以液相層析串聯式質譜儀檢測河水、底泥、魚體肌肉與肝臟之硝化/氧合多環芳香烴與個人保健品

Abstract

硝化/氧合多環芳香烴(nitrated and oxygenated polycyclic aromatic hydrocarbons)為高致突變性與致癌性的多環芳香烴衍生物，並且可以經由吸附大氣中懸浮微粒沉降進入環境水體中；個人保健用品(personal care products)例如：鎮痛解熱劑(analgesics)、防曬劑(UV filters)、美容用品保存劑 (cosmetic preservatives)、驅蟲劑(例如DEET)與咖啡因(caffeine)等，則因為大量使用也易於在環境中發現。本研究針對河水、底泥與魚體組織(肌肉與肝臟)中之5種硝化多環芳香烴、3種氧合多環芳香烴、13種個人保健品成分開發分析方法並加以驗證。河水樣本以PolarPlus C18之圓盤式萃取匣搭配自動固相萃取設備進行前處理；底泥與魚體組織以基質固相散布法萃取(matrix solid-phase dispersion)，以SiliaBond C18為吸附劑並以甲醇與丙酮沖提，沖提之樣本以氧化鋁萃取匣進行淨化。樣本在濃縮之後以極致液相層析�串聯式質譜儀（ultra performance liquid chromatography/tandem mass spectrometry, UPLC-MS/MS）搭配同位素稀釋技術定量分析。本研究使用Kinetex PFP層析管柱分離形成正電荷離子待測物，搭配10-mM醋酸銨加上0.25%乙酸水溶液(水相)與甲醇(有機相)做為移動相；形成負電荷離子之待測物以CORTECS C18層析管柱分離，搭配0.25%乙酸水溶液(水相)與甲醇(有機相)做為移動相。帶正電之待測物以電灑游離（electrospray ionization, ESI+）與大氣壓光游離法（atmospheric pressure photoionization, APPI+）為其游離源；而帶負電之分析物則以（ESI-）游離之。 電灑游離法可適用於本研究之所有個人保健用品，以及1-硝基芘(1-nitropyrene)、2-硝基芴(2-nitrofluorene) 與5,12-萘并萘醌(5,12-naphthacenequinone)，且提供高於大氣壓光游離法2.39-36.8倍之訊號強度。然而，7-硝基苯[a]蒽(7-nitrobenz[a]anthracene)與 9-硝基蒽(9-nitroanthracene)只能以大氣壓光游離法游離之；相較氯苯(chlorobenzene)與甲苯(toluene)，苯甲醚(anisole)最適合做為大氣壓光游離法之摻雜劑，分析硝化/氧合多環芳香烴。極致液相層析將層析時間縮短至8分鐘內（包括管柱再平衡時間）。地表水之萃取效率介於61.8-126%，底泥、魚之肌肉與肝臟之萃取效率分別為30.6-67.8%、21.5-70.5%與31.8-127%。地表水與底泥之基質效應因子介於21-92.9%與88.6-147%之間；肌肉與肝臟之基質效應因子除乙醯胺酚(acetaminophen)為28%與10%，其餘介於75.1-161%與31.4-81.7%之間。地表水中待測物方法偵測極限為0.14-6.88 ng/L，底泥、肌肉與肝臟則為0.56-14.7 ng/g（濕重）、1.18-23.9 ng/g（濕重）與1.13-19.4 ng/g（濕重）。此方法測試三種添加濃度之定量偏差與相對標準偏差，地表水樣本分別小於30%與25%，而底泥、肌肉與肝臟樣本分別小於20%與15%。 本分析方法用於調查基隆河河水、底泥與吳郭魚組織中硝化/氧合多環芳香烴與個人保健用品之含量。所有河水樣本中均測得乙醯胺酚、異布洛芬(ibuprofen)凱妥普洛芬(ketoprofen)、那普洛辛(naproxen)、羥苯甲酮(oxybenzone)、二苯基酮 (benzophenone)、DEET與咖啡因，其中以咖啡因(506 ± 751 ng/L ，n = 6)與DEET(119 ± 44.3 ng/L，n = 6)濃度最高。咖啡因、羥苯甲酮、二苯基酮、羥苯甲酸甲酯(methyl paraben)與凱妥普洛芬皆可見於所有底泥樣本，二苯基酮(benzophenone) (258 ± 64.8 ng/g w.w., n = 6)為底泥中濃度最高之待測物。乙醯胺酚、DEET、二苯基酮、羥苯甲酸甲酯與凱妥普洛芬可在大多數的魚體組織中發現，在肌肉樣本中濃度最高的是二苯基酮(132 ± 46.4 ng/g w.w., n = 15) ，而在肝臟中濃度最高的則是乙醯胺酚(325 ± 162 ng/g w.w., n = 15)。本次調查發現，鎮痛解熱劑、防曬劑與DEET較易累積在肝臟中，而羥苯甲酸甲酯與咖啡因則傾向於累積於肌肉中。此外，硝化/氧合多環芳香烴在水體、底泥、魚體組織皆無發現。根據本研究之生物累積係數(bioaccumulation factor, BAF)討論，擁有較高辛醇－水分佈係數(log Kow)之待測物，如二苯基酮、羥苯甲酮、凱妥普洛芬與羥苯甲酸甲酯，有較高的生物累積係數(412 ± 132 至28,142 ± 22,756)；乙醯胺酚與咖啡因之辛醇－水分佈係數雖然遠低於上述待測物，但也擁有高生物累積係數(492 ± 667 至 18,513 ± 14,281)。由此可見，大量使用之個人保健用品可能累積於魚肉和其肝臟，甚至可能進入食物鏈，其潛在之生態和健康風險值得進一步關注。

Nitrated and oxygenated polycyclic aromatic hydrocarbons (NPAHs and OPAHs) are highly mutagenic and carcinogenic; they can adsorb to air particulates for long-range transport, then enter the aquatic environment through atmospheric deposition. Some personal care products (PCPs), such as analgesics, UV filters, cosmetic preservatives, DEET, and caffeine, are used at a large scale. This study developed and validated an analytical method for analyzing five NPAHs, three OPAHs, and 13 personal care product ingredients in water, sediment and fish tissues (muscle and liver). Analytes in water samples were extracted using PolarPlus C18 disks with high-flow automated solid-phase extraction; sediment, muscle and liver were treated with matrix solid-phase dispersion (MSPD) on SiliaBond C18, and eluents of methanol and acetone were directly passed through alumina cartridges for cleanup. After concentrations, the eluents were injected onto ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) with multiple reaction monitoring and analytes were quantified with isotope-dilution techniques. Analytes forming positive ions were separated on a Kinetex PFP column (50 × 2.1 mm, 2.7 μm) using positive electrospray ionization (ESI+) and positive atmospheric pressure photoionization (APPI+), with 10-mM ammonium acetate/0.25% acetic acid(aq) and methanol for gradient elution; analytes forming negative ions were separated on a CORTECS C18 column (30 × 2.1 mm, 1.6 μm) using negative ESI (ESI-), and the mobile phases were 0.04% acetic acid(aq) and methanol. ESI provided better signal intensities on all PCPs (2.39-36.8 times higher) than APPI, and was also suitable for analyzing 1-nitropyrene, 2-nitrofluorene and 5,12-naphthacenequinone; however, 7-nitrobenz[a]anthracene and 9-nitroanthracene can only be detected using APPI, and the use of anisole as the dopant offered better signal intensity than that using chlorobenzene and toluene. Each chromatographic separation was less than eight minutes including column re-equilibrium. The extraction efficiency of the analytes from surface water were 61.8-126%.; the extraction efficiency of MSPD on sediment, muscle and liver were 30.6-67.8%, 21.5-70.5%, and 31.8-127%, respectively. Matrix effect factors of surface water and sediment were 21-92.9% and 88.6-147%, respectively; matrix effect factors of from muscle and liver were 75.1-161%, and 31.4-81.7%, respectively, except that acetaminophen were lower than 28% and 10%., respectively. The limits of detection (LODs) of analytes in water ranged from 0.14-6.88 ng/L, and the LODs in sediment, muscle and liver ranged from 0.56-14.7 ng/g wet weight (w.w.), 1.18-23.9 ng/g (w.w.) and 1.13-19.4 ng/g (w.w.), respectively. This study tested method accuracy and precision at three spiked levels, and most of the quantitative biases and relative standard deviations were lower than 30% and 25% on water samples, respectively; most of those on sediment, muscle and liver samples were lower than 20% and 15% respectively. This method was applied to investigate the NPAHs/OPAHs and PCPs in river water, sediment and tilapia (Oreochromis spp.) in the Kee-Lung River, Taipei. Acetaminophen, ibuprofen, ketoprofen, naproxen, oxybenzone, benzophenone, caffeine, and DEET were detected in all of the water samples; the most abundant analytes were caffeine (506 ± 751 ng/L, n = 6) and DEET (119 ± 44.3ng/L, n = 6), respectively. Caffeine, oxybenzone, benzophenone, methyl paraben and ketoprofen were detected in all sediment samples; benzophenone was the most abundant compound (258 ± 64.8 ng/g w.w., n =6). Acetaminophen, DEET, benzophenone, methyl paraben and ketoprofen were detected in most of fish tissues. Benzophenone was the the highest among the analytes in fish muscle (132 ± 46.4 ng/g w.w., n = 15), and acetaminophen (325 ± 162 ng/g w.w., n = 15) was the highest in liver. Analgesics, UV filters and DEET in fish liver were found to be higher than that in muscle, but caffeine and methyl paraben tended to accumnlate in muscle. The NPAHs and OPAHs were not detected in water, sediment and fish tissues. According to bioaccumulation factors (BAFs) of the analytes in our study, benzophenone, oxybenzone, ketoprofen and methyl paraben which have high log Kow, tended to have higher BAFs; however, although acetaminophen and caffeine have much lower log Kow than above analytes, they still had high BAFs in fish muscle and liver. The bioaccumulation of PCPs in fish muscle and liver, which implies the potential for entering the food chain, may pose ecological and health risks.