BrukerTechnicalPaPerSerieSPharmaceutical Applications of EPR Spectroscopy

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Bruker Technical PaPer SerieS

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ePR (electron Paramagnetic Resonance) is a spectroscopic technique that detects species that have unpaired electrons. It is also often called eSR (electron Spin Resonance). a surprisingly large number of materials have unpaired electrons. These include free radicals, many transition metal ions, and defects in materials. Free radicals are often short-lived, but still play crucial roles in many processes, such as photosynthesis, oxidation, catalysis, and polymerization reactions. In fact, free radicals are far more common than most people may realize.

For example, you most likely come in contact with free radicals multiple times before you even leave your house in the morning. about 20 percent of the air in your bedroom is paramagnetic molecular oxygen as a triplet state, so there are free radicals present as soon as you take a deep breath upon waking up. Coffee has carbon-based radicals from roasting, and tea has manganese, iron, and catechols. Free radicals are created if you toast your bagel. Some toothpastes contain manganese, and your hair contains the pigment melanin.

However, the toxicity of free radicals can be used to our advantage, particularly when we get sick. Invading microbes need to be neutralized and our white blood cells release reactive oxygen species, or ROS to kill the invading pathogens. ROS are free radicals and they’re very highly reactive. Their activities are beneficial, but can also be toxic. These ROS are detectable by ePR, which can tell us how much and what type of free radicals are detected.

How does EPR work?ePR is a magnetic resonance technique similar to NMR (Nuclear Magnetic Resonance). However, instead of measuring the nuclear transitions in our sample, we are detecting the transitions of unpaired electrons in an applied magnetic field. Like a proton, the electron has “spin,” which gives it a magnetic property known as a “magnetic moment.” The magnetic moment makes the electron behave like a tiny bar magnet similar to one you might put on your refrigerator. When we supply an external magnetic field, the unpaired electrons can either orient in a direction parallel or antiparallel to the direction of the magnetic field. This creates two distinct energy levels for the unpaired electrons and allows us to measure them as they are driven between the two levels.Initially, there will be more electrons in the lower energy level (i.e., antiparallel to the field) than in the upper level (parallel). We use a fixed frequency of

microwave irradiation to excite some of the electrons in the lower energy level to the upper energy level. In order for the transition to occur, we must also have the external magnetic field at a specific strength, such that the energy level separation between the lower and upper states is exactly matched by our microwave frequency. In order to achieve this condition, we sweep the external magnet’s field while exposing the sample to a fixed frequency of microwave irradiation. The condition where the magnetic field and the microwave frequency are “just right” to produce an ePR resonance—or absorption—is known as the “resonance condition.”

Only ePR detects unpaired electrons unambiguously. Other techniques, such as fluorescence, may provide indirect evidence of free radicals, but ePR alone yields incontrovertible evidence of their presence. In addition, ePR has the unique power to identify the paramagnetic species that is detected. ePR spectra are very sensitive to local environments. Therefore, the technique sheds light on the molecular structure near the unpaired electron. Sometimes, the ePR spectra exhibit dramatic lineshape changes, giving insight into dynamic processes such as molecular motions or fluidity.

The ePR spin-trapping technique, which detects short-lived, reactive free radicals, very nicely illustrates how EPR detection and identification of radicals can be exploited. This technique has been vital in the biomedical field for elucidating the role of free radicals in many pathologies and toxicities. ePR spin-labeling is a technique used by biochemists whereby a paramagnetic molecule (i.e., the spin label) is used to “tag” macromolecules in specific regions. From the ePR spectra reported by the spin label, they can determine the type of environmentin which the spin label is located.

eSeeM and eNDOR are two ePR methods that measure the interactions of the electron with the surrounding nuclei. They are extremely powerful techniques for probing the structure of “active sites” in metalloproteins. another important application for quantitative ePR is radiation dosimetry. among its uses are dose measurements for sterilization of medical goods and foods, detection of irradiated foods, and the dating of early human artifacts.

But perhaps one of the most important industries ePR has found a strong foothold in is pharmaceutical, where ePR can be used for:

• detecting and evaluating degradation

Pharmaceutical Applications of EPR

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• optimizing stability and shelf-life• reaction monitoring• sterilization; and• paramagnetic impurity profiling

Detecting and evaluating degradationelements like oxygen, moisture, sterilization processes, impurities, and excipient interactions are important factors in considering the stability of pharmaceutical products. all these factors may cause degradation of active pharmaceutical ingredients (aPIs), excipients, or drug formulations. Ultimately, degradation processes involve free radicals, so understanding free radicals and transition metals as degradation mechanisms is important—and ePR can help.

The ePR intensity of the detected signal is used to calculate the free radical concentration, which correlates with the level of degradation. In other words, quantitative ePR measurements provide and determine the extent of degradation of your sample.

ePR can identify the radical intermediates and help determine the reaction mechanism. Quantitative analysis shows the extent of free radical formation on exposure to a defined amount of pro-oxidants, such as light, temperature, etc. This can be used to determine and predict long-term stability characteristics of the sample.

Optimizing stability and shelf-lifeOxidation is one of the major causes of direct instability, so the ability to probe and understand oxidative degradation is extremely important.

Forced oxidation, also known as “stress testing,” is routinely used in pharmaceutical development to predict the stability of drug products that affects purity, effectiveness, and safety. In stress testing, the drug product is often exposed to heat, light or chemical agents with the goals being to understand degradation pathways, determine intrinsic stability and shelf-life, develop stable formulations, and evaluate antioxidant efficiency.

FIGURE 1 is an example demonstrating the efficiency of two antioxidants labeled a and B. as you can see from the figure, the free radical concentration determined by ePR decayed much faster in the drug formulation mixed with antioxidant a compared to antioxidant B. at the end of the stress testing experiment, the final radical concentration was about three times less than antioxidant B—meaning

antioxidant a is approximately three times more efficient than B.

In a shelf-life case study of tablet formulations, ePR was used to detect forced degradation of five samples that were placed at elevated temperatures of 40 degrees Celsius.

These five tablet formulations were stress tested over a time course of 10 days. as you can see from the bar diagram (FIgURe 2), increasing free radical concentration was detected in tablets 3-5, meaning that those established fomulations are susceptible to oxidation, indicating a reduced shelf-life. On the other hand, tablets 1 and 2 with low radical formulation have much better potential for drug development.

antioxidant a is more effective

than antioxidant B at quenching the

free radicals in the drug formulation.

Figure 1

Figure 2

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Reaction monitoringPharmaceutical regulations now require more fundamental understanding of the chemistry involved in aPI production, including reactive, intermediate identification. The surge of new molecules that exhibits specific properties is vital to pharmaceutical development, and reaction monitoring is a critical step in optimizing the synthesis of these new drugs. additionally, understanding reaction mechanism can lead to cost savings and a high-quality final product. Chemistries involving radicals and transition metals are integral components of maximizing product yield and minimizing environmental footprint.

ePR can help accomplish all of this in terms of reaction monitoring by:

• Identifying reaction interme-diates to obtain mechanistic information

• answering key chemical questions, such as reaction yield and reaction kinetics

• generating data to build kinetics models

• Quantifying free radicals and metals over the course of the reaction

SterilizationProper sterilization is important for ensuring safety in manufacturing and packaging of the pharmaceuticals. The most commonly used sterilization processes are gamma or electron beam irradiation, dry heat or pressure. However, the process can generate free radicals that are responsible for degradation of the irradiated materials, and cause alteration of the physico-chemical properties of the sterilized product. The process can also decrease drug potency by partial decomposition during sterilization, and may be a toxicological hazard.

Identifying the structure of radicals provides a better understanding of the mechanism of radiolysis. Additionally, quantification of radical amount enables you to establish a threshold for the radiosterilization of these drugs.

ePR spectroscopy is the only technique for non-invasive detection of free radicals. By quantifying free radicals, you can assess the level of degradation during the sterilization processes.

Paramagnetic impurity profilingall drugs contain impurities that can arise from aPIs or excipients, or both. They can also be introduced into the drug process during formulation processes,

packaging and storage. Impurities have many unwanted effects, including decreasing the potency, lowering the product shelf life and inducing toxicity.

Organic impurities are often free radicals from byproducts, intermediates, or degradation processes. Inorganic impurities include transition metals. ePR spectroscopy can detect traces of impurities down to parts-per-billion levels. Manganese present at trace levels in calcium hydrogen phosphate that is commonly used as a sealer in tablets can be easily detected and quantified by EPR (see FIGURE 3).

a case study published a few years ago by genentech Inc. showed amino acid oxidation in a formulation induced by autoxidation of polysorbate 20, known as PS20, typically used as a stabilizer. This is an example where degradation of excipients leads to oxidation over time. The process of degradation of PS20 by autoxidation and catalyzed by transition metals results in side-chain cleavage and free radical formation. EPR detected, identified and quantified the free radical impurities.

What triggers autoxidation is the interaction between organic peroxides found in PS20 with concentrations between 11 and 44 ppm and iron found in the protein drug in concentrations of 0.03 and 0.08 ppm. This metal catalyst oxidation is a radical chain reaction and it goes through initiation and propagation steps with formation of three different radical species—alkyl, alkoxyl, and peroxyl radicals—that were detected, identified, and quantified by EPR.

EMXnano: The standard for benchtop EPRBruker offers a benchtop ePR spectrometer—the eMXnano—that is compact compared to the traditional floor models. It does not require any special electrical service or water cooling, and is very easy to place in your lab.

It was designed with the goal of giving researchers that are new to ePR the ability to incorporate this

Figure 3

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technique easily into their applications. The eMXnano offers a wide range of features to pursue the previously mentioned pharmaceutical applications or to develop your own new applications. among these features are automatic tuning, which makes changing samples fast and convenient; data collection, processing and storage, which can be automated; and there is an integrated motorized amplitude and field reference standard to ensure reproducibility.

It is fully calibrated for quantitative analysis with dedicated application workflows—there is no need for a calibration curve. Workflows include:

• Spin count with an easy to use interface allows direct conversion of the measured ePR signal to the concentration of radicals for transition metals in the sample.

• Spin fit facilitates identification of the EPR active species, even in multi component samples, pro-viding concentrations of individual components.

• Species identification is made easy with the spin fit library of commonly encountered EPR active species. You can also create your own new entries for your own library.

Bruker also offers a full range of optional accessories. For photo stability studies, there is a UV irradiation system that provides in-situ irradiation of the sample in the microwave cavity with simultaneous ePR detection. The system is closed so it is much safer, meaning you don’t need to where UV goggles when performing experiments.

For thermal stability studes, Bruker offers a variable temperature accessory that has a range from 100 K to 425 K. The digital temperature control systems make use of liquid or gaseous nitrogen for stable temperature control. easy and quick sample

exchange at any temperature offers safe operation and high measurement throughput.For reaction monitoring, there is a flow-through cell, which is optimized to offer high sensitivity for all samples. Sample exchange is via static injection, continuous flow or auto sampler.

For aqueous samples, a flat cell is an option that maximizes the usable sample volume for low concentration measurements. There is optical access, which enables the irradiation with the optional UV system. Similarly, for biological tissue samples, the tissue sample is designed for the study of plant, animal and human tissues. As with the flat sell, it also enables irradiation with the optional UV system.

For physiological sample conditioning, the optional gas and temperature controller maintains physiological or pathological conditions when studying biological samples by detecting reactive oxygen species. The software controls the temperature in the range from +10 to +50 degrees Celsius with an accuracy of +/- 0.1. The gas mixtures can be controlled between oxygen, carbon dioxide, and nitrogen.

Giving researchers the power and flexibility needed in an easy to use compact unit, the eMXnano brings affordable high-end ePR performance to labs. For the first time in this instrument category, quantitative EPR is offered at your fingertip.

No matter what the focus of your application, the crucial performance requirements of an ePR spectrometer are sensitivity and stability. The latest generation magnet and microwave technology delivers class-leading performance, enabling the eMXnano to combine ease of use with highest quality ePR data. ¤

About this technicAl pAperThis white paper was adapted from a Bruker Corporation webinar titled, “Pharmaceutical applications of electron Paramagnetic Resonance (ePR) Spectroscopy.” Both webinar speakers are scientists at Bruker BioSpin in Billerica, Mass.

Dr. Ralph Weber, Senior applications Scientist, joined Bruker in 1989. He is responsible for much of the documentation for ePR and also offers customer support for pulse, high frequency, and imaging applications. He is currently co-principal investigator on a five-year NIH grant to develop pre-clinical ePR imaging technology and to promote its use in the pharmaceutical industry.

Dr. Kalina Ranguelova has been an ePR applications Scientist in Bruker BioSpin Corporation since 2011. Her current focus is detection and identification of free radicals in biological systems and pharmaceuticals using spin traps and spin probes. She has publications in many journals, and has presented at many international meetings related to free radical research in biology and protein chemistry.

You can view the webinar in its full on-demand via Bruker’s website:www.bruker.com/serv ice/educat ion-t ra in ing/webinars/epr-webinars/pharmaceut ical-appl icat ions-of-electron-paramagnetic-resonance-epr-spectroscopy.html