INTRODUCTION

Forensic and toxicology laboratories around the world are using headspace analysis to analyse biological fluids for volatile components. The determination of blood alcohol components (BAC) is a routinely performed headspace analysis, frequently used in the suspicion of driving under influence (DUI). Using a headspace injection of the sample onto two columns, one for quantitation and the other for confirmation of the volatile compounds, who are finally detected with separate flame ionization detectors. (FID)

This application note presents the results of the determination of blood alcohol using headspace sampling. The HT3 headspace sampler is perfectly capable for the analysis of several volatile compounds in blood (ethanol), including acetaldehyde and acetone (metabolized components of ethanol in blood), Isopropanol (absorbed or inhaled from rubbing alcohol vapours). These compounds are quantitated by using an internal standard n-propanol. Figure 1 shows the SCION Instruments HT3 headspace sampler used throughout this application with  the 8X00 GC platform.

Figure 1. SCION Instruments HT3 Headspace Sampler and the SCION Instruments 8X00 GC platform 

INSTRUMENTS PARAMETERS

The HT3 headspace sampler was connected to a SCION Instruments 8X00-GC, equipped with dual FID’s. Table 1 is showing the analytical conditions of the GC system. The static loop instruments conditions are presented in table 2.

Table 1: Analytical settings GC

Table 2: Static loop instruments conditions HT3

EXPERIMENTAL

Blood samples are mixed with an internal standard with buffering agent. The internal standard solution of 0,015% v/v% n-propanol is dissolved in 0,5M ammonium sulphate and deionized water.

A set of standards is prepared for the components ethanol, methanol, acetaldehyde, isopropanol and acetone in concentration range of 0.40, 0.20, 0.10, 0.05, 0.025, 0.010, 0.0025% v/v. These concentrations correspond to blood ethanol concentrations of 0.32, 0.16, 0.08, 0.04, 0.02, 0.008 and 0.002g/dL. The concentration of the compounds in each standard expressed in DUI blood alcohol mg/dL is listed in table 3.

Standards were prepared by pipetting a known amount of neat solvents up into 100mL volumetric flasks, containing the 0,015%n-propanol internal standard solution. A refrigerated syringe was used to pipet the acetaldehyde, for reducing the error during sample preparation of the  volatile compound. 1mL of these standard solutions is transferred into a HT3 22mL sample vial with a graduated pipet, capped with a Teflon lined silicon septa and headspace crimp caps and properly crimped.

Standards from table 3 are injected to generate correlation coefficients  of the analysis.

Table 3: Concentrations of the standards

RESULTS AND DISCUSSION

A sample chromatogram is presented in figure 2, showing the chromatogram of the 0.008g/dL ethanol standard on the quantitation column.

Figure 2:  Chromatogram 0.008g/dL ethanol standard on the quantitation column.

Table 4: Precision of the quantitation column.

Figure 2 is showing a good separation between the components, within 3 minutes analysis time. Precision of the assay is determined by injecting seven times the same sample (0,008 g/dl ethanol standard).  The precision for each component in the standard is presented in Table 4 for the quantitation column. A sample chromatogram is presented in figure 3, showing the chromatogram of the 0.008g/dL ethanol standard on the quantitation column. This chromatogram shows a good separation between the components, within 3 minutes analysis time.

Figure 3:  Chromatogram 0,008g/dL ethanol standard on the confirmation column.

Table 5: Precision of the confirmation column.

Precision of the assay is determined by injecting seven times the same sample (0,008 g/dL ethanol standard).  The precision for each component in the standard is presented in Table 5 for the confirmation column.

Calibration curves

Table 6 presents the correlation coefficient for a seven point calibration curve from standards prepared at 0.32%, 0.16%, 0.08%, 0.04% and 0.02%, 0.008% and 0.002%. set of parameters followed in this paper provides a correlation coefficient of 1.0000 for a seven point calibration curve ranging from 0.002g/dL to 0.32g/dL ethanol concentration.  The correlation curves for methanol, acetone and isopropanol were all greater than 0.999 for this method for their corresponding concentration. The correlation coefficient for acetaldehyde was greater than 0.998, and was expected because of the volatility of the compound during sample preparation.

Table 6: Correlation coefficients for a seven point calibration curve.

CONCLUSION

The determination  of blood alcohol, in association with the SCION Instruments 8X00 GC is easy to perform with the HT3 SCION Instruments Headspace Sampler.

The correlations coefficients of the seven point calibration curves are for most components greater than 0.999. Exceptional is acetaldehyde with a correlation coefficient of 0.998, due to the volatility of the compound during standard preparation.

The internal standard method precision was 0.8% for 0.008 mg/dL ethanol standard, measured in septenary, prepared by graduated pipet with 2% RSD.

The HT3 with 2 capillary columns produced blood alcohol data with excellent linearity and precision for ethanol concentrations in the range of 0.32 g/dL to 0.002g/dL, within a analysis time of 3 minutes using n-propanol as internal standard.

Although the 4X6-GC series is not shown in this application note, it is also possible to perform this method on the SCION instruments 4X6 GC series. Ordering information can be found in the table below. For customisation please contact your local sales representative.

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Download complete Application Note here: Blood alcohol determination with HT3 Headspace Analyzer

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