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Synchrotron radiation micro-computed tomography-guided chromatographic analysis displays the material distribution in tablets Liu Zhang, Li Wu, Caifen Wang, Guoqing Zhang, Lin Yu, Hai Yan Li, Abi Maharjan, Yan Tang, Dunwei He, Peter York, Huimin Sun, Xianzhen Yin, Jiwen Zhang, and Lixin Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04726 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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Analytical Chemistry

Synchrotron radiation micro-computed tomography-guided chromatographic analysis displays the material distribution in tablets Liu Zhang,†, ‡, # Li Wu,§, ‡, # Caifen Wang,‡, # Guoqing Zhang,‡ Lin Yu,†, ‡ Haiyan Li,‡ Abi Maharjan,‡ Yan Tang,‡ Dunwei He,⊥ Peter York,‡,∇ Huimin Sun,¶ Xianzhen Yin,‡, * Jiwen Zhang,§, ‡, * Lixin Sun†, * †

Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China; § School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, Yantai University, Yantai 264005, China; ‡ Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; ⊥ Shandong Hi-Qual Pharmatech Co., Ltd; Zibo 255035, China; ∇ Institute of Pharmaceutical Innovation, University of Bradford, Bradford, West Yorkshire BD7 1DP, United Kingdom; ¶ National Institutes for Food and Drug Control, Beijing 100050, China; # The authors contribute equally to the research. *

Corresponding Author

Prof. Lixin Sun Tel/Fax: +86-24-23986365; E-mail: [email protected]; Prof. Jiwen Zhang Tel/Fax: +86-21-20231980; E-mail: [email protected]; Assoc. Prof. Xianzhen Yin Tel/Fax: +86-21-20231980; E-mail: [email protected]

ABSTRACT: One unusual and challenging scientific field that has received only cursory attention to date is the three-dimensional (3D) microstructure and spatial distribution of drug(s) and formulation materials in solid dosage forms. This study aims to provide deeper insight to the relationships between the microstructure of multiple-unit pellet system (MUPS) tablets and the spatial distribution of the active pharmaceutical ingredient (API) and excipients to facilitate the design of quantitative models for drug delivery systems. Synchrotron radiation X-ray micro-computed tomography (SR-µCT) was established as a 3D structure elucidation technique, which, in conjunction with liquid chromatography coupled to mass spectrometry (LC-MS) or liquid chromatographyevaporative light-scattering detector (LC-ELSD) enables chemical analysis of tablets. Based on the specific interior construction of theophylline MUPS tablets, the spatial distribution of materials was acquired by quantifying micro-region samples that had been validated by SR-µCT for their locations in the MUPS tablets. The 3D structure of the MUPS tablets was catalogued as three structural domains: a matrix layer (ML), a protective cushion layer (PCL) and pellets (PL). Compared with the components in the ML, components in the PL had a larger proportion of theophylline, sucrose, and diethyl phthalate and a smaller proportion of lactose and sodium lauryl sulfate, whereas glyceryl monostearate was found to account for a large portion of the PCL. Microstructural characterization-guided zonal chemical determination represents a new approach for quality assessment and the development of drug delivery systems with in-depth insight into their constituent layers on a new scale. KEY WORDS: Material distribution; drug delivery system; SR-µCT; microstructure.

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The ability to understand, evaluate, and most importantly control material distribution in a drug delivery system is critical to formulation development, process design and optimal therapeutic function in the pharmaceutical sciences. Drug delivery systems consist of the drug substance as well as a number of functional ingredients for desirable characteristics.1 Multiple-unit pellet system (MUPS) tablets, a sophisticated delivery system using coated pellets for controlled drug release with a more complex mechanical form, have taken on a very important role in the pharmaceutical industry.2 Both industry and academia are actively pursuing the development of such formulations since this drug delivery strategy has the

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potential of providing unique features in the designed formulation. In essence, the development of a drug delivery system needs to address the structural design and the spatial distribution control of the active pharmaceutical ingredient (API) and excipients, both of which are directly related to the quality attribute and therapeutic performance. Furthermore, MUPS tablets are multi-particulate in nature, and the total drug dose is divided into many units, which have a far more complex structure and material spatial distribution. The quality by design (QbD) initiative of the FDA requires a process to be controllable and predictable.3 Theories and methods to characterize the internal structure and to map the material distribution

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in MUPS tablets will facilitate the implementation of QbD approaches to guarantee high quality and therapeutic efficacy. Systematic studies of three-dimensional (3D) material distribution and dosage form structures are rare due to the lack of appropriate analytical methodologies. Near infrared spectroscopy (NIR) and Raman imaging have been used to study the distribution of chemicals within pharmaceutical products.4, 5 However, these technologies can be used to research only the surface of a sample or require the sample to be sectioned to generate optically flat surfaces to acquire internal information about the component distribution. Terahertz time-domain reflection spectroscopy (THz-TDRS), using THz radiation in the lower end of the far infrared region, has been employed to study coated tablet.6 Because of the lack of data processing methods, however, THz spectra cannot provide a visual representation of or satisfactory structural information about the specimen. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) characterized the chemical distribution of materials and material transfer in two-dimensional (2D) membrane by the FTIR abundance distribution of the chemical group in the membrane.7, 8 Existing research using X-ray diffractometry and micro-focus X-ray (MFX) imaging to evaluate the material distribution in formulations have been confined to study the single component9 10 without an in-depth investigation of the relationship between the structure and material distribution of drug delivery systems. Therefore, synchrotron radiation micro-computed tomography (SR-µCT) is proposed here as an advanced tomographic imaging technology which employs bremsstrahlung radiation to obtain the 3D interior construction of a solid sample non-invasively.11 In contrast to conventional techniques, the SR-µCT allows noninvasive scanning, quantitative visualization and evaluation of the 3D structures with high-speed imaging, high intensity of X-ray beam and high spatial resolution.12 Within pharmaceutical material science and drug delivery research, the first 3D structural insight of orifices in osmotic pump tablets was provided by SR-µCT to improve the design of advanced osmotic pumps for controlled drug release.13 Also the changes of internal structure of tablet cores during the drug release process were investigated using SR-µCT to determine the intrinsic drug release mechanism of the osmotic drug delivery system.14 For the individual pellet microstructures of multiple-pellet formulations, relationships between the microstructure and drug release from single pellets were creatively studied based on the powerful SR-µCT technique.15 Microstructural investigation using SR-µCT was also helpful in identifying the tastemasking mechanism of acetaminophen in coated microspheres16 and crystal form identification in polymorphic mixtures.17 Nevertheless, these studies have been mostly focused on morphological models. It is especially of interest to apply SR-µCT for both the actual microstructure and quantitative material distribution in drug delivery systems. To gain integrated information on the material distribution and microstructure in drug delivery systems, appropriate analytical approaches with high efficiency and low solvent and sample consumption need to be combined. High-performance liquid chromatography (HPLC) is the most popular technique for pharmaceutical analysis by far.18 The sensitivity of detection using mass spectrometers (MSs) implemented with an electrospray ionization (ESI) source for pharmaceutical assays of raw materials has been widely reported.19, 20 In addition, organic material without UV absorption or fluorescence properties in pharmaceutical product can be detected by an evapo-

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rative light-scattering detector (ELSD) based on volatile nature. An inventive combination of SR-µCT with quantitative determination is expected to have unprecedented value in the investigated fields. Based on the SR-µCT high resolution 3D analysis, microstructural information of the MUPS tablet can be obtained non-invasively. From a phase-contrast methodology to distinguish materials of different densities, the highly-resolved 3D data also provide the capability to differentiate and identify the substructures of pellets. Owing to the slight difference in density among APIs and excipients, the low chemical contrast SRµCT data provides only limited information about material distribution. However when combined with SR-µCT 3D structural analysis, the data enable the subsample to be spatially localized and to determine which functional substructure of this subsample belongs to. Chemical analysis provides additional details of the relationships between the microstructure and material distribution based on the specificity and high sensitivity of chromatographic analyses. In our study, subsamples from the micro-regions of the MUPS tablets were collected and classified according to their structure, as determined by SR-µCT. Material distribution along with internal construction was elucidated after using chromatographic techniques, i.e. HPLC-ESI-MS/MS, HPLC-ELSD, which were employed to quantify drug and excipients levels in the subsamples. The findings demonstrated the feasibility of this methodology in revealing the material spatial distribution in a drug delivery system in situ. EXPERIMENTAL SECTION Materials. Theophylline tablets were manufactured by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). Theophylline (≥99.0 %, THEO), ammonium acetate and formic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ultra-pure water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). Sucrose (≥99.5 %, SUC) and lactose (≥99.5 %, LAC) were produced by Shanghai Yunhong Pharmaceutical Excipients Co., Ltd. (Shanghai, China). Diethyl phthalate (≥99.5 %, DEP), sodium lauryl sulfate (≥99.0 %, SLS) and glyceryl monostearate (≥99.0 %, GEM) were supplied by Sichuan Hanhua Pharmaceutical Excipients Co., Ltd. (Sichuan, China). All analytical reagents were of HPLC grade and were used without further purification. Sampling from the Tablet for SR-µCT Detection. When SR-µCT is used to characterize a material, the sample size is roughly proportional to the image resolution, and the sample must be entirely covered within the imaging area. A tablet was broken into pieces, and identifiable subsamples from microregions in the MUPS tablet in a size range of 100-200 µm were selected randomly. To investigate the location of subsamples within the microstructure of the MUPS tablet and accommodate the sample size within a fixed holder system, three differently sized hard gelatin capsule shells were prepared, each of which contained four subsamples. The subsamples in each capsule were easily distinguished by their size and shape, as shown in Figure 1A, and were assigned an identification number. These capsules were closed to form a specimen for SR-µCT scanning. Figure 1B shows a 2D synchrotron Xray tomographic image of how the components of the capsule shells nested together. Before analysis, the height of the acquisition window used to cover the subsamples in the capsule was adjusted, and the CT scan was performed.

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Figure 1. Preparation and identification of subsamples for the theophylline multiple-unit pellet system (MUPS) tablet. The subsamples in each capsule can be easily distinguished by size and shape (A), and the prepared specimen for the SR-µCT scan was shown in a tomographic image (B).

SR-µCT Scan. Individual tablets of the MUPS and its prepared subsamples were examined. SR-µCT tomographic images of the samples were acquired using the BL13W1 beam line at Shanghai Synchrotron Radiation Facility (SSRF). The tablet and its subsamples were fixed on the sample stage with double-sided adhesive tape and were examined in different SR-µCT scans. X-rays were derived from an electron storage ring with an average beam current of 180 mA, and an accelerated energy of 3.5 GeV. The imaging parameters were optimized according to the preliminary experiments and references.21 The tablet and its subsamples were scanned with photon energies of 16.0 keV and 13.0 keV, respectively. After penetration through the sample, the X-rays were converted into visible light by a YAG: Ce scintillator (200 µm thickness). Projections were magnified by diffraction-limited microscope optics (1.25 × magnification for the tablet and 2 × magnification for the subsamples) and digitized in highresolution with a physical pixel size of 5.2 µm for the tablet and 3.25 µm for the subsamples, respectively (ORCA Flash 4.0 Scientific CMOS, Hamamatsu K.K., Shizuoka Pref., Japan). The exposure time and the sample-to-detector distance were set as 1 s and 20 cm, respectively, for the tablet and 0.8 s and 5 cm, respectively, for the subsamples after a series of confirmatory studies. For each acquisition, 900 projection images were captured with an angular step size of 0.15° for 180°. Flat-field images (i.e., X-ray illumination on the beam path without the sample) and dark-field images (i.e., X-ray illumination off) were also collected during each acquisition procedure to correct the electronic noise and variations in the X-ray source brightness. Imaging acquisition time for each CT scan was 30 min. 3D Structure Reconstruction. Hundreds of projected tomographic images of the tablet and its subsamples were reconstructed using software developed by SSRF to perform a direct filtered back-projection algorithm.22 PITRE (Phasesensitive X-ray Image Processing and Tomography REconstruction) for extraction of diffraction-enhanced imaging and PITRE_BM (PITRE Batch Manager) for tasks created via PITRE23 were applied to execute phase retrieval for propagation-based phase-contrast tomography to obtain projection data. After sinogram generation, 2D slices of the specimen were obtained. To enhance the quality of the reconstructed slices, propagation-based phase-contrast extraction was conducted. Then, all the images of slices of the tablet were processed by commercially available Amira software (version 6.01, FEI, USA) to acquire the full reconstructed 3D images of the tablet. For the slices of the subsamples in different ranges were first analyzed using Image-Pro Plus software (Version 7.0, Media Cybernetics, Inc., USA) to separate every subsample. Next, Amira software was employed to produce reconstructed 3D images of the subsamples.

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Depending on the magnitude of X-ray absorption for different materials of the MUPS tablet, the differences in gray value among materials were determined, and micro-structures of interest were extracted from the 3D models by segmentation. The 3D rendered data were analyzed with commercially available Image-Pro Analyzer 3D software (Version 7.0, Media Cybernetics, Inc., USA) to obtain the quantitative data of the structural volume. Quantitative Determination. Depending on the information (Table S1) detailed on the label of the MUPS tablet, chromatographic quantitative methods using HPLC-MS/MS or HPLC-ELSD for ingredients in the MUPS tablet were developed (Table S2). The THEO, SUC, LAC, SLS and DEP contents in the tablet and its subsamples were quantified on a highly sensitive HPLC-MS/MS system which consisted of an Agilent 1260 HPLC coupled to an Agilent G6460A triple quadrupole mass spectrometer (Agilent Technologies, Torrance, USA). Mass spectrometric detection was performed using a jet stream ESI source operating in the positive or negative ion mode. The instrument was optimized for THEO, SUC, LAC, SLS, and DEP by infusing a solution of each compound prepared in the corresponding solvent. Nitrogen was used as both the drying gas under a flow of 10 L/min at 300 °C and the sheath gas under a flow of 8 L/min at 350 °C. The pressure of the curtain gas and nebulizer was 15 psi and 45 psi, respectively. The instrument was operated with a capillary voltage of 3.5 kV. Multiple reaction monitoring (MRM) transitions were employed for data acquisition. Instrument control, data acquisition and quantification were performed by Agilent MassHunter Workstation software B.03.01. The optimized fragmentor voltage, collision energy (CE) and other detection parameters for each sample were listed in Table S2. Glyceryl monostearate, an organic material without appreciable UV absorbance or fluorescence properties, was detected using ELSD. The chromatographic experiments were performed on an Agilent 1290 HPLC system equipped with an Agilent G4218A ELSD with a drift tube temperature of 70 °C and a nebulizer gas pressure of 3.3 bar. Data were collected by Chemstation version B.04.03 software. The chromatographic conditions and detection parameters were optimized as shown in Table S2. All the methods were validated for the quantification of the corresponding ingredients in tablet samples. RESULTS AND DISCUSSION Tablet Structure Characterization by SR-µCT. The use of non-destructive X-ray micro-tomography allows characterization of the density distribution of an individual tablet and then further characterize its architecture. 3D images of the MUPS tablets are presented in the front and back directions (Figure 2, A) and 2D slices of the radial middle layer and the axial middle layer of the tablet are displayed (Figure 2, B). Pellets with a recognized circular shape are clearly visible in the images of slices based on the inherently different X-ray absorption of different compounds. Interestingly, the slice of the radial middle layer shows that pellets are distributed uniformly and unregularly in the radial direction of the MUPS tablet, whereas an enrichment zone of pellets in the axial direction is observed in the slice of the axial middle layer. This unusual inhomogeneous distribution of pellets in the MUPS tablet, which cannot be observed without the powerful 3D visualization tool, clearly has a profound significance for pharmaceutical design and drug efficacy.

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Partial magnification of the slice (Figure 2, C) indicate the MUPS tablet consists of three domains: a matrix layer (ML), a protective cushion layer (PCL) and pellets (PL). In addition, the ML and PCL structural domains without specific shape are shown as lighter areas with a relatively high gray value and darker areas with a low gray value, respectively. The PCL, which frequently wrap around the PL may be designed to serve as a blocker controlling the release of the drug in the PL. Furthermore, the PL, a spherical unit, consist of at least 3 lam-

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inar layers from the center to the outside which are termed here as the pellet core (PC), the drug layer (DL) and the coating layer (CL). The size of the PL for pharmaceutical use is determined between 0.5 and 1.2 mm from the virtual slices that can be dynamically transformed in Amira. With an average thickness approximately 100 µm, the CL is relatively uniform with a high degree of compactness (no gaps were found in CL). However, the PCs in different PL are not very uniform with detectable gaps.

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Figure 2. 3D images in the front and back directions (A), 2D slices in the radial and axial directions (B) and partial magnification of a 2D slice of the theophylline multiple-unit pellet system (MUPS) tablet (C). The tablet consists of three domains: a matrix layer (ML), a protective cushion layer (PCL) and pellets (PL), and the PL consist of at least 3 laminar layers from the center to the outside: the pellet core (PC), the drug layer (DL) and the coating layer (CL). 290

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Classification and Location of the Subsamples in the Tablet. The CT image values (gray levels) provide information on the material X-ray attenuation coefficient at each point in the image. Depending on the typical structure and the gray value of different materials contained in the subsamples, all the subsamples from the MUPS tablets could be classified into three groups: sample in the matrix layer (SML, Figure 3, A, B), sample containing the protective cushion layer (SCL, Figure 3, C, D) and sample of pellets (SPL, Figure 3, E, F). As shown in Figure 3, complete 3D images (Figure 3, A, C and E) and cross-sectional views (Figure 3, B, D and F) of representative subsamples are displayed in an attempt to characterize and compare these three types, and each subsample is identified using a specific code.

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SMLs have a relatively higher gray value arising from the ML. The different shades observed in the cross-sectional view of SMLs confirm that they are a mixture of multiple components. In particular, SCLs are those samples containing part of the relatively lower gray value domain of the PCL. The obvious contrast between the bright and dark areas suggests the diversity of the ingredients. Importantly, because of the indistinguishability of the two structural domains outside the PL (ML and PCL) when observed by the naked eye or an optical microscope, obtaining a subsample from the PCL alone is difficult. SPLs, a pellet or a part of a pellet with a curved surface, are to some extent easily distinguished. From the cross-section of SPLs, the color of the visible round CL is deeper than that of the PC, reflecting the differences between the CL and PC in composition.

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Analytical Chemistry

Figure 3. Classification of subsamples depends on the typical structure and the gray value. Three type of subsamples were identified: sample in the matrix layer (SML, A and B), sample containing the protective cushion layer (SCL, C and D) and sample of pellets (SPL, E and F). Complete 3D images (A, C and E) and cross-sectional views (B, D and F) are displayed to characterize and compare these three types.

Quantitative Assay. When the test specimen was the theoMUPS tablet can guide the determination of material in the phylline MUPS tablet, a fine powder obtained by grinding subsamples. In particular, GEM, which has a relatively high samples from at least 6 tablets was used. An appropriate content compared to the other lower density excipients (Table amount of the obtained powder was accurately weighed using 345 S1), is presumed to be contained in the PCL and thus need to an electronic balance (ME5, Sartorius, Germany) and disbe determined in the SCLs. The average proportion of each solved in the corresponding solvent in Table S3 to determine ingredient in the SMLs and SPLs demonstrate that compared the content of the ingredients (n=6). For subsamples of the with the content of individual excipients, the content of the MUPS tablet, different processing steps were employed to drug THEO is relatively high in both of the types, and the extract the corresponding ingredients contained in the different 350 SPLs contain slightly higher content of THEO than the SMLs. types of subsamples. To ensure that the ingredients in different In addition, the SMLs have a significantly higher content of LAC and a lower content of SUC than the SPLs. Furthermore, types of subsample were fully extracted and accurately measured, the solvents and concentration of each test solution were the SMLs contain almost no DEP, whereas the SPLs consist of thoroughly investigated, and they are listed in Table S3. Test hardly any SLS, and GEM is almost undetectable in the SMLs solutions of all the samples were centrifuged (12000 r/min, 5 355 and SPLs. There are multiple reasons to expect that the conmin), and the upper solutions were injected into the HPLCtents of some ingredients will fluctuate considerably in indiMS/MS system or HPLC-ELSD system. vidual subsamples, even though they originated from the same structural domain, including inhomogeneity caused by artifiThe contents of THEO, SUC, LAC, SLS, DEP and GEM in cial mixing, the complexity of ingredients and the small size the MUPS tablet, SMLs and SPLs were determined, and the 360 of subsamples. results are listed in Table 1. Quantitative analysis of the Table 1. Content of theophylline (THEO), sucrose (SUC), lactose (LAC), sodium lauryl sulfate (SLS), diethyl phthalate (DEP) and glyceryl monostearate (GEM) determined in the theophylline tablet, sample in the matrix layer (SML) and sample of pellets (SPL). (n=6, mean ± SD) Sample

THEO

SUC

LAC

SLS

DEP

GEM

Table

30.89 ± 1.10

22.34 ± 0.35

9.33 ± 0.44

0.67 ± 0.10

0.43 ± 0.02

16.78 ± 1.03

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SML

38.71 ± 5.04

20.92 ± 4.63

15.92 ± 2.06

1.85 ± 0.82

0.01 ± 0.01

0.00

SPL

40.81 ± 5.00

32.01 ± 10.19

4.10 ± 1.41

0.04 ± 0.01

0.71 ± 0.40

0.00

Because of the low density of GEM and its high content in the MUPS tablet (Table 1), SCLs were processed to measure the levels of GEM. The treatment process and quantitative method for the detection of GEM and other ingredients in a tablet sample were different; thus, the sample used for quantifying GEM could not be used in the detection of other ingredients. Based on this premise, the GEM content in the low gray value domain of the PCL contained in the SCLs needed to be further analyzed. Determination of GEM by HPLC-ELSD for SCL, volume measurement rely on Image pro Analyzer 3D software and calculation of GEM content in low gray value domain using the equation (1) and (2) were performed. The results are tabulated in Table 2.  = 10 ⁄ ×  C=

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 ⁄ 

× 100 %

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(1) (2) 400

and VCL is the volume of the low gray value domain in the SCL, which is calculated using Image-Pro Analyzer 3D software. The combination of the SR-µCT technique and the chromatographic method enables accurately calculation of GEM content in the PCL (low gray value domain) by analyzing SCLs, which are samples with impure structures. The results show that GEM takes up a large portion in the PCL. Additionally, the fluctuating values of GEM content in different SCL samples are attributed to several factors of which the difference of compaction degree in different regions of the MUPS tablet is an important issue. After segmentation based on the gray value of the subsamples, individual material volumes were calculated and the GEM content in the PCL was correlated with chromatographic analyses. The SR-µCT characterization of the MUPS tablet with comprehensive architectural detail was also used to localize the subsamples in the tablet. Classification of the subsamples based on their location in the MUPS tablet not only provided targeted quantitative determination, but also enhanced the reliability of the experimental approaches.

where M is the weight of GEM in the SCL; A is the peak area of GEM; a and b are the intercept and slope of the linear formula of GEM, respectively; M, A, a and b are determined and calculated via the HPLC-ELSD method; Vs is the volume of solvent used to dissolve the SCL; C represents the GEM 405 content in the low gray value domain; ρ is the density of GEM; Table 2. Analysis and determination of glyceryl monostearate (GEM) content in samples containing the protective cushion layer (SCLs) via HPLC-ELSD and SR-µCT. Type and number

Weight /mg

GEM content (M) /mg

Volume of low gray value area in SCL (VCL) /µm3

GEM content in low gray value area (C) /%

SCL1

0.413

0.053

8.65 × 107

63.86

SCL2

0.455

0.144

2.05 × 108

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0.107

1.67 × 10

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8

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SCL3

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0.682

SCL4

0.570

0.125

1.68 × 10

SCL5

0.447

0.095

1.36 × 108

72.76

SCL6

0.456

0.061

7.08 × 107

89.72

Spatial Distribution Analysis of Prescriptions. The diversity in component and contents of materials among the three types of subsamples originating from different structures in the MUPS tablet reflects their various roles and functions. The distribution of THEO in the different structural domains, likely due to the combined effects of the structure and excipients, undoubtedly relate to designing drug release behavior. SUC (sucrose power) and LAC in the ML are most likely used as diluents, whereas SUC (sugar spheres) detected in the PL may function as the porous inert core of a pellet, especially for a multiple-unit sustained release formulation. It is reasonable that SLS, a surfactant, can be detected only outside the PL, and the detection of DEP in the PL demonstrates its pharmaceutical function of improving plastic molding properties and its use as a cellulose resin plasticizer in the film coatings of PL. GEM, an excipient with multiple pharmaceutical functions including as a lubricant, emulsifier, stabilizer and controlled release material,24 present in the PCL which wraps around the PL is undoubtedly serving as part of the sustained release skeleton for the tablet to delay the release of the drug in the PL. Because of the complexity of the composition of a drug delivery system, establishing a quantitative method for all the materials is challenging and unnecessary. The investigated MUPS

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GEM content in low gray value area /% (Mean ± SD)

73.88 ± 9.15

tablet also contains acacia, starch, magnesium stearate, talc, white beeswax, cetyl alcohol, myristyl alcohol and cellulose acetate phthalate, the detection of which has not yet been integrated into the assay. Considering both the pharmaceutical function of excipients and the X-ray absorption signal observed in different structures, it is cellulose acetate phthalate which possesses the lowest density, is most likely locate in the CL of the PL.25 The three main structures are visualized in a single slice extracted from the whole slice of a radial middle layer of the MUPS tablet (Figure 4). The morphology, material distribution, component difference and other actual characteristics of the different structures in the MUPS tablet are presented from a 2D perspective in situ and independently. When segmented from the complete tablet image, the ML is seen to contain many spherical voids. The lighter color of the ML is a consequence of the high-density composition materials THEO, SUC, LAC, SLS, etc. The PCL with a noticeable dark gray color in the image of the whole tablet slice is extracted to be layer as a waistcoat like coating to pellets. The lower absorption of Xrays with a lower gray value of the PCL also demonstrates a high content of low-density GEM. The characteristics of the various structures in PL, such as the loose and porous pellet

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Analytical Chemistry core and the dense coating layer, are also fully captured in SRµCT analysis. In our research, the unique architecture of the MUPS tablet and material spatial distribution profile have

prominent significance for the sustained release behavior of the drug, which is beyond the scope of the present report but deserves further investigation.

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Figure 4. Presentation of the components in a two-dimensional single slice containing the matrix layer, protective cushion layer and pellets extracted from the whole slice of a radial middle layer of the MUPS tablet.

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CONCLUSION Material spatial distribution in solid dosage forms, especially in those with complex architectures, has specific functional significance for drug delivery. In most cases, relationships between material distribution and microstructure of dosage forms have not yet been adequately investigated. In this research, a novel methodology has been established for the first time to combine spatial structural detail and material composition of a complex drug delivery system via the combination of SR-µCT and chromatography. In contrast to conventional morphological characterization, the complexity of the internal hierarchical structure determined by SR-µCT can provide insight into the material distribution in small units of drug delivery systems. By combining SR-µCT data with chromatographic analyses, this research offers new capabilities in determining material distribution profiles in structural domains. The general benefits of visualizing internal architecture as a result of a production process, linked to simple sample pretreatment for chromatographic determination based upon particle sample material density differentiation and volume calibration of SRµCT, provide new knowledge to aid the architecture based dosage form design and development. The evidence and findings show the feasibility of this powerful approach in revealing the 3D material distribution in situ of the microstructure in drug delivery systems.

ASSOCIATED CONTENT

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The supporting information is available free of charge on the ACS Publications website. The supporting information displayed the prescription of the theophylline multiple-unit pellet system tablet and the optimized determination conditions for theophylline and the excipients. (PDF)

AUTHOR INFORMATION Corresponding Author

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* Prof. Lixin Sun School of Pharmacy, Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China; Tel: +86-24-23986365; E-mail: [email protected]; * Prof. Jiwen Zhang Center for Drug Delivery System, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No. 501 of Haike Road, Shanghai 201203, China; Tel/Fax +86-21-20231980; E-mail: [email protected]; * Assoc. Prof. Xianzhen Yin Center for Drug Delivery System, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No. 501 of Haike Road, Shanghai 201203, China; Tel/Fax +86-21-20231980; E-mail: [email protected]

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The manuscript was written and discussed through contributions of all authors. L.Z. and C.W. are specialized in quantitative analysis, and X.Y. and L.W. contributed to the SR-µCT experiments.

Supporting Information L.S. and J.Z. guided the research. All authors have given approval to the final version of the manuscript.

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ACKNOWLEDGMENT The authors are grateful for the financial support from the National Natural Science Foundation of China (No.81573392, 81773645), National Science, Intergovernment Key International Scientific and Technological Innovation Cooperation (2016YFE0125100), and Technology Major Project (2017ZX09101001-006) and China Postdoctoral Science Foundation (2017M610284). Our thanks go to the BL13W1SSRF beamline of the SSRF for the precious beamtime and for the help from the team.

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