LC/MS Analysis of Opioid Glucuronide Metabolites in Urine on Ascentis® Express F5 after Solid Phase Extraction (SPE) using Supel™-Select HLB
Materials
analytical column
SPE tube or plate
standard
Morphine-3-β-D-glucuronide solution
1 mg/mL in methanol with 0.05% NaOH, ampule of 1 mL, certified reference material, Cerilliant®Hydromorphone solution
1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Buprenorphine glucuronide solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Buprenorphine solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Codeine solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Codeine-6-β-D-glucuronide solution
100 μg/mL in methanol: water (2:8), ampule of 1 mL, certified reference material, Cerilliant®Hydrocodone solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Hydromorphone-3-β-D-glucuronide
100 μg/mL in methanol: water (1:1), ampule of 1 mL, certified reference material, Cerilliant®Morphine solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Norbuprenorphine glucuronide solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Norbuprenorphine solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Oxycodone solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Oxymorphone-D3 solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Oxymorphone-3-β-D-glucuronide solution
100 μg/mL in methanol: water (1:1), ampule of 1 mL, certified reference material, Cerilliant®CONDITIONS
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
Description
Analysis Note
Sample pretreatment: β-glucuronidase hydrolysis
The workflow for the LC/MS analysis of native drugs and associated metabolites from urine is shown here. The convenient liquid form of beta-glucuronidase from Sigma was used for hydrolysis of the glucuronide metabolites. SPE using Supel-Select HLB yielded clean extracts. The Ascentis Express F5 columns gave highly efficient, well-resolved peaks and rapid separation. Ultra high purity LC-MS solvents and additives provided trouble-free operation, and Cerilliant CRMs gave reliable quantification.
Legal Information
Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany
Supel is a trademark of Sigma-Aldrich Co. LLC