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Literature Analysis: Mass Spectrometry

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    Separations and Mass Spectrometry: LC-MS for In Vivo Neurochemical Analysis

    Out-of-class Questions – Complete prior to in-class discussion

    Article: Peng Song, Omar S. Mabrouk, Neil D. Hershey, and Robert T. Kennedy, “In Vivo Neurochemical Monitoring Using Benzoyl Chloride Derivatization and Liquid Chromatography-Mass Spectrometry,” Analytical Chemistry 2012, 84, 412-419.

    Summary: This paper describes a method for separating and detecting 17 neurotransmitters, metabolites, and amino acids in less than 8 minutes using liquid chromatography-mass spectrometry (LC-MS). These neurochemicals are collected from living rats using microdialysis probes implanted into the rat brains. Prior to separation by HPLC, these molecules are derivatized with benzyl chloride. After successful optimization of separation conditions, the researchers determined the effects of two drugs (bicuculine and neostigmine) on neurochemical concentrations in different brain regions. Carefully read the paper and answer the following questions before our in-class discussion.

    1. We have not discussed microdialysis in class. Read the introduction to the paper and Demonstration 27-1 on page 755 of your textbook (Harris, 8th Ed.), which discusses microdialysis. In your own words describe how microdialysis works. Uses sketches if necessary.

       

       

       

       

       

       

       

       

       

       

       

    1. List a few applications of microdialysis in neuroscience.

       

       

       

       

       

    1. Look at the structures and reaction scheme in Figure 1. Draw the structures of benzoylated GABA and benzoylated DOPAC.

       

       

       

       

       

       

       

       

    1. Describe the HPLC stationary and mobile phases. Are these normal phase or reversed phase separations? Are they isocratic separations or is a mobile phase gradient used?

       

       

       

       

       

       

       

       

    1. What type of ionization source was used in the mass spectrometer? What type of mass separator was used?

       

       

       

       

       

       

       

       

    1. Why is acetylcholine the first compound to elute from the HPLC column?

       

       

       

       

       

       

       

       

       

    1. Why are norepinephrine (NE) and dopamine (DA) the last compounds to elute from the column?

       

       

       

       

       

       

       

       

    1. What was the m/z of the most abundant fragment for most of the analytes? What is the structure of this fragment?

       

       

       

       

       

       

       

     

     

    NAMES:___________________________________

    Separations and Mass Spectrometry: LC-MS for In Vivo Neurochemical Analysis

    In-class Discussion Questions

    Article: Peng Song, Omar S. Mabrouk, Neil D. Hershey, and Robert T. Kennedy, “In Vivo Neurochemical Monitoring Using Benzoyl Chloride Derivatization and Liquid Chromatography-Mass Spectrometry,” Analytical Chemistry 2012, 84, 412-419.

    1. This study uses electrospray ionization, a triple quadrupole mass separator, and multiple reaction monitoring.
      1. Describe the processes that are occurring in each of the three quadrupoles.

         

         

         

         

         

         

         

      2. In class we discussed selected ion monitoring, extracted ion monitoring, and selected reaction monitoring. How do you think multiple reaction monitoring differs from those we discussed in class?

         

         

         

         

         

         

      3. Why do you think the authors chose this ion monitoring mode?

         

         

         

         

         

    1. Why were the neurotransmitters and metabolites derivatized with benzoyl chloride? What improvements in the analysis were facilitated by benzoyl derivatization?

       

       

       

       

       

       

       

       

       

       

       

       

    1. Look at the structures in Figure 1 and the chromatogram in Figure 2. Considering the benzoyl chloride derivatization chemistry, explain the general trend in retention times for the species shown in Figure 2.

       

       

       

       

       

       

       

       

       

       

       

       

    1. What was the internal standard, and how do you think internal standardization was implemented in these studies?

       

       

       

       

       

       

       

       

       

       

       

       

    1. The data collected in Figures 4 and 5 were collected with different microdialysis sampling rates. What were the sampling rates, and why were different sampling rates used?

       

       

       

       

       

       

       

       

       

       

       

       

       

    1. Earlier this semester we discussed how electrochemistry (fast-scan cyclic voltammetry - FSCV) can be used to analyze neurotransmitters in the brains of living rats. Compare and contrast the FSCV method with the LC-MS method described here. What were the strengths and weaknesses of each method? If you wanted to do in vivo neurochemical analysis, which method would you choose? Why?

       

       

       

       

       

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