University of Kentucky Veterinary Diagnostic Laboratory, Toxicology Laboratory, University of Kentucky, 1490 Bull Lea Rd, Lexington, KY 40511, USA.
U.S. Food and Drug Administration, Center for Veterinary Medicine, 8401 Muirkirk Rd, Laurel, MD 20708, USA.
J Anal Toxicol. 2023 May 19;47(5):429-435. doi: 10.1093/jat/bkad017.
Anti-coagulant rodenticides (ARs) are commonly utilized for controlling rodent populations; however, non-target companion and wildlife animals are also exposed. A method was developed for quantitation of seven ARs (chlorophacinone, coumachlor, bromadiolone, brodifacoum, difethialone, diphacinone and warfarin) and dicoumarol (a naturally occurring anti-coagulant) in animal serum. Analytes were extracted with 10% (v/v) acetone in methanol and analyzed by reverse phase high-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (negative mode) combined with multiple reaction monitoring. In-house method validation in the originating laboratory using non-blinded samples revealed method limits of quantitation at 2.5 ng/mL for all analytes. The inter-assay accuracy ranged from 99% to 104%, and the relative standard deviation ranged from 3.5% to 20.5%. Method performance was then verified in the originating laboratory during an exercise organized by an independent party using blinded samples. The method was successfully transferred to two naïve laboratories and further evaluated for reproducibility among three laboratories by means of Horwitz ratio (HorRat(R)) values. Such extensive validation provides a high degree of confidence that the method is rugged, robust, and will perform as expected if used by others in the future.
抗凝血灭鼠剂（ARs）常用于控制鼠类种群；然而，非目标伴生动物和野生动物也会接触到这些药剂。本研究建立了一种同时检测七种 ARs（氯鼠酮、氯敌鼠、溴鼠灵、大隆、敌鼠酮、双香豆素和华法林）和一种天然抗凝血剂（双香豆素）在动物血清中的含量的方法。分析物用 10%（v/v）甲醇中的丙酮提取，采用电喷雾正离子化（ESI+）源和多反应监测模式的反相高效液相色谱-串联质谱法（LC-MS/MS）进行分析。在原始实验室采用非盲法样本进行实验室内部验证，结果表明所有分析物的定量限（LOQ）均为 2.5ng/mL。批内准确度的范围为 99%至 104%，相对标准偏差的范围为 3.5%至 20.5%。随后，在一个由独立第三方组织的盲样实验中，在原始实验室中对该方法进行了验证。该方法成功转移至两个新的实验室，并通过 Horwitz 比值（HorRat(R)）值在三个实验室间进一步评估了其重现性。这种广泛的验证提供了高度的置信度，即该方法坚固耐用，在未来如果被其他人使用，将按预期运行。
