Jonathan P. Danaceau, Erin E. Chambers and Kenneth J. Fountain
Waters Corporation (Milford, MA, USA)
Application Advantages â– Analyze all metabolites without enzymatic hydrolysis â– Suitable for a comprehensive drug group consisting of 26 opioids and opioid analgesic compounds â– Quick and easy sample preparation process â– All analytes and metabolites have a linear response â– Linearity, accuracy and precision are improved compared to the dilution method â– Matrix effect is reduced
Waters Solutions
ACQUITY UPLC System®
ACQUITY UPLC BEH, 2.1 x 100 mm, 1.7 μm column
Oasis® MCX μElution board
Xevo® TQD Mass Spectrometer
MassLynx TM software
Key words
Opioids, opioids, UPLC, toxicology, SPE, sample preparation
Introduction
Analysis of natural and synthetic opioids has always been an important aspect of forensic toxicology. In the past, the sample was usually hydrolyzed with an acid or an enzyme and the glucuronide metabolite was released before GC/MS analysis 1 . With the advent of LC/MS/MS technology, glucuronide metabolites can now be directly analyzed 2-5 . Direct analysis of glucuronide metabolites eliminates the risk of false negatives due to incomplete hydrolysis, since enzymatic efficiency is highly dependent on the enzyme used and the drug substrate analyzed 6 .
Unlike the general matrix, the urine sample can be analyzed by the “dilute injection†method, in which the sample is diluted with the internal standard mixed solution and directly injected into the LC/MS/MS system for analysis 2,4 . However, the drawback of this technique is that the various matrix components contained in the urine sample may interfere with the MS signal. In addition, this technique cannot change the sample concentration. This may result in inaccurate quantification of certain glucuronide metabolites eluting under higher aqueous conditions, as glucuronide metabolites may desolventize as well as various opioids under these conditions. Unable to generate strong MS/MS product fragment ions.
This application note highlights a method for the analysis of 26 opioids that is directly analyzed by UPLC®/MS/MS after extraction of the sample using a hybrid SPE. Direct analysis of glucuronide metabolites eliminates the need for enzymatic or chemical hydrolysis steps. The results show that the hybrid SPE greatly improves linearity, accuracy and precision compared to the simple "dilution injection" method and reduces matrix effects. In addition, this experiment also analyzed previously validated samples for further evaluation of the above methods.
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