International Journal of Pharmaceutics 489 (2015) 170–176
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Crystallization speed of salbutamol as a function of relative humidity and temperature Sarah Zellnitz a, * , Olga Narygina b , Christian Resch c , Hartmuth Schroettner d, Nora Anne Urbanetz a a
Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, Graz, Austria PANalytical B.V., Almelo, Lelyweg 1, Almelo, The Netherlands Anton Paar GmbH, Anton-Paar-Straße 20, Graz, Austria d Austrian Centre for Electron Microscopy and Nanoanalysis, TU Graz, Steyrergasse 17/III, Graz, Austria b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 March 2015 Received in revised form 29 April 2015 Accepted 30 April 2015 Available online 2 May 2015
Spray dried salbutamol sulphate and salbutamol base particles are amorphous as a result of spray drying. As there is always the risk of recrystallization of amorphous material, the aim of this work is the evaluation of the temperature and humidity dependent recrystallization of spray dried salbutamol sulphate and base. Therefore in-situ Powder X-ray Diffraction (PXRD) studies of the crystallization process at various temperature (25 and 35 C) and humidity (60%, 70%, 80%, 90% relative humidity) conditions were performed. It was shown that the crystallization speed of salbutamol sulphate and base is a non-linear function of both temperature and relative humidity. The higher the relative humidity the higher is the crystallization speed. At 60% relative humidity salbutamol base as well as salbutamol sulphate were found to be amorphous even after 12 h, however samples changed optically. At 70% and 90% RH recrystallization of salbutamol base is completed after 3 h and 30 min and recrystallization of salbutamol sulphate after 4 h and 1 h, respectively. Higher temperature (35 C) also leads to increased crystallization speeds at all tested values of relative humidity. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Inhalation Powder X-ray diffraction Salbutamol sulphate Salbutamol base Spray drying Amorphicity
1. Introduction Salbutamol, a b2-adrenoreceptor agonist, is a common drug used for the treatment of bronchial asthma via inhalation. In order to reach their target site, the tiny airways of the lung, drug particles administered using Dry Powder Inhalers (DPIs) have to exhibit an aerodynamic diameter of 1 mm–5 mm (Newmann and Clarke, 1983; Telko and Dsc, 2005; Timsina et al., 1994; Zanen et al., 1992). Therefore, the powder has to be micronized before application. One of the methods to obtain micron-sized drug particles for pulmonary drug delivery is spray drying (Chawla et al., 1994; Chougule et al., 2007; Vidgrén et al., 1987; Zhao et al., 2008). Chawla et al. reported that spray drying of salbutamol sulphate on a Buechi 190 mini spray dryer leads to spherical shaped particles sufficiently small in diameter (median diameter 4.5 mm) to be used in dry powder inhalation formulations. Spray drying did not alter salbutamol sulphate chemically but reduced the crystallinity of the resulting drug particles (Chawla et al., 1994). Further, Gorny et al. and Littringer et al. reported that spray drying of salbutamol
* Corresponding author. Tel.: +43 316 873 30981; fax: +43 316 873 1030981. E-mail address: [email protected] (S. Zellnitz). http://dx.doi.org/10.1016/j.ijpharm.2015.04.079 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
sulphate from aqueous solutions results in amorphous particles (Gorny et al., 2007; Littringer et al., 2013). The challenge when working with amorphous products is the stability of the products as there is always the risk of recrystallization to the more stable crystalline state. Crystallization of the amorphous particles may be triggered by exposure to humid air or by heating of the sample (Columbano et al., 2002). Therefore, when working with amorphous particles it is necessary to know the temperature (T) and relative humidity (RH) conditions that trigger recrystallization of the particles. By knowing these critical conditions and controlling the storage conditions, it can be ensured that the particles remain in their amorphous state. Alternatively crystallization of the amorphous particles can be intentionally achieved by exposure to humid air or by heating of the sample (Columbano et al., 2002). Insitu powder XRD is a powerful method to investigate and monitor crystallization processes and to determine crystallization speed, degree of crystallinity and the resulting crystal phases. The study presents an investigation of the impact of relative humidity (RH) and temperature (T) on the crystallization process of spray dried salbutamol sulphate and salbutamol base. The sample conditions in the present study were controlled using a sample conditioning stage with a multi-sample holder offering space for eight samples (MHC-trans Multi-sample
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Humidity Chamber, Anton Paar GmbH, Graz, Austria). As humidity driven changes in pharmaceutical substances are usually slow and extremely sensitive to slight differences of the process parameters, the capability of this sample stage to investigate eight samples in parallel saves time and provides highly comparable results for all samples. 2. Materials and methods
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chute (Vibri, Sympatec, Clausthal-Zellerfeld, Germany). Data evaluation was performed using the software Windox 5 (Sympatec, Clausthal-Zellerfeld, Germany). 2.3.2. Scanning electron microscopy (SEM) Morphology of the spray dried samples was examined using a scanning electron microscope (SEM) (Zeiss Ultra 55, Zeiss, Oberkochen, Germany) operating at 5 kV. Samples were gold palladium sputtered prior to analysis.
2.1. Materials 2.4. Powder X-ray diffraction (PXRD) Crystalline salbutamol sulphate (USP25 quality) and crystalline salbutamol base were purchased from Selectchemie (Zuerich, Switzerland) and used as received. Pure ethanol was obtained from VWR International GmbH (Vienna, Austria). 2.2. Spray drying Amorphous salbutamol sulphate and salbutamol base particles were prepared on a Nano Spray Dryer B-90 (Buechi Labortechnik AG, Flawil, Switzerland) equipped with the long version of the drying chamber. Aqueous salbutamol sulphate solutions used for spray drying were prepared with purified water (TKA Micro Pure UV ultrapure water system, TKA Wasseraufbereitungssysteme GmbH, Niederelbert, Germany). To get particles in the size range of 1 mm–5 mm the spray drying parameters were chosen according to the work of Littringer et al. (2013): sprayhead mesh, 5.5 mm; feed concentration, 7.5% (m/m); inlet temperature, 120 C; spraying intensity, 30% and flow rate, 110 L/min. Salbutamol base was spray dried from pure ethanolic solution. The spray drying parameters were: sprayhead mesh, 5.5 mm; feed concentration, 3% (m/m); inlet temperature, 100 C; spraying intensity, 30% and flow rate, 110 L/min. The spray dried particles were stored in a desiccator over silica gel prior to analysis. The crystalline starting materials as well as the amorphous spray dried products were used as samples in the present study. 2.3. Sample characterization 2.3.1. Laser light diffraction The particle size distribution of the starting materials and the spray dried products was determined by laser light diffraction (Helos/KR, Sympatec, Clausthal-Zellerfeld, Germany). Powder dispersion was achieved with a dry dispersing unit (Rodos/L, Sympatec, Clausthal-Zellerfeld, Germany) at a dispersing pressure of 2.0 bar. Particles were fed into the dispersing unit by a vibrating
For powder X-ray diffraction (XRD) measurements at various temperature and relative humidity conditions a PANalytical empyrean diffractometer (PANalytical B.V., Almelo, Netherlands) equipped with a Cu(Ka) X-ray tube, a focusing X-ray mirror, an Anton Paar MHC-trans multi-sample humidity chamber (Anton Paar GmbH, Graz, Austria) and a PIXcel1D detector. The MHC_trans chamber is designed for XRD measurements in transmission geometry in the range of 2–55 2u. The software packages data collector v5.0, HighScore plus v4.0 and data viewer v1.5 were used for the measurements and data analysis. 2.5. Quantification of the amorphous content of salbutamol sulphate using powder X-ray diffraction 2.5.1. Reference sample set For the quantification of the amorphous/crystalline ratio during crystallization of spray dried salbutamol sulphate reference samples with known amorphous/crystalline ratio were prepared. The pure starting material was considered as 100% crystalline and the pure spray dried product as 100% amorphous. Reference samples were prepared by mixing 25, 50 and 75% (m/m) of the spray dried material with the starting material. From each mixture, two reference samples were prepared. The respective amounts of amorphous spray dried material and crystalline starting material were filled in a vial and stirred/shaked for a few minutes by hand. Sample exposure to humid air was minimized to the smallest possible level during sample preparation. Between experiments, samples were kept in a desiccator over silica gel. In order to ensure a constant irradiated sample volume for the XRD measurements, the amount of material filled in the MHC-trans sample cups was weighed. The representative data set measured from the eight reference samples is shown in Fig. 1. XRD patterns from reference samples of the same composition are nearly identical. This confirms that by loading the same sample amounts in a sample cup, reproducible results can be obtained. The data set, covering 2u range from 6 to 40 (Fig. 1), was used to quantify amorphous/
Fig. 1. Full range XRD patterns from the reference sample set with the same sample weight per sample cup.
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crystalline ratio for the experimental run with spray-dried salbutamol sulphate at 25 C and 70% RH. Two more reference data sets, covering 2u ranges of 15–20 and 18.2–20.2 , were measured (data not shown here) and used for the quantification of amorphous/crystalline ratio for the experimental runs at 25 C at 80% RH and 90% RH, respectively (Table 1). The quantification of the amorphous/crystalline ratio was performed using Partial Least Squares Regression (PLSR) method implemented in the PANalytical HighScore 4.0 package (De Jong, 1993; Degen et al., 2014). The detection limit of in-situ powder XRD is within 0.2–0.3 wt%. The presence of crystalline phase of this wt percentage or above is detected. 2.5.2. Partial least squares regression (PLSR) method PLSR method is a statistical method commonly used in spectroscopy (NIR – near infrared) and nuclear magnetic resonance (NMR) but only recently in the XRD field. When applied to XRD it enables extraction of “hidden” properties directly from XRD patterns. Detailed description of the PLSR method applied to XRD, as well as its implementation in PANalytical software HighScore, is described elsewhere (Degen et al., 2014; Koenig et al., 2014). In the case study PLSR was applied to calculate the amorphous/ crystalline ratio for salbutamol sulphate as a function of T/RH. For each experimental run at 25 C and 70%, 80% and 90% RH a PLSR model using corresponding data set of the reference XRD patterns was optimized and crossed-checked. In Fig. 2 representative optimization plot and obtained PLSR calibration model are shown. This model was used to quantify the amorphous/crystalline ratio for the experimental run at 25 C and 70% RH. As seen from Fig. 2a, the optimal number of PLSR factors was found to be 3 with the scaling mode “standardize” (De Jong, 1993). The root-mean-square error of prediction (RMSEP) was found to be 3.31 wt%. 2.6. Test execution – crystallization of spray dried salbutamol sulphate and salbutamol base samples at different RH–T conditions Table 1 gives a list of the measurements performed. For each run fresh sample loadings (with the same sample amount) were prepared: two loadings of both, spray dried salbutamol sulphate and salbutamol base, and one loading of both starting materials (salbutamol sulphate and salbutamol base). In total for each T–RH run six samples were prepared. For each T–RH run all samples were measured at starting conditions, 25 C and 10% RH. Then within few minutes T and RH were changed to the certain desired setpoints and measurements of all samples were repeated at each set of T–RH conditions over a few hours or till all samples were fully crystalline. 3. Results and discussion 3.1. Particle size and morphology of spray dried salbutamol base and sulphate samples Fig. 3 shows SEM images of spray dried salbutamol sulphate and spray dried salbutamol base. Both images show that spray drying with the set conditions resulted in spherical shaped particles with Table 1 List of measurements performed and measurement conditions. Temperature/ C
Relative humidity/%
Scan range/( 2u)
25 25 25 25 35 35
60 70 80 90 70 90
5–40 5–40 15–20 18.8–20.2 5–40 18.8–20.2
appropriate size for inhalation. Spray dried salbutamol base particles do not appear perfectly smooth, it seems that there are needle-like structures present on the surface (Fig. 3b). By contrast, the surface of spray dried salbutamol sulphate particles looks smooth (Fig. 3a), especially the surface of smaller particles. With increasing particle size the surface of spray dried salbutamol sulphate particles becomes more corrugated. This can be explained by the drying process of the particles. During drying a shell on the surface of the droplet is formed. Inside, water is still present and continues to vaporize. As a consequence pressure builds up inside and inflates the particle. The shells of the larger particles collapse after inflation, giving the particles a corrugated surface, whereas the shell of smaller particles has a higher mechanical stability and does not collapse. Therefore the particle surface remains smooth (Littringer et al., 2013). Table 2 gives the characteristic particle sizes and the span determined via laser diffraction. The crystalline starting materials of salbutamol sulphate and salbutamol base show particle sizes within the same range. Particle size distributions of spray dried salbutamol sulphate and spray dried salbutamol base confirm what was assumed from SEM images. Spray drying under the set conditions resulted in particles of appropriate size suitable for inhalation. Moreover, the characteristic diameters for spray dried salbutamol sulphate and base are in the same range. Except for crystalline salbutamol sulphate starting material the span of all samples is almost the same; for salbutamol sulphate starting material the span is slightly higher. 3.2. Crystallization of spray-dried salbutamol sulphate at different RH–T conditions 3.2.1. Crystallization of spray-dried salbutamol sulphate at 25 C and 60% RH Fig. 4 shows three representative XRD patterns of spray dried salbutamol sulphate held at 25 C and 60% RH for 12 h. As seen from the figure the sample was purely amorphous (no Bragg peaks present) in the beginning of the experimental run. After 12 h at 25 C and 60% RH sample remained amorphous, however the diffraction intensity decreased. Second measured sample of salbutamol sulphate (data are not shown) also did not show formation of a crystalline phase within 12-h exposure to 60% RH at 25 C. Although no crystallization was observed, both samples changed optically, (Fig. 5). As a result of humidity exposure, the spray dried powder samples became like a solidified gel and the volume of the samples seemed to be reduced due to fusion of the discrete particles. The XRD data, measured at 60% RH, (Fig. 4), reveal that the macroscopic change started earlier than the crystallization. Comparing the XRD patterns measured just after reaching 60% relative humidity at 25 C and after 1 h, a slight decrease in diffraction intensity can be detected. Assuming that the decrease of diffraction intensity corresponds to the visual change of the sample, it may be concluded that the visual change of the sample already starts within the first hour of exposure to 60% RH. 3.2.2. Crystallization of spray-dried salbutamol sulphate at 25 and 35 C and 70–90% RH In the following section, the experimental results of the runs at 25 C and relative humidity of 70–90% will be discussed. At a relative humidity above 60% RH the crystallization speed of spray dried salbutamol sulphate significantly increases. At 70% RH samples start to crystallize after 1.5 h (Fig. 6) and are completely crystallized after 5 h (Fig. 8). The crystallization speed increases even further with the further increase of relative humidity (Fig. 8). The optical sample changes at 25 C and 70% RH (Figs. 7 and 8) are even more pronounced compared to the optical changes
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Fig. 2. (a) Optimization plot showing two tested scaling modes, the number of factors used and the RMSEP for a test set of 1 salbutamol XRD scan, and (b) the PLSR calibration model (blue squares) and its cross-validation (red squares). This calibration model was used to quantify amorphous/crystalline ratio for the experiment run at 25 C and 70% RH. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. SEM pictures of spray dried (a) salbutamol sulphate and (b) salbutamol base.
observed at 25 C and 60% RH (Fig. 5). The samples shrink even more and sample cracking is visible (Fig. 7). It has been observed that at the same T–RH conditions two loadings of the spray dried salbutamol sulphate show different crystallization speed (data not shown). This is not surprising as the crystallization process and especially the nucleation is dependent on multiple factors like the presence of crystallization seeds, mechanical stress, environmental conditions etc. Therefore, a big advantage of the experimental setup used in the present work is
that various samples can be measured at the same time under the same environmental conditions (T, RH). Consequently, the effect of temperature and humidity on the crystallization of the samples can be studied at exactly the same conditions and results can be accurately compared among each other. Results in Fig. 8 indicate that the crystallization speed of amorphous salbutamol sulphate increases with increasing relative humidity. Crystallization takes longest at 70% RH, is faster at 80% RH and fastest at 90% RH. At 80% and 90% RH recrystallization of the
Table 2 Characteristic particle sizes and spans for salbutamol sulphate and base starting material and spray dried products. Sample Salbutamol Salbutamol Salbutamol Salbutamol
sulphate starting material base starting material sulphate spray dried base spray dried
X10/mm
X50/mm
X90/mm
SPAN
2.16 0.04 2.45 0.08 0.48 0.08 0.56 0.01
12.4 0.15 17.12 0.20 2.81 0.17 2.27 0.01
30.27 0.36 34.45 0.19 5.78 0.04 4.96 0.05
2.27 1.87 1.89 1.94
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Fig. 7. Image of 6 samples after 6 h at 25 C and 70% RH. Fig. 4. XRD patterns of spray-dried salbutamol, repeated measurements over 12 h.
is completed much faster. The required time until full sample crystallization is well in accordance with the results in Fig. 7. When increasing the temperature from 25 C to 35 C and the relative humidity from 70% to 90% crystallization speed further increases and recrystallization starts almost immediately and is complete after 15 min. Accordingly, temperature as well as relative humidity significantly increases crystallization speed. 3.3. Crystallization of salbutamol base
Fig. 5. Image of 6 samples after 12 h at 25 C and 60% RH.
amorphous content starts immediately whereas a reduction of the amorphous content and thus crystallization is deferred at 70% RH. In order to evaluate the effect of temperature on the crystallization speed of salbutamol sulphate, two additional experimental runs were performed at 35 C and 70% and 90% RH, indicating even faster crystallization of spray dried salbutamol sulphate (Fig. 9) compared to the same conditions at 25 C (Fig. 8). A quick way to estimate the crystallization speed from a series of diffraction patterns is by means of the peak-to-background ratio, i.e., the ratio between the net height of the Bragg-peaks and the background intensity. Fig. 9 shows the crystallization speed of spray dried salbutamol sulphate at different T–RH conditions. The crystallization of salbutamol sulphate at 70% RH and 25 C starts latest (after 10 min) and takes longest (about 100 min). When either increasing the relative humidity to 90% at 25 C or increasing the temperature to 35 C at 70 % RH, recrystallization starts earlier (after 30 min) and
Beyond the determination of the crystallization speed of salbutamol sulphate, also the crystallization speed of salbutamol base was determined by plotting peak-to-background ratio as a function of time. Crystallization of spray dried salbutamol base was analyzed at 25 and 35 C at 70 and 90% RH. The results are shown in Fig. 10. As well as spray dried salbutamol sulphate, spray dried salbutamol base crystallized very slowly at 60% relative humidity and 25 C. Almost no change was observed over 12 h (data not shown). At higher humidity and temperature values crystallization speed increases. Salbutamol base crystallizes faster than the sulphate but the same trends concerning the influence of relative humidity and temperature on the crystallization speed could be observed. At 70% RH and 25 C recrystallization of salbutamol base starts at the latest (after about 500 min) and takes the longest time. When increasing the RH from 70% to 90% at 25 C, recrystallization starts much earlier and is completed within a few minutes. Increasing the temperature at 70% from 25 C to 35 C also significantly increases crystallization speed. At 70% RH and 35 C recrystallization of salbutamol base starts after a few minutes and is also completed after several minutes. When further increasing the RH to 90% at 35 C, recrystallization starts almost immediately and is complete after several minutes. Thus, the same trend as with
Fig. 6. Representative set of XRD patterns of spray-dried salbutamol sulphate kept at 25 C and 70% RH for 6 h. Patterns are shifted vertically for clarity.
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Fig. 8. Crystallization speed of spray dried salbutamol sulphate (n = 2) at 25 C and relative humidity of 70%, 80% and 90% RH. The data points represent the average amount of amorphous salbutamol sulphate as a function of time at variable
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salbutamol sulphate is observed, the crystallization speed increases with increasing temperature and increasing relative humidity. Water vapor sorption experiments of spray dried salbutamol sulphate particles reported by Littringer et al. showed a continuous increase in mass of spray dried salbutamol sulphate particles attributed to the sorption of water in the RH range of 0–60% measured via water vapor sorption. Although the amount of water absorbed was quite high the samples remained amorphous. At 60% RH the mass suddenly decreased, which was attributed to crystallization of the samples. (Littringer et al., 2013) Therefore, the dramatic change in crystallization speed between 60% and 70% RH can be attributed to the fact that the water content at equilibrium at 60% RH is not high enough in order to facilitate recrystallization whereas it is sufficient to initiate recrystallization at relative humidities above 60%. The same behavior was also observed for spray dried salbutamol base samples (data not shown). Comparing the peak-to-background ratios in Fig. 9 (salbutamol sulphate) and Fig. 10 (salbutamol base), results show that spray dried salbutamol base recrystallizes faster than spray dried salbutamol sulphate. This could be observed for all investigated T–RH conditions. For both, spray dried amorphous salbutamol sulphate and base, it could be observed that increasing the RH from 70% to 90% RH at 25 C leads to a pronounced increase in the crystallization speed. An increase in RH from 70% to 90% RH at 35 C also increases crystallization speed of salbutamol sulphate and base, but the increase is less pronounced at 35 C than at 25 C as crystallization at 35 C happens quite fast anyway. 4. Conclusion and outlook
Fig. 9. Crystallization speed of spray dried salbutamol sulphate (n = 2) at different T–RH conditions. The relative peak-to-background ratio is plotted as a function of time.
Fig. 10. Crystallization speed of spray dried salbutamol base (n = 2) at different T– RH conditions. The peak-to-background ratio is plotted as a function of time.
This study showed that the PLSR method has proven to be suitable to quantify the amount of amorphous content in salbutamol sulphate samples. The quantification could be realized by extracting the amorphous/crystalline ratio as a function of time at various relative humidity conditions. The use of the MHC-trans multi-sample humidity chamber allowed measurements under controlled temperature and humidity conditions and most importantly the sample sets could be measured under exactly the same temperature and humidity conditions. As a result, a valid comparison of the crystallization behavior of the different samples could be achieved. Investigations on the influence of relative humidity on the crystallization behavior of salbutamol revealed that the crystallization speed of amorphous spray dried salbutamol sulphate and amorphous spray dried salbutamol base strongly depends on the relative humidity. The higher the relative humidity the higher is the crystallization speed. Salbutamol base crystallized faster than salbutamol sulphate. At 60% relative humidity salbutamol base as well as salbutamol sulphate remain amorphous even after 12 h, but at 70% RH the crystallization process of salbutamol base is complete within 3 h and at 90% RH the complete crystallization only takes about 30 min. For comparison, the crystallization process of salbutamol sulphate at 70% and 90% RH is complete within 4 h and 1 h, respectively. The crystallization speed is also strongly dependent on the temperature. Higher temperature (35 C) leads to increased crystallization speeds. This is true at all tested values of relative humidity (70–90% RH). Hence, for spray dried salbutamol sulphate and base samples the critical conditions for recrystallization determined within this study are 60% RH and 25 C. At these conditions, the XRD patterns showed no crystallization peaks over 12 h. Additional long-time measurements at various T–RH levels are necessary to determine the full kinetics of the recrystallization of spray-dried particles and to find out whether there are threshold T–RH conditions below which no crystallization occurs.
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Acknowledgements The authors acknowledge the financial support of this research project by the German Research Foundation (DFG) for financial support within the priority program SPP 1486 “Particles in Contact”. References Chawla, A., Taylor, K.M.G., Newton, J.M., Johnson, M.C.R., 1994. Production of spray dried salbutamol sulphate for use in dry powder aerosol formulation. Int. J. Pharm. 108, 233–240. Chougule, M.B., Padhi, B.K., Jinturkar, K.a., Misra, A., 2007. Development of dry powder inhalers. Recent Pat. Drug Deliv. Formul. 1, 11–21. Columbano, A., Buckton, G., Wikeley, P., 2002. A study of the crystallisation of amorphous salbutamol sulphate using water vapour sorption and near infrared spectroscopy. Int. J. Pharm. 237, 171–178. De Jong, S., 1993. SIMPLS: an alternative approach to partial least squares regression. Chemometr. Intell. Lab. Syst. 18, 251–263. Degen, T., Sadki, M., Bron, E., Koenig, U., Nénert, G., 2014. The HighScore suite. Powder Diffr. 29, S13–S18. doi:http://dx.doi.org/10.1017/S0885715614000840. Gorny, M., Jakobs, M., Mykhaylova, V., Urbanetz, N.A., 2007. Quantifying the degree of disorder in micronized salbutamol sulfate using moisture sorption analysis.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Crystallization speed of salbutamol as a function of relative humidity and temperature Sarah Zellnitz a, * , Olga Narygina b , Christian Resch c , Hartmuth Schroettner d, Nora Anne Urbanetz a a
Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, Graz, Austria PANalytical B.V., Almelo, Lelyweg 1, Almelo, The Netherlands Anton Paar GmbH, Anton-Paar-Straße 20, Graz, Austria d Austrian Centre for Electron Microscopy and Nanoanalysis, TU Graz, Steyrergasse 17/III, Graz, Austria b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 March 2015 Received in revised form 29 April 2015 Accepted 30 April 2015 Available online 2 May 2015
Spray dried salbutamol sulphate and salbutamol base particles are amorphous as a result of spray drying. As there is always the risk of recrystallization of amorphous material, the aim of this work is the evaluation of the temperature and humidity dependent recrystallization of spray dried salbutamol sulphate and base. Therefore in-situ Powder X-ray Diffraction (PXRD) studies of the crystallization process at various temperature (25 and 35 C) and humidity (60%, 70%, 80%, 90% relative humidity) conditions were performed. It was shown that the crystallization speed of salbutamol sulphate and base is a non-linear function of both temperature and relative humidity. The higher the relative humidity the higher is the crystallization speed. At 60% relative humidity salbutamol base as well as salbutamol sulphate were found to be amorphous even after 12 h, however samples changed optically. At 70% and 90% RH recrystallization of salbutamol base is completed after 3 h and 30 min and recrystallization of salbutamol sulphate after 4 h and 1 h, respectively. Higher temperature (35 C) also leads to increased crystallization speeds at all tested values of relative humidity. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Inhalation Powder X-ray diffraction Salbutamol sulphate Salbutamol base Spray drying Amorphicity
1. Introduction Salbutamol, a b2-adrenoreceptor agonist, is a common drug used for the treatment of bronchial asthma via inhalation. In order to reach their target site, the tiny airways of the lung, drug particles administered using Dry Powder Inhalers (DPIs) have to exhibit an aerodynamic diameter of 1 mm–5 mm (Newmann and Clarke, 1983; Telko and Dsc, 2005; Timsina et al., 1994; Zanen et al., 1992). Therefore, the powder has to be micronized before application. One of the methods to obtain micron-sized drug particles for pulmonary drug delivery is spray drying (Chawla et al., 1994; Chougule et al., 2007; Vidgrén et al., 1987; Zhao et al., 2008). Chawla et al. reported that spray drying of salbutamol sulphate on a Buechi 190 mini spray dryer leads to spherical shaped particles sufficiently small in diameter (median diameter 4.5 mm) to be used in dry powder inhalation formulations. Spray drying did not alter salbutamol sulphate chemically but reduced the crystallinity of the resulting drug particles (Chawla et al., 1994). Further, Gorny et al. and Littringer et al. reported that spray drying of salbutamol
* Corresponding author. Tel.: +43 316 873 30981; fax: +43 316 873 1030981. E-mail address: [email protected] (S. Zellnitz). http://dx.doi.org/10.1016/j.ijpharm.2015.04.079 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
sulphate from aqueous solutions results in amorphous particles (Gorny et al., 2007; Littringer et al., 2013). The challenge when working with amorphous products is the stability of the products as there is always the risk of recrystallization to the more stable crystalline state. Crystallization of the amorphous particles may be triggered by exposure to humid air or by heating of the sample (Columbano et al., 2002). Therefore, when working with amorphous particles it is necessary to know the temperature (T) and relative humidity (RH) conditions that trigger recrystallization of the particles. By knowing these critical conditions and controlling the storage conditions, it can be ensured that the particles remain in their amorphous state. Alternatively crystallization of the amorphous particles can be intentionally achieved by exposure to humid air or by heating of the sample (Columbano et al., 2002). Insitu powder XRD is a powerful method to investigate and monitor crystallization processes and to determine crystallization speed, degree of crystallinity and the resulting crystal phases. The study presents an investigation of the impact of relative humidity (RH) and temperature (T) on the crystallization process of spray dried salbutamol sulphate and salbutamol base. The sample conditions in the present study were controlled using a sample conditioning stage with a multi-sample holder offering space for eight samples (MHC-trans Multi-sample
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Humidity Chamber, Anton Paar GmbH, Graz, Austria). As humidity driven changes in pharmaceutical substances are usually slow and extremely sensitive to slight differences of the process parameters, the capability of this sample stage to investigate eight samples in parallel saves time and provides highly comparable results for all samples. 2. Materials and methods
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chute (Vibri, Sympatec, Clausthal-Zellerfeld, Germany). Data evaluation was performed using the software Windox 5 (Sympatec, Clausthal-Zellerfeld, Germany). 2.3.2. Scanning electron microscopy (SEM) Morphology of the spray dried samples was examined using a scanning electron microscope (SEM) (Zeiss Ultra 55, Zeiss, Oberkochen, Germany) operating at 5 kV. Samples were gold palladium sputtered prior to analysis.
2.1. Materials 2.4. Powder X-ray diffraction (PXRD) Crystalline salbutamol sulphate (USP25 quality) and crystalline salbutamol base were purchased from Selectchemie (Zuerich, Switzerland) and used as received. Pure ethanol was obtained from VWR International GmbH (Vienna, Austria). 2.2. Spray drying Amorphous salbutamol sulphate and salbutamol base particles were prepared on a Nano Spray Dryer B-90 (Buechi Labortechnik AG, Flawil, Switzerland) equipped with the long version of the drying chamber. Aqueous salbutamol sulphate solutions used for spray drying were prepared with purified water (TKA Micro Pure UV ultrapure water system, TKA Wasseraufbereitungssysteme GmbH, Niederelbert, Germany). To get particles in the size range of 1 mm–5 mm the spray drying parameters were chosen according to the work of Littringer et al. (2013): sprayhead mesh, 5.5 mm; feed concentration, 7.5% (m/m); inlet temperature, 120 C; spraying intensity, 30% and flow rate, 110 L/min. Salbutamol base was spray dried from pure ethanolic solution. The spray drying parameters were: sprayhead mesh, 5.5 mm; feed concentration, 3% (m/m); inlet temperature, 100 C; spraying intensity, 30% and flow rate, 110 L/min. The spray dried particles were stored in a desiccator over silica gel prior to analysis. The crystalline starting materials as well as the amorphous spray dried products were used as samples in the present study. 2.3. Sample characterization 2.3.1. Laser light diffraction The particle size distribution of the starting materials and the spray dried products was determined by laser light diffraction (Helos/KR, Sympatec, Clausthal-Zellerfeld, Germany). Powder dispersion was achieved with a dry dispersing unit (Rodos/L, Sympatec, Clausthal-Zellerfeld, Germany) at a dispersing pressure of 2.0 bar. Particles were fed into the dispersing unit by a vibrating
For powder X-ray diffraction (XRD) measurements at various temperature and relative humidity conditions a PANalytical empyrean diffractometer (PANalytical B.V., Almelo, Netherlands) equipped with a Cu(Ka) X-ray tube, a focusing X-ray mirror, an Anton Paar MHC-trans multi-sample humidity chamber (Anton Paar GmbH, Graz, Austria) and a PIXcel1D detector. The MHC_trans chamber is designed for XRD measurements in transmission geometry in the range of 2–55 2u. The software packages data collector v5.0, HighScore plus v4.0 and data viewer v1.5 were used for the measurements and data analysis. 2.5. Quantification of the amorphous content of salbutamol sulphate using powder X-ray diffraction 2.5.1. Reference sample set For the quantification of the amorphous/crystalline ratio during crystallization of spray dried salbutamol sulphate reference samples with known amorphous/crystalline ratio were prepared. The pure starting material was considered as 100% crystalline and the pure spray dried product as 100% amorphous. Reference samples were prepared by mixing 25, 50 and 75% (m/m) of the spray dried material with the starting material. From each mixture, two reference samples were prepared. The respective amounts of amorphous spray dried material and crystalline starting material were filled in a vial and stirred/shaked for a few minutes by hand. Sample exposure to humid air was minimized to the smallest possible level during sample preparation. Between experiments, samples were kept in a desiccator over silica gel. In order to ensure a constant irradiated sample volume for the XRD measurements, the amount of material filled in the MHC-trans sample cups was weighed. The representative data set measured from the eight reference samples is shown in Fig. 1. XRD patterns from reference samples of the same composition are nearly identical. This confirms that by loading the same sample amounts in a sample cup, reproducible results can be obtained. The data set, covering 2u range from 6 to 40 (Fig. 1), was used to quantify amorphous/
Fig. 1. Full range XRD patterns from the reference sample set with the same sample weight per sample cup.
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crystalline ratio for the experimental run with spray-dried salbutamol sulphate at 25 C and 70% RH. Two more reference data sets, covering 2u ranges of 15–20 and 18.2–20.2 , were measured (data not shown here) and used for the quantification of amorphous/crystalline ratio for the experimental runs at 25 C at 80% RH and 90% RH, respectively (Table 1). The quantification of the amorphous/crystalline ratio was performed using Partial Least Squares Regression (PLSR) method implemented in the PANalytical HighScore 4.0 package (De Jong, 1993; Degen et al., 2014). The detection limit of in-situ powder XRD is within 0.2–0.3 wt%. The presence of crystalline phase of this wt percentage or above is detected. 2.5.2. Partial least squares regression (PLSR) method PLSR method is a statistical method commonly used in spectroscopy (NIR – near infrared) and nuclear magnetic resonance (NMR) but only recently in the XRD field. When applied to XRD it enables extraction of “hidden” properties directly from XRD patterns. Detailed description of the PLSR method applied to XRD, as well as its implementation in PANalytical software HighScore, is described elsewhere (Degen et al., 2014; Koenig et al., 2014). In the case study PLSR was applied to calculate the amorphous/ crystalline ratio for salbutamol sulphate as a function of T/RH. For each experimental run at 25 C and 70%, 80% and 90% RH a PLSR model using corresponding data set of the reference XRD patterns was optimized and crossed-checked. In Fig. 2 representative optimization plot and obtained PLSR calibration model are shown. This model was used to quantify the amorphous/crystalline ratio for the experimental run at 25 C and 70% RH. As seen from Fig. 2a, the optimal number of PLSR factors was found to be 3 with the scaling mode “standardize” (De Jong, 1993). The root-mean-square error of prediction (RMSEP) was found to be 3.31 wt%. 2.6. Test execution – crystallization of spray dried salbutamol sulphate and salbutamol base samples at different RH–T conditions Table 1 gives a list of the measurements performed. For each run fresh sample loadings (with the same sample amount) were prepared: two loadings of both, spray dried salbutamol sulphate and salbutamol base, and one loading of both starting materials (salbutamol sulphate and salbutamol base). In total for each T–RH run six samples were prepared. For each T–RH run all samples were measured at starting conditions, 25 C and 10% RH. Then within few minutes T and RH were changed to the certain desired setpoints and measurements of all samples were repeated at each set of T–RH conditions over a few hours or till all samples were fully crystalline. 3. Results and discussion 3.1. Particle size and morphology of spray dried salbutamol base and sulphate samples Fig. 3 shows SEM images of spray dried salbutamol sulphate and spray dried salbutamol base. Both images show that spray drying with the set conditions resulted in spherical shaped particles with Table 1 List of measurements performed and measurement conditions. Temperature/ C
Relative humidity/%
Scan range/( 2u)
25 25 25 25 35 35
60 70 80 90 70 90
5–40 5–40 15–20 18.8–20.2 5–40 18.8–20.2
appropriate size for inhalation. Spray dried salbutamol base particles do not appear perfectly smooth, it seems that there are needle-like structures present on the surface (Fig. 3b). By contrast, the surface of spray dried salbutamol sulphate particles looks smooth (Fig. 3a), especially the surface of smaller particles. With increasing particle size the surface of spray dried salbutamol sulphate particles becomes more corrugated. This can be explained by the drying process of the particles. During drying a shell on the surface of the droplet is formed. Inside, water is still present and continues to vaporize. As a consequence pressure builds up inside and inflates the particle. The shells of the larger particles collapse after inflation, giving the particles a corrugated surface, whereas the shell of smaller particles has a higher mechanical stability and does not collapse. Therefore the particle surface remains smooth (Littringer et al., 2013). Table 2 gives the characteristic particle sizes and the span determined via laser diffraction. The crystalline starting materials of salbutamol sulphate and salbutamol base show particle sizes within the same range. Particle size distributions of spray dried salbutamol sulphate and spray dried salbutamol base confirm what was assumed from SEM images. Spray drying under the set conditions resulted in particles of appropriate size suitable for inhalation. Moreover, the characteristic diameters for spray dried salbutamol sulphate and base are in the same range. Except for crystalline salbutamol sulphate starting material the span of all samples is almost the same; for salbutamol sulphate starting material the span is slightly higher. 3.2. Crystallization of spray-dried salbutamol sulphate at different RH–T conditions 3.2.1. Crystallization of spray-dried salbutamol sulphate at 25 C and 60% RH Fig. 4 shows three representative XRD patterns of spray dried salbutamol sulphate held at 25 C and 60% RH for 12 h. As seen from the figure the sample was purely amorphous (no Bragg peaks present) in the beginning of the experimental run. After 12 h at 25 C and 60% RH sample remained amorphous, however the diffraction intensity decreased. Second measured sample of salbutamol sulphate (data are not shown) also did not show formation of a crystalline phase within 12-h exposure to 60% RH at 25 C. Although no crystallization was observed, both samples changed optically, (Fig. 5). As a result of humidity exposure, the spray dried powder samples became like a solidified gel and the volume of the samples seemed to be reduced due to fusion of the discrete particles. The XRD data, measured at 60% RH, (Fig. 4), reveal that the macroscopic change started earlier than the crystallization. Comparing the XRD patterns measured just after reaching 60% relative humidity at 25 C and after 1 h, a slight decrease in diffraction intensity can be detected. Assuming that the decrease of diffraction intensity corresponds to the visual change of the sample, it may be concluded that the visual change of the sample already starts within the first hour of exposure to 60% RH. 3.2.2. Crystallization of spray-dried salbutamol sulphate at 25 and 35 C and 70–90% RH In the following section, the experimental results of the runs at 25 C and relative humidity of 70–90% will be discussed. At a relative humidity above 60% RH the crystallization speed of spray dried salbutamol sulphate significantly increases. At 70% RH samples start to crystallize after 1.5 h (Fig. 6) and are completely crystallized after 5 h (Fig. 8). The crystallization speed increases even further with the further increase of relative humidity (Fig. 8). The optical sample changes at 25 C and 70% RH (Figs. 7 and 8) are even more pronounced compared to the optical changes
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Fig. 2. (a) Optimization plot showing two tested scaling modes, the number of factors used and the RMSEP for a test set of 1 salbutamol XRD scan, and (b) the PLSR calibration model (blue squares) and its cross-validation (red squares). This calibration model was used to quantify amorphous/crystalline ratio for the experiment run at 25 C and 70% RH. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. SEM pictures of spray dried (a) salbutamol sulphate and (b) salbutamol base.
observed at 25 C and 60% RH (Fig. 5). The samples shrink even more and sample cracking is visible (Fig. 7). It has been observed that at the same T–RH conditions two loadings of the spray dried salbutamol sulphate show different crystallization speed (data not shown). This is not surprising as the crystallization process and especially the nucleation is dependent on multiple factors like the presence of crystallization seeds, mechanical stress, environmental conditions etc. Therefore, a big advantage of the experimental setup used in the present work is
that various samples can be measured at the same time under the same environmental conditions (T, RH). Consequently, the effect of temperature and humidity on the crystallization of the samples can be studied at exactly the same conditions and results can be accurately compared among each other. Results in Fig. 8 indicate that the crystallization speed of amorphous salbutamol sulphate increases with increasing relative humidity. Crystallization takes longest at 70% RH, is faster at 80% RH and fastest at 90% RH. At 80% and 90% RH recrystallization of the
Table 2 Characteristic particle sizes and spans for salbutamol sulphate and base starting material and spray dried products. Sample Salbutamol Salbutamol Salbutamol Salbutamol
sulphate starting material base starting material sulphate spray dried base spray dried
X10/mm
X50/mm
X90/mm
SPAN
2.16 0.04 2.45 0.08 0.48 0.08 0.56 0.01
12.4 0.15 17.12 0.20 2.81 0.17 2.27 0.01
30.27 0.36 34.45 0.19 5.78 0.04 4.96 0.05
2.27 1.87 1.89 1.94
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Fig. 7. Image of 6 samples after 6 h at 25 C and 70% RH. Fig. 4. XRD patterns of spray-dried salbutamol, repeated measurements over 12 h.
is completed much faster. The required time until full sample crystallization is well in accordance with the results in Fig. 7. When increasing the temperature from 25 C to 35 C and the relative humidity from 70% to 90% crystallization speed further increases and recrystallization starts almost immediately and is complete after 15 min. Accordingly, temperature as well as relative humidity significantly increases crystallization speed. 3.3. Crystallization of salbutamol base
Fig. 5. Image of 6 samples after 12 h at 25 C and 60% RH.
amorphous content starts immediately whereas a reduction of the amorphous content and thus crystallization is deferred at 70% RH. In order to evaluate the effect of temperature on the crystallization speed of salbutamol sulphate, two additional experimental runs were performed at 35 C and 70% and 90% RH, indicating even faster crystallization of spray dried salbutamol sulphate (Fig. 9) compared to the same conditions at 25 C (Fig. 8). A quick way to estimate the crystallization speed from a series of diffraction patterns is by means of the peak-to-background ratio, i.e., the ratio between the net height of the Bragg-peaks and the background intensity. Fig. 9 shows the crystallization speed of spray dried salbutamol sulphate at different T–RH conditions. The crystallization of salbutamol sulphate at 70% RH and 25 C starts latest (after 10 min) and takes longest (about 100 min). When either increasing the relative humidity to 90% at 25 C or increasing the temperature to 35 C at 70 % RH, recrystallization starts earlier (after 30 min) and
Beyond the determination of the crystallization speed of salbutamol sulphate, also the crystallization speed of salbutamol base was determined by plotting peak-to-background ratio as a function of time. Crystallization of spray dried salbutamol base was analyzed at 25 and 35 C at 70 and 90% RH. The results are shown in Fig. 10. As well as spray dried salbutamol sulphate, spray dried salbutamol base crystallized very slowly at 60% relative humidity and 25 C. Almost no change was observed over 12 h (data not shown). At higher humidity and temperature values crystallization speed increases. Salbutamol base crystallizes faster than the sulphate but the same trends concerning the influence of relative humidity and temperature on the crystallization speed could be observed. At 70% RH and 25 C recrystallization of salbutamol base starts at the latest (after about 500 min) and takes the longest time. When increasing the RH from 70% to 90% at 25 C, recrystallization starts much earlier and is completed within a few minutes. Increasing the temperature at 70% from 25 C to 35 C also significantly increases crystallization speed. At 70% RH and 35 C recrystallization of salbutamol base starts after a few minutes and is also completed after several minutes. When further increasing the RH to 90% at 35 C, recrystallization starts almost immediately and is complete after several minutes. Thus, the same trend as with
Fig. 6. Representative set of XRD patterns of spray-dried salbutamol sulphate kept at 25 C and 70% RH for 6 h. Patterns are shifted vertically for clarity.
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Fig. 8. Crystallization speed of spray dried salbutamol sulphate (n = 2) at 25 C and relative humidity of 70%, 80% and 90% RH. The data points represent the average amount of amorphous salbutamol sulphate as a function of time at variable
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salbutamol sulphate is observed, the crystallization speed increases with increasing temperature and increasing relative humidity. Water vapor sorption experiments of spray dried salbutamol sulphate particles reported by Littringer et al. showed a continuous increase in mass of spray dried salbutamol sulphate particles attributed to the sorption of water in the RH range of 0–60% measured via water vapor sorption. Although the amount of water absorbed was quite high the samples remained amorphous. At 60% RH the mass suddenly decreased, which was attributed to crystallization of the samples. (Littringer et al., 2013) Therefore, the dramatic change in crystallization speed between 60% and 70% RH can be attributed to the fact that the water content at equilibrium at 60% RH is not high enough in order to facilitate recrystallization whereas it is sufficient to initiate recrystallization at relative humidities above 60%. The same behavior was also observed for spray dried salbutamol base samples (data not shown). Comparing the peak-to-background ratios in Fig. 9 (salbutamol sulphate) and Fig. 10 (salbutamol base), results show that spray dried salbutamol base recrystallizes faster than spray dried salbutamol sulphate. This could be observed for all investigated T–RH conditions. For both, spray dried amorphous salbutamol sulphate and base, it could be observed that increasing the RH from 70% to 90% RH at 25 C leads to a pronounced increase in the crystallization speed. An increase in RH from 70% to 90% RH at 35 C also increases crystallization speed of salbutamol sulphate and base, but the increase is less pronounced at 35 C than at 25 C as crystallization at 35 C happens quite fast anyway. 4. Conclusion and outlook
Fig. 9. Crystallization speed of spray dried salbutamol sulphate (n = 2) at different T–RH conditions. The relative peak-to-background ratio is plotted as a function of time.
Fig. 10. Crystallization speed of spray dried salbutamol base (n = 2) at different T– RH conditions. The peak-to-background ratio is plotted as a function of time.
This study showed that the PLSR method has proven to be suitable to quantify the amount of amorphous content in salbutamol sulphate samples. The quantification could be realized by extracting the amorphous/crystalline ratio as a function of time at various relative humidity conditions. The use of the MHC-trans multi-sample humidity chamber allowed measurements under controlled temperature and humidity conditions and most importantly the sample sets could be measured under exactly the same temperature and humidity conditions. As a result, a valid comparison of the crystallization behavior of the different samples could be achieved. Investigations on the influence of relative humidity on the crystallization behavior of salbutamol revealed that the crystallization speed of amorphous spray dried salbutamol sulphate and amorphous spray dried salbutamol base strongly depends on the relative humidity. The higher the relative humidity the higher is the crystallization speed. Salbutamol base crystallized faster than salbutamol sulphate. At 60% relative humidity salbutamol base as well as salbutamol sulphate remain amorphous even after 12 h, but at 70% RH the crystallization process of salbutamol base is complete within 3 h and at 90% RH the complete crystallization only takes about 30 min. For comparison, the crystallization process of salbutamol sulphate at 70% and 90% RH is complete within 4 h and 1 h, respectively. The crystallization speed is also strongly dependent on the temperature. Higher temperature (35 C) leads to increased crystallization speeds. This is true at all tested values of relative humidity (70–90% RH). Hence, for spray dried salbutamol sulphate and base samples the critical conditions for recrystallization determined within this study are 60% RH and 25 C. At these conditions, the XRD patterns showed no crystallization peaks over 12 h. Additional long-time measurements at various T–RH levels are necessary to determine the full kinetics of the recrystallization of spray-dried particles and to find out whether there are threshold T–RH conditions below which no crystallization occurs.
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