LC/MS Analysis of Ethanol Metabolites in Urine on Ascentis^® Express OH5 after Dilution with Deuterated Internal Standards

Ascentis Express OH5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53757-U)

[A] 5 mM ammonium formate and 0.1% formic acid in 95:5, acetonitrile:water; [B] 5 mM ammonium formate and 0.1% formic acid in 80:20, acetonitrile:water

0 to 100% B in 1 min, held at 100% B for 4 min, to 0% B, held for 5 min

0.4 mL/min

1450 psi (100 bar)

30 °C

MS, ESI(-), 125.0 m/z, 126.1 m/z, 221.1 m/z, 226.2 m/z

5 μL

urine spiked with ethyl sulfate and ethyl-β-D-glucuronide, both at 50 ng/mL, diluted 20:1 in acetonitrile containing deuterated internal standards at 100 ng/mL

Ethyl sulphate (EtS) and ethyl glucuronide (EtG) are direct ethanol metabolites and may indicate recent alcohol consumption. The two compounds differ in their pathways for formation and degradation. Being polar compounds, they are poorly retained by C18 phases and elute early in the chromatogram along with matrix compounds. This leads to poor or unreliable quantification. The method shown here uses HILIC mode on an Ascentis Express OH5 column to retain both analytes well, making it very likely to be more robust and reliable, as well as highly MS-friendly. Cerilliant CRMs provided reliable quantification. The internal standard was needed for accurate quantification. External calibration resulted in a large excess of sulfate.

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