Method Guidance for Morphologi 4-ID Spectrometer Varieties

Introduction

Building on customer feedback, in 2020 a new Raman spectrometer was introduced for the Morphologi 4-ID (M4-ID) system. This new spectrometer provides signal-to-noise improvements, enabling Morphologically-Directed Raman spectroscopy (MDRS) measurement times to be shortened, and improving performance for weak scatter applications.

This technical note provides guidance on method transfer and method development using Morphologi 4-ID systems fitted with the new spectrometer. For the purposes of this document, the first spectrometer type (part numbers MOR28XX) is referred to as type 1, and the second spectrometer (part numbers MOR29XX) is referred to as type 2.

The following areas are covered:

• Summary of spectrometer differences
• Guidance for creating new MDRS methods on a type 2 spectrometer M4-ID system
• Guidance for transferring MDRS methods to a type 2 spectrometer M4-ID system
  

Summary of Spectrometer Differences

The Morphologi 4-ID system has an integrated 785 nm Ocean Optics Raman spectrometer to enable the chemical identification of individual particles. Only the Raman spectrometer itself has been changed, with the type 2 spectrometer fitted the same way within the Raman box enclosure of the M4-ID. The type 2 spectrometer has an increased average signal throughput compared to the type 1 spectrometer. This means that when using the same spectral acquisition settings, users should see higher average signal-to-noise ratios. The laser spot size and wavelength range remain unchanged at 2 µm and 150-2800 cm-1 respectively. If transferring a MDRS method, the selected laser power percentage should not need to be changed.

When developing or transferring a MDRS method on a M4-ID fitted with the type 2 spectrometer consideration of the following chemical measurement parameters is recommended:

• Exposure time
• Particle selection
• Chemical library
• Collection of background spectrum for subtraction (if applicable)
• Correlation scores

These will be covered in more detail in the following two sections.

Guidance for Creating New MDRS Methods

The guidance in this section is intended for customers developing a new MDRS method on a M4-ID system fitted with the type 2 spectrometer who are familiar with results from the type 1 spectrometer. For general MDRS method development guidance please refer to the list of resources in this document’s Appendix.

The main consideration when creating a new MDRS measurement is exposure time, with the higher average signal-to-noise ratio from the type 2 spectrometer providing an opportunity to reduce chemical measurement times. This signal-to-noise improvement will also benefit applications where the chemical identification of materials that have weak Raman scattering efficiencies is required, such as in protein biotherapeutics. Figure 1, which shows an overlay of Raman spectra acquired from the same API particle in a nasal spray sample using the type 1 and type 2 spectrometer, highlights the difference in signal-to-noise ratio.

Figure 1: Overlay of 60 second spectra acquired from a nasal spray API particle using the type 2 spectrometer (red line) and type 1 spectrometer (blue line). A quartz substrate was used, with no background subtraction applied. 150-2000 cm-1 wavelength range shown.

Testing indicates that for most applications the exposure, and thus measurement, time can be reduced by approximately 50%. Figure 2 shows that 15 second spectra acquired from different samples on the type 2 spectrometer are comparable to 30 second spectra acquired from these samples on the type 1 spectrometer.

Figure 2: a) Overlay of 15 second spectrum acquired from a nasal spray API particle using the type 2 spectrometer (red line) and 30 second spectrum acquired using the type 1 spectrometer (blue line);
b) Overlay of 15 second spectrum acquired from a lactose particle using the type 2 spectrometer (red line) and 30 second spectrum acquired using the type 1 spectrometer (blue line).

The exposure time required may depend on the Raman scattering efficiency of the materials of interest, the particle size range of interest and the sample preparation method. During method development successively shorter acquisition times should be tested across the range of particle sizes being targeted in the MDRS measurement, with a focus on the smallest particle sizes. Acquisition from smaller particles will have a lower signal-to-noise ratio, thus the exposure time needs to be sufficient for the smallest particles of interest.

It is recommended that Raman spectra to be used in the reference library and for background subtraction should be acquired using the same spectrometer settings (exposure time and laser power). Once all the chemical parameters have been defined and verified, they can be added to the final SOP. It is recommended to validate the complete method for robustness and reproducibility.

Guidance for Transferring MDRS Methods

To investigate likely impacts when transferring an existing MDRS method, three separate preparations of a nasal spray sample were analyzed with the 50x objective using the same SOP on the M4-ID with both the type 1 and type 2 spectrometer. Only smaller particles were targeted as this is where the higher signal-to-noise ratio was expected to have the greatest effect on the correlation scores. The SOP was configured to collect Raman spectra from >1000 particles with a Circular Equivalent Diameter (CED) between 1 and 3 µm using a 60 second exposure time.

References were created by collecting and averaging three spectra from a single API particle using each spectrometer type. The mean spectra (Figure 1) became the type 2 and type 1 reference libraries and were used to correlate to the particle spectra over the 1350-1800 cm-1 wavelength range. Particles were classified as API if they had a score of 0.7 or higher to the reference spectrum.

The results from the three measurements per spectrometer type were evaluated and then combined to give one record for each spectrometer. Overall, there was a ~15% increase in the number of spectra classified as API acquired from the type 2 spectrometer in comparison to the type 1 spectrometer at equivalent exposure times. Figure 3 shows an overlay of the number-weighted and volume-weighted CED distributions for the particles classified as API from the two spectrometers. The distributions reveal an increase in particles classified as API closer to 1 µm, highlighting the subtle shift towards to smaller particle sizes.

Figure 3: a) Overlay of the number-weighted CED distributions for particles in the 1-3 µm particle size range classified as API in a nasal spray using the type 2 spectrometer (red line) and type 1 spectrometer (black line); b) Overlay of the volume-weighted CED distributions for particles in the 1-3 µm particle size range classified as API in a nasal spray using the type 2 spectrometer (red line) and type 1 spectrometer (black line).

For most applications where the chemical identification of very small (< 3 µm) particles is required transferring to a type 2 spectrometer is expected to be beneficial, with an increased number of particles being classified at equivalent exposure times, providing further insight into the sample. To achieve equivalent results and/or take advantage of the improvements to reduce the measurement time, a shorter exposure time is recommended. As discussed earlier, testing suggests that halving the exposure time (if not already short) will give similar results. For best results, the reference and background spectra should be recollected using the type 2 spectrometer.

Appendix – MDRS Method Development Resources

Below is a list of suggested Malvern Panalytical resources to aid general MDRS method development:

• Built in Help in the Morphologi 4 software (navigate to Help à Help Topics)
• Webinar: Advanced image analysis for particle characterization: Theory and best practice guide – Advanced image analysis for particle characterization: Theory and best practice guide | Malvern Panalytical
• Webinar: What is that particle? Where did it come from? Characterizing foreign particulate matter in pharmaceutical products – What is that particle? Where did it come from? Characterizing foreign particulate matter in pharmaceutical products | Malvern Panalytical