Determination of Perfluorooctanoic Acids in Different Water Samples by Electrospray Extraction Ionization Tandem Mass Spectrometry
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摘要:
全氟辛酸（PFOA）是一种持久性有机污染物，长期环境暴露会对生态系统与人类健康造成极大的风险，因此，发展快速定量监测水中PFOA的方法至关重要。本研究采用电喷雾萃取电离-串联质谱（EESI-MS/MS）法检测自来水、江水、河水中的痕量PFOA。结果表明，在优化的实验条件下，PFOA在1～500 ng/L浓度范围内的线性关系良好，线性相关系数（R2）为0.9976，方法检出限（LOD）为0.57 ng/L，定量限（LOQ）为 1.73 ng/L，回收率为92.1%～105.2%，相对标准偏差（RSD，n=6）为1.89%～5.45%。本方法具有灵敏度高、准确性好、分析速度快等优点，在监测环境水体污染物方面具有良好的应用前景。
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关键词:
- 电喷雾萃取电离-串联质谱（EESI-MS/MS） /
- 快速检测 /
- 全氟辛酸（PFOA） /
- 水
Abstract:Water is a vital resource on the earth, serving as the foundation of life and playing a crucial role in our daily activities. Perfluorooctanoic acid (PFOA) is an organic compound, which poses a significant threat to the environment due to its persistence and bioaccumulation. PFOA exhibits various toxicities, including neurotoxicity, immunotoxicity, and carcinogenicity. It has been detected in worldwide of air and water samples, as it can easily move through different environmental media such as water, soil, and the atmosphere. The widespread distribution of PFOA in these media poses potential risks to both ecosystems and human health over the long term. Therefore, it is of utmost importance to develop a rapid detection method for accurately monitoring PFOA levels in water. In this study, the electrospray extraction ionization-tandem mass spectrometry (EESI-MS/MS) technique was utilized to directly and rapidly detect PFOA in tap water and river water. To achieve the highest ionization efficiency of PFOA in EESI-MS/MS across different water samples, the experimental parameters such as ion transfer tube temperature, spray voltage, electrospray solvent flow rate, and sample flow rate were optimized. The optimal conditions were temperature of 200 °C for the ion transfer tube, spray voltage of 2 kV, electrospray solvent flow rate of 4 µL/min, and sample flow rate of 6 µL/min. The experiments were conducted under negative ion mode with a mass scan range of m/z 50-500. Methanol solution was used as the electrospray solvent, and collision-induced dissociation (CID) was employed. The secondary characteristic fragment ion m/z 369 of m/z 413 for qualitative and quantitative analysis, enabling direct and rapid detection of PFOA in various water samples. The experimental results demonstrated good linearity of PFOA in the concentration range of 1-500 ng/L, with a linear correlation coefficient (R2) of 0.9976. The detection limit (LOD) of the proposed method is 0.57 ng/L, and the limit of quantification (LOQ) is 1.73 ng/L. The recovery rate ranges from 92.1% to 105.2%, with a relative standard deviation (RSD, n=6) of 1.89%-5.45%. In summary, this method has the advantages of high sensitivity, accuracy, and fast analysis speed, without the need of sample pretreatment. The established EESI-MS/MS method can be effectively applied for direct and rapid detection of PFOA in different water samples, showing promising prospects for monitoring environmental water pollutants. Moreover, it provides valuable references and technical means for the detection of other pollutants.
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水是生命之源，普通成年人每天正常的饮水量约为1 500～2 000 mL。环境中的化学物质能够通过饮用水进入人体，饮水暴露剂量主要取决于饮用水中污染物浓度、饮水量等因素，饮用水是否卫生、安全，直接影响人们的身体健康[1]。
全氟化合物（PFCs）是一类普遍存在的持久性和难降解的有机污染物，具有神经毒性、免疫毒性、生殖毒性和致癌性等，在天然水体中的含量范围约为几至几百ng/L[2]，对人体健康造成严重威胁，已成为生活用水中新的潜在隐患。PFCs存在于不同的环境介质中，对全球范围内的生物有毒害作用[3-6]。其中，全氟辛酸 （PFOA）是生物群和人类生活过程中含量最丰富、最常见的一种PFCs，具有分布广、毒性大、环境持久性和生物累积性等特征，能够对生态环境和人类健康造成严重和不可逆转的危害[7-10]。此外，PFOA具有较强的化学稳定性、热稳定性及生物惰性等特点[11-13]，在环境中具有高度的移动性，广泛分布于水、土壤、大气等环境介质中[14-18]。人体暴露PFOA的途径主要是饮水摄入，其会对机体免疫器官、免疫功能以及免疫因子产生影响，进而导致相关的免疫疾病。因此，监测不同水样中PFOA含量意义重大。2019年，斯德哥尔摩公约批准增列PFOA等相关化合物为持久性有机污染物（POPs）受控物质；2020年，欧盟POPs法规将PFOA列入管控范围；近年来，我国对PFOA实施严格管控，水质参考指标，即PFOA在生活饮用水的水质限值为80 ng/L[19-20]。
目前，检测PFOA的方法主要有高效液相色谱-串联质谱（HPLC-MS/MS）法[21-27]、气相色谱-质谱（GC-MS）法[28-29]等。这些方法虽然具有高灵敏度、高准确度等优点，但存在费时费力、操作步骤繁琐等缺点，无法实现PFOA的直接快速检测。
基于此，本研究拟利用自制的电喷雾萃取电离源（EESI）结合LTQ-XL型线性离子阱质谱仪，快速检测不同水样中的PFOA。希望为不同水样中PFOA的直接检测和快速监测提供技术手段，为环境水体中其他污染物的监管提供理论支持。
1. 实验部分
1.1 主要仪器与装置
电喷雾萃取电离源：本实验室自制；LTQ-XL型线性离子阱质谱仪：美国Thermo Scientific公司产品，配有Xcalibur数据处理系统；微量注射泵：英国保定兰格恒流泵有限公司产品；KQ3200DE数控超声波清洗器：昆山市超声仪器有限公司产品；精密电子天平：瑞士Mettler Toledo公司产品。
EESI源主要由相互独立的电喷雾通道和样品通道2部分构成[30]，示于图1。EESI源工作条件[31-32]：电喷雾通道及样品通道与质谱进样口之间角度α=150°，距离b=4 mm；电喷雾通道与样品通道之间角度β=60°，间距a=1.5 mm。
1.2 主要材料与试剂
全氟辛酸：北京曼哈格生物技术有限公司产品；甲醇（色谱纯）：德国默克公司产品；高纯氮气（99.999%）：南昌国滕气体有限公司产品；超纯水：北京费尔德科学仪器有限公司产品。
用甲醇配制50 mg/L全氟辛酸标准储备液和1 mg/L 全氟辛酸标准使用液；萃取剂为甲醇；实际环境水样取自某市自来水、江水、河水。
1.3 质谱条件
负离子检测模式，质量扫描范围m/z 50～500，电离电压2.0 kV，离子传输管温度200 ℃，毛细管电压−35 V，透镜电压−110 V，电喷雾溶剂流速4 μL/min，样品流速6 μL/min，雾化气（N2，纯度99.999%）压强0.6 MPa。在进行PFOA标准溶液多级串联质谱检测时，母离子隔离宽1.5 u，碰撞能量6%，活化值Q为0.25，碰撞时间30 ms。其他检测参数由LTQ-Tune 系统自动优化。
2. 结果与讨论
2.1 PFOA标准溶液的EESI-MS/MS分析
EESI-MS/MS基于电喷雾通道喷出的甲醇溶液（即初级带电液滴）与样品通道喷出的样品液滴在三维空间中进行碰撞，进而实现了液-液萃取与电荷转移，并完成去溶剂化过程，供质谱检测[33-34]。采用EESI-MS/MS分析PFOA标准溶液，一级质谱图示于图2a。进一步结合碰撞诱导解离（CID）实现对PFOA的定性检测，二级碎片离子信号m/z 369、169[23, 35]为PFOA的质子化分子离子，示于图2b。
2.2 EESI-MS/MS参数的优化
为了使EESI-MS/MS对PFOA的离子化效率达到最佳，优化了离子传输管温度、喷雾电压、电喷雾溶剂流速、样品流速等参数。在负离子模式下，均选择 m/z 413的二级特征碎片离子m/z 369 的质谱响应强度来表示对PFOA的检测效率。
2.2.1 离子传输管温度
离子传输管温度会影响离子的去溶剂化效果，进而影响目标信号的强度，其优化结果示于图3a。离子传输管温度在100～400 ℃范围内, 目标离子的质谱响应强度呈先升高后降低的趋势，接近200 ℃时，响应强度最大。因此，选择离子传输管温度为200 ℃。
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图 3 不同实验条件对PFOA信号强度的影响注：a. 离子传输管温度；b. 喷雾电压；c. 电喷雾溶剂流速；d. 样品流速Figure 3. Effects of different experiment conditions on PFOA signal intensity2.2.2 喷雾电压
本实验考察了喷雾电压在1.5～4.5 kV范围内对质谱响应强度的影响，结果示于图3b。当喷雾电压大于 2.0 kV时，质谱响应强度呈下降趋势，推测原因为喷雾电压过高导致喷雾口发生电晕放电，降低了离子的形成效率，使目标信号强度降低。因此，选择喷雾电压为2.0 kV。
2.2.3 电喷雾溶剂流速
本实验考察了电喷雾溶剂流速在2～14 µL/min范围内对质谱响应强度的影响，结果示于图3c。当流速大于4 µL/min时，信号强度降低，表明流速过高会导致离子化效率降低。因此，选择电喷雾溶剂流速为4 µL/min。
2.2.4 样品流速
本实验考察了样品流速在2～14 µL/min范围内对质谱响应强度的影响，结果示于图3d。可见，目标物信号强度呈先升高后降低的趋势，当样品流速大于6 µL/min时，信号强度降低。推测原因为样品流速过大会减弱样品的雾化效率，进而降低目标离子的质谱响应强度。因此，选择样品流速为6 µL/min。
2.3 PFOA的定量分析
2.3.1 线性关系、检出限与定量限
配制1、10、50、100、250、500 ng/L PFOA水溶液，在优化的实验条件下进行直接质谱分析，并选择 m/z 413 的二级特征碎片离子m/z 369 作为定量离子进行分析。以PFOA水溶液的质量浓度为横坐标，以目标离子的5次平均质谱信号强度为纵坐标绘制标准曲线，线性回归方程为y=10.3660x+173.6172，示于图4。PFOA在1～500 ng/L浓度范围内呈现良好的线性关系，线性相关系数（R2）为0.9976。当信噪比（S/N）＝3.3时，根据LOD＝3.3σ/b[36]（σ为空白标准偏差，b为斜率）计算检出限为0.57 ng/L；当S/N＝10时，根据LQD＝10σ/b[36]计算定量限为1.73 ng/L。
2.3.2 精密度和准确度
选择不同水样作为基质评价方法的精密度和准确度，分别配制3、30、300 ng/L加标溶液，在优化的实验条件下，采用EESI-MS/MS测定不同水样中的PFOA，重复测定6次，结果列于表1。 PFOA在自来水中的回收率为92.1%～104.9%, 相对标准偏差（RSD）为1.89%～4.49%；在江水中的回收率为99.2%～105.2%，RSD为2.99%～5.45%；在河水中的回收率为92.3%～102.6%，RSD为 2.82%～3.68%。
表 1 加标样品的分析结果（n=6）Table 1. Analytical results of spiked samples (n=6)样本
Sample样本本底值
Sample background value/(ng/L)加标量
Added value/(ng/L)测量值
Measured value/(ng/L)回收率
Recovery rate/%相对标准偏差
RSD/%自来水 ND 3 2.76 92.1 4.49 30 27.96 93.2 4.45 300 314.82 104.9 1.89 江水 29.86 3 32.83 99.2 3.67 30 61.43 105.2 5.45 300 341.02 103.7 2.99 河水 51.78 3 54.55 92.3 3.68 30 81.93 100.5 2.97 300 359.51 102.6 2.82 2.3.3 实际样品分析
采用EESI-MS/MS技术直接检测自来水、江水、河水中的PFOA，以m/z 413为母离子，通过碰撞诱导解离（CID）进行检测，结果示于图5。结果表明，自来水中未检出PFOA；江水、河水中PFOA的残留水平分别低于30、52 ng/L。此外，将本研究方法与高效液相色谱-串联质谱（HPLC-MS/MS）法进行对比，结果列于表2。可见，本方法可用于直接快速检测不同水样中痕量PFOA。
表 2 EESI-MS/MS和HPLC-MS/MS对环境水体的分析结果Table 2. Analysis results of environmental water by EESI-MS/MS and HPLC-MS/MS方法
Method样品
SamplePFOA含量
Content of PFOA/(ng/L)线性范围
Linear range/(ng/L)检出限
LOD/(ng/L)单个样品分析时间
Analysis time of single sample/minEESI-MS/MS 自来水 ND 1～500 0.57 1 江水 29.86 河水 51.78 HPLC-MS/MS 自来水 ND 50～1000a 10a 5a 江水 29.42 河水 54.19 注：a表示数据引用自参考文献[37] 3. 结论
本研究采用 EESI-MS/MS技术快速定性、定量分析不同水样中的PFOA，并优化了离子传输管温度、喷雾电压、电喷雾溶剂流速、样品流速等实验条件。结果表明，本方法可快速灵敏、高效准确地检测不同水样中的PFOA，在监测环境水体污染物方面具有良好的应用前景，适用于不同环境中PFOA的直接快速检测。
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图 3 不同实验条件对PFOA信号强度的影响
注：a. 离子传输管温度；b. 喷雾电压；c. 电喷雾溶剂流速；d. 样品流速
Figure 3. Effects of different experiment conditions on PFOA signal intensity
表 1 加标样品的分析结果（n=6）
Table 1 Analytical results of spiked samples (n=6)
样本
Sample样本本底值
Sample background value/(ng/L)加标量
Added value/(ng/L)测量值
Measured value/(ng/L)回收率
Recovery rate/%相对标准偏差
RSD/%自来水 ND 3 2.76 92.1 4.49 30 27.96 93.2 4.45 300 314.82 104.9 1.89 江水 29.86 3 32.83 99.2 3.67 30 61.43 105.2 5.45 300 341.02 103.7 2.99 河水 51.78 3 54.55 92.3 3.68 30 81.93 100.5 2.97 300 359.51 102.6 2.82 
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表 2 EESI-MS/MS和HPLC-MS/MS对环境水体的分析结果
Table 2 Analysis results of environmental water by EESI-MS/MS and HPLC-MS/MS
方法
Method样品
SamplePFOA含量
Content of PFOA/(ng/L)线性范围
Linear range/(ng/L)检出限
LOD/(ng/L)单个样品分析时间
Analysis time of single sample/minEESI-MS/MS 自来水 ND 1～500 0.57 1 江水 29.86 河水 51.78 HPLC-MS/MS 自来水 ND 50～1000a 10a 5a 江水 29.42 河水 54.19 注：a表示数据引用自参考文献[37]
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