Development and Validation of a Colorimetric Screening Method and a Rapid HS GCFID Confirmation Method for Chloroform in Toxicological Specimens: Application to Three Fatal Poisoning Cases

Ann Pharm Fr. 2026 May 13:S0003-4509(26)00071-4. doi: 10.1016/j.pharma.2026.05.003. Online ahead of print.

ABSTRACT

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 0.1 mg/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 30 mg/dL for CHCl₃ with a LOD of 0.12 mg/dL, and a LOQ of 0.25 mg/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 a pink color, together with quantitative analysis of two case studies, confirmed chloroform at a lethal blood concentration of 24.2 mg/dL and 25.7 mg/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.

PMID:42134740 | DOI:10.1016/j.pharma.2026.05.003