Laboratory of Food Analysis, Department of Bioanalysis, Ghent University, Gent, Belgium.
Anal Chim Acta. 2012 Sep 5;741:58-69. doi: 10.1016/j.aca.2012.06.038. Epub 2012 Jul 1.
Detection of mycotoxin biomarkers in urine of humans and animals provides a direct approach for assessing exposure to these mycotoxins as opposed to the indirect approach of food analysis, which in most cases is affected by the heterogeneity of the toxin in the food samples. Seven (7) mycotoxins and their metabolites (total 18 analytes) were selected and an LC-MS/MS method for their determination in human urine was developed and validated. The method consisted of direct analysis of two mycotoxin conjugates, deoxynivalenol-glucuronide and zearalenone-glucuronide without beta glucuronidase digestion of the urine samples. Since high method sensitivity is of utmost importance in such study, critical factors which could improve the analyte recovery and method sensitivity were investigated by a D-optimal experimental design. Urine samples (10 mL) were first extracted with 15 mL ethyl acetate/formic acid (99/1, v/v) followed by SAX SPE clean-up of the acidified aqueous fraction. Both extracts were combined and analyzed using an LC-MS/MS system operated in the positive ionization mode. A total run time of 28 min was adopted with all the 18 analytes eluting within 15 min. The method was validated by taking into consideration the guidelines specified in Commission Decision 2002/657/EC and 401/2006/EC. Forty samples obtained from volunteers within the laboratory research group were analyzed as part of a pilot study. All results were expressed per mg creatinine. A total of 9 samples were found contaminated with one or more of the following analytes: DON, OTA, OTα, 4-OH OTA, ZEN, CIT and β-ZOL. One-eighth (5/40) of the samples were contaminated with DON in the range of 3.7-67 ng mg(-1) creatinine. Samples with detectable levels of DON did not show any co-occurrence of DON-3Glu. One sample was found to be contaminated with 4-OH OTA (<LOQ), co-occurring with only OTA (0.2 ng mg(-1) creatinine). OTα (up to 4.4 ng mg(-1) creatinine) was detected in three other samples co-occurring with low levels of OTA (up to 0.3 ng mg(-1) creatinine) and no 4-OH OTA detected. ZEN was detected in 10% (4/40) of the samples analyzed. Three samples were contaminated with β-ZOL (3.3-20 ng mg(-1) creatinine), co-occurring with ZEN (<LOQ-10.8 ng mg(-1) creatinine). The ratio of ZEN/β-ZOL varied for all the three samples. α-ZOL was not detected in any of the 40 samples. CIT was detected in one sample at 4.5 ng mg(-1) creatinine. This is the first study carried out with a small group of the Belgian population to assess exposure to mycotoxins using biomarkers.
尿液中真菌毒素生物标志物的检测为评估人类和动物接触这些真菌毒素提供了一种直接的方法，而不是通过食物分析的间接方法，因为在大多数情况下，食物样本中的毒素异质性会影响分析结果。本研究选择了 7 种（7）种真菌毒素及其代谢物（共 18 种分析物），并开发和验证了一种用于人尿液中这些物质测定的 LC-MS/MS 方法。该方法包括直接分析两种真菌毒素缀合物，即脱氧雪腐镰刀菌烯醇-葡糖苷酸和玉米赤霉烯酮-葡糖苷酸，而无需对尿液样品进行β-葡糖苷酸酶消化。由于在这种研究中，方法的灵敏度至关重要，因此通过最优化实验设计研究了可以提高分析物回收率和方法灵敏度的关键因素。首先用 15 毫升乙酸乙酯/甲酸（99/1，v/v）提取 10 毫升尿液，然后用 SAX SPE 对酸化的水相部分进行净化。将两种提取物合并，并使用 LC-MS/MS 系统在正离子模式下进行分析。采用总运行时间为 28 分钟，所有 18 种分析物在 15 分钟内洗脱完毕。本方法的验证考虑了委员会 2002/657/EC 和 401/2006/EC 规定的准则。作为一项初步研究的一部分，对实验室研究小组的 40 名志愿者的样本进行了分析。所有结果均以每毫克肌酐表示。共有 9 份样本被发现受到以下一种或多种分析物的污染：DON、OTA、OTα、4-OH OTA、ZEN、CIT 和β-ZOL。八分之一（5/40）的样本中 DON 的含量范围为 3.7-67ngmg(-1)肌酐。DON 可检测水平的样本均未显示 DON-3Glu 的共同出现。一份样本被发现被 4-OH OTA（<LOQ）污染，仅与 OTA（0.2ngmg(-1)肌酐）共存。在另外 3 个样本中检测到 OTα（高达 4.4ngmg(-1)肌酐），与低水平的 OTA（高达 0.3ngmg(-1)肌酐）共存，未检测到 4-OH OTA。在分析的 40 个样本中，有 10%（4/40）检测到 ZEN。三个样本被 β-ZOL 污染（3.3-20ngmg(-1)肌酐），与 ZEN（<LOQ-10.8ngmg(-1)肌酐）共存。所有三个样本的 ZEN/β-ZOL 比值均有所不同。在所有 40 个样本中均未检测到α-ZOL。CIT 在一个样本中的含量为 4.5ngmg(-1)肌酐。这是首次使用生物标志物对比利时人群进行的一项评估接触真菌毒素的小型研究。
