Product Note R35-08/16. BRAVO The must have handheld Raman solution for Pharmaceutical Industry. Advantages

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1 Product Note R35-08/16 BRAVO The must have handheld Raman solution for Pharmaceutical Industry Advantages Patented fluorescence mitigation Large spectral range Laser class 1M safe operation Intuitive and guided touchscreen operation Highest wavelength accuracy and precision Enhanced material distinction Compliant to latest Pharma regulations Raman Intensity Calcium Carbonate Corn Starch Croscarmellose Sodium Lactose Microcrystalline Cellulose Talc Process and cost optimizations challenge our daily business. By introducing handheld Raman spectrometers that enable to probe materials like active pharmaceutical ingredients or excipients directly through transparent packaging at any location new opportunities emerged. With BRAVO the dedicated handheld Raman solution for the Pharmaceutical industry combining outstanding performance and ease of use Bruker started a new era in material control. SSE TM Patented Fluorescence Mitigation Sequentially Shifted Excitation allows measuring more materials compared to conventional handheld Raman spectrometers by mitigation of fluorescence while keeping the performance at least as high as for benchtop systems (Figure 1) Raman Shift cm Figure 1: BRAVO SSE TM spectra of typical Pharmaceutical excipients. Duo LASER TM Excitation Large Spectral Range BRAVO allows recording a large spectral range from 300 cm -1 to 3200 cm -1 Raman shift and hence provides even information from the CH-stretching range (Figure 1). 500 Laser class 1M Safe Operation As a laser class 1M product operation of BRAVO does not require any laser safety equipment, restricted area or laser safety officer.

2 Home screen with status informa on and overview of workflows Fully configurable can retrieve all material informa on with one scan. Clear result presenta on and further func onali es as batch scan mode and advanced result presenta on available. Figure 2: Verification workflow Intuitive and Guided Touch screen Operation Likewise the operation of a smartphone the user is guided through a clearly laid out user interface designed for the needs of material inspection. BRAVO provides highest standards and sophisticated workflows for efficient operation (Figure 2). Material, Method and Library Setup BRAVO makes building libraries quick and easy by an intuitive workflow in combination with Bruker s powerful OPUS software suite. Due to the high precision optics of BRAVO best results in the market using an HQI approach are achieved enhancing the distinction of even very similar materials. In Table 1 results from the verification of celluloses are presented comparing an HQI approach and an HQI approach in combination with SSE TM. It can be clearly seen that an Sample evaluated towards MCC microcrystalline cellulose (MCC) Hydroxy propyl methyl cellulose (HPMC) Hydroxy propyl cellulose (HPC) Sodium carboxy methyl cellulose (SCMC) Correlation (no SSE TM ) with SSE TM Table 1: Spectra of diffrent cellulose materials tested against a method for the verification for MCC. The calculated correlation is displayed with and without the use of the SSE method. HQI approach with BRAVO already results in a successful distinction which can be significantly increased further by combination with SSE TM. IntelliTip TM - Automated Tip Recognition When acquiring library spectra BRAVO stores tip information automatically and advises during the verification process which tip to be used. IntelliTip TM hence always ensures that the proper tip according to the material to be measured is applied in order to receive best spectrum quality at any time. Validation BRAVO and all of its software are fully compliant to the latest Pharmaceutical regulations like 21 CFR Part 11, EP and USP Due to the robust and precise optics BRAVO s wavelength precision and accuracy by far exceed the recently specified regulatory requirements for handheld Raman spectrometers and outperforms those of other handheld Raman devices. In particular it can be noted that BRAVO s performance is comparable to high performance benchtop Raman spectrometers and matches even the more stringent regulatory requirements of this instrument class. Reference standards for comprehensive system tests of BRAVO are according to ASTM 1840 as specified by the USP and EP regulations and of course a comprehensive validation manual including certificates and step by step instructions for IQ, OQ and PQ procedures is available. Bruker Optics Bruker Optics is continually improving its products and reserves the right to change specifications without notice Bruker Optics BOPT

3 Product Note R34-05/15 BRAVO Advanced Data Acquisition and Evaluation in Handheld Raman Spectroscopy The main benefit of handheld spectrometers is that the instrument itself can be taken to the sample and thus can be employed for various applications with high degree of flexibility. For routine applications like materials identification and verification, it is highly desirable to have as many functions as possible automated. A good example is the quality control of incoming goods using handheld Raman spectrometers, which allows analyzing materials directly through packaging [1]. There are also always many advanced applications where the instrument requirements will be user or application dependent. Previously most small handheld Raman systems lacked the flexibility to allow the user to optimize the data collection for the desired analysis. The handheld Raman spectrometer BRAVO features an advanced operation mode via remote control utilizing Bruker s comprehensive spectroscopy software OPUS. The remote control is accomplished via Wi-Fi or an Ethernet connection using a docking station. Consequently, in any case where a mobile or handheld instrument needs to be run untethered to electrical power supply, and has to require flexible data acquisition BRAVO is a very powerful solution. With BRAVO there is the direct option to initiate measurements from the OPUS software and to subsequently run user defined evaluation processes. In addition, it is possible to set parameters such as integration time and number of co-added measurements manually. The settings can be stored in experiment files which can be assigned to a user or specific application. What possibilities are coming up? BRAVO measures Raman spectra in a quality typically only obtainable from powerful benchtop spectrometers [2]. This means that acquired spectra can be used for any kind of evaluation such as quantification or complex identification methods. At this stage, not only the mathematically processed data used for on board verifications based on the Sequentially Shifted Excitation (SSE TM ) method is available but also the raw data resulting from each laser excitation is accessible, which is often preferred for scientific applications [3]. The spectroscopy software suite OPUS combines a wide range of functionality for analytical and research applications with an intuitive design and highest degree of flexibility. Furthermore, additional software packages are available to meet the needs of specific applications [4]. The following examples provide insight into the many possibilities based on the remote control option and comprehensive OPUS spectroscopy software.

4 OPUS SEARCH The OPUS SEARCH package is an advanced library searching tool capable of mixture analysis and represents a typical approach to identify the spectra of unknown materials. A typical example is given in Figure 1. Libraries can be setup by the user and many comprehensive databases from common suppliers are available. Typical applications include forensic investigations, detection of hazardous materials by first responders, materials characterization in the industrial environment, and analysis of objects in the field of art and cultural heritage [5]. OPUS IDENT The OPUS IDENT package is an excellent comprehensive tool for reliable identification of raw materials, intermediates and finished products offering the highest flexibility for method setup. The combination of user defined data pretreatment, selection of various methods for data evaluation and control of many more parameters make it the ultimate easy-to-use program for dedicated quality control solutions. Figure 2 shows the example of the differentiation of very similar oil samples using principle component analysis. OPUS QUANT OPUS QUANT is a state-of-the-art software package for setting up and validating quantification models utilizing advanced PLS based methods. Analogous to the setup of an IDENT method, the user is guided step-by-step in generating the quantification models. In Figure 3, an example of a calibration is given to determine the relative anhydrite content in gypsum PC Fig. 2: 3D score plot of an IDENT analysis of similar oil samples measured with the BRAVO. The colored spheres represent the areas in which the identification was successful, respectively. Very similar materials (yellow and blue spheres) can be addressed in sub libraries or assigned to one class. Prediction (%) anhydrite content cross validation PC True (%) PC3 0.5 Figure 1: Example of the analysis of an unknown liquid (red spectrum). The mixture analysis based on the analysis of multiple libraries reveals the presence of two materials, polyethylene glycol (PEG) and the synthetic cannabinoid JWH-018. The calculated composition spectrum (dashed line) matches the query spectrum. Fig. 3: Calibration model for the relative anhydrite content in gypsum based on the evaluation of the symmetric SO 4 vibration. References [1] F. Fromm, New Possibilities in Raw Material ID Using Handheld Raman Spectroscopy, Pharmaceutical Business Review, October [2] Bruker Product Note T30 03/16, Accuracy is crucial: The starting point for a robust transfer of methods. [3] Bruker Product Note T29 12/15, Efficient mitigation of fluorescence in Raman spectroscopy using SSE TM. [4] [5] Conti et al., Portable Sequentially Shifted Excitation Raman spectroscopy as an innovative tool for in situ chemical interrogation of painted surfaces, Analyst (2016). Bruker Optics Bruker Optics is continually improving its products and reserves the right to change specifications without notice Bruker Optics BOPT

5 Product Note T30 03/16 BRAVO - Accuracy is crucial: The starting point for a robust transfer of methods Handheld devices nowadays make Raman spectroscopy available for various routine applications operated with a high degree of automation. Some decades ago this could not be imagined as for example it often was a challenge to ensure and achieve the required Raman shift accuracy during operation. With new arising developments, i.e. very stable diode lasers, Raman spectroscopy is getting more and more robust and user-friendly. An important issue for handheld Raman spectrometers is the capability of a robust transfer of libraries between different instruments of the same type. In other words it is required that the same results are obtained with a common set of library data for various instruments. Prerequisite is a thorough calibration which guarantees a high accuracy and sets every spectrometer to a well-defined state. Of course, the second requirement is the capability of a spectrometer maintaining its performance during operation which is related to a high precision. Especially this issue is of high importance as handheld instruments are not operated under laboratory conditions but instead being exposed to manifold varying environmental conditions. Thus, a high accuracy combined with a high precision form the basis for a reliable in field operation using library data being generated in a centralized manner on multiple spectrometers. With BRAVO special care is taken to make the highest standards only common for benchtop instrumentation available in handheld Raman spectroscopy. X-axis calibration is based on measurements of the emission of a neon lamp and Raman shift standards as defined by the ASTM [1]. Multiple Raman lines are evaluated to ensure a comprehensive calibration valid across the entire spectral range. To demonstrate the high level of accuracy achieved table 1 compares peak positions of cyclohexane to ASTM literature values based on measurements performed at multiple BRAVO spectrometers. Notably, all Raman lines match very closely their literature values and the individual deviations are well below 1 cm -1 which is exceptional for handheld spectrometers. To emphasize this results it needs to be mentioned that cyclohexane was not used for spectrometer calibration. For the Pharmaceutical industry the chapters USP 1120 by the United States and EP by the European Pharmacopeia define the minimum system requirements and guidelines for Raman instrumentation being operated in validated environments [2, 3].For example the EP as well defines acceptable tolerances for the x-axis accuracy which are given in table 1 for benchtop and handheld instrumentation. It is obvious that the BRAVO spectrometer

6 Table 1: Cyclohexane - ASTM values [1] Cyclohexane BRAVO (average +/- std. dev.) Deviation to ASTM / / / / / / / / / / 3.0 Allowed deviations according to EP (benchtop/handheld) Table 1: Comparison of Raman peak positions (unit: cm -1 ) of cyclohexane to ASTM literature values as determined by BRAVO. The positions are shown as average values and standard deviation based on single measurements at 20 different BRAVO spectrometers, respectively. is capable to be operated in line to requirements for benchtop instrumentation and easily exceeds the softened values for handheld instrumentation. OPUS the overall performance of the spectrometer can be easily monitored running comprehensive system tests according to current Pharmaceutical regulations. A common way to compare spectral data for library matching is to calculate the correlation coefficient between the measured spectrum and reference data. The correlation coefficient calculates to a number ranging from 1 to -1 representing a qualitative measure of identity in which 1 represents a perfect match. In reality this parameter is not only influenced by the similarity of the reference and analyzed material but depends as well on other parameters such as background signals, i.e. fluorescence. In general it is aimed to add as much as possible significance to this value in means of selectivity towards other materials, for example an adequate data pretreatment is recommended [4]. Regarding this aspect BRAVO s patented SSE TM mitigates fluorescence signals in an active manner [5]. A further influence on the correlation is given by the accuracy of a spectrometer which is illustrated in figure 1. Here the correlation coefficient of two Raman spectra of calcite is calculated in the range of 400 cm -1 to 1800 cm -1 as function of a constant deviation in x-axis accuracy across the entire spectral range. It is evident that for a deviation of 2 cm -1 the typically applied threshold of 0.95 is barely reached. To ensure the required x-axis accuracy for BRAVO not only at an instrument test but continuously during operation a neon spectrum is generated with every Raman spectrum measured using an inbuilt neon lamp. Of course, the same care is taken at y-axis calibration stage using a NIST SRM relative intensity correction standard for Raman spectroscopy. Finally, with the spectroscopy software Correlation coefficient References typical threshold calcite (400 cm -1 to 1800 cm -1 ) Deviation (cm -1 ) Figure 1: Correlation coefficient of two spectra of calcite in the range of 400 cm -1 to 1800 cm -1 as function of their relative deviation in x- axis accuracy (constant shift across the spectral range). [1] ASTM E (2014), Standard Guide for Raman Shift Standards for Spectrometer Calibration. [2] <1120> Raman Spectroscopy, United States Pharmacopeia. [3] Raman Spectroscopy, EUROPEAN PHARMACOPEIA 8.7. [4] J. Kauffman et al., Spectral Processing for Raman Library Searching, Amer. Pharm. Rev. 14, (2011). [5] Bruker Product Note T29 12/15, Efficient mitigation of fluorescence in Raman spectroscopy using SSE TM. Bruker Optics Bruker Optics is continually improving its products and reserves the right to change specifications without notice Bruker Optics BOPT

7 Product Note T29 12/15 BRAVO Efficient mitigation of fluorescence in Raman spectroscopy using SSE TM For many years, Raman spectroscopy has been utilized as an important tool for materials characterization in the analytical laboratory. With new developments in laser technology and spectrometer design, handheld Raman spectrometers can now be designed and built for practical everyday use in a vast array of applications [1]. The BRAVO by Bruker takes handheld Raman spectroscopy to a new level, overcoming the limitations of previously available systems. These previous limitations included i.e., limited wavelength accuracy [2], non-safe laser usage, and fluorescence interference. The wavelength accuracy of the BRAVO is significantly better than competitive devices resulting in the highest data consistency. The BRAVO is also a Class 1M laser safe device. Lastly, the BRAVO incorporates new technology called Sequentially Shifted Excitation (SSE TM ) to mitigate fluorescence. The general task of a handheld instrument is obvious it is a spectrometer which can be employed anywhere with the highest degree of flexibility, where the sample in question does not need to be transferred to a laboratory for reliable and rapid identification. A requirement that goes hand-in-hand with the automation of measurements and data analysis in handheld devices is to achieve a maximum robustness with respect to factors affecting the reliability of results. This starts with minimizing the risk for operating errors with intelligent software that provides an intuitive guided workflow for acquiring and processing the data. One of the most challenging aspects of Raman analysis is overcoming fluorescence interference. Fluorescence illumination is a highly efficient process that frequently yields a strong signal that can overwhelm the desired Raman spectrum. Additionally, a fluorescence background can limit reproducibility, which is required for conducting analyses in the materials identification and quantification process. The patented Sequentially Shifted Excitation (SSE TM ) method used by BRAVO detects and removes the fluorescence signal leaving only the desired Raman spectrum [3,4]. How does SSE TM work? In order to extract the Raman signal from an acquired spectrum, the BRAVO software must recognize and differentiate the Raman spectrum from other interfering signals (fluorescence) automatically. To make this possible the wavelength of the lasers used for excitation are sequentially shifted during the measurement. As a consequence, the position of the Raman signal on the detector is deliberately changed and the signals at constant wavelength, such as fluorescence, remain at the same position. The SSE TM algorithm extracts the moving signals which relate directly to the Raman signature (see Fig. 1) yielding the desired spectrum.

8 Raman intensity (arb. units) SSE TM processed talc in PE bag raw data TM at SSE absorp on PE signals Raman intensity (arb. units) BRAVO sodium alginate 785 nm excita on Raman shi (cm -1 ) Figure 1: Raman spectra of talc in a PE bag at sequentially shifted excitation (raw data at SSE TM ) and after fluorescence mitigation (SSE TM processed). Raman lines of talc indicated by dashed lines are moving as the laser wavelength is changed. The SSE TM processed spectrum only contains the moving signals extracted from the three spectra Raman shi (cm -1 ) Figure 2: Comparison of Raman spectra of sodium alginate acquired using a conventional dispersive Raman instrument at 785 nm excitation (red) and BRAVO (blue). What are the benefits of SSE TM? One of the main advantages of SSE TM is that after the data is processed, the resulting spectrum is free of fluorescence and is most suitable for unambiguous identification. Without SSE TM, a data pretreatment such as baseline correction would be necessary in order to attempt to remove the interfering fluorescence signal. The possible information loss because of a data pretreatment could yield invalid results [5]. The SSE TM method is most sensitive in retaining the intrinsic bandshape and positions of bands related to the material of interest. This allows not only the quick and accurate identification of unknown compounds, but also the characterization of contaminants and other trace compounds. Figure 2 shows an example where sodium alginate acquired with 785 nm excitation has an intense fluorescence background dominating the spectrum of interest (red spectrum). After application of the SSE TM technique with the BRAVO (blue spectrum), the resulting spectrum can be ready analyzed and the compound reliably identified. Some handheld Raman devices employ 1064 nm lasers, because fluorescence is generally less common with NIR excitation. However, the Raman scattering efficiency decreases significantly as longer wavelength lasers are used. The resulting lower signal needs to be compensated for with increased measuring time and/or higher laser power. Additionally, many samples undergo transient heating with 1064 nm excitation. SSE TM handles fluorescence while maintaining high sensitivity and under the safest conditions: The BRAVO is a class 1M system in all modes of operation. Consequently, whenever fluorescence or background signals are present, SSE TM offers significant benefits for unambiguous material identification of the broadest range of materials. References [1] S. Assi, Identification of counterfeit drugs using dual laser handheld Raman, Eur. Pharm. Rev. 20, 20 (2015) [2] Product Note T30: BRAVO - Accuracy is crucial: The starting point for a robust transfer of methods [3] J. B. Cooper et al., Sequentially Shifted Excitation Raman Spectroscopy: Novel Algorithm and Instrumentation for Fluorescence-Free Raman Spectroscopy in Spectral Space, Appl. Spectrosc. 67, 973 (2013) [4] J. B. Cooper et al., Spatially compressed dual-wavelength excitation Raman spectrometer, Appl. Opt. 53, 3333 (2014) [5] J. Kauffmann et al., Spectral Preprocessing for Raman Library Searching, Amer. Pharm. Rev. 14, (2011) Bruker Optics Bruker Optics is continually improving its products and reserves the right to change specifications without notice Bruker Optics BOPT