Rapid Quantitation of Alcohol Metabolite Markers in Urine

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct metabolites of ethanol. Despite the fact that just a small portion of the ethanol consumed by humans is transformed into this pair of compounds, they are employed as biomarkers for consumption or abstinence from alcohol, for example, in tests determining fitness to drive.

EVOQ triple quadrupole MS with Elute UHPLC

Figure 1. EVOQ triple quadrupole MS with Elute UHPLC

It is still possible to detect these compounds in urine, even once ethanol concentration can no longer be measured, since their window of detection in this form is around 3-5 days. The typical cut-off value for EtG is approximately 100 ng/mL.

With EtG, there is a risk of both false-negative results, which can happen as a result of bacterial degradation, for example, with a urinary tract infection, and false-positive results, which can happen following the use of ethanol-based mouthwash or hand-sanitizers.

This is why the inclusion of EtS in the assay is vital. A specific detection process capable of picking up both EtG and EtS can be achieved through High Performance Liquid chromatography (HPLC) along with a triple quadrupole mass spectrometer.

Experimental

Samples

Recipe (Munich) provided urine calibrators at six diverse concentrations (Table 2), as well as a quality control sample. Analysis was carried out on two samples of a round robin test arranged by the “Society of Toxicological and Forensic Chemistry” (GTFCh).

Additionally, urine samples of two people (both social drinkers; individual 1 female, 30 y, 61 kg; individual 2 male, 37 y, 64 kg) were gathered 3 and 24 hours after each had ingested one glass of wine (200 mL) and one glass of spirits (20 mL).

Sample Preparation

Urine samples were placed in a centrifuge at 4000 rpm for 10 min. 100 µL urine, 20 µL internal standard solutions (5 µg/ mL EtG-d5 and EtS-d5) and 250 µL methanol were added to an Eppendorf tube.

Following vortexing, the tubes were placed in the centrifuge and 270 µL supernatant were transmitted into an HPLC vial. Evaporation of the supernatant took place in a vacuum concentrator at 45 °C and the residue re-formed in 600 µL 0.1% formic acid.

Instrumentation  
UHPLC: Bruker Elute UHPLC
Column: Restek, Ultra Biphenyl, 3 µm, 2.1 x 100 mm with guard column 2.1 x 10 mm
Mobile phase A: Water, 2 mM ammonium formate, 0.1% formic acid
Mobile phase B: Methanol, 2 mM ammonium formate, 0.1% formic acid
Gradient: 0.0 min 2% B 1.0 min 80% B 2.0 min 80% B 2.01 min 2% B 3.0 min 2% B
Flow rate: 400 µL/min
Injection volume: 10 µL
Column oven: 40 °C
Mass spectrometer: EVOQ Elite triple quadrupole mass spectrometer
Ion source: VIP H-ESI negative, 4000 V
Probe gas: 50 units at 300 °C
Cone gas: 25 units at 350 °C
Nebulizing gas: 50 units
Active exhaust: on
Collision gas: Argon, 1.5 mTorr
MRM transitions: see Table 1

 

Calibration curves of EtG (left) and EtS (right) over the range 80 - 9621 ng/mL (EtG) and 27 - 4732 ng/mL (EtS)

Figure 2. Calibration curves of EtG (left) and EtS (right) over the range 80 - 9621 ng/mL (EtG) and 27 - 4732 ng/mL (EtS)

Results and Discussion

Calibration curves demonstrated exceptional linearity (r² ≥ 0.999) and a high standard of accuracy with bias < ±8% for both EtG and EtS (Figure 2, Table 2). Strong accuracy was also found in the quality control sample, with a deviation of 9.4% for both analytes.

Replicate injections (n=5) of the two round robin test samples revealed outstanding accuracy, with RSD ≤ 2.8% for each compound. The quantitative results all fell inside the accepted range (Table 3).

EtG and EtS were each distinguished in the two genuine samples. The concentrations actually surpassed the calibration range in the samples collected 3 hours after the ingestion of alcoholic drinks. 24 hours after the ingestion, both analytes could still be detected in the two samples (Table 4).

The innovative Elute UHPLC system delivered outstanding chromatographic peak shape and a FWHM (full width half maximum) of just 3-4 seconds, as presented in Fig. 3.

Overlaid chromatograms of the five replicates of round robin test sample 1; left peak EtS, right peak EtG

Figure 3. Overlaid chromatograms of the five replicates of round robin test sample 1; left peak EtS, right peak EtG

Table 1. MRM transitions

Compound Rt
(min)
Precursor
Ion
Product
Ion 1
CID 1
(V)
Product
Ion 2
CID 2
(V)
Ethyl glucuronide 1.34 221.0 75.2 11 85.2 14
Ethyl glucuronide-d5 1.33 226.1 75.0 11    
Ethyl sulfate 1.08 125.0 97.1 12 80.1 26
Ethyl sulfate-d5 1.07 130.0 98.0 12    

 

Table 2. Calibration and QC of EtG and EtS

  Ethyl glucuronide Ethyl sulfate
Sample Target concentration [ng/mL] Calculated concentration [ng/mL] Bias [%] Target concentration [ng/mL] Calculated concentration [ng/mL] Bias [%]
Calibrator 1 80.1 80 -0.1 27.8 27 -2.5
Calibrator 2 191 192 0.6 75.9 82 7.6
Calibrator 3 582 570 -2.1 244 236 -3.2
Calibrator 4 1464 1505 2.8 580 594 2.4
Calibrator 5 4873 4754 -2.4 1933 1860 -3.8
Calibrator 6 9621 9736 1.2 4732 4705 -0.6
Quality control 111 121 9.4 48 53 9.4

 

Table 3. Results of round robin test

Sample Analyte Target concentration [ng/mL] Accepted Range [ng/mL] Calculated concentration [ng/mL] (mean, n = 5) Accuracy Bias [%] Precision RSD [%] (n = 5)
Round robin test sample 1 EtG 609 399 – 819 613 0.7 2.3
EtS 886 596 – 1176 856 -3.4 2.4
Round robin test sample 2 EtG 1240 840 – 1640 1558 25.6 2.8
EtS 790 528 – 1052 807 2.2 2.6

 

Table 4. Results of real samples; * concentration above calibration range

Sample Concentration EtG [ng/mL] Concentration EtS [ng/mL]
Individual 1: 3 h approx. 19,000* approx. 9,000*
Individual 1: 24 h 93 96
Individual 2: 3 h approx. 19,000* approx. 8,600*
Individual 2: 24 h 179 124

 

Conclusion

A rapid, 3 minute method to detect and quantify EtG and EtS in urine with the new Bruker Elute UHPLC coupled to the EVOQ Elite triple quadrupole MS was demonstrated. The technique offers outstanding linearity and excellent accuracy. Both EtG and EtS were distinguished in the two genuine samples.

About Bruker Daltonics

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Last updated: Apr 2, 2019 at 10:11 AM

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