Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
Laboratory of Toxicology, National Institute of Criminalistics and Criminology, Brussels, Belgium.
Clin Chem. 2018 Aug;64(8):1221-1229. doi: 10.1373/clinchem.2018.289496. Epub 2018 May 18.
Detection of new highly potent synthetic opioids is challenging as new compounds enter the market. Here we present a novel screening method for the detection of opiates and (synthetic) opioids based on their activity.
A cell-based system was set up in which activation of the μ-opioid receptor (MOR) led to recruitment of β-arrestin 2, resulting in functional complementation of a split NanoLuc luciferase and allowing readout via bioluminescence. Assay performance was evaluated on 107 postmortem blood samples. Blood (500 μL) was extracted via solid-phase extraction. Following evaporation and reconstitution in 100 μL of Opti-MEM I, 20 μL was analyzed in the bioassay.
In 8 samples containing synthetic opioids, in which no positive signal was obtained in the bioassay, quadrupole time-of-flight mass spectrometry revealed the MOR antagonist naloxone, which can prevent receptor activation. Hence, further evaluation did not include these samples. For U-47700 (74.5-547 ng/mL) and furanyl fentanyl (<1-38.8 ng/mL), detection was 100% (8/8) for U-47700 and 95% (21/22) for furanyl fentanyl. An analytical specificity of 93% (55/59) was obtained for the opioid negatives. From an additional 10 samples found to contain other opioids, 5 were correctly scored positive. Nondetection in 5 cases could be explained by very low concentrations (<1 ng/mL alfentanil/sufentanil) or presence of inactive enantiomers.
The MOR reporter assay allows rapid identification of opioid activity in blood. Although the cooccurrence of opioid antagonists is currently a limitation, the bioassay's high detection capability, specificity, and untargeted nature may render it a useful first-line screening tool to investigate potential opioid intoxications.
由于新化合物不断进入市场，检测新型高活性合成阿片类药物具有挑战性。在此，我们提出了一种基于其活性检测阿片类药物和（合成）阿片类药物的新型筛选方法。
建立了一种基于细胞的系统，其中μ-阿片受体（MOR）的激活导致β-arrestin 2 的募集，从而导致分裂 NanoLuc 荧光素酶的功能互补，并通过生物发光进行读出。在 107 份尸检血液样本中评估了该方法的性能。通过固相萃取提取 500 μL 血液。蒸发后，在 100 μL Opti-MEM I 中重悬，用 20 μL 在生物测定中进行分析。
在 8 份含有合成阿片类药物的样本中，生物测定未得到阳性信号，而四极杆飞行时间质谱揭示了可以阻止受体激活的 MOR 拮抗剂纳洛酮。因此，进一步的评估不包括这些样本。对于 U-47700（74.5-547 ng/mL）和呋喃芬太尼（<1-38.8 ng/mL），U-47700 的检测率为 100%（8/8），呋喃芬太尼的检测率为 95%（21/22）。阿片类阴性的分析特异性为 93%（55/59）。在另外 10 份被发现含有其他阿片类药物的样本中，有 5 份被正确标记为阳性。在 5 例未检测到的情况下，可能是由于浓度非常低（<1 ng/mL 阿芬太尼/舒芬太尼）或存在无活性对映体。
MOR 报告基因检测可快速鉴定血液中的阿片类药物活性。尽管阿片类拮抗剂的共同存在目前是一个限制，但生物测定的高检测能力、特异性和非靶向性质可能使其成为一种有用的一线筛查工具，用于调查潜在的阿片类药物中毒。
