以超高效能液相層析/串聯式飛行時間質譜術篩查血漿中環境汙染物

Abstract

人們可能從環境中暴露到各種有害汙染物質並造成不良健康效應。不論暴露途徑為何，人體生物偵測可以得到暴露化學物質於人體內濃度。利用極致高效液相層析高解析質譜儀進行無選擇性的資料蒐集可以用來篩查關切的化學物質。此研究發展並建立以非標的物方式偵測血漿樣品中的環境汙染物篩查平台。 血漿樣品與1%甲酸乙腈溶液混合並經由96孔Ostro萃取盤濾除蛋白質與磷脂質，濃縮回溶後的樣品利用正離子與負離子電灑游離法與超高效能液相層析/四極柱飛行時間串聯質譜儀進行分析，儀器分析時間為20分鐘。化合物於Phenomenex Luna Omega Polar C18管柱進行分離分析，質譜以全離子碎片模式 (三種撞擊能量0, 20, 40伏特) 收集質荷比 70至1100的資料。方法以樣品前處理、管柱選擇與品質管制評估進行最佳化。 此研究分析500個來自中研院人體生物資料庫的血漿樣品，受試者為30至70歲正常台灣人。血漿樣品包含品管樣品皆添加6種同位素標記標準品以評估批次內與批次間樣品的分析再現性。除了13C4-PFBA受到質量接近的干擾物影響，大部分同位素內標的質量精確度落在5 ppm以內；同位素標準品的積分面積批次內相對標準偏差落在5.9% 至18.9% 之間 (n = 43及26)，批次間相對標準偏差落在22.0-43.1%之間 (n = 12)；於2個月12批次的樣品分析期間 (n = 572)，正電分析模式下同位素標準品的滯留時間差異在0.3分鐘內，於負電模式下在1分鐘內。 500個樣品分析資料與3500個化合物資料庫進行二次質譜的比對，比對標準為質量準確度在10 ppm以內，除母離子之外至少再比對到一個同位素或加成物、以及至少有一個子離子與母離子共層析疊合(coelution)。ToxPi計分系統被用做試探性比對到的化合物質排序，包含利用化合物偵測頻率、訊號豐度、暴露資料及毒性資料來進行化學物質排序。於此研究樣品中比對到計分較高的關切物質為 (1) 對羥基苯甲酸酯類 (parabens); (2) 塑化劑: 己二酸酯、鄰苯二甲酸酯、磷酸酯阻燃劑; (3) 全氟碳化合物及 (4) 其他: 殺蟎劑 (metolcarb)、8-羥基喹啉及、對特辛基苯酚等。 此研究於樣品前處理及儀器分析上建立了人體血漿樣品的篩查平台，提供一個新的篩查工具來調查人體常暴露到的關切化學物質且收集到的樣品資料具有可回溯的特性。所得到的排序化合物清單可幫助暴露評估、化學物控制及人體健康。

People may expose to many concerned chemicals from the environment and cause health effects. Biomonitoring of human specimens can reflect internal doses of exposed chemicals regardless of the exposure route. With data-independent acquisition using a UHPLC-HRMS, we can screen concerned compounds without chemical standards. This study developed and established a platform for identifying environmental pollutants in human plasma using a non-targeted approach. Plasma samples were mixed with 1% formic acid/acetonitrile, then were extracted with Waters Ostro 96-well plates for removing proteins and phospholipids; The concentrated and reconstituted filtrates were analyzed with an Agilent UHPLC-QTOF MS in both positive and negative electrospray ionization. Compounds were separated on a Phenomenex Luna Omega Polar C18 column (100 × 2.1 mm, 1.6 μm) within 20 minutes. Information at all ion MS/MS (AIM) mode from m/z 70 to m/z 1100 were collected at three collision energies (CE = 0, 20, and 40 V). The analytical method was optimized on sample preparation, column selection, and quality assurance. This study analyzed 500 plasma samples obtained from the Taiwan Biobank, and the age of subjects was from 30 to 70 years old. Samples including quality control samples were pre-spiked with six stable isotope-labeled surrogate standards for evaluating the reproducibility within and between batches of analysis. Most of the mass accuracy was within 5 ppm except for 13C4-perfluorobutanoic acid, which resulted from a very close m/z in backgrounds. The %RSD of peak areas of the six surrogates were 5.9%-18.9% within batches (n = 43 or 26) and were 22.0-43.1% between batches (n = 12). The differences of retention time of the three surrogate isotope standards were lower than 0.3 minute in positive ion mode and lower than 1 minute in negative ion mode in 12 batches through two-month analysis. The 500-sample data were compared with MS/MS spectral libraries of about 3,500 suspect chemicals. The criteria for tentative identification were a compound precursor ion co-eluted with at least one of the product ions, mass accuracy within  10 ppm, and at least one isotope or adduct founded. A novel ToxPi scoring system was used for the prioritization of tentatively identified compounds based on detection frequency, abundance, exposure, and bioactivity data. The concerned chemicals tentatively identified in this study with higher priority scores were (1) Parabens: propylparaben, butylparaben, isobutylparaben, and methylparaben; (2) Plasticizer: tri-(2-chloroisopropyl) phosphate (TCPP), tributylphosphate (TBP), Tris(2-butoxyethyl) phosphate (TBEP), dinonyl phthalate (DNP), di(2-ethylhexyl)phthalate (DEHP) and di (2-ethylhexyl) adipate (DEHA); (3) Perfluorochemicals (PFCs): perfluorooctanoic acid (PFOA), perfluoro-octanesulfonic acid (PFOS), and perfluorodecanoic acid (PFDA); (4) Others: 8-hydroxyquinoline, metolcarb, and 4-tert-Octylphenol. This research set up the procedure of suspect screening on human plasma on both sample preparation and instrumental analysis with UHPLC-QTOF MS, offering a new screening tool to find the chemicals that people are commonly exposed to with a “known unknown” pattern and retrospective data. The prioritized chemicals, which could be utilized to facilitate the exposure assessment, chemical regulations, and human health.