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BackFall 2000SUMMER MENTORING PROGRAM - 2000 RESEARCH ARTICLESThe Stability of Gamma Hydroxybutyric Acid and Gamma Butyrolactone in Wine and VodkaBristol Rush, Krystal Bagley, Deanne Grant, Emil Bobyock, Anthony Macherone and G. John DiGregorioGamma hydroxybutyric acid (GHB, Figure 1) is a naturally occurring, normal constituent of the human body found primarily in the basal ganglia. 
Figure 1. Gamma Hydroxybutyric Acid. Pharmacologically, GHB acts as a neurotransmitter antagonist and is metabolized to succinic semialdehyde and gamma aminobutyric acid (GABA). Therapeutically, it has been used as a general anesthetic and was purported to be a growth hormone stimulator by the health food industry. GHB can be readily synthesized from gamma butyrolactone (GBL, Figure 2). Large doses of either GHB or GBL have psychotropic effects and, in recent years, the use of both as recreational drugs has become increasingly popular. 
Figure 2. Gamma Butyrolactone. Although distribution of GHB was banned by the FDA in the 1990's, access to the drug was provided through mail order and Internet sources. GBL, on the other hand, continued to be legally distributed in products such as acetone-free nail polish remover and health food supplements until mid 2000. GHB and GBL have been illicitly distributed as "Liquid-X", "Ecstasy" "Grievous Bodily Harm" or "G-riffic". Taken internally, they produce a broad range of dose-dependent neurologic effects including dizziness, euphoria, depression, drowsiness, amnesia and coma - the latter three have been maliciously exploited and further earned these compounds a reputation as "date-rape" drugs. Death has been known to occur especially when GHB or GBL is combined with alcohol. Many cases come into the lab presenting a beverage presumed to contain GHB and/or GBL. Most of these samples have forensic implications and accurate identification and quantitation of the compound(s) present in the solution is essential. Information such as this may assist the police or attorneys in their effort to prove or disprove culpability during an investigation or trial. Unfortunately, the majority of the samples presented to the lab are collected days, perhaps even weeks, before analysis and the stability of GHB and GBL in a beverage matrix over time is not well understood. Ostensibly, there is pH dependence for the conversion from one compound to the other: GBL is the acid catalyzed condensation product of GHB and; GHB is the base-induced hydrolysis product of GBL. Additionally, the amount of time the compound is exposed to the matrix may also affect its stability. The question therefore is: how sensitive are GHB and GBL to the pH of the beverage and the time frame that they are exposed to the matrix. Will conversion from one species to the other occur as a function of pH and/or time? To investigate these questions, a series of experiments (Table 1) were undertaken in which red table wine (pH = 3.6) and eighty-proof vodka (pH = 3.4) were spiked separately with GHB and GBL. Aliquots of each permutation were independently analyzed by GC-MS for both GHB and GBL at time zero, 30 minutes after spiking, 24 hours later and finally, one week after spiking. 
Table 1. Experimental Series. The pH of the beverages remained essentially constant throughout the course of the experiment fluctuating by ± 0.1 pH units over an eight-week period. Analysis of blank (not-spiked) red wine revealed that it contained a basal amount of both GHB (~5ug/mL) and GBL (~10ug/mL) the origin of which is most likely the natural products contained in the fermentation mixture. GHB was not detected in the vodka blank although a small amount of GBL (~2ug/mL) was found to be naturally present possibly as an artifact of the distillation process. No conversion from GHB to GBL, or vice-versa, occurred in the wine matrix over one-weeks time and GBL was not formed from GHB in the vodka matrix. However, our results indicate that GBL to GHB conversion does occur in vodka. GHB was observed at time zero and throughout subsequent analysis in a vodka sample spiked only with GBL. The average concentration of GHB detected in the sample was approximately half that of the GBL originally spiked. This result is counter-intuitive. One would not predict GHB to be formed from GBL at low pH. The observation suggests another mechanism, yet to be determined, is responsible for the conversion. It may be that the acid structure (GHB) is more stable at lower pH. However, the pH of the wine is nearly equivalent to that of the vodka, substantiating the assumption that a similar GBL to GHB conversion should occur in wine. The absence of this reaction may indicate that wine possesses an inherent mechanism that inhibits species conversion. Preliminary results of this research offer new perspectives into the stability of GHB and GBL in beverages of moderately low pH. There appears to be no simple dependence on pH to convert one compound to the other. It further appears that a period of one week is not enough to effect conversion. Although both are of similar pH, the observation of GBL to GHB conversion in vodka, but not in wine, suggests that these seemingly simple molecules demonstrate complex and unpredictable behavior in composite organic matrices. Perhaps the most intriguing aspect of this project was the unexpected discovery that GHB and/or GBL were naturally present in these beverages. Further work will be done to evaluate other beverages for the presence of naturally occurring GHB and GBL as well as continued research into the stability of these compounds as a function of pH, time and beverage type. MALDI-TOF Analysis of Amiodarone in Human Heart TissueAndrea Garcia, Catherine Bentzley, Anthony Macherone and G. John DiGregorioAmiodarone is an iodine-rich, benzofuran derivative classified as a class III anti-arrhythmic drug used to treat life threatening ventricular arrhythmias. Amiodarone acts on cardiac sodium and potassium channels to prolong the action potential and refractory period thereby reducing the overall heart rate. Many people are prescribed amiodarone prior to receiving a heart transplant and the medication is discontinued after the transplant surgery. However, amiodarone is highly fat-soluble and a large reserve is held in adipose and other tissue even after discontinuation of the drug regime. Hence, over time, residual amiodarone leaches out of these reserves and begins to act on the new heart. In some cases, a pacemaker is required until cessation of the amiodarone action occurs. Presently, heart transplant recipients are required to have a monthly biopsy of the new heart tissue for pathological determination of rejection. There exist no criteria for the determination of residual amiodarone levels in the heart and therefore there is no method to determine when the amiodarone has been completely washed out of the body, nor does there exist a method to accurately identify a time frame when the pace maker can be safely removed from the new heart. The goal of this research is therefore to develop a suitable assay using Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) to quantitate amiodarone levels in heart tissue biopsies obtained from transplant recipients. The results will be used to establish correlations between residual amiodarone leaching out of peripheral tissue into the new heart and clinical cardiac effects. MALDI-TOF involves the ablation of an analyte doped in a matrix fixed to a gold-coated, stainless steel backing plate. In the presence of an electric field, a nitrogen pulse-laser (337 nm) is used to strike the sample and causes the matrix and analyte to desorb, ionize and become accelerated down a time of flight tube. Ideally, all ions enter the flight tube with the same kinetic energy. Therefore, the time it takes the ion to travel the flight tube can be used to determine the mass of that ion. Standards of amiodarone are prepared and doped into 6-aza-2-thiothymine (ATT) used as the matrix. Explanted heart tissue is prepared by rinsing in water and vortexing with ATT. The standards and the supernatant from the samples are deposited onto the backing plate and allowed to air dry. Analysis of the spectra obtained from the standard solutions demonstrates well-resolved peaks for both amiodarone and ATT - the most intense occurring at m/z 288, which is characteristic of 2(ATT+H)+1. This peak is of uniform intensity in each standard/sample and can therefore be used as an internal standard. Amiodarone appears at m/z 644 and is readily distinguished from other spectral features. Quantitation of amiodarone in each tissue sample is reproducibly achieved via interpolation of an appropriate calibration curve. Figure 1 illustrates a MALDI-TOF spectrum obtained from explanted heart tissue known (by previous HPLC analysis) to contain amiodarone. 
Figure 1. MALDI-TOF Spectrum The results of this preliminary work indicate that MALDI-TOF may be a viable technique for the analysis of amiodarone in human heart tissue biopsies. SUMMER MENTORING PROGRAM - 2000 RESEARCH ABSTRACTCarboxyhemoglobin StabilizationFelicia Corsaro-Barbieri, Jonathan Treitel, Tuyet-Trinh Truong, Anthony Macherone and G. John DiGregorioCarboxyhemoglobin (COHb) is a normal component of circulating human blood. Typical COHb concentrations range from 1% to 8%. Elevated levels may result through environmental exposure or hemolytic disease. Due to in vitro COHb instability, accurate analysis requires proper sample preparation and handling. It would be desirable to “fix” the COHb content in blood at the time of collection so that the concentration at time of analysis is equal to that at sampling. In this investigation, two methods were explored as a means of COHb stabilization. First, the use of free heme as a carbon monoxide scavenger was investigated; although the method is chemically valid, the high absorptivities of the compounds involved precluded quantitative spectrophotometric analysis. Second, the stability of COHb in blood samples was monitored by spectrophotometric measurement of the percent COHb saturation over time. These samples were diluted in a oxygen-free borate buffer solution, (with and without the addition of sodium dithionite and/or carbon dioxide) and stored in sealed vials. A difference in the stability of samples of different COHb content was observed. Differences in the stability of samples prepared in buffer alone, in dithionite-treated buffer and in carbon dioxide-saturated buffer were also observed. Initial results indicate that maximum stability for samples in this study is achieved in dithionite-treated borate buffer solution. Other research abstracts from the 2000 Summer Mentoring Program will be presented in future editions of the Rieders Renaissance Report. FEATURE ARTICLEThe Fredric Rieders "Renaissance Recognition AwardMichael F. RiedersWhen Fredric Rieders began as a student at Thomas Jefferson University in 1949, dedication to academic excellence was expected. Long hours, late nights and hard work were the norm and support from the University community was essential for success. Most students focused on academic standing and distinguished themselves through competing for high test scores, achievement of multiple research publications and a first class thesis. A few rare students went above and beyond academic achievement and made a significant and lasting contribution to the Jefferson community that was remembered and appreciated by their peers and faculty. The "Renaissance Recognition Award" was conceived by Dr. Fredric Rieders in order to recognize students who have distinguished themselves accordingly. The award is given to those students who, in the eyes of their peers and faculty, have made an extraordinary contribution to the Thomas Jefferson University College of Graduate Studies community that far exceeds academic achievement. Dr. Rieders believes that it is important to recognize that graduate education encompasses more than excellence in academic achievement, more than high quality research, more than producing a thesis that adds to the global body of knowledge. A true renaissance scientist enhances the quality of, and contributes to, the human aspects pertaining to the activities surrounding a graduate education. The "Renaissance Recognition Award" is a cash award with a certificate of recognition and a small gift to commemorate the occasion and is funded through an investment account of The Fredric Rieders Family Renaissance Foundation. Dr. Rieders has personally presented the award in the previous two years' ceremonies at the Alumni Day Celebration. Awardees have included Amy Shoonover, M.S., William A. Knapp, M.S., Bernadette Mandes, M.S., Providence Tara Santangelo, M.S. and Oscar Baylin Goodman, Jr., Ph.D. SEMINAR SCHEDULE
UPCOMING EVENTSUnexpected Suspicious Poisoning Deaths.On December 7, 2000 the Fredric Rieders Family Renaissance Foundation is presenting a symposium focused on Unexpected Suspicious Poisoning Deaths to be presented from 1:00pm to 6:00pm at the National Medical Services Facility in Willow Grove, PA. Attendance is by invitation only. Continuing Education credit may be provided by the Coroners Education Board of Pennsylvania. The Speakers will include Drs. Fredric Rieders, Randall Baselt, Michael Baden and G. John DiGregorio.The agenda for the symposium is as follows:
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