| 摘要: | 對-羥基苯甲酸酯 (Alkyl p-hydroxybenzoates, 簡稱parabens) 因其無色無味、價格低廉、不易硬化或泥漿化，自20世紀以來被當作防腐劑之用途，常添加於化妝品、食品及藥物中，被人類廣泛使用。但近年來的相關研究顯示，parabens可能具有雌激素活性、干擾內分泌系統，並影響人類生殖系統功能，被列為潛在內分泌干擾物質，故檢測parabens在人體尿液中的含量是勢在必行的。
本研究開發出一套有效、溶劑使用量少、對環境友善且可快速檢測的方法來檢測人體尿液中的parabens。利用超音波輔助乳化微萃取法 (Ultrasound-assisted emulsification microextraction, USAEME) 進行樣品前處理，搭配線上衍生 (on-line derivatization) GC-MS和超高效液相層析高解析串聯質譜儀 (UHPLC-QTOF-MS) 進行定性與定量。
本方法利用實驗統計設計 (statistical experimental design) Box-Behnken design (BBD) 及變異數分析 (Analysis of variance, ANOVA) 來做萃取最佳化探討。最佳化後萃取條件為：將1 mL酵素水解後的尿液 (含有0.1 g的氯化鈉)，快速注入200 μL萃取劑乙酸乙酯，經由超音波震盪1分鐘後，以7000 rpm 離心10分鐘，取出上層液並吹氮回溶，使用GC-MS分析時取出10 μL並添加醋酸酐衍生試劑，再進入GC-MS中進行線上衍生並偵測；而使用UHPLC-QTOF-MS檢測時，只需直接進樣2 μL 就可以進行分析。
以GC-MS分析4種parabens的偵測極限為 0.01 - 0.02 ng/mL；在inter-days及intra-day的測試中，所得之相對標準偏差 (RSDs) 小於8 %；以UHPLC-QTOF-MS分析9種parabens時的偵測極限為0.12-0.25 ng/mL ，所得之相對標準偏差小於 10 %，顯示此萃取方法及以兩種儀器分析皆具有良好的再現性及穩定性。在收集的人體尿液中，利用GC-MS檢測時，大部分樣品皆被測得MeP、EtP、PrP、BuP，其在男性尿液的濃度介於 0.3 - 124.5 ng/mL 間；女性尿液的濃度介於 27.6 – 246.8 ng/mL 間，此結果顯示在女性尿液中，對-羥基苯甲酸酯濃度較高，這可能與其生活方式息息相關；而利用UHPLC-QTOF-MS檢測時，除了檢測到常見的四種parabens外，還發現其中一支樣品含有微量的PeP與HxP，由此結果可發現仍然有少許被禁用和毒性較高的parabens可能在台灣被使用中。
;Alkyl p-hydroxybenzoates, also known as parabens, was added in cosmetic, pharmaceutical and food as preservative since the 20th century. Because of its odorless, low prices and difficult hardening, they have widely been used by various applications. Recent studies have concluded that parabens might disrupt the endocrine system, affect funtions of human reproductive systems, and are classified as a potential endocrine disrupter. Therefore, it is important to develop a method to analyze paranbens in human urine.
This study demonstrates an efficient, less solvent used, eco-friendly and rapid analysis method for the determination of parabens in human urine. The method involved the use of ultrasound-assisted emulsification microextection (USAEME) coupled with on-line derivatization GC-MS or UHPLC-QTOF-MS.
The parameters of extaction method were optimized by Box-Behnken design and analysis of variance. The optimal conditions of USAEME were: after hydrolysis of 1 mL urine sample, 200 μL ethyl acetate (as an extractant) rapid injected into urine containing 0.1 g of sodium chloride in a conical bottom tube. After ultrasonication for 1 min and centrifugation at 7000 rpm for 10 min, the upper layer was taken and dried under nitrogen and reconsitituted with methanol. For using GC-MS analysis, a fraction of 10 μL was mixed with 1.0 μL acetic anhydride reagent and directly injected to GC-MS system to obtain on-line derivatization. For using UHPLC-QTOF-MS analysis, a fraction of 2 μL was directly injected into UHPLC system..
The detection limits with GC-MS was 0.01 - 0.02 ng/mL. Intra-day and inter-days precision (% RSD) for four analytes were less than 8 %; the detection limits with UHPLC-QTOF-MS was 0.12-0.25 ng/mL, the RSD for nine analytes were less than 10 %, indicated that these methods show a good stability and reproducibility. In real samples, MeP、EtP、PrP and BuP were detected in most urine samples by GC-MS, the total concentrations ranged from 0.3 to 124.5 ng/mL for male; and from 27.6 to 246.8 ng/mL for female. Female urine samples showed higher total concentrations for the four target parabens, which may indicate higher exposure due to lifestyle. Moreover, small amount of PeP and HxP was detected in one urine sample by UHPLC-QTOF-MS, this results indicate that some disabled and higher toxicity parabens were in use in Taiwan. |
