Understanding the structure of a pharmaceutical compound at an atomic scale is a key step towards rationally predicting and controlling the substance’s properties. For crystalline substances, often the preferred method of choice for structure determination is single crystal X-ray diffraction (SCXRD). However the technique requires crystals that are sufficiently large, and ideally crystals should be at least several micrometres thick. Growing sufficiently large crystals for analysis by SCXRD can be very challenging for certain compounds.

This has been the case for bismuth subsalicylate (BSS) – the active ingredient of Pepto-Bismol. Pepto-Bismol has been consumed since 1900 to treat various stomach ailments such as diarrhoea and dyspepsia. Today it is one of the most common stomach remedies in parts of the world such as North America. Despite its rather long history and current continued use, the molecular structure of BSS has remained unknown due to reasons such as the small size of the crystals, as well as its intricate structure as we later discovered.  

In our study we applied electron diffraction to investigate the structure of BSS. In an electron diffraction experiment an electron beam is instead scattered by the sample rather than X-rays. The technique allows for studies to be performed on much smaller crystal specimens that are only a few tens of nanometres thick. Crystals that contain organic molecules, however, were historically difficult to study by electron diffraction because often the crystals would be damaged by the electron beam. Recent developments in both the hardware and software have now made it possible to collect higher quality electron diffraction data. Data sets can be acquired within a just a few minutes or even seconds now, which significantly reduces damage to the crystals during the measurement and preserves the structure of interest.

Electron diffraction data were collected on several highly ordered crystals of BSS. However the completeness of each individual data set was rather low due to the low symmetry of the crystals. Therefore we applied hierarchical clustering to merge select datasets to improve the overall data completeness. The structure of BSS was finally determined from the merged electron diffraction data set (Figure 2). In the crystal structure bismuth cations (Bi³⁺) are bonded to bridging oxygen anions (μ-O^2-) which form a rod-shaped inorganic building unit (IBU) which extends along the a-axis. These inorganic rods are linked to one another along the b-axis by the organic salicylate anions (Hsal^-) resulting in a coordination polymer with a layered structure. The extended layered structure with the outer coating of organic salicylate ligands helps to explain the very poor solubility of BSS in water and its resistance to acid environments.

Although the crystal structure of BSS was finally determined, we realized that crystals of BSS from different sources were not quite the same. Crystals from one source were highly ordered, while crystals from another source seemed less ordered as indicated by the low data resolution of the electron diffraction data, as well as the presence of diffuse scattering. In order to understand the structural disorder, BSS crystals were investigated by high resolution scanning transmission electron microscopy (STEM). Integrated Differential Phase Contrast (iDPC) STEM images clearly shows the orientation of the IBUs in the layers (Figure 3). It was revealed that some parts of the crystal had regular arrangements of the IBUs thus indicating stacking of the layers as in the ordered structure (Figure 3, area 1). However in other parts of the crystal it was seen that the IBUs had the opposite orientation indicating that neighbouring layers had opposite orientations. In some sections the orientation of the neighbouring stacked layers seemed random (Figure3, area 2), while in other sections the orientations alternated (Figure 3, area 3).

Through these recently developed electron microscopy techniques a structural understanding of commercial BSS has been established. We envision that both electron diffraction and STEM imaging will play larger roles in structural characterization of pharmaceutical compounds in the near future.

This work on BSS began in 2017 thanks to inspiration from Prof. Tomislav Friŝĉić who brought to our attention that surprisingly the structure of one of the most commercially significant bismuth compounds was still unknown. For a number of years we had worked on and off in this project without any major breakthroughs, which in the end we realized was in part due to the disorder in the crystals we were investigating. During those years significant developments were made in the field of electron crystallography which were pivotal to this work. These included the development of automated electron diffraction data collection procedures, hierarchical clustering of datasets, and iDPC imaging. Also of vital importance to finally determining the structure was the perseverance and determination of the researchers and students working in this research project (Figure 4).

Read our article in Nature Communications:

• Erik Svensson Grape, Victoria Rooth, Mathias Nero, Tom Willhammar & A. Ken Inge, Structure of the active pharmaceutical ingredient bismuth subsalicylate, Nat Commun 13, 1984 (2022). https://doi.org/10.1038/s41467-022-29566-0

We thank the reviewers for their comments which led to improvements to the article.