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Fluorescence Studies of the Dehydration of Cefadroxil Monohydrate*
Fluorescence Studies of the Dehydration of Cefadroxil Monohydrate HARRY G. BRITTAIN Center for Pharmaceutical Physics, 10 Charles Road, Milford, New Jersey 08848
Received 20 November 2006; revised 9 January 2007; accepted 19 January 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20953
ABSTRACT: Although cefadroxil does not exhibit the phenomenon of photoluminescence when dissolved in a fluid medium, the compound has been found to exhibit fluorescence in its solid-state monohydrate crystal form. The monohydrate was found to exhibit complicated photoluminescence, where two different sets of emission spectra could be obtained upon irradiation with an appropriate excitation wavelength. One of these photophysical systems became strongly suppressed when the monohydrate was half-dehydrated, and only one of the photophysical systems could be observed in this hemihydrate. In the fully dehydrated state, both photophysical pathways became almost totally suppressed, so that the nonsolvated cefadroxil became effectively nonfluorescent. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:2757–2764, 2007
Keywords: dehydration; fluorescence spectroscopy; hydrate; physical characterization; polymorphism; solid state
INTRODUCTION It is well established that many compounds of pharmaceutical interest are capable of forming crystal structures where water molecules become incorporated in the crystal lattice. Morris1 has provided a useful categorization of hydrate types, with the two most appropriate to pharmaceutics being the isolated lattice type (water molecules occupying discrete positions in the crystal structure) and the lattice channel type (water molecules located in channels formed in the interior of the crystal). The properties of these hydrate types are congruent with the classification used by Byrn et al.,2 who divided hydrates into those for which dehydraPart 5 in the series, ‘‘Photoluminescence of Pharmaceutical Materials in the Solid State.’’ Correspondence to: Harry G. Brittain (Telephone: 908-9963509; Fax: 908-996-3560; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 2757–2764 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
tion caused a change in the X-ray powder diffraction pattern (i.e., the isolated lattice hydrates) and each of which could be dehydrated without an accompanying change in the X-ray powder diffraction pattern (i.e., the lattice channel hydrates). The dehydration associated with hydrates of thiamine hydrochloride,3 naproxen sodium,4 sulfathiazole and nitrofurantoin,5 azithromycin,6 fluconazole,7 and theophylline5,8 have been studied by the classical techniques of X-ray diffraction and thermal analysis. In many of these works, spectroscopic methodology was also used to obtain even more detailed information about the dehydration mechanisms and about the nature of intermediate species. When the quality of the information derived from studies such as these was sufficient, the kinetics of the dehydration process was evaluated.9–11 Many cephalosporin antibiotics are obtained as lattice channel hydrates, making studies of their dehydration mechanisms somewhat more difficult
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owing to the lack of structural changes associated with the hydration and dehydration processes. The channels in cefazolin sodium have been reported to run perpendicular to the short a-axis of the unit cell, and are approximately ellipsoidal, ˚ and a minor axis of having a long axis of 14.3 A ˚ .12 The sodium ions and the water molecules 7.1 A were found to be disordered in the structure, and thermal displacement of the water caused a contraction along the b-axis. Following its isomorphic dehydration, the a-form of cefazolin sodium was destabilized owing to the disruption of hydrogen-bonding network in the lattice channels.13 Given the difficulty of obtaining unequivocal information on the dehydration of channel hydrates, methodology is continually sought that can yield information about the dehydration processes in lattice channel structures, such as the use of hydrogen/deuterium exchange to evaluate the water dynamics in such structures.14 In a previous work, the utility of solid-state fluorescence spectroscopy was demonstrated as a means to study the formation of carbamazepine dihydrate from its anhydrous Form-III phase in aqueous slurries.15 Molecular fluorescence represents transitions among electronic states that are themselves derived from molecular orbitals, so phase transformations can cause perturbations of the molecular orbitals and hence the fluorescence dynamics. When these perturbations cause specific changes in the fluorescence intensity or maxima in spectral bands, fluorescence spectroscopy can be used to study phase transformations in real time. Cefadroxil, (6R,7R)-7-{[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino}-3-methyl-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, is commercially available as a monohydrate crystal form, although hemihydrate forms are also known.16,17 It has been found that although cefadroxil does not exhibit fluorescence when dissolved in a fluid medium, cefadroxil monohydrate does exhibit moderately weak fluorescence in the solid state. In the present work, the solidstate fluorescence spectroscopy of cefadroxil monohydrate has been studied, and has also been used to study the processes that accompany its thermally induced dehydration. The trends observed in the fluorescence results indicated the existence of an isolatable, half-dehydrated cefadroxil monohydrate, whose X-ray powder diffraction was the same as that of the initial starting material.
EXPERIMENTAL SECTION Materials Cefadroxil monohydrate was obtained from Sigma-Aldrich (Milwaukee, WI), and was recrystallized twice from water before use. Crystallization of the compound was effected from concentrated solutions through an evaporative process, and the product was harvested prior to complete drying of the solution. The phase identity of the crystallized product as the authentic monohydrate was established by means of X-ray powder diffraction. Partially and fully dehydrated products of the recrystallized material were obtained by isothermally heating 600 mg aliquots of crystallized substance to the desired degree of weight loss in an Ohaus MB45 moisture determination system. The physical or spectroscopic properties of the dehydrated products were immediately obtained once the desired degree of dehydration had been attained.
Instrumentation and Techniques X-ray powder diffraction patterns were obtained using a Rigaku MiniFlex powder diffraction system, equipped with a horizontal goniometer in the u/2u mode. The X-ray source was nickel˚ ). filtered Ka emission of copper (1.54056 A Samples were packed into an aluminum holder using a back-fill procedure, and were scanned over the range of 50 to 6 degrees 2u, at a scan rate of 0.5 degrees 2u/min. Using a data acquisition rate of 1 point per second, the scanning parameters equate to a step size of 0.0084 degrees 2u. Calibration of each powder pattern was effected using the characteristic scattering peaks of aluminum at 44.738 and 38.472 degrees 2u. The solid-state fluorescence excitation and emission spectra were obtained for samples packed into 5-mm glass NMR tubes. Low-resolution (bandpass approximately 3 nm) exploratory spectra were obtained on a Perkin-Elmer LS 5B luminescence spectrometer, whose sample compartment was modified to enable measurements to be made on samples contained in the NMR tubes. The liquid cell holder was removed, and replaced by an aluminum block that had a hole drilled through its length to permit kinematic placement of the sample tube. The block had an additional lateral removal of metal that permitted irradiation of the sample and subsequent fluorescence detection at right-angles.
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High-resolution excitation spectra of the solid samples were subsequently obtained on a spectrometer specially constructed for this purpose. Sample excitation was effected by using a 0.5 m monochromator (Spex model 1870) to discriminate the output of a 300 W xenon arc lamp. The grating used in this device was blazed at 300 nm, ruled at 1200 g/mm, and the monochromator was characterized by a dispersion of 1.2 nm/mm slit width. The input and output slits were set at 0.5 mm, which set the resolution of the spectra at 0.6 nm. The fluorescence was allowed to pass through a long-pass filter (1% w/v solution of sodium nitrite in a 1 cm cell) to remove scattering light, and then detected by an end-on photomultiplier tube (Thorn EMI type 6256QB having S-11 response). After current-to-voltage conversion, the spectra were electronically acquired using a data acquisition rate of 1 point per second. High-resolution emission spectra of the solid samples were obtained on a separate spectrometer specially constructed for this purpose. Sample excitation was effected by using a combination of glass and solution filters18 to isolate the desired output of a 250 W xenon arc lamp. The sample was irradiated using front-face excitation, and the fluorescence analyzed by a 0.5 m monochromator (Spex model 1870) having a grating blazed at 500 nm and ruled at 1200 g/mm. The dispersion of this monochromator was 1.2 nm/mm slit width, so with the input and output slits set at 0.5 mm, the resolution of the spectra was 0.8 nm. The fluorescence was detected by an end-on photomultiplier tube (Thorn EMI type 9558QB having S-20 response), and after current-to-voltage conversion, the spectra were electronically acquired using a data acquisition rate of 1 point per second. The time dependence of the fluorescence associated with cefadroxil monohydrate being heated under isothermal conditions was followed using a Turner model 111 fluorimeter that was modified in the following manner. The sample was placed on the operating area of an Instec microscopy hot stage, and isothermally heated at the desired temperature for approximately 1 h. The stage required 30–45 s to reach operating temperature, and the experiment was considered to begin once a constant temperature was achieved. To observe the fluorescence, the sample was irradiated from underneath by the filtered output of a 250 W xenon arc lamp, where the excitation wavelength was isolated using an appropriate combination of glass and solution filters.18 The luminescence was collected above the sample using a front-surface DOI 10.1002/jps
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mirror positioned at a 458 angle so that the fluorescence was reflected onto the entrance slit of the fluorimeter. After being passed through a 425 nm long-pass glass filter, the fluorescence intensity was continually detected by the Turner fluorimeter. The output from the fluorimeter was subsequently acquired using an analog-to-digital converter, at a data acquisition rate of 1 point per second.
RESULTS AND DISCUSSION The crystal structure of the channel monohydrate of cefadroxil has been published in at least two articles19,20 with the substance being reported to crystallize in the orthorhombic P212121 space group. Averaging the results from both articles, the unit cell dimensions can be stated as ˚ , b ¼ 11.216 A ˚ , and c ¼ 14.436 A ˚ . In a ¼ 11.052 A the crystal structure, the cefadroxil molecules exist as zwitterions, and assume a folded conformation that is characteristic of other b-lactam compounds containing a 7b-phenylglycyl side chain. Four cefadroxil molecules are found per unit cell, and the overall crystal packing consists of an intricate hydrogen-bonding network in which the water molecules play an important role and constitute 4.72% of the total weight.
Thermal Analysis and X-Ray Diffraction Studies Samples of cefadroxil monohydrate were isothermally heated at temperatures of 70, 90, and 1108C, and the percent weight loss monitored over a 30 min time period. These data are plotted in Figure 1. Interestingly, the sample heated at 708C did not reach full dehydration, but instead the weight loss leveled off at a value that corresponded to a loss of approximately one-half of the water of hydration. On the other hand, the sample heated at 1108C was fully dehydrated at the end of the heating period. The sample heated at 908C did not achieve full dehydration within the 30-min time. However, this sample did become more than 50% dehydrated and appeared to be trending toward eventual full dehydration. That cefadroxil monohydrate can be classified as a lattice hydrate type is demonstrated in the X-ray diffraction patterns of Figure 2, where it can be noted that all of the scattering peaks present in the pattern of the initial monohydrate are present in the patterns of the half dehydrated and fully
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Figure 1. Isothermal heating profiles of cefadroxil monohydrate, heated at temperatures of 708C (&), 908C (*), and 1108C (*).
dehydrated materials. A closer examination of the patterns, however, reveals the existence of subtle structural changes associated with the dehydration that are revealed in the relative intensities of corresponding peaks. For example, the strong
Figure 2. X-ray powder diffraction patterns of cefadroxil monohydrate (lowest trace), cefadroxil monohydrate approximately 50% dehydrated (middle trace), and fully dehydrated cefadroxil monohydrate (upper trace).
scattering peak at 14.6 degrees 2u undergoes a significant increase in intensity when the initial monohydrate is half dehydrated, but undergoes no change in intensity when the substance is fully dehydrated. On the other hand, the scattering peak at 11.3 degrees 2u undergoes no change in intensity when the initial monohydrate is half dehydrated, but does significantly increase in intensity when the substance is fully dehydrated. The peak at 12.4 degrees 2u decreases in intensity with the 50% dehydration, and then undergoes a small increase in intensity when the sample becomes fully dehydrated. Other scattering peaks (e.g., the scattering peak at 10.1 degrees 2u) do not change intensity at all during the dehydration process. The results of the thermal analysis and X-ray diffraction experiments indicate that while cefadroxil monohydrate is capable of undergoing an isomorphic dehydration, there seems to be an intermediate species that exists when the monohydrate is half dehydrated.
Solid-State Fluorescence Studies The solid-state excitation and emission spectra of luminescent molecules are generally found to be substantially different relative to those measured for the same substance in a condensed or dissolved state.21,22 This effect originates subsequent to the initial excitation process, where rapid intermolecular energy transfer among molecules in their excited states causes the excitation to become delocalized. The effect of this delocalization is to extend the excited state molecular orbital over the molecules involved in the energy transfer. Interactions of this type can result in splitting of the single-molecule energy levels into bundled sets of levels (i.e., Davydov splitting), as well displacement of states to energies lower than those of the free molecule. When such effects are operative, the solid-state luminescence spectroscopy will usually bear little resemblance to the solutionphase spectroscopy, with both the excitation and emission spectra being considerably red-shifted relative to the analogous solution-phase spectra.23 The low-resolution fluorescence excitation and emission spectra of cefadroxil monohydrate are shown in Figure 3. The excitation spectrum was obtained using an emission-monitoring wavelength of 500 nm, and was found to consist of two overlapping bands. The main peak was noted to have a maximum at 392 nm, but a partially
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Figure 3. Low-resolution excitation (emission monitored at 500 nm; solid trace) and emission (excitation at 375 nm; dashed trace) spectra of cefadroxil monohydrate.
resolved shoulder was observable around 360 nm. The emission spectrum was obtained using excitation at 375 nm, and consisted of a band having a maximum at 461 nm. This emission band also appeared to contain an unresolved feature at approximately 540 nm. To investigate the fluorescence spectroscopy in more detail, the excitation and emission spectra were acquired under high-resolution conditions. The excitation of cefadroxil monohydrate using highly resolved incident energy permitted the observation of different emission bands, where excitation at 360 nm led to observation of an emission band having a maximum at 458 nm (see Fig. 4a) and where excitation at 390 nm led to observation of an emission band having a maximum at 539 nm (see Fig. 4b). For the remainder of this discussion, these two spectroscopic patterns will be identified as the EX360-EM460 and the EX390-EM540 systems. It was found that the fluorescence intensity of EX360-EM460 system was approximately twice that of the EX390EM540 system. From these spectroscopic results, it appears that at least two pathways of energy transfer are available to cefadroxil molecules in the monohydrate structure. When cefadroxil monohydrate is irradiated using broadband excitation and the emission is monitored under equivalent lowresolution conditions, both modes of fluorescence DOI 10.1002/jps
Figure 4. (a) High-resolution excitation (emission monitored at 460 nm; solid trace) and emission (excitation at 360 nm; dashed trace) spectra of the cefadroxil monohydrate EX360-EM460 system. Also shown are (b) the high-resolution excitation (emission monitored at 540 nm; solid trace) and emission (excitation at 390 nm; dashed trace) spectra of the cefadroxil monohydrate EX390-EM540 system.
are activated and the resulting spectra represent the sum of these. However, when the wavelength bandwidth of the excitation energy is sufficiently narrowed, the two modes can be activated separately. Since the weight loss cefadroxil monohydrate heated at 708C for 30 min appeared to level off when the sample had lost approximately half of the water in its channels (i.e., weight loss of 2.4%), the high-resolution excitation and emission spectra of this material was obtained. As shown in Figure 5, excitation at 390 nm (i.e., into the EX390-EM540 system) led to observation of an emission band having a maximum at 540 nm. Interestingly, the fluorescence of the EX360EM460 system proved to be barely detectable, while the overall fluorescence intensity of the EX390-EM540 system increased by approximately 35% relative to the intensity of this system in the initial monohydrate sample. These observations provide strong evidence for the dual
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Figure 5. High-resolution excitation (emission monitored at 540 nm; solid trace) and emission (excitation at 390 nm; dashed trace) spectra of half-dehydrated cefadroxil monohydrate.
obtained. For this sample, no fluorescence from the EX360-EM460 system could be detected at all, and the fluorescence associated with the EX390EM540 system was extremely weak. As shown in Figure 6, excitation at 390 nm led to observation of a very weak emission band having a maximum at 540 nm. Using a hot stage luminescence spectroscopy, the evolution of fluorescence intensities as a function of isothermal heating time was followed. Owing to the spectral monitoring at elevated temperatures, the overall fluorescence intensity of all monitored samples was depressed relative to the intensities of samples whose fluorescence was obtained at ambient temperatures. However, it still proved possible to measure the family of intensity curves that have been illustrated in Figure 7. For all three heating temperatures, the intensity of the EX360-EM460 system decreased monotonically to undetectable levels. On the other hand, the intensity of the EX390-EM540 system actually increased during the initial stages of the isothermal heating before eventually decreasing
emission model that was noted for the initial monohydrate sample. Finally, the high-resolution excitation and emission spectra of fully dehydrated cefadroxil monohydrate that had been heated at 1108C for 30 min (i.e., weight loss of approximately 5%) was
Figure 6. High-resolution excitation (emission monitored at 540 nm; solid trace) and emission (excitation at 390 nm; dashed trace) spectra of the fully dehydrated cefadroxil monohydrate.
Figure 7. Fluorescence intensities measured in real time for samples of cefadroxil monohydrated heated isothermally at 70, 90, and 1108C. For each profile, the intensity of the EX360-EM460 system is shown as the solid trace, while the intensity of the EX390EM540 system is shown as the dashed trace.
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Figure 8. Dependence of cefadroxil fluorescence intensities as a function of total weight loss in isothermally heated samples, where full dehydration corresponds to an approximate 5% weight loss. The intensity of the EX360-EM460 system is shown as the solid trace, while the intensity of the EX390-EM540 system is shown as the dashed trace.
to barely detectable levels at the end of the heating process. Correlation of the time evolution of thermally induced weight loss data from Figure 1 with the time evolution of thermally induced changes in fluorescence intensity data of Figure 7 enabled the development of profiles for the dependence of EX360-EM460 and EX390-EM540 system emission intensities on the cefadroxil percentage water content that are shown in Figure 8. The plots clearly show that the emission intensity of the EX360-EM460 system decreases to nearly zero when the weight loss equaled approximately 2.5%, or when the monohydrate has been dehydrated to that of a hemihydrate. At the same time, the emission intensity of the EX390-EM540 system reached its maximal value when the weight loss equaled approximately 2.5% (or that of a hemihydrate), and decreased to nearly zero when the sample had been completely dehydrated to a nonsolvated state.
CONCLUSIONS Although not photoluminescent in its dissolved state, cefadroxil was found to exhibit fluorescence DOI 10.1002/jps
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in the monohydrate crystal form (even when partly dehydrated). The thermal analysis and Xray diffraction studies indicated that cefadroxil monohydrate appeared to undergo a relatively simple thermally induced dehydration, but the solid-state fluorescence spectroscopy studies point toward the existence of a cefadroxil hemihydrate that could be obtained by appropriate thermal dehydration. The existence of cefadroxil hemihydrates has been disclosed,16,17 and the present photoluminescence work has indicated that there is yet another route to the hemihydrate state that was not discernable from the conduct of more traditional methods of preparation. The body of fluorescence spectral results indicates the existence of two major photophysical pathways for delocalization of excitation in the cefadroxil monohydrate crystal, each of which may be selectively activated by irradiation with the proper excitation wavelength. One of these photophysical systems appears to dominate the spectroscopy of the monohydrate, but is eliminated once the monohydrate is dehydrated to a hemihydrate. The other photophysical pathway exists in the monohydrate structure, and becomes the sole mechanism for observable fluorescence once the cefadroxil monohydrate is partially dehydrated to the hemihydrate. In the fully dehydrated state, both photophysical pathways are destroyed, and the cefadroxil becomes effectively nonfluorescent.
REFERENCES 1. Morris KR. 1999. Structural aspects of hydrates and solvates. In: Brittain HG, editor. Polymorphism in pharmaceutical solids. New York: Marcel Dekker. pp 125–181. 2. Byrn SR, Pfeiffer RR, Stowell JG. 1999. Hydrates and solvates. West Lafayette: SSCI Inc. pp. 233–247. 3. Te RL, Griesser UJ, Morris KR, Byrn SR, Stowell JG. 2003. X-ray diffraction and solid-state NMR investigation of the single-crystal to single-crystal dehydration of thiamine hydrochloride monohydrate. Cryst Growth Design 3:997–1004. 4. Kim Y-S, Paskow HC, Rousseau RW. 2005. Propagation of solid-state transformations by dehydration and stabilization of pseudopolymorphic crystals of sodium naproxen. Cryst Growth Design 5:1623–1632. 5. Karjalainen M, Airaksinen S, Rantanen J, Aaltonen J, Yliruusi J. 2005. Characterization of polymorphic solid-state changes using variable temperature
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X-ray powder diffraction. J Pharm Biomed Anal 39: 27–32. Blanco M, Valdes D, Llorente I, Bayod M. 2005. Application of NIR spectroscopy in polymorphic analysis: Study of pseudopolymorph stability. J Pharm Sci 94:1336–1342. Alkhamis KA, Salem MS, Obaidat RM. 2006. Comparison between dehydration and desolvation kinetics of fluconazole monohydrate and fluconazole ethylacetate solvate using three different methods. J Pharm Sci 95:859–870. Nunes C, Mahendrasingam A, Suryanarayanan R. 2006. Investigation of the multi-step dehydration reaction of theophylline monohydrate using 2dimensional powder X-ray diffractometry. Pharm Res 23:2393–2404. Dong Z, Salsbury JS, Zhou D, Munson EJ, Schroeder SA, Prakash I, Vyzovkin S, Wight CA, Grant DJW. 2002. Dehydration kinetics of neotame monohydrate. J Pharm Sci 91:1423–1431. Zhou D, Schmitt EA, Zhang GGZ, Law D, Wight CA, Vyzovkin S, Grant DJW. 2003. Model-free treatment of the dehydration kinetics of nedocromil sodium trihydrate. J Pharm Sci 92:1367–1376. Sheth AR, Zhou D, Muller FX, Grant DJW. 2004. Dehydration kinetics of piroxicam monohydrate and relationship to lattice energy and structure. J Pharm Sci 93:3013–3026. Stephenson GA, Diseroad BA. 2000. Structural relationship and desolvation behavior of cromolyn, cefazolin, and fenoprofen sodium hydrates. Int J Pharm 198:167–177. Mimura H, Gato K, Kitamura S, Kitagawa T, Kohda S. 2002. Effect of water content on the
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solid-state stability in two isomorphic clathrates of cephalosporin: Cefazolin sodium pentahydrate (a form) and FK041 hydrate. Chem Pharm Bull 50:766–770. Ahlqvist MUA, Taylor LS. 2002. Water dynamics in channel hydrates investigated using H/D exchange. Int J Pharm 241:253–261. Brittain HG. 2004. Fluorescence studies of the transformation of carbamazepine anhydrate form-III to its dihydrate phase. J Pharm Sci 93: 375–383. Marsili L. 1990. Solvate of cefadroxil. United States Patent 4,962,195, issued October 9, 1990. Kumar Y, Singh SK. 2002. Process for preparing crystalline cefadroxil hemihydrate from cefadroxil dimethylformamide solvate. United States Patent 6,337,396, issued January 8, 2002. Parker CA. 1968. Photoluminescence of solutions. Amsterdam: Elsevier. pp 186–191. Shin W, Cho SW. 1992. Structure of cefadroxil monohydrate. Acta Cryst C48:1454–1456. Seetharaman J, Rajan SS, Srinivasan R. 1993. Crystal structure of cefadroxil. J Chem Cryst 23: 235–238. McClure DS. 1959. Electronic spectra of molecules and ions in crystals. New York: Academic Press. pp 1–46. Brittain HG. 2006. Luminescence spectroscopy. In: Brittain HG, editor. Spectroscopy of pharmaceutical solids. New York: Taylor and Francis. pp 151–204. Brittain HG, Elder BJ, Isbester PK, Salerno AH. 2005. Solid-state fluorescence studies of some polymorphs of diflunisal. Pharm Res 22:999–1006.
DOI 10.1002/jps
Received 20 November 2006; revised 9 January 2007; accepted 19 January 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20953
ABSTRACT: Although cefadroxil does not exhibit the phenomenon of photoluminescence when dissolved in a fluid medium, the compound has been found to exhibit fluorescence in its solid-state monohydrate crystal form. The monohydrate was found to exhibit complicated photoluminescence, where two different sets of emission spectra could be obtained upon irradiation with an appropriate excitation wavelength. One of these photophysical systems became strongly suppressed when the monohydrate was half-dehydrated, and only one of the photophysical systems could be observed in this hemihydrate. In the fully dehydrated state, both photophysical pathways became almost totally suppressed, so that the nonsolvated cefadroxil became effectively nonfluorescent. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:2757–2764, 2007
Keywords: dehydration; fluorescence spectroscopy; hydrate; physical characterization; polymorphism; solid state
INTRODUCTION It is well established that many compounds of pharmaceutical interest are capable of forming crystal structures where water molecules become incorporated in the crystal lattice. Morris1 has provided a useful categorization of hydrate types, with the two most appropriate to pharmaceutics being the isolated lattice type (water molecules occupying discrete positions in the crystal structure) and the lattice channel type (water molecules located in channels formed in the interior of the crystal). The properties of these hydrate types are congruent with the classification used by Byrn et al.,2 who divided hydrates into those for which dehydraPart 5 in the series, ‘‘Photoluminescence of Pharmaceutical Materials in the Solid State.’’ Correspondence to: Harry G. Brittain (Telephone: 908-9963509; Fax: 908-996-3560; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 2757–2764 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
tion caused a change in the X-ray powder diffraction pattern (i.e., the isolated lattice hydrates) and each of which could be dehydrated without an accompanying change in the X-ray powder diffraction pattern (i.e., the lattice channel hydrates). The dehydration associated with hydrates of thiamine hydrochloride,3 naproxen sodium,4 sulfathiazole and nitrofurantoin,5 azithromycin,6 fluconazole,7 and theophylline5,8 have been studied by the classical techniques of X-ray diffraction and thermal analysis. In many of these works, spectroscopic methodology was also used to obtain even more detailed information about the dehydration mechanisms and about the nature of intermediate species. When the quality of the information derived from studies such as these was sufficient, the kinetics of the dehydration process was evaluated.9–11 Many cephalosporin antibiotics are obtained as lattice channel hydrates, making studies of their dehydration mechanisms somewhat more difficult
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owing to the lack of structural changes associated with the hydration and dehydration processes. The channels in cefazolin sodium have been reported to run perpendicular to the short a-axis of the unit cell, and are approximately ellipsoidal, ˚ and a minor axis of having a long axis of 14.3 A ˚ .12 The sodium ions and the water molecules 7.1 A were found to be disordered in the structure, and thermal displacement of the water caused a contraction along the b-axis. Following its isomorphic dehydration, the a-form of cefazolin sodium was destabilized owing to the disruption of hydrogen-bonding network in the lattice channels.13 Given the difficulty of obtaining unequivocal information on the dehydration of channel hydrates, methodology is continually sought that can yield information about the dehydration processes in lattice channel structures, such as the use of hydrogen/deuterium exchange to evaluate the water dynamics in such structures.14 In a previous work, the utility of solid-state fluorescence spectroscopy was demonstrated as a means to study the formation of carbamazepine dihydrate from its anhydrous Form-III phase in aqueous slurries.15 Molecular fluorescence represents transitions among electronic states that are themselves derived from molecular orbitals, so phase transformations can cause perturbations of the molecular orbitals and hence the fluorescence dynamics. When these perturbations cause specific changes in the fluorescence intensity or maxima in spectral bands, fluorescence spectroscopy can be used to study phase transformations in real time. Cefadroxil, (6R,7R)-7-{[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino}-3-methyl-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, is commercially available as a monohydrate crystal form, although hemihydrate forms are also known.16,17 It has been found that although cefadroxil does not exhibit fluorescence when dissolved in a fluid medium, cefadroxil monohydrate does exhibit moderately weak fluorescence in the solid state. In the present work, the solidstate fluorescence spectroscopy of cefadroxil monohydrate has been studied, and has also been used to study the processes that accompany its thermally induced dehydration. The trends observed in the fluorescence results indicated the existence of an isolatable, half-dehydrated cefadroxil monohydrate, whose X-ray powder diffraction was the same as that of the initial starting material.
EXPERIMENTAL SECTION Materials Cefadroxil monohydrate was obtained from Sigma-Aldrich (Milwaukee, WI), and was recrystallized twice from water before use. Crystallization of the compound was effected from concentrated solutions through an evaporative process, and the product was harvested prior to complete drying of the solution. The phase identity of the crystallized product as the authentic monohydrate was established by means of X-ray powder diffraction. Partially and fully dehydrated products of the recrystallized material were obtained by isothermally heating 600 mg aliquots of crystallized substance to the desired degree of weight loss in an Ohaus MB45 moisture determination system. The physical or spectroscopic properties of the dehydrated products were immediately obtained once the desired degree of dehydration had been attained.
Instrumentation and Techniques X-ray powder diffraction patterns were obtained using a Rigaku MiniFlex powder diffraction system, equipped with a horizontal goniometer in the u/2u mode. The X-ray source was nickel˚ ). filtered Ka emission of copper (1.54056 A Samples were packed into an aluminum holder using a back-fill procedure, and were scanned over the range of 50 to 6 degrees 2u, at a scan rate of 0.5 degrees 2u/min. Using a data acquisition rate of 1 point per second, the scanning parameters equate to a step size of 0.0084 degrees 2u. Calibration of each powder pattern was effected using the characteristic scattering peaks of aluminum at 44.738 and 38.472 degrees 2u. The solid-state fluorescence excitation and emission spectra were obtained for samples packed into 5-mm glass NMR tubes. Low-resolution (bandpass approximately 3 nm) exploratory spectra were obtained on a Perkin-Elmer LS 5B luminescence spectrometer, whose sample compartment was modified to enable measurements to be made on samples contained in the NMR tubes. The liquid cell holder was removed, and replaced by an aluminum block that had a hole drilled through its length to permit kinematic placement of the sample tube. The block had an additional lateral removal of metal that permitted irradiation of the sample and subsequent fluorescence detection at right-angles.
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High-resolution excitation spectra of the solid samples were subsequently obtained on a spectrometer specially constructed for this purpose. Sample excitation was effected by using a 0.5 m monochromator (Spex model 1870) to discriminate the output of a 300 W xenon arc lamp. The grating used in this device was blazed at 300 nm, ruled at 1200 g/mm, and the monochromator was characterized by a dispersion of 1.2 nm/mm slit width. The input and output slits were set at 0.5 mm, which set the resolution of the spectra at 0.6 nm. The fluorescence was allowed to pass through a long-pass filter (1% w/v solution of sodium nitrite in a 1 cm cell) to remove scattering light, and then detected by an end-on photomultiplier tube (Thorn EMI type 6256QB having S-11 response). After current-to-voltage conversion, the spectra were electronically acquired using a data acquisition rate of 1 point per second. High-resolution emission spectra of the solid samples were obtained on a separate spectrometer specially constructed for this purpose. Sample excitation was effected by using a combination of glass and solution filters18 to isolate the desired output of a 250 W xenon arc lamp. The sample was irradiated using front-face excitation, and the fluorescence analyzed by a 0.5 m monochromator (Spex model 1870) having a grating blazed at 500 nm and ruled at 1200 g/mm. The dispersion of this monochromator was 1.2 nm/mm slit width, so with the input and output slits set at 0.5 mm, the resolution of the spectra was 0.8 nm. The fluorescence was detected by an end-on photomultiplier tube (Thorn EMI type 9558QB having S-20 response), and after current-to-voltage conversion, the spectra were electronically acquired using a data acquisition rate of 1 point per second. The time dependence of the fluorescence associated with cefadroxil monohydrate being heated under isothermal conditions was followed using a Turner model 111 fluorimeter that was modified in the following manner. The sample was placed on the operating area of an Instec microscopy hot stage, and isothermally heated at the desired temperature for approximately 1 h. The stage required 30–45 s to reach operating temperature, and the experiment was considered to begin once a constant temperature was achieved. To observe the fluorescence, the sample was irradiated from underneath by the filtered output of a 250 W xenon arc lamp, where the excitation wavelength was isolated using an appropriate combination of glass and solution filters.18 The luminescence was collected above the sample using a front-surface DOI 10.1002/jps
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mirror positioned at a 458 angle so that the fluorescence was reflected onto the entrance slit of the fluorimeter. After being passed through a 425 nm long-pass glass filter, the fluorescence intensity was continually detected by the Turner fluorimeter. The output from the fluorimeter was subsequently acquired using an analog-to-digital converter, at a data acquisition rate of 1 point per second.
RESULTS AND DISCUSSION The crystal structure of the channel monohydrate of cefadroxil has been published in at least two articles19,20 with the substance being reported to crystallize in the orthorhombic P212121 space group. Averaging the results from both articles, the unit cell dimensions can be stated as ˚ , b ¼ 11.216 A ˚ , and c ¼ 14.436 A ˚ . In a ¼ 11.052 A the crystal structure, the cefadroxil molecules exist as zwitterions, and assume a folded conformation that is characteristic of other b-lactam compounds containing a 7b-phenylglycyl side chain. Four cefadroxil molecules are found per unit cell, and the overall crystal packing consists of an intricate hydrogen-bonding network in which the water molecules play an important role and constitute 4.72% of the total weight.
Thermal Analysis and X-Ray Diffraction Studies Samples of cefadroxil monohydrate were isothermally heated at temperatures of 70, 90, and 1108C, and the percent weight loss monitored over a 30 min time period. These data are plotted in Figure 1. Interestingly, the sample heated at 708C did not reach full dehydration, but instead the weight loss leveled off at a value that corresponded to a loss of approximately one-half of the water of hydration. On the other hand, the sample heated at 1108C was fully dehydrated at the end of the heating period. The sample heated at 908C did not achieve full dehydration within the 30-min time. However, this sample did become more than 50% dehydrated and appeared to be trending toward eventual full dehydration. That cefadroxil monohydrate can be classified as a lattice hydrate type is demonstrated in the X-ray diffraction patterns of Figure 2, where it can be noted that all of the scattering peaks present in the pattern of the initial monohydrate are present in the patterns of the half dehydrated and fully
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Figure 1. Isothermal heating profiles of cefadroxil monohydrate, heated at temperatures of 708C (&), 908C (*), and 1108C (*).
dehydrated materials. A closer examination of the patterns, however, reveals the existence of subtle structural changes associated with the dehydration that are revealed in the relative intensities of corresponding peaks. For example, the strong
Figure 2. X-ray powder diffraction patterns of cefadroxil monohydrate (lowest trace), cefadroxil monohydrate approximately 50% dehydrated (middle trace), and fully dehydrated cefadroxil monohydrate (upper trace).
scattering peak at 14.6 degrees 2u undergoes a significant increase in intensity when the initial monohydrate is half dehydrated, but undergoes no change in intensity when the substance is fully dehydrated. On the other hand, the scattering peak at 11.3 degrees 2u undergoes no change in intensity when the initial monohydrate is half dehydrated, but does significantly increase in intensity when the substance is fully dehydrated. The peak at 12.4 degrees 2u decreases in intensity with the 50% dehydration, and then undergoes a small increase in intensity when the sample becomes fully dehydrated. Other scattering peaks (e.g., the scattering peak at 10.1 degrees 2u) do not change intensity at all during the dehydration process. The results of the thermal analysis and X-ray diffraction experiments indicate that while cefadroxil monohydrate is capable of undergoing an isomorphic dehydration, there seems to be an intermediate species that exists when the monohydrate is half dehydrated.
Solid-State Fluorescence Studies The solid-state excitation and emission spectra of luminescent molecules are generally found to be substantially different relative to those measured for the same substance in a condensed or dissolved state.21,22 This effect originates subsequent to the initial excitation process, where rapid intermolecular energy transfer among molecules in their excited states causes the excitation to become delocalized. The effect of this delocalization is to extend the excited state molecular orbital over the molecules involved in the energy transfer. Interactions of this type can result in splitting of the single-molecule energy levels into bundled sets of levels (i.e., Davydov splitting), as well displacement of states to energies lower than those of the free molecule. When such effects are operative, the solid-state luminescence spectroscopy will usually bear little resemblance to the solutionphase spectroscopy, with both the excitation and emission spectra being considerably red-shifted relative to the analogous solution-phase spectra.23 The low-resolution fluorescence excitation and emission spectra of cefadroxil monohydrate are shown in Figure 3. The excitation spectrum was obtained using an emission-monitoring wavelength of 500 nm, and was found to consist of two overlapping bands. The main peak was noted to have a maximum at 392 nm, but a partially
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Figure 3. Low-resolution excitation (emission monitored at 500 nm; solid trace) and emission (excitation at 375 nm; dashed trace) spectra of cefadroxil monohydrate.
resolved shoulder was observable around 360 nm. The emission spectrum was obtained using excitation at 375 nm, and consisted of a band having a maximum at 461 nm. This emission band also appeared to contain an unresolved feature at approximately 540 nm. To investigate the fluorescence spectroscopy in more detail, the excitation and emission spectra were acquired under high-resolution conditions. The excitation of cefadroxil monohydrate using highly resolved incident energy permitted the observation of different emission bands, where excitation at 360 nm led to observation of an emission band having a maximum at 458 nm (see Fig. 4a) and where excitation at 390 nm led to observation of an emission band having a maximum at 539 nm (see Fig. 4b). For the remainder of this discussion, these two spectroscopic patterns will be identified as the EX360-EM460 and the EX390-EM540 systems. It was found that the fluorescence intensity of EX360-EM460 system was approximately twice that of the EX390EM540 system. From these spectroscopic results, it appears that at least two pathways of energy transfer are available to cefadroxil molecules in the monohydrate structure. When cefadroxil monohydrate is irradiated using broadband excitation and the emission is monitored under equivalent lowresolution conditions, both modes of fluorescence DOI 10.1002/jps
Figure 4. (a) High-resolution excitation (emission monitored at 460 nm; solid trace) and emission (excitation at 360 nm; dashed trace) spectra of the cefadroxil monohydrate EX360-EM460 system. Also shown are (b) the high-resolution excitation (emission monitored at 540 nm; solid trace) and emission (excitation at 390 nm; dashed trace) spectra of the cefadroxil monohydrate EX390-EM540 system.
are activated and the resulting spectra represent the sum of these. However, when the wavelength bandwidth of the excitation energy is sufficiently narrowed, the two modes can be activated separately. Since the weight loss cefadroxil monohydrate heated at 708C for 30 min appeared to level off when the sample had lost approximately half of the water in its channels (i.e., weight loss of 2.4%), the high-resolution excitation and emission spectra of this material was obtained. As shown in Figure 5, excitation at 390 nm (i.e., into the EX390-EM540 system) led to observation of an emission band having a maximum at 540 nm. Interestingly, the fluorescence of the EX360EM460 system proved to be barely detectable, while the overall fluorescence intensity of the EX390-EM540 system increased by approximately 35% relative to the intensity of this system in the initial monohydrate sample. These observations provide strong evidence for the dual
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Figure 5. High-resolution excitation (emission monitored at 540 nm; solid trace) and emission (excitation at 390 nm; dashed trace) spectra of half-dehydrated cefadroxil monohydrate.
obtained. For this sample, no fluorescence from the EX360-EM460 system could be detected at all, and the fluorescence associated with the EX390EM540 system was extremely weak. As shown in Figure 6, excitation at 390 nm led to observation of a very weak emission band having a maximum at 540 nm. Using a hot stage luminescence spectroscopy, the evolution of fluorescence intensities as a function of isothermal heating time was followed. Owing to the spectral monitoring at elevated temperatures, the overall fluorescence intensity of all monitored samples was depressed relative to the intensities of samples whose fluorescence was obtained at ambient temperatures. However, it still proved possible to measure the family of intensity curves that have been illustrated in Figure 7. For all three heating temperatures, the intensity of the EX360-EM460 system decreased monotonically to undetectable levels. On the other hand, the intensity of the EX390-EM540 system actually increased during the initial stages of the isothermal heating before eventually decreasing
emission model that was noted for the initial monohydrate sample. Finally, the high-resolution excitation and emission spectra of fully dehydrated cefadroxil monohydrate that had been heated at 1108C for 30 min (i.e., weight loss of approximately 5%) was
Figure 6. High-resolution excitation (emission monitored at 540 nm; solid trace) and emission (excitation at 390 nm; dashed trace) spectra of the fully dehydrated cefadroxil monohydrate.
Figure 7. Fluorescence intensities measured in real time for samples of cefadroxil monohydrated heated isothermally at 70, 90, and 1108C. For each profile, the intensity of the EX360-EM460 system is shown as the solid trace, while the intensity of the EX390EM540 system is shown as the dashed trace.
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Figure 8. Dependence of cefadroxil fluorescence intensities as a function of total weight loss in isothermally heated samples, where full dehydration corresponds to an approximate 5% weight loss. The intensity of the EX360-EM460 system is shown as the solid trace, while the intensity of the EX390-EM540 system is shown as the dashed trace.
to barely detectable levels at the end of the heating process. Correlation of the time evolution of thermally induced weight loss data from Figure 1 with the time evolution of thermally induced changes in fluorescence intensity data of Figure 7 enabled the development of profiles for the dependence of EX360-EM460 and EX390-EM540 system emission intensities on the cefadroxil percentage water content that are shown in Figure 8. The plots clearly show that the emission intensity of the EX360-EM460 system decreases to nearly zero when the weight loss equaled approximately 2.5%, or when the monohydrate has been dehydrated to that of a hemihydrate. At the same time, the emission intensity of the EX390-EM540 system reached its maximal value when the weight loss equaled approximately 2.5% (or that of a hemihydrate), and decreased to nearly zero when the sample had been completely dehydrated to a nonsolvated state.
CONCLUSIONS Although not photoluminescent in its dissolved state, cefadroxil was found to exhibit fluorescence DOI 10.1002/jps
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in the monohydrate crystal form (even when partly dehydrated). The thermal analysis and Xray diffraction studies indicated that cefadroxil monohydrate appeared to undergo a relatively simple thermally induced dehydration, but the solid-state fluorescence spectroscopy studies point toward the existence of a cefadroxil hemihydrate that could be obtained by appropriate thermal dehydration. The existence of cefadroxil hemihydrates has been disclosed,16,17 and the present photoluminescence work has indicated that there is yet another route to the hemihydrate state that was not discernable from the conduct of more traditional methods of preparation. The body of fluorescence spectral results indicates the existence of two major photophysical pathways for delocalization of excitation in the cefadroxil monohydrate crystal, each of which may be selectively activated by irradiation with the proper excitation wavelength. One of these photophysical systems appears to dominate the spectroscopy of the monohydrate, but is eliminated once the monohydrate is dehydrated to a hemihydrate. The other photophysical pathway exists in the monohydrate structure, and becomes the sole mechanism for observable fluorescence once the cefadroxil monohydrate is partially dehydrated to the hemihydrate. In the fully dehydrated state, both photophysical pathways are destroyed, and the cefadroxil becomes effectively nonfluorescent.
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