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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

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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

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

Description
    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®
    See All (14)

SPE tube or plate

Product No.
Description
Pricing

analytical column

Product No.
Description
Pricing

standard

Product No.
Description
Pricing

M-031

Morphine-3-β-D-glucuronide solution, 1 mg/mL in methanol with 0.05% NaOH, ampule of 1 mL, certified reference material, Cerilliant®

H-004

Hydromorphone solution, 1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

B-035

Buprenorphine glucuronide solution, 100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

B-044

Buprenorphine solution, 1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

C-015

Codeine solution, 100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

C-087

Codeine-6-β-D-glucuronide solution, 100 μg/mL in methanol: water (2:8), ampule of 1 mL, certified reference material, Cerilliant®

H-003

Hydrocodone solution, 1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

H-051

Hydromorphone-3-β-D-glucuronide, 100 μg/mL in methanol: water (1:1), ampule of 1 mL, certified reference material, Cerilliant®

M-030

Morphine solution, 100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

N-045

Norbuprenorphine glucuronide solution, 100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

N-059

Norbuprenorphine solution, 1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

O-002

Oxycodone solution, 1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

O-003

Oxymorphone-D3 solution, 100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

O-030

Oxymorphone-3-β-D-glucuronide solution, 100 μg/mL in methanol: water (1:1), ampule of 1 mL, certified reference material, Cerilliant®

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