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Quantitation of Urinary Ethyl Glucuronide

and Ethyl Sulfate Using Ultrahigh Resolution

LC-MS

Forensic Toxicology Use Only

Kent Johnson, Fortes Laboratories, Portland, OR; Marta Kozak, Thermo Fisher Scientific, San Jose, CA

Introduction

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are

sensitive and specific urinary biomarkers of recent alcohol

intake that are of great interest in today’s forensic

toxicology laboratories.

Goal

To demonstrate the quantitation of EtG and EtS in urine

using a liquid chromatography-mass spectrometry

(LC-MS) method with ultrahigh resolution on the Thermo

Scientific Exactive benchtop mass spectrometer.

Experimental

Calibration Standards and Sample Preparation

Calibration standards were prepared by spiking blank

urine with EtG and EtS to final concentrations ranging

from 25 ng/mL to 20,000 ng/mL.

Calibration standards and urine samples were spiked

with internal standards (EtG-d5 and EtS-d5) and diluted

10 times with an LC mobile phase prior to injection onto

the analytical column.

Commercial QC samples were used to obtain method

accuracy and precision.

HPLC

HPLC analysis was performed using a Thermo Scientific

Accela liquid chromatography system with a Thermo

Scientific Hypersil GOLD C18 column (50 x 2.1 mm;

5 µm). A diluted sample of 20 µL was analyzed with a

6-minute gradient method.

Mass Spectrometry

MS analysis was carried out on the Exactive™ benchtop

LC-MS instrument equipped with an electrospray

ionization (ESI) source. Full scan data with resolution of

100,000 FWHM at

m/z

200 was acquired.

Results and Discussion

Figure 1 shows the linear calibration curves for EtG

(100-20,000 ng/mL) and EtS (100-20,000 ng/mL).

Figure 2 shows chromatograms of EtG and EtS at

25 ng/mL and the respective deuterated internal standards.

Chromatograms for compound detection and quantitation

are reconstructed with a mass tolerance of 5 ppm.

Conclusion

The Exactive benchtop LC-MS instrument provides

excellent quantitative analysis of EtG and EtS in a

6-minute method. When applied to real samples, the

method meets the demands of today’s forensic toxicology

laboratories with exceptional performance.

Method Performance Summary

Target Analytes

Ethyl glucuronide

Ethyl sulfate

enirU

enirU

xirtaM

LOD

25 ng/mL

25 ng/mL or less

LOQ

100 ng/mL

100 ng/mL

Recovery

> 85%

> 85%

Precision

< 15%

< 15%

Assay Linearity

100 – 20,000 ng/mL

100 – 20,000 ng/mL

%1 <

%1 <

QOLL ta revoyrraC

Sample Volume

100 µL

100 µL

Analysis Time

6 minutes

6 minutes

Key Words

• Exactive

• Accela HPLC

• EtG / EtS

• Pain

Management

• Forensic

Toxicology

Application

Note: 488b

1.8

2.0

2.2

2.4

2.6

2.8

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

RelativeAbundance

RT:1.94

MA:10638

1.74

1.80

2.13

2.48

2.35

2.50

2.56

2.88

2.61

RT:1.94

MA:611241

2.29 2.35

2.63

2.23

2.81

2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

RelativeAbundance

RT:2.59

MA:19113

2.45

3.33

2.33

3.39

2.74

3.21

2.91 3.01

3.50 3.53 3.86

RT:2.56

MA:756109

2.96

2.77

12.2

33.2

3.44

25 ng/mL standards (LOD)

StE

GtE

EtG-D5

EtS-D5

Figure 2: LOD chromatograms of EtG and EtS at

25 ng/mL with deuterated internal standards.

0

2000

4000

6000

8000 10000 12000 14000 16000 18000 20000 22000

ng/mL

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

AreaRatio

EtG 100- 20,000 ng/mL

EtS 100-20,000 ng/mL

0

2000

4000

6000

8000 10000 12000 14000 16000 18000 20000 22000

ng/mL

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

AreaRatio

Figure 1: Linear calibration curves for EtG (100-20,000 ng/mL) and EtS

(100-20,000 ng/mL).

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