Analysis of 2,4-Dinitrophenol in Postmortem Blood and Urine by Gas Chromatography-Mass Spectrometry: Method Development and Validation and Report of Three Fatalities in the United States.

2,4-dinitrophenol (2,4-DNP) is a compound used in the early 1900s as a weight-loss drug but later prohibited due to its severe adverse effects, including death. It has however been attracting interest, due to its weight-loss properties, and appears to be re-emerging in forensic casework. As 2,4-DNP is available for use in industry and as a pesticide and easily accessible online, the dissemination of this drug can be fast. The compound exerts its effects through inhibition of ATP synthesis, and corresponding thermogenic energy loss which can be fatal. A method for qualitative and quantitative analysis of 2,4-DNP in blood and urine specimens using GC-MS with hydrogen as carrier gas is described. The method was validated and displayed acceptable performance parameters with linearity (R2 higher than 0.998), inter-assay imprecision (lower than 10.6%), intra-assay imprecision (lower than 10.7%), and extraction efficiency (92.1%). Stability of 2,4-DNP in blood and urine was studied, and the drug was stable up to 30 days refrigeration or frozen. Six cases in United States suspected to be related to 2,4-DNP were analyzed. Three cases were found to be positive for 2,4-DNP. Concentrations of 2,4-DNP were in the range of 61.6-220 mg/L in urine and <3-114 mg/L in blood. Based on our findings, we suggest that medical examiners and forensic toxicologists be aware of the reappearance of 2,4-DNP, including this compound as a target in death investigations related to weight-loss drugs.

Voltammetric and amperometric determination of 2,4-dinitrophenol metabolites.

Methods for determination of 2-amino-4-nitrophenol and 4-amino-2-nitrophenol, metabolites of 2,4-dinitrophenol, were developed using differential pulse (DP) voltammetry and HPLC with amperometric and spectrophotometric detection. The applicability of these methods was tested by the determination of the analytes in model samples of urine after preliminary separation by solid-phase extraction. Voltammetry enabled parallel determination of both analytes, but its application in real matrix was severely limited due to the interference of other compounds present in urine. HPLC allowed the determination in real urine matrix down to micromolar concentrations; amperometric detection proved to be more sensitive and selective than the spectrophotometric one.

Two deaths attributed to the use of 2,4-dinitrophenol.

We report the cases of two individuals, one in Tacoma, WA, and the second in San Diego, CA, whose deaths were attributed to ingestion of 2,4-dinitrophenol (2,4-DNP). 2,4-DNP has historically been used as a herbicide and fungicide. By uncoupling mitochondrial oxidative phosphorylation, the drug causes a marked increase in fat metabolism that has led to its use to aid weight loss. Both cases reported here involved its use for this purpose. Features common to both cases included markedly elevated body temperature, rapid pulse and respiration, yellow coloring of the viscera at autopsy, history of use of weight loss or body building supplements, and presence of a yellow powder at the decedent's residence. Because of its acidic nature, the drug is not detected in the basic drug fraction of most analytical protocols, but it is recovered in the acid/neutral fraction of biological extracts and can be measured by high-performance liquid chromatography or gas chromatography-mass spectrometry. The concentration of 2,4-DNP in the admission blood samples of the two deaths reported here were 36.1 and 28 mg/L, respectively. Death in both cases was attributed to 2,4-DNP toxicity. Review of information available on the internet suggests that, although banned, 2,4-DNP is still illicitly promoted for weight loss.