Detecting fentanyl, norfentanyl, and fentanyl analog metabolites in synthetic urine using biocompatible-solid phase microextraction and direct analysis in real time-mass spectrometry
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Abstract
Since the late 1990s, the United States has been facing an epidemic involving an increasing number of opioid overdose deaths. This rapid increase calls for the development and optimization of rapid analytical techniques. Direct Analysis in Real Time (DART®) is an ambient ionization technique that, when coupled with mass spectrometry (MS), can quickly and accurately detect compounds in the solid, liquid, or gaseous state. It replaces traditional analytical techniques such as Liquid Chromatography (LC) and Gas Chromatography (GC), allowing for a higher throughput of samples. Sample preparation is not required for DART®-MS analysis, but it can improve the overall signal intensity of each analyte that may be present in a sample by removing complex matrix interference. Biocompatible Solid Phase Microextraction (Bio-SPME) is a sample preparation technique that allows for sample clean up and analyte pre-concentration in one step, prior to Direct Analysis in Real Time-Mass Spectrometry (DART-MS). Given the increase of synthetic opioid-overdose deaths in the past several years, the development and use of rapid analytical techniques is necessary for crime laboratories and medical examiner offices to consider implementing.
This project involved the optimization of Bio-SPME sample preparation and DART®-MS analytical conditions to successfully detect mixtures of fentanyl, norfentanyl, and fentanyl analog metabolites spiked into synthetic, drug-free urine. Octadecyl (C18) Bio-SPME fibers were selected for this method, and it was determined that a 30-minute fiber extraction resulted in the greatest signal intensity of each of the extraction times analyzed. A 2.5-millimeter (mm) Internal Diameter (I.D.) ceramic cap allowed for the greatest signal intensity of analytes when compared to the 0.5 mm I.D. ceramic cap. Multi-analyte Multiple Reaction Monitoring (MRM) methods that included two transitions per analyte were created, and a DART® source heater temperature of 250°C provided for the greatest signal intensity for each analyte. Additionally, altering the pH of the urine to 10 allowed for increased fentanyl and norfentanyl adsorption onto the Bio-SPME fiber. This method successfully detected each analyte at 500 ng/mL in the urine, however, it was determined that quantitation would be affected because competitive adsorption was observed in mixtures containing more than two compounds. Limited quantitative work was performed and demonstrated that norfentanyl can be quantitated in urine between 10 and 250 ng/mL, when it was the only analyte present in the urine. These optimized analytical conditions and multi-analyte MRM have demonstrated the rapid detection of fentanyl, norfentanyl, and fentanyl analog metabolite mixtures in urine.