Blood alcohol determination by HT3 Automatic Static/Dynamic Headspace Sampler

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

Part Settings
Injector
(split splitless)
200°C
10:1
Column 1: SCION-WAXMS
2: SCION-WAXMS
Oven Program 40°C
Carrier Helium
Column flow 12 ml/min
Detector FID with ceramic jet, 250°C
Air: 300 ml/min, Fuel gas (H2): 35 ml/min, Make up (N2): 23 ml/min
Inj. Volume 1 ml
Software Compass CDS

Table 2: Static loop instruments conditions HT3

Variable Value Variable Value
Constant Heat Time On Mixing time 0.0 min
GC Cycle Time 3 mins Mixing level Level 5
Valve Oven Temp 200°C Mixer stabilize time 0.50 min
Transfer Line Temp 200°C Pressurise 10 psig
Standby Flow Rate 200 mL/min Pressurise Time 2.0 min
Platen/Sample Temp 70°C Pressurise Equil Time 0.20 min
Platen Temp Equil. Time 1.00 min Loop Fill Pressure 5 psig
Sample Equil. Time 10.00 min Loop Fill Time 0.20 min
Mixer Off Inject Time 0.50 min

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

  Standard Concentration (g/dL)        
Compounds 1 2 3 4 5 6 7
Methanol 0.32 0.16 0.08 0.04 0.02 0.008 0.002
Acetaldehyde 0.08 0.04 0.02 0.01 0.005 0.004 0.0005
Ethanol 0.32 0.16 0.08 0.04 0.02 0.008 0.002
Acetone 0.08 0.04 0.02 0.01 0.005 0.004 0.0005
Isopropanol 0.20 0.10 0.05 0.025 0.0125 0.005 0.00125

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.

  Methanol   Acetaldehyde
  Ethanol   Isopropanol   Acetone   n-Propanol (IS)
Area IS Ratio Area IS Ratio Area IS Ratio Area IS Ratio Area IS Ratio Area
1 37.9879 0.1973 42.1094 0.2187 69.6079 0.3615 72.3644 0.3758 46.8225 0.2431 192.5791
2 40.5376 0.2037 42.6875 0.2145 73.1351 0.3674 74.4834 0.3742 47.3482 0.2379 199.0514
3 39.7432 0.1951 42.6868 0.2095 73.7363 0.3619 76.3297 0.3747 48.0622 0.2359 203.7249
4 40.1865 0.2081 40.7507 0.2111 71.4818 0.3702 71.7019 0.3713 45.2439 0.2343 193.0856
5 40.2395 0.1972 41.9664 0.2056 74.3102 0.3641 76.0220 0.3725 47.3413 0.2320 204.0724
6 38.6157 0.2034 40.9111 0.2154 69.5918 0.3665 70.5772 0.3717 45.1332 0.2377 189.8920
7 37.6379 0.2025 39.1901 0.2109 67.9005 0.3653 69.1451 0.3720 43.4294 0.2337 185.8657
Average 39.2783 0.2010 41.4717 0.2122 71.3948 0.3653 72.9462 0.3732 46.1972 0.2364 195.4673
%RSD 3.0 2.3 3.1 2.0 3.4 0.8 3.8 0.5 3.6 1.6 3.6

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.

  Acetaldehyde   Methanol   Ethanol   Acetone   Isopropanol   n-Propanol (IS)
Area IS Ratio Area IS Ratio Area IS Ratio Area IS Ratio Area IS Ratio Area
1 43.6512 0.2628 41.0730 0.2473 63.3810 0.3816 47.4316 0.2856 68.8978 0.4148 166.1035
2 44.2913 0.2567 43.7153 0.2533 66.6057 0.3860 47.8574 0.2773 70.9253 0.4110 172.5634
3 44.2272 0.2506 42.8884 0.2430 67.2499 0.3811 48.6533 0.2757 72.7133 0.4121 176.4668
4 42.4985 0.2548 43.2528 0.2593 64.9051 0.3892 45.7679 0.2744 68.2796 0.4094 166.7748
5 43.6353 43.3207 43.3207 0.2433 72.5385 0.4074 47.9482 0.2693 72.7095 0.4083 178.0594
6 42.6253 41.4251 41.4251 0.2527 62.8839 0.3836 45.5205 0.2777 66.7594 0.4072 163.9421
7 40.8633 0.2560 40.4448 0.2534 61.4196 0.3847 43.8038 0.2744 65.2469 0.4087 159.6378
Average 43.1132 0.2551 42.3029 0.2503 65.5691 0.3876 46.7118 0.2763 69.3617 0.4102 169.0783
%RSD 2.8 2.3 3.1 2.4 5.6 2.4 3.7 1.8 4.2 0.6 4.0

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.

Compound Correlation Coefficients Concentration range (%)
Methanol 0.9995 0.002 to 0.32
Acetaldehyde 0.9983 0.0005 to 0.08
Ethanol 1.0000 0.002 to 0.32
Acetone 0.9999 0.005 to 0.08
Iso Propanol 0.9995 0.00125 to 0.20

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.

Part number SCION HT3 Headspace sampler
SC149300000 HT3 Headspace Autosampler 110V.
SC149300100 HT3 Headspace Autosampler 230V.
SC14930000S HT3 Dynamic Headspace Autosampler 110V.
SC14930010S HT3 Dynamic Headspace Autosampler 230V.

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