Industries

Aerospace & Defense

EAG is your R&D partner, working with aerospace and defense manufacturers and suppliers to create faster, safer and more reliable aircraft and components. We use the most advanced…

EAG is your R&D partner, working with aerospace and defense manufacturers and suppliers to create faster, safer and more reliable aircraft and components. We use the most advanced analytical methodologies to answer complex engineering, materials and manufacturing problems, from evaluating the purity of new materials to confirming the characteristics of ceramic matrix composites. For research, development and production, we work alongside your technical staff to formulate and troubleshoot the next generation of aerospace and defense materials.

Chemicals & Basic Materials

For over 50 years, EAG’s scientists have applied advanced analytical techniques to solve tough problems and support R&D activities for manufacturers and users of a wide variety…

For over 50 years, EAG’s scientists have applied advanced analytical techniques to solve tough problems and support R&D activities for manufacturers and users of a wide variety of chemicals and materials. EAG offers reliable analytical results, development support for novel products, and answers to complex problems for manufacturers of chemicals used in metals, fibers, plastics, coatings, adhesives, construction materials, textiles, lubricants and more.

Consumer Products

Consumer products manufacturers and partners turn to EAG to answer R&D questions, ensure supply chain quality, respond to regulatory requirements and ensure well-supported legal…

Consumer products manufacturers and partners turn to EAG to answer R&D questions, ensure supply chain quality, respond to regulatory requirements and ensure well-supported legal arguments. EAG scientists have helped all types of consumer products companies accelerate innovation, resolve manufacturing concerns and comply with evolving regulations.

Food & Agriculture

Drive R&D productivity and keep pace with evolving environmental regulations. From agrochemical development to evaluating innovative packaging solutions, EAG scientists support…

Drive R&D productivity and keep pace with evolving environmental regulations.

From agrochemical development to evaluating innovative packaging solutions, EAG scientists support the agriculture/food s industry with scientific expertise required to enhance shelf life, ensure product safety and understand environmental impact. Whether in need of food quality investigations or help supporting regulatory filing of new chemicals, ask EAG. WE KNOW HOW.

Law

When a business’s brand and reputation is at stake, clients turn to EAG’s litigation support services for dependable scientific answers. Since 1959, our scientific expertise,…

When a business’s brand and reputation is at stake, clients turn to EAG’s litigation support services for dependable scientific answers. Since 1959, our scientific expertise, testing and testimony have supported legal strategies for intellectual property, product liability and insurance cases. Our scientists offer expertise in technology, life sciences, electronics, industrial and consumer products, having tested a broad array of materials for legal challenges. We understand the unique needs of legal projects – speed, effective communication, reliability and confidentiality.

Medical Device & Diagnostics

From initial concept and prototypes to verification/validation, submission and launch, EAG Laboratories is your partner in medical device development. From implant…

From initial concept and prototypes to verification/validation, submission and launch, EAG Laboratories is your partner in medical device development. From implant biocompatibility studies to surface chemistry for contact lenses, we have the experience and techniques to characterize, test and assure purity of medical devices and their materials, components, products and packaging. By utilizing our unique expertise in materials sciences, chemistry, electronics and regulatory support, EAG empowers device companies to create new ways to heal and improve patient quality of life.

Pharmaceuticals

Overcome R&D roadblocks with technical and regulatory know-how. For over two decades, EAG scientists have helped solve complex issues for the pharmaceutical Industry. Using…

Overcome R&D roadblocks with technical and regulatory know-how. For over two decades, EAG scientists have helped solve complex issues for the pharmaceutical Industry. Using multidisciplinary science and engineering, we help improve product development efficiency, avoid regulatory setbacks and uncover sources of problematic manufacturing issues. From GLP and cGMP studies to contaminant and packaging investigations, the scientists of EAG support every phase of the product lifecycle.

Technology

For the high-tech industry, EAG is the leader in materials characterization, surface analysis, microscopy and electronics testing. Our scientists and engineers troubleshoot and…

For the high-tech industry, EAG is the leader in materials characterization, surface analysis, microscopy and electronics testing. Our scientists and engineers troubleshoot and solve issues with consumer electronics, chip packages, printed circuit boards, displays, sensors, high-speed communications and more. We meet the demanding needs of technology innovation, enabling our clients to have confidence in their research & development, supply chains, and ultimately their manufacturing processes

Services

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.

How do you drive R&D productivity and keep up with regulations?

Modification of an Analytical Method for the Analysis of Clopyralid in Animal Tissues and the Pitfalls Encountered

APPLICATION NOTE

By Stanley R. Shaer¹, Mark Fortenberry¹, Brian Bothwell¹, Darcy Shackelford² and Ed Olberding³

¹EAG Laboratories, ²Dow AgroSciences, Inc., ³Haystack Analytics, LLC

ABSTRACT

Purpose: The objective of the study was to convert a method  utilizing analyte derivatization with GC/MS detection to a direct  analysis by LC/MS-MS.

Methods: The original analytical method consisting of a basic hydrolysis/extraction technique followed by HLB solid-phase extraction (SPE) column (Waters, 200 mg/6 mL) cleanup, propylation, and detection by capillary gas chromatography with negative-ion chemical ionization mass spectrometry (GC-NCIMS) was modified for direct analysis by LC/MS-MS. The original extraction procedure and a modification of the HLB SPE clean-up procedure were utilized to provide successful analysis by LC/MSMS without matrix enhancement/suppression or interferences. Conversion of a method from one utilizing derivatization followed by GC/MS analysis to that of a direct detection by LC/MS-MS can pose unique analytical challenges to obtain comparable sensitivity and specificity. This paper will give an overview of the unique recovery, interference and sensitivity issues overcome during the process.

For the final method, residues of clopyralid are extracted from animal tissue samples with 2.5N NaOH with heating at approximately 105 °C for a minimum of 2 hours. Optional cleanup for poultry liver is affected by partitioning the basic extract with dichloromethane (DCM). An aliquot of the extract is acidified with HCl and submitted to a polymeric reversed-phase solid phase extraction column (Waters, HLB SPE) cleanup and elution with DCM. After removal of the DCM using nitrogen blow down, the sample is reconstituted in 10:90, acetonitrile:0.1% formic acid. The final extract is filtered through a 0.2 m PTFE syringe filter and then analyzed by liquid chromatography coupled with negativeion electrospray ionization tandem mass spectrometry (ESI LC/ MS-MS).

CHALLENGES

LC/MS-MS Parameters: The first step in developing a new method was to set up the instrument detection system. Since the original method was capillary gas chromatography with negative-ion chemical ionization mass spectrometry, the LC/MSMS parameters had to be developed. The clopyralid dissolved in methanol was infused directly into the triple quad detector and a mass spectrum was generated in the negative-ion mode using electrospray. The clopyralid structure is presented in Table 1 and the spectrum in Figure 1. The most intense molecular ion (M-H)- 190 m/z with the corresponding product ion of 146 m/z was used for quantitation (Figure 2). Both EPA and European guidelines require that a confirmation transition be monitored to confirm the identity of the molecule being detected. A second product ion generated from the same molecular ion is preferred. However, no other product ions were generated in sufficient quantities to be a satisfactory confirmation transition. Since the molecule contained chlorine, it was decided to use the +2 molecular ion [(M-H)- +2] 192 m/z and its corresponding product ion 148 m/z (Figure 3).

Table 1: Identity and structure of Clopyralid
Table 1: Identity and structure of Clopyralid

Figure 1: Negative-Ion electrospray mass spectrum for Clopyralid
Figure 1: Negative-Ion electrospray mass spectrum for Clopyralid

Figure 2: Product-ion mass spectrum of m/z 190 showing a major transition ion at m/z 146
Figure 2: Product-ion mass spectrum of m/z 190 showing a major transition ion at m/z 146

Figure 3: Product-ion mass spectrum of m/z 192 showinga major transition ion at m/z 148
Figure 3: Product-ion mass spectrum of m/z 192 showinga major transition ion at m/z 148

Once the quantitation and confirmation transitions were selected, the HPLC parameters needed to be established to obtain adequate sensitivity and separation from sample matrix. HPLC parameters utilizing a methanol/water system with 0.1% formic acid a modifier was developed. This system seemed to be adequate until issues were encountered with the poultry liver matrix which contained negative peaks on both sides of the clopyralid peak (Figure 4). To further clean up the sample, a dichloromethane partitioning step on the basic extract was added which removed the negative peak eluting before the clopyralid peak (Figure 5).

Figure 4: Poultry liver sample fortified with Clopyralid at 0.010 mg/kg (LOQ) (68447 102) without DCM cleanup. Mobile Phase A: 0.1% Formic acid in water, Mobile Phase B: 0.1% Formic acid in MeOH, 35 μL injection
Figure 4: Poultry liver sample fortified with Clopyralid at 0.010 mg/kg (LOQ) (68447 102) without DCM cleanup. Mobile Phase A: 0.1% Formic acid in water, Mobile Phase B: 0.1% Formic acid in MeOH, 35 μL injection

Figure 5: Poultry liver sample fortied with Clopyralid at 0.010 mg/kg (LOQ) (68447 102D) with DCM cleanup. Mobile Phase A: 0.01% Formic acid in water Mobile Phase B: 0.01% Formic acid in 60:40, MeOH:ACN, 15 μL injection
Figure 5: Poultry liver sample fortied with Clopyralid at 0.010 mg/kg (LOQ) (68447 102D) with DCM cleanup.Mobile Phase A: 0.01% Formic acid in water. Mobile Phase B: 0.01% Formic acid in 60:40, MeOH:ACN, 15 μL injection

To further affect separation from the negative peak eluting after of the clopyralid, the organic phase was modified to 60:40, methanol:acetonitrile. To increase the sensitivity and reduce the baseline noise, the modifier was reduced to 0.01% formic acid. As can be seen in Figure 5 the sensitive was increase by 48% even though the injection volume was reduced from 35 uL to 15 uL.

Recovery Issues: During the development of the cleanup method, tests were conducted to determine if any analyte losses would be incurred during the blow down step. No losses were seen when reagent spikes were blown down under a stream of nitrogen at a water bath temperature of 40°C even after continuing the blow down for 15 minutes after all dichloromethane had been removed. However, low recoveries were seen during the blow down step in the presence of matrix with post SPE spikes. The use of vortexing and sonication to dissolve the residues yielded variable recoveries. When multiple samples were analyzed in one set they were placed in a test tube rack during sonication. The samples remained clear (Figure 6, right test tube) even after prolonged sonication and vortexing and recoveries were low. While troubling shooting this step a single sample was handheld and placed in the sonicator and the sample became cloudy.

Figure 6: Example of proper dissolution of the final sample
Figure 6: Example of proper dissolution of the final sample

As the sample was rotated and tilted to assure the solvent came in contact with the entire inner surface of the test tube during sonication the sample became more cloudy (Figure 6, left test tube). Further investigation determined that eluting the SPE with dichloromethane was actually dissolving a portion of the plastic that contained the HLB column bed. As the dichloromethane was evaporating under nitrogen blow down, the plastic was trapping the clopyralid on the sides of the test tube.

Since the plastic was not soluble in the 90% aqueous final diluent, simple vortexing or sonication in a vertical position was not sufficient to loosen the plastic residue and liberate the clopyralid. Thus, it was a necessity to sonicate each sample by hand, insuring the solvent came in contact with the entire inner surface of the test tube to physically remove the plastic from the tube surface allowing the clopyralid to dissolve in the solvent. This is the most critical step in the procedure and accounts for the majority of analyte losses. Having a cloudy final solution is an indication of proper dissolution of the sample. Filtration through a 0.2-m PTFE syringe filter removes all cloudiness.

Results: The method validation study was conducted to determine the recovery levels and the precision of the method for the determination of residues of clopyralid in animal matrices. The performance of the analytical method was determined with each set of samples by fortifying aliquots of the appropriate control matrix with a clopyralid solution and analyzing the set following the procedures described within this report. Samples were fortified at the limit of detection (LOD) of 0.003 mg/kg, the limit of quantitation (LOQ) of 0.010 mg/kg, and at 1.0 mg/kg. Samples fortified at the LOD were analyzed only to demonstrate observable peaks at the LOD level; the results were not included in average percent recovery calculations. An unfortified control matrix and reagent blank were also included in each set.

Table 2a: Recovery of Clopyralid from poultry liver
Table 2a: Recovery of Clopyralid from poultry liver

Table 2b: Recovery of Clopyralid from poultry live
Table 2b: Recovery of Clopyralid from poultry liver

The results of the quantitation and confirmatory recoveries for poultry liver are listed in Table 2. For the quantitation results, the individual recoveries for all samples fell within the range of 70 to 110% and the average recoveries at each fortification level also fell within the range of 70 to 110%. The average recoveries for all fortification levels fell within the range of 70 to 110%. Relative standard deviations at each fortification level were all less than 20%.

The method is selective for the determination of clopyralid by virtue of the chromatographic separation and MS/MS detection system used. Using published guidelines (1), when detection is by tandem mass spectrometry methods, confirmation of the presence of the analyte should require the observation of a precursor ion plus one structurally significant product ion observed at the same retention time. By monitoring multiple MS/ MS ion transitions for each analyte, the confirmation ratios were calculated for clopyralid in each sample set and compared to the average for the calibration standards. The results are listed in Table 2. The confirmation ratios in all samples (accept the LOD) were within ±20% of the average found for the standards, indicating that the method is selective for the determination of clopyralid in poultry liver.

Conclusions: The analytical method for the determination of clopyralid in animal matrices has been demonstrated to be satisfactory in terms of accuracy, precision, linearity, and selectivity. The method was validated over the concentration range of 0.003-1.0 mg/kg for poultry liver with a limit of quantitation of 0.010 mg/kg.

 

REFERENCES

  1. Baldwin, R.; Bethem, R. A.; Boyd, R. K.; Budde, W. L.; Cairns, T.; Gibbons, R. D.; Henion, J. D.; Kaiser, M. A.; Lewis, D. L.; Matusik, J. E.; Sphon, J. A.; Stephany, R. W.; Trubey, R. K.; J. Am. Soc. Mass Spectrom. 1997, 8, 1180-1190.

 

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