Development and validation of a colorimetric screening method and a rapid HS-GC-FID confirmation method for...

The present study reports the development and validation of a simple and rapid colorimetric method for chloroform (CHCl₃) detection in biological specimens, coupled with a dual-column (HS-GC-FID) confirmation method for quantitative analysis. The colorimetric method is based on a modified Fujiwara reaction yielding a characteristic pink coloration, with a detection limit of 0.1mg/dL. For the HS-GC-FID method, samples were diluted with an aqueous n-propanol internal standard (IS) and analyzed using a dual capillary column with front and back detectors. The method exhibited linearity from 0.25 to 30mg/dL for CHCl₃ with a LOD of 0.12mg/dL, and a LOQ of 0.25mg/dL. The accuracy (bias) range for CHCl₃ is -10.0% to 12.67, and the precision (%CV) ranges from 2.29 to 18.0%. No carryover was observed. Established methods were applied in a real homicidal forensic case study investigation. Colorimetric screening, indicated by the appearance of pink color, together with quantitative analysis of two case studies, confirmed chloroform at a lethal blood concentration of 24.2 and 25.7mg/dL, establishing acute CHCl₃ poisoning as the cause of death. This study presents the development and application of a rapid, robust, and sensitive analytical methodology for the unequivocal confirmation of CHCl₃.