Jonathan P. Danaceau, Erin E. Chambers and Kenneth J. Fountain

Waters Corporation (Milford, MA, USA)

Application Advantages â–  Analysis of glucuronide metabolites without enzymatic hydrolysis â–  Suitable for a comprehensive drug group consisting of 22 opioids and opioid analgesic compounds â–  Compared with traditional LLE, SPE or protein precipitation methods, sample preparation Quick and easy â–  Removed endogenous phospholipids â–  Linear response to all analytes and metabolites

Waters Solutions
OstroTM 96-well plate
ACQUITY UPLC® BEH Column
ACQUITY UPLC System
Xevo® TQD Mass Spectrometer

Keywords <br> opioids, opiates, UPLC, forensic toxicology, blood

About <br> natural and synthetic opioids has been an important aspect of forensic toxicology. A high proportion of crimes and/or deaths are caused by misuse or abuse of narcotic analgesics (such as oxycodone and hydrocodone) and illegal opioid heroin. Forensic laboratories often need to analyze different drugs in a whole blood sample to determine the exact cause of death, or to determine whether to drive under the action of a drug, or for other criminal or research purposes. 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 . By enzymatic hydrolysis of the glucuronide metabolite conversion processing step to its free form increases analysis time and cost, and can not ensure consistent degree of hydrolysis ^2. With the advent of modern UPLC®/MS/MS technology, glucuronide metabolites can now be directly analyzed ^3-6 .

Whole blood analysis can be performed using a variety of sample preparation methods, including liquid-liquid extraction (LLE) and solid phase extraction (SPE). One of the simplest methods is protein precipitation after cell lysis. In this study, a rapid and direct sample preparation method was introduced. The whole blood sample was pretreated with an Ostro sample preparation plate to lyse the cells, which were then precipitated with acetonitrile and eluted with a 96-well plate. All sample pretreatments were performed in Ostro plate wells without centrifugation or transfer of samples from individual centrifuge tubes.

After the sample preparation was completed, UPLC/MS/MS analysis was performed on 22 opioids and their metabolites. Glucuronide metabolites can be analyzed directly without enzymatic or chemical hydrolysis. The calibration curve is linear and the corresponding detection limits are easily reached.

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