easy use; sample preparation only requires the filling and positioning of the measuring sample holder. After examination the sample can be regained for further use. Some of the problematic features of Raman spectra are as follows.
Estimation of Polymorphic Modifications as Impurities in Drugs
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Figure 7.2.A. 1700-300 cm -1 region of the FT-Raman spectra of cimetidine modifications A-E [11] and a hydrate H 9 Too strong an irradiating laser beam can easily burn the sample. To avoid this, one has to position the sample to a lower beam power (approx. 100 mW). The setting of the measuring power requires special care, and has to be done step-by-step. During this procedure the analyst should watch the
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baseline at about 3500 cm-J. A lift of the baseline in this region shows that the irradiating beam is too strong, and overheating of the sample has started and a lower power should be used. 9 Between a few very strong bands there are really small peaks in the Raman spectrum. This is only a virtual problem, as the signal to noise ratio, i.e. the baseline reproducibility of the spectrum, can be improved by increasing the number of scans. The above depicted small peaks emerge reproducibly from the baseline and in a properly chosen spectrum window they can be also be analysed. 9 The intensity of the scattered light is very small, approximately 10 -6 fold of the irradiating beam, which can be easily exceeded by the slightest fluorescence that overloads the amplifier of the instrument. A sample contaminated with fluorescent impurities cannot be analysed by Raman spectroscopy; while in the course of a structure elucidating Raman examination one can get rid of the fluorescent contaminant by performing a recrystallisation with clarification, in a morphological examination this is impossible since dissolution of the material destroys the morphological composition.
7.2.4. Quantitative Determination of Morphological Impurities Concerning the quantitative examinations of morphological impurities in bulk drug substances the following should be noted. In the trade of pharmaceutical bulk materials the requirements regarding the level of morphological impurities are usually not specified. In the majority of cases no well-defined limits and procedures are reported in the literature either. In some cases 10%, in other cases only 1-2% morphological impurity is tolerated. In general the tolerated morphological cross-contamination is about 3-5%. The examples given below are taken mostly from the author's experience.
7.2.4.1. Measurements Based on Well-Resolved Bands Obviously, the determination of the morphological composition of a mixture from their FT-IR or FT-Raman spectra rests on the availability of those of the pure modifications. In the simplest cases one or more perfectly resolved and properly intense bands can be found in the spectrum of the contaminant modification which do not overlap with any bands of the main component. Whether such diagnostically useful bands can be found depends on some degree of luck. If a diagnostic band exists, a calibration can be carried out with model mixtures using one well resolved band of the main component as an internal standard for the examination.
Estimation of Polymorphic Modifications as Impurities in Drugs
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For example, modification A offamotidine may contain modification B as an impurity. As seen in Fig. 7.2.B, modification B has a sharp and intense IR band at 3506 cm-1 while the most intense band of modification A can be found at 3452 cm -1 [21]. This band of form A can be used as an internal standard for quantitative examinations. In a spectrum where the noise of the baseline was eliminated by accumulating a properly large number of scans (about 50 scans on the given equipment), 0.5% of modification B can be reliably detected in form A [22]. Cimetidine has six well-reproducible [11] modifications amongst which form A is used in the therapy. Fig. 7.2.A shows the region between 1700 and 300 cm -~ of the Raman spectra of these modifications. The morphological purity of modification A can be checked near 510 cm -~, where modification A has no scattering, while all other possible forms exhibit one or two bands. With the help of model mixtures, 5% of modification B proved to be well detectable in this way.
7.2.4.2. Measurements Using Spectrum Manipulations In everyday practice one can hardly find adequately separated and intense spectral bands in the spectra of the modifications. The strong bands usually overlap with smaller ones whereby morphological impurity usually manifests itself only in the form of slight deformations in the IR or Raman bands of the main component. Such spectra can be evaluated either through spectral subtraction or via derivatisaton. In the case of spectral subtraction the IR spectra of the sample as well as that of the morphologically homogeneous main component, usually presented in transmittance, must be transformed into absorbance, then stored in the proper files of the memory of the computer. Ideally, spectral subtraction results in the perfect elimination of the bands due to the main component, giving a residual low-intensity curve which must be examined whether it contains diagnostic bands due to the contaminating modification. Morphological impurity can be declared only on the positive identification of the most intense 3-4 bands of the contaminating modification in the residual spectrum. It is worth noting that the infrared spectra of solid samples can be slightly influenced both by minor chemical contaminants and by the mechanical circumstances of the preparation of the pellet. This slight deviancy of the spectra sometimes results in sharp and relatively intense peaks in the difference spectra after spectral subtraction. Consideration of these peaks instead of the known main bands of the possible contaminant modification can misguide the spectroscopist. Scanning the second derivative of the infrared spectrum of the morphologically contaminated sample can also be useful: in the second
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Figure 7.2.B. Infrared spectra of famotidine. Curve 1" famotidine A; curve 2: famotidine B; curve 3" famotidine A spiked with 1% of form B
derivative positive peaks detect both the bands of the main component and that of the contaminant modification. Identification of the sharpest and most significant bands of the contaminant can verify the presence of the undesired form. An example is given in Fig. 7.2.C. The quantitation of morphological impurities can be performed by comparing the manipulated spectra with that of identically processed model mixtures.
Estimation of Polymorphic Modifications as Impurities in Drugs
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Figure 7.2.C. Infrared spectra of cimetidine A (1), cimetidine A spiked with 5% B modification (2), cimetidine B (4) and the 2nd derivative spectrum of 2 (3). Maxima and shoulders indicating the presence of modification B are marked with arrows
7.2.4.3. Evaluation of Complete Spectrum Curves with Mathematical Methods There are cases where the modifications exhibit decidedly different spectra, but these spectra do not contain sufficiently strong and well-resolved bands. The examination of morphological purity can then be carried out using curvefitting procedures. Just as in NIR spectroscopy, the same suitable spectral region of the pure modifications and the mixture is selected and stored in
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memory. There are software programs which can compose the mixture spectrum of the sample from the standard spectra of the modifications through least squares curve fitting methods. This procedure gives the relative weights of the composing spectra in the sample of unknown composition. As it is reported by Jalsovszky et al. [23] the calculated composition correlates well with the composition of the model mixture: by applying the partial least squares method, even 2% morphological cross-contamination can be determined with less than 10% relative error.
References 1. J. Anwar, S.E. Tarling and P. Barnes, J. Pharm. Sci. 78, 337-342 (1989) 2. C.M. Deely, R.A. Spragg and T.L. Threlfall, Spectrochim. Acta 47/A, 1217-1223 (1991) 3. W.P. Findlay and D.E. Bugay, J. Pharm. Biomed. Anal. 16, 921-930 (1998) 4. A.M. Tudor, M.C. Davies, C.D. Melia, D.C. Lee, R.C. Mitchell, P.J. Hendra and S.J. Church, Spectrochim. Acta 47/A, 1389-1393 (1991) 5. European Pharmacopoeia 1997, Council of Europe, Strasbourg (1997) 6. S. Byrn, R. Pfeiffer, M. Ganey, C. Hoiberg and G. Poochikian, Pharm. Res. 12, 945-954 (1995) 7. J. Ketakolainen, A. Poso, V. Viitasaari, J. Gynther, J. Pirttim~iki, E. Laine and P. Paronen, Pharm. Res. 12, 299-304 (1995) 8. Unpublished results 9. R.J. Markovich, C.A. Evans, C.B. Coscolluela, S.A. Zibas and J. Rosen, J. Pharm. Biomed. Anal. 16, 661-673 (1997) 10. A. Kiss and J. R6p~si, Analyst 118, 661-664 (1993) 11. B. Hegedfis and S. G6r6g, J. Pharm. Biomed. Anal. 3, 303-313 (1985) 12. J. Fahrenfort, Spectrochim. Acta 17, 698-709 (1961) 13. A. Salari and R.E. Young, Int. J. Pharm. 163, 157-166 (1998) 14. M.P. Fuller and P.R. Griffith, Anal. Chem. 50, 1906-1910 (1987) 15. K.J. Hartauer, E.S. Miller and J.K. Guillory, Int. J. Pharm. 85, 163-174 (1992) 16. A.M.C. Davies and P. Williams, Near Infrared Spectroscopy: The Future Waves, NIR Publications (1996) 17. T. Norris, P.K. Aldridge and S.S. Sekulic, Analyst 122, 549-552 (1997) 18. P. Ghetti, A. Ghidini and R. Stradi, Boll. Chim. Farm. 133, 689-697 (1994) 19. J.R. Ferraro and K. Nakamoto, Introductory Raman Spectroscopy, Academic Press (1994)
Estimation of Polymorphic Modifications as Impurities in Drugs
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20. D.B. Chase and J.F. Rabolt, Fourier Transform Raman Spectroscopy, Academic Press (1994) 21. B. Hegedfis, P. Bod, K. Hars~nyi, I. Pdter, A. Kfilm~n and L. P~rk~nyi, J. Pharm. Biomed. Anal. 7, 563-569 (1989) 22. Under publication 23. G. Jalsovszky, O. Egyed, S. Holly and B. Hegedfis, Appl. Spectrosc. 49, 1142-1145 (1995)
Bd.la Heged{_.is
7.2.1. Introduction The last decades gave rapid development in the sensitivity and efficiency of chromatographic procedures as well as in such highly sophisticated structure elucidating techniques as NMR and MS. This development toppled the previous monopoly of vibrational and UV-VIS spectroscopy in organic chemical research. However, vibrational spectroscopy is regaining prestige due to its specific utility in the fast-spreading morphological research of bulk drugs: it is probably the most straightforward method that provides the kind of spectral fingerprint of the molecule that is sensitive to morphological differences stemming from differences in hydrogen bonds or the rotational hindrance of a group. There are of course exceptions where vibrational spectra show no difference between modifications. For example the polymorphism of enalapril maleate [1] comes from the different packing parameters of conformationally identical molecules. In such cases the application of other instrumental techniques like X-ray diffraction or ~3C-NMR solid state spectroscopy are recommended. Over the last few years FT-Raman spectroscopy has been rapidly gaining weight besides the long-established and universally employed FT-IR spectroscopy. Since FT-IR and FT-Raman spectroscopy are both vibrational methods, but utilise different selection rules, they are useful complementary tools as is reflected by their growing simultaneous application in morphological research [2-4].
7.2.2. FT-IR Measurements
7.2.2.1. Standard FT-IR Spectroscopy The principles and manual methods of the preparation of infrared spectra with proper spectral resolution and intensities using both the KBr and nujol technique are described in the European Pharmacopoeia 1997 [5]. Such spectra are needed for the examination of the morphological purity of bulk drugs. In
Estimation of Polymorphic Modifications as Impurities in Drugs
581
this section we only touch upon those aspects of FT-IR where the application of the standard methodology can give misleading or false results and therefore a specialised sample preparation procedure must be employed. (a) The sample is hygroscopic. Bulk drug substances are often salts with a hygroscopic character. These materials are rather unfavourable in respect of controlling their morphological purity by infrared spectroscopy. First, the salt character of the sample generally broadens the infrared absorption bands. Secondly, the broad diffuse absorption band of protonated nitrogens covers the infrared spectrum in the region of 3000-2000 cm -1. In this case the pellet must be prepared under flushing dry nitrogen and measurement must commence as soon as possible. The application of nujol mull can be another means to obtain IR spectra of such materials, but in such cases the strong absorption bands of the suspending nujol will cover other parts of the spectrum in the 3000-2800 cm -1 and 1500-1300 cm -1 regions. (b) The morphological composition of the sample changes under mechanical treatment. The recrystallisation of metastable modifications into more stable forms provoked by mechanical influences is well known among bulk drug substances. For example, cefixamine trihydrate turns into a mixture of crystalline and amorphous forms on grinding [6]. Similarly, cyclophosphamide monohydrate looses its crystal water and changes its morphological composition on such mechanical treatments as grinding and tabletting [7]. To take an example from our own experience, modification C of mebendazole is a widely used drug in the veterinary practice. In the presence of traces of modification A, modification C tends to recrystallise into the more stable, but inefficient modification A. The latter reaches a well detectable level over a 1-2-min stirring period which is normally needed for the preparation of a homogeneous KBr pellet. If the usual amount of sample is increased 4-5fold and the homogenising procedure is reduced to a slight agitation with the tip of a spatula, any undesired changing of the sample can be avoided. The above examples involve interconversions between existing modifications, but it is important to note that mechanical treatment may also cause pseudo-polymorphism in materials which have secondary structures in the solid state, like proteins, hyaluronates, gelatines etc. [8]. As the destruction of the secondary structure is a random phenomenon, the same sample can give different spectra as a function of stirring time and local pressure values. These problems suggest the application of other methods instead of the usual infrared method. (c) The morphological features of the sample change without mechanical influences. Unstable samples may change morphologically with some statistical probability which depends on the light and heat influences reaching the sample [9]. Thus an undesired transition of the sample is more likely to occur as the time between sample preparation and measurement increases. Such a
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procedure was detected and followed by the combined use of IR and DSC in the case of six interconverting mexiletine hydrochloride modifications [10]. The time-dependent spontaneous recrystallisation of amorphous indomethacin was reported by Byrn et al. [6]. This uncertainty is one of the reasons behind the fast spread of in situ techniques (see NIR) which do not require any sample preparation. A rare phenomenon we had the fortune to encounter in our practice involves morphological change induced by the infrared radiation of the FTIR instrument [ 11]. A KBr pellet containing a relatively loose crystal-hydrate modification of cimetidine was left in the infrared beam of the instrument for some hours, while its spectrum was repeatedly recorded. As a result of this prolonged irradiation the monohydrate modification of cimetidine turned quantitatively into form A.
7.2.2.2. Other Techniques The rapid and diverse developments in instrumental analytical methods have also affected infrared spectroscopy, particularly in the spread of different reflection and combined methods. The basic principle of the ATR (attenuated total reflectance) technique, first applied by Fahrenfort in 1961 [12] relies on the phenomenon that in the course of total reflection the reflecting beam penetrates to some lxm depth into the material touching the outer surface of the reflecting plate. This coaction of the infrared radiation with the outer material results in the attenuation of light. In practice the measurement can be performed with the help of a special accessory, consisting of a quite large ZnSe (or other special material) crystal with a perfectly planparallel flat measuring surface and an inlet and an outlet plate of 45 ~ As the above depicted coaction attenuates the measuring beam to an extent that depends on the length of the coated measuring crystal, the light leaving the crystal shows a wavelength-intensity profile very similar (but not identical) to the usual infrared spectrum in the analytical region (4000-400 cm-1). The above-noted problem with the recrystallisation of metastable formulations during sample preparation can be easily circumvented in this way, as this examination requires no other sample preparation than the setting of the sample on the measuring crystal. The ATR method is a powerful tool for the examination of bulks, liquids and ointments. The examination of the morphological composition of ganciclovir bulk samples with the help of the ATR technique was reported by Salari et al. [13]. In a paper of Markovich et al. [9] the examination of the solid state amorphous to crystalline transition of an azetidinone derivative drug substance was described. The ATR method proved to be well applicable in monitoring the slow crystallisation of the active agent.
Estimation of Polymorphic Modifications as Impurities in Drugs
583
The DRFTIS (diffuse reflectance Fourier transform infrared spectroscopy) technique is also applicable in the analytical infrared spectrum region. This method was first described by Fuller and Griffith in 1978 [ 14]. Its essence also lies in the attenuation of the infrared beam reflected from the sample. The DRFTIS method is also an easily performed technique, as the sample preparation only requires pouring the sample, or its powdered mixture with KBr, into the sample holder and flattening its surface. This flat surface of the sample has to be irradiated by the beam. The reflected radiation, containing the spectral information, is collected by a special optical arrangement and is worked up with the help of a standard software. This method can be a useful tool in the examination of the morphological composition of a drug substance, as demonstrated by Hartauer et al. [ 15] in the case of sulphamethoxazole. These authors report less than 4% relative differences between the measured and the theoretical values of the morphological composition of the samples. Its ease and rapidity makes the NIR (near infrared reflection) technique [16] an indispensable tool in the fast identification, and morphological purity control of bulk drugs. This method covers the 10 000-4000/cm region of the spectrum, which contains mainly overtones and combination bands. Nevertheless, owing to the computerised handling of the whole spectrum, NIR is a rather sensitive and reliable method. In the last few years combined measuring heads which are equipped with flexible fibre optics gave a further momentum to this technique. Norris et al. [17] report the application of a NIR equipment with an on-line fibre optic probe in the examination of the polymorphic conversion of trovafloxacin mesylate modifications in crystal slurry in organic solvents. The latest infrared microscopes, being the most multifunctional and most sensitive tools in morphological research, should also be mentioned [ 18]. They have a heatable stage and are equipped with a multichannel detector unit which facilitates the examination of the full spectra of separate spots of 20 • 20 txm as the function of temperature and time both in transmission and in reflection. The spread of these instruments in quality control laboratories is expected.
7.2.3. FT-Raman Measurements The principles of Raman spectroscopy [19], named after its Nobel awarded discoverer, Sir Chandrasekhara Venkata Raman, were first published in 1928. This technique which measures vibrational spectral transitions of the atoms and atomic groups of the molecules in the 4000-100 cm -1 spectral region, is an important complementary method to infrared spectroscopy. The widespread use of Raman spectroscopy had for a long time been hindered by various technical obstacles: at the beginning by the lack of sufficient light
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sources, later, when visible light lasers were used, frequently by strong fluorescence. It was the development of a new type of laser (see below), and its application in the well known Michaelson infrared interferometers that gave a real breakthrough in the utility of Raman spectroscopy after 1986. The Nd:YAG (an yttrium aluminium garnet crystal doped with triplyionised neodymium) laser produces a near infrared light beam of 9395 cm - 1 (1064 nm wavelength), and its output is up to 1000 mW power or more. The greatest advantage of this laser comes from its low photon energy, which makes it practically inactive in the field of fluorescence. As the latest instruments are equipped with efficient optical filters cutting off the 105-108 times more intense Rayleigh component from the reflected light, all the technical obstacles were removed, and an efficient method was established to perform measurements based on the well known Fourier transform method. On a modern FT-Raman equipment (very often combined with an FT-IR unit) Raman spectra can be recorded with arbitrary spectral resolution and a large number of scans. Details of the technical realisation and the measurement procedures are discussed in excellent handbooks [19,20]. Although the FT-Raman method has not yet been introduced in the field of quality control of bulk drugs by the pharmacopoeias, in the following some important practical aspects of this method will shortly be noted. Some advantageous features of Raman spectroscopy are as follows. 9 The greatest advantage of Raman spectroscopy lies in its high selectivity in respect of the polarity or polarisability of the given groups. This means that highly polarisable groups, including symmetrical vibrations which are inactive in the infrared spectra, are very good Raman scatterers, while the Raman spectrum is nearly blind in respect of highly polarised groups, like a loose O-H or a N+-H group, which are very intense in the IR spectra, sometimes covering the 3700-2000 cm -1 region. This characteristic gives a fairly flat baseline in the Raman spectrum, consequently baseline correction - in contrast to infrared spectra- is normally unnecessary. 9 Raman bandwidths are smaller than the corresponding infrared band widths. This results in reduced overlaps and more well-separated symmetrical bands, which can be used in the evaluation of the spectra. As a characteristic example the FT-Raman spectra of five polymorphic modifications and a monohydrate of cimetidine are presented in Fig. 7.2.A. 9 A further advantage of FT-Raman spectroscopy is its fast and easy use; sample preparation only requires the filling and positioning of the measuring sample holder. After examination the sample can be regained for further use. Some of the problematic features of Raman spectra are as follows.
Estimation of Polymorphic Modifications as Impurities in Drugs
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Figure 7.2.A. 1700-300 cm -1 region of the FT-Raman spectra of cimetidine modifications A-E [11] and a hydrate H 9 Too strong an irradiating laser beam can easily burn the sample. To avoid this, one has to position the sample to a lower beam power (approx. 100 mW). The setting of the measuring power requires special care, and has to be done step-by-step. During this procedure the analyst should watch the
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baseline at about 3500 cm-J. A lift of the baseline in this region shows that the irradiating beam is too strong, and overheating of the sample has started and a lower power should be used. 9 Between a few very strong bands there are really small peaks in the Raman spectrum. This is only a virtual problem, as the signal to noise ratio, i.e. the baseline reproducibility of the spectrum, can be improved by increasing the number of scans. The above depicted small peaks emerge reproducibly from the baseline and in a properly chosen spectrum window they can be also be analysed. 9 The intensity of the scattered light is very small, approximately 10 -6 fold of the irradiating beam, which can be easily exceeded by the slightest fluorescence that overloads the amplifier of the instrument. A sample contaminated with fluorescent impurities cannot be analysed by Raman spectroscopy; while in the course of a structure elucidating Raman examination one can get rid of the fluorescent contaminant by performing a recrystallisation with clarification, in a morphological examination this is impossible since dissolution of the material destroys the morphological composition.
7.2.4. Quantitative Determination of Morphological Impurities Concerning the quantitative examinations of morphological impurities in bulk drug substances the following should be noted. In the trade of pharmaceutical bulk materials the requirements regarding the level of morphological impurities are usually not specified. In the majority of cases no well-defined limits and procedures are reported in the literature either. In some cases 10%, in other cases only 1-2% morphological impurity is tolerated. In general the tolerated morphological cross-contamination is about 3-5%. The examples given below are taken mostly from the author's experience.
7.2.4.1. Measurements Based on Well-Resolved Bands Obviously, the determination of the morphological composition of a mixture from their FT-IR or FT-Raman spectra rests on the availability of those of the pure modifications. In the simplest cases one or more perfectly resolved and properly intense bands can be found in the spectrum of the contaminant modification which do not overlap with any bands of the main component. Whether such diagnostically useful bands can be found depends on some degree of luck. If a diagnostic band exists, a calibration can be carried out with model mixtures using one well resolved band of the main component as an internal standard for the examination.
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For example, modification A offamotidine may contain modification B as an impurity. As seen in Fig. 7.2.B, modification B has a sharp and intense IR band at 3506 cm-1 while the most intense band of modification A can be found at 3452 cm -1 [21]. This band of form A can be used as an internal standard for quantitative examinations. In a spectrum where the noise of the baseline was eliminated by accumulating a properly large number of scans (about 50 scans on the given equipment), 0.5% of modification B can be reliably detected in form A [22]. Cimetidine has six well-reproducible [11] modifications amongst which form A is used in the therapy. Fig. 7.2.A shows the region between 1700 and 300 cm -~ of the Raman spectra of these modifications. The morphological purity of modification A can be checked near 510 cm -~, where modification A has no scattering, while all other possible forms exhibit one or two bands. With the help of model mixtures, 5% of modification B proved to be well detectable in this way.
7.2.4.2. Measurements Using Spectrum Manipulations In everyday practice one can hardly find adequately separated and intense spectral bands in the spectra of the modifications. The strong bands usually overlap with smaller ones whereby morphological impurity usually manifests itself only in the form of slight deformations in the IR or Raman bands of the main component. Such spectra can be evaluated either through spectral subtraction or via derivatisaton. In the case of spectral subtraction the IR spectra of the sample as well as that of the morphologically homogeneous main component, usually presented in transmittance, must be transformed into absorbance, then stored in the proper files of the memory of the computer. Ideally, spectral subtraction results in the perfect elimination of the bands due to the main component, giving a residual low-intensity curve which must be examined whether it contains diagnostic bands due to the contaminating modification. Morphological impurity can be declared only on the positive identification of the most intense 3-4 bands of the contaminating modification in the residual spectrum. It is worth noting that the infrared spectra of solid samples can be slightly influenced both by minor chemical contaminants and by the mechanical circumstances of the preparation of the pellet. This slight deviancy of the spectra sometimes results in sharp and relatively intense peaks in the difference spectra after spectral subtraction. Consideration of these peaks instead of the known main bands of the possible contaminant modification can misguide the spectroscopist. Scanning the second derivative of the infrared spectrum of the morphologically contaminated sample can also be useful: in the second
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%T
3
3600.0
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Figure 7.2.B. Infrared spectra of famotidine. Curve 1" famotidine A; curve 2: famotidine B; curve 3" famotidine A spiked with 1% of form B
derivative positive peaks detect both the bands of the main component and that of the contaminant modification. Identification of the sharpest and most significant bands of the contaminant can verify the presence of the undesired form. An example is given in Fig. 7.2.C. The quantitation of morphological impurities can be performed by comparing the manipulated spectra with that of identically processed model mixtures.
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2
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Figure 7.2.C. Infrared spectra of cimetidine A (1), cimetidine A spiked with 5% B modification (2), cimetidine B (4) and the 2nd derivative spectrum of 2 (3). Maxima and shoulders indicating the presence of modification B are marked with arrows
7.2.4.3. Evaluation of Complete Spectrum Curves with Mathematical Methods There are cases where the modifications exhibit decidedly different spectra, but these spectra do not contain sufficiently strong and well-resolved bands. The examination of morphological purity can then be carried out using curvefitting procedures. Just as in NIR spectroscopy, the same suitable spectral region of the pure modifications and the mixture is selected and stored in
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memory. There are software programs which can compose the mixture spectrum of the sample from the standard spectra of the modifications through least squares curve fitting methods. This procedure gives the relative weights of the composing spectra in the sample of unknown composition. As it is reported by Jalsovszky et al. [23] the calculated composition correlates well with the composition of the model mixture: by applying the partial least squares method, even 2% morphological cross-contamination can be determined with less than 10% relative error.
References 1. J. Anwar, S.E. Tarling and P. Barnes, J. Pharm. Sci. 78, 337-342 (1989) 2. C.M. Deely, R.A. Spragg and T.L. Threlfall, Spectrochim. Acta 47/A, 1217-1223 (1991) 3. W.P. Findlay and D.E. Bugay, J. Pharm. Biomed. Anal. 16, 921-930 (1998) 4. A.M. Tudor, M.C. Davies, C.D. Melia, D.C. Lee, R.C. Mitchell, P.J. Hendra and S.J. Church, Spectrochim. Acta 47/A, 1389-1393 (1991) 5. European Pharmacopoeia 1997, Council of Europe, Strasbourg (1997) 6. S. Byrn, R. Pfeiffer, M. Ganey, C. Hoiberg and G. Poochikian, Pharm. Res. 12, 945-954 (1995) 7. J. Ketakolainen, A. Poso, V. Viitasaari, J. Gynther, J. Pirttim~iki, E. Laine and P. Paronen, Pharm. Res. 12, 299-304 (1995) 8. Unpublished results 9. R.J. Markovich, C.A. Evans, C.B. Coscolluela, S.A. Zibas and J. Rosen, J. Pharm. Biomed. Anal. 16, 661-673 (1997) 10. A. Kiss and J. R6p~si, Analyst 118, 661-664 (1993) 11. B. Hegedfis and S. G6r6g, J. Pharm. Biomed. Anal. 3, 303-313 (1985) 12. J. Fahrenfort, Spectrochim. Acta 17, 698-709 (1961) 13. A. Salari and R.E. Young, Int. J. Pharm. 163, 157-166 (1998) 14. M.P. Fuller and P.R. Griffith, Anal. Chem. 50, 1906-1910 (1987) 15. K.J. Hartauer, E.S. Miller and J.K. Guillory, Int. J. Pharm. 85, 163-174 (1992) 16. A.M.C. Davies and P. Williams, Near Infrared Spectroscopy: The Future Waves, NIR Publications (1996) 17. T. Norris, P.K. Aldridge and S.S. Sekulic, Analyst 122, 549-552 (1997) 18. P. Ghetti, A. Ghidini and R. Stradi, Boll. Chim. Farm. 133, 689-697 (1994) 19. J.R. Ferraro and K. Nakamoto, Introductory Raman Spectroscopy, Academic Press (1994)
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20. D.B. Chase and J.F. Rabolt, Fourier Transform Raman Spectroscopy, Academic Press (1994) 21. B. Hegedfis, P. Bod, K. Hars~nyi, I. Pdter, A. Kfilm~n and L. P~rk~nyi, J. Pharm. Biomed. Anal. 7, 563-569 (1989) 22. Under publication 23. G. Jalsovszky, O. Egyed, S. Holly and B. Hegedfis, Appl. Spectrosc. 49, 1142-1145 (1995)