The morphological analysis of crystalline methadone: a novel combination of microscopy techniques




methadone, birefringence, Michel-Levy birefringence colour chart, recrystallization methods, retardation, 3-D imaging, confocal microscopy, SEM, polarized light microscopy


The aim: to evaluate combined microscopy techniques for determining the morphological and optical properties of methadone hydrochloride (MDN) crystals.

Materials and methods: MDN crystal formation was optimized using a closed container method and crystals were characterized using polarized light microscope (PLM), scanning electron microscopy (SEM) and confocal microscopy (CM). SEM and CM were used to determine MDN crystal thickness and study its relationship with crystal retardation colours using the Michel-Levy Birefringence approach.

Results: Dimensions (mean±SD) of diamond shaped MDN crystals were confirmed using SEM and CM. Crystals were 46.4±15.2 Vs 32.0±8.3 µm long, 28.03±8.2 Vs 20.85±5.5 µm wide, and 6.62±2.9 Vs 9.6±4.6 µm thick, respectively. There were significant differences between SEM and CM thickness measurements (U=1283, p<0.05), as the SEM exhibited thinner diamond crystals. The combined use of PLM and Michel-Levy chart enabled the observation of a predominantly yellow coloured MDN crystal, mean thickness at (428 nm) mean retardation value.

Conclusion: The SEM was superior and successfully determined MDN crystal dimensions for the first time, whilst the CM results were affected by the Rhodamine dye staining process used for visualisation. The qualitative analysis of the crystallinity status of methadone hydrochloride optimally achieved using a combination of PLM and SEM techniques

Supporting Agency

  • Ministry of Higher Education and Scientific Research of Iraq through an educational scholarship to Noor R.T. Al-Hasani

Author Biographies

Noor R. Al-Hasani, King’s College London; University of Baghdad

Assistant Professor in Clinical Pharmacy

Department of Pharmacy

Department of Basic Sciences

College of Dentistry

Paul G. Royall, King’s College London

Senior Lecturer in Pharmaceutics

Department of Pharmacy

Neil Rayment, King’s College London

Research Fellow

Department of Nutrition

Kim Wolff, King’s College London

Professor of Analytical, Forensic and Addiction Science

King’s Forensics

Department of Analytical Environmental & Forensic Science


  1. Bishara, R. H. (1974). Methadone Hydrochloride. Analytical Profiles of Drug Substances. Vol. 3. Academic Press, 365–439. doi:
  2. Moffat, A. C., Osselton, M. D., Widdop, B., Watts, J. (2019). Clarke's analysis of drugs and poisons. Vol. 3. London: Pharmaceutical press.
  3. Pan, P.-P., Wang, J., Luo, J., Wang, S.-H., Zhou, Y.-F., Chen, S.-Z., Du, Z. (2017). Silibinin affects the pharmacokinetics of methadone in rats. Drug Testing and Analysis, 10(3), 557 –561. doi:
  4. Sun, H.-M., Li, X.-Y., Chow, E. P. F., Li, T., Xian, Y., Lu, Y.-H. et. al. (2015). Methadone maintenance treatment programme reduces criminal activity and improves social well-being of drug users in China: a systematic review and meta-analysis. BMJ Open, 5 (1), e005997–e005997. doi:
  5. Russolillo, A., Moniruzzaman, A., Somers, J. M. (2018). Methadone maintenance treatment and mortality in people with criminal convictions: A population-based retrospective cohort study from Canada. PLOS Medicine, 15 (7), e1002625. doi:
  6. Bretteville-Jensen, A. L., Lillehagen, M., Gjersing, L., Andreas, J. B. (2015). Illicit use of opioid substitution drugs: Prevalence, user characteristics, and the association with non-fatal overdoses. Drug and Alcohol Dependence, 147, 89–96. doi:
  7. Harris, M., Rhodes, T. (2013). Methadone diversion as a protective strategy: The harm reduction potential of “generous constraints.” International Journal of Drug Policy, 24 (6), e43–e50. doi:
  8. Winstock, A. R., Lea, T. (2009). Diversion and Injection of Methadone and Buprenorphine Among Clients in Public Opioid Treatment Clinics in New South Wales, Australia. Substance Use & Misuse, 45 (1-2), 240–252. doi:
  9. Betancourt, A. O., Gosselin, P. M., Vinson, R. K. (2012). New immediate release formulation for deterring abuse of methadone. Pharmaceutical Development and Technology, 18 (2), 535–543. doi:
  10. Shaw, I. F., Berk, J. (1976). U.S. Patent No. 3,980,766. Washington: U.S. Patent and Trademark Office; published: 14.09.1976.
  11. Elie, L. E., Baron, M. G., Croxton, R. S., Elie, M. P. (2012). Investigation into the suitability of capillary tubes for microcrystalline testing. Drug Testing and Analysis, 5 (7), 573–580. doi:
  12. Elie, L., Baron, M., Croxton, R., Elie, M. (2012). Microcrystalline identification of selected designer drugs. Forensic Science International, 214 (1-3), 182–188. doi:
  13. Kuś, P., Rojkiewicz, M., Kusz, J., Książek, M., Sochanik, A. (2019). Spectroscopic characterization and crystal structures of four hydrochloride cathinones: N-ethyl-2-amino-1-phenylhexan-1-one (hexen, NEH), N-methyl-2-amino-1-(4-methylphenyl)-3-methoxypropan-1-one (mexedrone), N-ethyl-2-amino-1-(3,4-methylenedioxyphenyl)pentan-1-one (ephylone) and N-butyl-2-amino-1-(4-chlorophenyl)propan-1-one (4-chlorobutylcathinone). Forensic Toxicology, 37 (2), 456–464. doi:
  14. Hubach, C. E., Jones, F. T. (1950). Methadone Hydrochloride Optical Properties, Microchemical Reactions, and X-Ray Diffraction Data. Analytical Chemistry, 22 (4), 595–598. doi:
  15. Bibi, S., Kaur, R., Henriksen-Lacey, M., McNeil, S. E., Wilkhu, J., Lattmann, E. et. al. (2011). Microscopy imaging of liposomes: From coverslips to environmental SEM. International Journal of Pharmaceutics, 417 (1-2), 138–150. doi:
  16. Kölemek, H., Bulduk, İ., Ergün, Y., Konuk, M., Korcan, S. E., Liman, R., Çoban, F. K. (2019). Synthesis of Morphine Loaded Hydroxyapatite Nanoparticles (HAPs) and Determination of Genotoxic Effect for Using Pain Management. Journal of Pharmaceutical Research International, 25 (6), 1–13. doi:
  17. Kania, A., Talik, E., Szubka, M., Ryba-Romanowski, W., Niewiadomski, A., Miga, S., Pawlik, M. (2016). Characterization of Bi2WO6 single crystals by X-ray diffraction, scanning electron microscopy, X-ray photoelectron spectroscopy and optical absorption. Journal of Alloys and Compounds, 654, 467–474. doi:
  18. Singh, M. R., Chakraborty, J., Nere, N., Tung, H.-H., Bordawekar, S., Ramkrishna, D. (2012). Image-Analysis-Based Method for 3D Crystal Morphology Measurement and Polymorph Identification Using Confocal Microscopy. Crystal Growth & Design, 12 (7), 3735–3748. doi:
  19. Khodaei, M., Esmaeili, A. (2019). New and Enzymatic Targeted Magnetic Macromolecular Nanodrug System Which Delivers Methadone and Rifampin Simultaneously. ACS Biomaterials Science & Engineering, 6 (1), 246–255. doi:
  20. Warren, F. J., Royall, P. G., Butterworth, P. J., Ellis, P. R. (2012). Immersion mode material pocket dynamic mechanical analysis (IMP-DMA): A novel tool to study gelatinisation of purified starches and starch-containing plant materials. Carbohydrate Polymers, 90 (1), 628–636. doi:
  21. Jaffe, M., Hammond, W., Tolias, P., Arinzeh, T. (Eds.). (2012). Characterization of biomaterials. Elsevier, 344.
  22. Carlton, R. A. (2011). Polarized Light Microscopy. Pharmaceutical microscopy. Springer Science & Business Media, 7–64. doi:
  23. Frandsen, A. F. (2016). Polarized light microscopy (No. KSC-E-DAA-TN37401).
  24. Klang, V., Valenta, C., Matsko, N. B. (2013). Electron microscopy of pharmaceutical systems. Micron, 44, 45–74. doi:
  25. Ren, F., Su, J., Xiong, H., Tian, Y., Ren, G., Jing, Q. (2016). Characterization of ibuprofen microparticle and improvement of the dissolution. Pharmaceutical Development and Technology, 22 (1), 63–68. doi:
  26. Wei, L., Yang, Y., Shi, K., Wu, J., Zhao, W., Mo, J. (2016). Preparation and Characterization of Loperamide-Loaded Dynasan 114 Solid Lipid Nanoparticles for Increased Oral Absorption In the Treatment of Diarrhea. Frontiers in Pharmacology, 7. doi:
  27. Furrer, P., Gurny, R. (2010). Recent advances in confocal microscopy for studying drug delivery to the eye: Concepts and pharmaceutical applications. European Journal of Pharmaceutics and Biopharmaceutics, 74 (1), 33–40. doi:
  28. Prasad, V., Semwogerere, D., Weeks, E. R. (2007). Confocal microscopy of colloids. Journal of Physics: Condensed Matter, 19 (11), 113102. doi:
  29. Korlach, J., Schwille, P., Webb, W. W., Feigenson, G. W. (1999). Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proceedings of the National Academy of Sciences, 96 (15), 8461–8466. doi:
  30. Mullin, J. W. (2001). Crystallization. Elsevier. doi:
  31. Agio, M., Alù, A. (Eds.). (2013). Optical antennas. Cambridge University Press. doi:
  32. Houck, M. M., Siegel, J. A. (2015). Friction ridge examination. Fundamentals of forensic science. San Diego: Elsevier Ltd, 493–518. doi:
  33. Patzelt, W., Leitz, E. (1985). Polarized Light Microscopy-Principles. Instruments, Applications. Wetzlar: Ernst Leitz Wetzlar GmbH, 103.
  34. Pygall, S. R., Whetstone, J., Timmins, P., Melia, C. D. (2007). Pharmaceutical applications of confocal laser scanning microscopy: The physical characterisation of pharmaceutical systems. Advanced Drug Delivery Reviews, 59 (14), 1434–1452. doi:
  35. Beaufort, L., Barbarin, N., Gally, Y. (2014). Optical measurements to determine the thickness of calcite crystals and the mass of thin carbonate particles such as coccoliths. Nature Protocols, 9 (3), 633–642. doi:




How to Cite

Al-Hasani, N. R., Royall, P. G., Rayment, N., & Wolff, K. (2022). The morphological analysis of crystalline methadone: a novel combination of microscopy techniques. ScienceRise: Pharmaceutical Science, (4 (38), 44–52.



Pharmaceutical Science