建立尿液中游離雙酚A的偵測方法且應用於2005-2008台灣一般民眾的尿液分析

Method Establishment and Application for Determination of Free Bisphenol A in urine from the General Population of Taiwan, 2005-2008

蔡宛伶(Wan-Ling Tsai)

指導教授 ： 彭瓊瑜

高雄醫學大學/健康科學院/公共衛生學系公共衛生學碩士班/碩士(2018年)

摘要

背景
雙酚A（bisphenol a, BPA）是一種具有內分泌干擾特性的合成化學品，用來當作生產聚碳酸酯和環氧樹脂的原料單體，廣泛應用於一般消費品當中如水瓶、食品容器和罐頭內襯中，另外在嬰兒奶瓶、玩具、牙科填補用具及熱感應收據當中也都含有雙酚A，所以在一般環境當中人們很容易暴露於雙酚A當中，且雙酚A在許多流行病學研究當中顯示會對人體造成許多健康危害，所以越來越受到大家的重視；雙酚A主要經由尿液代謝出人體外，故本研究的目的是建立分析尿液中游離BPA的方法，並了解台灣一般人群中尿液BPA的分佈情況。

方法
本研究使用液相層析串聯質譜儀（LC-MS / MS）分析尿液中BPA濃度，分析BPA需要先找到液相層析和質譜的最佳化條件，並且建立良好的QA/QC程序，以確定數據的準確性。使用固相萃取管(SPE)進行尿液中BPA的萃取，萃取後經由離心機濃縮。回收效率以添加BPA的人工尿液和真人混和尿液進行測試。建立的BPA萃取分析方法應用於2005年至2008年“台灣營養與健康調查”（NAHSIT）的518份尿液樣本中分析游離BPA。

結果
BPA分析的最佳液相條件為梯度洗脫：60-100 ％甲醇（0-4分鐘），100％甲醇（4-9分鐘），100-60％甲醇（9-11分鐘）和60％甲醇（11-12分鐘）；流速為0.5 ml/min。質譜條件多反應監測（MRM）系統，其參數為： BPA的Q1為227、Q3為212和133。此條件下BPA的檢量線R2為0.999，表示具有良好的線性關係；LOD、LOQ分別為0.07、0.23 ng/ml。將該方法應用於台灣一般人群的尿液中BPA濃度結果測得檢出率為49%，樣本的幾何平均濃度(幾何標準差)分別為1.88（2.13）μg/ L(未調整肌肝酸)和2.11（2.17）μg/ g-creatinine (肌肝酸調整)。

結論
雖然此建立分析尿液中游離雙酚A的方法有良好的線性關係，但此方法的最低偵測極限(LOD)不夠低，所以還是無法檢測到台灣大部分人群尿液中雙酚A的濃度，故此分析方法還需進一步的討論與改進。

關鍵字

雙酚A ； 液相層析串聯質譜儀 ； 尿液分析 ； 基質效應 ； 固相萃取

並列摘要

Objective
Bisphenol A (BPA) is a synthetic chemical with endocrine disrupting properties, used as a raw material for the production of polycarbonates and epoxy resins, which were widespread used in general consumer products such as water bottles, food containers, and can lining. The purposes of this study were to establish a method for the analysis of free BPA in urine and to understand the distribution of BPA in the general population in Taiwan.

Methods
A liquid chromatography tandem mass spectrometry (LC-MS /MS) system was used in this study. The concentration of 1 ppm was used to find the best conditions in liquid chromatography and mass spectrometry. The recovery rate was determined by analysis of artificial urine and pool urine spiked with BPA standard. Samples were purified with solid-phase extraction (SPE) cartridges to remove unwanted or interfering chemicals. The established method was used to analyze 518 urine samples which were from the project “Nutrition and Health Survey in Taiwan” (NAHSIT) in 2005-2008.

Results
The optimum LC conditions for BPA analysis were gradient elution, the program was as follows: 60-100% methanol (0-4 min), 100% methanol (4-9 min), 100-60% methanol (9-11 min), and 60% methanol (11-12 min). The flow rate was 0.5 ml/min. The multiple reaction monitoring (MRM) is optimal MS parameters for mass spectrometry using 227-212 and 227-133 for Bisphenol A molecular weight monitoring. On C8 column the method had good linearity with R2 of 0.999, LOD、LOQ were 0.07 and 0.23 ng/ml separately. This method was used to determine urinary BPA levels in the general population in Taiwan. The detection rate was 49%. Of the detected samples, GM(GSD) were 1.88 (2.13) μg/L and 2.11 (2.17) μg/g creatinine.

Conclusion
The established method had good linearity; however, the LOD of this methods is not low enough to determine free BPA for most of the population in Taiwan. Further improvement in the method is needed.

並列關鍵字

Bisphenol a (BPA) ； LC-MS/MS ； urine analysis ； matrix effect ； solid-phase extraction

參考文獻

1. Li DK, Zhou Z, Miao M, He Y, Wang J, Ferber J, Herrinton LJ, Gao E, Yuan W: Urine bisphenol-A (BPA) level in relation to semen quality. Fertil Steril 2011, 95(2):625-630 e621-624.

2. Diamanti-Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM, Zoeller RT, Gore AC: Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr Rev 2009, 30(4):293-342.

3. Shen YP, Zheng YM, Jiang JT, Liu YM, Luo XM, Shen Z, Chen X, Wang Y, Dai YH, Zhao J et al: Higher Urinary Bisphenol A Concentration Is Associated with Unexplained Recurrent Miscarriage Risk: Evidence from a Case-Control Study in Eastern China. Plos One 2015, 10(5).

4. Huang YQ, Wong CKC, Zheng JS, Bouwman H, Barra R, Wahlstrom B, Neretin L, Wong MH: Bisphenol A (BPA) in China: A review of sources, environmental levels, and potential human health impacts. Environ Int 2012, 42:91-99.

5. Konieczna A, Rutkowska A, Rachon D: Health risk of exposure to Bisphenol A (BPA). Rocz Panstw Zakl Hig 2015, 66(1):5-11.