Table 1: Positive ions acquired in multiple reaction monitoring (MRM) mode.
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This application note describes the extraction of both ethyl glucuronide (EtG) and the lower concentration metabolite ethyl
sulphate (EtS) from human urine using EVOLUTE AX columns followed by LC-MS-MS analysis. EtG and EtS are specific metabolites
formed within the body after the ingestion of alcohol. EVOLUTE AX is a resin-based mixedmode sorbent with an optimized combination
of non-polar (hydrophobic), polar (hydrophilic) and weak anion exchange interactions ideally constructed to extract acidic
analytes such as ethyl glucuronide.
Extraction Conditions
Sample pre-treatment: Dilute urine samples with acetonitrile (1:9, v/v)
Column conditioning: Methanol (3 mL)
Column equilibration 1: Water (3 mL)
Column equilibration 2: Acetonitrile (3 mL)
Sample loading: Load pre-treated sample (2 mL total volume) onto EVOLUTE AX 100 mg/3 mL column (part number 6130010-B)
Interference elution 1: Acetonitrile (3 mL)
Interference elution 2: Methanol (3 mL)
Analyte elution: 2% HCl in acetonitrile (3 mL)
Evaporation: Evaporate to dryness at 40 °C
Reconstitution: Reconstitute in HPLC grade water (250 µL), vortex mix and add acetonitrile (250 µL), vortex mix
Analytical Conditions
Instrument: Waters Acquity UPLC with 20 µL injection loop interfaced to Premier XE triple quadrupole mass spectrometer equipped with
an electrospray interface for mass analysis.
Column: UPLC HSS C18 (150 mm × 1.8 µm × 2.1 mm i.d.)
Mobile phase: A = 0.05% Formic acid (aq) B = Acetonitrile
Flow rate: 0.2 mL/min
Injection volume: 10 µL (partial loop with overfill)
Sample temp: 10 °C
Column temp: 50 °C
Desolvation temp: 450 °C
Source temp: 100 °C
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