APPLICATION NOTE 10713

Rapid, automated, and accurate determination of blood

alcohol concentration (BAC) by headspace coupled to
gas chromatography and flame ionization detection

Authors Goal
Jane Cooper, Giulia Riccardino, and The aim of this work is to demonstrate the performance of the new Thermo
Cristian Cojocariu Scientific™ TriPlus 500™ headspace (HS) autosampler coupled to a Thermo
Thermo Fisher Scientific, Runcorn, UK Scientific™ TRACE™ 1310 Gas Chromatograph, using the Thermo Scientific™
Chromeleon™ Chromatography Data System (CDS) for fast, accurate, and
routine determination of blood alcohol concentration (BAC).
Keywords
Blood alcohol concentration, Introduction
BAC, headspace analysis, valve Blood alcohol concentration analysis is one of the most common tests
and loop, ethanol, human blood, performed in forensic science. For the purposes of law enforcement, BAC is
headspace gas chromatography, used to define the level of intoxication and can also provide a rough measure
HS-GC, flame ionization detector, of impairment. Many countries forbid operation of motor vehicles or heavy
FID, TriPlus 500 HS, robustness machinery by anyone with alcohol concentration above a legal limit, usually
expressed in grams per deciliter (g/dL). BAC legal limits vary in different
countries: 0.08 g/dL in the majority of USA states, England, and Wales;
0.05 g/dL in Italy and Scotland; 0.02 g/dL in Sweden and Norway. In some
cases, zero tolerance BAC laws are enforced, either for all (e.g. Brazil,
Hungary, Kuwait), for specific age groups (e.g. under 20 years old in Japan),
for a specific time after gaining a driving license (e.g. drivers in their first two
years after gaining a license in Italy), or for those in some jobs (e.g. military).
 BAC analysis is routinely carried out using the headspace The vial was sealed (caps P/N 20-MCBC-ST3) and mixed
sampling technique coupled to gas chromatography prior to analysis.
(GC) with flame ionization detection (FID) or mass
spectrometry (MS) detection, as this is a simple and fast Instrument and method setup
analytical technique allowing for high sample throughput. A Thermo Scientific™ TriPlus 500™ HS autosampler
The main challenges related to BAC determination that coupled to a Thermo Scientific™ TRACE™ 1310 Gas
can lead to inaccurate results are carryover from previous Chromatograph with a Thermo Scientific™ Instant-
injections, resulting in elevated and in some cases false Connect Split/Splitless Injector (SSL) and two Thermo
positive results and non-linear ethanol calibration, due Scientific™ Instant-Connect Flame Ionization Detectors
to poor instrument performance or inadequate method (FID) were used for all experiments (Figure 1).
optimization.1

Forenisc toxicology laboratories require accurate,

reliable results, that are obtained timely and robustly
24/7. Reduced/limited sample preparation, minimizing
preparation errors, and increasing sample throughput is
also preferred. Lack of analytical robustness can result
in biased results and delayed turnaround times with
increased analysis costs.

Experimental
Sample preparation
Blood alcohol mix resolution control standards,
0.1 g/dL (P/N 36256) containing eight target components
in water (acetaldehyde, acetone, acetonitrile, ethanol,
ethyl acetate, 2-propanol (isopropanol), methanol
and methyl ethyl ketone), were acquired from Restek
(Bellefonte, PA, USA).

Whole blood certified control samples containing 0.02,

0.05, 0.08, and 0.3 g/dL ethanol (P/Ns WH02-030, Figure 1. TriPlus 500 HS autosampler (240 vial configuration)
WH05-030, WH08-030, and WH30-030) and whole connected to the Trace 1310 GC, offering the highest sample
throughput in the most compact design
blood blank check samples (P/N 11WH025) were
acquired from ACQ Science (Rottenburg, Germany).
Chromatographic separation of the target analytes was
For targeted blood alcohol quantitative analysis, achieved using two capillary GC columns, a Thermo
methanol, ethanol, acetone, isopropanol, acetonitrile, Scientific™ TraceGOLD™ TG-ALC1, 30 m × 0.32 mm i.d.
ethyl acetate, and 1-propanol (internal standard) × 1.8 µm, film capillary column (P/N 26074-3390)
individual stock standards at 10 g/dL in water (LC/MS and Thermo Scientific™ TraceGOLD™ TG-ALC2,
grade) were prepared. Diluting from the individual stock 30 m × 0.32 mm i.d. × 1.2 µm, film capillary column
standards, mixed calibration working standards over (P/N 26073-2260).
5 levels (ranging from 0.01 to 0.2 g/dL) were prepared
in water. Diluting from the 1-propanol (internal standard) A dual-column, dual-FID configuration was implemented
stock standard, a 0.2 g/dL working internal standard was (Figure 2) through a Thermo Scientific™ Microfluidic
prepared in water. 3-port column connector (P/N 60201-398), connected
to the flow from the HS via a guard column, Thermo
Standards/blood samples or water blanks (500 µL) were Scientific™ GuardGOLD™, 5 m × 0.32 mm i.d. (P/N
transferred to a 10 mL crimp headspace vial (P/N 10-CV) 26050-0532). This configuration, using columns with
containing 60 µL 1-propanol (0.2 g/dL, internal standard). different chemistries and selectivities, results in different

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target compound elution order and retention times, Table 1. GC-FID conditions
which enables compound identification and confirmation
for BAC analysis. TRACE 1310 GC system parameters

Injection Mode: Split

Split Ratio: 20:1
FID 1 FID 2 Carrier Gas, Carrier Mode,
He, constant flow, 15
Flow (mL/min):
TraceGOLD TG-ALC1,
Column 1: 30 m × 0.32 mm i.d. × 1.8 µm,
film capillary column (P/N 26074-3390)
TraceGOLD TG-ALC2,
Column 2: 30 m × 0.32 mm i.d. × 1.2 µm,
TriPlus 500 HS film capillary column (P/N 26073-2260)
Guard Column (to connect GuardGOLD, 5 m x 0.32 mm i.d.
HS to the microfluidic device) (P/N 26050-0532)
Oven Temperature Program

Temperature 1 (°C): 50
Hold Time (min): 5
Column 1 Column 2
TraceGold TraceGold FID
TG-ALC 1 TG-ALC 2 Temperature (°C): 300
Microfluidic 3-port Air Flow (mL/min): 350
connector H2 Flow (mL/min): 35
Figure 2. Dual-FID configuration implemented through a microfluidic N2 Flow (mL/min): 40
3-port connector, connected to the TriPlus 500 HS autosampler via
Acquisition Rate (Hz): 25
a guard column

Additional HS-GC-FID parameters are detailed in Table 2. Headspace conditions

Tables 1 and 2. Conditions were optimized to reduce
TriPlus 500 HS autosampler parameters
analysis time, improve sample throughput, and
maintain robust analytical performance, with a total Incubation Temperature (°C) 70
GC runtime of 5 min (last peak eluting at 2.6 min). Incubation Time (min) 15
The TriPlus 500 HS autosampler sample overlapping Vial Shaking Fast
capability and the automatic cycle time optimization Vial Pressurization Mode Pressure
are essential for long, unattended sample sequences, Vial Pressure (kPa) (Auxiliary Gas Nitrogen) 100
supported by the vial capacity expandable up to Vial Pressure Equilibration Time (min) 0.20
240 vials. Loop Size (mL) 1
Loop/Sample Path Temperature (°C) 70
Data processing Loop Filling Pressure (kPa) 50
Data were acquired, processed, and reported using Loop Equilibration Time (min) 0.10
Thermo Scientific™ Chromeleon™ Chromatography Data Needle Purge Flow Level 4
System (CDS), version 7.2. Chromeleon CDS allows the Injection Mode Standard
analyst to setup acquisition, processing, and reporting Injection Time (min) 0.5
methods with easy data reviewing and flexible data
reporting.

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 Results and discussion
BAC analysis requires analytical methods that are fast,
reliable, and cost effective. Analytical performance
was tested for the detailed HS-GC-FID configuration,
including chromatographic separation of target analytes,
compound linearity, peak area repeatability, recovery,
carryover, and quantitation of BAC in blood samples.

Chromatography
Chromatographic resolution and chromatographic peak
shape are vital in determining peak area, and in turn,
the precise concentration of the target analytes.1 The
chromatographic separation of the components in the
blood alcohol mix resolution control standard at 0.1 g/dL
in water, applying the HS-GC-FID conditions described in
Tables 1 and 2, on both analytical columns, is illustrated
in Figure 3.

Chromatographic separations on both analytical columns

for a mixed alcohol working calibration standard at
0.1 g/dL in water, containing methanol, ethanol, acetone,
isopropanol, acetonitrile, ethyl acetate, 1-propanol (as
internal standard) are shown in Figures 4 and 5.

Chromatographic separations on both analytical columns

for a whole blood control standard at 0.08 g/dL ethanol
(1-propanol as internal standard) are shown in Figures 6
and 7.

As the two dedicated GC capillary columns have

different stationary phases with different polarity, different
retention times and elution order are achieved (e.g.,
acetone and acetonitrile co-elutes on the TraceGOLD
TG-ALC1 column but they are well separated on the
TraceGOLD TG-ALC2 column), enabling confident Figure 3. Chromatographic separation of target components in a
blood alcohol mix resolution control standard at 0.1 g/dL in water
compound identification and confirmation. Excellent peak (1-propanol as internal standard) on the TraceGOLD TG-ALC1 (A)
shape is achieved, with peak asymmetry values between and TraceGOLD TG-ALC2 (B) capillary columns
0.94 and 1.04 for all target compounds in a 0.1 g/dL
mixed alcohol calibration working standard, as illustrated
for ethanol in Figures 4b and 5b. Excellent peak shapes
are also observed for ethanol in blood samples with
peak asymmetry values of 1.01 and 1.03, as illustrated in
Figures 6B and 7B for the 0.08 g/dL whole blood control
sample.

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Figure 4. Chromatographic separation of target compounds in a mixed alcohol calibration working standard at 0.1 g/dL in water, plus
internal standard (1-propanol) on the TraceGOLD TG-ALC1 capillary column (A), peak asymmetry for ethanol, with a tailing factor (Tf) of
1.03 indicating an almost perfect Gaussian peak (B)

Figure 5. Chromatographic separation of target compounds in a mixed alcohol calibration working standard at 0.1 g/dL in water, plus
internal standard (1-propanol) on the TraceGOLD TG-ALC2 capillary column (A), peak asymmetry for ethanol, with a tailing factor (Tf) of
1.04 indicating an almost perfect Gaussian peak (B)

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 Figure 6. Chromatographic separation of ethanol and 1-propanol (internal standard) in a whole blood control sample,
0.08 g/dL ethanol, on the TraceGOLD TG-ALC1 capillary column (A), peak asymmetry for ethanol, with a tailing factor
(Tf ) of 1.01 indicating an almost perfect Gaussian peak (B)

Figure 7. Chromatographic separation of ethanol and 1-propanol (internal standard) in a whole blood control sample,
0.08 g/dL ethanol, on the TraceGOLD TG-ALC2 capillary column (A), peak asymmetry for ethanol, with a tailing factor
(Tf ) of 1.03 indicating an almost perfect Gaussian peak (B)

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Linearity of response Additionally, linearity for ethanol was tested using
Obtaining linear response is critical for accurate and whole blood control samples over five levels ranging
precise compound quantification. To assess compound from 0.02 to 0.3 g/dL, with demonstrated coefficient
linearity, a five-level calibration curve (with concentrations of determination R2 of 0.9993, and average calibration
ranging from 0.01 to 0.2 g/dL) was used with internal factor %RSD of 3.1 (Figure 9).
standard (1-propanol) correction of the responses.
Peak area repeatability
Excellent linearity was obtained for all target compounds Confident quantification of BAC in routine testing relies
with coefficient of determinations R2 >0.998, and average on obtaining stable analyte response in solvent standards
calibration factors %RSD ≤6. A detailed report of linearity and ultimately in blood samples. Repeatability of absolute
assessment for all target compounds is given in Tables peak area responses was tested in solvent standards
3A and 3B. The calibration curve for ethanol is shown as well as in blood samples. Repeatability in mixed
in Figure 8, using the TraceGOLD TG-ALC2 capillary alcohol standards was assessed by carrying out n=15
column. The peak area repeatability (as %RSD) of the consecutive analysis of mixed alcohol standards at 0.04
internal standard (1-propanol) for n=5 injections was <2.7 and 0.1 g/dL in water. Additionally, ethanol peak area
on both columns, indicating outstanding precision of the repeatability was evaluated from n=7 injections of whole
method. blood certified control samples at 0.3 g/dL.

Table 3A. Calibration linearity assessment, showing retention Table 3B. Calibration linearity assessment, showing retention
time, average calibration factor (ACF) %RSD, and coefficient of time, average calibration factor (ACF) %RSD, and coefficient of
determination (R2) for the target compounds using the TraceGOLD determination (R2) for the target compounds using the TraceGOLD
TG-ALC1 capillary column TG-ALC2 capillary column

TraceGOLD TG-ALC1 TraceGOLD TG-ALC2

Compound Compound
RT (min) ACF %RSD R2 RT (min) ACF %RSD R2
Methanol 0.68 3.3 0.9995 Methanol 0.93 3.2 0.9983
Ethanol 0.87 3.4 0.9996 Ethanol 1.26 2.5 0.9997
Acetone + Acetone 1.36 5.2 0.9987
1.34 5.8 0.9986
Acetonitrile Isopropanol 1.50 3.9 0.9991
Isopropanol 1.07 2.3 0.9998 Acetonitrile 1.97 6.0 0.9984
Ethyl acetate 2.60 3.6 0.9995 Ethyl acetate 2.18 3.7 0.9992

A B

0.08 g/dL
1-propanol USA /
%RSD = 2.7 0.05 g/dL England
n=5 Italy /
0.02 g/dL Scotland ethanol
Sweden / ACF %RSD = 2.5
Norway R2 = 0.9997

Figure 8. Average peak area for 1-propanol (internal standard), annotated with the peak area repeatability (as %RSD) (A), the linearity
response for ethanol over a concentration range of 0.01 to 0.2 g/dL on the TraceGOLD TG-ALC2 capillary column, annotated with the
coefficient of determination (R2) and the average calibration factor (ACF) (as %RSD). The blue arrows indicate blood alcohol legal limits for
vehicle operation in different countries (B).

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 direct connection between the sampling loop and the
chromatographic column.

0.02 to 0.3 g/dLethanol
Quantification of BAC in real blood samples
To test the method performance, whole blood control
samples over five levels ranging from 0.02 to 0.3 g/dL
were analyzed and quantified against the mixed alcohol
working standards, with internal standard correction of
ACF %RSD = 3.1
the responses.
R2 = 0.9993
Accurate recovery values are critical in BAC analysis to
clearly define the level of intoxication, provide a rough
measure of impairment, and accurately determine
compliance against legal levels. Excellent recovery of
Figure 9. Linearity response of ethanol in whole blood control
samples over five levels (ranging from 0.02 to 0.3 g/dL), on the ethanol from whole blood certified control samples
TraceGOLD TG-ALC2 capillary column, annotated with the (between 93% and 107%) are shown in Table 5 and
coefficient of determination (R2) and the average calibration factor
Figure 10.
(ACF) (as %RSD).

The results of these experiments demonstrate excellent Carryover assessment

precision, with peak area %RSD between 0.7 and 3.2 Compound carryover (contamination from either
(Table 4), assisted by the advanced features of the previously injected standards or blood samples) is one
TriPlus 500 HS autosampler such as the optimized of the known issues when performing HS-GC analysis of
pneumatic control, the sample path inertness, and the BAC and can cause erroneous, false positive results.

Table 4. Assessment of precision (as repeatability of peak area responses) in mixed alcohol standards (n=15) at 0.1 and 0.04 g/dL in water,
and whole blood control samples (n=7) at 0.3 g/dL ethanol

TraceGOLD TG-ALC2 TraceGOLD TG-ALC1

Compound 0.1 g/dL (water) 0.04 g/dL (water) 0.3 g/dL (blood) 0.3 g/dL (blood)
n=15 n=15 n=7 n=7
%RSD %RSD %RSD %RSD
Methanol 0.9 1.5 - -
Ethanol 0.8 1.5 1.5 1.5
Acetone 0.7 2.2 - -
Isopropanol 0.8 1.0 - -
Acetonitrile 0.8 1.7 - -
Ethyl acetate 1.1 3.2 - -

Table 5. Calculated ethanol concentration in whole blood certified control samples, versus the specified ethanol concentrations, and the
associated recovery values

Specified TraceGOLD TG-ALC2 TraceGOLD TG-ALC1

concentration Concentration Concentration
(g/dL) Recovery (%) Recovery (%)
(g/dL) (g/dL)
0.30 0.303 101 0.321 107
0.15 0.154 102 0.154 103
0.08 0.076 94 0.076 95
0.05 0.047 94 0.047 93
0.02 0.020 101 0.020 100

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To assess potential carryover from solvent standards, To assess potential carryover from blood samples,
injections of a mixed alcohol standard (n=15) at 0.1 g/dL in injections of whole blood control sample (n=7) at 0.3 g/dL
water, using the TraceGOLD TG-ALC2 capillary column, ethanol, using the TraceGOLD TG-ALC1 and TraceGOLD
were carried out followed by a blank (water) injection. TG-ALC2 capillary columns were carried out followed by
No detectable carryover was achieved as shown in blank (n=3) water injections. No detectable carryover for
Figure 11A for ethanol. ethanol was achieved as shown in Figure 11B and 11C,
respectively.

Figure 10. For whole blood certified control samples: ethanol recovery using the TraceGOLD TG-ALC1 (brown) and the
TraceGOLD TG-ALC2 (green) capillary columns (A); overlaid chromatograms using the TraceGOLD TG-ALC1 (B-1), and the
TraceGOLD TG-ALC2 (B-2) capillary columns for whole blood certified control samples

Figure 11. Ethanol carryover (%) was assessed from solvent standards with injections of a mixed alcohol standard (n=15, 0.1 g/dL in water)
using the TraceGOLD TG-ALC2 capillary column (A), followed by a blank (water) injection; from blood samples with injections of a whole
blood control sample (n=7, 0.3 g/dL ethanol) using the TraceGold TG-ALC2 (B) and TG-ALC1 (C) capillary columns, followed by blank (n=3)
water injections.
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Conclusions • Ethanol recoveries of 94% and 107% were achieved for
• The data demonstrated that the TriPlus 500 HS the analysis of whole blood control samples.
autosampler can provide high level performance for
• Aided by the efficient pneumatic purging of the TriPlus
reliable quantitation of blood alcohol content and fulfills
500 HS autosampler and the short inert sample path,
the needs of forensics laboratories for fast, accurate,
no detectable carryover was achieved for the target
and high-throughput routine analysis.
compounds in the blank samples analyzed after the
• Using a dual column / FID configuration compound injection of a mixed alcohol standard (at 0.1 g/dL
separation in <5 min was achieved, with excellent peak concentration in water) and a whole blood control
shapes (peak asymmetry values between 0.94 and sample (at 0.3 g/dL ethanol).
1.04). Such a short run time allows for high-throughput
• Chromeleon CDS software offers an ideal solution for
analysis, aided by automatically optimized overlapped
the targeted analysis of BAC with user-friendly data
headspace incubation cycles and unattended analysis
processing for high-throughput analysis, with easy data
of up to 240 samples.
reviewing and flexible data reporting.
• Compound linearity obtained for methanol, ethanol,
References
acetone, isopropanol, acetonitrile, and ethyl acetate in 1. Boswell, H.A and Frank, F.L. Uncertainty of Blood Alcohol Concentration (BAC) results
aqueous standards over a calibration range of 0.01 to as Related to Instrumental Conditions: Optimization and Robustness of BAC analysis
headspace parameters. Chromatography, 2015, 2, 691–708.
0.2 g/dL resulted in coefficients of determination
2. Calculate MDL using Environmental Protection Agency (EPA) Method for Detection of
R2 >0.998 and average calibration factor %RSD of ≤6, MDL, 40 CFR part 136. Appendix B, Revision 1.11.
indicating an excellent linear response for all the target
analytes.

• Excellent precision was achieved for the analysis of

0.04 g/dL standards (n=15) with peak area %RSD for
all compounds between 0.7 and 3.2, assisted by the
advanced pneumatic control and high sample path
inertness of the TriPlus 500 HS autosampler.

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