LC/MS Analysis of Opioid Glucuronide Metabolites in Urine on Ascentis^® Express F5 after Solid Phase Extraction (SPE) using Supel^™-Select HLB

sample preparation

Solid Phase Extraction

sample/matrix

urine spiked with drugs and metabolites at 300 ng/mL

SPE well plate

Supel-Select HLB 96-well plate, 60 mg/well (575662-U)

condition

1 mL methanol followed by 1 mL water

sample addition

1 mL hydrolyzed urine

washing

1 mL water followed by 1 mL 25% methanol

elution

1 mL acetonitrile

column

Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 μm particles (53569-U)

mobile phase

[A] 5 mM ammonium formate, pH 4.2 (adjusted with formic acid); [B] 5 mM ammonium formate in (95:5 acetonitrile:water) pH 5.2 (adjusted with formic acid)

gradient

15% B held for 1 min, to 70% B in 2 min, held at 70% B for 7 min

flow rate

0.4 mL/min

column temp.

35 °C

detector

ESI(+) TOF, full scan, m/z 100-1000

injection

3 μL

sample

300 ng/mL