Article pubs.acs.org/molecularpharmaceutics
Investigating the Recrystallization Behavior of Amorphous Paracetamol by Variable Temperature Raman Studies and Surface Raman Mapping Jagadeesh Babu Nanubolu and Jonathan C. Burley* Laboratory of Biophysics and Surface Analysis, School of Pharmacy, Boots Science Building, University of Nottingham, Nottingham, U.K., NG7 2RD S Supporting Information *
ABSTRACT: In situ Raman spectroscopy and Raman mapping are used to monitor the crystallization of amorphous paracetamol in both covered and uncovered geometries, for which different crystallization pathways have been reported previously. The results suggest that surface crystallization predominates in the uncovered samples, leading to forms I and II, whereas in the covered samples bulk crystallization dominates and leads to form III. KEYWORDS: Raman spectroscopy, variable temperature, amorphous, polymorph, glassy, supercooled liquid, glass transition, peak width, peak position, phase transition, disorder, PCA, MCR, covered, uncovered, surface, bulk, crystallization, Raman mapping
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INTRODUCTION Amorphous dosage forms are of particular interest in the pharmaceutical industry owing to their rapid dissolution rates and enhanced solubility over crystalline forms.1 However they have stability issues whereby they often convert into crystalline forms during drug processing and scale-up.2 A further concern with amorphous substances is that they are known to exist in multiple amorphous states or forms each of them exhibiting varying amounts of randomness or disorder in the solid state.3 Such multiple amorphous forms are routinely observed when different amorphization techniques are employed for the same compound.4 This means that the material produced under melt quenching can be quite different from the material prepared by spray drying or any other methods like milling, cryomilling, freeze-drying or melt extrusion. The different nature of these amorphous substances is reflected in the physical property variations such as water uptake hygroscopic nature, glass transition temperature, recrystallization tendency and also overall stability.5 For example, amorphous trehalose prepared by spray drying technique was shown to absorb more water compared to amorphous trehalose prepared by dehydration.5a © 2012 American Chemical Society
Another interesting example is ursodeoxycholic acid, which showed a large difference in the recrystallization process. Milled amorphous material recrystallized in two days while the meltquenched amorphous material was stable and did not crystallize even after a month’s time.5b,c Sheth et al.6 showed that milling different polymorphs of piroxicam can also lead to significant differences in the resulting amorphous solids. The amorphous material obtained from the alpha crystalline form showed a higher tendency to recrystallize as the alpha form. Similarly, milling the beta crystalline form resulted in amorphous material which preferentially reverts to the beta crystalline form upon heating. The current understanding on this phenomenon is that the material prepared by direct solid state transformation of crystalline to amorphous state can retain the memory of the long-range order of the parent crystalline form, likely via the presence of small seed Received: Revised: Accepted: Published: 1544
January 19, 2012 April 3, 2012 April 27, 2012 April 30, 2012 dx.doi.org/10.1021/mp300035g | Mol. Pharmaceutics 2012, 9, 1544−1558
Molecular Pharmaceutics
Article
crystals7 which are far below the experimental detection limits. Based on this assumption, one may reason out why other techniques like freeze-drying, spray drying or melt quenching, which involve a complete destruction of the crystal structure, frequently produce an amorphous material that shows properties different from that of milled samples.4−6 Amorphous griseofulvin5d is an interesting example to recall in this context which showed a difference of almost 60 °C in the recrystallization temperature. Milled samples recrystallized at 65 °C while melt quenched samples did not recrystallize until the temperature was raised to 131 °C. In view of these substantial differences arising from various amorphous samples, a thorough screen by multiple amorphization techniques or methods is always useful, and in fact suggested, to assess risks associated with the stability, recrystallization and decomposition behavior of drugs prior to finalizing a preferred method and a desired amorphous formulation for the drug development. The glass transition temperature (Tg) is one of the important parameters to understand the thermodynamic stability of a particular amorphous formulation. Below this temperature, the material exists as amorphous solid in the glassy state, and above Tg the material exists as amorphous liquid in the supercooled liquid state.8 In the amorphous solid, vibration is the main degree of freedom, whereas in the amorphous liquid, rotational, translational and torsional degrees of freedom are also available. Recrystallization is therefore much faster in the amorphous liquid than in the amorphous solid. The glass transition temperature can be characterized by thermal methods (DSC, MDSC, DMTA)9−11 and spectroscopic methods (IR, NMR, Dielectric relaxation).12−14 A higher glass transition temperature typically delays the recrystallization process and helps in improving the stability of an amorphous formulation. Nevertheless, a few examples demonstrated crystallization even though they were stored at temperatures below their glass transition temperature, i.e. crystallization was noticed directly from the amorphous solid state. Such cases are known to be observed upon prolonged storage, when the humidity levels are high15 or promoted by surface nucleation phenomena.16 Amorphous nifedipine is one such example which crystallizes well below its glassy transition temperature, but its chemical derivative felodipine is found to be stable below Tg.15a,b Paracetamol (acetaminophen) is a common analgesic. As a pure material, it has been shown to adopt three crystalline forms (polymorphs), in addition to a liquid melt which can be supercooled, and an amorphous solid form.17−19 The order of stability of the solid forms at standard temperature and pressure is form I > form II > form III > amorphous.20,21 Despite its apparently simple chemical structure and well-characterized solid forms, paracetamol exhibits rather peculiar and poorly understood crystallization behavior. Apparently minor changes in the experimental conditions frequently lead to different crystallization pathways.22,23 A recent paper by Qi et al.23 found that slow cooled amorphous paracetamol recrystallized as the metastable form III, while the quench cooled paracetamol crystallized as the stable form I. Furthermore, the pans in which these DSC experiments were carried out had an impact on their crystal transformations. In the case of a hermetically sealed pan, form III simply melted upon heating, whereas in a pinhole pan, a transformation from form III to II occurred followed by melting of II. They reasoned that the thermal history is more important than the subsequent heating rate, which appeared to have limited influence on the recrystallization profile.23
Di Martino et al.22 had earlier reached a similar conclusion on the nature of recrystallization behavior when they could never identify form III of paracetamol other than between the slide and the coverslip during thermomicroscopy experiments or in a closed pan during DSC analysis. A recent article by Perrin et al.17c also indicated the requirement of inert (air-free) conditions for obtaining form III. The necessity of closed conditions seems to be obligatory for the formation of form III. Note however that a pure sample of form III has been prepared from a melt−cool recrystallization route in a solid-state NMR rotor of 4 mm internal diameter (strictly not under inert conditions), which allowed determination of the number of molecules in the asymmetric unit.21 In addition to the above examples which showed recrystallization of amorphous paracetamol to either form III or I, a few researchers18a including ourselves have occasionally noticed the crystallization of form II from amorphous samples. Despite the widespread evidence for the unusual presence of multiple crystallization pathways for amorphous paracetamol in different experimental configurations, there exists in the literature no real theory for why this might be the case. Likewise to the best of our knowledge no experimental evidence exists for any differences in the amorphous solid or liquid states that might account for this strong missing link between minor changes in experimental configuration and the crystallization pathway. The aim therefore of the current work is to begin to fill this gap by spectroscopic probing of paracetamol as it crystallizes from various amorphous samples prepared under different experimental setups. We have employed confocal Raman microscopy for characterization in order to gain direct molecular-level information on this intriguing phenomenon.
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EXPERIMENTAL SECTION Materials and Methods. Paracetamol was purchased from Sigma-Aldrich. The white crystalline powder was characterized by DSC and PXRD. Its melting point at 169 °C and a good resemblance of powder’s XRD pattern to the simulated pattern of HXACAN04 crystal structure from the Cambridge Structural Database24 confirmed it as crystalline form I. There were no extra peaks in the diffraction pattern which confirmed the material to be pure within the detection limits (∼99.5%). Sample Preparation for Variable Temperature and Isothermal Crystallizations. In the covered experimental configuration, approximately 5 mg of paracetamol was sandwiched between a micro slide and a rectangular coverslip of dimensions 20 × 20 mm. The coverslip was gently pressed to ensure that the sample spread uniformly, such that it had good thermal contact with the heating element during experiments. In the uncovered configuration, 5 mg of paracetamol was placed on a micro slide without the coverslip on top. Samples were initially melted at high temperature (190 °C) and were quickly cooled to low temperature (−100 °C, temperature below Tg) or room temperature (25 °C, temperature just above Tg) in different experimental setups with a constant cooling rate of 30 °C min−1, the maximum cooling rate available in this experimental configuration. Melt quenched samples were confirmed as amorphous by low-wavenumber Raman spectra. The amorphous samples were then subjected to crystallization under heating and isothermal conditions. For this, LTS350 thermal stage manufactured by Linkam Scientific Industries Ltd. was used. The stage was designed to operate in the temperature range −196 to 420 °C. Temperatures were controlled with TMS94 temperature controller unit and 1545
dx.doi.org/10.1021/mp300035g | Mol. Pharmaceutics 2012, 9, 1544−1558
Molecular Pharmaceutics
Article
complex spectroscopic data with many variables into fewer principal components projecting all the essential information of the original data. The spectral data of covered and uncovered samples were split into two regions, phonon (20−400 cm−1) and molecular (400 to 3800 cm−1), and PCA was carried out on the split data. The first four principal components (PC1, PC2, PC3 and PC4), which account for 98% of the variance of the data, were analyzed and are presented below. The highest variance (approximately 80−90%) is captured on the first principal component axis followed by a subsequent variance (5−10%) on the second principal axis and so on. The results of PCA are analyzed as loadings which indicate peakwise information and a scores plot that provides changes occurring in the variable temperature experiment. Raman Surface Mapping. To facilitate an ultrafast spectral data acquisition in fraction of seconds, a high speed electron multiplying charge coupled device (EMCCD) detector was used. The enhanced sensitivity of the detector through the electron amplification process significantly reduced the data acquisition times without affecting the signal-to-noise ratio of the spectrum, and a high level coordination was ensured between the high speed detector and the sample stage movement to initiate the ultrafast mapping. The confocal aperture was set to 150 μm for obtaining good spectral and spatial resolution during surface mapping. Covered and uncovered amorphous samples were prepared as discussed before. Mapping was performed at regular time intervals to capture events during the crystallization. For the covered material, a total of 10,201 spectra were collected in 50 min on a sample area of 25000 × 25000 μm with a spatial resolution of 250 μm between spectra and 0.1 s data acquisition time for each spectrum. For the uncovered material, a total of 14,520 spectra were collected in 15 min to map a sample area of 6000 × 6000 μm with a spatial resolution of 50 μm between spectra. The data acquisition time for each spectrum was only 0.01 s. The justification for employing different data acquisition methods (0.1 s for covered and 0.01 s for uncovered) is as follows. In the uncovered sample, Raman scattering signals were directly collected by the lens, however, they had to pass through the coverslip in the covered samples before reaching the lens. As a result, the signal strength was weakened to some extent. To overcome this, longer data acquisition time was required for covered samples. The multivariate curve resolution (MCR) method was employed to deconvolute the spectroscopic data into individual spectral components.25 For this a separate MCR-ALS module (alternating least-squares algorithm)28 was installed in R and routine data processing treatments like median centering and variance scaling were performed. Results of MCR analysis are presented as loadings which indicate peakwise information of each phase and a scores plot that provides the spatial distribution of the corresponding phase in the mapped area. False color images were generated in ctioga2 0.2 version.27b Computations. Gaussian 03 was used for calculating Raman spectra of paracetamol.30a Jobs were submitted to a high performance computing facility provided in the University of Nottingham. The initial geometry was chosen from the crystal structure of paracetamol form I, and it was optimized to the nearest local minimum by DFT calculations at the B3LYP/ 6-31G(d,p) level. The vibrational frequencies were then computed on the energy minimized molecule. The atomic motion of each vibrational mode was visualized in the Gabedit software.30b The calculated Raman spectrum was corrected
LNP94 cooling system attached to it. The precise control of liquid nitrogen flow into the stage enables rapid cooling rates, which are essential to avoid recrystallization during the preparation of amorphous samples. In the variable temperature experiments, the heating rate employed was 1 °C min−1 for achieving each destination temperature and the temperature was held constant during the spectral data collection. The time assigned for each spectrum was set to 2 s, and each spectrum was collected twice to allow automated filtering of cosmic radiation. The spectral range was selected from 20 to 3800 cm−1. In the isothermal conditions, temperature was held constant and Raman spectra were recorded constantly with time progress. The spectral resolution in terms of both intensity counts and peak widths is good. The standard deviation for a given peak in these experiments is noted to be within the acceptable limit of ±0.1 cm−1, and other systematic errors are estimated to be within the typical range for Raman spectroscopy measurements.25 The good agreement between transition temperatures observed by Raman and DSC indicates that any potential effect of laser-induced heating of the sample is negligible.26 Confocal Raman Microscopy. The Raman microscope LabRAM HR was purchased from HORIBA Jobin Yvon. The system is a dispersive Raman spectrometer. It operates in the back scattering configuration. It features the use of high quality edge filters to discard much of the Rayleigh scattering and thereby facilitate access to the low-wavenumber region (
