Quantitative Determination of Nitrous Oxide in Human Blood by HS-GC–MS: Forensic Application of Two Fatal Poisoning Cases

Nitrous oxide (N2O), also known as laughing gas, has a euphoric effect and is becoming increasingly popular as a recreational inhalant drug. Deaths caused by recreational nitrous oxide abuse are rare, but may still occur. Although some methods for the quantification of N2O by GC-MS have been reported, elimination of carbon dioxide interference and the choice of a suitable internal standard remain current limitations to accurate N2O quantification. Here, a validated method using headspace-gas chromatography-mass spectrometry (HS-GC-MS) is described that allows the quantification of N2O in human blood samples: sodium hydroxide is used to remove carbon dioxide, and n-pentane is chosen as a suitable internal standard. Collectively, the validation results show a good linear relationship of N2O in blood within the concentration range of 0.02 similar to 0.5 mL/mL and an LOD of 0.005 mL/mL. Subsequent application of the validated method to two real mortality cases due to N2O intoxication provided reference values for blood concentrations in forensic cases. Other biological specimens (gaseous samples and tissues) of the deceased were also analyzed to demonstrate that the deaths were caused by asphyxia due to the inhalation of N2O.