The combination of differential scanning calorimetry and x-ray diffraction is a gold standard for investigated solid-state alterations of lipid-based pharmaceutical products. The combined methodology is a strong tool for screening the polymorphism, crystal growth and face transition of lipids combined with the screening, the terminal behavior and the visibility of the drug with lipid matrix. This methodology can be used to understand the interaction between the solid state of lipid and the performance of his pharmaceutical product.
Such products can be ranged from nano lipid formulations to matrix tablets. The procedure will be performed by Gerfried Luschin-Ebengreuth. Gerfried is a scientist from our lab.
To begin switch on the nitrogen gas supply and set the pressure between 0.2 and 0.5 bar. Power up the differential scanning calorimetry or DSC instrument and the automatic sample changer. Open the software and click the yes button to activate the standby mode, and then click on view signals.
Allow a calibration of the device for at least one hour. Purge the furnace with nitrogen. Click on the new method icon and go to method definition.
When the overview window opens, activate the temperature modulation option. Go to header tab and select the method by clicking on sample. Go to the tab temperature program.
Select purge two MFC and protective MFC both at 50 milliliters per minute. Set standby at 20 degree Celsius, heating cycle at five kelvin per minute from 20 degree Celsius to above the melting temperature of lipid isothermal holding at this temperature for five minutes. Cooling cycle to zero degree Celsius at five Kelvin per minute.
Set the final emergency reset temperature at a temperature 10 degree Celsius above the highest temperature of the program and final standby temperature at 20 degrees Celsius. Go to the calibration tab and select the appropriate temperature and sensitivity file. Save the method.
Weigh three to four milligrams of each sample into aluminum crucibles and record the exact weight loaded into each crucible. Seal the aluminum crucible with a pierced lid. Place the crucibles in the auto sampler tray and activate the auto sampler mode in the software and load related method for each sample.
Fill out the sample position, sample name and weight of each sample, and the position of reference crucible in the sample tray view window. Start the measurement. Open the raw data using the software for data analysis and plant the temperature versus heat flow by clicking on the button X time, X temperature.
On the popped up window, click on hide isothermal segments. On the left side of the screen, select only the curves that are to be analyzed. 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.
Click on the curve followed by evaluation and area. To calculate the enthalpy of fusion as the area under the curve of endotherms. Select the integration boundaries by moving the vertical lines around two to three degrees Celsius before and after the onset and endpoint of the peak.
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. Determine the onset of melting temperature or T-onset by clicking on the curve to be analyzed. And then on evaluation and onset.
Select the quantification boundaries by moving the vertical lines to the most straight section of the curve. This is usually around five to 10 degrees Celsius before and after the peak. Then determine the melting temperature or TM by clicking on the curve to be analyzed.
Followed by evaluation and peak, the obtained value is the peak maximum. Use an x-ray scattering system composing of a point focusing camera fixed to a sealed tube x-ray generator and equipped with a control unit and related software. Use two linearly positioned sensitive detectors to cover both small and wide angle X-ray scattering regions.
Switch on the cooling water system on the control unit, the vacuum pump, the gas valves, and the power and safety control system. Then switch on the voltage control and purging valves for the detectors at a gas flow between 10 to 20 milliliters per minute. Switch on the x-ray tube and standby option and wait approximately 10 minutes.
Switch off the standby mode and power up the x-ray tube to full power, which is greater than 50 kilovolts. Wait for at least 30 minutes. Start the control software and click on reset TPF.
Then choose Tungsten filter and set position. Go to position to fix the position of the Tungsten filter. Fill the samples into special glass capillaries with an external diameter of approximately two millimeters avoiding any air entrapment in the capillaries.
Seal the glass capillary with wax and place it carefully into the capillary holder. Switch on the motor for sample rotation and close the vacuum valve and wait until the pressure is beneath five millibars. In the software, select a position resolution of 1024 for both detectors and set the exposure time to 1, 200 seconds.
Click on the tap tools to set up the energy limits, then click on set energy limits and resolutions and restart. Set up the energy limits to a suitable range between 400 to 900. Open the safety shutter and start the measurement.
Ensure that the measurement window shows a maximum of 80 counts per second. If this is not given, adapt the filter position. Export the data as P00 files.
The data consists of the intensity of transmission and absorption versus the channel number. 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. Create a plot of normalized intensity versus two times the diffraction angle.
Use the function of screen reader to find diffraction peaks in both small angle x-ray scattering or SAXS and wide range x-ray scattering or WAXS regions. Apply brags equation to compute the diffraction peaks for which the maximum intensity was reached into interplay or spacings. Calculate the ratios of the peak position of the SAXS region to find out the crystal symmetry of the lipids and use the main diffraction peak of the SAXS region to quantify the crystallite thickness.
Fit the peak into a Gaussian function via classical leased squares and obtain the full width at half maximum by clicking on analysis peaks and baselines, peak analyzer, open dialogue. On the popped up window, select the option Fit Peaks Pro. Select a constant baseline with Y equal zero.
Select the main diffraction peak of the SAXS region. Click on fit control to select the peak fit parameters. Choose the Gauss Amp function.
Set the parameters Y zero XC1 and A1 as fixed and fit the data. Obtain the full width at half maximum from the fit. Finally, use the surer equation to compute the crystalline thickness.
The tune fork and chair configurations of a Triacylglycerol molecule, the sub cell, the Lamella, and the crystalline platelet are shown here. The representative image shows the short spacing and long spacing patterns of the alpha form of Tripalmitin and WAXS and SAXS regions. The same for the beta form is shown in this image.
The active pharmaceutical ingredient or API crystals coded with glycerol monostearate are measured via DSC and x-ray directly after coating and after three months of storage under accelerated conditions. DSC data of samples after three months of storage under accelerated show an endotherm at T-onset equals 55.7 degrees Celsius with two overlapped events at 60.2 degrees Celsius for the melting of remaining alpha form and at TM equals 63.8 degrees Celsius as the main event for the melting of beta form. The polymorphic transition is confirmed with the x-ray data by detecting the short D spacings typical for the beta form combined with the reduction in the Lamella thickness due to the molecular tilting.
Significant alteration in the release profiles was also observed which can be explained by the evident polymorphic transformation of alpha form to beta form with a denser sub cell arrangement resulting in a water repellent surface. DSC data, X-ray data, and an API release profile of API Crystals coded with PG13C16C18 partial directly after coding and after three months of storage under accelerated conditions are shown here. DSC analysis of stored samples revealed unchanged thermograms, which depict no polymorphism and no phase separation.
The stable solid state of PG3C16C18 partial was confirmed by the x-ray patterns. The WAXS region shows a peak corresponding to a short spacing of D equals 4.15 angstrom and T0 in stored samples associated with the alpha form. The SAXS region revealed an unaltered main peak at a long D spacing of D equals 63.7 angstrom corresponding to a lamella structure with two L configuration.
The stable solid-state of the lipid matrix results in the stable release profile of API from coding. To provide high quality x-ray data, it is important to avoid entrapment between particles when filling the capillaries. Also to select an exposure time that is based on the type of sample and the available equipment.
During the development of novel pharmaceutical products, screening the solid-state of lipids and their interaction with the API is used to select the suitable manufacturing process and to predict the product stability.
