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UHPLC/MS/MS Analysis of Benzodiazepines and Glucuronide Metabolites in Urine on Titan™ C18 after SPE using Supel™-Select HLB, β-Glucuronidase Enzyme Digestion

UHPLC/MS/MS Analysis of Benzodiazepines and Glucuronide Metabolites in Urine on Titan™ C18 after SPE using Supel™-Select HLB, β-Glucuronidase Enzyme Digestion

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)
column temp. 35 C
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
injection 2 µl
detector ESI(+), MRM

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.
Categories Analytical Chromatography, Pharmaceutical Analysis, SPE Applications, Antiepileptic/antiseizure, Drugs of abuse, Anxiolytics/sedatives, Anticonvulsant/antiepiletic/antiseizure, Neurotransmitters
Featured Industry Forensics and Toxicology
Legal Information Supel is a trademark of Sigma-Aldrich Co. LLC, Titan is a trademark of Sigma-Aldrich Co. LLC
suitability application for UHPLC

Materials

     
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