An LC-MS/MS method has been developed for the sensitive and selective determination of 35 mycotoxins (biomarkers of exposure) in pig urine samples. Sample preparation includes creatinine adjustment (with the developed LC-UV method) with enzymatic hydrolysis of pig urine samples followed by liquid-liquid (LLE) extraction. The LLE protocol, as well as enzymatic hydrolysis for indirect mycotoxin glucuronides determination, was optimized in this study. Additionally, two other sample preparation protocols were compared with the developed LLE method: immunoaffinity columns and solid-phase extraction cartridges (Oasis HLB). The detection and quantification of the biomarkers were performed using triple quadrupole mass spectrometry.

The method was validated with regard to the guidelines specified by the EMEA (European Medicines Agency). The extraction recoveries were higher than 60% for 77% of the analytes studied, with the intra- and inter-day relative standard deviation being lower than 20% for most of the compounds at four different concentration levels. The limits of quantification ranged from 0.1 ng/mL for zearalenone and sterigmatocystin to 8 ng/mL for nivalenol. To the best knowledge of the authors, the matrix effect was evaluated for the first time in this study for six different urine samples, and the coefficient of variation was found to be lower than 15% for most analytes studied. Finally, the developed method was applied to analyse 56 pig urine samples. Deoxynivalenol (1–20 ng/mL), zearalenone (0.1–1.5 ng/mL) and ochratoxin A (1.5–15 ng/mL) were the main analytes detected in these samples. Moreover, the co-occurrence of alternariol monomethyl ether and alternariol in pig urine is reported herein for the first time.

已开发出一种 LC-MS/MS 方法，用于灵敏和选择性测定猪尿样品中的 35 种真菌毒素（暴露生物标志物）。样品制备包括肌酐调整（使用已开发的 LC-UV 方法），对猪尿样品进行酶水解，然后进行液-液 (LLE) 提取。本研究对 LLE 方案以及用于间接测定霉菌毒素葡萄糖醛酸苷的酶水解进行了优化。此外，将另外两种样品制备方案与开发的 LLE 方法进行了比较：免疫亲和柱和固相萃取小柱 (Oasis HLB)。使用三重四极杆质谱法进行生物标志物的检测和定量。

该方法根据 EMEA（欧洲药品管理局）指定的指南进行了验证。所研究的 77% 的分析物的萃取回收率高于 60%，而四种不同浓度水平的大多数化合物的日内和日间相对标准偏差低于 20%。定量限范围从玉米赤霉烯酮和杂菌素的 0.1 ng/mL 到雪霉烯醇的 8 ng/mL。据作者所知，本研究首次评估了六种不同尿液样本的基质效应，发现大多数研究分析物的变异系数低于 15%。最后，将开发的方法应用于分析 56 份猪尿液样本。脱氧雪腐镰刀菌烯醇 (1–20 ng/mL)、玉米赤霉烯酮 (0.1–1.5 ng/mL) 和赭曲霉毒素 A (1. 5–15 ng/mL) 是这些样品中检测到的主要分析物。此外，本文首次报道了猪尿中交链孢单甲醚和交链交酯共存的情况。