B-305 A Novel Dilute-n-shoot Method for Quantitating Isotonitazene and Desmetonitazene in Human Urine By LC/MS

Abstract

Abstract Background Isonitazene is a benzimidazole derived opioid analgesic drug related to etonitazene, which has been sold as a designer drug. According to doctors isotonitazene has the potency of 20–200 times of fentanyl. The side effects of isotonitazene are like fentanyl and has been linked to many fatalities in Europe and the US. It has led to a number of overdoses through secondhand contact, by smelling or coming in contact with skin. It is slowly replacing fentanyl as the deadliest street drug in the US. We developed a dilute-n-shoot method that would test for isotonitazene and its metabolite metodesnitazene in human urine. This method used filtration to remove unwanted particulates and protein. It reduces the number of steps needed to prepare samples for liquid chromatography/mass spectrometry analysis compared to methods that require centrifugation. This assay allows for easier training of staff. In addition, it is easily adaptable to increase sample load. Method Isotonitazene and metodesnitazene urine samples were prepared for LC/MS analysis by diluting them 1:20 in 0.1% formic acid in water. These samples were spiked with internal standard and filtered through a 0.2 μm Captiva 96-well filter plate. Isotonitazene and metodesnitazene concentrations were measured using LC/MS in the positive ion mode. The separation was obtained using a phenylhexyl UPLC column, 50 × 2.1 mm, 1.8 μm, along with a 50:50 acetonitrile/methanol/water formic acid gradient mobile phase. Results The separation was completed in a proximately 4.5 min with good resolution between isotonitazene and metodesnitazene. Validation studies demonstrated that this method was linear from 2 to 100 ng/mL, with r2 ≥ 0.990 for both isotonitazene and metodesnitazene. Interday and intraday precision for both analytes were acceptable, with CV values < 5.0%. Accuracy measurements at 5, 25 and 80 ng/mL gave results where the percent difference was ≤20% for the 4 ng/mL concentration and ≤15% for the 25 and 80 ng/mL. A study of interfering compounds showed that there were no interferences that would affect the accuracy of isotonitazene and metodesnitazene. Recovery studies showed that > 90% of each analyte was recovered from the filtration step. The LOQ was determined to be 2 ng/mL and the LOD was determined to be 0.5 ng/mL. The reportable range was determined to be 2 to 200 ng/mL. The results of this validation will be discussed in detail in this poster. Conclusion The method was linear and precise for both analytes over a range of 2 to 100 ng/mL. The method was reproducible and easy to use. Isotonitazene and metodesnitazene could easily be quantitated in human urine using this method.