Abstract
The Opioid Epidemic continues to increase throughout the United States. According to the CDC, 66 % of all drug overdose deaths in 2016 involved an opioid [1]. This calculates to roughly 116 deaths every day from opioid related overdoses. After becoming addicted to prescription opioids, users may unfortunately turn to illicit alternatives such as heroin. To compound the issue, heroin has increasingly been found to be mixed with other synthetic opioids such as fentanyl, which is 100 times more potent than morphine. In order to respond effectively to this epidemic, forensic, health care, and law enforcement scientists need access to fast methods for assessing and monitoring which opioids are involved.
Automating the entire hydrolysis, extraction, and subsequent analysis by LC-MS/MS provides the critically needed high throughput analysis for opioids in urine. Using the novel GERSTEL MPS robotic autosampler, syringe transfer of all liquids involved in the enzymatic hydrolysis procedure, controlled incubation of the samples for a defi ned period of time, as well as extraction of the subsequent hydrolyzed urine samples using dispersive solid phase extraction were performed. The resulting eluents from the automated extractions were then introduced into the new Agilent Ultivo LC-MS/MS instrument.
Introduction
A variety of sample handling steps are required prior to the analysis of urine samples to accurately determine analyte concentrations. These steps typically begin with the enzymatic hydrolysis of analytes from their conjugated forms to the native drug using enzymes such as beta-glucuronidase. The genetically modifi ed, pure beta-glucuronidase IMCSzyme can hydrolyze multiple drug classes within 30 minutes with high effi ciency [2]. To ensure that the hydrolysis process is complete and reproducible, the pH, temperature and duration must be controlled and optimized for the enzyme used. To achieve the very low limits of detection necessary for drug compounds and their metabolites, it is often necessary to remove interfering matrix. Interferences can be produced as a result of the hydrolysis procedure or occur naturally from the biological nature of the urine samples themselves. Solid phase extraction (SPE) is a widely used, proven method for sample preparation and sample clean-up of hydrolyzed urine samples in the fi eld of forensic analysis. Disposable Pipette Extraction (DPX) was developed as an alternative to traditional SPE, combining effi cient and rapid extraction with signifi cantly reduced solvent consumption. DPX relies on dispersive solid-phase extraction devices that use sorbent loosely contained in a pipette tip to effi ciently mix with the sample solution. The main advantages of DPX technology are: Rapid extraction, high recoveries, negligible solvent waste is generated, and the extractions can be fully automated and coupled to GC/MS or LC/MS injection.
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