G006299

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

application for SPE, application for HPLC

Properties

• technique(s): HPLC: suitable; solid phase extraction (SPE): suitable
• test parameters: 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
• suitability: application for HPLC; application for SPE
• application(s): clinical; forensics and toxicology

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

Analyte

Oxymorphone-3-β-D-glucuronide solution, 100 μg/mL in methanol: water (1:1), ampule of 1 mL, certified reference material, Cerilliant^®
Morphine-3-β-D-glucuronide solution, 1 mg/mL in methanol with 0.05% NaOH, 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^®
Codeine-6-β-D-glucuronide solution, 100 μg/mL in methanol: water (2:8), ampule of 1 mL, certified reference material, Cerilliant^®
Morphine solution, 100 μg/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^®
Hydromorphone solution, 1 mg/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^®
Codeine solution, 100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant^®
Hydrocodone 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^®
Buprenorphine 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^®
Buprenorphine solution, 1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant^®
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