氣相層析與極致液相層析/串聯式質譜儀分析全氟碳化物之比較

Comparison of Gas Chromatography and Ultra-performance Liquid Chromatography Coupled with Tandem Mass Spectrometry for Determining Perfluorinated Chemicals

賴怡潔(Yi-Chieh Lai)

指導教授 ： 陳家揚

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01753

摘要

全氟碳化物 (perfluorinated chemicals, PFCs) 因具有防水耐油的特性而被廣泛使用在工業製造過程及民生消費產品中。由於全氟碳化物具持久性及生物累積特性，在環境中的危害也相對地提高，其中以全氟辛烷磺酸 (perfluorooctyl sulfonate) 和全氟辛酸 (perfluorooctanoic acid) 最為嚴重。過去文獻也已指出全氟碳化物會對人體可能導致生殖及肝臟毒性、免疫抑制、甚至可能影響孩童的生長發育。然而，室內環境為現代人長時間久留之處，且許多中性 (neutral) 及離子型 (ionic) 全氟碳化物可能會藉由室內傢俱、噴霧式產品進而存在於人類居住的環境中而被吸入或藉由手觸碰到灰塵後而攝入，因此存在於室內的全氟碳化物不容忽視。
　　本研究比較了氣相及液相層析儀搭配不同游離源對於五種中性及九種離子型全氟碳化物的游離效能，並針對可能影響待測物訊號強度的參數及移動相等因素作進一步探討。五種中性全氟碳化物使用氣相層析/串聯式質譜儀 (gas chromatography coupled with tandem mass spectrometry, GC-MS/MS) 搭配電子游離 (electron ionization, EI) 及化學游離 (chemical ionization, CI) 分析，或在極致液相層析/串聯式質譜儀 (ultra-performance liquid chromatography coupled with tandem mass spectrometry, UPLC-MS/MS) 搭配電灑游離 (electrospray ionization, ESI) 、大氣壓力光游離 (atmospheric pressure photoionization, APPI) 以及大氣壓力化學游離 (atmospheric pressure chemical ionization, APCI) 分析。氣相層析使用HP-5ms (5%-苯基)-甲基聚硅氧烷管柱 (30 m × 0.25 mm i.d., 0.25 μm) ，而液相層析則使用兩種Kinetex C18管柱 (50 × 2.1 mm, 2.6 μm 及 30 × 2.1 mm, 1.7 μm)，在每分鐘0.5 mL的流速及層析溫度40°C下，使用梯度流析。另一方面，九種離子型全氟碳化物以UPLC-MS/MS使用ESI負離子模式 (-) 分析，並比較待測物於兩種Kinetex C18管柱 (50 × 2.1 mm, 2.6 μm 及 30 × 2.1 mm, 1.7 μm) ，在每分鐘0.5 mL的流速下，使用梯度流析的結果。對於全氟碳化物，本研究於UPLC系統進樣1微升之1 ng/μL中性全氟碳化物標準品的丙酮/甲醇(1:9, v/v) 溶液，而在GC系統則進樣1微升之1 ng/μL待測物的丙酮溶液；離子型全氟碳化物則於UPLC-MS/MS進樣2微升之0.1 ng/μL甲醇溶液。本研究最適化每種化合物之質譜/質譜參數、游離源參數以及層析條件等之後才進行比較。另外，每次實驗結果皆為三重覆，並加裝隔離管柱 (isolator column, 50 × 2.1 mm) 以避免UPLC系統潛在的全氟碳化物干擾。
　　在選擇性反應偵測 (selected reaction monitoring, SRM) 模式下，本研究選用兩種方法進行化合物定量及定性，其中一種為選擇最佳之偵測母離子 (precursor ion) 及由其產生之兩個子離子 (product ion) 分別做為定量和定性，另一種方式則是選擇兩個最佳之偵測母離子，再藉由此兩個母離子各別產生的一個子離子做定量和定性。本研究藉由降低EI的電子能量 (electron energy) 至30-59 eV的範圍中以得到訊號最佳的母離子，但相較之下，CI的訊號強度仍高於EI (最高達87倍)，唯一的例外為全氟辛烷磺胺 (perfluorooctane sulfonamide, PFOSA) ，不論在CI或EI中所產生的訊號皆較弱，且GC的層析峰 (peak) 會有拖尾現象。而相較於APPI-和APCI-系統下偵測的中性全氟碳化物，ESI- 提供了較佳的訊號強度 (至少8倍以上)，尤其是氟調聚合醇 (fluorotelomer alcohols, FTOHs)。ESI所使用的層析條件對於兩類型的全氟碳化物皆容易形成較尖的層析峰，但APPI和APCI則只能呈現明顯的全氟磺胺類 (perfluoroalkyl sulfonamides, PFASAs) 的層析峰。此外，中性及離子型的全氟碳化物在1.7 μm 的C18管柱下能夠產生較佳的訊號強度 (相較於2.6 μm的管柱最高達三倍) 且較窄的層析峰，尤其是perfluorohexanoic acid (相較於2.6 μm的管柱縮短了四分之一峰寬)。
甲醇為層析中性和離子型全氟碳化物的有機移動相首選，添加10 mM N-甲基嗎啡琳 (N-methylmorpholine) 於水相移動相則適合離子型全氟碳化物。本研究原本希冀利用添加0.04% 醋酸 (acetic acid), 5 mM 醋酸胺 (ammonium acetate) 和10 mM N-甲基嗎啡琳 (N-methylmorpholine) 於水相移動相中以增加中性全氟碳化物的訊號感度，但結果顯示Milli-Q純水仍為較佳的水相移動相。此外，相較於沒有添加dopant於APPI模式時，添加5% 流動相流速的甲苯 (toluene) 及10% 流動相流速的苯甲醚 (anisole) 最能提升中性全氟碳化物訊號強度 (最高達14.4倍及8.1倍)，尤其是FTOHs。
　　因此，本研究認為ESI可能是較合適的離子源，因為它能夠同時提供五種中性全氟碳化物較佳的訊號感度。本研究所開發的分析方法相較於GC-MS/MS及傳統LC-MS/MS能夠大幅降低液相層析時間至4.0分鐘 (中性全氟碳化物) 與5.6分鐘 (離子型全氟碳化物)，且具有良好的再現性 (相對標準偏差皆小於15%)。

關鍵字

全氟碳化物 ； 極致液相層析/串聯式質譜儀 ； 氣相層析/串聯式質譜儀 ； 電子游離 ； 化學游離 ； 電灑游離 ； 大氣壓力光游 ； 大氣壓力化學游離

並列摘要

Perfluorinated chemicals (PFCs) are widely used in furniture, carpets, sprayers, and clothes lint because of water- and oil- repellent properties. PFCs may result in several adverse health effects such as immunosuppression, developmental and hepatic toxicity. Neutral PFCs (fluorotelomer alcohols and perfluoroalkyl sulfonamides) could degrade into ionic PFCs (perfluoroalkyl carboxylates and perfluoroalkyl sulfonates) by biotic and abiotic transformation.
This study compared the performance of different ionization sources on analyzing five neutral and nine ionic PFCs with gas chromatography (GC) and ultra-performance liquid chromatography (UPLC) coupled with a triple-quadrupole tandem mass spectrometer (MS/MS) at selected reaction monitoring (SRM) mode. Atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) were utilized when the MS/MS coupled with UPLC and chemical ionization (CI) and electron ionization (EI) were the choices for GC. The five neutral PFCs were separated on a Kinetex C18 column (50 × 2.1 mm, 2.6 μm and 30 × 2.1 mm, 1.7 μm) at 40°C at the UPLC system with a flow rate of 0.5 mL/min and on a HP-5ms (5%-phenyl)-methylpolysiloxane column (30 m × 0.25 mm i.d., 0.25 μm) at the GC system. The nine ionic PFCs were separated on a Kinetex C18 column (30 × 2.1 mm, 1.7 μm) at 40°C and Kinetex C18 column (50 × 2.1 mm, 2.6 μm) at 45°C with a flow rate of 0.5 mL/min at the UPLC system, respectively. In addition, an isolator column (50 × 2.1 mm) was installed on the UPLC to avoid potential interferences of PFCs from the UPLC system. One microliter of 1 ng/μL neutral PFCs standards in acetone/methanol (1:9, v/v) was injected onto the GC system and one microliter of the same concentration of these analytes in acetone was injected onto the UPLC system. For ionic PFCs, two microliters of 0.1 ng/μL analyte standards in methanol were injected onto the UPLC system.
Two criteria were used for quantification and confirmation in this study, either one precursor ion with two product ions or two precursor/product ion transitions. The best electron ionization energy for forming the molecular ions of neutral PFCs was between 30 and 59 eV at the EI mode. CI provided a better signal intensity (up to eighty-seven times) than EI for most neutral PFCs except for perfluorooctane sulfonamide (PFOSA), which was in weak signal intensity and a tailing peak. Negative ESI provided greater signal intensity (at least eight times higher) than negative APPI and negative APCI for neutral PFCs, especially on fluorotelomer alcohols (FTOHs). In addition, the signal intensities of neutral and ionic PFCs were higher (up to around three times) and the peaks of these analytes were narrower on the 1.7 μm Kinetex C18 column than those on the 2.6 μm Kinetex C18 column, especially for PFHxA (only one forth of peak width at the 1.7-μm column relative to that at the 2.6-μm one).
Methanol as the organic mobile phase was the first choice for neutral and ionic PFCs; 10 mM N-methylmorpholine(aq) was used as the aqueous mobile phase for ionic PFCs; Milli-Q water produced much better signal intensities (2-25,000 times) for neutral PFCs than those of 0.04% acetic acid(aq) and 5 mM ammonium acetate(aq). Comparing with signals without dopants at APPI mode, dopants (at 5 or 10% of the mobile phase flow rate) of toluene and anisole increased the signals (up to 14.4 times and 8.1 times) at the APPI mode for neutral PFCs , especially on FTOHs.
ESI would be the most suitable ionization source among the testing ones for analyzing the five neutral PFCs simultaneously because it proved satisfactory responses for all of those analytes. Comparing with the GC/CI-MS/MS and traditional LC-MS/MS, use of UPLC/(-)ESI-MS/MS significantly reduced the total chromatographic time to 5.6 min for ionic PFCs and 4.0 min for neutral PFCs, and also provided reproducible signal responses (%RSDs were lower than 15%).

並列關鍵字

perfluorinated chemicals (PFCs) ； ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) ； gas chromatography-tandem mass spectrometry (GC-MS/MS) ； electron ionization (EI) ； chemical ionization (CI) ； electrospray ionization (ESI) ； atmospheric pressure photoionization (APPI) ； atmospheric pressure chemical ionization (APCI)

參考文獻

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