Morphological Analysis of Spherical Adsorptive Carbon Granules Using Three-Dimensional X-Ray Micro-computed Tomography

Abstract

The purpose of this study was to clarify applicability of three-dimensional X-ray micro-computed tomography (3D X-ray micro-CT) to elucidate interior morphology of spherical adsorptive carbon fine granules. Scanning of small single spherical granule hold on the rotating sample stage provided the structural information without particular preparation (e.g., slicing) that can affect the definite morphology. The three model formulations with similar appearance showed different internal structure in the 3D images, including large hollow in one of them. Other formulations showed some small empty or higher density area in the filled granules, suggesting uneven distribution of carbon. The results indicated relevance of the X-ray micro-CT analysis on the physical characterization of the spherical adsorptive carbon granule formulations.

Introduction

An orally administered spherical adsorptive carbon granule formulation was originally developed in 1991 to reduce uremic toxins in the gastrointestinal tract of patients with chronic renal failure.^1,2) The small black granules, 0.2–0.4 mm in diameter, mainly comprised porous microcrystalline carbon derived from petroleum pitch and small amounts of excipients.^1,3) The orally administered activated carbon particles are insoluble in water and organic solvents and adsorb uremic toxins produced and/or secreted in the gastrointestinal tract, thereby reducing the systemic absorption of uremic toxins.⁴⁾ A bland and two generic granule products are available in the market in Japan, in addition to some capsule and tablet formulations. The granule formulations were reported to have some differences in their physicochemical properties (e.g., in vitro adsorption of indicator chemicals) that may affect their clinical efficacy.⁵⁾ To ensure therapeutic equivalence, the Expert Committee on Quality of Generic Products recommended that manufacturers of generic products optimize their processes using in vitro adsorption of several major index toxins as key parameters.⁶⁾

Understanding the physical properties of granules is crucial to the development and production of complex formulations. Different spherical adsorptive carbon granule products have different internal structures (e.g., filled or hollow) because of their different manufacturing methods.⁵⁾ However, the small particle size perturbs the preparation of appropriately sliced, undamaged samples required for analysis by optical microscopy and scanning electron microscope (SEM). Thus, in this study the applicability of micro-computed tomography (CT) analysis in determining the morphology of particulate carbon granules was studied.^7–14)

Experimental

A bland (Kremezin^®, Kureha Co., Japan) and two generic (products of Nichi-iko Pharmaceutical Co., Japan and Mylan Seiyaku Ltd., Japan) spherical adsorptive carbon fine granule formulations purchased in 2009 and 2014 were subjected to the structural analysis. The three products are randomly assigned to the formulations A–C. The three-dimensional (3D) X-ray micro-CT scan was performed using a system (TDM1000H-Sμ, Yamato Scientific Co., Tokyo, Japan) with a 50 kV (0.086 mA) microfocus X-ray source. One relatively large granule was held using a double-sided adhesive tape (Nicetack, Nichiban Co., Japan) on top of a metal stick oriented perpendicular to the axis of rotation in an atmospheric environment. The X-ray shadow images were acquired in 1,200 views, one frame per view, with a pixel resolution of 0.49 µm (approximate scan time of 60 min). The X-ray shadow images were reconstructed into 3D cross sections using the VGStudio MAX 2.1 program (Volume Graphics GmbH, Heidelberg, Germany), and all subsequent analyses were based on the volume data set. Five cross-sectional 2D images at equally spaced planes of the small solids viewed from each of the three angles (XY, XZ, and YZ) were used to obtain the number of compartments in each circular area (0.482-mm diameter).

Results and Discussion

The formulations showed similar appearance fine granules (Fig. 1). Reconstitution of the X-ray scan data resulted in 3D images of the granules (Fig. 2). The white and black area in the images suggested carbon matrix and vacant regions in the granules. Formulations A (a, d) and C (c, f) showed a largely homogenous solid matrix throughout the granule with a brighter outer perimeter, suggesting a higher density solid on the outer surface. Some granules showed small pores and bright dots, which suggests a partially uneven distribution of carbon or other components in the granules. The granule surface and fine pores contacting gastrointestinal fluid should mainly contribute to in vitro adsorption of toxins. One of the formulations showed a solid outer layer and a large hollow cavity with some debris-like reticular solid inside (B: b, e). Images of multiple granules indicated similar thickness of the outer carbon layer. The cavity inside the granule appeared to be larger than that reported from optical microscopy images.⁵⁾ Furthermore, the formulations obtained in 2009 (a, b, c) and 2014 (d, e, f) showed similar CT images. Analysis of more granules should be required to know the granule-to-granule and batch-to-batch variation of the internal morphology.

Fig. 1. Appearance of Spherical Adsorptive Carbon Granules in Formulations A

Fig. 2. 3D Images of the Granules Sliced after Their Structural Reconstruction

Each figure shows 3D image of Formulation A (a, d), B (b, e), C (c, f) purchased in 2009 (a–c) and 2014 (e–f).

Our non-destructive X-ray micro-CT analysis showed different morphologies for small spherical adsorptive carbon granules in different products, which is largely in accordance with previous reports.⁵⁾ The filled and hollow cores should explain the products’ differences in specific gravity and resulting behavior upon suspension in water; however, similarity in structure and composition is not a prerequisite for therapeutic equivalence between the innovator and generic products. This analysis also provides information on uneven densities in the core matrix of the granules. Appropriate sample holding and precise controlling of stage rotation during multiple scans to maintain the coordinate position of the small granules should contribute to data reconstitution into clear 3D images. Among the several methods used to hold the granules, double-sided adhesive tape provided the most stable sample position on the metal shaft of the sample stage compared with other methods (e.g., use of gel grew, data not shown).

Micro-CT analysis provides valuable information both in optimization of the process parameters in the development stage and monitoring of the product through their lifecycle. Although the CT analysis requires long scan times, availability of 3D structural information at arbitrary positions and angles in the absence of sample preparation should be a major advantage of micro-CT over other methods used to study the structure of pharmaceutical solids (e.g., SEM and optical microscopy). The X-ray micro CT analysis, however, does not provide information on the precise structure of micropores contributing to absorb toxin. Analysis by SEM or synchrotron X-ray micro-CT should provide much precise information required for such purpose.¹⁴⁾ Understanding the mechanisms and target size ranges of various analytical methods should be inevitable for their complementary applications.

Conflict of Interest

The authors declare no conflict of interest.

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