大鼠尿液中食品加工衍生含氮危害物之極致液相層析串聯質譜分析方法開發

異環胺、亞硝胺及丙烯醯胺等三類致癌物為生活飲食中常見之食品製程衍生含氮危害物，常於煎、炒、烤、炸、滷等高溫長時間的烹調下生成。據文獻指出，大量攝入這類食品製程衍生含氮危害物可能提升人體罹患大腸癌、膀胱癌及乳癌等多種癌症的風險，故目前已有多數物質被國際癌症研究機構(International Agency For Research On Cancer, IARC)歸類為二級致癌物。然多數文獻僅偏重於單一物質之討論，同時分析三類致癌物之研究較缺乏，且動物試驗大多給與高劑量暴露，與人體實際暴露情形不符。為了解異環胺、亞硝胺及丙烯醯胺在人體內之實際代謝情形並開發尿液之同時監測方法，本研究選用24隻Sprague-Dawley遠交群大鼠(簡稱SD大鼠)進行分組動物實驗，以0.00 (控制組)、0.01、0.05、0.10mg/kg之低劑量將12種異環胺、6種亞硝胺及丙烯醯胺採管餵方式進行單次共同暴露，收集96小時內之大鼠尿液作為後續研究之分析樣品，以模擬人體同時暴露到三類致癌物後在體內之代謝情境並分析之。本研究以極致液相層析串聯質譜法(Ultra-performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)進行尿液中食品加工衍生含氮危害物之定量分析方法開發，於前處理過程中運用酵素水解法還原葡萄醣醛酸之代謝產物以得知總代謝量，同時比較固相萃取法(Solid-Phase Extraction, SPE)及支撐式液相萃取技術(Supported Liquid Extraction, SLE)之淨化效果，並製作基質匹配檢量線針對最低定量極限(Lower limit of quantitation, LLOQ)及低(6 ng/mL)、中(40 ng/mL)、高(80 ng/mL)三個確效濃度點進行方法確效。結果顯示相較於SPE，SLE於分析物之回收率及尿液基質效應的降低皆有更良好的表現，並於酵素水解測試觀察到尿液中異環胺之葡萄醣醛酸代謝產物存在。此方法於多數分析物之定量皆展現良好的靈敏度及穩定性，各物質基質匹配檢量線之決定係數(coefficient of determination, r2)皆大於0.990，顯示其線性關係良好，偵測極限及定量極限介於0.1至2.5 ng/mL及 2至10 ng/mL之間，除丙烯醯胺、一項異環胺 (MeIQ)及一項亞硝胺 (NMOR)等少數物質外，於各確效濃度點皆能獲得良好的同日及異日間之精確度(%RSD 0.0-18.2 %)及準確度(88-126%)。真實樣品除少數亞硝胺外，多數暴露之異環胺、亞硝胺及丙烯醯胺皆能於大鼠尿液中偵測到其濃度，並於暴露後6小時之尿液中達到高峰，與各物質之理論半衰期及過往文獻之相對暴露代謝比例相符。此研究成功建立一套穩定分析尿液中微量異環胺、亞硝胺及丙烯醯胺等食品製程衍生含氮危害物之定量方法，同時比較了SLE及SPE兩萃取技術之適用性，可望提供後續進行生物監測、暴露風險評估等相關研究參考。

關鍵字

異環胺；丙烯醯胺；亞硝胺；極致液相層析串聯質譜法；低劑量暴露

並列摘要

Heterocyclic amines (HCAs), N-nitrosamines (NAs) and acrylamide (AA) are three categories of process-induced nitrogen hazards (PINHs) that are commonly found on people’s daily diet. Researchers had indicated that these substances might increase the risk of many cancers, such as colorectal cancer, bladder cancer, and breast cancer by proceeding a lot of animal tests. Consequently, some of HCAs, NAs, and AA had already been classified as Group 2A or 2B carcinogens by the International Agency for Research on Cancer (IARC). However, although a large amount of individual research about HCAs, NAs or AA had been well studied for the past few years, the co-exposure study is still limited, and the usage of the dose was usually very high, which is not in line with the actual exposure situation. To establish a stable method to simultaneously quantify the PINHs in urine, this research utilized 24 male Sprague-Dawley rats (SD rats) which aged at 5 weeks as an animal model and gave the relatively low doses of 0.00 (control), 0.01, 0.05 and 0.10 mg/kg b.w. of twelve HCAs, six NAs, and one AA by oral gavage to obtain the exposed urine sample between 96 h for analysis and simulate the real exposure situation at the same time. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) was used to quantitate PINHs in urine samples. Solid-phase extraction (SPE) and supported liquid extraction (SLE) were used to compare the recovery and matrix effect for pretreating urine samples. To realize the total contents of the PINHs in urine, enzymatic hydrolysis was also conducted before the extraction to hydrolyze glucuronide metabolites. The validation of the method was completed by the establishment of the matrix-matched calibration curve and validated four concentration levels: (lower limit of quantitation) LLOQ, low QC (6 ng/mL), medium QC (40 ng/mL), and high QC (80 ng/mL). The results indicated that compared to SPE, the SLE technique showed the ideal outcomes in higher recoveries and lower matrix effects. And the glucuronide metabolites of HCAs were also found in rats’ urine through the test of the efficiency of enzymatic hydrolysis. The validated method demonstrated the great sensitives and stability on most of the analytes. Which the matrix-matched calibration curve showed good linearity with the coefﬁcient of determination (r2) greater than 0.990. And the (limits of detection) LODs and LLOQs of most of the PINHs were within the range of 0.03-0.70 ng/mL and 0.10-2.00 ng/mL, respectively. Besides, except for a few analytes (AA, MeIQ, and NMOR), most analytes displayed great within-run and between-run precisions (%RSD 0.0-18.2 % ) and accuracies (88-126%). When applied the method to SD rat urine samples, except for some of NAs, almost all of the PINHs administrated to SD rats were detected in the exposed urine with the maximal concentrations observed within the samples collected in the first 6 hours after dosing, which matched the half-lives and the relative metabolic ratio of PINHs reported in the previous researches. This study not only successfully simultaneously quantified and compared the extraction efficiency of SPE an SLE of the PINHs in real urine samples by UPLC-MS/MS, but the method also demonstrated the good feasibility and sensitivity for the detection of trace levels (ng/mL) of PINHs. That made this study be a usable reference to conduct further researches such as human biomonitoring or risk assessment of the exposure of PINHs and benefited public health.

並列關鍵字

Heterocyclic amines ； N-nitrosamines ； Acrylamide ； UPLC–MS/MS ； Low dose animal study

參考文獻

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大鼠尿液中食品加工衍生含氮危害物之極致液相層析串聯質譜分析方法開發

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