利用極致液相層析-三段四極柱串聯式質譜儀同時分析人體尿液中的 10 種黴菌毒素之方法開發

Abstract

黴菌毒素是由黴菌自然產生的次級代謝物，常見於受污染的食品中，例如穀類、咖啡豆以及發酵製品。這些毒素可能引起多種不良健康效應，包括肝毒性、腎毒性以及致癌性。雖然多國已進行黴菌毒素暴露的生物監測研究，並透過尿液及血液樣本進行分析，其中以歐洲的研究最為豐富，而目前僅有兩篇論文分析台灣人口體內黃麴毒素的暴露情形，並無檢測其他不同黴菌毒素的原型物。因此，本研究旨在開發並驗證一套分析方法，利用極致液相層析-三段四極柱串聯式質譜儀（UHPLC-MS/MS），以定量檢測人類尿液中10種黴菌毒素。該方法進一步應用於分析自彰化基督教醫院招募、年滿20歲之受試者的尿液樣本，以評估人體暴露程度。此次目標檢測的10種黴菌毒素包括NIV、DON、AFM1、AFB1、FB1、AOH、CIT、ZEN、OTA及STER。液相層析採用ZORBAX Eclipse Plus C18管柱，流動相為含0.2%醋酸的（A）水及（B）甲醇，梯度洗脫於7分鐘內完成。檢測採正負離子模式的電灑游離（ESI）進行分析。考量尿液基質之複雜性，本研究優化了固相萃取（SPE）程序，使用Sep-Pak C18 1 cc Vac萃取匣進行樣本前處理以降低基質干擾。方法驗證內容包括檢量線、日內與日間準確度及精密度，以及殘留效應之評估。根據方法驗證結果AFM1、ZEN及STER之最低定量濃度（LLOQ）均為0.5 ng/mL；而NIV、DON、AFB1、FB1、AOH、CIT及OTA之最低定量濃度則為5 ng/mL。在所分析的94筆尿液樣本中，共有42筆檢出黴菌毒素，檢出物包括DON、OTA、CIT、ZEN、AFM1及AFB1，檢出率介於1.06%至25.53%之間。其中以DON的檢出率最高，濃度範圍為1.15至15.37 ng/mg c。為探討黴菌毒素暴露與腎臟健康之間的關聯，本研究進行了卡方檢定及費雪精確檢定等統計分析。結果顯示，黴菌毒素暴露與腎絲球過濾率（GFR）分期或尿白蛋白排泄分類之間，均未呈現統計上顯著相關，部分結果中OR有輕微正相關。本研究為首次針對台灣一般成人族群進行多種黴菌毒素原型物的暴露調查，以填補此領域之研究缺口。研究結果提供了重要的基礎數據，可作為未來食品安全監測、風險評估及政策制定之參考。

Mycotoxins are secondary metabolites naturally produced by fungi and are commonly found in contaminated foods such as cereals, coffee beans, and fermented products. These toxins may cause various adverse health effects, including hepatotoxicity, nephrotoxicity, and carcinogenicity. Although many countries have conducted biomonitoring studies on mycotoxin exposure through the analysis of urine and blood samples, the majority of such research has been conducted in Europe. Currently, only two published studies have analyzed aflatoxin exposure in the Taiwanese population, and none have investigated the presence of other types of mycotoxins. Therefore, this study aimed to develop and validate an analytical method for quantifying 10 mycotoxins in human urine using ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-MS/MS). This method was subsequently applied to analyze urine samples from participants aged 20 years and older, recruited from Changhua Christian Hospital, to assess human exposure levels. The 10 targeted mycotoxins were NIV, DON, AFM1, AFB1, FB1, AOH, CIT, ZEN, OTA, and STER. Chromatographic separation was achieved using a ZORBAX Eclipse Plus C18 column, with a mobile phase consisting of (A) water and (B) methanol, both containing 0.2% acetic acid. Gradient elution was completed within 7 minutes. Electrospray ionization (ESI) was used in both positive and negative ionization modes for analyte detection. Due to the complexity of the urine matrix, a solid-phase extraction (SPE) procedure using Sep-Pak C18 1 cc Vac Cartridges was optimized to reduce matrix interferences prior to analysis. Method validation included the assessment of calibration curves, intra-day and inter-day accuracy and precision, and carryover effects. The lower limits of quantification (LLOQ) were 0.5 ng/mL for AFM1, ZEN, and STER, and 5 ng/mL for NIV, DON, AFB1, FB1, AOH, CIT, and OTA. Of the 94 urine samples analyzed, mycotoxins were detected in 42, including DON, OTA, CIT, ZEN, AFM1, and AFB1, with detection frequencies ranging from 1.06% to 25.53%. DON showed the highest detection frequency, with concentrations ranging from 1.15 to 15.37 ng/mg creatinine. To assess the association between mycotoxin exposure and renal health, statistical analyses including Chi-square and Fisher’s exact tests were performed. No statistically significant associations were found between mycotoxin exposure and either glomerular filtration rate (GFR) stages or urinary albumin excretion categories, although some odds ratios (OR) indicated a slight positive association. This study represents the first investigation into the exposure of multiple parent-form mycotoxins among the general adult population in Taiwan, thereby filling a research gap in this field. The findings provide valuable baseline data that can support future food safety monitoring, risk assessment, and policy-making.