The G. John DiGregorio Summer Science Program Research
2012 Final Research Projects:
1.The Analysis of Degradation Products of Phenethylamine Drugs
Arcadia Mentor: Sarah Muller
Students: Alex Middleberg, Dominique Mason, and Kevin Smallwood
Final Poster Coming Soon
Many new recreational drugs with hallucinogenic and stimulant properties have appeared on the market in the last two years. Adverse effects of the use of these compounds have included overdose, abuse, and violent and delusional acts. Since the legal or scheduling status of these products is often unclear, they are often advertised as being “legal highs.” The products are typically derivatives of illegal substances and therefore produce similar effects. The illicit drug industry seeks to make these derivatives available to users before they become scheduled substances. During the 1980’s, Dr. Alexander Shulgin, a pharmacologist and recreational drug pioneer, synthesized psychoactive drugs in various forms including the drugs classified as the 2C series.
Currently, the substance 2, 5-dimethoxy-4-ethylphenethylamine, or more commonly known as 2C-E, is gaining popularity among users because it is not yet scheduled in many states, and is therefore easily obtainable. 2CE and other similar compounds generally elicit effects that include pupil dilation, facial flushing, diaphoresis, bruxism, facial grimacing, tachycardia, tachypnea, emotional liability, and subjective symptoms such as accelerated internal clock, and detachment from surroundings. These legal highs are stimulating hallucinogens that are thought to be non-addictive, yet have recently caused deaths when users overdose.
Recent research, focused on developing an analytical method to measure the stability of 2C-E in oral fluid. This research determined that the substance degrades in specimens, especially in the presence of carbonates. Additionally, the compounds have limited stability in biological samples, degrading by as much as 20% in a week. The discovery occurred when preparing the drug sample to be analyzed with carbonate buffer for analysis on Gas Chromatography Mass Spectrometry (GC/MS). The stability of 2C-E is an important issue when attempting to detect this and related drugs (2C-D, 2C-I, 2C-P, and 2C-T-7) in the urine of a suspected user because carbonates are naturally present in urine. This indicates that when ingested, 2C-E may degrade and be undetected by toxicologists. Understanding if and how the drug breaks down may lead to possible toxicological markers to detect the presence of 2C-E drugs in urine, and some understanding of how long they may be detectable in stored samples. Furthermore, knowing the identity of the degradation product may provide an alternative analytical target. The goal of this research will be to identify what the degradation products are and to attempt to prevent 2C-E degradation in the presence of carbonate in urine.
2. Method Development and Validation for the Analysis of Fatty Acid Hydrolase (FAAH) Inhibitors
Arcadia Mentor: Anisha Paul
Students: Davon Whitest, Colette Trouillot, and Bryan Melilli
Final Poster Coming Soon
Synthetic cannabinoids, commonly labeled as “legal high” products are generally found in herbal incenses. The “legal high” products are sold over the internet and produce similar effects to marijuana. The Federal Government as well as several states are in the process of trying to add these emerging recreational drugs to the list of controlled substances as they become recognized and identified. Another approach taken by the drug manufacturers has been to change the biochemistry of the naturally occurring compounds in the human brain to produce the cannabis like effect without using one of the scheduled cannabinoid active drugs.
One way they are doing this is by making compounds that inhibit the fatty acid amide hydrolase enzyme (FAAH), which is responsible for the degradation of the endogenous cannabinoid anandamide. Examples of these FAAH inhibitors are the “URB” compounds that are the focus of this study. The naturally occurring fatty acid amide anandamide binds to CB receptors in the human brain and can produce anxiolytic, antidepressant and analgesic effects. As URB compounds inhibit this enzyme, and make it perform reactions more slowly, levels of anandamide in the brain can become elevated and may increase these effects which are desired by the user.
For the purpose of this study methods for the simultaneous analysis of URB-447, URB-596, URB-602, URB-754, and URB 937 will be developed and characterized. The first phase of this study will be optimizing and validating a screening process to identify URB compounds in biological samples utilizing Gas Chromatography Mass Spectrometry (GC/MS) as well as Triple Quadrupole Liquid Chromatography Mass Spectrometry (LC/MS-MS). The stability of the compounds by GC/MS and LC/MS-MS will be evaluated also. The second phase of the project will be to study how the drugs are metabolized in the body, using a human liver microsome model. This part of the study will help the laboratory determine which analytes to test for in blood or urine samples to demonstrate exposure or use.
3.Evaluation of Solid Phase Microextraction Pipette Tips for Screening of Synthetic Cannabinoids in Serum
Arcadia Mentor: Apisri Ieamniramit
Students: Phoebe Ingraham, Lauren Wray, and Borat Tan
Final Poster Coming Soon
Synthetic cannabinoids are an important emerging class of recreational drugs, producing marijuana-like effects in users. Being able to detect their use in biological matrices including urine, blood, saliva and hair is important for forensic applications. Traditionally liquid/liquid or solid phase extraction methods have been evaluated for the detection of these compounds. Furthermore, within classes of drugs, a variety of acid-base behaviors and functional groups exist which can strongly affect solubility or affinity to a sorbent. As a result, previous extraction methods may have limitations such as incomplete recovery, multiple steps involved, or producing unclean extracts, which impacts subsequent chromatography.
Solid Phase Micro Extraction (SPME) using micropipette tips is a novel and useful technique for preparing samples prior to analysis by chromatography and mass spectrometry. This project will focus on the development of a drug screening method for the analysis of synthetic cannabinoids and their metabolites in serum using pipette tips with C18 adsorbent in the bottom of the tip. These unique microscale solid phase extraction tips have been used successfully in methods with limited sample size. The advantages of using C18 tips also include high efficiency, simplicity, and the use of only a small amount of reagents and sample.
This project will use BioBasic C18 Thermo Scientific HyperSep Microscale SPME Tips. The HyperSep BioBasic C18 Columns/Tips feature a highly retentive alkyl-bonded phase for nonpolar to moderately polar compounds like synthetic cannabinoids. The C18 phase consists of trifunctional octadecyl which is a hydrophobic reversed phase. Biobasic columns are designed specifically for chromatography of proteins, peptides, nucleic acids and other biomolecules. The 300 Angstrom pore size, high purity silica and stable bonding chemistry of BioBasic packing make them ideal for life science applications. Furthermore, BioBasic columns have extra dense bonding chemistry which results in a highly stable, reproducible surface for reliable results. Analysis will be performed using Triple Quadrupole Liquid Chromatography Mass Spectrometry (LC/MS-MS).
This particular project will focus on recovering and analyzing the synthetic cannabinoids JWH-210, JWH-120, AM-2201, AM-1248, and XLR-14 in plasma. Analytical parameters that must be determined include the limit of detection, limit of quantitation, percent recovery, interday precision, intraday precision, and linear dynamic range.
5.Method Development and Validation of Dimethylamylamine in Nutritional Supplements Using Gas Chromatography Mass Spectrometry
Arcadia Mentor: Brittany Pasierb
Students: Stephen Cancel, Victoria Spadafora, and Jasmine Oates
Final Poster Coming Soon
Methylhexanamine also known as DMAA was first synthesized and patented in 1944 by Eli Lilly as a nasal decongestant, but currently has no recognized medicinal use in the United States. Since 2006, DMAA has been marketed as a nutritional and anabolic supplement and is commonly found in products such as Oxyelite, Jack3d, and Geranamine. These products can be purchased from health and nutrition stores and are marketed as “fat burning” nutritional supplements. Because of its stimulant like effects, DMAA is considered a performance enhancing supplement generally used in pre workout regimes or prior to sports related competitions.
As a central nervous system stimulant, DMAA is believed to have a role in cathecholamine release. However, unlike other stimulants it does not directly interact with norepinephrine receptors. Studies indicate DMAA has less stimulating effects than amphetamine in addition to reduced toxicity, adding to its overall appeal as a natural alternative to prescription stimulants. These factors contribute to the supplement being used by many athletes and people heavily involved with fitness. In 2011, DMAA was associated with two deaths within the military population. The toxicological reports confirmed both decedents had DMAA in their system, although the cause of death resulted from a heart attack in both cases. Chemically DMAA exists as four enatiomers, which makes its analysis more challenging.
This project will characterize DMAA using Gas Chromatography Mass Spectrometry (GC/MS) and Liquid Chromatography Mass Spectrometry (LC/MS), including its stability, and potential for degrading or changing chemically during the analytical procedure. It will also characterize the methods ability to be used to quantify DMAA in biological fluid, using standard calibration and quantitation techniques. Once the method has been validated, products known to contain DMAA will be purchased from health and nutrition stores, and other on-line sources to determine the exact concentration of this compound within these products. Additionally, analysis will be carried out using samples obtained from patients using the nutritional supplement, to determine DMAA concentrations in biological specimens.