Further elucidation of the metabolism of 2,4-dinitrophenol in a case of unexpected death

Abstract 2,4-Dinitrophenol (2,4-DNP) was discovered and popularized in the 1930s as a weight-loss drug. Due to multiple adverse effects, including death, its use as a prescription drug was banned in 1938. However, toxicity cases have risen over the past two decades as 2,4-DNP is easily obtained illegally online. In a fatal case at our hospital involving rapid, unexplained deterioration, 2,4-DNP was identified through a full toxicological analysis of blood and urine. An LC-MS/MS method for detecting and quantifying 2,4-DNP in plasma and urine was developed and validated according to European Medicine Agency (EMA) criteria. It also allowed quantification of its main metabolites 2-amino-4-nitrophenol (2A-4NP) and 4-amino-2-nitrophenol (4A-2NP) in urine. In plasma, 2A-4NP was only semi-quantifiable; 4A-2NP was undetected, likely due to matrix effects reducing sensitivity. Results obtained with and without enzymatic hydrolysis showed that 2,4-DNP-glucuronide and 2A-4NP plus its glucuronide are the primary metabolites, while 4A-2NP and its glucuronide are less prominent. No sulfate conjugates were detected. This study is the first to compare sample preparation with and without enzymatic hydrolysis, offering new insights into 2,4-DNP metabolism and the relative importance of its major metabolites and their glucuronides.