Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

The overall goal of this procedure is to perform chemically selective imaging on the surface of a tablet undergoing pharmaceutical dissolution using in situ car dissolution analysis. This is accomplished by first tuning the car's laser system to a frequency selective for the chemical of interest in the tablet and setting up the UV absorption spectrometer to record spectra. The second step is to place a tablet into the car's dissolution flow cell and screw tightly closed.

Next, the car's dissolution flow cell is placed on the microscope stage and piping connects it to the pump and dissolution medium. The final step is to simultaneously record a car's dissolution video while pumping dissolution medium and recording UV absorption spectra. Ultimately in situ car's dissolution analysis is used to show that surface changes during dissolution can be visualized and correlated with changes in dissolution rate.

The main advantage of this technique over existing techniques, such as pharmacopia dissolution methods, is that init car's dissolution analysis provides in-depth information about the surface of a tablet, which can change during dissolution. To begin, turn on the car's laser and the deuterium lamp ultraviolet or UV light source and allow it to warm up. Open the shutter on the deuterium lamp UV light source by setting the shutter switch to open.

Next, turn on the microscope control PC and open the microscope control software. Turn on the UV spectrometer PC and open the spectrometer control software. Select the desired microscope objective.

Set the filters in the filter set turret to transmit excitation lasers, and reflect the car's signal. Place appropriate filters in front of the photo multiplier tube detector to transmit the car's signal and filter unwanted light. For system testing, turn on the peristaltic pump and pump dissolution medium through the V-shaped UV flow cell for a few minutes to clear previous liquid from the piping.

Determine the flow rate of the pump by weighing the amount of dissolution medium pumped two minutes. Adjust the pump speed until the desired flow rate is reached. For UV dissolution measurement in the UV spectrometer control software, click the file menu.

Then click New absorbance measurement to open a window, which lists all available spectrometers. Click on the correct UV spectrometer and then click next to open a window, which displays the data acquisition parameters. Define both the integration time and the spectral averaging.

Click the button labeled next to bring up the screen used to record the reference spectrum. Click on the button that appears as a yellow light bulb to record a reference spectrum pump dissolution medium continuously. During this measurement, close the shutter on the deuterium lamp UV light source by setting the switch to closed.

Click the button labeled next to bring up the screen used to record the dark spectrum. Click on the button that appears as a gray light bulb to record a dark spectrum pump dissolution medium continuously. During this measurement, click on the button that says finish to begin the UV absorbance measurements in the cars microscope control software, click on the button that selects an XYT measurement.

Click the dropdown box and select the image size in pixels. Drag the imaging speed slider to either the fast, medium, or slow position. Click the arrows labeled zoom to adjust the zoom level.

Then click the dropdown box and select the objective used. Click the input box and type the amount of frames required for the car's dissolution video Using the optical parametric oscillator or OPO controller, adjust the settings of the OPO such as temperature pazo position, and LEO filter position until maximal laser output at the desired ramen frequency is reached. Place a tablet into the sample holder of the custom built car's flow cell and screw the sample holder tightly closed.

To prevent leakage, attach the piping to the car's flow cell, connecting the car's flow cell to the beaker containing the dissolution medium and the peristaltic pump. Place the car's flow cell containing a tablet on the microscope stage. Check that the car's flow cell is connected to the dissolution medium beaker.

The peristaltic pump, the V-shaped UV flow cell and the waste collection beaker. Click the XY repeat button to start the microscope system scanning in a continuous scan mode. Adjust the focus of the microscope by moving the objective until the surface of the tablet is in the field of view on the microscope control computer screen.

Click on the slider in the microscope control software labeled PMT. Adjust the detector sensitivity by increasing or decreasing PMT voltage until a satisfactory image is visible on the screen. Click stop in the microscope control software to stop the continuous scan simultaneously.

Start pumping dissolution medium. Start recording a single XYT scan and start collecting UV absorbent spectra. During the dissolution experiment, monitor the video recording and manually adjust the microscope focus.

To ensure the tablet is continually in focus. Stop the peristaltic pump by turning it off. Click the file menu and then click save as video on the microscope control software to save the XYT scan as a video on the spectrometer control software, click the file menu save, and then click stop export.

To stop the collection of UV absorption spectra, remove the car's flow cell from the microscope stage and remove the tablet from the car's flow cell. As a final step, wash the car's flow cell using water and ethanol and then dry using tissue paper. Selected frames from the car's dissolution video are shown here at the beginning of dissolution.

There are areas of green showing the theophylline content of the tablet. The dark areas are where there is only ethyl cellulose present on the surface of the tablet. After about 60 seconds, the beginning of theophylline monohydrate crystal growth is observed on the surface as narrow needle shaped crystals growing outwards from at least one crystal nucleus at the center of the frame.

The monohydrate crystal growth can be clearly seen after 130 seconds. At this time, the monohydrate crystal has not spread entirely across the surface of the tablet. The presence of the ethyl cellulose regions may physically block the lengthening of the monohydrate needles.

After 250 seconds, it can be seen that the monohydrate coverage of the surface is not as prominent, suggesting that the monohydrate crystals are beginning to dissolve the UV dissolution profile for the dissolution of the theophylline anhy with ethyl cellulose tablet shows that dissolution of theophylline anhydrate quickly reaches a maximum concentration of around 90 micrograms per milliliter within 120 seconds. After this time point, the dissolution rate begins to decrease. After watching this video, you should have a good understanding on how to perform in situ dissolution analysis using cos microscopy.

This technique allows enhanced understanding of the dissolution process by monitoring the surface of a dissolving tablet using chemically selective imaging.