相干反斯托克斯拉曼散射（CARS）显微可视化药片剂溶出过程中
Summary
相干反斯托克斯拉曼散射（CARS）显微术是结合本征流通过溶解的设置，以允许在原位和药物片剂进行溶出度的表面的实时可视化。使用这种定制的设置，因此能够使用内联紫外吸收光谱记录药物溶出曲线相关CARS视频。
Abstract
传统的药物溶出试验测定药物通过测定溶出介质的药物含量溶解在一段时间的量。这种方法提供了有关正在发生的事情溶解药片表面上几乎没有直接的信息。作为片剂表面的组成和结构可以在溶解过程中改变，这是必不可少的溶出度试验中对其进行监视。在这项工作中相干反斯托克斯拉曼散射显微镜被溶解时用于图像片的表面，同时用紫外吸收光谱是同时提供溶解药物浓度的在线分析含有茶碱无水物和乙基纤维素的50％的混合物的片剂。该测量结果表明， 在原 CARS显微镜能够成像的选择性茶碱在乙基纤维素的存在。此外，无水茶碱转化为茶碱一水合物溶解过程中，用针形哭stals溶解过程中不断增长的数位板表面。茶碱无水物到一水合物的转化，结合的药物减少暴露于流动介质中的溶解导致降低溶出率。我们的研究结果表明， 在原位 CARS显微镜结合内嵌紫外吸收光谱是能够监测药物片剂溶出度和关联面的变化与改变溶解速度。
Introduction
在口服药物剂型，如片剂和胶囊的发展有较强的重视溶出度测试。口服剂型都必须溶解它们可以被吸收为治疗功效之前。难溶性药物一般都达到足够的浓度，这使得溶出度测试尤为重要1的问题。药典溶出度方法是最常用的溶出分析。在大多数情况下，这需要制备药物片剂或胶囊中，然后放入流动的溶解介质的烧杯中。溶解的药物浓度，然后通过使用标准的光谱技术，如UV吸收光谱仪2分析该溶出介质的样品来确定。这些传统的药物溶出方法不提供样品的任何直接分析或可能被对剂型的溶解表面发生任何变化。样品的溶解过程中直接分析可以提供关于溶解剂型的详细信息，并可能识别导致溶出试验失败的问题。
溶解剂型的直接分析，需要在它们能够监测溶解过程的原位分析技术的使用。溶解过程中就地记录的分析技术不能被溶解介质的存在的影响，该技术需要一个高的时间分辨率，以可靠地测量在秒数量级的变化，以溶解剂型。衰减全反射紫外光谱法已被证明是适合的溶解过程中测量的变化，但缺少由成像技术3中提供的空间分辨率。如扫描电子显微镜（SEM）和自发拉曼映射传统药物成像技术都具有限制因素防止它们的使用在原地解散。
扫描电镜成像是能够成像的药物剂型的表面的高分辨率快速成像技术。然而，通常在真空条件下进行的扫描电镜成像，需要样品涂层使得它在原位溶解成像不适宜。光纤耦合自发拉曼光谱结合了流通池和UV流通过吸收光谱，目前已进行溶解过程中监测各种药物系统在原位 ，包括茶碱4，卡马西平，和吲哚美辛5。拉曼光谱是能够识别溶解过程中发生的表面变化，但它提供了关于在地表变化没有发生空间信息。自发拉曼映射使用拉曼光谱，并提供有关样品的表面空间信息，但成像只需要几分钟的顺序来小时，这取决于图像区域，使得它在原地解散成像不适合。
相干反斯托克斯拉曼散射（CARS）显微术是一种快速成像技术，结合内嵌紫外吸收光谱，它使我们能够开发就地解散分析能力的技术。 CARS显微镜提供了快速而不受溶出介质中的存在使得其在原位溶解分析一个合适的技术影响化学选择性成像。 CARS技术大致分为两个组的基础上，激光器的脉冲持续时间;其中之一是窄带车（皮秒脉冲激光器），而另一个是宽带CARS（飞秒脉冲激光器）。一个典型的CARS显微镜系统由两个脉冲激光源，在倒置显微镜。以产生CARS信号时，脉冲激光器中的一个需要是可调谐的，以便有两个激光器相匹配的拉曼振动之间的频率差。此外，两个激光器必须重叠在空间（空间）和时间（时间），以从两个激光器到达样品在同一时间同一区域的脉冲。作为拉曼振动是特定化学和CARS信号仅在显微镜的聚焦体积内产生，CARS显微镜能够化学选择性成像，分辨率降低到衍射极限的。
使用单一的拉曼振动模式窄带CARS显微镜允许大约快100倍成像相比，自发拉曼成像技术，6。宽带CARS显微镜图像在更宽的光谱范围（600-3,200 -1对比〜4厘米-1），但具有较低的频谱分辨率（大约10 -1对比〜4厘米-1）和较慢的成像速度（50毫秒/像素对比〜5微秒/像素）则为窄带CARS显微镜7。
窄带CARS显微镜已被用于图像DRUG释放一些药物系统。在药物制剂领域，Kang 等 8-10成像药物加载的聚合物膜。最初它们成像的装药，其随后从静态溶出介质中的药物释放的成像分布。 Jurna 等 11和Windbergs 等 12更进一步和成像首先在随后使用动态溶出介质成像的药物溶解脂质剂型的茶碱分布。
我们已经开发了一种新的分析方法同时监测在数位板上进行溶解与窄带CARS显微镜的同时，录制溶解的药物浓度，用紫外吸收光谱表面的变化。我们说明了如何使用包含模型药物茶碱结合乙基纤维素进行溶解，以水为溶出介质，这种方法的成像片。
Protocol
Representative Results
Discussion
When performing CARS microscopic dissolution experiments there are a few critical aspects that need to be monitored during the experiment. Firstly, introducing the dissolution medium to the CARS flow cell causes the focus to move. This means that the image is immediately lost and it takes a few microns of objective adjustment to find the surface again. Secondly, there is risk of liquid leakage from the CARS flow cell if the glass cover breaks during the experiment. This can potentially cause liquid damage to the optics, so it is important to listen for any cracking sound that could mean the glass has broken. Finally, there is also a small chance that the piping can become blocked due to particulate matter in the system during the experiment, this can be seen as a sudden unusual change in the UV spectra and also through periodically checking the flow during the experiment.
Particulate blockage of the piping is mainly an issue with tablets that have been designed to disintegrate during dissolution. This is one of the limitations for this technique as this system requires the surface of the tablet to remain intact throughout the dissolution to allow imaging. In addition to disintegrating tablets, it is currently not possible to image tablets that are designed to swell during dissolution as this can lead to breakage of the CARS flow cell.
Imaging tablets during dissolution provides a greater understanding of what is occurring on the surface of a dissolving tablet. Conventional pharmaceutical dissolution methods focus only on the drug content dissolved in the dissolution medium which can identify whether the tablet passes or fails the required standard. However, in the case of a failed test it is difficult to determine what caused the failure. The case of a failed dissolution test is potentially where in situ dissolution analysis using CARS microscopy can provide answers.
Future applications for in situ dissolution analysis using CARS microscopy could include investigations using more complicated tablets containing more than one drug or excipient, in particular non-swelling sustained or controlled release dosage forms during formulation development. Additionally, it could be possible to investigate samples using biorelevant dissolution media creating conditions more closely related to in vivo.
In conclusion, this work shows that CARS microscopy is capable of rapid chemically specific imaging based on Raman vibrational frequencies allowing selective imaging of the drug in a tablet containing both drug and excipient. Additionally, CARS microscopy combined with inline UV absorption spectroscopy is a powerful tool capable of monitoring the surface of tablets undergoing dissolution and correlating surface changes seen using CARS with changes in dissolution rate.
Disclosures
The authors have nothing to disclose.
Acknowledgements
房颤是由荷兰技术基金会污水处理厂，这是苏国的应用科学分工，经济事务部的技术方案支持。 （STW OTP 11114）。
Materials
| Name of the Material/Equipment | Company | Catlog number | Comments/Description | Website |
| Paladin 1064nm laser | Coherent | N/A | Prototype model not for sale | http://www.coherent.com/ |
| Levante Emerald Optical parametric oscillator | APE Berlin | N/A | http://www.ape-berlin.de/en/products/levante/levante-emerald-opo#block-views-products-block-1 | |
| IX 71 Microscope | Olympus | N/A | http://www.olympusamerica.com/seg_section/product.asp?product=1023 | |
| Fluoview 300 scanning unit | Olympus | N/A | http://www.olympusamerica.com/seg_section/seg_product_print.asp?product=133 | |
| Photon multiplier tube R3896 | Hamamatsu | N/A | https://www.hamamatsu.com/jp/en/R3896.html | |
| Free standing optics / filters | Thorlabs and Chroma | N/A | http://www.chroma.com/ | |
| http://www.thorlabs.de/index.cfm? | ||||
| Reglo peristaltic pump | ISMATEC | N/A | http://www.ismatec.com/int_e/pumps/t_reglo/reglo.htm | |
| USB2000+ spectrometer | Ocean Optics | N/A | http://www.oceanoptics.com/products/usb2000+.asp | |
| DT-MINI-2-GS light source | Ocean Optics | N/A | http://www.oceanoptics.com/Products/dtmini.asp | |
| FIA-Z-SMA-TEF Z shaped flow cell | Ocean Optics | N/A | http://www.oceanoptics.com/Products/fiazsmaflowcells.asp | |
| QP400-2-SR-BX optical fiber | Ocean Optics | N/A | http://www.oceanoptics.com/Products/premgradesol.asp | |
| Plastic piping | ISMATEC | N/A | http://www.ismatec.com/int_e/tubing/misc/tubing_home.htm | |
| CARS dissolution tablet flow cell | N/A | N/A | Homebuilt at university – designed to hold 12mm diameter, 3mm thick tablets. The flowcell has a channel depth of around 0.5mm. | |
| Glass beakers | VWR | D108980 | https://us.vwr.com/store/catalog/product.jsp?product_id=4537423 | |
| Theophylline anhydrate | BASF | 30058079 | http://www.basf.com/group/corporate/en/brand/THEOPHYLLINE | |
| ethylcellulose | Colorcon | N/A | http://www.colorcon.com/products-formulation/all-products/film-coatings/sustained-release/ethocel | |
References
- Ku, M. Use of the Biopharmaceutical Classification System in Early Drug Development. The AAPS Journal. 10, 208-212 (2008).
- . The United States Pharmacopeia. United States Pharmacopeial Convention 32nd ed. , 1-8 (2009).
- Florence, A. J., Johnston, A. Applications of ATR UV/vis spectroscopy in physical form characterisation of pharmaceuticals. Spectrosc. Eur. 4, (2004).
- Aaltonen, J., et al. In situ measurement of solvent-mediated phase transformations during dissolution testing. J. Pharm. Sci. 95, 2730-2737 (2006).
- Savolainen, M., et al. Better understanding of dissolution behaviour of amorphous drugs by in situ solid-state analysis using Raman spectroscopy. European Journal of Pharmaceutics and Biopharmaceutics. 71, 71-79 (2009).
- Slipchenko, M. N., et al. Vibrational imaging of tablets by epi-detected stimulated Raman scattering microscopy. Analyst. 135, 2613-2619 (2010).
- Parekh, S. H., Lee, Y. J., Aamer, K. A., Cicerone, M. T. Label-Free Cellular Imaging by Broadband Coherent Anti-Stokes Raman Scattering Microscopy. Biophys. J. 99, 2695-2704 (2010).
- Kang, E., et al. In Situ Visualization of Paclitaxel Distribution and Release by Coherent Anti-Stokes Raman Scattering Microscopy. Anal. Chem. 78, 8036-8043 (2006).
- Kang, E., Robinson, J., Park, K., Cheng, J. -. X. Paclitaxel distribution in poly(ethylene glycol)/poly(lactide-co-glycolic acid) blends and its release visualized by coherent anti-Stokes Raman scattering microscopy. J. Controlled Release. 122, 261-268 (2007).
- Kang, E., et al. Application of coherent anti-stokes Raman scattering microscopy to image the changes in a paclitaxel-poly(styrene-b-isobutylene-b-styrene) matrix pre- and post-drug elution. Journal of Biomedical Materials Research Part A. 87A, 913-920 (2008).
- Jurna, M., et al. Coherent anti-Stokes Raman scattering microscopy to monitor drug dissolution in different oral pharmaceutical tablets. Journal of Innovative Optical Health Sciences. 2, 37-43 (2009).
- Windbergs, M., et al. Chemical Imaging of Oral Solid Dosage Forms and Changes upon Dissolution Using Coherent Anti-Stokes Raman Scattering Microscopy. Anal. Chem. 81, 2085-2091 (2009).
- Fussell, A., Garbacik, E., Offerhaus, H., Kleinebudde, P., Strachan, C. In situ dissolution analysis using coherent anti-Stokes Raman scattering (CARS) and hyperspectral CARS microscopy. European Journal of Pharmaceutics and Biopharmaceutics. 85, 1141-1147 (2013).
- Lua, Y. -. Y., Cao, X., Rohrs, B. R., Aldrich, D. S. Surface Characterizations of Spin-Coated Films of Ethylcellulose and Hydroxypropyl Methylcellulose Blends. . Langmuir. 23, 4286-4292 (2007).
- Skoog, F. J., Holler, S. R., Crouch, . Principles of Instrument analysis. 6 ed. , (2007).
- Rodríguez-Hornedo, N., Lechuga-Ballesteros, D., Hsiu-Jean, W. Phase transition and heterogeneous/epitaxial nucleation of hydrated and anhydrous theophylline crystals. Int. J. Pharm. 85, 149-162 (1992).
