June 2019

Medical Mass Spectrometry Vol. 3 No. 1Research Paper®

Quantiﬁcation of ethanol in whole blood by extraction using NeedlEx andgas chromatography/mass spectrometry
Masae Iwai*, Fumio Kondo, Takayoshi Suzuki, Tadashi Ogawa, Hiroshi Seno
Department of Legal Medicine, Aichi Medical University School of Medicine,1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195, Japan
Abstract

In this study, a new quantiﬁcation method was developed for analysis of ethanol in whole blood samples byneedle extraction (NeedlEx ) and gas chromatography/mass spectrometry (GC/MS). Whole blood samples (0.1 mL) con®

taining ethanol and ethanol-d6 (internal standard) were incubated at room temperature in a 10-mL headspace vial. The®

NeedlEx for alcohols with a gas aspirating pump was exposed to the headspace of vial to allow adsorption alcohols before
GC/MS. The GC separation of the compounds was achieved on a fused-silica capillary column Rtx-5 ms (30 m × 0.25 mmi.d.; 0.25-μm ﬁlm thickness) with MS detection operated in electron impact ionization ion source mode. The regression
equations showed good linearity (r＞0.998) from 0.1 to 5.0 mg/mL for whole blood. The accuracies and precisions were99.0‒112% and 3.0‒7.0%, respectively. The method was successfully applied to actual analyses of autopsy samples. The
present results on the analysis of ethanol by NeedlEx -GC/MS suggest its applicability to a number of other volatile com®

pounds in clinical and forensic toxicology.
®

Key words: ethanol, NeedlEx , whole blood, gas chromatography/mass spectrometry

Introduction

technique that combines the chromatographic separation
power of gas chromatography with the high detection speci-

Ethanol is a most popular beverage, but its excessive
consumption causes poisonings, fatal accidents, and many1)
violent crimes . Blood ethanol quantiﬁcations are manda-

ﬁcity power of mass spectrometry. Thus, this approach has2,3)

tory in clinical and forensic toxicological investigations .
Headspace gas chromatography with ﬂame-ionization

NeedlEx , a new extraction needle, is a device that concentrates volatile organic compounds (VOCs), such as alco-detection (GC-FID) has been the gold standard for ethanol

hols, organic solvents, amines, and fatty acids. Several

analysis, because of its easiness, sensitivity, accuracy, andreports have described the application of NeedlEx in the8‒12)

been widely used in various analytical works.

®

®

4)

13‒16)

relative speciﬁcity . However, this method has inherent

analysis of VOCs in biological samples

drawbacks in that it depends on only the retention time of5‒7)
the target compound for identiﬁcation . Gas chromatogra-

the best of our knowledge, there are no reports of quantiﬁ®
cation of ethanol in whole blood by NeedlEx -GC/MS.

. However, tophy/mass spectrometry (GC/MS) is a powerful analytical

Accordingly, in this study, we developed and validated anew quantiﬁcation method for ethanol in whole blood. This

*Corresponding author

method was successfully applied for the quantiﬁcation ofMasae Iwai

ethanol in whole blood samples collected from autopsy

Department of Legal Medicine, Aichi Medical University
School of Medicine, 1‒1 Yazakokarimata, Nagakute, Aichi

cases.

480‒1195, JapanTel.: ＋81‒561‒62‒3311, Fax: ＋81‒561‒63‒8120Materials and Methods

Received December 13, 2018. Accepted January 31, 2019.
Epub April 2, 2019.

NeedlEx for alcohols, which adsorbs monohydric alcohols with up to 3 carbon atoms, and solvents, such as ace-

DOI: 10.24508/mms.2019.06.003

tone and diethyl ether, with boiling points below 100°
C,

Reagents and chemicals

E-mail: masaoshi@aichi-med-u.ac.jp®

30June 2019

Medical Mass Spectrometry Vol. 3 No. 1

were purchased from Shinwa Chemical Industries (Kyoto,Japan). Ethanol and ethanol-d6 [internal standard (IS)] weresource and interface temperatures were 200 and 240°
C,purchased from Wako Pure Chemicals (Osaka, Japan) andMerck KGaA (Darmstadt, Germany), respectively. All

injection port was kept at 200°
C. The analysis was carriedout with a scanning speed of 833 u/s. For single-scan analy-

other reagents were of analytical grade. Laboratory distilledwater was puriﬁed using a Synergy UV (Millipore, Mol-and interface temperatures were maintained at 200°
C andsheim, France). Other common chemicals used in this study

280°
C, respectively. The data were acquired by usingwere of the highest purity commercially available. Human

GCMS solution software (Shimadzu, Kyoto, Japan). The
ions observed as the base peak (ethanol: m/z 31, ethanol-d6:

respectively, and electron ionization at 70 eV was used. The

sis, the scan range was m/z 20 to 250. The MS ion source

whole blood to be used as blank samples was obtained from
Tennessee Blood Services (Memphis, TN, USA).

m/z 33) were used for quantiﬁcation, and the second andthird most abundant ions (ethanol: m/z 45, 46, ethanol-d6:

Calibration standards and validation samplesm/z 49, 51) were used as qualiﬁers.

The IS solution was prepared in water at a concentrationof 1.0 mg/mL. The concentrations of calibration standard

Method validation

solutions were 0.1, 0.2, 0.5, 1.0, 2.5, and 5.0 mg/mL in

The method has been validated according to the US FDA17)
guidelines on bioanalytical method validation . Calibra-blank whole blood. Validation samples were adjusted to0.1, 0.3, 2.0, and 4.0 mg/mL.

tion curves were constructed by plotting the peak area ratioof each analyte to the IS (y-axis) versus the concentration ofExtraction procedure

the corresponding analytes (x-axis). The slope and y-inter-

Whole blood samples (0.1 mL) and 0.1 mL IS solution(1.0 mg/mL ethanol-d6) were pipetted into the 10-mL head-

cept of the regression line were obtained by six different

space vial; the vial was covered with a rubber stopper, andinjections of each calibrator. The lower limit of quantitation(LOQ) was deﬁned as the lowest quantitative concentra-

an aluminum cap was pressurized with a crimper and seal.

tion. The intra- and interday accuracies and precisions wereThe samples were incubated at room temperature fordetermined by conducting 5 experiments in the course of a®

15 min. A NeedlEx for alcohols was then connected to agas aspirating pump (Model AP-20 KITAGAWA; Komyo

single day and by conducting an experiment on 5 different
days, respectively. The precisions were expressed as per-Rikagaku Kogyo K. K., Kawasaki, Japan) to draw the gas

cent coefﬁcients of variation. The accuracies and precisions

present in the headspace of the vial. After drawing the®
10 mL gas, the NeedlEx was attached to a Luer-lock typeshould be within 20%.

gas-tight syringe. The NeedlEx was then inserted into theForensic autopsy samples

injection port of the GC/MS, and helium gas (0.2 mL) was

Femoral vein and right heart blood samples wereobtained from autopsy cadavers at Aichi Medical Univer-®

®

drawn through the NeedlEx by pulling the plunger of thegas-tight syringe. After 10 s, the helium collected in the gastight syringe was injected by pushing the plunger, whichsity in 2017. Whole blood samples were collected in 5- or15-mL tubes and stored at −80°
C until analysis. The alco-

resulted in desorption of the analytes from the media in thehol content examinations of these samples were ofﬁcially®

NeedlEx .

determined by judicial authorities.

GC/MS conditions

Results and Discussion

GC/MS analysis was performed on a Shimadzu GCMS
QP2020 (Shimadzu, Kyoto, Japan). A 30-m Rtx-5 ms col-GC/MS analysis
GC separation was optimized and carried out asdescribed in the Materials and Methods section. MS ion-umn (Restek, PA, USA) with 0.25 mm internal diameterand 0.25 μm ﬁlm thickness was used. The initial oven tem-ization parameters were optimized by injecting ethanol andethanol-d6 at a concentration of 1.0 mg/mL. The fragment

perature was 40°
C. After holding at the initial temperature

ion at m/z 31 observed as the base peak of ethanol corre＋
sponded [M‒CH3] , and the second and third most abun-for 10 min, the temperature was increased up to 250°
C at arate of 20°
C/min. Helium was used as a carrier gas at aﬂow-rate of 1.5 mL/min with a 40 : 1 split ratio. The ion＋＋

dant ions at m/z 45 and 46 were [M‒H] and [M] , respec31

June 2019

Medical Mass Spectrometry Vol. 3 No. 1tively.
Selected ion chromatograms of blank human whole

0.998 (Fig. 2), thus establishing a high degree of linearity
within this range. The LOQ was set at 0.1 mg/mL, as indi-blood samples without spiking (Fig. 1a) and a blank humancated that blood alcohol concentration less than 0.1 mg/mL

whole blood sample spiked with 1.0 mg/mL ethanol andethanol-d6 (Fig. 1b) are shown. The peaks of ethanol andshould be reported negative in postmortem toxicology .
The intra- and interday accuracies and precisions were1)

ethanol-d6 in Fig. 1b were clearly visible, with retention

evaluated at four levels, as summarized in Table 1. The
accuracies and precisions were 99.0‒112% and 3.0‒7.0%,

times of 1.0 min, and there were no peaks that interferedwith the measurements. It should be mentioned that the ethanol-d6 gave peaks at m/z 33, 49 and 51, despite the fact

respectively. Their values were acceptable at all concentrations. Overall efﬁciencies including both extraction recov-

that six deuterium atoms are present in the molecule. Thisery and matrix effect were determined at the low (0.3 mg/is due to the rapid exchange of a deuterium at the hydroxymL) and high concentrations (4.0 mg/mL) by comparing

moiety for a hydrogen atom immediately after the contact

whole blood samples with solutions of reference standard18)

with water .

ethanol dissolved in water (n＝5). They were 108% forMethod validation

0.3 mg/mL and 104% for 4.0 mg/mL, which showed almost
the same results as described in previous reports by head-

The method for ethanol quantiﬁcation was validated by

space GC/MS

characterizing a series of parameters, such as linearity,accuracy, and precision (intra- and interday). The calibra-Applications

12,19).

tion curve showed a good linear relationship in the range

We analyzed the 17 blood samples collected at autopsies

from 0.1 to 5.0 mg/mL, with a correlation coefﬁcient ofin 2017. These samples were taken from femoral veins (n＝4) and right hearts (n＝13) and were previously checked tocontain ethanol by GC-FID. Figure 3 shows representative

Fig. 2.

Calibration curve of ethanol in whole blood. Results are means of six different injections for eachcalibrator.

Table 1.

Intra- and interday accuracy and precision data
for ethanol in whole blood

Spiked concentration(mg/mL)

Fig. 1.

SIM chromatograms of blank human whole blood
samples spiked without (a) and with (b) 1.0 mg/mL
ethanol and 1.0 mg/mL ethanol-d6.

a32

aa

IntradayInterdayAccuracy(%)

Precision(%CV)

Accuracy(%)

Precision
(%CV)

0.1

111

3.01123.90.3

104

4.0

99.6

6.3

2.0

105

3.7103

5.7

4.0

103

6.4

99.0

7.0

Values are the means of ﬁve replicate determinations.

June 2019

Medical Mass Spectrometry Vol. 3 No. 1®
involving NeedlEx -GC/MS for the quantiﬁcation of etha-

nol in whole blood. The applicability of this method wasdemonstrated by measuring the concentrations of ethanol inwhole blood samples from autopsies. This method wasfound to be suitable for forensic and clinical toxicology
analyses. We are currently trying this technique for thedetection of other VOCs in human body ﬂuids.

Conﬂict of Interest
The authors have no conﬂicts of interest directly relevant
Fig. 3.

to the content of this article.

SIM chromatograms obtained from an autopsy
blood sample.

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Table 2.

Quantiﬁed concentrations of ethanol in blood
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