The Analysis of 2,4-Dinitrophenol in Biological Specimens

2,4-dinitrophenol (2,4-DNP) is a synthetic compound used primarily as an ingredient in dyes, wood preservatives, photographic developers, pesticides, and explosives. It was used in the 1930s as a weight loss medication following the discovery that it uncouples oxidative phosphorylation and increases lipid metabolism. By 1938, the U.S. Food and Drug Administration banned the use of 2,4-DNP for weight loss purposes.;In August 2006, a 26 year old female in the County of Sacramento died from 2,4-DNP poisoning. The Sacramento County District Attorney's Laboratory of Forensic Services (Sacramento County Laboratory) received a blood kit containing the decedent's femoral blood for alcohol and toxicology testing. Based on the Coroner's request, the sample was analyzed for acid-neutral drugs using gas chromatography/mass spectrometry (GC-MS). The results showed a large unidentified peak co-eluting with the internal standard, barbital. A library database search tentatively identified the peak as 2,4-dinitrophenol. The sample was sent to an outside laboratory, which confirmed 2,4-DNP at a concentration of 170 mg/L.;The goal of this project was to develop a suitable and reliable method for the detection and quantitation of 2,4-DNP in blood and tissue samples. This was accomplished by modifying the Sacramento County Laboratory's acid-neutral extraction procedure to include the use of deuterated 2,4-dinitrophenol (2,4-DNP-D3) as the internal standard and derivatization using BSTFA+1%TMCS. A range of GC-MS parameters were evaluated and ultimately a 4:1 split injection and a 2 ul injection volume worked best for achieving reproducibility. The mass spectrometer was run in selected ion monitoring (SIM) mode and monitored for 2,4-DNP ions with a m/z at 241, 195, and 137. 2,4-DNP-D3 was monitored for ions with a m/z at 244, 198, and 140. The method under these parameters proved to be both precise and accurate.;A stability study was conducted for 2,4-DNP in samples stored in different conditions for a period of time. After one year, the concentration of 2,4-DNP in the samples stored at ambient temperature (21°C), under refrigeration (2°C), and frozen (-18°C) dropped approximately 14%, 12%, and 11%, respectively.;The Coroner's Office had additional samples from the 2006 case that they provided for 2,4-DNP analysis once the method was validated. The case samples were analyzed multiple times for their 2,4-DNP concentration to determine the biological disposition of the drug in the decedent's body. The average 2,4-DNP concentration for each of the case samples was as follows: gastric contents at 634 mg/L, duodenum contents at 575 mg/L, liver at 6.7 mg/L, vitreous humor at 10.7 mg/L, bile at 102 mg/L, heart blood (red/grey top tube) at 149 mg/L, heart blood (bottle) at 145 mg/L, femoral blood (grey top tube) at 121 mg/L, femoral blood (from the blood kit originally submitted to the Sacramento County Laboratory) at 113 mg/L, antemortem serum (green top tube) at 231 mg/L, antemortem serum (yellow top tube) at 246 mg/L, and antemortem blood (purple top tube) at 172 mg/L. In this research project a method for the detection and quantitation of 2,4-dinitrophenol is presented along with the biological disposition of the drug in the body of a deceased female.