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Forensic laboratories face the need to analyze many samples of human blood and body fluids for alcohol content. The most important compound in blood-alcohol (BAC) analysis is ethanol, but the detection of several other significant alcohols and their metabolites (methanol, iso-propanol, acetone, acetaldehyde, etc.) is fundamental because they can be present in blood and can eventually interfere with ethanol quantification.
Because of the large number of blood alcohol analyses to be performed for legal or medical purposes, particular attention must be focused on the methodology to be used. Important factors to consider are sample throughput, resolution, and reliability. Therefore, an effective method for these analyses should be fast, precise, and accurate.
Gas chromatographic (GC) analysis provides the best level of sensitivity and accuracy in analyzing for these volatile compounds. Direct injection of biological samples into GC columns has been used in the past as the method of sample introduction, but this could cause column contamination and therefore worsen performance.
The use of headspace sampling instead of direct injection prevents the buildup of nonvolatile contamination at the head of the column and helps to reduce the need for inlet maintenance and extends column lifetime. The dual-column GC system is the best choice for providing blood alcohol confirmation in a few minutes.
Methodology
Instrumentation: GC and headspace autosampler
The work was carried out with a GC equipped with an HT2000H headspace autosampler (HTA s.r.l., Brescia, Italy). The analysis was performed with two different application-specific columns for blood alcohol testing (MEGA-ALC 1 0.15 mm × 0.85 μm × 10 m and MEGA-ALC 2 0.15 mm × 0.85 μm × 10 m) (MEGA S.N.C., Milan, Italy). The instrument settings are described in Table 1.
Table 1 – Instrument settings
Materials
Blood alcohol standard
- Blood Alcohol Mix Resolution Control Standard was used: eight components, 100 mg/dL, water, 1 mL/ampule (Restek [Bellefonte, CA], cat. #36256).
Figure 1 – GC-FID chromatogram obtained with headspace injection (performed with HT2000H autosampler) of a blood alcohol mix standard in an application-specific column for blood alcohol testing (MEGA-ALC 1 0.15 mm × 0.85 μm × 10 m).
Figure 2 – GC-FID chromatogram obtained with headspace injection (performed with HT2000H autosampler) of a blood alcohol mix standard in an application-specific column for blood alcohols testing (MEGA-ALC 2 0.15 mm × 0.85 μm × 10 m).
Results
The results are shown in Figures 1 and 2.
Conclusion
Successful blood alcohol analysis can be performed with fast analysis times and good reproducibility using the HT2000H autosampler and gas chromatograph equipped with MEGA-ALC 1 0.15 mm × 0.85 μm × 10 m and MEGA-ALC 2 0.15 mm × 0.85 μm × 10 m GC columns. Both tested columns provided good resolution of all the blood alcohol analytes. The elution order of the eight components was different between the two phases. This selectivity difference is important for confirmation analysis. The 0.15-mm-i.d. size is one of the best compromises between Fast and conventional GC, allowing users to save time while maintaining resolution power and loading capability for analysis.
The HT2000H autosampler allows users to perform very reproducible injections, as demonstrated by the low peak area RSD achieved (below 5% for all the analytes).
For more information, please contact HTA s.r.l., via del mella, 77-79, 25131 Brescia, Italy; tel.: +39 030 3582920; fax: +39 030 3582930; e-mail: [email protected] ; www.hta-it.com.
Please see our Gas Chromatography Equipment section to find manufacturers that sell these products