Chemical Physics Letters 390 (2004) 20–24 Using terahertz pulsed spectroscopy to study crystallinity Clare J. Strachan a, Thomas Rades a, David A. Newnham b, Keith C. Gordon c, a School of Pharmacy, University of Otago, P.O. Box 913, Dunedin 9001, New Zealand b TeraView Limited, 302/304 Cambridge Science Park, Milton Road, Cambridge CB4 0WG, UK c Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand d Cavendish Laboratory, University of Cambridge, Madingley Road, Cambridge CB3 0HE, UK Received 27 February 2004; in final form 25 March 2004 The application of terahertz pulsed spectroscopy to polymorphic, liquid crystalline and amorphous forms of pharmaceutical compounds has been investigated. The different polymorphic forms of carbamazepine and enalapril maleate exhibit distinct tera-hertz absorbance spectra. In contrast to crystalline indomethacin and fenoprofen calcium, amorphous indomethacin and liquidcrystalline fenoprofen calcium show no absorption modes, which is likely to be due to a lack of order. These findings suggest that themodes observed are due to crystalline phonon and possibly hydrogen-bonding vibrations. The large spectral differences betweendifferent forms of the compounds studied is evidence that terahertz pulsed spectroscopy is well-suited to distinguishing crystallinitydifferences in pharmaceutical compounds.
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both situations variation in drug crystallinity must beinvestigated and monitored.
Polymorphism and crystallinity changes are enduring Terahertz radiation ($0.1–3 THz, corresponding to issues in the pharmaceutical industry. Eight out of the 3.3–100 cmÀ1) can induce low frequency bond vibra- ten most commonly prescribed drugs in the United tions, crystalline phonon vibrations, hydrogen-bonding States in 2001 are known to exhibit polymorphism or stretches, torsion vibrations and in gases molecular ro- hydrate formation. Crystallinity variations in a phar- tations [6]. Detection of these modes is likely to yield maceutical substance may exhibit physicochemical dif- rich information when characterising materials. How- ferences that impact at therapeutic, manufacturing, ever, until recently experimental difficulties, especially commercial and legal levels [1–3]. While polymorphism with regard to sources and detectors, inhibited the use of is usually undesired, a metastable polymorphic form or the terahertz regime to investigate material properties.
an amorphous form of a drug may sometimes be used Recent advances are now allowing terahertz technology advantageously, for example to increase solubility of a to be applied to many fields such as the semiconductor, poorly soluble compound or to improve flow properties, medical, defence and space industries [7,8]. In particular important in tablet or capsule manufacturing [4,5]. In terahertz pulsed spectroscopy (TPS), which producesbroadband pulses on the femtosecond time scale, showsseveral application advantages. Its coherent natureprovides high sensitivity with room temperature sources and detectors over a relatively broad frequency range.
Corresponding author. Fax: +44-0-1223-435382.
TPS measurements allow both the absorption coefficient [email protected] (P.F. Taday).
and refractive index of a material to be calculated, and 0009-2614/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.cplett.2004.03.117 C.J. Strachan et al. / Chemical Physics Letters 390 (2004) 20–24 time-resolved studies on the sub-picosecond time scale pharmaceutical compounds, demonstrating the tech- potentially allow insight into dynamic systems [7,8]. In nique’s applicably to a broad range of solid-state forms addition the low energy of terahertz radiation minimises TPS has been applied to biologically active com- pounds, e.g. to illustrate low frequency vibrational modes of amino acids, proteins and DNA [9–11], and todifferentiate isomeric configurations of retinal chromo- phores [12]. Benzoic acid and some monosubstitutedderivatives, including salicylic acid and acetylsalicylic Carbamazepine (5H-dibenz[b, f]azepine-5-carboxam- acid (aspirin) have also been differentiated using TPS ide, purity >99%) and enalapril maleate (N-N-[(S)- 1-ethoxycarbonyl-3-phenyl-propyl]-L-alanyl-L-proline The ability of TPS to probe lattice and hydrogen- hydrogen maleate, purity >99%) were obtained from bonding vibrations [6] makes it an ideal technique to Salutas Pharma GmbH (Barleben, Germany). Feno- investigate pharmaceutical polymorphism and crystal- profen calcium dihydrate (calcium methyl-3-phenoxy- linity. TPS has been used to probe such vibrations in polycrystalline and amorphous saccharides [14]. In a Chemical Manufacturing Corp. (New Jersey, USA).
recent study by Taday et al. [15], two polymorphs of Indomethacin (1-[19]-5-methoxy-2-methylindole-3-ace- ranitidine hydrochloride were shown to give distinct tic acid) was purchased from Sigma Chemical Co. (St.
terahertz spectra, with only one peak at the same fre- Louis, MO, USA). Polyethylene (PE) powder (Inducos quency between 0 and 3 THz. To our knowledge, no 13/1, particle size <80 lm) was obtained from Induchem other studies using TPS to investigate pharmaceutical crystallinity and polymorphism have been published.
Carbamazepine (CBZ), enalapril maleate (EM), in- domethacin (IM) and fenoprofen calcium (FC) (Fig. 1)are pharmaceutical examples that collectively exist in CBZ was supplied as form III (P -monoclinic), and polymorphic, liquid crystalline and amorphous states.
this form was used without further purification. Form I CBZ is known to exist in four [16] and EM in two an- (triclinic) was obtained by heating form III to 170 °C for hydrous crystalline forms [17,18]. IM can be produced in 2 h, as described by Lefebvre et al. [22] and McMahon et both a crystalline and amorphous form [19,20], and FC al. [23]. EM designated as form II was used as received.
may form a crystalline dihydrate or a supercooled Form I of EM was prepared by crystallisation from thermotropic liquid crystalline state [21]. This Letter ethyl acetate in the presence of methanol (3.5% w/w) as extends the work of Taday et al. [15] by investigating the described by Ip et al. [24]. Crystalline FC dihydrate was ability of TPS to differentiate crystalline, amorphous used as received. The supercooled thermotropic liquid and supercooled liquid crystalline forms of these four crystalline form of FC was prepared by heating the Fig. 1. Molecular structures of: (a) carbamazepine; (b) enalapril maleate; (c) indomethacin; (d) fenoprofen calcium.
C.J. Strachan et al. / Chemical Physics Letters 390 (2004) 20–24 crystalline powder in beakers open to air in a preheatedoven for 45 min at 125 °C to remove the water of crystallization [25]. The samples were then brought backto room temperature over silica gel. Crystalline IM (c-form) was used as received. Amorphous IM wasproduced by melting the crystalline form at 165 °C, followed by quench cooling in liquid nitrogen and sub- sequent warming back to room temperature in a dessi- cator over silica gel [20]. All samples were gently ground using a pestle and mortar to reduce particle size as much as possible and therefore minimize Mie scattering, and their solid-state form was confirmed by X-ray powderdiffraction. Samples were stored at 4 °C over silica gel.
Sample tablets were prepared by mixing the phar- maceutical solid powder with PE powder (EM 25% w/w; CBZ 50% w/w or FC and IM 75% w/w in PE) with apestle and mortar using geometric dilution. PE is a Fig. 2. Absorbance spectra of CBZ form III (solid line) and form I tablet binder and diluent with negligible absorption in the terahertz regime. Circular tablets (300 mg, 13 mmdiameter) were formed with a hydraulic press using1 ton compression (Specac Ltd., UK). Samples were also compressed into tablets using 2 and 5 ton compres-sion, with no spectral changes observed for any of the compounds. Samples were prepared and measured intriplicate.
All measurements were made using a TPITM spectra 1000 transmission spectrometer (TeraView Limited, Cambridge, UK). Samples were measured at an instru-ment resolution of 2–3 cmÀ1 over the range from 2 to 75 cmÀ1. Data was acquired and processed using OPUSTM Fig. 3. Absorbance spectra of EM form I (solid line) and form II Figs. 2–5 show the terahertz absorption spectra for the different solid-state forms of CBZ, EM, IM, and FC respectively. It is evident from the figures that differencesin the solid-state forms for all four compounds give rise to marked differences in the terahertz absorption spectrabetween 2 and 75 cmÀ1.
Comparison of the spectra of CBZ forms III and I (Fig. 2) show peaks that are polymorph-distinct. Thespectrum of form III exhibits major peaks at 41, 60 and 68 cmÀ1, and a smaller peak at 47 cmÀ1 while the form I spectrum has prominent peaks at 31, 44, 52 and 70 cmÀ1, with a low intensity peak at 23 cmÀ1. The mid-infrared (IR) and Raman spectra of forms III and I aresimilar. Polymorph sensitive absorptions however, can be observed at 1390 and 1680 cmÀ1 in the IR spectra [16]. Computational studies have shown that these modes are associated with the CONH2 moiety. Both Fig. 4. Absorbance spectra of IM crystalline (solid line) and amor- forms exhibit dimer formation with hydrogen-bonding C.J. Strachan et al. / Chemical Physics Letters 390 (2004) 20–24 It is interesting to note the increasing absorbance for amorphous indomethacin with increasing frequency.
Previous experimental work on both PE and lactose with various particle sizes (data not shown) has dem- onstrated that as particle size approaches the incident radiation wavelength, attenuation of the terahertz ra- diation occurs, as would be expected for Mie scattering.
The average particle size of the crystalline IM sample was very small (<80 lm). As the amorphous IM was formed by quench cooling of the melt, with a solid mass resulting from the process it was necessary to createparticles by grinding this mass with a pestle and mortar.
Care was taken in this process to avoid recrystallisation of the amorphous drug making it difficult to reduce the particle size to the same size range as that of the crys- talline form. It is thus likely that some particles of the Fig. 5. Absorbance spectra of FC crystalline hydrate (solid line) and amorphous form of the drug remained sufficiently large liquid crystalline anhydrate (dashed) 75% in PE.
to induce Mie scattering, causing the absorbance of thesample to increase with wavenumber. However, withlonger wavelengths, larger particle sizes are required for between the CONH2 groups. Differences in the crystal Mie scattering than is the case for shorter wavelength structure associated with this hydrogen-bonding are techniques, and thus it is likely that TPS can toler- likely to be responsible for the differences observed in ate much larger particles before particle size influ- ences the spectra obtained than for example, near-IR EM forms I and II also show pronounced spectral differences (Fig. 3). Form I has peaks which occur at 23, FC was chosen as a further pharmaceutical example 39 and 69 cmÀ1, with form II exhibiting peaks at 20, 27, due to its ability to exist in a thermotropic liquid crys- and 57 cmÀ1. In addition both forms have a common talline state that can be cooled to room temperature.
mode at approximately 44 cmÀ1. Forms I and II of EM The liquid crystalline state has previously been identified exhibit nearly identical mid-IR and Raman spectra. This as hexagonal [21]. The crystalline hydrate exhibited has been attributed to very similar crystal packing, hy- spectral peaks at 17, 27, 52 and approximately 66 cmÀ1.
The liquid crystalline form, however, lacked any distinct However, differences have been observed between the peaks in the spectral region studied. Comparison of the two forms of EM in the far-IR Raman spectra of forms crystalline and liquid crystalline samples suggests that I and II between 25 and 100 cmÀ1 [27]. The TPS results all modes present in the crystalline sample are due to presented in this Letter suggest that the terahertz regime long-range order resulting in phonon modes and/or is better suited than the mid-IR region to differentiate hydrogen-bonding between the water and FC molecules.
polymorphs when dealing with organic crystals showing The liquid crystalline form exists as a hexagonal close- packed thermotropic phase formed by 1.7 nm diameter IM was studied in both the c-crystalline and amor- rods [25]. The absence of any terahertz signature in the phous forms. The crystalline form shows peaks at 41, 50 liquid crystalline form would suggest that either the and 66 cmÀ1. There are no distinct peaks in the spectrum 1.7 nm order is insufficient to sustain a phonon mode in of the amorphous form. Diffuse, unstructured absorp- the spectral region studied or the modes observed in the tion was observed in the amorphous from. A similar crystalline form are a consequence of interactions with observation was made by Walther et al. [14] when the water molecules in the crystal as no solvent mole- comparing polycrystalline and amorphous forms of the cules are present in the thermotropic mesophase.
saccharides glucose, fructose and sucrose. Bertie et al.
[28] observed a broad featureless spectrum of amor-phous ice when comparing crystalline and amorphous forms, however the spectrum only extended down to130 cmÀ1. Such spectra suggest that the observed signals The pharmaceutical examples investigated in this with the crystalline IM are due to intermolecular study show that a variety of different crystalline and vibration modes of long-range order. If the modes amorphous forms of organic molecules are readily dif- present in the crystalline sample were due to intramo- ferentiated by their terahertz absorption spectra. The lecular vibrations, one would also expect to see these absence of distinct modes in the amorphous and liquid crystalline samples suggests that the absorbances in C.J. Strachan et al. / Chemical Physics Letters 390 (2004) 20–24 more ordered samples are due to crystalline phonon [12] M. Walther, B. Fischer, M. Schall, H. Helm, P. Uhd Jepsen, vibrations. Terahertz spectroscopy easily differentiates [13] M. Walther, P. Plochoka, B. Fischer, H. Helm, P. Uhd Jepsen, different crystalline polymorphs, even when the crystal- Biopolymers (Biospectroscopy) 67 (2002) 310.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2002, p. 2996–30000066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.9.2996–3000.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved. 16S rRNA Mutation-Mediated Tetracycline Resistance inMonique M. Gerrits,1 Marcel R. de Zoete,1 Niek L. A. Arents,2Ernst J. Kuipers,1 and Johannes G. Kusters1* Department of Gastroenterology and

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