Improved Separation of Blood Alcohols Using Zebron™ ZB-BAC1 and BAC2 GC Columns

Significance of the Topic

The accurate and rapid determination of blood alcohol content (BAC) is critical in forensic and clinical settings to support legal decisions, ensure public safety, and monitor metabolic conditions. Efficient separation and quantitation of ethanol and related volatile compounds minimize false positives and increase laboratory throughput.

Objectives and Overview of the Study

This study evaluates the performance of two novel GC phases, Zebron ZB-BAC1 and ZB-BAC2, for headspace-GC-FID analysis of five blood alcohol analytes. Key goals include achieving fast run times, high reproducibility, and sensitive detection across a calibration range of 0.025–0.400% vol. in water, reflecting common forensic BAC limits.

Methodology and Instrumentation

Blood alcohol standards (methanol, acetaldehyde, ethanol, acetone, isopropanol) were prepared at 0.025–0.400% in 5 mL water with internal standards (t-butanol, n-propanol, 2-butanol) held at 0.100%. Headspace vials were heated at 60 °C for 13 minutes, and 1.0 mL injections were split 0.8:1 into two FIDs. The GC oven was held isothermal at 40 °C for 5 minutes. Chromatograms were used to assess retention times, resolution, precision, and signal-to-noise ratios for LOD/LOQ determination.

Used Instrumentation

• Gas chromatograph: Agilent HP6890 with flame ionization detector (FID at 250 °C)
• Autosampler: Overbrook Scientific HT-200H headspace autosampler (40 mL/min fill, 30 mL/min injection)
• Zebron ZB-BAC1 column (30 m × 0.53 mm ID × 3.00 μm)
• Zebron ZB-BAC2 column (30 m × 0.53 mm ID × 2.00 μm)
• Carrier gas: Helium at constant flow of 12.9 mL/min

Main Results and Discussion

Both phases achieved baseline resolution of all five analytes in under 5 minutes, with ethanol separation in <2 minutes on ZB-BAC1 and <2.2 minutes on ZB-BAC2. Using t-butanol as internal standard reduced run times to 1.7 minutes. Precision across replicates at 0.025% and 0.100% levels showed absolute and relative RSDs of ~1–3% irrespective of the internal standard used. Calibration curves exhibited excellent linearity (R² 0.9980–0.9998). LODs for ethanol ranged from 0.0002–0.0003% (2.2–2.6 ppm) and LOQs from 0.0007–0.0009% (7.4–8.8 ppm), far below the 0.08% (800 ppm) legal threshold.

Benefits and Practical Applications

• High throughput: run times as short as 1.7 minutes
• Robust quantitation with low RSDs and sub-ppm detection limits
• Dual-column confirmation reduces risk of co-elution errors
• Straightforward integration into routine forensic and clinical workflows

Future Trends and Opportunities

Further improvements may include coupling these phases with mass spectrometry for enhanced specificity, development of faster or trap-based headspace modules, automation of sample preparation, and expansion of volatile biomarker panels for broader diagnostic applications.

Conclusion

Zebron ZB-BAC1 and ZB-BAC2 columns, paired with headspace-GC-FID and the HT-200H autosampler, deliver rapid, reproducible, and highly sensitive blood alcohol analysis. Their complementary selectivities facilitate confirmatory testing and support high-throughput forensic and clinical laboratories.

References

1. 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.