Quantitation and Confirmation of Blood Ethanol Content using a

Quantitation and Confirmation of
Blood Ethanol Content using a New
GC/FID/MS Blood Alcohol Analyzer
Application Note
Forensics and Toxicology
Authors
Abstract
Fred Feyerherm
This application note highlights the development of a method for determining blood
Agilent Technologies, Inc.
alcohol concentration using an Agilent 7890B GC with FID coupled to an Agilent
Houston, TX
5977A MSD. The combination of detection by FID and MSD provides precise quanti-
Jessica Westland and Craig Marvin
tation of alcohol concentration along with spectral confirmation of alcohol
Agilent Technologies, Inc.
presence within a complex blood sample matrix.
Wilmington, DE
Introduction
Experimental
Blood alcohol concentration (BAC) corresponds directly to the
level of impairment an intoxicated driver has when operating a
vehicle. To address driving while intoxicated cases, law
enforcement agencies have established threshold values for
BAC. Breathalyzer and field sobriety tests provide subjective
indication of impairment. Defensibility in court requires quantitation of ethanol content making BAC the most widely run test
in toxicology laboratories.
This experiment was performed on an Agilent 7890B GC
equipped with a split/splitless inlet and coupled to an FID, an
Agilent 5977A GC/MSD, and an Agilent 7697A Headspace
Sampler. Figure 1 depicts the experimental setup. A capillary
flow technology (CFT) two-way splitter was used to split the
flow from the GC column to the FID and MS detectors. This
arrangement provided accurate and reproducible flow to both
detectors (Figure 2).
Due to the number of samples received and their relative short
hold times, toxicology laboratories require rapid, accurate, and
reliable tests for BAC. Headspace gas chromatography is
widely used by law enforcement laboratories [1]. While this
technique meets many of these lab’s requirements, the possibility of false positives through sample carry-over or co-elution
of a contaminant has elevated the demand for mass spectral
confirmation of ethanol presence above the routine analysis by
gas chromatography coupled with dual flame ionization detection (GC-FID). The use of headspace GC coupled with a flame
ionization detector (FID) and a mass spectrometer (MS) provides simultaneous quantitation and spectral confirmation of
ethanol presence in blood.
The software used was Agilent MassHunter GC/MS
Aquisition B.07.00.SP2 and MSD ChemStation Enhanced Data
Analysis F.01.00.1903.
HSS
transfer line
S/S
Inlet
AUX
EPC
DB-A
Column
This application tests the combination of an Agilent 7697A
Headspace Sampler, 7890B GC with FID and a 5977A GC/MS
for the separation, quantitation, and confirmation of alcohol
compounds in blood. The purpose of this study was to illustrate that the addition of mass spectrometry validates alcohol
identification and adds legal defensibility to the data. Agilent
instrumentation was used for accurate and precise results, in
rapid, high capacity analyses.
Agilent
5977B MSD
Figure 1.
FID
Purged
splitter
Agilent 7697A
Headspace
Sampler
Agilent 7890B Column
GC/FID/MS configuration for blood alcohol.
Effluent splitter (2-way)
DB-ALC1
column in
Aux EPC in
MSD out
FID out
Competitive advantage
Figure 2.
2
Reproducible flow to both detectors using a CFT two-way purged
splitter.
Results and Discussion
Instrument conditions
DB-ALC-1
Carrier
Helium
Oven
55 °C Isothermal
Inlet
Capillary Split/Splitless with EPC
Inlet liner
Ultra inert (p/n 5190-4047)
GC
Agilent 7890B GC
Detector
FID with EPC
MSD
Agilent 5977A Mass Spectrometer
Sampler
Agilent 7697A Headspace Sampler
Transfer line
Deactivated fused silica, 0.53 mm id
CFT device
2-way purged splitter
GC septum
Bleed and temperature optimized, BTO 11 mm septa
(p/n 5183-4757)
Gold seals
Ultra Inert Gold Seals (p/n 5190-6145)
CFT ferrules
Flexible metal ferrules (p/n G3188-27502 for 0.32-id column,
p/n G3188-26503 for 0.53 mm id tubing), internal nut
(p/n GB2855-20530)
Inlet/FID
85:15 Vespel graphite ferrules (p/n 5062-3514, 10 pk)
Figure 3 shows the chromatograms from the DB-ALC1 column
for separation and elution order of analytes for the multicomponent mix using combined FID and MS signals. Figure 4
depicts the retention time alignment for the multicomponent
mix. Under the analytical conditions for baseline separation of
ethanol, other possible sample constituents such as
methanol, 2-propanol, and acetone, was achieved in less than
three (3) minutes.
×105
18
16
14
12
10
8
6
4
2
Acetone
TIC
2-Propanol
Abundance
Column
Ethanol
Methanol
Time 0.2
×105
20
Abundance
MSD flow rate 1.25 mL/min
Sample preparation
Ethanol reference standards were prepared by the addition of
500 µL of each reference standard solution to 4.5 mL distilled
water and 5 µL diluted internal standard.
N-Propanol
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
FID
16
12
8
4
0
Time 0.2
Figure 3.
The stock internal standard solution (ISTD) was prepared by
performing a 1:10 dilution of n-propanol in distilled water to a
final working concentration of nominally 0.08 g/dL.
Performance of this method was evaluated through the
analysis of real world samples.
×105
Chromatographic separation of multicomponent mix at 0.4% g/dL.
TIC
Abundance
20
Ethanol calibrators were prepared in a separate mix.
15
Ethanol
1.199
Methanol
10 1.010
Acetone
1.624
2-Propanol
1.397
N-Propanol
1.761
5
Abundance
Time 1.0
×105
FID
12
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.624
1.397
1.200
10
1.761
8 1.010
6
4
2
Time 1.0
Figure 4.
3
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
Retention time alignment for FID and MS Chromatograms for
multicomponent mix.
To verify recovery, n-propanol was added to the calibration
standards and samples. System calibration was performed for
ethanol from 0.02 to 0.4 g/dL, at seven levels with 10 replicates per level. Figure 5 shows the calibration curves generated for ethanol on both detectors. As illustrated, the calibration curves were linear for both FID and MS, with R2 of 0.9991
and 0.9989 respectively.
Sample carryover can be of concern with running high concentration BAC samples. This study not only focused on testing carryover from sample to sample, but carryover from possible sample residue remaining in the headspace sampler. The
verification was performed by analyzing a multicomponent
mix with a high concentration of ethanol (0.4 g/dL), followed
by the analysis of an ISTD blank. Figure 6 verifies the absence
of ethanol in the ISTD blank, demonstrating that there is no
sample carryover with this method.
FID
×10 5
45
Multicomponent mix at 0.4% w/v
40
×10 5
18 TIC
16
14
12
10
8
6
4
2
30
2
R = 0.999
25
20
15
10
5
0
0.05
0.20
0.25
0.30
0.10
0.15
Ethanol concentration (% w/v)
Time 0.2
×10 5
FID
20
0.35
TIC
×10 5
35
Abundance
0
Response
30
25
Ethanol
Methanol
0.4
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.2
2.4
2.6
16
12
8
R = 0.9989
0
15
0.20
0.25
0.30
0.10
0.15
Ethanol concentration (% w/v)
0.35
Calibration curves for ethanol detection with FID and MS.
Time 0.4
×10 4
8 FID
7
6
5
4
3
2
1
0
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
n-Propanol
Abundance
Figure 5.
0.05
Abundance
5
0
ISTD blank after 0.4% multicomponent mix
×10 5
14 TIC
12
10
8
6
4
2
10
0
0.6
n-Propanol
4
2
20
Acetone
2-Propanol
Abundance
Response
35
Figure 6.
4
The top chromatograms show the analysis of ethanol in the high
concentration standard (4 g/dL). The bottom chromatograms
verify the absence of ethanol carry over from the 4 g/dL standard
in the ISTD blank
Agilent ChemStation software offers custom report options
for data presentation and comparison. The sample report
shown in Figure 7 includes quantitation results from a sample
analysis, the FID response is used for quantitation, and a
comparison of collected MS spectra to NIST spectral data is
displayed. This report provides an easy review of concentration data, and visual confirmation of the presence of ethanol
in the sample analyzed. The system is designed so that retention times match for each component on the FID and MSD
channels.
Conclusion
This study confirms the rapid, robust, and accurate BAC
analysis using the 7890B GC configured with an FID and
5977A MSD. The direct coupling of the 7697A Headspace
Sampler transfer line to the split/splitless inlet and EPC controlled vial sampling at pressures above ambient provided
reproducible performance across a wide calibration range,
and eliminated carryover. EPC controlled CFT provided reproducible split of column flow between the FID and MSD to
allow for simultaneous detection and spectral confirmation of
ethanol presence in a single injection. The system showed no
carryover from sample to sample, even after challenging the
system with a high ethanol concentration injection.
Additionally, this method provides defensible data with quantitation using FID detection and spectral confirmation by MS
confirmation for ethanol and other targets. The flexible
custom reporting options also provide concentration information, example chromatography, and a comparison of collected
and reference library spectra. Overall, use of the Agilent
7890B GC/FID, coupled to an Agilent 5977A MSD and 7697A
Headspace Autosampler, is an excellent tool demonstrating
precise, accurate, reproducible, and defensible data in the
detection and quantitation of BAC for law enforcement.
FID for
quantitation
Collected
spectra
NIST spectral
confirmation
Figure 7.
Agilent ChemStation custom report.
5
References
1.
J.L. Westland, F.L. Dorman, Forensic Sci. Int., 231(2013),
pp. 50-56.
2.
H. Boswell, F. Dorman "Determine Blood Alcohol with
Dual Column/Dual FID for Precision and Reproducibility"
Agilent Technologies publication 5991-3671EN.
For More Information
These data represent typical results. For more information on
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www.agilent.com/chem.
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Information, descriptions, and specifications in this publication are subject to change
without notice.
© Agilent Technologies, Inc., 2014
Printed in the USA
May 19, 2014
5991-4059EN

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