Long-Term Stability of 13 Fentanyl Analogs in Blood

Fentanyl analogs continue to play a major role in proliferating the opioid epidemic in the USA. With high rates of overdose deaths, forensic laboratories experience backlogs, which may lead to false-negative results due to drug instability. To address this issue, a quantitative method was validated for fentanyl analogs (3-methylfentanyl, 4-anilino-N-phenethylpiperidine (4-ANPP), 4-fluoro-isobutyrylfentanyl (4-FIBF), acetylfentanyl, acrylfentanyl, butyrylfentanyl, carfentanil, cyclopropylfentanyl, fentanyl, furanylfentanyl, methoxyacetylfentanyl, p-fluorofentanyl and valerylfentanyl) in blood using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) and used to assess long-term stability under various temperature conditions (-20°C, 4°C, ∼25°C and 35°C) for 9 months. Authentic specimens were also analyzed 6 months apart for applicability to postmortem blood. Method validation resulted in calibration ranges of 1-100 ng/mL and limits of detection of 0.5 ng/mL. Precision and bias were acceptable (within ±7.2% coefficient of variation (CV) and ±15.2%, respectively). Matrix effects exhibited ion enhancement for all analytes, except carfentanil and 4-ANPP in low-quality control (>25%). For long-term stability, fentanyl analogs (except acrylfentanyl) remained stable under room temperature and refrigerated conditions at low and high concentrations (81.3-112.5% target) for 9 months. While most fentanyl analogs remained stable frozen, degradation was observed after 2 weeks (four freeze/thaw cycles). At elevated temperatures, most analytes were stable for 1 week (74.2-112.6% target). Acrylfentanyl was unstable after 24 h under elevated (70% loss) and room temperatures (53-60% loss), 48-72 h when refrigerated (28-40% loss) and 4 weeks when frozen (22% loss). In authentic bloods (n = 7), initial furanylfentanyl (FuF) and 4-ANPP concentrations were 1.1-3.6 and 1.4-6.4 ng/mL, respectively. Percentage loss of FuF and 4-ANPP over 6 months were 16.3-37.4% and 0.2-26.8%, respectively. Samples suspected to contain fentanyl analogs are recommended to be stored refrigerated or frozen with limited freeze/thaw cycles. Due to instability, in the event of an acrylfentanyl overdose, samples should be analyzed immediately or stored frozen with analysis within 1 month.