EtG/EtS on Raptor EtG/EtS: Updated Method Conditions (Mobile Phase and Gradient)

	Peaks	t_R (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
1.	Ethyl-β-D-glucuronide-d5 (EtG-d5)	1.21	200	226.2	85.0	-
2.	Ethyl-β-D-glucuronide (EtG)	1.23	500	221.2	75.1	85.1
3.	Ethyl sulfate-d5 (EtS-d5)	2.36	50	130.1	98.0	-
4.	Ethyl sulfate (EtS)	2.38	500	125.1	97.1	80.0

Column

Raptor EtG/EtS (cat.# 9325A12)

Dimensions:

100 mm x 2.1 mm ID

Particle Size:

2.7 µm

Pore Size:

90 Å

Guard Column:

UltraShield UHPLC precolumn filter, 0.2 μm frit (cat.# 25809)

Temp.:

35 °C

Standard/Sample

Ethyl-β-D-glucuronide-d5 (EtG-d5) (cat.# 34102)

Ethyl-β-D-glucuronide (EtG) (cat.# 34101)

Ethyl sulfate-d5 sodium salt (EtS-d5) (cat.# 34104)

Ethyl sulfate sodium salt (EtS) (cat.# 34103)

Diluent:

0.01% Formic acid in water

Conc.:

500 ng/mL (40x acetonitrile precipitation sample prep)

Inj. Vol.:

10 µL

Mobile Phase

0.01% Formic acid in water

0.1% Formic acid in acetonitrile

Time (min)	Flow (mL/min)	%A	%B
0.00	0.5	95	5
3.00	0.5	65	35
3.01	0.5	95	5
4.50	0.5	95	5

Detector	MS/MS
Ion Source:	Electrospray
Ion Mode:	ESI-
Mode:	MRM
Instrument	HPLC
Sample Preparation	A 500 ng/mL standard was prepared in urine. A 50 μL aliquot was mixed with 10 μL of internal standard (20 μg/mL EtG-d5 and 5 μg/mL EtS-d5 in water) and 150 µL of acetonitrile by vortexing at 3000 rpm for 10 seconds and centrifuged at 4300 rpm for 10 minutes at 10 °C. After centrifugation, 100 µL of the supernatant was diluted with 900 µL (40x dilution) of 0.01% formic acid in water. The sample was then vortexed at 3000 rpm for 10 seconds and injected for LC-MS/MS analysis.

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