Chemical Analysis Facility Core

Director: Brian Buckley, Ph.D.

The EOHSI Chemical Analysis Facility Core (CAF) is a state-of-the-art chromatographic and mass spectrometric facility designed to support the Institute’s investigators and collaborators with methods development for assays of environmental contaminants or their metabolites. The CAF Laboratory is directed by Dr. Brian Buckley and specializes in the development and evaluation of novel analytical methodologies which may be applied to current and future EOHSI projects. The CAF also provides expert consultation on sample collection and analysis, quality assurance, data interpretation, instrument acquisition, and sample preparation. Laboratory personnel are also responsible for the training of students on analytical instrumentation and sample analysis. The CAF has both inorganic and organic capabilities.

Video Tour - LCMS and GCMS Labs

Organic Analysis Laboratory

Organic-Analysis

A majority of the effort in methods development for organic analysis has been in support of our researchers in their studies of neurotoxicology. The laboratory recently created new methods for analysis of pesticides in cell culture, catecholamines in plasma, and PCBs in serum. In addition we have utilized the new HPLC/MS technology to evaluate degradation products of drugs used in emergency response kits as well as pharmaceuticals found in drinking water. Our drinking water work for pharmaceuticals was featured on ABC World News in October 2008.

Inorganic Analysis Laboratory

Inorganic-Analysis

A majority of the effort in methods development for inorganic analysis has been directed toward differentiation of various metabolites of toxic metals found in human biological samples. For example, we have utilized a method for quantifying the toxic forms of arsenic and have developed a new method for separation and quantitation of two organometallic forms of chromium. Biomonitoring work has been performed on populations with known or suspected exposures to these contaminants in their environment. Direct support was provided to Rutgers University by collecting and analyzing turf field samples for heavy metals and organic contaminants when the safety of the material was called into question. A similar response with rapid analysis was provided for the city of Newark.

New Capabilities

new-icpms

Within the inorganic analysis specialty lies a unique capability, specifically inorganic and organometallic speciation using a high resolution magnetic sector ICP/MS. The magnetic sector instrument has already proven to be 2 to 3 orders of magnitude more sensitive than our quadrupole instrument for many elements and operates at higher resolution, removing many of the isobar interferences in our current assays. For example the chromium isotope at a mass to charge of 52 suffers from isobars of argon carbide and argon oxide. Operating the magnetic sector instrument at 4000 we can completely remove this isobar interference from our chromium measurements. Coupled with the separation capabilities of an ion chromatograph, it allows us to speciate or differentially quantify both chromium III and chromium VI in drinking water to levels well below 10 ppb. The ICP/MS we use for this measurement is a Nu Instruments Attom and the ion chromatograph is a Metrohm.

In addition to quantitation and speciation at much lower levels than previously possible, the higher resolution ICP/MS allows us greater precision in isotopic ratio measurements. This is important because stable isotope labels can be used to identify an element introduced into an experiment rather than one already present. For example if we wish to study the uptake of magnesium in a cell culture we can use the labeled form or the isotopically enriched form to discriminate from the magnesium already present in the cell. With the quadrupole instrument our isotopic precision was roughly 2%. With a high resolution instrument our precision is below half a percent.

We’ve just begun to explore the capabilities of the high resolution ICP/MS with the ion chromatograph interface for many biological and environmental applications.

The instrument was purchased with funds from the National Institutes of Health shared instrumentation grant program.

Sample Preparation Laboratory

sample-prep

The EOHSI Analytical Core Laboratory receives samples for analysis from a variety of sources including investigators from the university community and state agencies.  Samples are typically received by laboratory staff; inspected for damage; logged into the laboratory data base; assigned a unique sample number; labeled properly; and stored at the appropriate temperature to await preparation and analysis.  All aspects of sample receipt adhere to written Standard Operating Procedures (SOPs) and established Quality Assurance protocols.  Additionally, rigorous chain-of-custody paperwork and methodology are maintained.

Samples may be received in a variety of forms including soil, water, and biological materials, as well as less typical matrices (e.g., artificial turf, garden hose, and plush children’s toys to name a few recent novel matrices).  Depending on the type of sample and the method of analysis, samples will proceed along the sample preparation continuum.  Typically, the Analytical Core Laboratory will analyze several QA/QC samples for each batch of samples being prepared for analysis, including preparation blanks (used to determine level of background interference); laboratory control samples (used to monitor the accuracy of the analytical method); matrix spike samples (used to evaluate the effect of the sample matrix on the accuracy of the analytical method); and sample duplicates (used to evaluate the precision of the analytical method).

Under the direction of Dr. Buckley, the CAF has twenty years of experience in developing novel analytical methods.  Sample preparation methodology may include microwave assisted acid digestion for inorganic analysis and solvent extraction for organic analysis.

Prior to the analysis of a batch of samples, standard calibration curves of signal response vs. analyte concentration are generated for each analytical instrument.   The number and concentration of calibration standards required to generate the calibration curve are specific to the various analytical methods.  The principal analytical methods employed by the CAF include gas chromatography (GC); gas chromatography-mass spectrometry (GC-MS); and inductively coupled plasma-mass spectrometry (ICP-MS).

CAF Research

Digestion and analysis of normal and neoplastic prostate continues to determine if a cadmium/zinc exchange in neoplastic prostates in search for a valuable biomarker for prostatic neoplasms as well as for other neoplastic tissues. This work is very difficult because of the low concentration of the metal in the tissue, the inhomogeneity of the metal throughout the tissue, and the very small size of the tissue samples that can be collected from a biopsy gun. The most recent results suggest that zinc concentrations can be accurately measured in a biopsy size tissue sample.

The ICPMS was also utilized to analyze environmental samples acquired from a home where a child in the home was found to have a high lead burden in the blood and the samples were collected to try to determine the source of the contamination. The situation was unusual because the home was newly constructed and lead paint was not a primary suspect. High soil concentrations (> 200 ppm) suggest that the contamination could be coming from the soil.

Work also continued on extraction and speciation of methyl mercury from mercury contaminated soils. In a contract from the New Jersey Department of Environmental Protection (NJDEP) the CAF was asked to develop a method to measure each of the prevalent mercury species in contaminated soil so that a standard method and subsequently a standard reference material could be developed for mercury speciation assays. Microwave extraction techniques were used for specie intact extraction of each mercury species and mixed polar and acidic phases under tightly controlled conditions yielded better than 98% recovery of the two primary species, spiked on to the soil samples. Ambient species, as expected, are much tougher to extract and may not be available for species intact extraction.

The liquid chromatograph ion trap mass spectrometer (LC/ITMS) continued to be one of the most utilized analytical techniques in the core. A second LC/ITMS was recently added to the CAF to alliviate the sparse availability of analysis time. A third instrument is anticipated, dedicated to the sequencing of peptides as part of EOHSI’s new proteomics initiative.

Dr. C.S. Yang’s group used the LC/ITMS in the analysis of glucoronides of catechines and their metabolites. LC/ITMS and LC/ITMS/MS characterization of glucoronidation of green tea catechins by liver and small intestine microsomes from mice treated with green tea was studied. LC/ITMS/MS experiments were also done to determine urinary and synthetic methylated catechins. In addition, the LC/MS and LC/MS/MS method for determination of urinary and plasma levels of polyphenols (EG, quercetin, resveratrol, etc) after uptake of grape fluid by human and mice was performed. There were three papers published this year from the Yang group on this work that utilized LC/ITMS.

Dr. J.Y. Hong’s group investigated the metabolism of PhIP using human liver or prostate microsomes. In rats, PhIP induces tumors in the colon and prostate. A novel method utilizing HPLC with mass spectrometric and fluorescence detection was employed for compound identification and quantization. PhIP was incubated with microsomes prepared from human liver and prostate; PhIP and its metabolite was extracted , separated by HPLC then analyzed using ESI positive mode with Selective Ion Monitoring and MS/MS. This method was found to be highly sensitive, selective and with simple sample preparation protocol and the results were published in Analytical Biochemistry.

In addition the LC/ITMS was utilized in the study of the metabolism of thujone (the active component of absinthe) and its metabolites. Dr. Paul Thomas’ group investigates the effect of thujone on rat hepatic microsomal cytochromes P450. Currently, thujone and its metabolites are separated by HPLC then detected by atmospheric pressure chemical ionization (APCI) in multiple mass spectrometric positive ion mode APCI/MS/MS. They have also begun to use LC/MS to identify quinine and its metabolites. Quinine was incubated with microsomes, then products were separated on HPLC and some of the metabolites were collected. Quinine and collected metabolites were analyzed by LC/MS in positive mode using APCI.

The CAF continues to explore the source of unknown contaminants in blank water samples using the gas chromatography ion trap mass spectrometer (GC/ITMS). As part of an NJDEP funded project researchers are isolating as many of the possible sources of contamination as possible. The need for doing such work is a function of the method’s sensitivity. The necessity of this work was demonstrated when an unknown peak was found in some of the mass spectral chromatograms of water samples acquired from a supply in Toms River, New Jersey. These samples were run by the NJ Department of Health and Senior Services (DHSS) lab and the unknown is cause for great concern. When samples obtained from the same supply were run here, researchers also saw this same unknown. Careful chromatography manipulation showed that there were actually two compounds within the one chromatographic peak and further analysis of the blank samples demonstrated that these compounds were found in any sample that was processed for analysis. Core personnel are currently trying to isolate the source of this peak as well as its identity.

The new analysis methods using GCITMS has mandated the development of new extraction techniques so that samples other than water can be analyzed for semi-volatile organics. A new analytical method for the analysis of PCBs and chlorinated pesticides was developed for the analysis of biological samples of adipose tissue by GCITMS. The samples were extracted using various mixtures of hexane and methylene chloride and the microwave extraction system. The microwave extraction technique allowed for a simpler one-step extraction protocol with greater extraction efficiencies.

Looking to the future seems very promising for the CAF. Preliminary results are already in on a successful protein sequence, a new arsenic speciation method and the use of stable isotopes of mercury for mercury tracers throughout a biological system. Although CAF personnel are constantly looking for new methods development challenges, researchers are very excited about the projects currently under development and the early data that have been collected.

Links

For more information about the CAF, contact Dr. Brian Buckley
email: bbuckley@eohsi.rutgers.edu | phone: 848-445-0202

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