Automating Drug Metabolism Studies MetaboLynx Automating Drug Metabolism Studies The next slide give a tour through he MetaboLynx software and highlight some of the key features and the benefits those features bring to the analysis of drug metabolites. For those who want to be able to use the MetaboLynx software ‘live’ there are some basic instructions on how to access the displayed windows within MassLynx.

What is MetaboLynx? MetaboLynx is the application manager for automated processing and reporting of metabolism data It is an extension of MassLynx designed specifically for this application MetaboLynx accelerates the processing and reporting of drug metabolism data It can help to carry out data manipulation in hours (minutes) where it would previously have taken days (hours) MetaboLynx has been designed to automate these processes. It is an option which can be loaded as an extension of MassLynx and will accelerate the processing and reporting of drug metabolism data

What is required? Many metabolites can be predicted Processing is time-consuming and repetitive NEED AUTOMATED PROCESSING Not all metabolites can be predicted Need to search data for unknowns They may be hidden by abundant endogenous peaks Data processing is extremely labour-intensive May need to acquire MS/MS data Set-up, processing and reporting is time-consuming NEED AUTOMATED METHOD SET-UP AND PROCESSING Where can Micromass help? Often drug metabolites can be predicted by a knowledge of the parent drug structure and of metabolic pathways. If using manual processing this would mean plotting an extracted mass chromatogram for each expected metabolite, integrating those chromatograms and then producing background-subtracted spectra for each detected peak. It is also necessary to check a control or blank sample (matrix with no added drug) analysis to ensure that none of the detected peaks are due to compounds found it the matrix. To do this manually for a large number of compounds is repetitive and time-consuming. Not all metabolites can be predicted and it is often necessary to search through a data file looking for low level metabolites which are not visible in the TIC as they are hidden by more abundant matrix peaks. This means it is necessary to go through a data file scan-by-scan or chromatogram-by-chromatogram to find these metabolites. This can obviously take many hours. In addition, once in development, it is necessary to generate product ion spectra to confirm or to identify a metabolite. Setting up and processing MS/MS data is also time-consuming. Automating these steps can reduce the time taken from days to hours or from hours to minutes.

Acquire control & metabolized samples Search expected & unexpected MetaboLynx Flow Path LC/MS Report LC/MS results Acquire control & metabolized samples Create MS/MS method MetaboLynx Auto-start MS/MS Search expected & unexpected Process MS/MS data Append to report Control comparison

MetaboLynx Set-up Flexible parameters - discovery or development Batch or single sample analysis Expected metabolites menu Unexpected metabolites Control sample Search data using MS, PDA or analog radioactivity detection, fluorescence, etc. Exact mass measurement and elemental composition Isotope cluster search chlorine, bromine, isotopic labeling Automated MS/MS set-up

MetaboLynx Set-up - Peak Detection

MetaboLynx Set-up - Expected Metabolites

Unexpected Metabolites Small peaks in complex matrices Requires intensive data mining Greatest time-saving through automation Using chromatogram peak detection User defined step size and thresholds

Control Comparison Direct subtraction not feasible Acquire control and experimental sample Process control and experimental sample Identify peaks by mass, RT and response Specify windows for RT and response Filter out matching peaks Utility depends on control sample

MetaboLynx Set-up - Isotope Clusters

Isotope Cluster Plot (Br)

Data Visualization - MetaboLynx Browser

Automated LC-MS/MS Creates and starts LC-MS/MS acquisition builds MS/MS method based on browser report creates new sample list creates new file name(s) and sample text automatically starts MS/MS (product ion) acquisition can choose full automation or manual intervention processes MS/MS data adds data to LC-MS report file and browser

MetaboLynx Browser - MS/MS

Instrument Compatibility ZMD / ZQ (single quad) LC-MS Quattro micro / LC / Ultima (triple quad) LC-MS/MS processing of precursor and neutral loss data LCT (oa-TOF) exact mass LC-MS Q-TOF micro / II / Ultima exact mass LC-MS and LC-MS/MS GCT (GC-TOF)

MetaboLynx - Summary Automation of metabolism studies Flexibility for metabolite screening or low level detection Speed sample throughput in discovery data mining in development Data visualisation and reporting Not just metabolism Impurity studies Further developments on-going