Undergraduate Research Topics with Dr. Dubay:

Project 1. Determination of the concentration of analgesics or other drugs that can be found in plasma after administration.

The ability of the body to clear analgesics or other drugs after administration is important to understand. Many new analgesics have not been studied thoroughly. I would like to have students work on the identification and quantitation of these compounds from plasma samples.

Steps required:

1. Isolation of the analytes from the plasma: Most organic compounds are either bound to proteins or freely available in the plasma. The drugs of interest are frequently readily bound to proteins so quantitation requires that the protein bound drug be released. The binding is not covalent so this can be done. The drug must the be isolated from the proteins and other organic materials found in the plasma. This success of this isolation is critical to the success of the analysis. Methodology will be developed to free the analyte for protein binding and isolate it from the plasma. This will be a crucial step to a successful method.

2. Choosing an internal standard that can be used for quantitation of the drug under investigation. There is no absolute response factor that can be used in the analysis that will allow the concentration of the drug to be calculated. Thus, a very similar compound needs to be used as an internal standard. The internal standard must not be present in the plasma.

3. An analytical separation of the analyte and the internal standard will be developed. This must provide good separation and peak shape for each compound.

4. Establishing a standard curve between the drug of interest and the internal standard. The literature should be consulted to determine the concentration expected for the analyte in plasma. If it is known, a concentration for the internal standard is chosen in the middle of that range. Six samples of the analyte are prepared over the concentration range of interest. To each of the six samples the internal standard is added at the level chosen. Each sample is then injected three times onto the instrument. The areas of the individual components will be determined. A plot is made of the ratios of the areas to determine if there is linearity over the range of interest.

5. The method is then tested. Plasma samples are spiked with the analyte and the internal standard. The spiked plasma is then processing using the method developed here.

Please, not that the methods developed here can be applied to the identification of a wide variety of compounds. This overall approach can be applied to drugs of abuse, steroids, antibiotics, cardiovascular treatments, etc.

Project 2. Determination of the concentration of nutritional supplements that are present in complex sample matrices by LC, GC, or MS.

Nutritional supplements are widely used by the general public. They are heavily advertised as being highly beneficial but what do we know about these products?

1. Are the products processed safely and do they provide a reproducible supply of the beneficial ingredients?

2. Do these products have other impurities that could be unsafe to the consumer?

3. Are generic products supplied by the major retailers like Target, WalMart, etc., as good as those from other manufacturers? Do they have the correct amounts present?

4. Are there any components that have a shorter shelf life than others? Does this affect the quality of the product? Are the more unstable components important markers of the quality of the product?

5. What governmental agencies are in charge of making sure the compounds present are safe?

A student can address some of these questions and others that can be asked about nutritional supplements. The first step in the process will be a good literature search to learn more about what is known of these products. A target supplement/s will be chosen for investigation. When it is determined what is know, it will be possible to make some decisions about what experiments need to be undertaken to extend our knowledge of these products.

Ultimately, the investigator will choose one or more products for study. The method of analysis will include use of instrumentation in our laboratory. The laboratory is well equipped with mass spectrometry systems that are coupled to chromatography and this can be a powerful tool for quantitating a particular compounds. In this study the presence of any impurities related to the parent compound/s will also be investigated.

Project 3. Identification of Steroids in the Blood or Urine:

Steroids are ubiquitous in our bodies. Some steroids are produced in the body for a wide range of biochemical functions. The concentrations of steroids are affected by the use of birth control pills and a wide range of drug therapies. There are also illegal or improper uses of steroids by athletes and members of the general population. Thus, the analytical chemist must determine with great certainty and accuracy which steroids are present and at what concentrations they are found. This will enable us to evaluate whether the presence of the steroids is normal and acceptable or whether they are being abused for some other purpose.

The questions that will be asked in this study are:

1. What steroids are present in the blood?

2. At what concentrations are they found in the blood?

3. What challenges are faced in the analysis?

4. What are the best methods for analyzing the steroids?

Project 4. Protein-MS Lab

Introduction:

The purpose of this laboratory is to introduce the student to the use of mass spectrometry for the identification of proteins. Proteins are critical regulators of biochemical processes within the body. Interference in the normal behavior of proteins leads to many different types of pathologies. In addition, abnormal reaction or modification of proteins also changes the normal biochemistry and can lead to cancer and other diseases. As a result, the identification and quantitation of proteins is important to understanding many diseases. This laboratory will then introduce the student to an important technique that is commonly used in biochemistry laboratories around the world.

Proteins can be very large and there can be a significant amount of heterogeneity in proteins. Determination of the molecular weight of these large molecules can vary as a result of the heterogeneity present. Analytical instrumentation is very useful but it still has some difficulties with the accurate assignment of the large masses of proteins. So measurement of the intact molecular weight of proteins is not sufficient for identification. Enzymatic digestion of proteins followed by mass spectrometry analysis at high resolution can allow a positive identification of the protein present.

The student working on this laboratory will learn to use modern methods for protein identification including mass spectrometry, liquid chromatography, trypsin digestion, etc.