Abstract This presented study describes a method based on high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) using N-(2-iodoacetyl)-1-pyrenemethylamine (NIPA) as a novel fluorescence labeling reagent for the determination of thyreostats in bovine milk. Five thyreostats, belonging to the group of imidazole and thiouracil, were investigated in this work: tapazole (TAP), thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU) and phenylthiouracial (PhTU). Thyreostats were specifically purified by a silver ion solid phase extraction (Ag-SPE) cartridge and then labeled using NIPA. The labeled derivatives showed excellent fluorescence property with maximum excitation and emission wavelengths of 330 nm and 375 nm, respectively. The labeled derivatives were separated on a reversed-phase Eclipse SB-C18 column within 12 min. Excellent linearity (R2 > 0.995) of all thyreostats was achieved with the limits of detection (LODs) and the limits of quantitation (LOQs) in the low micrograms per liter range of 0.21–0.30 μg/L and 0.70–1.00 μg/L, respectively. Satisfactory recoveries in the range of 93.5–98.0% were obtained for all thyreostats. The developed method has been successfully applied to analyze thyreostats in bovine milk with good applicability. Thirty bovine milk samples have been investigated, and varying levels of thiouracil were detected in thirteen of these samples. The highest level in the raw milk reached a value of 4.5 μg/L. To our best knowledge, this study is the first to report the presence of naturally occurring thiouracil in milk by HPLC-FLD analysis.

中文翻译：

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高效液相色谱结合荧光检测法测定牛奶中天然存在的甲状腺素抑制剂

摘要 本研究描述了一种基于高效液相色谱结合荧光检测 (HPLC-FLD) 的方法，该方法使用 N-(2-碘乙酰基)-1-芘甲胺 (NIPA) 作为一种新型荧光标记试剂，用于测定牛体内的甲状腺素抑制因子。牛奶。在这项工作中研究了属于咪唑和硫尿嘧啶组的五种甲状腺激素：他唑 (TAP)、硫尿嘧啶 (TU)、甲基硫尿嘧啶 (MTU)、丙硫氧嘧啶 (PTU) 和苯基硫尿嘧啶 (PhTU)。晶闸管通过银离子固相萃取 (Ag-SPE) 小柱进行专门纯化，然后使用 NIPA 进行标记。标记的衍生物显示出优异的荧光特性，最大激发和发射波长分别为 330 nm 和 375 nm。标记的衍生物在 12 分钟内在反相 Eclipse SB-C18 柱上分离。检测限 (LOD) 和定量限 (LOQ) 在 0.21–0.30 μg/L 和 0.70–1.00 μg/L 的低微克/升范围内实现了所有甲状腺素调节器的出色线性 (R2 > 0.995)，分别。所有晶闸管均获得了 93.5-98.0% 范围内的令人满意的回收率。所建立的方法已成功应用于牛乳中甲状腺激素的分析，具有良好的适用性。对 30 个牛奶样品进行了调查，其中 13 个样品中检测到不同水平的硫尿嘧啶。原料奶中的最高含量达到了 4.5 μg/L。据我们所知，这项研究是第一个通过 HPLC-FLD 分析报告牛奶中存在天然硫尿嘧啶的研究。