The Research Center Pharmaceutical Engineering (RCPE) is funded within the framework of COMET - Competence Centers for Excellent Technologies by BMK, BMDW, Land Steiermark and SFG. The COMET program is managed by the FFG.

1. Differential scanning calorimetry (DSC)
<ol>
	<li>Instrument preparation
	<ol>
		<li>Use a differential scanning calorimeter equipped with an intracooler, an autosampler, and the software for instrument control and data analysis.</li>
		<li>Switch on the nitrogen gas supply and set the pressure between 0.2&#8211;0.5 bar and power up the DSC instrument and the automatic sample changer.</li>
		<li>Open the software and activate the standby mode by clicking on the Yes button. Allow equilibration of the device for at least one hour</li>
		<li>Purge the furnace with nitrogen, click on the New Method icon and go to Method Definition. Activate the Temperature Modulation option in the overview window.&#160; Go to header tab and select the method by clicking on &#8220;sample&#8221;.</li>
		<li>Go to the tab Temperature Program, select Purge 2 MFC and on Protective MFC, both at 50 mL/min.</li>
		<li>Insert the following measurement method: Standby at 20 &#176;C, heating cycle at 5 K/min from 20 &#176;C to above the melting temperature of lipid, isothermal holding at this temperature for 5 min, cooling cycle to 0 &#176;C at 5 K/min to -20 &#176;C, final emergency reset temperature at a temperature 10 degrees &#176;C above the highest temperature of the program, and final standby temperature at 20 &#176;C.</li>
		<li>Go to the calibration tab and select the appropriate temperature and sensitivity file. Save the method</li>
	</ol>
	</li>
	<li>Sample preparation and measurement
	<ol>
		<li>Weigh 3&#8211;4 mg of each sample into aluminum crucibles. Record the exact weight loaded into each crucible and seal the aluminum crucible with a pierced lid.</li>
		<li>Place the crucibles in the autosampler tray and activate the autosampler mode in the software and load related method for each sample.</li>
		<li>Fill out the sample position, sample name, and weight of each sample, and the position of reference crucible in the Sample Tray View window and start the measurements.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Open the raw data using the software for data analysis and plot the temperature versus heat flow, by clicking on the button &#8220;X-time / X-temperature&#8221;</li>
		<li>On the popped out window, click on &#8220;Hide isothermal segments&#8221;. On the left side of the screen, select only the curves that are to be analyzed (e.g. unclick the &#8220;Additional&#8221; data).</li>
		<li>Check the thermal behavior of lipids as endothermic and exothermic events of absorbed or released energy in the form of heat, respectively, as a function of temperature.</li>
		<li>Click on the curve, followed by Evaluation and Area, to calculate the enthalpy of fusion as the area under the curve of endotherms.</li>
		<li>Select the integration boundaries by moving the vertical lines around 2 to 3 degrees Celsius before and after the onset and endpoint of the peak.</li>
		<li>Select a linear baseline for the peak integration. The area between the curve and the baseline is proportional to the change in enthalpy. Click on Apply to finish the calculation. Similarly, calculate the enthalpy of crystallization as the area under the curve of exotherms</li>
		<li>Determine the onset of melting temperature (To) by clicking on the curve to be analyzed and then on Evaluation and Onset.</li>
		<li>Select the quantification boundaries by moving the vertical lines to the most straight section of the curve. This is usually around 5-10&#176;C before and after the peak. Then, determine the melting temperature by clicking on the curve to be analyzed, followed by Evaluation and Peak. The obtained value is the peak maximum.</li>
	</ol>
	</li>
</ol>
2. Small- and wide-angle x-ray scattering (SWAXS)
<ol>
	<li>Instrument preparation
	<ol>
		<li>Use an x-ray scattering system, composing a point-focusing camera fixed to a sealed-tube x-ray generator and equipped with a control unit and related software.</li>
		<li>Use cooper (&#955; = 1.54 &#197;) at 50 kV and 1 mA as an x-ray source and two linearly positioned sensitive detectors to cover both small- and wide-angle x-ray scattering regions.</li>
		<li>Ensure safety requirements to protect from x-ray exposure.</li>
		<li>&#160;Switch on the cooling water system on the control unit, the vacuum pump, the gas valves, and the power and safety control system.</li>
		<li>Switch on the voltage control and purging valves for the detectors at a gas flow between 10&#8211;20 mL/min.</li>
		<li>Switch on the x-ray tube and the standby option and wait approximately 10 min. Switch off the standby mode and power up the x-ray tube to full power (&#62;50kV) and wait at least 30 minutes.</li>
		<li>Start the control software and click on RESET TPF. Choose Tugsten filter and set position. Go to Position to fix the position of the Tungsten filter</li>
	</ol>
	</li>
	<li>Sample preparation and measurement
	<ol>
		<li>Make sure that the samples are available as fine powder. If necessary, grind the samples gently at low temperatures to provide a fine powder.</li>
		<li>Fill the samples into special glass capillaries with an external diameter of approximately 2 mm, avoiding any air entrapment in the capillaries. Seal the glass capillary with wax and place it carefully into the capillary holder.</li>
		<li>Switch on the motor for sample rotation and close the vacuum valve until the pressure is beneath 5 mbar.</li>
		<li>In the software, fix the measurements setting by selecting a position resolution of 1024. Fix the exposure time to 1200 s.</li>
		<li>Setup the energy limits by clicking on the tap Tools, then click on energy and resolution, and click on restart. Setup the energy limits to a suitable range between 400-900.</li>
		<li>Open the safety shutter and start the measurements. Ensure that the measurement window shows a maximum of 80 counts per second. If this is not given, adapt the filter position.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Export the data as p00 files. The data consists of the intensity of transmission and absorption versus the channel number and the diffraction angle.</li>
		<li>Transfer the data for evaluation to a statistical software and correct the data by normalizing the intensities using the scattering mass measured with the Tungsten filter.</li>
		<li>Create a plot of normalized intensity versus two times the diffraction angle [(2&#920;) 2xtheta].</li>
		<li>Use the function of &#8220;screen reader&#8221; to find diffraction peaks in SAXS and WAXS regions.</li>
		<li>Apply Bragg&#39;s equation to compute the diffraction peaks with maximum intensity into short and long d-spacing for WAXS and SAXS regions, respectively.</li>
		<li>Calculate the ratios of the peak position of the SAXS region to find out the crystal symmetry of the lipids (e.g., lamellar, hexagonal, cubic).</li>
		<li>Use the main diffraction peak of the SAXS region to quantify the crystallite thickness (D). Fit the peak into a Gaussian function via classical least squares and obtain the FWHM by clicking on Analysis, Peaks and baselines, Peak analyzer, Open dialog.</li>
		<li>On the popped out window, select the option &#8220;Fit Peaks Pro&#8221;. Select a constant baseline with y = 0, and select the main diffraction peak of the SAXS region and click on &#8220;Fit control&#8221; to select the peak fit parameters.</li>
		<li>Choose the GaussAmp function. Set the parameters y_0, xc_1 and A_1 as fixed and obtain the FWHM from the fit. Use the Scherrer equation to compute the crystallite thickness.</li>
	</ol>
	</li>
</ol>
3. Dissolution tests
<ol>
	<li>API release from coated multiparticulate systems
	<ol>
		<li>Use USP apparatus 2 (paddle) for dissolution studies.</li>
		<li>Fill the dissolution test vessels with phosphate buffer pH 6.8, and heat to 37 &#176;C.</li>
		<li>Weigh triplicates of samples of coated particles equivalent to a single dose of API, and place the samples into dissolution test vessels.</li>
		<li>Start the paddle at a speed of 100 rpm.</li>
		<li>Set the auto-sampler to take samples of 1 mL at the following sampling points: 30 min, 60 min, 90 min, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, and 24 h.</li>
		<li>Analyze the samples via a suitable HPLC method5,7,17.</li>
		<li>Analyze the data by plotting the cumulative API release versus time.</li>
		<li>Carry out the experiments for stored samples under long-term (25 &#176;C, 60% relative humidity) and accelerated (40 &#176;C and 70% relative humidity).</li>
	</ol>
	</li>
	<li>API release from solid lipid-nanoparticles (SLN)
	<ol>
		<li>Prepare simulated lung fluid (SLF) by mixing 0.02% (w/w) of dipalmitoylphosphatidylcholine in Dulbecco&#39;s phosphate buffer saline (D-PBS), with the following composition: KCl (2.683 mM), KH2PO4 (1.47 mM), NaCl (136.893 mM), Na2HPO4&#183;2H2O (8.058 mM), CaCl2&#183;2H2O (0.884 mM), and MgCl2&#183;6H2O (0.492 mM). Pre-heat it at 37 &#176;C.</li>
		<li>Use dialysis cassettes with a cellulose membrane bag of a controlled cut-off of 7,000 Da in triplicate for each sample.</li>
		<li>Assign one dialysis cassette for each sampling time: 0.5 h, 1.5 h, 3 h, 5 h, 7 h, and 24 h. Hydrate the dialysis cassettes for 2 min by immersing them in SLF. Then, carefully dry their surface with soft paper towels.</li>
		<li>Inject 3 mL of the sample (lipid-nanosuspension), equivalent to 600 &#181;g of dexamethasone, into each cassette.</li>
		<li>Immerse each cassette in 200 mL of SLF at 37 &#176;C (sink conditions) and agitate the system at 125 rpm.</li>
		<li>Take 200 mg samples from inside the cassette using a syringe at each determined sampling time.</li>
		<li>Determine the API content using the developed HPLC-MS method18.</li>
		<li>Calculate the API released from SLN by mass balance, according to18, briefly as the difference between the total amount of API in SLN and the remaining amount of API after sampling.</li>
		<li>Repeat the process for stored samples.</li>
	</ol>
	</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+and+structural+behaviour+of+crude+palm+oil:+Crystallisation+at+very+low+cooling+rate.">Thermal and structural behaviour of crude palm oil: Crystallisation at very low cooling rate.</a> European Journal of Lipid Science and Technology. 109 (4), 410-421 (2007).</li><li>Askin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simultaneous+differential+scanning+calorimetry-X-ray+diffraction+study+of+olanzapine+crystallization+from+amorphous+solid+dispersions.">A simultaneous differential scanning calorimetry-X-ray diffraction study of olanzapine crystallization from amorphous solid dispersions.</a> Molecular Pharmaceutics. 17 (11), 4364-4374 (2020).</li><li>Clout, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+differential+scanning+calorimetry+-+synchrotron+X-ray+powder+diffraction:+A+powerful+technique+for+physical+form+characterization+in+pharmaceutical+materials.">Simultaneous differential scanning calorimetry - synchrotron X-ray powder diffraction: A powerful technique for physical form characterization in pharmaceutical materials.</a> Analytical Chemistry. 88 (20), 10111-10117 (2016).</li><li>Jendrzejewska, I., Goryczka, T., Pietrasik, E., Klimontko, J., Jampilek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+and+thermal+analysis+of+selected+drugs+containing+acetaminophen.">X-ray and thermal analysis of selected drugs containing acetaminophen.</a> Molecules. 25 (24), 5909 (2020).</li><li>Righetti, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+Polymers+Investigated+by+Temperature-Modulated+DSC.">Crystallization of Polymers Investigated by Temperature-Modulated DSC.</a> Materials. 10 (4), 442 (2017).</li><li>Sauer, B. B., Kampert, W. G., Neal Blanchard, E., Threefoot, S. A., Hsiao, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+modulated+DSC+studies+of+melting+and+crystallization+in+polymers+exhibiting+multiple+endotherms.">Temperature modulated DSC studies of melting and crystallization in polymers exhibiting multiple endotherms.</a> Polymer. 41 (3), 1099-1108 (2000).</li><li>Ali, F., Kumar, R., Lal Sahu, P., Singh, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+characterization+and+compatibility+study+of+roflumilast+with+various+pharmaceutical+excipients.">Physicochemical characterization and compatibility study of roflumilast with various pharmaceutical excipients.</a> Journal of Thermal Analysis and Calorimetry. 130, 1627-1641 (2017).</li></ol>

Authors disclose any and all conflicts of interest.

Powder x-ray diffraction and DSC were described in this manuscript as gold standards for the solid-state analysis of LBEs. Powder x-ray diffraction has the outstanding advantage of processing the measurements in situ, with minimum solid-state manipulation of samples during the measurements. Moreover, the same-filled capillaries can be stored under different conditions after initial measurements to investigate the solid-state alteration during storage. In this work, we focused on the wide- and small-angle regions...

Lipids are a class of materials that contain long-chain aliphatic hydrocarbons and their derivatives. They cover a broad range of chemical structures, including fatty acids, acylglycerols, sterols and sterol esters, waxes, phospholipids, and sphingolipids1. The use of lipids as pharmaceutical excipients started in 1960 for embedding drugs in a wax matrix to provide sustained release formulations2. Since then, lipid-based excipients (LBEs) have gained extensive attention for diverse applications, such as modified drug release, taste-masking, drug encapsulation, and enhanced drug bioavailability. LBEs can be applied in a broad range of pharmaceutical dosage forms via versatile manufacturing processes, namely, hot-melt coating, spray-drying, solid lipid extrusion, 3D printing, tableting, and high-pressure homogenization, among others. Dosage forms such as tablets, orally disintegrating films, multiparticulate systems, nano and microparticles, pellets, and 3D-printed forms are the result2,3,4.
LBEs possess the &#34;General Recognized as Safe&#34; status, low toxicity, good biocompatibility, and improved patient tolerance. Their natural origin and broad availability allow them to empower green and sustainable pharmaceutical manufacturing. Nevertheless, the use of LBEs has been associated with unstable dosage forms. Alterations in the properties of lipid-based products after storage have been widely reported. The solid-state of LBEs and the existence of lipid polymorphism are considered the main reasons for the instability of lipid-based dosage forms5,6,7,8.
The mechanical and physical properties of lipids are closely related to their crystallization properties and the structure of their crystal network, which shows distinct hierarchies of structural organization. When lipids are used in the manufacturing of pharmaceutical products, the crystal structure is affected by the process parameters applied, such as temperature, organic solvents, shear, and mechanical forces, which in turn affects the performance of the pharmaceutical product5,7,9,10,11,12. To understand this structure-function relationship, it is important to know the fundaments of lipid crystallization and crystal structure and analytical methods to screen them.
At the molecular level, the smallest unit of a lipid crystal is termed a &#34;unit cell.&#34; A regular three-dimensional repetition of unit cells builds the crystal lattices, with stronger molecular interactions alongside their lateral directions than the longitudinal ones, explaining the layered construction of lipid crystals. The repeated cross-sectional packing of hydrocarbon chains is known as sub-cell1,12,13 (Figure 1). Lamellae are the lateral packing of lipid molecules. In the crystal package, the interfaces between different lamellae are made of methyl end groups, whereas the polar glycerol groups are placed at the interior parts of the lamella14. To differentiate each fatty-acid chain in the lamella, the term leaflet is employed, which represents a sublayer composed of single fatty-acid chains. Acylglycerols can be arranged in double (2L) or triple (3L) leaflet chain lengths14. The surface energy of the lamellae drives them to epitaxially stack to each other, to provide nano-crystallites. Different processing factors such as cooling temperature and rate affect the number of stacked lamellae and thereby, the crystallite thickness (~10-100 nm). Aggregation of crystallites leads to the formation of spherulites in micro-scale, and the aggregation of spherulites provides the crystal network of LBEs with defined macroscopic behavior13.
Solid-state transitions start at the molecular level. The geometrical transition from one sub-cell to another is called polymorphism. Three major polymorphs of &#945;-, &#946;&#39;-, and &#946;-form are usually found in acylglycerols, ordered according to increased stability. Tilting of the lamella with respect to end-groups occurs during polymorphic transitions1,13. Storage and melt-mediated polymorphic transitions are experienced by LBEs. Storage transitions occur when the metastable form is stored below its melting temperature, whereas melt-mediated transitions happen as the temperature rises above the melting point of a metastable form provoking melting and successive crystallization of the more stable form.
Furthermore, phase separation and crystal growth can also occur. Phase separation is driven by initial multiphasic crystallization and growth of one phase or more. Particle-particle interactions, including sintering, molecular interactions, microstructural features, and foreign components, can also trigger crystal growth1,5.
Monitoring the solid-state transitions of LBEs and their impact on the performance of dosage forms is of significant importance. Among others, differential scanning calorimetry (DSC) and x-ray diffraction, specifically simultaneous small and wide-angle X-ray scattering (SWAXS), are two gold standards for assessing lipid solid-state.
DSC is commonly used to measure the enthalpy changes of the material of interest associated with the heat flow as a function of time and temperature. The method is widely used for the screening of thermal behavior of lipids, such as possible pathways of melting and crystallization, corresponding temperature and enthalpy of different polymorphic forms, as well as minor and main fractions of lipid compositions. These data can be used to depict the heterogeneity, multiple phases, and lipid polymorphism5,7,13.
X-ray diffraction techniques are the most powerful methods for structure determination in the solid state. Possessing ordered nanostructure with repeated lamellae, the reflection of x-ray beam from lipid crystals can be investigated using Bragg&#39;s law:
d = &#955;/2sin&#952;&#160; &#160; &#160;(Equation 1)
where &#955; is the x-ray wavelength of 1.542 &#197;, &#952; is the diffraction angle of the scattered beam, and d is the interplanar spacing of repeated layers, defined as lamella length in lipids. The small-angle region of the x-ray can be perfectly used to detect the long-spacing pattern and calculate the lamella length (d). The larger the repeated distance d, the smaller the scattering angle (1-15&#176;, small-angle region) since d is inversely proportional to sin &#952;. The sub-cell arrangement of lipids can be characterized as the short-spacing pattern in the wide-angle region of the x-ray diffraction. Both the long- and short-spacing patterns of lipids (lamella length and sub-cell arrangement) can be used to indicate the monotropic polymorphic transformation. For example, the &#945;-form (hexagonal) can be altered to &#946; (triclinic) due to a change in the angle of tilt of the chains, with alterations in the lamella length (long-spacing pattern, in the small-angle region, 1-15&#176;) and in the cross-sectional packing mode (short-spacing pattern, in the wide-angle region, 16-25&#176;) (Figure 2).
The information obtained from the SAXS region can further be used to investigate the crystal growth by measuring its thickness (D) via the Scherrer equation15:
D = K&#955;/FWHMcos&#952;&#160; &#160; &#160;(Equation 2)
Where, FWHM is the width in radians of the diffraction maximum measured at a half-way height between the background and the peak, generally known as full-width at half-maximum (FWHM); &#952; is the diffraction angle; &#955; is the X-ray wavelength (1.542 &#197;) and K (Scherrer constant) is a dimensionless number that provides information about the shape of the crystal (in case of the absence of detailed shape information K = 0.9 is a good approximation). Please note that the Scherrer equation can be used to estimate mean crystal sizes of up to about 100 nm since the peak broadening is inversely proportional to the crystallite size. Therefore, its application is useful for determining the thickness of nanoplatelets and, indirectly, the number of aggregated lamellae. Examples of using this well-known approach for screening the crystal properties of lipids in the pharmaceutical formulation development and the corresponding instability in product performance can be found in5,12,16,17,18.
Monitoring the solid-state of LBEs within each developmental stage through well-established analytical techniques provide an effective strategy for designing high-performance manufacturing processes and stable lipid-based pharmaceutical products.
This publication presents the critical application of a comprehensive solid-state analysis of LBEs for monitoring the changes in solid-state and its correlation to the alteration in the release profile of active pharmaceutical ingredient (API) from the pharmaceutical dosage form. Multiparticulate systems based on lipid-coated API crystals via hot-melt coating, and nano-lipid suspensions produced via high-pressure homogenization are taken as case studies. The focus of this publication is the application of powder x-ray diffraction and DSC as analytical tools. The first two examples show the effect of polymorphic transformation and crystal growth, respectively, on the alteration in API release from coated samples. The last example reveals the correlation between the stable solid-state of lipid and the stable performance of the pharmaceutical product in lipid-coated multiparticulate systems and in nano-lipid suspensions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Cassettes with a cellulose membrane bag with a cut-off of 7000 Da, Thermo Scientific Slide-A-Lyzer 7K</td>
			<td>Fisher Scientific Inco, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control software of x-ray system</td>
			<td>HECUS dedicated house equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control unit of x-ray system</td>
			<td>HECUS dedicated house equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Differential scanning calorimeter (DSC) aluminum crucibles and lids</td>
			<td>Netzsch, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Differential scanning calorimeter DSC 204 F1 Phoenix (NETZSCH, Germany).</td>
			<td>Netzsch, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dipalmitoylphosphatidylcholine (DPPC)</td>
			<td>Sigma-Aldrich</td>
			<td>850355P</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissolution paddle apparatus II, Erweka DT 828 LH</td>
			<td>Erweka GmbH, Langen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dynasan 116</td>
			<td>IOI OLEO</td>
			<td></td>
			<td>Tripalmitin</td>
		</tr>
		<tr>
			<td>Geleol</td>
			<td>Gattefosse</td>
			<td></td>
			<td>Glyceryl monosterarate&#160;</td>
		</tr>
		<tr>
			<td>KCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>529552</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Kolliphor P 188</td>
			<td>BASF Chem Trade</td>
			<td></td>
			<td>Poloxamer 188&#160;</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Netzsch DSC 204F1 Software Version 8.0.1</td>
			<td>Netzsch, Germany</td>
			<td>6.239.2-64.51.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Origin Pro (OriginLab, Northampton, MA) (statistical software</td>
			<td>OriginLab, Northampton, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proteous Analysis Software</td>
			<td>Netzsch, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 65</td>
			<td></td>
			<td></td>
			<td>Polysorbate 65</td>
		</tr>
		<tr>
			<td>Witepsol PMF 1683</td>
			<td>IOI OLEO</td>
			<td></td>
			<td>Triglycerol ester of stearatic/palmitic acid (partially esterified)</td>
		</tr>
		<tr>
			<td>Witepsol PMF 282</td>
			<td>IOI OLEO</td>
			<td></td>
			<td>Diglycerol ester of stearic acid&#160;</td>
		</tr>
		<tr>
			<td>X-ray HECUS system composed of a point-focusing camera and two linearly positioned sensitive detectors</td>
			<td>HECUS dedicated house equipment</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a package of established analytical tools to investigate the solid-state alteration of lipid-based excipients

Lipid-based excipients (LBEs) are low-toxic, biocompatible, and natural-based, and their application supports the sustainability of pharmaceutical manufacturing. However, the major challenge is their unstable solid-state, affecting the stability of the pharmaceutical product. Critical physical properties of lipids for their processing-such as melt temperature and viscosity, rheology, etc.-are related to their molecular structure and their crystallinity. Additives, as well as thermal and mechanical stress involved in the manufacturing process, affect the solid-state of lipids and thus the performance of pharmaceutical products thereof. Therefore, understanding the alteration in the solid-state is crucial. In this work, the combination of powder x-ray diffraction and differential scanning calorimetry (DSC) is introduced as the gold standard for the characterization of lipids' solid state. X-ray diffraction is the most efficient method to screen polymorphism and crystal growth. The polymorphic arrangement and the lamella length are characterized in the wide- and small-angle regions of x-ray diffraction, respectively. The small-angle x-ray scattering (SAXS) region can be further used to investigate crystal growth. Phase transition and separation can be indicated. DSC is used to screen the thermal behavior of lipids, estimate the miscibility of additives and/or active pharmaceutical ingredients (API) in the lipid matrix, and provide phase diagrams. Four case studies are presented in which LBEs are either used as a coating material or as an encapsulation matrix to provide lipid-coated multiparticulate systems and lipid nanosuspensions, respectively. The lipid solid-state and its potential alteration during storage are investigated and correlated to the alteration in the API release. Qualitative microscopical methods such as polarized light microscopy and scanning electron microscopy are complementary tools to investigate micro-level crystallization. Further analytical methods should be added based on the selected manufacturing process. The structure-function-processability relationship should be understood carefully to design robust and stable lipid-based pharmaceutical products.

Correlation between polymorphic transition of lipid and API release in lipid-coated API crystals: 
API crystals coated with glycerol monostearate are measured via DSC and x-ray directly after coating and after 3 months of storage under accelerated conditions (40 &#176;C, 75% relative humidity)7. Glyceryl monostearate is a multiphasic system containing 40%-55% monoglycerides, 30%-45% diglycerides, and 5%-15% glycerides, mainly tristearin19. The...

Watch this Scientific Journal Video about A Package of Established Analytical Tools to Investigate the Solid-State Alteration of Lipid-Based Excipients at JoVE.com

A Package of Established Analytical Tools to Investigate the Solid-State Alteration of Lipid-Based Excipients

This publication shows the application of x-ray diffraction and differential scanning calorimetry as gold standards for investigating the solid-state of lipid-based excipients (LBEs). Understanding the solid-state alteration in LBEs and its effect on the performance of pharmaceutical products thereof is the key factor for manufacturing robust lipid-based dosage forms.

Lipid-based excipients (LBEs) are low-toxic, biocompatible, and natural-based, and their application supports the sustainability of pharmaceutical ...

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1.9K Views.  GmbH. This publication shows the application of x-ray diffraction and differential scanning calorimetry as gold standards for investigating the solid-state of lipid-based excipients (LBEs). Understanding the solid-state alteration in LBEs and its effect on the performance of pharmaceutical products thereof is the key factor for manufacturing robust lipid-based dosage forms.

Video: A Package of Established Analytical Tools to Investigate the Solid-State Alteration of Lipid-Based Excipients

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Combining Solid-state and Solution-based Techniques: Synthesis and Reactivity of Chalcogenidoplumbates(II or IV)

The phases of &#34;PbCh2&#34; (Ch = Se, Te) are obtained from solid-state syntheses (i.e., by the fusion of the elements under inert conditions in silica glass ampules). Reduction of such phases by elemental alkaline metals in amines affords crystalline chalcogenidoplumbate(II) salts comprised of [PbTe3]2- or [Pb2Ch3]2- anions, depending upon which sequestering agent for the cations is present: crown ethers, like 18-crown-6, or cryptands, like [2.2.2]crypt. Reactions of solutions of such anions with transition-metal compounds yield (poly-)chalcogenide anions or transition-metal chalcogenide clusters, including one with a &#181;-PbSe ligand (i.e., the heaviest-known CO homolog).
In contrast, the solid-state synthesis of a phase of the nominal composition &#34;K2PbSe2&#34; by successive reactions of the elements and by the subsequent solvothermal treatment in amines yields the first non-oxide/halide inorganic lead(IV) compound: a salt of the ortho-selenidoplumbate(IV) anion [PbSe4]4-. This was unexpected due to the redox potentials of Pb(IV) and Se(-II). Such methods can further be applied to other elemental combinations, leading to the formation of solutions with binary [HgTe2]2- or [BiSe3]3- anions, or to large-scale syntheses of K2Hg2Se3 or K3BiSe3 via the solid-state route.
All compounds are characterized by single-crystal X-ray diffraction and elemental analysis; solutions of plumbate salts can be investigated by 205Pb and 77Se or 127Te NMR techniques. Quantum chemical calculations using density functional theory methods enable energy comparisons. They further allow for insights into the electronic configuration and thus, the bonding situation. Molecular Rh-containing Chevrel-type compounds were found to exhibit delocalized mixed valence, whereas similar telluridopalladate anions are electron-precise; the cluster with the &#181;-PbSe ligand is energetically favored over a hypothetical CO analog, in line with the unsuccessful attempt at its synthesis. The stability of formal Pb(IV) within the [PbSe4]4- anion is mainly due to a suitable stabilization within the crystal lattice.

Combining Solid-state and Solution-based Techniques: Synthesis and Reactivity of ChalcogenidoplumbatesII or IV

The synthesis of chalcogenidoplumbates(II,IV) via the in situ reduction of nominal "PbCh2" (Ch = Chalcogen) and via a solid-state reaction and subsequent solvothermal reactions is presented. Additionally, reactivities of plumbate(II) solutions are portrayed, which yield the heaviest-known CO homolog known to date: the &#181;-PbSe ligand.

The phases of &#34;PbCh2&#34; (Ch = Se, Te) are obtained from solid-state syntheses (i.e., by the fusion of the elements under inert conditions in silica ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Failure of Cleaning Verification in Pharmaceutical Industry Due to Uncleanliness of Stainless Steel Surface

The aim of this work is to identify the parameters that affect the recovery of pharmaceutical residues from the surface of stainless steel coupons. A series of factors were assessed, including drug product spike levels, spiking procedure, drug-excipient ratios, analyst-to-analyst variability, intraday variability, and cleaning procedure of the coupons. The lack of a well-defined procedure that consistently cleaned the coupon surface was identified as the major contributor to low and variable recoveries. Assessment of cleaning the surface of the coupons with clean-in-place solutions (CIP) gave high recovery (&#62;90%) and reproducible results (Srel&#8804;4%) regardless of the conditions that were assessed previously. The approach was successfully applied for cleaning verification of small molecules (MW &#60;1,000 Da) as well as large biomolecules (MW up to 50,000 Da).

The lack of a well-defined procedure that consistently cleaned coupon surfaces was identified as the major contributor to low and variable recoveries in cleaning verification. This manuscript describes the correct protocol of cleaning stainless steel coupons.

The aim of this work is to identify the parameters that affect the recovery of pharmaceutical residues from the surface of stainless steel coupons. A ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

High-Contrast and Fast Photorheological Switching of a Twist-Bend Nematic Liquid Crystal

Smart viscoelastic materials that respond to specific stimuli are one of the most attractive classes of materials important to future technologies, such as on-demand switchable adhesion technologies, actuators, molecular clutches, and nano-/microscopic mass transporters. Recently it was found that through a special solid-liquid transition, rheological properties can exhibit significant changes, thus providing suitable smart viscoelastic materials. However, designing materials with such a property is complex, and forward and backward switching times are usually long. Therefore, it is important to explore new working mechanisms to realize solid-liquid transitions, shorten the switching time, and enhance the contrast of rheological properties during switching. Here, a light-induced crystal-liquid phase transition is observed, which is characterized by means of polarizing light microscopy (POM), photorheometry, photo-differential scanning calorimetry (photo-DSC), and X-ray diffraction (XRD). The light-induced crystal-liquid phase transition presents key features such as (1) fast switching of crystal-liquid phases for both forward and backward reactions and (2) a high contrast ratio of viscoelasticity. In the characterization, POM is advantageous in offering information on the spatial distribution of LC molecule orientations, determining the type of liquid crystalline phases appearing in the material, and studying the orientation of LCs. Photorheometry allows measurement of a material&#8217;s rheological properties under light stimuli and can reveal the photorheological switching properties of materials. Photo-DSC is a technique to investigate thermodynamic information of materials in darkness and under light irradiation. Lastly, XRD allows studying of microscopic structures of materials. The goal of this article is to clearly present how to prepare and measure the discussed properties of a photorheological material.

This protocol demonstrates the preparation of a photorheological material that exhibits a solid phase, various liquid crystalline phases, and an isotropic liquid phase by increasing temperature. Presented here are methods for measuring the structure-viscoelasticity relationship of the material.

Smart viscoelastic materials that respond to specific stimuli are one of the most attractive classes of materials important to future technologies, such ...

Analyzing Melts and Fluids from Ab Initio Molecular Dynamics Simulations with the UMD Package

We have developed a Python-based open-source package to analyze the results stemming from ab initio molecular-dynamics simulations of fluids. The package is best suited for applications on natural systems, like silicate and oxide melts, water-based fluids, and various supercritical fluids. The package is a collection of Python scripts that include two major libraries dealing with file formats and with crystallography. All the scripts are run at the command line. We propose a simplified format to store the atomic trajectories and relevant thermodynamic information of the simulations, which is saved in UMD files, standing for Universal Molecular Dynamics. The UMD package allows the computation of a series of structural, transport and thermodynamic properties. Starting with the pair-distribution function it defines bond lengths, builds an interatomic connectivity matrix, and eventually determines the chemical speciation. Determining the lifetime of the chemical species allows running a full statistical analysis. Then dedicated scripts compute the mean-square displacements for the atoms as well as for the chemical species. The implemented self-correlation analysis of the atomic velocities yields the diffusion coefficients and the vibrational spectrum. The same analysis applied on the stresses yields the viscosity. The package is available via the GitHub website and via its own dedicated page of the ERC IMPACT project as open-access package.

Melts and fluids are ubiquitous vectors of mass transport in natural systems. We have developed an open-source package to analyze ab initio molecular-dynamics simulations of such systems. We compute structural (bonding, clusterization, chemical speciation), transport (diffusion, viscosity) and thermodynamic properties (vibrational spectrum).

We have developed a Python-based open-source package to analyze the results stemming from ab initio molecular-dynamics simulations of fluids. The package ...

Applying Hyperspectral Reflectance Imaging to Investigate the Palettes and the Techniques of Painters

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are well-established techniques for the investigation of works of art with particular attention to paintings. Most modern museums put at the disposal of their research groups portable equipment that, together with the intrinsic non-invasiveness of RS and FORS, makes possible the in situ collection of reflectance spectra from the surface of artefacts. The comparison, performed by experts in pigments and painting materials, of the experimental data with databases of reference spectra drives the characterization of the palettes and of the techniques used by the artists. However, this approach requires specific skills and it is time consuming especially if the number of the spectra to be investigated becomes large as is the case of Hyperspectral Reflectance Imaging (HRI) datasets. The HRI experimental setups are multi-dimensional cameras that associate the spectral information, given by the reflectance spectra, with the spatial localization of the spectra over the painted surface. The resulting datasets are 3D-cubes (called hypercubes or data-cubes) where the first two dimensions locate the spectrum over the painting and the third is the spectrum itself (i.e., the reflectance of that point of the painted surface versus the wavelength in the operative range of the detector). The capability of the detector to simultaneously collect a great number of spectra (typically much more than 10,000 for each hypercube) makes the HRI datasets large reservoirs of information and justifies the need for the development of robust and, possibly, automated protocols to analyze the data. After the description of the procedure designed for the data acquisition, we present an analysis method that systematically exploits the potential of the hypercubes. Based on Spectral Angle Mapper (SAM) and on the manipulation of the collected spectra, the algorithm handles and analyzes thousands of spectra while at the same time it supports&#160;the user to unveil the features of the samples under investigation. The power of the approach is illustrated by applying it to Quarto Stato, the iconic masterpiece by Giuseppe Pellizza da Volpedo, held in the Museo del Novecento in Milan (Italy).

Hyperspectral Reflectance Imaging hypercubes include remarkable information into a large amount of data. Therefore, the request for automated protocols to manage and study the datasets is widely justified. The combination of Spectral Angle Mapper, data manipulation, and a user-adjustable analysis method constitutes a key-turn for exploring the experimental results.

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are well-established techniques for the investigation of works of art with ...

Screening of Coatings for an All-Solid-State Battery Using In Situ Transmission Electron Microscopy

With the ever-increasing use of Li-ion batteries, especially due to their adoption in electric vehicles, their safety is in prime focus. Thus, the all-solid-state batteries (ASSBs) that use solid electrolytes instead of liquid electrolytes, which reduce the risk of flammability, have been the center stage of battery research for the last few years. However, in the ASSB, the ion transportation through the solid-solid electrolyte-electrode interface poses a challenge due to contact and chemical/electrochemical stability issues. Applying a suitable coating around the electrode and/or electrolyte particles offers a convenient solution, leading to better performance. For this, researchers are screening potential electronic/ionic conductive and nonconductive coatings to find the best coatings with suitable thickness for long-term chemical, electrochemical, and mechanical stability. Operando transmission electron microscopy (TEM) couples high spatial resolution with high temporal resolution to allow visualization of dynamic processes, and thus is an ideal tool to evaluate electrode/electrolyte coatings via studying (de)lithiation at a single particle level in real-time. However, the accumulated electron dose during a typical high-resolution in situ work may affect the electrochemical pathways, evaluation of which can be time-consuming. The current protocol presents an alternative procedure in which the potential coatings are applied on Si nanoparticles and are subjected to (de)lithiation during operando TEM experiments. The high volume changes of Si nanoparticles during (de)lithiation allow monitoring of the coating behavior at a relatively low magnification. Thus, the whole process is very electron-dose efficient and offers quick screening of potential coatings.

Screening of Coatings for an All-Solid-State Battery Using In Situ Transmission Electron Microscopy

Utilizing the volume change of Si nanoparticles during (de)lithiation, the present protocol describes a screening method of potential coatings for all-solid-state batteries using in situ transmission electron microscopy.