8 Evaluation of N-desbutylbupivacaine Interference in Fentanyl/Norfentanyl Detection in Three Matrices: Comparative Analysis of Immunoassay and Liquid Chromatography-Tandem Mass Spectrometry Methods

Introduction Norfentanyl, a primary metabolite of fentanyl, is often targeted in laboratory tests designed to detect fentanyl exposure. N-desbutylbupivacaine (NDB) is a metabolite of bupivacaine, a common epidural anesthetic that has been shown to cause isobaric interference in mass spectrometry analysis for norfentanyl, potentially leading to false positives or inaccurate quantification. This is particularly of concern in neonates, who may receive NDB via placental transfer during labor, and are assessed after birth for fentanyl exposure. This study systematically investigated the extent of NDB interference in fentanyl detection across three biological matrices (umbilical cord tissue, meconium, and urine) using liquid chromatography tandem mass spectrometry (LC-MS/MS) and immunoassay screening (urine only). Methods NDB was spiked into drug-free urine at 0, 5, 10, 20, 100, and 500 ng/mL. Samples were analyzed using the ARK™ Fentanyl II Assay (fentanyl cutoff = 1 ng/mL). LC-MS/MS analysis was performed using three different in-house LC-MS/MS methods [Assay 1: fentanyl confirmation assay (fentanyl/norfentanyl cutoff = 1 ng/mL), Assay 2: pain management panel (fentanyl/norfentanyl cutoff = 2 ng/mL), Assay 3: emergency toxicology screening panel (fentanyl cutoff = 2 ng/mL)]. Homogenized cord tissue and meconium samples were spiked with NDB at concentrations of 1000 ng/g and 1500 ng/g, respectively. Samples were analyzed using in-house LC-MS/MS panels that target fentanyl (cutoff = 0.5 ng/g for cord, 10 ng/g for meconium). Results No crossreactivity was observed using the ARK Fentanyl II Assay. Analysis of the same samples by LC-MS/MS showed different impact of NDB on each method. In Assay 1, NDB eluted right before norfentanyl causing the ion ratio to deviate from the acceptable range. In Assay 2, NDB did not cause an ion ratio flag, but artificially increased the detected norfentanyl concentration. In Assay 3, NDB did not interfere with norfentanyl but did cause an interfering peak for lidocaine. No interference from NDB was observed in the cord or meconium assays. Conclusion Fentanyl results from NDB-containing specimens are unlikely to be affected by NDB interference with either immunoassay or LC-MS/MS, suggesting fentanyl results are reliable when norfentanyl cannot be reported due to NDB interference. Disparity in effects of NDB on norfentanyl detection and quantitation in three different LC-MS/MS methods demonstrate the importance of assessing NDB interference during method development to avoid reporting false positives or incorrect concentrations.