Metrohm Instant Raman Analyzers (MIRA): Calibration, Verification, and Performance Validation

This article describes Instrument Calibration, System Verification, and Performance Validation with respect to both Raman theory and government norms and standards and outlines these tests in the System Suitability Test (SST) for Mira P.

Raman spectroscopy arises as a result of inelastic scattering of incident laser photons on a material, generating Raman scattered light with energy that is shifted from that of incident photons. This is expressed as a spectrum by plotting the Raman signal (Intensity, y-axis) against shifted energies, denoted as wavenumbers (cm^–1) on the x-axis. In general, largely varying Raman spectra can be experienced as a result of the efficiency of optical transmission and detector response, which can be resolved by standardization and calibration.

USP, in collaboration with National Institute of Standards and Technology (NIST), American Society for Testing and Materials (ASTM), European Pharmacopoeia (EP), and research groups, outlines the routines and Standard Reference Materials (SRM) for the standardization of Raman spectrometers.

Figure 1. Mira P Raman analyzer.

Instrument Calibration

Calibration of Raman instruments “corrects instrument-induced spectral artifacts,” as mandated by USP <1120> and <858>. This validates repeatability and measurement accuracy, and on a larger scale, it leads to universal Raman libraries, spectra, and instruments. Instrument calibration necessitates three components:

• Wavenumber (cm^–1) calibration or correction of absolute peak positions on the x-axis of a Raman spectrum
• Laser calibration
• Relative intensity or intensity calibration response expressed on the y-axis

X-Axis Calibration (TA Standard)

Raman Shift Standards for Spectrometer Calibration, ASTM E1840-96, are used for establishing the integrity of peak positions on the x-axis of Raman spectra. Accurate wavenumber shifts with standard deviations of <1 cm^–1 have been cautiously determined for a small number of chemicals, selected to represent a complete spectral range for Raman spectrometers (350–2300 cm^–1 for handheld instruments).

In Metrohm Raman, a 1:1 v/v mixture of Toluene:Acetonitrile is used for x-axis calibration, with an acceptance criterion of ±2 cm^–1, surpassing criteria for handheld Raman systems (±2.5 established by ASTM E1840).

Raman spectroscopy is a trademark technology for the identification and verification of materials. Every material has unique Raman spectra, where peaks represent the distinctive chemical bonds in a substance. At the most basic level, x-axis calibration guarantees accurate identification of material through the mapping of standard wavenumber peaks on the Raman shift axis to previously reported values.

Figure 2. Certification for TA Standard.

Y-Axis Calibration

Although y-axis calibration is less insightful, by no means is it less significant. The use of Raman spectroscopy for the identification of materials is dependent on the comparison of measured spectra within established spectral libraries. Apart from absolute peak position, peak shape and height have an impact on accurate library comparison and must be standardized.

As mandated by USP 29-NF 24 <1120> and as outlined in NIST 2241, Raman spectrometers are standardized through intensity correction. In Metrohm Raman, an “NIST traceable” method is used for y-axis correction, which involves the application of a traceable correction to an established standard to all spectra collected by a system, thereby establishing an internal standard to which all future measurements are corrected.

In particular, NIST SRM 2241 is a chromium-doped green glass standard unique for Raman spectroscopy with an excitation wavelength of 785 nm. This is the established “true spectrum” to which all other measured spectra are compared. Fundamentally, measurement of the ratio between the true and measured spectra is performed and the ratio is applied as a correction to all future acquisitions. Following this, NIST SRM correction is applied to each system using spectral processing algorithms, and it is validated with the same TA calibration standard used for x-axis calibration at Metrohm Raman.

Figure 3. Raman spectral library matching.

The use of a particular NIST calibration standard for Raman instruments that run at a laser wavelength of 785 nm indicates that it is essential to perform laser calibration for each instrument.

System Verification

Verification of general system fitness and calibration routines guarantees that an instrument performs accurately and in agreement with the claims of the manufacturer. Metrohm Raman SST report’s system verification portion includes verification of the fitness and alignment of individual components, as outlined in USP <1120>. The verification routine involves the evaluation and reporting of laser and raster fitness, voltage inputs, signal/noise, admissible loads, and the display.

Figure 4. Mira P with Calibrate/Verify Accessory.

Performance Validation

For a majority of the Raman instruments, calibration and verification are adequate for qualification. Mira P developed by Metrohm Raman is a handheld system that is exclusively designed for application in regulated industries such as cosmetic, pharmaceutical, and food industries. The Calibrate Verify Attachment (CVA), which is a new accessory for this sophisticated system, adds one more level of confidence to AIQ, as mandated by USP 42-NF 4 In-process Revision <858>.

USP<858> outlines AIQ very specifically in terms of admissible accuracy and precision, procedures, range, and limits for the validation and verification of Raman spectrometers. Below is the validation routine followed by Metrohm Raman:

• Confirm system performance over time
• Show that system calibration and verification is carried out with suitable sensitivity, accuracy, and precision
• Confirm wavenumber calibration
• Stick to standards mandated by EP 2.2.48 and 21 CFR Part 211

Calibrate/Validate Accessory (CVA)

CVA includes not only the TA standard mentioned above but also a second accepted standard for AIQ of Raman instruments — polystyrene.

As outlined by USP and EP 2.2.48, polystyrene is a nontoxic, stable organic substance approved (NIST SRN 706a) for the validation of Raman instruments. The fitness of a system can be reconfirmed through wavenumber calibration. This is a robust test, and user adherence to 21 CFR Part 211 is implicit upon successful AIQ with polystyrene.

Figure 5. CVA rendering.

SST Report

Each time a system is subjected to calibration, the resulting SST can be archived as part of a traceable audit trail. Following is a sample SST with annotation:

• The Test Created field is a record of the actual date and time of calibration
• Overall Status is just a Pass/Fail indication of the fitness of the system
• Package Version denotes the existing firmware on the device
• ·The service life of the Calibration Standard is two years
• Verification results sum up the specific values reported in subsequent sections

Calibration

The absolute wavenumber peak position in the combined spectra of a 50:50 mixture of Toluene:Acetonitrile is confirmed through wavenumber verification. TA is an ASTM standard for calibrating Raman shift frequencies and represents peaks over the entire Raman spectral range of 400–2300 cm^–1. In general, this is a component alignment test; it particularly guarantees integrity of peak positions.

The health of y-axis calibration and correction is reflected by intensity verification. Y-axis calibration involves the test of the efficiency of each optical component in a system, such as filters, lenses, and detectors. Intensity verification also involves the assessment of correction algorithms used by the manufacturer. The peak ratios for toluene (left) and acetonitrile (right) described below suggest that suitable correction algorithms have been used.

Verification

The health of the laser and inherent instrument noise are evaluated by performing performance tests. Suitable values for Maximum Intensity, in accordance with the height of the 1003.6 cm^–1 toluene peak, suggest that the instrument is passing and detecting light in an accurate manner. Noise measurements are obtained from a non-Raman-active portion of the sensor to reflect the actual spectral noise of the CCD. These tests ascertain whether the signal and noise performance of the instrument is within admissible parameters established by the manufacturer as recommended by USP 40-NF 35 <1058>.

Validation

Figure 6. SST Passed Screenshot from Mira P.

Voltage Verification routines evaluate the health of a system, from an electrical perspective. Information related to all electrical subsystems can be obtained here.

The Raster Test is exclusive to Metrohm Raman spectrometers, which are provided with Orbital Raster Scan (ORS) technology. This is the original RPM of the raster during the testing. In case the raster test turns out to be a failure, the laser interlock gets tripped and the laser does not function.

The System Clock shows that the system is in accordance with real time (UTC, confirmed each time the instrument is connected to a computer), thereby guaranteeing data integrity.

The TA Spectrum is the original spectrum obtained by the instrument at the time of calibration. It offers instantaneous visual confirmation of the quality of collected data and can be used to confirm various measurements reported in this article.

An added layer of guarantee that a system is working properly is Performance Verification. This calibration validates the accuracy of Raman peak positions within admissible range based on EP 2.2.48. Again, the original Polystyrene Spectrum that was obtained at the time of Instrument Calibration is included to enable visual confirmation of reported numerical results.

This information has been sourced, reviewed and adapted from materials provided by Metrohm AG.

For more information on this source, please visit Metrohm AG.