Complex mixtures, such as those encountered in biology, are often analyzed by LC-MS because of the tremendous resolving power of combining these techniques. Powerful LC-MS platforms have become the norm for metabolomics for example. Even though mass spectrometers are providing ever higher resolution, improved separations are often found to improve the detection of metabolites by reducing ionization suppression and simplifying spectra interpretation. Improving the resolution of HPLC can be achieved by using smaller particles or longer columns. Both approaches require higher pressure for driving mobile phase flow so that pressure available can be considered a limiting factor in resolution. Even with high resolution, the broad polarity range of metabolomics samples makes it difficult to resolve all compounds in a mixture. In this work we describe use of an HPLC system capable of generating 40,000 psi for analysis of metabolomic mixtures. We also describe derivatization strategies that improve separation and detection of polar compounds when using reversed phase columns with ESI-MS. We discuss the use of these methods in monitoring the brain chemistry of living subjects. We show that using microdialysis coupled to LC-MS/MS it is possible detect changes in neurotransmitters and related metabolites associated with disease states, behavior, and drug effects.
Robert T. Kennedy
Robert T. Kennedy, Ph.D.
Professional Preparation Undergraduate Institution: University of Florida B.S., 1984
Graduate Institution: University of North Carolina-Chapel Hill Ph.D., 1988
Post-Doctoral Research: University of North Carolina-Chapel Hill 1988-1991
Appointments 8/2015-present Chair, Chemistry Department, University of Michigan
8/2002 – present Hobart H. Willard Distinguished University Professor of Chemistry, University of Michigan
8/2002 – present Professor of Pharmacology, University of Michigan
8/1998 – 7/2002 Professor of Chemistry, University of Florida
8/1996 – 8/1998 Associate Professor of Chemistry, University of Florida
8/1991 – 8/1996 Assistant Professor of Chemistry, University of Florida
Research Interests
Bioanalytical chemistry. Development of microfluidics, chromatography, electrophoresis, and mass spectrometry methods and instruments. Application of methods to in vivo neuroscience, hormone secretion, metabolomics, high-throughput screening, and enzyme evolution.
Other Experience and Service
Scientific Advisory Board of Aclara BioSciences (1995-2000) and CellBiosciences (2002-2007)
Sandlin ZD, Shou MS, Shackman JG, & Kennedy RT (2005) “Microfluidic electrophoresis chip coupled to microdialysis for in vivo monitoring of amino acid neurotransmitters.” Chem. 77:7702-7708.
Song P, Mabrouk OS, Hershey ND, & Kennedy RT. (2012) “In vivo neurochemical monitoring using benzoyl chloride derivatization and liquid chromatography mass spectrometry” Chem. 84:412-419.
Hamid AA, Pettibone JR, Mabrouk OS, Hetrick VL, Schmidt R, Vander Weele CM, Kennedy RT, Aragona BJ, Berke JD. (2016) “Mesolimbic dopamine signals the value of work” Neuro. 19:117-26.
Huang L, Shen H, Atkinson MA, & Kennedy RT (1995) “Detection of exocytosis at individual pancreatic beta-cells by amperometry at a chemically-modified microelectrode.” PNAS 92:9608-9612.
Roper MG, Shackman JG, Dahlgren GM, & Kennedy RT (2003) “Microfluidic chip for continuous monitoring of hormone secretion from live cells using an electrophoresis-based immunoassay.” Chem. 75:4711-4717.
Gee KR, Zhou ZL, Qian WJ, & Kennedy R (2002) “Detection and imaging of zinc secretion from pancreatic beta-cells using a new fluorescent zinc indicator.” Am. Chem. Soc. 124:776-778.
Ueki K, et al. (2006) “Total insulin andIGF-I resistance in pancreatic beta cells causes overt diabetes.” Gen. 38:583-588.
Lorenz MA, El Azzouny MA, Kennedy RT, Burant CF. (2013) “Metabolome Response to Glucose in the β-Cell Line INS-1 832/13.” Biol. Chem. 288:10923-35.
Schultz, NM, & Kennedy RT, (1993) “Rapid Immunoassays using Capillary Electrophoresis with Fluorescence Detection.” Chem. 65:3161-3165.
German I, Buchanan DD, & Kennedy RT (1998) “Aptamers as ligands in affinity probe capillary electrophoresis.” Chem. 70:4540-4545.
Jin S, Anderson GJ, Kennedy RT. (2013) “Western blotting using microchip electrophoresis interfaced to a protein capture membrane” Chem. 85:6073-9.
Sun S, Slaney TR, & Kennedy RT (2012) “Label free screening of enzyme inhibitors at femtomole scale using segmented flow ESI-MS”, Chem. 84:5794-800.
LC School Reviews
“I now have a better understanding of how HPLC works and why column choice is so important.”
“It is a fantastic course for learning the theory of LC and method development”
“The instructor, Merlin Bicking, is very helpful. It was great to see how wiling he is to answer questions and all the variety of methods he worked on.”
“I am just beginning my position from out of school. I found the course very insightful in terms of preparation for my future career”
“It is very useful for me because having hands on experience, which helped me to understand more of HPLC.”
“Great class. Would easily take again and recommend Dr. Merlin anytime. Would love to take his other courses. If he had a book would quickly buy for reference.”
Robert T. Kennedy, Ph.D.
Abstract:
LC-MS for Dynamic Metabolomics of the Brain.
Robert T. Kennedy
Professional Preparation
Undergraduate Institution: University of Florida B.S., 1984
Graduate Institution: University of North Carolina-Chapel Hill Ph.D., 1988
Post-Doctoral Research: University of North Carolina-Chapel Hill 1988-1991
Appointments
8/2015-present Chair, Chemistry Department, University of Michigan
8/2002 – present Hobart H. Willard Distinguished University Professor of Chemistry, University of Michigan
8/2002 – present Professor of Pharmacology, University of Michigan
8/1998 – 7/2002 Professor of Chemistry, University of Florida
8/1996 – 8/1998 Associate Professor of Chemistry, University of Florida
8/1991 – 8/1996 Assistant Professor of Chemistry, University of Florida
Research Interests
Bioanalytical chemistry. Development of microfluidics, chromatography, electrophoresis, and mass spectrometry methods and instruments. Application of methods to in vivo neuroscience, hormone secretion, metabolomics, high-throughput screening, and enzyme evolution.
Other Experience and Service
Honors and Awards
Publications (Selected from over 250)
http://www.ncbi.nlm.nih.gov/sites/myncbi/robert.kennedy.1/bibliography/40512700/public/?sort=date&direction=ascending
LC School Reviews
“I now have a better understanding of how HPLC works and why column choice is so important.”
“It is a fantastic course for learning the theory of LC and method development”
“The instructor, Merlin Bicking, is very helpful. It was great to see how wiling he is to answer questions and all the variety of methods he worked on.”
“I am just beginning my position from out of school. I found the course very insightful in terms of preparation for my future career”
“It is very useful for me because having hands on experience, which helped me to understand more of HPLC.”
“Great class. Would easily take again and recommend Dr. Merlin anytime. Would love to take his other courses. If he had a book would quickly buy for reference.”