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.

中文翻译：

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13 种芬太尼类似物在血液中的长期稳定性

芬太尼类似物在美国阿片类药物流行的扩散中继续发挥重要作用。由于药物过量致死率很高，法医实验室会出现积压，这可能会因药物不稳定而导致假阴性结果。为了解决这个问题，对芬太尼类似物（3-甲基芬太尼、4-苯胺基-N-苯乙基哌啶 (4-ANPP)、4-氟-异丁酰芬太尼 (4-FIBF)、乙酰芬太尼、丙烯酰芬太尼、丁酰芬太尼、卡芬太尼、环丙基芬太尼、芬太尼、呋喃芬太尼、甲氧基乙酰基芬太尼、对氟芬太尼和戊基芬太尼）使用液相色谱-四极杆飞行时间质谱（LC-QTOF-MS）在血液中并用于评估各种温度条件下的长期稳定性（– 20°C、4°C、~25°C 和 35°C）9 个月。真实标本也每隔 6 个月进行一次分析，以确定其对死后血液的适用性。方法验证的校准范围为 1–100 ng/mL，检测限为 0.5 ng/mL。精度和偏差是可以接受的（分别在 ±7.2% 变异系数 (CV) 和 ±15.2% 范围内）。除了低质量对照中的卡芬太尼和 4-ANPP（>25%）外，基质效应对所有分析物都表现出离子增强。对于长期稳定性，芬太尼类似物（丙烯酰芬太尼除外）在室温和冷藏条件下在低浓度和高浓度（81.3-112.5% 目标值）下保持稳定 9 个月。虽然大多数芬太尼类似物在冷冻状态下保持稳定，但在 2 周后观察到降解（四个冷冻/解冻循环）。在升高的温度下，大多数分析物可稳定 1 周（74.2–112.6% 目标值）。丙烯芬太尼在升高（70% 损失）和室温（53-60% 损失）、冷藏 48-72 小时（28-40% 损失）和冷冻 4 周（22% 损失）下 24 小时后不稳定。在真实血液 (n = 7) 中，初始呋喃芬太尼 (FuF) 和 4-ANPP 浓度分别为 1.1-3.6 和 1.4-6.4 ng/mL。6 个月内 FuF 和 4-ANPP 的百分比损失分别为 16.3-37.4% 和 0.2-26.8%。建议将怀疑含有芬太尼类似物的样品冷藏或冷冻，并进行有限的冷冻/解冻循环。由于不稳定性，如果丙烯芬太尼过量，应立即分析样品或冷冻保存，并在 1 个月内进行分析。呋喃芬太尼 (FuF) 和 4-ANPP 的初始浓度分别为 1.1-3.6 和 1.4-6.4 ng/mL。6 个月内 FuF 和 4-ANPP 的百分比损失分别为 16.3-37.4% 和 0.2-26.8%。建议将怀疑含有芬太尼类似物的样品冷藏或冷冻，并进行有限的冷冻/解冻循环。由于不稳定性，如果丙烯芬太尼过量，应立即分析样品或冷冻保存，并在 1 个月内进行分析。呋喃芬太尼 (FuF) 和 4-ANPP 的初始浓度分别为 1.1-3.6 和 1.4-6.4 ng/mL。6 个月内 FuF 和 4-ANPP 的百分比损失分别为 16.3-37.4% 和 0.2-26.8%。建议将怀疑含有芬太尼类似物的样品冷藏或冷冻，并进行有限的冷冻/解冻循环。由于不稳定性，如果丙烯芬太尼过量，应立即分析样品或冷冻保存，并在 1 个月内进行分析。