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Pharmaceutical & Biopharmaceutical Development Services

EAG brings unparalleled expertise to the development and commercialization of small molecule drugs, biopharmaceuticals, antibody-drug conjugates (ADCs), drug-device combination…

EAG brings unparalleled expertise to the development and commercialization of small molecule drugs, biopharmaceuticals, antibody-drug conjugates (ADCs), drug-device combination products and other therapies. From designing IND-enabling studies to delivering full CMC analytical and QC support, we join your R&D team as a true partner. EAG scientists take time to understand both your commercial goals and the unique characteristics of your compound. We provide expert guidance to balance regulatory expectations with expediency and cost, and approach technical challenges with flexibility and resolve.

Materials Testing & Analysis

When it comes to understanding the physical structure, chemical properties and composition of materials, no scientific services company offers the breadth of experience, diversity…

When it comes to understanding the physical structure, chemical properties and composition of materials, no scientific services company offers the breadth of experience, diversity of analytical techniques or technical ingenuity of EAG. From polymers to composites, thin films to superalloys—we know how to leverage materials sciences to gain a competitive edge. At EAG, we don’t just perform testing, we drive commercial success—through thoughtfully designed investigations, technically superior analyses and expert interpretation of data.

Environmental Testing & Regulatory Compliance

Having helped develop the test methods that shape current regulatory guidelines, EAG chemists, biologists and toxicologists have evaluated the environmental impact of thousands of…

Having helped develop the test methods that shape current regulatory guidelines, EAG chemists, biologists and toxicologists have evaluated the environmental impact of thousands of active ingredients and formulations—from pesticides and pharmaceuticals to industrial chemicals and consumer products. Whether you are exploring “what if” scenarios, registering a new active ingredient or formulation, responding to a data call-in or seeking to understand the latest guidance, turn to EAG for technical excellence, sound advice, GLP-compliant study execution and expert interpretation.

Microelectronics Test & Engineering

Whether connecting the internet of things, guiding surgical lasers or powering the latest smart phone, integrated circuits and microelectronics touch nearly every aspect of human…

Whether connecting the internet of things, guiding surgical lasers or powering the latest smart phone, integrated circuits and microelectronics touch nearly every aspect of human life. In the world of technology, innovation and continuous improvement are imperatives—and being able to quickly and reliably test, debug, diagnose failures and take corrective action can make the difference between a doomed product launch and building a successful global brand. EAG offers you the world’s largest and most diverse collection of specialized analytical instrumentation, capacity to perform a variety of microelectronic tests in parallel, and the multi-disciplinary expertise required to draw true insight from data.

Custom Synthesis & Radiolabeling

No contract service provider has more experience performing custom synthesis and producing isotopically labeled compounds to support product development in life science, chemical…

No contract service provider has more experience performing custom synthesis and producing isotopically labeled compounds to support product development in life science, chemical and related industries than we do. From 14C and 3H radiolabeled clinical trial materials synthesized under cGMP, to stable-labeled active ingredients for metabolism and environmental fate/effects testing, turn to EAG. We have extensive experience with multi-step and other complex synthesis projects, and our comprehensive, in-house analytical services ensure quick turnaround of purity and structural confirmation.

Crop Biotechnology & Development

EAG combines biotechnology and protein characterization expertise with more than 50 years' experience analyzing chemical compounds in plant and environmental matrices to address…

EAG combines biotechnology and protein characterization expertise with more than 50 years’ experience analyzing chemical compounds in plant and environmental matrices to address the growing needs of the biotechnology crop industry. We offer a wide range of techniques required to fully characterize the event insertion and expressed proteins, as well as the various studies required to confirm the food, feed and environmental safety of products that represent the trait. From early-stage protein confirmation to GLP-compliant EDSP and allergenicity testing, we help you make faster, more informed development decisions and comply with evolving global regulations of genetically engineered crops.

Litigation Support & Expert Testimony

When you need solid science and investigative engineering to address product failures, inform legal strategy, protect intellectual property or address product liability disputes,…

When you need solid science and investigative engineering to address product failures, inform legal strategy, protect intellectual property or address product liability disputes, turn to EAG. We’ve provided technical consulting, analysis and expert testimony for hundreds of cases involving the aerospace, transportation, medical device, electronics, industrial and consumer product industries. Our team of experts understands the legal process and your need for responsiveness, effective communication, scientifically defensible opinion and confidentiality. From professional consulting to data review to trial preparation and expert witness testimony, ask EAG.

Techniques

Chromatography

Using an array of advanced separation techniques and innovative technology, we conduct highly precise analytical chromatography for various industries. Whether you want a closer…

Using an array of advanced separation techniques and innovative technology, we conduct highly precise analytical chromatography for various industries. Whether you want a closer look at the purity of your pharmaceutical or need to better understand an agrochemical’s components, EAG has the expertise to separate and evaluate any compound.

Mass Spectrometry

Need to evaluate the molecular structure of a compound or identify its origins? EAG knows how. With state-of-the-art tools, we can separate, vaporize and ionize the atoms and…

Need to evaluate the molecular structure of a compound or identify its origins? EAG knows how. With state-of-the-art tools, we can separate, vaporize and ionize the atoms and molecules in almost any pure or complex material to detect and obtain mass spectra of the components. We rely on decades of experience in mass spectrometry to provide our clients with precise analyses and the best detection limits.

Imaging

EAG is a world leader in high-resolution imaging down to the atomic level. We offer unmatched analytical know-how, generating extremely detailed surface and near surface images…

EAG is a world leader in high-resolution imaging down to the atomic level. We offer unmatched analytical know-how, generating extremely detailed surface and near surface images for various industries, from consumer electronics to nanotechnology. Using state-of-the-art equipment and innovative techniques, we conduct expert imaging to aid in failure analysis, dimensional analysis, process characterization, particle identification and more. If you want to investigate a material with angstrom scale resolution, you can count on EAG to get the job done quickly and precisely.

Spectroscopy

EAG offers a vast array of spectroscopic techniques to clients in various industries, from defense contractors to technology pioneers. We combine unparalleled expertise and…

EAG offers a vast array of spectroscopic techniques to clients in various industries, from defense contractors to technology pioneers. We combine unparalleled expertise and methodology with cutting-edge technology to analyze your organic, inorganic, metallic and composite materials for identification, compositional, structural and contaminant information. Whether you need expert spectroscopic analysis to improve your production process or to surmount a technical challenge, EAG is up to the task.

Physical/Chemical Characterization

Need to identify your unique material? Want to analyze the thermal properties of a sample, or measure the success of a process step? If it has to be done quickly and it has to be…

Need to identify your unique material? Want to analyze the thermal properties of a sample, or measure the success of a process step? If it has to be done quickly and it has to be done right, you can count on EAG. We offer a range of adaptable techniques and innovative methods to evaluate the physical and chemical characteristics of any compound. Our highly precise testing and analytical services will improve your production process, expedite R&D and help you conquer any technical challenge.

About

A Global Scientific Services Company

One of the most respected names in contract research and testing, EAG Laboratories is a global scientific services company operating at the intersection of science, technology and…

One of the most respected names in contract research and testing, EAG Laboratories is a global scientific services company operating at the intersection of science, technology and business. The scientists and engineers of EAG apply multi-disciplinary expertise, advanced analytical techniques and “we know how” resolve to answer complex questions that drive commerce around the world.

Our Customers

Science and technology transcend industry boundaries, and so does demand for EAG’s expertise. We partner with companies across a broad spectrum of high-tech, high-impact and…

Science and technology transcend industry boundaries, and so does demand for EAG’s expertise. We partner with companies across a broad spectrum of high-tech, high-impact and highly regulated industries. We help our customers innovate new and improved products, investigate manufacturing problems, perform advanced analyses to determine safety, efficacy and regulatory compliance, and protect their brands.

Our Company Culture

EAG’s corporate culture is firmly rooted in four guiding principles: “foster a growth mindset,” “find a better way,” “earn more loyal customers,” and “win…

EAG’s corporate culture is firmly rooted in four guiding principles: “foster a growth mindset,” “find a better way,” “earn more loyal customers,” and “win together.” Across all of our 20+ locations, you will find a true passion for science and the power of science to improve the world we live in. Hear what some of our ~1200 scientists, engineers and support personnel say about what it means to be part of EAG Laboratories.

Careers

EAG is growing, and we are always looking for talented, problem-solving oriented individuals to join our company. If you have a “we know how” spirit, we want to hear from you.…

EAG is growing, and we are always looking for talented, problem-solving oriented individuals to join our company. If you have a “we know how” spirit, we want to hear from you. Browse current openings now, and re-visit our careers page often.

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Correlation of the Mass Spectrometric Analysis of Heat-Treated Glutaraldehyde Preparations to Their 235 nm / 280 nm UV Absorbance Ratio

WHITE PAPER

By Richard Dunphy, Leonard Rosik

Pharmaceutical Laboratory

Abstract

The 235 nm/280 nm UV absorbance ratio (UV Ratio) is widely used as a measure of the degree of polymerization of glutaraldehyde. Conventional practice is to measure the UV absorbance of bulk material at 235 nm (C=C absorption) and at 280 nm (C=O absorption). Gel permeation chromatography of preparations of heat-treated glutaraldehyde (HTGA) did not have sufficient mass discrimination to explain the variation in the UV Ratio of HTGA. Preparations of HTGA were analyzed by direct infusion ion trap mass spectrometry (IT-MS) and time of flight mass spectrometry (TOF-MS). Structures were assigned to the masses found. The structures of the UV-absorbing species were used to calculate absorbance contributions from each species found in the preparations at greater than 5% relative abundance. The contributions were weighted and summed. A MS-based UV Ratio was calculated and compared to the experimentally determined UV Ratio for the HTGA preparations. A very good correlation was obtained.

Introduction

Polymerized glutaraldehyde has been used as a fixative agent for electron microscopy and to immobilize proteins for decades (1-5). The solution chemistry of glutaraldehyde has been reviewed (2). The UV Ratio is a parameter that has been used classically to describe the extent of polymerization of the material (3,4,7,8). An additional analytical method was developed to better characterize the polymeric mixture and correlate to this absorbance ratio. Development data from a low molecular weight SEC method resulted in nearly the same molecular weight values for all four of the HTGA samples examined (Mp = 377 to 384 Da) which had UV Ratios of from 2 to 20. This suggested that the UV Ratio (degree of polymerization) was not simply molecular weight dependent but was also dependent on the structures of the oligomers present. The existing literature seems to support this (1,3,6-9). Direct infusion mass spectra of the HTGA preparations were acquired. A simple weighted average molecular weight was calculated for the samples (based on the masses seen and their relative abundances in each sample) and was found to not correlate to the UV Ratio, as was seen for the SEC results. From the MS data the structures were assigned for the various types of oligomers present. Based on these structures and the published values for the mass extinction coefficients of the chromophores (1) in the oligomers a MS-based UV Ratio was calculated for the bulk HTGA.

Experimental

The HTGA samples were prepared in water and heat treated. The samples had varying degrees of polymerization as indicated by their UV Ratio, as shown in Table 1. The HTGA samples were diluted 1:1 with a 0.1% formic acid aqueous solution for mass spectrometric analysis.

Table 1: HTGA Samples

Table 1: HTGA Samples

The diluted samples were analyzed on a Thermo Fleet Ion Trap Mass Spectrometer by direct infusion into the electrospray ionization (ESI) source using the onboard syringe pump at a flow rate ~ 15 µL/min. Data was collected for the range of m/z 50 to 1000 for two minutes and the data from the entire infusion was averaged. Earlier SEC work with these samples had indicated that the vast majority of the sample would be below 1000 Da. The MS thus obtained for 74, 78-2, 78-1, and 78-3 are shown in Figure 1, Figure 2, Figure 3, and Figure 4 respectively. The majority of the structures could be assigned from this data. Note that due to the sodium content of the samples the ions seen are sodiated cations that are +23 u from the actual neutral masses of the oligomers of interest.

Some peaks seen were not readily assigned based on these spectra. The samples were also analyzed by Waters Micromass LCT Premier Time of Flight Mass Spectrometer in order to obtain empirical formulae for the ions of interest and thus allow complete structural assignments. Direct infusion into the electrospray ionization (ESI) source using a syringe pump at a flow rate ~ 15 µL/min. Data was collected for the range of m/z 50 to 1000 for two minutes and the data from the entire infusion was averaged.

Figure 1: Mass Spectrum of Sample 74

Figure 1: Mass Spectrum of Sample 74

Figure 2: Mass Spectrum of Sample 78-2

Figure 2: Mass Spectrum of Sample 78-2

Figure 3: Mass Spectrum of Sample 78-1

Figure 3: Mass Spectrum of Sample 78-1

Figure 4: Mass Spectrum of Sample 78-3

Figure 4: Mass Spectrum of Sample 78-3
Results and Discussion

The acetal family of oligomers were the most predominant series of compounds seen in the aqueous samples. Their general structure is shown in Figure 5 along with the mass assignments and two example structures. These oligomers have no chromophores and so do not contribute to the UV Ratio. This is why the UV Ratio does not correlate to the average molecular weight of the sample. These compounds therefore were not included in the calculation.

Figure 5: The Acetal Family of Oligomers

Figure 5: The Acetal Family of Oligomers

The aldol condensation family of oligomers were also seen in the samples. Their general structure is shown in Figure 6 along with the mass assignments and two example structures.

Figure 6: The Aldol Condensation Family of Oligomers

Figure 6: The Aldol Condensation Family of Oligomers

The aldol condensation family of oligomers shown above in Figure 6 can also undergo the elimination of water to give compounds with some of the aldol linkages dehydrated to yield unsaturated aldehydes. Their general structure is shown in Figure 7 and Table 2, along with the mass assignments and two example structures.

Figure 7: The Partially Dehydrated Aldol Condensation Family of Oligomers

Figure 7: The Partially Dehydrated Aldol Condensation Family of Oligomers

Table 2

Table 2

The aldol condensation oligomers shown in Figure 6 have suffered aerobic oxidation to the diacids. Their general structure is shown in Figure 8 along with the mass assignments and two example structures.

The aldol condensation family of oligomers shown above in Figure 6 can also undergo the elimination of water to give compounds with some of the aldol linkages dehydrated to yield unsaturated aldehydes. Their general structure is shown in Figure 7 and Table 2, along with the mass assignments and two example structures.

Figure 8: The Oxidized Aldol Condensation Family of Oligomers

Figure 8: The Oxidized Aldol Condensation Family of Oligomers

The above assignments are in agreement with previous UV, IR, and NMR studies (1,2,6,9). There were no significant peaks found in the sample spectra that corresponded to oxidized and partially dehydrated oligomers. This was also the case for mono-oxidized and partially dehydrated species.

Calculation of the MS-Based 235 nm/280 nm UV Absorbance Ratio

The structural assignments are in agreement with previous UV, IR, and NMR studies (1,2,6,9). Once the structures for all of the significant peaks have been assigned then the UV absorbance contributions for each molecule can be calculated. The aldehydes moieties absorb at 280 nm and the carbon-carbon double bond absorbs at 235 nm. The mass extinction coefficients for the unconjugated aldehyde group, the conjugated aldehyde group, and the conjugated carbon-carbon double bond have been given as 0.042, 0.41, and 18.6 L/g cm (1). Therefore, for each relevant mass (structure that has a UV absorbance) the number of each kind of contributing chromophore can be determined by inspection. Those contributions to the 280 nm or the 235 nm absorptions can then be summed. These summed contributions can then be weighted with the relative abundance of the peak in the MS to give weighted 280 nm and 235 nm absorptions. The weighted absorptions can then be summed9 for all the relevant molecules in a sample and the UV Ratio calculated. The MS-based UV Ratio can then be correlated to the experimentally determined UV Ratio by regression. Summaries of this calculation for the samples 3.5% HTGA, 15% HTGA-1, 15% HTGA-2, and 15% HTGA-3 are shown in Figure 9, Figure 10, Figure 11, and Figure 12 respectively. A co-efficient of determination (R2) value of 0.9944 was obtained and is show in Figure 13.

Figure 9: MS-Based UV Ratio of Sample 74

Figure 9: MS-Based UV Ratio of Sample 74

Figure 10: MS-Based UV Ratio of Sample 78-2

Figure 10: MS-Based UV Ratio of Sample 78-2

Figure 11: MS-Based UV Ratio of Sample 78-1

Figure 11: MS-Based UV Ratio of Sample 78-1

Figure 12: MS-Based UV Ratio of Sample 78-3

Figure 12: MS-Based UV Ratio of Sample 78-3

Figure 13: Correlation of Experimental UV Ratio to the MS-Based UV Ratio

Figure 13: Correlation of Experimental UV Ratio to the MS-Based UV Ratio
Conclusions

Preparations of HTGA were analyzed by direct infusion MS and structures were assigned to the masses found. The structures of the UV-absorbing species were used to calculate absorbance contributions from each species found in the preparations at greater than 5% relative abundance. The contributions were weighted and summed and a MS-based UV Ratio was calculated and compared to the experimentally determined UV Ratio for the HTGA preparations. Very good correlation was obtained.

References
  1. Margel S. and Rembaum A., Macromolecules 1980, 13, 19-24.
  2. Mignealt I. et al., BioTechniques 2004, 37, 790-802.
  3. Prento P., Histochemical Journal 1995, 27, 906-913.
  4. Jones G.J., Journal of Histochemistry and Cytochemistry 1974, 22, 911-913.
  5. Goff C.W. and Oster M.O., Journal of Histochemistry and Cytochemistry 1974, 22, 913-915.
  6. Aso C. and Aito Y., Die Makromolekulare Chemie 1962, 58, 195-203.
  7. Anderson P.J., Journal of Histochemistry and Cytochemistry 1967, 15, 652-661.
  8. Frigerio N.A. and Shaw M.J., Journal of Histochemistry and Cytochemistry 1969, 17, 176-181.
  9. Hardy P.M. et al., Chemical Communications 1969, 565-566.