17th International Mass Spectrometry Conference :: Prague, 2006
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|Session:||High Throughput Analysis: From Discovery to Clinical Applications|
|Presentation date:||Mon, Aug 28, 2006|
|Presentation time:||09:50 – 11:20|
Kate Yu1, Peter Alden1, Robert S. Plumb1, Li Di2, Susan Li2, Edward Kerns2, Paul Chilvers31 Waters Corporation, Milford, United States
Correspondence address: Kate Yu, Waters Corporation, Pharmaceutical Market Development, 34 Maple Street, Milford, 01757 United States.
Keywords: Automation; Chromatography, Liquid (LC); MS/MS, Liquid Chromatography; Pharmaceutical.
Novel aspect: Automatic UPLC/MS/MS multi-mode ionization method development and quantitative analysis protocol applied to microsome stability test.
The application of a combined ESI and APCI ionization source (ESCi) will increase the throughput and sample coverage by eliminating the need to physically change the ionization source and to repeat injections for failed samples. The stability test screens compound stability in microsomes etc. providing important information about potential liabilities of drug candidates. The characteristics for analysis carried in drug discovery are: many new compounds to be analyzed for screening purpose; typically, each solution will contain a single test compound; many injections are required. Each injection contains a single analyte. To apply ESCi™ routinely in drug discovery lab, an automated protocol is crucial for minimizing the instrument time required for analysts.
The goal for this project was to develop an automated ESCi-LC/MS/MS analytical protocol for the in vitro metabolic stability test using the 96 well plate format to demonstrate the advantages of ESCi™ multimode ionization for higher throughput and broader compound coverage in drug discovery.
The system used was an UPLC-triple quadrupole mass spectrometer. The microsome incubation was based on a published method. The quantitative analysis was completely automated. For the 96 well plate microsome stability test, a few standards with known metabolism were incorporated as QC checks. A 2.1x50 mm UPLC column with 1.7 µm particle size was used for the LC separation, the flow rate was 0.6 ml/minute.
For ESCi™ MS and MSMS scan experiments, the purpose was to choose the optimum ionization mode as well as the optimum MRM parameters. This type of experiment is typically qualitative. We were able to obtain spectra with sufficient quality to allow optimization to be completed automatically. For ESCi™ MRM experiments, the number of data points obtained varied depending on the number of channels to be monitored in a single injection. Less data points were obtained when more MRM channels were to be monitored. Mixture analysis can be useful for cassette dosing experiments. For the microsomal stability test, a typical experiment is carried out for a single unknown compound per well. Therefore, the need is simply to analyze a single compound per injection. With the ESCi™ capability, compounds in a 96 well plate can be analyzed in a single analytical batch with both ESI and APCI.