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The Analytical Center is a state-of-the-art chromatographic and mass spectrometric facility designed to support the Institute's investigators with methods development for assays of environmental contaminants or their metabolites and other biological species. Brian Buckley, Ph.D. is the Center's Director. It is a core responsibility of the Analytical Center to continue to evaluate new analytical methodologies for potential application to EOHSI projects and to generate new methods as the need arises. In addition to methods development, the Analytical Center provides expert consultation on sample collection, quality assurance, data interpretation, instrumentation acquisition and sample preparation. Center personnel are also responsible for student training on the use of the analytical instrumentation as well as sample analysis and have more than 40 years of cumulative analytical experience.

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 his 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 Analytical Center 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 center. A second LC/ITMS was added to the analytical center this year to alliviate the sparse availability of analysis time. A third instrument is anticipated in the next year, 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 this year. There were three papers published this year from the Yang group on this work that utilized LCITMS.

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 continues. Dr. Paul Thomas' group investigates the effect of -thujone on rat hepatic microsomal cytochromes P450. Currently, the -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 than 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 Center continues to explore the source of unknown contaminants 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, reseatchers 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. Center 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 Analytical Center. 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 the Analytical Center's personnel is 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.

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