Winter 2001
SUMMER MENTORING PROGRAM - 2000
RESEARCH ABSTRACT
Catecholamine Extraction Methodology
James A. King, Marena T. LaFontaine, Marcus Jackson, Anthony Macherone and G. John DiGregorio
Epinephrine, a physiologically active catecholamine, is derived directly from norepinephrine by the action of phenylethanolamine-N-methyltransferase. Systemic secretion from the adrenal medulla of epinephrine into the blood stream is paramount to stimulating the sympathetic nervous system as part of the fight-or-flight reaction. Epinephrine and norepinephrine are metabolized by catecholamine-O-methyltransferase to form metanephrine and normetanephrine, respectively. In vivo, this enzymatic metabolism occurs quite rapidly and the half-life of epinephrine and norepinephrine has been reported as approximately two minutes Berne, Robert M., Levy, Matthew N. Principles of Physiology 2 Ed. Mosby-Year Book, Inc., New York, (1996) while the half-life of metanephrine and normetanephrine is much greater Macherone, AJ., Rieders, F., Selenski, C. Epinephrine Poisoning Part II: Stability of Metanephrine and Normetanephrine in Exhumed Tissue. In: Proceedings of the American Academy of Forensic Science; 2000 Feb. 21-26; Reno, NV:272.
Persons acting out of pseudo-altruistic intent or blatantly committing murder may insidiously administer epinephrine, or other catecholamines such as dopamine, as a means to cause sudden death via ventricular fibrillation in their intended victim. Recently, Dr. Michael Swango, a physician and convicted serial murderer, admitted to using epinephrine to induce death in several of his patients. Thus interest to detect and quantitate catecholamines in suspicious, unexpected deaths has grown dramatically in the forensic arena. Previously, our lab reported an effective and accurate method to extract metanephrine and normetanephrine from embalmed and non-embalmed post-mortem tissue homogenates Rieders, F., Macherone, AJ., Selenski, C. Vickery, W. Epinephrine Poisoning Part I: Extraction and Analysis of Metanephrine and Normetanephrine from Exhumed Tissue. In: Proceedings of the American Academy of Forensic Science; 2000 Feb. 21 - 26; Reno, NV:269. The limitation to the method was that it was unable to extract epinephrine, norepinephrine or any of the 3,4-dihydroxy catecholamine compounds. Therefore, in this past summers work we attempted to develop a procedure to simultaneously extract five catecholamine compounds, including 3,4-dihydroxy catecholamines and 3-methoxy-4-hydroxy catecholamines, from various biological matrices for qualitative and quantitative analysis by liquid chromatography with electrochemical detection. The 3,4-dihydroxy catecholamines compounds used in the study were dopamine, epinephrine, norepinephrine and 3,4-dihydroxybenzylamine as an internal standard. The 3-methoxy-4-hydroxy catecholamines included metanephrine, normetanephrine and their internal standard 3-methoxy-4-hydroxybenzylamine. Extraction methodologies included a proprietary catecholamine extraction from ESA, an extraction procedure developed by Dr. Kevin Ballard, National Medical Services, Inc. and the development of an original extraction procedure.
The ESA extraction does not extract metanephrine or normetanephrine and therefore is not applicable for this study.
The procedure uses heptafluorobutyric acid (HFBA) as the ion-pair reagent, which proved to be incompatible with our analytical finish. The HFBA produces a peak, which interferes with epinephrine and norepinephrine. HFBA also competes with the ion-pair reagent used in the analytical finish. The HFBA could not be removed easily.
The analytical finish is an HPLC method, which uses octanesulfonate as the ion-pair reagent and methanol as the organic component of the mobile phase. We have been developing an extraction method, which uses octanesulfonate as the ion-pair reagent and acetonitrile as the organic component of the mobile phase.
The procedure can extract the catecholamines and is compatible with our analytical finish. The norepinephrine elutes easily and work needs to be done to keep the recovery of norepinephrine high and also getting all of the 4-hydroxy-3-methoxy-benzylamine (HMBA) out. HMBA is an internal standard.
1. Berne, Robert M., Levy, Matthew N. Principles of Physiology 2 Ed. Mosby-Year Book, Inc., New York, (1996)
2. Macherone, AJ., Rieders, F., Selenski, C. Epinephrine Poisoning Part II: Stability of Metanephrine and Normetanephrine in Exhumed Tissue. In: Proceedings of the American Academy of Forensic Science;
2000 Feb. 21-26; Reno, NV:272
3. Rieders, F., Macherone, AJ., Selenski, C. Vickery, W. Epinephrine Poisoning Part I: Extraction and Analysis of Metanephrine and Normetanephrine from Exhumed Tissue. In: Proceedings of the American
Academy of Forensic Science; 2000 Feb. 21 - 26; Reno, NV:269
