Donna Zwirner Waters, Milford, MA 01757 Office: 800-252-4752 x8477 FAX: E-mail: donna_zwirner@waters.com Web: Job Title: Field Chemistry Specialist M.Sc. in Molecular and Cell Biology from Brandeis University, Waltham, MA

Speaker: Donna Zwirner, Waters, Milford, MA 01757-3696

Topic: A Systematic Approach to Reducing Matrix Effects in LC/MS/MS Analyses

Place: Building 549, Conference Room A, NCI at Frederick, Frederick, MD

Time: Tuesday, January 20, 2009, at 2:00 PM

Abstract: The MS response obtained from an analyte in neat solution can differ significantly from that same analyte in a matrix. Matrix effects, resulting from co-eluting matrix components that compete form ionization capacity, manifest themselves as suppression or enhancement of the analyte signal. Matrix effects can be infinitely variable and difficult to control or predict. They are caused by numerous factors, including but not limited to, endogenous phospholipids. Other sources of variability in matrix effects include subject differences, possibly due to diet or other factors, and concentration of the endogenous phospholipids. The severity and nature (suppression versus enhancement) of matrix effects observed can be a function of the concentration of the lipids at the elution time of the analyte. Matrix effects can also be compounded by co-eluting metabolites, impurities or degradation products. All of the above can cause significant errors in the accuracy and precision of bioanalytical methods. These effects should be evaluated as a part of quantitative LC-ESI-MS/MS method development, validation, and routine use. We believe that all available tools, or combinations of tools, should be employed to reduce these effects. In this seminar, we compare different sample preparation methods, investigate the influence of mobile phase pH on both matrix components and several basic analytes, and we compare HPLC and UPLC™ analysis for sensitivity and the presence of matrix effects. We chose to focus on basic analytes in a pharmaceutical bio-analysis environment for this research. The amount of specific matrix components, or classes of matrix components, remaining in the extracts was measured by LC/MS/MS (electrospray ionization) using multiple reaction monitoring (MRM). Upon high energy in-source CID, the lipids implicated in matrix effects yield a characteristic fragment ion at m/z 184, corresponding to the trimethylammonium-ethyl phosphate cation. It is this ion that allows us to monitor multiple phospholipids and lysophospholipids with a single MRM transition, thus maximizing the duty cycle in the mass spectrometer. The relative levels of phospholipids present in our samples were measured by monitoring the transition 184.3 -> 184.3. The cone voltage was set to 90 V and the collision energy set to 3 eV so that no further fragmentation of the diagnostic ion would occur. Based on our research, we conclude that mixed-mode solid phase extraction (SPE), appropriate mobile phase pH and UPLC offer the best solution for controlling matrix effects in bioanalytical analyses.

The slides to this seminar are in a 1.4 Megabyte PDF file, which can be opened and read by using the free Adobe Acrobat Reader®.

Accompanying Poster: "A Systematic Approach to Reducing Matrix Effects in Bioanalytical Studies" , authored by E. Chambers, D. M. Diehl, and J. R. Mazzeo, Waters Corporation, Milford, MA 01757 (the poster is in a 0.3 Megabyte PDF file, which can be opened and read by using the free Adobe Acrobat Reader®)