UHPLC/MS/MS Analysis of Benzodiazepines and Glucuronide Metabolites in Urine on Titan C18 after SPE using Supel™-Select HLB, β-Glucuronidase Enzyme Digestion
Materials
SPE tube or plate
enzyme
standard
Oxazepam solution
1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Nordiazepam-d5 solution
1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Nordiazepam solution
1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Alprazolam-d5 solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Alprazolam solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®7-Aminoflunitrazepam solution
1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®7-Aminoclonazepam solution
1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®7-Aminoclonazepam-d4 solution
100 μg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®7-Aminoflunitrazepam-d7 solution
1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®Diazepam-d5 solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Diazepam solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Flurazepam solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Flunitrazepam solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Flunitrazepam-d7
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Lorazepam glucuronide solution
100 μg/mL in acetonitrile: water (1:1), ampule of 1 mL, certified reference material, Cerilliant®Lorazepam solution
1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®Midazolam solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Nitrazepam solution
1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®Oxazepam glucuronide solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Oxazepam-d5 solution
100 μg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Temazepam glucuronide lithium salt solution
100 μg/mL in methanol (as free carboxylate), ampule of 1 mL, certified reference material, Cerilliant®Temazepam solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®CONDITIONS
sample preparation
SPE (Solid Phase Extraction)
SPE well plate
Supel-Select HLB 96-well plate, 60 mg/well (575662-U)
condition
1 mL acetonitrile, 1 mL water
sample addition
1.1 mL enzyme-digested urine
washing
1 mL water, 1 mL 25% methanol
elution
1 mL acetonitrile
column
Titan C18, 5 cm x 2.1 mm I.D., 1.9 μm particles (577122-U)
mobile phase
[A]: 5 mM ammonium formate [B]: 5 mM ammmonium formate in acetonitrile:water (95:5)
gradient
10 to 90% B in 2.5 min, held for 0.5 min
flow rate
0.3 mL/min
column temp.
35 °C
detector
ESI(+), MRM
injection
2 μL
Description
Analysis Note
Urine samples were spiked with various glucuronide metabolites of benzodiazepines along with the parent drugs. The glucuronide metabolites were converted back to the parent drug using β-glucuronidase, then processed using SPE prior to LC/MS analysis. The Supel-Select HLB provided clean extracts and the Titan C18 provided rapid resolution of parent drugs and metabolites. Cerilliant CRMs provided reliable quantitation.
Legal Information
Supel is a trademark of Sigma-Aldrich Co. LLC
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