Journal Pre-proof

Quantitation and Identification of Ethanol and Inhalant Compounds in

Whole Blood Using Static Headspace Gas Chromatography Vacuum
Ultraviolet Spectroscopy

James A. Diekmann III , Jack Cochran , James Alex Hodgson ,

Dr. Jonathan Smuts

PII: S0021-9673(19)31015-5
DOI: https://doi.org/10.1016/j.chroma.2019.460607
Reference: CHROMA 460607

To appear in: Journal of Chromatography A

Received date: 8 July 2019

Revised date: 30 September 2019
Accepted date: 7 October 2019

Please cite this article as: James A. Diekmann III , Jack Cochran , James Alex Hodgson ,
Dr. Jonathan Smuts , Quantitation and Identification of Ethanol and Inhalant Compounds in Whole
Blood Using Static Headspace Gas Chromatography Vacuum Ultraviolet Spectroscopy, Journal of
Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460607

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Highlights

 Gas chromatography – vacuum ultraviolet spectroscopy for blood alcohol/inhalants

 Vacuum ultraviolet spectroscopy provides authoritative identification of compounds
 Spectral deconvolution allows for accurate quantitation of coeluting peaks

1
 Quantitation and Identification of Ethanol and Inhalant Compounds
in Whole Blood Using Static Headspace Gas Chromatography Vacuum
Ultraviolet Spectroscopy

James A. Diekmann III, Jack Cochran, James Alex Hodgson, Dr. Jonathan
Smuts

1500 Arrow Point Dr

Bldg 8, Suite 805
Cedar Park, Texas 78613, US

Abstract: Gas chromatography (GC) and vacuum ultraviolet spectroscopy

(VUV) are powerful and complementary techniques for the analysis of
small molecules in forensics. Most notably, flame ionization detection
(FID) is commonly used with GC to identify and quantify volatile
compounds. An FID's price point and ease of use makes it an attractive
approach for routine laboratories that are in high demand for forensics
analysis, but with the contingency that an FID relies on retention time
for identification and quantification. A new and innovative method
using static headspace gas chromatography coupled with vacuum
ultraviolet (VUV) spectroscopy was developed for the quantitative
determination of ethanol in blood and identification of inhalants. This
study investigates the possibility of using VUV as an alternative
technique to traditional methods that use FID and mass spectrometry
(MS) in toxicology and forensic analysis. VUV brings both
identification and quantitation based on Beer-Lambert's Law while using
a simple single-column solution. This paper investigates 25 compounds,
including ethanol, methanol, acetone, benzene, and toluene using a 130-
240 nm wavelength range for identification and quantification using GC-
VUV, even when coelutions occur. Ethanol was examined under a
concentration range of 9 to 495 mg/dl, and the method was found to be
linear with an r2 = 0.997 and a LOD of 3.1 mg/dl. Ethanol was fully
separated from other volatile organic compounds (VOCs) as well as
endogenous materials present in blood. Nonaromatic VOCs were analyzed
at concentration ranges of 2.4 to 99 mg/dl with LODs around 0.2 mg/dl;
aromatic VOCs were analyzed from 0.5 to 24 mg/dl with LODs ~ 0.1 mg/dl.

Keywords: Gas Chromatography, Vacuum ultraviolet detection, Blood

alcohol analysis, Forensic science, Toxicology application, Ethanol

2
 1. Introduction

Alcoholic beverages are consumed worldwide and are arguably one of the most prolific legal substances. Unfortunately, the side effects
of alcohol consumption have led to fatalities across the globe. According to the Center for Disease Control, in 2016 nearly 10,500 people
died in alcohol impaired car crashes, accounting for 28% of all traffic-related deaths in the United States [1]. To minimize the risk of death
associated with vehicular manslaughter, many municipal governments regulate its consumption. For example, the US actively enforces
existing 0.08% blood alcohol concentration (BAC) laws, and minimum legal drinking age and zero tolerance laws for drivers younger than
21 years old [2,3].
In forensic and toxicology laboratories, static headspace GC is commonly used as an analytical tool for identifying and quantifying
alcohol in blood but can also be used to screen inhalants of abuse, which can impair driving[4]. Specifically, two FIDs are used with
parallel dual columns, providing phase selectivity for resolving coelutions [5]. Although FID can’t identify compounds, the robust retention
times, high sensitivity, ease of use, and wide linear range of detection make FID a reliable detector for BAC determination and inhalant
screening. Since FIDs are non-selective detectors, compound identification is reliant on retention times and require full compound speciation
for accurate quantification. In cases where coelutions are unresolved from the parallel dual column technique, MS is required [6–9]. But
even MS has its shortcomings for identifying isobaric compounds.
Until recently, use of far-UV spectroscopy (<200 nm) as a detection tool was limited because vacuum was required to remove strongly
absorbing compounds in ambient air, notably oxygen and water. The advantages of this spectral region (130-240 nm), dubbed the VUV,
have long been recognized [10–15] as a potentially powerful tool in gas chromatography. A new VUV absorption spectrometer (VUV
Analytics, Inc., Austin, TX) was developed for GC [16] that circumvents this requirement and monitors molecular absorption at ambient
pressure from 130-240 nm with 0.05 nm resolution. Nearly all compounds absorb in this electromagnetic region and yield unique spectral
fingerprints, especially for small molecules.
The power of GC-VUV has already been demonstrated for a variety of applications, including multiclass pesticides [17], fatty acid
methyl esters in food oils [18], polychlorinated biphenyls [19], breath analysis [20], and pharmaceuticals [21], among many other areas of
interest [22,23]. The novelty of GC-VUV is its ability to spectroscopically deconvolve isobaric compounds, and its non-destructive nature
provides first principle detection of analytes, yielding authoritative spectral confirmation[24].
This study explores a single column static headspace GC-VUV method as an alternative to traditional approaches that require dual FID
parallel columns analysis and MS. VUV absorbance spectroscopy simplifies the GC configuration and can be used to determine the BAC
levels of ethanol as well as screen for inhalants of abuse. GC-VUV provides both qualitative and quantitative information for a variety of
small molecules found in routine blood analysis, including esters, aldehydes, ketones, alcohols, and aromatics.

2. Experimental

2.1 Materials

All standards and solvents were obtained from Sigma-Aldrich (St. Louis, MO), Fisher Scientific (Pittsburgh, PA), and Chem Service
(West Chester, PA). Organic-free water was used as the diluent matrix for each sample. Ethanol-free whole bovine blood (Fisher Scientific,
Pittsburgh, PA) was used as a substitute for human blood due to legal issues associated with handling human biologicals in a non-medically
certified laboratory; it also closely simulates human blood, providing a suitable environment for this study [25]. Anhydrous sodium chloride
(Sigma-Aldrich, St. Louis, MO) was added to samples to increase the sensitivity of static headspace by lowering the partitioning coefficients,
creating a more pronounced response for polar compounds that have greater affinity towards water [26].

2.2 Instrumentation

All samples were run on an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA) with a Gerstel MPS2
autosampler (Gerstel, Inc., Linthicum, MD). The column used was a Rxi-624 Sil MS (30m x 0.25mmid x 1.4µm) (Restek Corporation,
Bellefonte, PA). Data was collected on a VGA-100 VUV absorbance detector (VUV Analytics, Inc., Cedar Park, TX). Table 1 details the
method run conditions used for all analyses.

3
2.3 Constructing a 25-Component Standard

A mixture of 25 compounds prepared in dimethyl sulfoxide (DMSO) was created to simulate VOCs present in blood alcohol analysis
and inhalant screening. Using this mixture, an eight-point calibration curve of ethanol was constructed from a concentration range of 9-450
mg/dl. Likewise, non-aromatic VOCs and aromatic VOCs had concentration ranges of 2.4-99 mg/dl and 0.5-24 mg/dl, respectively. The
balance between these concentration ranges was chosen such that all compounds went into solution.

2.4 Sample Preparation

Each 20ml headspace vial was prepared with 0.69±0.01g of NaCl and 2ml of organic-free water.. To simulate blood alcohol
concentration, each vial of 200µl ethanol-free blood was spiked with varying levels of a 25-component solution containing ethanol, spanning
a range from 9-450 mg/dl. The concentration range for ethanol was chosen such that it complied with Texas state law of intoxication of
0.08% (80 mg/dl). The remaining analyte levels were comparable to levels that could be seen in blood analysis.

3. Results & Discussion

3.1 Blanks

Blanks were constructed in a 20ml headspace vial with 2ml of organic-free water, 200µl of ethanol-free blood, and 0.69±0.01g of
NaCl.. Blanks were run after each duplicate calibration level. There was no carryover effect from ethanol when blank samples were
analyzed after each replicate set and after the maximum concentration of 495 mg/dl. Minimal amounts of carryover were observed for high
boiling point compounds. In particular, aromatic inhalants such as toluene, ethylbenzene, and xylenes were present. This carryover could be
attributed to the syringe temperature (90ºC) being significantly below the boiling points of these compounds. Overall, the si gnal-to-noise
ratios (S/N) of these inhalants were much less than 3, indicating an insignificant contribution to the inhalant areas measured. This carryover
did not affect ethanol or other compound present in this study.

3.2 Analysis of Ethanol

An eight-point calibration curve covering the blood concentration range of 9-495 mg/dl was constructed for ethanol using spectral data
collected from 130-240nm (Figure 1). To ensure the blood matrix has no significant effect on the quantitation of ethanol, a calibration curve
of DMSO was substituted as the blood matrix and evaluated against the blood calibration curve . For both calibration curves,
each level was run in duplicate. Ethanol in the blood and DMSO matrices was found to be linear with an r2 of 0.997 and 0.998, respectively.
The slopes for the blood and DMSO matrices are 5.56 and 5.37, respectively, with a 3.5% difference in slopes, suggesting that the blood
matrix has minimal effect on ethanol’s recovery.
Figure 2 shows a chromatogram where acetaldehyde, methanol, and ethanol elute. . In Figure 3 acetaldehyde, methanol, ethanol, and
5 other compound spectra are shown; each absorbance spectrum, like all non-enantiomeric absorbance spectra, is unique. Under these
chromatographic conditions, there were no interferences with ethanol from endogenous materials present in blood, the 24 VOCs in the spike
solution, or the internal standards.
Table 2 shows the repeatability (n=7), intra-day coefficient of variation (n=7), and inter-day coefficient of variation (n=21) evaluated
on three consecutive days at concentrations of 19, 78, and 300 mg/dl. The method yielded reproducible, sensitive, and robust results of
(mean ± S.D.) 20 ± 1, 80 ± 5, and 300 ± 20 mg/dl, respectively. Method sensitivity was determined by the limit of quantification (7 mg/dl,
S/N = 10) and the limit of detection (2 mg/dl, S/N = 3). This method could be useful in forensic cases where intoxications levels around
legal limits must be determined.

3.3 Screening for 24 BAC and Inhalant Related Compounds

Twenty-four compounds were used to demonstrate VUV capability of screening a wide variety of inhalant and BAC compounds.
Traditional methods that examine inhalant analysis take upward to 15 minutes [27], but this method could easily be optimized to only
examine a subset of BAC compounds, which current routine methods examine.

4
 Ethanol-free blood was spiked with VOCs from Table 3. Calibration curves were generated for all 24 compounds over various
concentration ranges, as displayed in Table 3, as well as their limits of detection (LOD). The r2 for most BAC and non-aromatic inhalants is
above 0.99. Higher variance and lower r2 values than those of BAC compounds were observed for some of the inhalants. This is probably
due to their lower volatility and thus larger partition coefficients, which is known to cause higher variability in headspace sampling.
Figure 2 shows the full chromatogram of the 24 VOCs eluting between 1.25 to 8.75 minutes. No other compounds eluted beyond 8.75
minutes. Three peaks contained coelutions: n-propanol and halothane at 3.80 minutes, ethyl acetate and 2-butanone at 4.20 minutes, and m-
xylene and p-xylene at 7.74 minutes. These coelutions were spectral deconvoluted for identification and quantification (Figure 4).
Carryover was observed but did not affect any VOCs measured in this study. While quantitation can be performed on these inhalant
and BAC compounds, an in-depth validation study was not performed because most forensic laboratories are only required to identify these
compounds in a blood stream.

3.4 Acetaldehyde Formation

In previous studies, it has been reported that acetaldehyde formation can occur when enzymes in blood interact with ethanol [28]. In a
separate study, the linearity of acetaldehyde was measured in a DMSO matrix and diluted in 2ml of organic-free water. Acetaldehyde
showed good linearity, with an r2 of 0.992. Since acetaldehyde can form as a contaminant, this compound was excluded from the 25-
compound mixture. Consequently, acetaldehyde formation can be monitored in the blood matrix. The amount of ethanol degraded to
acetaldehyde was found to be insignificant, suggesting little impact on the accuracy and repeatability for ethanol analysis in blood using this
approach..

4. Conclusion

In this investigation, static headspace GC-VUV absorption spectroscopy was used to identify and quantitate 25 VOCs in bovine blood.
Bovine blood closely models a human blood matrix, allowing for a quick preliminary study of how GC-VUV would perform with blood
alcohol analysis and inhalant screening. The absorbance spectrum for each VOC had unique spectral features, which allows for easy
identification, even when coelutions occur. Although minimal amounts of acetaldehyde were generated, there was no impact to ethanol’s
measurement nor to any of the 24 VOCs identified and quantified in this short-term study. Furthermore, the unknown present in this analysis
did not affect any of the results.
Traditional approaches use parallel dual-column GC with FID - a two column, two FID set-up - for quantification and, when
necessary, a mass spectrometer for identification of unknowns and coelutions. GC-VUV can perform the same analysis by using a single-
column setup at ambient pressure. This proves to be advantageous because the GC setup is simplified, and VUV spectroscopy can
unequivocally confirm and quantitate compounds regardless of separation in a single injection and can easily identify small molecules.
GC-VUV reported good results, lending this approach as a feasible alternative to traditional methods in the forensic and toxicology
community for BAC and inhalant analysis in blood.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

5
 5. Acknowledgements

This project was funded by VUV Analytics, Inc., Cedar Park, Texas, USA. J.A.Diekmann wants to thank VUV Analytics for technical
support and critical review of the manuscript and invaluable discussions.

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7
Figure 1 shows an 8-level calibration curve of ethanol in a whole bovine blood matrix, ranging from
9-495 mg/dl.

Figure 2 shows 25 VOCs eluting between 1.25 to 8.75 minutes. No other compounds eluted beyond
8.75 minutes.

8
Figure 3 shows 8 spectra: acetaldehyde, methanol, ethanol, chloroform, ethyl acetate, 2-butanone,
benzene and o-xylene. Each spectrum is distinctly different from one another, which holds true for
the remaining 18 VOCs identified in this study.

Figure 4 shows a chromatographic (left) and spectroscopic (right) coelution of 2-butanone and ethyl
acetate.

9
Table 1 shows the headspace, GC, and VUV method conditions used in this study.

Instrument Operating Parameters

Auto-Sampler Gerstel MPS

Syringe Temp (°C) 90°C

Incubation Temp
80°C
(°C)

Incubation Time
15 min
(min)

Injection Volume
250 µl
(µl)

GC Agilent 6890

Oven – Initial Temp

35°C
(°C)

Oven – Initial Time

1.00 min
(min)

Oven – Rate 1
15°C/min
(°C/min)

Oven – Final Temp

245°C/min
(°C/min)

Oven – Final Time

No Hold
(min)

Inlet – Mode Split

Inlet – Temp (°C) 250°C

Inlet – Split Ratio 5:1

Column Rxi-624 Sil MS (30m, 0.25mmid, 1.4µm)

Flow (ml/min) 2.0 ml/min (Constant flow)

Detector VGA-100

Transfer Line Temp

275°C
(°C)

Flow Cell Temp (°C) 275°C

Make-up Gas
0.25 Psi
Pressure (Psi)

Acquisition Rate
4.5 Hz
(Hz)

Acquisition Range
125-240 nm
(nm)

10
Table II. Short-Term Repeatability of Ethanol in Whole Blood

19 mg/dl Whole Blood 78 mg/dl Whole Blood 300 mg/dl Whole Blood

Day 1 Day 2 Day 3 All Days Day 1 Day 2 Day 3 All Days Day 1 Day 2 Day 3 All Days

Concentration (mg/dl) 19 19 19 19 78 78 78 78 300 300 300 300

Sample size (n) 7 7 7 21 7 7 7 21 7 7 7 21

Mean (µ) 19 20 21 20 77 80 84 80 280 300 300 300

Minimum 19 20 20 19 68 74 73 68 270 240 280 240

Maximum 21 21 22 22 81 85 89 89 300 340 320 340

Stand. Dev. (σ) 0.86 0.58 0.57 1.0 4.5 3.3 5.2 4.9 9.2 31 13 21

CV (RSD%) 4.4 2.9 2.7 5.5 5.8 4.2 6.2 7.2 3.2 10 4.4 8.8

11
Table III. Compound Information in Blood Matrix

Compounds (min) Conc. Range (mg/dl) LOD (mg/dl)

Methanol 2.00 38 to 99 8.8 0.981

Ethanol 2.58 9 to 495 3.1 0.997

Propionaldehyde 2.84 2.4 to 99 0.63 0.998

Acetone 2.90 2.4 to 99 0.34 0.996

2-Propanol 2.99 2.4 to 99 0.54 0.998

Acetonitrile 3.11 11 to 99 5.5 0.998

Dichloromethane 3.25 2.4 to 99 0.63 0.994

t-Butanol 3.32 2.4 to 99 0.70 0.998

n-Hexane 3.66 2.4 to 99 2.8 0.822

n-Propanol 3.80 2.4 to 99 1.3 0.997

Halothane 3.80 2.4 to 99 1.0 0.983

2-Butanone 4.20 2.4 to 99 0.36 0.998

Ethyl Acetate 4.20 2.4 to 99 0.44 0.998

2-Butanol 4.31 11 to 99 4.5 0.998

Chloroform 4.44 2.4 to 99 0.89 0.991

Isobutyl Alcohol 4.74 2.4 to 99 0.37 0.998

Benzene 4.85 0.5 to 24 0.10 0.987

Trichloroethene 5.34 0.5 to 24 0.12 0.981

Isoamyl Alcohol 6.22 2.4 to 99 0.44 0.997

Toluene 6.35 0.5 to 24 0.098 0.982

Tetrachloroethene 6.78 0.5 to 24 0.11 0.975

Ethylbenzene 7.64 0.5 to 24 0.11 0.977

m-Xylene 7.74 0.5 to 24 0.12 0.972

p-Xylene 7.74 0.5 to 24 0.11 0.979

o-Xylene 8.10 0.5 to 24 0.10 0.979

12