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Mar 15, 2009 (Vol. 29, No. 6)

Desorption Electrospray Ionization

A DESI-MS Workflow Has Applications in Drug Candidate Qualification

  • Experiment

    Male Sprague-Dalley rats were given 50  mg/kg clozapine PO and sacrificed 30 minutes post-dose. The brain was extracted, immediately frozen over liquid nitrogen, and cut into 10 µm slices using a cryo-microtome. Each tissue section was thaw-mounted onto a plain microscope glass slide and analyzed without further processing.

    Tissue-imaging experiments were conducted using a prototype version of an automated, two-dimensional DESI ion source like the one depicted in Figure 1b.  The data was acquired using a Thermo-Fisher Scientific LTQ linear ion trap mass spectrometer using positive ion detection.  The DESI ion source parameters were as follows: tip to surface distance: 2 mm; incident angle: 55°; solvent flow rate: 2 µL/min; and and nebulizing gas back pressure: 100 psi (7 bar). Raw data files acquired in Xcalibur™ were processed using FireFly (Figure 1d). 

  • Drug Imaging

    Click Image To Enlarge +
    Figure 2. A–B

    In the drug candidate qualification process, pharmacokinetic screening, metabolic stability with metabolite identification, enzyme induction and inhibition, and excretion studies play a major role. For tissue-distribution studies, whole-body autoradiography (WBA) is commonly employed. It has the advantage of being quantitative in nature but suffers from several disadvantages. 

    In particular, WBA requires the use of radioactive compounds and turnaround times can be up to several weeks. DESI-MS imaging presents an alternative approach for tissue-distribution studies by allowing for direct analysis and imaging of intact tissue sections without the use of radio-active labels. Sample turnaround times of just a few hours are another benefit. A typical mass spectrum recorded on a 10 µm thick rat-lung tissue section is presented in Figure 2a

    The lower m/z range represents the region where most drugs and metabolites are detected. The upper portion of the m/z range represents the lipid and peptide region. Figure 2b shows the DESI mass spectrum in the lower m/z range of the lung tissue. Of particular interest are the peaks at m/z 327 and m/z 313, which represent the presence of the drug clozapine and its metabolite, desmethylclozapine, respectively. Among other possibilities, the remaining peaks include endogenous small molecules carrying a positive charge.

    Figure 3 shows the optical image and the DESI image from a rat brain section taken from an animal that had been dosed at 50 mg/kg of clozapine via oral gavage and the brain removed 30 minutes post-dose. The tissue was imaged in the MS/MS mode, and the DESI product ion spectra resulting from fragmentation of clozapine at m/z 327.1 (M+H)+ were recorded with a pixel size of 245 µm x 245 µm.

    The Omni Spray 2D Ion Source has  advantages and presents possibilities for the drug discovery scientists’ toolbox. Utilizing the Omni Spray 2D Ion Source for tissue-distribution studies has the potential to eliminate the need for expensive synthetic molecules while increasing sample throughput. DESI has also been used for exploring the impact of drugs and disease on endogenous compounds, revealing counterfeit drug formulations, and identifying thin layer chromatography spots. In addition, the technology has shown potential in pharmacokinetic screening and cleaning validation.  

  • Click Image To Enlarge +
    Figure 3. A and B


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