CORE--ANALYTICAL

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The Analytical Core will provide LC-MS (liquid chromatography-mass spectrometry), LC-MS-MS (liquid chromatography-tandem mass spectrometry) and MS-MS (tandem mass spectrometry) analyses to support the efforts of Projects by Farnsworth, van Breeman, and Bolton within this Botanical Center. In Specific Aim 1 the botanical natural products isolated by Project by Farnsworth will be characterized with respect to molecular weight, elemental composition (based on exact mass measurements), structure (as indicated by MS-MS fragmentation patterns), and HPLC retention time. Part of this process will include dereplication which is the rapid identification of compounds previously reported to occur in botanicals so that the efficiency of Project by Farnsworth is enhanced. In addition, quantitative LC-MS-MS will be used to measure the concentrations of active compounds in the botanical extracts in support of the standardization effort of Project by Farnsworth, and in serum and urine samples in support of the Phase I clinical trial of hops in Project by van Breemer. In Specific Aim 2 botanical extracts will be screened using ultrafiltration LC-MS (a technique invented in this laboratory) for the rapid identification of ligands to the estrogen receptors and serotonin receptors. These high throughput screening assays will complement the bioassay efforts of Project by Bolton and enhance the productivity of the entire Botanical Center. In Specific Aim 3, LC-MS and LC-MS-MS assays will be developed based on the 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging assay, the measurement of malondialdehyde as an indicator of lipid peroxidation, and the measurement of 8-isoPGF2a which is a product of non-enzymatic decomposition of arachidonic acid used as a biomarker of oxidative stress. These assays will complement the antioxidant Aim 3 of Project by Bolton and Aim 2 of Project by van Breemer and will thereby help provide a broad picture of the antioxidant properties of the botanical dietary supplements under investigation. Finally, Specific Aim 4 the intestinal permeability of active botanical compounds will be assayed using the Caco-2 cell monolayer model of human intestinal absorption in combination with LC-MS and LC-MS-MS to support the preclinical development of active botanical dietary supplements for women's health. As shown by the synergy between this laboratory and all four Projects during the previous funding period, this Core laboratory will continue to enhance the productivity of the entire Botanical Center.