一、以極致效能液相層析/串聯式質譜儀分析臍帶血中全氟碳化合物之濃度。二、臍帶血中全氟碳化合物與胎兒出生結果之相關性

Part I. Determination of perfluorinated chemical levels in umbilical cord blood using ultra-performance liquid chromatography/tandem mass spectrometry,Part II. The association between perfluorinated chemical levels in umbilical cord blood and birth outcomes

溫婷雯(Ting-Wen Wen)

指導教授 ： 陳保中

國立臺灣大學/公共衛生學院/職業醫學與工業衛生研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02571

摘要

Part I.
目的：應用簡單且快速的前處理步驟，並搭配極致液相層析/串聯質譜儀（UPLC-MS/MS），建立血漿/血清中12 種全氟碳化合物定性兼定量之分析方法。本研究亦利用前述發展之分析方法進行台灣地區臍帶血檢體中全氟碳化合物的檢測分析，以建立本土全氟碳化合物的背景濃度。
方法：血漿/血清樣本前處理程序，為添加甲酸和甲醇溶液進行蛋白質沉澱後，添加穩定同位素標定內標準品，經由超音波震盪及高速離心，取上清液過濾後，以Waters 極致液相層析(Acquity UPLC) 結合Waters Quattro Premier XE 三段式四極棒質譜儀 (triple-quadrupole mass spectrometer) 分析。本研究使用BEH C18 管柱(2.1 × 50 mm, 1.7μm)，流速為0.5 μL/min，層析動相為10mM 甲基嗎啡啉與甲醇溶液。分析物之游離化方式，則採用電灑游離法 (electrospray ionization) 於負離子模式，進行選擇性反應偵測 (selected reaction monitoring, SRM)。
結果：添加全氟碳化合物於胎牛血清基質，分析方法之回收率為85%到104%，標準差範圍為0.02 到6.37%。同日異日檢量線之定量準確度與精確度方面，所有待測物的定量誤差範圍小於7%，變異係數則在0.022 到8.22%。本研究之定量極限定義為10 倍的訊號雜訊比。12 種全氟碳化合物的定量極限為0.009 到0.269 ng/mL ng/mL。本研究針對台灣地區444 個臍帶血血漿檢體進行12 種全氟碳化合物檢測。結果顯示，高檢出率（68%以上）的全氟十一酸（PFUA）、全氟辛磺酸（PFOS）、全氟壬酸（PFNA）以及全氟辛酸（PFOA），其最高濃度分別為35.1、22.6、21.5以及6.3 ng/mL。全氟己磺酸（PFHxS）僅21%的檢出率。全氟己酸（PFHxA）、全氟癸酸（PFDeA）、2-甲基-全氟辛磺乙酸胺（Me-PFOSA-AcOH）、2-乙基-全氟辛磺乙酸胺（Et-PFOSA-AcOH）、全氟辛磺酸胺（PFOSA）以及全氟十二酸（PFDoA）檢出率則小於10%。全氟庚酸（PFHpA）在所有臍帶血樣本中均小於定量極限。
結論：本研究開發了一個快速且具有足夠定量準確性和精確性的方法，可成功運用在分析血漿/血清中12 種全氟碳化合物的濃度。我們需要進一步關注全氟碳化合物特別是高檢出率與高濃度的全氟辛磺酸、全氟辛酸、全氟壬酸及全氟十一酸和本研究之研究族群未來生長發育之相關性。

Part II.
目的：本研究主要目的在於探討臍帶血中全氟碳化合物濃度包括全氟辛磺酸（PFOS），全氟辛酸（PFOA），全氟壬酸（PFNA）以及全氟十一酸（PFUA）與胎兒出生結果之相關性。
方法：本研究的研究對象包含439 位產後婦女，收案地點包括北台灣的四間診所和醫院。產後利用結構式問卷對母親進行訪談，並在生產時收集胎兒臍帶血。以極致效能液相層析/串聯式譜儀（UPLC-MS/MS）測量臍帶血血漿中全氟碳化合物之濃度。全氟碳化合物濃度經過log10 轉換以符合常態分布。使用線性迴歸分析臍帶血中全氟碳化合物和出生結果的關係。
結果：臍帶血中全氟辛磺酸，全氟辛酸，全氟壬酸以及全氟十一酸濃度中位數分別為1.22、2.27、2.55 以及6.41 ng/mL。在線性迴歸模式中，調整潛在干擾因子後發現只有PFOS 和懷孕周數 (每log10 單位：β = -1.82 週, 95% CI; -1.38 到-0.27週)，出生體重 (每log10 單位：β = -304 公克; 95% CI, -454 到-159 公克)，身長 (每log10 單位：β = -1.25 公分; 95% CI, -1.97 到0.53 公分)和頭圍 (每log10 單位：β =-0.93 公分; 95% CI, -1.4 到-0.42 公分)都呈現顯著負相關。PFNA 和身長 (每log10單位：β = 0.44 公分; 95% CI, 0.12 到0.76 公分)則呈現顯著正相關。
結論：本研究結果顯示臍帶血中全氟辛磺酸 (PFOS)濃度和新生兒出生結果呈現負向相關。需要全面且完整的研究進一步探討各種不同全氟碳化合物對人體健康的潛在影響。

關鍵字

蛋白質沉澱 ； 生長發育 ； 基質干擾 ； 台北出生世代研究 ； 全氟辛磺酸 ； 全氟壬酸 ； 出生體重 ； 身長

並列摘要

Part I.
Objectives: This study aimed at employing protein precipitation approach coupled with an ultra performance liquid chromatography － tandem mass spectrometry (UPLC/MSMS) to establish a qualitative and quantitative method for the determination of twelve perfluorinated compounds in plasma/serum samples. Using the developed method, the study also surveyed the levels of these PFCs within umbilical cord blood plasma in Taiwan Birth Panel Study (TBPS).
Methods: Sample preparation includes using protein precipitation with formic acid and methanol, mixing stable isotope labeled standard, and followed by sonication and centrifugation. Analyses were carried out using a Waters Acquity UPLC coupled with a Waters Quattro Premier XE triple-quadrupole mass spectrometer. The instrument is operated in selected reaction monitoring (SRM) with an electrospray ionization source in negative ionization mode. Use of a BEH C18 column (2.1x50 mm, 1.7 μm) with 10 mM methylmorphline/methanol gradient provides a fast chromatography separation (5.5 min) and robust peak shapes. Matrix-matched calibration with one internal standard was used to compensate the matrix effects.
Results: The recoveries of sample preparations of PFCs spiked into bovine serum ranged between 85 and 104% with relative standard deviations from 0.02 to 6.37%. Inter- and intra-day accuracy was less than 7% and inter- and intra-day coefficients of variations were within 0.02% to 8.22% for all analytes. The limits of quantitation (LOQs), defined as a signal-to-noise ratio of ten, ranged from 0.009 to 0.269 ng/mL for 12 PFCs. Four out of the twelve PFCs were detected in at least 68% of umbilical cord plasma samples (n=444) and their maximum concentration were 35.1 ng/mL for perfluoroundecanoic acid (PFUA), 22.6 ng/mL for perfluorooctane sulfonate (PFOS), 21.5 ng/mL for perfluorononanoic acid (PFNA), and 6.3 ng/mL for perfluorooctanoate (PFOA). Perfluorohexyl sulfonate (PFHxS) was found to be above the LOQs in 21% samples. Perfluorohexanoic acid (PFHxA), perfluorodecanoic acid (PFDeA), 2-(N-methyl-perfluorooctane sulfonamide) acetic acid (Me-PFOSA-AcOH), 2-(N-ethyl-perfluorooctane sulfonamido) acetic acid (Et-PFOSA-AcOH), perfluorooctane sulfonamide (PFOSA) and perfluorododecanoic acid (PFDoA) were detected in a few samples (< 10 %) and no perfluoroheptanoic acid (PFHpA) presented in any samples.
Conclusions: We have developed a simple and sensitive method for measuring trace levels of 12 PFCs in plasma/serum. PFCs are frequency detected in umbilical cord blood in TBPS especially PFOS, PFOA, PFNA and PFUA. The association between levels of these chemicals in umbilical cord blood and further development of these children requires further investigation.

Part II.
Objectives: The objective of this study was to explore the association between birth outcomes the concentration of PFCs including perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluoroundecanoic acid (PFUA) in umbilical cord blood plasma.
Ｍethods: The study population consisted of 439 postpartum women collected from four hospitals and clinics in northern Taiwan. We interviewed them by a structured questionnaire after delivery and collected cord blood at birth. The concentration of PFCs in umbilical cord blood was analyzed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). We examined the association between newborn birth outcomes and log10-transformed PFCs levels by linear regression models.
Results: The median PFOA, PFOS, PFNA and PFUA concentrations in cord plasma were 1.22、2.27、 2.55 and 6.41 ng/mL, respectively. After adjusting for potential confounders, only PFOS was negatively associated with gestational age (per log10 unit: β = -0.82 weeks, 95% CI; -1.38 to -0.27 weeks), birth weight (per log10 unit:β = -304 g; 95% CI, -454 to -159 g), length (per log10 unit: β = -1.25 cm; 95% CI, -1.97 to -0.53 cm), and head circumference (per log10 unit: β = -0.93 cm; 95% CI, -1.43 to -0.42 cm). PFNA was positively associated with length (per log10 unit: β = 0.44 cm; 95% CI, 0.12 to 0.76 cm).
Conclusion: Our data suggest inverse association between PFOS levels in umbilical cord blood plasma and birth outcomes. Further study with more comprehensive exposure data would be needed to find out potential effects of variety perfluorinated chemicals.

並列關鍵字

protein precipitation ； development ； matrix effect ； Taiwan Birth Panel Study ； perfluorooctyl sulfonate ； perfluorononanoic aci ； birth weight ； length

參考文獻

Part I. Determination of perfluorinated chemical levels in umbilical cord blood using ultra-performance liquid chromatography/tandem mass spectrometry

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