Critical Factors in Selecting an Internal Standard for Accurate Determination of Blood Alcohols in Post Mortem Samples

Significance of the topic

Accurate determination of blood alcohol content in post mortem samples is critical for forensic investigations and traffic‐fatality analysis. Traditional use of n-Propanol as an internal standard can be compromised by bacterial generation of this compound during decomposition, leading to biased ethanol quantitation.

Study objectives and overview

This study aimed to identify alternative internal standards for dual-column headspace GC analysis of blood alcohols in post mortem specimens. The performance of t-Butanol and 2-Butanol was compared to n-Propanol across three column sets: Restek Rtx-BAC1/2, J&W DB-ALC1/2, and Phenomenex Zebron ZB-BAC1/2.

Methodology

A five-point calibration (0.025 to 0.400 % v/v) was prepared in water, with all internal standards at 0.100 % v/v. Samples were analyzed isothermally at 40 °C by split injection. Linearity, limit of detection (LOD), limit of quantitation (LOQ), and reproducibility were evaluated for each column pair. The lowest calibration level (0.025 %) was used to assess precision.

Instrumentation

Agilent 6890 gas chromatograph equipped with Overbrook Scientific HT-200 autosampler and dual flame ionization detectors. Phenomenex Zebron ZB-BAC1 column (30 m × 0.53 mm × 3.0 µm) and ZB-BAC2 column (30 m × 0.53 mm × 2.0 µm) were used, connected via a guard column and Y-splitter. Helium carrier gas at 80 cm/s, split ratio 0.8 to 1, injector temperature 150 °C, detector temperature 250 °C.

Main results and discussion

Restek and J&W column pairs failed to resolve t-Butanol from acetone, causing co-elution and quantitation errors at 40 °C. Both column sets did separate 2-Butanol but required doubling of run time. In contrast, Zebron ZB-BAC1 and ZB-BAC2 fully resolved both t-Butanol and 2-Butanol from target analytes in under two minutes for t-Butanol and under five minutes for 2-Butanol, reducing total analysis time by approximately 9 %. Calibration for all analytes showed excellent linearity with R2 values from 0.9980 to 0.9998. LODs were below 0.0003 % and LOQs below 0.001 % for all compounds. Precision at the lowest level exhibited relative standard deviations under 3.5 %.

Benefits and practical applications

• Elimination of endogenous n-Propanol interference in post mortem samples.
• Faster run times and higher sample throughput.
• Improved peak resolution and analytical precision.
• Sensitivity and reproducibility meet forensic testing requirements.

Future trends and potential applications

Development of even shorter GC methods and integration with automated data analysis platforms will further enhance laboratory efficiency. Exploration of novel stationary phases and coupling with mass spectrometric detection may expand applicability to complex biological matrices beyond blood.

Conclusion

The Phenomenex Zebron ZB-BAC1 and ZB-BAC2 column pair offers an optimal solution for reliable, rapid blood alcohol analysis in post mortem and routine forensic testing when using t-Butanol or 2-Butanol as internal standards.

Reference

• 1. National Highway Traffic Safety Administration. Monitoring of Traffic Fatalities Related to Alcohol.
• 2. Nanikawa R.; Ameno K.; Hashimoto Y.; Hamada K. Medicolegal Studies on Alcohol Detected in Dead Bodies – Alcohol Levels in Skeletal Muscle. Forensic Sci. Int., 20, 133–140.