超声波有机溶剂萃取法和改进的Bligh-Dyer法提取甘油二烷基甘油四醚类化合物效果对比

王欢业，刘卫国,，张传伦

1. 中国科学院地球环境研究所 黄土与第四纪地质国家重点实验室，西安710061

2. 西安交通大学 人居环境与建筑工程学院，西安710049

3. 同济大学 海洋地质国家重点实验室，上海200092

超声波有机溶剂萃取法和改进的Bligh-Dyer法提取甘油二烷基甘油四醚类化合物效果对比

王欢业1，刘卫国1,2，张传伦3

1. 中国科学院地球环境研究所 黄土与第四纪地质国家重点实验室，西安710061

2. 西安交通大学 人居环境与建筑工程学院，西安710049

3. 同济大学 海洋地质国家重点实验室，上海200092

由古菌和细菌产生的甘油二烷基甘油四醚类化合物（GDGTs）是微生物学和古环境研究中的一类重要的生物标志化合物。有机溶剂萃取法和改进的Bligh-Dyer法（简称BD法）是两种常用的提取环境样品中GDGTs的方法，然而目前对这两种方法提取效果的对比研究还比较少。本研究分别采用超声波有机溶剂萃取法和BD法对两个土壤样品和两个湖泊沉积物样品的GDGTs进行了提取。对比分析表明，超声波有机溶剂萃取法提取出的核心脂形式的GDGTs（C-GDGTs）含量较高而BD法通常能提取出更多的完整极性膜脂形式的GDGTs（IP-GDGTs）。另外，两种方法得到的C-GDGTs中TEX86、MBT和CBT指标基本相同，但BD法提取的C-GDGTs的BIT值稍微偏高。这些结果将为土壤和湖泊沉积物样品GDGTs提取方法的选择以及不同研究间各指标的对比提供一定的依据。

甘油二烷基甘油四醚类化合物；超声波有机溶剂萃取法；Bligh-Dyer法；土壤；湖泊沉积物

甘油二烷基甘油四醚类化合物（GDGTs，glycerol dialkyl glycerol tetraethers）是新兴的有机生物标志物，它们广泛分布于土壤、泥炭、海洋、湖泊等各种环境中（Schouten et al，2013）。通常所说的GDGTs是以核心脂（Core lipids）形式存在的GDGTs，即C-GDGTs，其基本结构包括两个烷基长链和两个甘油分子，烷基长链末端通过醚键与甘油结合形成闭合双链环状大分子（Langworthy，1977；De Rosa and Gambacorta，1988；Schouten et al，2013）。目前，研究较多的GDGTs主要包括类异戊二烯（Isoprenoid）GDGTs和支链（Branched）GDGTs两大类，可分别简称为iGDGTs和bGDGTs（图1）。由于GDGTs广泛存在于各种环境中，且其分布对环境参数比较敏感，前人提出了一系列基于GDGTs的指标，如：重建水体古温度的TEX86指标（Schouten et al，2002）、重建土壤/大气温度的MBT/CBT指标（Weijers et al，2007）、重建土壤pH的CBT指标（Weijers et al，2007），以及重建陆源有机质输入的BIT指标（Hopmans et al，2004）等。这些指标在海洋和陆地古气候重建中发挥了非常重要的作用（王欢业等，2011；Schouten et al，2013）。

图1 本文讨论的主要GDGTs的结构图Fig.1 Chemical structures of GDGTs discussed in the text

在活体细胞中，磷酸、醣基或葡萄糖醛酸等组成的极性头基团会通过醚键键合在C-GDGTs两端的甘油基团上。这样，GDGTs是以完整极性膜脂（Intact polar lipids，简称IPLs）的形式存在的（Sturt et al，2004；Koga and Morii，2005；Lipp et al，2008；Pitcher et al，2009；Liu et al，2010；姚鹏和于志刚，2010；Peterse et al，2011；曹鹏等，2012），被称作intact polar GDGTs，即IPGDGTs。随着细胞的凋亡降解，IP-GDGTs会逐渐丢失极性头基团转化成C-GDGTs。因此，在有机地球化学研究和微生物学研究中IP-GDGTs常被认为来自现存活体细胞或者刚刚死亡的微生物（Lipp et al，2008；Lipp and Hinrichs，2009；Pitcher et al，2009；Liu et al，2011；Tierney et al，2012）。但是需要注意的是，也有一些研究表明某些IP-GDGTs在环境中可以保存很长时间，因而可能并非所有的IP-GDGTs都是原位产生的（Schouten et al，2010，2012）。

多种提取方法均可用来提取环境样品中的GDGTs（Huguet et al，2010）。其中，超声波有机溶剂萃取法和改进的Bligh-Dyer（Bligh and Dyer，1959）提取法（本文简称为BD法）是两种常用的方法（Hopmans et al，2004；Sturt et al，2004；Lipp and Hinrichs，2009；Liu et al，2011；Jia et al，2013；Wu et al，2013；Yang et al，2014；Dong et al，2015；Xing et al，2015），尤其是在同时分析C-GDGTs与IP-GDGTs 的研究中（Pitcher et al，2009，2011；Wei et al，2011；Ayari et al，2013；Liu et al，2013；Jia et al，2014；Lengger et al，2014）。为便于不同研究之间结果的对比，有必要对两种方法提取GDGTs产量和指标的可能差异进行有效评估。目前关于两种方法对GDGTs提取效果的对比研究还很少（Huguet et al，2010；Zhang et al，2012）。Huguet et al（2010）调查了多种提取方法对于古菌培养物、美国Hood Canal峡湾水体悬浮物和沉积物以及俄勒冈州土壤中iGDGTs的萃取效率，但未评估不同方法对GDGTs指标的影响。Zhang et al（2012）对比了超声波有机溶剂萃取法和BD法对珠江下游和珠江口水体悬浮物和沉积物bGDGTs的提取效率和GDGTs分布的影响，但未报道iGDGTs的提取效率以及TEX86指标的差异。另外，两项研究均没有涉及湖泊沉积物样品。本研究中，以青海湖和柴达木盆地地区两个湖泊沉积物样品和两个土壤样品为研究对象，对比超声波有机溶剂萃取法和BD法提取GDGTs对其含量和分布（指标）的可能影响。

1 材料与方法

本实验的样品采自青海省的青海湖和柴达木盆地地区。两个土壤样品（表层0 — 5 cm）TR1和TR2分别采自橡皮山和茶卡盐湖周边。两个湖泊沉积物样品CJW1和CJW2分别采自青海湖和托素湖。其中，CJW1为湖中心沉积物（3 — 5 cm），CJW2为湖边沉积物（0 — 5 cm）。样品在野外保存在液氮或干冰中运回实验室。

冷冻干燥并研磨后的样品分别用超声波有机溶剂萃取法和BD法抽提。超声波有机溶剂萃取法：称取5 g样品向其中加入一定量的C46内标，依次用甲醇、二氯甲烷/甲醇（1:1）、二氯甲烷、二氯甲烷/甲醇（1:1）和甲醇超声震荡15 min萃取其中的有机质，5次得到的总提取液在水浴中用氮气吹干。BD法：用体积比2:1:0.8的甲醇/二氯甲烷/磷酸盐缓冲溶液（pH 7.4）超声震荡萃取样品（5 g，并提前加有一定量的C46内标）3次，每次15 min。合并三次所得萃取液加入一定量的二氯甲烷和磷酸盐缓冲液调整三者比例为1:1:0.9。此时溶液出现较好分层，将底部的有机相转移到40 mL玻璃瓶中，残余相用二氯甲烷再萃取两次，均收集到40 mL玻璃瓶里。将几次收集的总萃取液在水浴中用氮气吹干。

用二氯甲烷重新溶解两种方法提取的各样品总提取物并平分为两份（分别记为F1和F2组分）。其中F1组分吹干后用99:1的正己烷/异丙醇溶解并过0.45 μm滤膜。F2组分按照Wei et al（2011）的方法进行酸水解，随后萃取得到水解后的有机组分，该组分吹干后也用99:1的正己烷/异丙醇溶解并过滤以备GDGTs测试。

样品的GDGTs测试所用仪器为高效液相色谱-大气压化学电离-质谱（HPLC-APCI-MS，HPLC型号为Agilent 1200，MS型号为6460三重四级杆）。分析方法修改自Hopmans et al（2000）和Schouten et al（2007）。进样量为2 μL。所用液相色谱柱为奥泰Prevail氰基柱（150 mm × 2.1 mm，3 μm）。洗脱流动相程序为：0 — 5 min，99:1正己烷/异丙醇；5 — 45 min，异丙醇比例线性升至1.8%，整个过程流动相流速为0.2 mL · min-1。每分析完一个样品，以 10%的异丙醇冲洗色谱柱15 min。质谱方法采用选择离子扫描（SIM）模式检测GDGTs质子化后特定质荷比的离子。通过对各GDGTs的[M+H]+离子峰面积进行积分并对比与C46内标[M+H]+离子峰面积的比值，可以对样品中GDGTs的含量进行定量（假设iGDGTs和bGDGTs均与C46内标具有相同的响应因子）。

F1组分中检测得到的GDGTs即为样品中的C-GDGTs。F2组分中检测得到的GDGTs为样品中IP-GDGTs酸解后生成的C-GDGTs加上样品中原始的C-GDGTs。进而，根据差减法可以计算IPGDGTs酸解生成的C-GDGTs的含量（Huguet et al，2010；Zhang et al，2012）。为与C-GDGTs区分，本文仍将由IP-GDGTs酸解生成的C-GDGTs称作PL-GDGTs。

表示bGDGTs甲基化指数的MBT指标和环化指数的CBT 指标按照 Weijers et al（2007）计算如下：

2 结果与讨论

2.1 GDGTs提取效率

为便于比较超声波有机溶剂萃取法与BD法对样品GDGTs的提取效率，用RCS/BD来表示两种方法提取GDGTs含量之比。对本研究中的两个土壤样品和两个湖泊沉积物样品，C-iGDGTs的RCS/BD均＞1.0（图2），平均为1.3，表明超声波有机溶剂萃取法提取C-iGDGTs的效率较高。Huguet et al（2010）对峡湾水体悬浮物、沉积物以及土壤的研究结果也表明超声波有机溶剂萃取法常能得到较多的C-iGDGTs。对于IP-iGDGTs，4个样品的RCS/BD均＜1.0，平均为0.6，表明BD法提取IP-iGDGTs的效率较高。另外，结果显示，两种方法对bGDGTs的提取效果与对iGDGTs的提取效果类似，C-bGDGTs的RCS/BD均≥1.0（图2），平均为1.1，而IP-bGDGTs的RCS/BD均≤1.0，平均为0.7。以上结果表明，超声波有机溶剂萃取法可以提取出较多的C-GDGTs而BD法通常能提取出更多的IPGDGTs。

图2 GDGTs各组分用超声波有机溶剂萃取法与BD法提取量比值（RCS/BD）Fig.2 The relative yield of GDGTs extracted by ultrasound-assisted organic solvent extraction vs. Bligh-Dyer extraction (RCS/BD)

与C-GDGTs相比，IP-GDGTs带有一定数量的极性头基团，这导致了IP-GDGTs极性较强。由于超声波有机溶剂萃取法所用的有机溶剂极性比BD法所用的甲醇/二氯甲烷/磷酸盐缓冲溶液极性要弱，根据相似相溶原理，超声波有机溶剂萃取法会对C-GDGTs有较高的提取效率，而BD法更适合提取极性较强的有机化合物。因此，对于只关心C-GDGTs的研究可以选用超声波有机溶剂萃取法，而对于注重IP-GDGTs提取效率的研究，应将BD法作为首选方法。但是需要注意的是，对于本实验选取的样品，两种方法提取的GDGTs含量并没有数量级上的差别。事实上，相对于GDGTs的提取效率， GDGTs的研究往往更关心的是GDGTs指标。因此，需要进一步对比超声波有机溶剂萃取法与BD法提取的GDGTs分布的差异。

2.2 GDGTs分布

首先，对比了C-GDGTs的分布（图3）。对于本研究的4个样品，两种方法提取C-GDGTs的TEX86值非常一致，其超声波有机溶剂萃取法与BD法的差值（即超声波有机溶剂萃取法TEX86-BD法TEX86）分别为0.00、-0.01、-0.01、0.01，平均值为-0.002，基本在仪器的分析误差之内（±0.01或更大）（Yang et al，2011；Wang et al，2012）。这些结果表明对湖泊沉积物和土壤样品采用两种不同的提取方法可能不会影响对C-iGDGTs的TEX86的计算。另外，两种方法得到的表示C-bGDGTs分布的MBT指标和CBT指标也比较接近，其差值分别为0.00、0.01、-0.01、0.00和-0.01、0.02、-0.01、-0.02，平均值分别为0.000和-0.008，表明对湖泊沉积物和土壤样品采用两种不同的提取方法可能也不会影响对C-bGDGTs的MBT和CBT的计算。Zhang et al（2012）对比这两种方法提取珠江下游和珠江口沉积物bGDGTs的结果也显示，超声波有机溶剂萃取法和BD法得到的大部分样品的C-bGDGTs的MBT和CBT并没有差异。

图3 超声波有机溶剂萃取法（圆圈）与BD法（三角）提取的C-GDGTs各指标对比Fig.3 Comparison of C-GDGT indices extracted using ultrasound-assisted organic solvent extraction (circles) and Bligh-Dyer extraction (triangles)

两种方法提取C-GDGTs的BIT值差别也较小（图3），4个样品的差值分别为-0.04、0.00、-0.05、-0.04，平均值为-0.031。但是，超声波有机溶剂萃取法得到的C-GDGTs的BIT似乎比BD法得到的值系统偏低。根据C-bGDGTs在色谱图中比C-iGDGTs具有更长的保留时间可以推测C-bGDGTs的极性比C-iGDGTs要强。因此，更适合提取极性较强化合物的BD法对C-bGDGTs的提取效率可能比对C-iGDGTs的提取效率要高，这会导致BD法得到的C-GDGTs的BIT值偏高。这一推测可以进一步通过RCS/BD的结果证实：C-bGDGTs的RCS/BD平均为1.1，小于C-iGDGTs的平均RCS/BD值（1.3）。

此外，还对比了IP-GDGTs的分布（图4）。两种方法提取IP-iGDGTs 组分的TEX86值较为一致，超声波有机溶剂萃取法与BD法的差值分别为0.02、-0.04、-0.02、-0.01，平均值为-0.012。两种方法提取IP-bGDGTs 组分的MBT和CBT具有一定差异，其差值分别为-0.01、-0.13、0.04、0.07和-0.48、0.13、0.06、0.39，平均值分别为-0.008和0.024。另外，两种方法得到的IP-GDGTs的BIT值也具有一定差异，其差值分别为0.00、0.00、0.12、-0.04，平均值为0.018。Zhang et al（2012）对珠江下游和珠江口沉积物的研究结果也表明用两种不同提取方法得到的IP-GDGTs的MBT、CBT和BIT值有所差异。需要指出的是，本文和Zhang et al（2012）中IP-GDGTs组分是由差减法得到的，但是该方法在计算GDGTs分布时可能误差较大（Lengger et al，2012），尤其是样品中IP-GDGTs相对于C-GDGTs含量较低的时候。因此，根据本文的结果，只能说两种方法提取湖泊沉积物和土壤样品引起的IP-GDGTs分布的差异可能不会太大，但是是否有差异或者具体有多大程度的差异仍需进一步的确认。

图4 超声波有机溶剂萃取法（圆圈）与BD法（三角）提取的IP-GDGTs各指标对比Fig.4 Comparison of IP-GDGT indices extracted using ultrasound-assisted organic solvent extraction (circles) and Bligh-Dyer extraction (triangles)

3 结论

本研究分别采用BD法和超声波有机溶剂萃取法提取了两个土壤样品和两个湖泊沉积物样品中的C-GDGTs和IP-GDGTs。结果显示：（1）超声波有机溶剂萃取法提取出的C-GDGTs含量较高而BD法通常能提取出更多的IP-GDGTs，但两者对GDGTs的提取效率并没有数量级上的差异。（2）两种方法得到的C-GDGTs中TEX86、MBT和CBT指标基本相同，但BD法得到的C-GDGTs的BIT稍微偏高。（3）两种方法提取IP-iGDGTs组分的TEX86值比较一致，而MBT、CBT和BIT指标具有一定差异（但不是很大）。考虑到本研究IP-GDGTs是由差减法计算的，尚无法确定两种提取方法会导致IP-GDGTs分布的何种差异。

致谢：感谢中国地质大学（武汉）蒋宏忱教授和杨渐博士等在野外采样工作中给予的帮助。

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Comparison of the ultrasound-assisted organic solvent extraction and modified Bligh-Dyer extraction for the analysis of glycerol dialkyl glycerol tetraethers from environmental samples

WANG Huanye1, LIU Weiguo1,2, ZHANG Chuanlun3
1. State Key Laboratory of Loess and Quaternary Geology, Institute of Earth Environment, Chinese Academy of Sciences, Xi’an 710061, China
2. School of Human Settlement and Civil Engineering, Xi’an Jiaotong University, Xi’an 710049, China
3. State Key Laboratory of Marine Geology, Tongji University, Shanghai 200092, China

Background, aim, and scope The microbial glycerol dialkyl glycerol tetraethers (GDGTs) are increasingly popular in the fi eld of organic geochemistry. These high-molecular-weight compounds produced by archaea and bacteria are sensitive to environmental variables, and therefore they contain important information on environmental parameters in paleoclimatic studies. Two extraction method, the ultrasonic-assisted organic solvent extraction and the modified Bligh-Dyer extraction (the BD extraction), are commonly applied forextracting GDGTs from environment samples. To date, however, only a few studies have compared the effect of the two extraction methods on the yield and distribution of GDGTs. In this study, we aimed to systematically compare the effect of the ultrasonic-assisted organic solvent extraction and the BD extraction on GDGTs extracted from sediment and soil samples, and to further provide some basis for the choice of extraction methods for GDGTs in soils and lake sediments, as well as for the comparison of GDGT distributions between different studies. Materials and methods We extracted GDGTs by the ultrasonic-assisted organic solvent extraction and the BD extraction from two soil samples and two lake sediment samples collected from the Qinghai province, China. GDGTs present as both core lipids (C-GDGTs) and intact polar lipids (IP-GDGTs) were analyzed as follows: each GDGT sample was separated into two halves, with one half subjecting to hydrolysis (the hydrolyzed fraction) and the other not (the non-hydrolyzed fraction); GDGTs of each fraction was directly measured on the high performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. Quantifi cation was performed by way of peak area integration of [M+H]+ions in the extracted ion chromatogram, and comparison with the internal standard. Ionization effi ciency for each GDGT was assumed identical. GDGTs in the non-hydrolyzed fraction were C-GDGTs, while the difference in yield of GDGTs between the hydrolyzed and non-hydrolyzed fractions is considered to be IP-GDGTs. Results (1) For iGDGTs, the yield of C-GDGTs using ultrasonic-assisted organic solvent extraction is higher than that using BD extraction (by 30% on average), while the yield of IP-GDGTs using ultrasonic-assisted organic solvent extraction is lower than that using BD extraction (by 40% on average). (2) For bGDGTs, the yield of C-GDGTs using ultrasonic-assisted organic solvent extraction is higher than that using BD extraction (by 10% on average), while the yield of IP-GDGTs using ultrasonic-assisted organic solvent extraction is lower than that using BD extraction (by 30% on average). (3) For C-GDGTs of the two soil samples and two lake sediment samples: the difference in TEX86values is 0.00,-0.01, -0.01 and 0.01, respectively; the difference in MBT values is 0.00, 0.01, -0.01 and 0.00, respectively; the difference in CBT values is -0.01, 0.02, -0.01 and -0.02, respectively. All these values are within the analytical error. However, the BIT values are systematically higher for the BD extraction (ca. 0.03 higher than those for the ultrasonic-assisted organic solvent extraction). Discussion Compared with C-GDGTs, the polarity of IPGDGTs is much stronger due to its polar headgroups. On the other hand, the polarity of the solvent used in the BD extraction (MeOH/dichloromethane/phosphate buffer) is much stronger than the organic solvent (MeOH/ dichloromethane) used in ultrasonic-assisted organic solvent extraction. According to the “Like Dissolves Like Theory”, the ultrasonic-assisted organic solvent extraction can extract more C-GDGTs while the BD extraction can extract more IP-GDGTs. For the TEX86index, it is calculated based on similar GDGTs with similar polarity, and therefore, the values of different methods showed little difference. For the MBT and CBT indices, the values of different methods also showed little difference, similar to the case for the TEX86index. For the BIT index, however, it is calculated based on two groups of GDGTs, i.e., iGDGTs and bGDGTs, and the polarity of bGDGTs is stronger than iGDGTs. Consequently, the BIT index should be higher if samples are extracted using a BD extraction. Conclusions The ultrasonic-assisted organic solvent extraction showed higher extraction effi ciency for C-GDGTs, while the BD extraction is more effective for IP-GDGTs. Moreover, the TEX86, MBT and CBT indices for C-GDGTs are not signifi cantly affected by method difference, while the BIT index of C-GDGTs is slightly higher using BD extraction. Recommendations and perspectives For C-GDGT investigations, the ultrasonic-assisted organic solvent extraction is the fi rst choice, while for studies concerning the yield of IP-GDGTs, the BD extraction should be used. Anyhow, the TEX86, MBT and CBT indices should not be signifi cantly biased if different extracting methods are used, and therefore, some of the GDGT results using different extracting methods might be comparable.

glycerol dialkyl glycerol tetraethers; ultrasonic-assisted organic solvent extraction; Bligh-Dyer extraction; soil; lake sediment

LIU Weiguo, E-mail: liuwg@loess.llqg.ac.cn

2016-11-15；录用日期：2017-02-14

Received Date: 2016-11-15; Accepted Date: 2017-02-14

中国科学院“西部之光”人才培养引进计划（XAB2015B01）

Foundation Item: CAS “Light of West China” Program (XAB2015B01)

刘卫国，E-mail: liuwg@loess.llqg.ac.cn

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: Wang H Y, Liu W G, Zhang C L, et al. 2017. Comparison of the ultrasound-assisted organic solvent extraction and modifi ed Bligh-Dyer extraction for the analysis of glycerol dialkyl glycerol tetraethers from environmental samples [J]. Journal of Earth Environment, 8(2): 176 – 184.