食品接觸包材中全氟和多氟烷基物質之高解析質譜鑑定策略

Abstract

全氟和多氟烷基物質 (PFAS) 因為其化學結構，具有防油防水的功能，並廣泛的應用在食品接觸包材、消防泡沫、紡織品以及各種工業產品上。PFAS會生物累積並危害人體健康，像是造成癌症、肝損傷、生殖毒性以及嬰幼兒發育毒性等。而食品接觸包材為人們暴露PFAS常見的來源之一。雖然現在國際間開始針對部分PFAS進行規範，但仍然有幾千種未知PFAS在產業中使用，本研究將進行非目標物分析，來了解目前台灣的食品接觸包材中含有哪些PFAS。 本研究使用超高效能液相層析儀搭配Agilent 6450四極桿/飛行時間質譜儀 (UHPLC-Q-TOF MS) ，管柱為Premier BEH C18 AX column (100 × 2.1 mm, 1.7 µm)，在ESI負電模式下，水相和有機相為5mM醋酸銨(以氨水調整至pH 7.0) 和甲醇，起始有機相為10%，18分鐘內有機相上升至100%並維持0.5分鐘，之後返回起始梯度，總層析時間22分鐘檢測100 ng/mL 32種PFAS混合標準品，其中26種PFAS標準品能得到完整波峰，包含9種全氟烷基羧酸 (PFCAs)、4種全氟烷基磺酸鹽 (PFSAs)、3種氟調聚物飽和酸 (FTCAs)、3種氟調聚物不飽和酸 (FTUCAs)、2種雙多氟烷基磷酸酯 (diPAPs) 以及5種全氟醚 (Perfluoroethers)，但2種多氟烷基磷酸酯 (PAPs) 與4種氟調聚物醇類 (FTOHs) 化合物無法得到完整波峰。之後使用此層析條件分別進行數據依賴擷取 (DDA) 和非數據依賴擷取 (DIA) 兩種不同資料收集模式，得到的資料再利用MSDIAL和FluoroMatch軟體進行分析比較各自的資料庫鑑定結果。 初步鑑定使用32種PFAS混和標準品進行測試。MSDIAL設定鑑定參數cut off 70%，在DIA比對到化合物總共111個，包含較高匹配 (比對到MS1和MS2) 31個、與建議 (僅比對到MS1或較低MS2比對分數) 80個；26個已知PFAS化合物都有鑑定到，但其中18種已知PFAS標準品有比對到同分異構物問題，表示比對結果需要人工再次確認才會比較準確。而在DDA比對到化合物總共69個，包含較高匹配21個與建議48個，已知PFAS中僅有6:2 diPAP和8:2 diPAP有比對到MS1及MS2，可能因為DDA資料收集涵蓋範圍從m/z 150到m/z 1200對Q-TOF MS來說較廣，使得各個PFAS被檢測並引發碰撞獲得MS2資訊的機會降低。 FluoroMatch相較於DIA在DDA有較好的分析結果。在DDA中能比對到1個分數A、82個分數B及25個分數C，已知PFAS標準品PFUnDA為分數A比對結果，但其餘已知PFAS僅為分數D，表示可能為PFAS，但結構不確定。DIA結果則沒有比對到分數A，只有1個分數B和1個分數C，而已知PFAS則都是分數D。但在DIA和DDA中都無法對PFAS標準品有良好的比對結果，可能與儀器MS2資料收集不足和背景雜訊較多有關。 19項100 cm2真實食品接觸包材樣本經過磨碎，使用10 mL甲醇在70℃下透過自動加壓流體萃取系統 (EDGE) 進行5分鐘萃取，重複萃取一次合併萃取液後上機分析。以DIA模式得到的資料使用MSDIAL進行分析，和液相層析串聯式質譜儀 (LC-QqQ MS) 所檢測到PFBA、PFHxA、PFBS、6:2 FTCA、6:2 PAP及8:2 PAP結果比較，發現在12項一般防油紙袋中有比對到已知PFBA和PFBS以及非目標物 (E)-2:2 FTUCA、五氟丙酸 (PFPrA) 和全氟丁烷磺醯胺乙醇 (FBSE)，3項微波爆米花紙袋中有比對到非目標物2:2 Fluorotelomer thia propanoic acid，而2-((3,3,3-Trifluoropropyl) thio) acetic acid和PFSM-N-MeFSAA (C4H6F3NO4S) 非目標物則是在一般防油紙袋和微波爆米花紙袋中都有發現，1項雞蛋紙盒中有比對到非目標物3- (3,3,3-Trifluoropropylsulfanyl) propanoic acid。其中PFPrA可能為PFBA和PFHxA降解產物，而 (E)-2:2 FTUCA可能為PAPs與6:2 FTCA的降解產物。FBSE為PFBS的衍生物。 本研究使用MSDIAL在DIA模式下成功從19項真實食品接觸包材中鑑定到PFBA、PFBS以及7種非目標PFAS；非目標PFAS中包含PFCAs、FTUCAs、PFBS衍生物以及其他較不常見的PFAS種類FT-thioethers和PFSM-N-MeFSAA，表示目前台灣食品接觸包材中仍有新興PFAS的疑慮。目前針對食品包材的非目標物分析還尚未有很多研究，因此本研究的非目標物分析能更了解DDA和DIA的差異以及不同資料庫比對方法，未來也可以作為目標物分析的輔助方法，用來監測其他種類食品接觸包材中殘留或新興的PFAS化合物。

Per- and polyfluoroalkyl substances (PFAS) are widely used in food contact packaging materials (FCMs), firefighting foams, textiles, and other industrial products owing to their hydrophilic and hydrophobic properties. PFAS exhibit high bioaccumulative potential and may induce cancer, liver damage, reproductive toxicity, and developmental toxicity. FCMs are a common source of PFAS exposure. Although the international community has begun to regulate PFAS, thousands of unknown PFAS are still being used in the industry. Therefore, this study performed suspect non-target analysis to determine the unknown PFAS used in FCMs in Taiwan. This study uses an ultra-high-performance liquid chromatograph coupled with an Agilent 6450 quadrupole/time-of-flight mass spectrometer (UHPLC-Q-TOF MS) with a Premier BEH C18 AX column (100 × 2.1 mm, 1.7 µm). In negative ESI mode, the mobile phases were 5 mM ammonium acetate (adjusted to pH 7.0 with ammonia) and methanol. The initial organic phase was 10%; after 18 min, the organic phase was increased to 100%, maintained for 0.5 minutes, and then returned to the initial gradient. The total chromatographic time was 22 min. Complete peaks were obtained for 26 of the 32 PFAS chemical standards at 100 ng/mL, including nine perfluoroalkyl carboxylic acids (PFCAs), four perfluoroalkyl sulfonates (PFSAs), three fluorotelomer saturated acids (FTCAs), three fluorotelomer unsaturated acids (FTUCAs), two di-polyfluoroalkyl phosphate esters (diPAPs), and five perfluoroethers, except for two polyfluoroalkyl phosphate esters (PAPs) and four fluorotelomer alcohols (FTOHs). Data-dependent acquisition (DDA) and data-independent acquisition (DIA) were performed, and the resultant data were processed using MSDIAL and FluoroMatch software to compare the respective database identification results. Initial identification was conducted using a 100 ng/mL standard solution of 32 PFAS. MSDIAL identification score was set at 70%. In the DIA mode, 111 compounds were identified, comprising 31 reference-matched PFAS (corresponding to both MS1 and MS2) and 80 suggested PFAS (corresponding solely to MS1 or exhibiting a low MS2 score). 26 known PFAS were identified at the appropriate retention time, but 18 of the known PFAS standards matched the isomers, which means that the comparison results need to be manually confirmed again to be more accurate. Conversely, 69 compounds were identified in the DDA mode, including 21 reference-matched compounds and 48 suggested PFAS. However, only two known PFAS, 6:2 diPAP, and 8:2 diPAP, were identified in MS1 and MS2, respectively. The dynamic range for DDA spanned from m/z 150 to m/z 1200, which is comparatively extensive for the quadrupole and diminishes the probability of initiating collisions to obtain MS2. For FluoroMatch, DDA has better analysis results than DIA. FluoroMatch annotated one at score A, 82 at score B, and 25 at score C with DDA; Perfluoroundecanoic acid (PFUnDA) among the known PFAS standards was identified at score A, while the others were only identified at score D, indicating potential PFAS with unconfirmed structures. For DIA, no chemicals were identified at score A, and only one each at scores B and C; all known PFAS were identified solely at score D. However, good comparison results could not be obtained for PFAS standards in both DIA and DDA, which may be related to insufficient MS2 data collection and high background noise. Nineteen 100 cm2 samples of FCMs were ground and then extracted with 10 mL methanol at 70°C using an energized dispersive guided extraction (EDGE) automated system for 2 cycles of 5 minutes. The data obtained in DIA mode were analyzed using MSDIAL. Compared with the ultraperformance liquid chromatography-triple quadrupole mass spectrometer (LC-QqQ MS), which detects PFBA, PFHxA, PFBS, 6:2 FTCA, 6:2 PAP, and 8:2 PAP, 12 general oil-proof paper bags were identified as PFBA and PFBS, as well as the non-target compounds (E)-2:2 FTUCA, Perfluoropropionic acid (PFPrA), and perfluorobutane sulfonamidoethanol (FBSE). Non-target compound 2:2 Fluorotelomer thia propanoic acid were identified in three microwave popcorn paper. In addition, the non-target compounds 2-((3,3,3-Trifluoropropyl) thio) acetic acid and PFSM-N-MeFSAA (C4H6F3NO4S) appeared in the above two categories. A non-target compound, 3- (3,3,3-Trifluoropropylsulfanyl) propanoic acid, was identified in the egg carton. In addition, PFPrA is the degradation product of PFBA and PFHxA, while (E)-2:2 FTUCA may be the degradation product of PAPs and 6:2 FTCA. FBSE is a derivative of the PFBS. In this study, MSDIAL in DIA mode successfully identified PFBA, PFBS, and seven non-targeted compounds from 19 FCMs. Non-targeted PFAS include PFCAs, FTUCAs, PFBS derivatives, and other less common PFAS types such as FT-thioethers and PFSM-N-MeFSAA, showing concerns about emerging PFAS in FCMs in Taiwan. Currently, there are few studies on non-target analysis of food packaging materials. Therefore, the non-targeted analysis of this study can better understand the differences between DDA and DIA, as well as the comparison methods of different databases. In the future, it can also serve as a complementary approach to targeted analysis for monitoring legacy and emerging PFAS in FCMs.