高效液相串联质谱法检定活性污泥中N-高丝氨酸内酯
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摘要
N-高丝氨酸内酯(AHLs)是革兰氏阴性菌群体感应系统中最重要的一类信号分子.本研究建立了高效液相串联质谱(HPLC-MS/MS)检测活性污泥中7种AHLs的方法.采用乙腈-水为流动相,选择电喷雾源正离子模式,经过梯度洗脱分离,在多反应监测模式下进行测定,分析时间为10 min.本方法在1—1000μg·L~(-1)范围内具有良好的线性关系,仪器检出限为1.0—2.0μg·L~(-1),仪器定量限为2.0—5.0μg·L~(-1),平均加标回收率在80.0%—113.4%之间.采用该方法对某污水处理厂和实验室序批式活性污泥装置(SBR)中活性污泥进行检测,均可检测到不同种类的AHLs,并具有良好的峰型及分离效果,说明方法可满足实际测定要求.
N-homoserine lactones( AHLs) are vital quorum-sensing signal molecules of gram-negative bacteria. A high performance liquid chromatography-tandem mass spectrometry( HPLC-MS/MS)method was established to determine the AHLs in activated sludge. A mixture of acetonitrile and water served as the mobile phase and an electrospray positive ion mode was set. After seperation by mobile phase with gradient elution,the sample was measured in multi-reaction monitoring mode with a detection time of 10 min. The results showed that the method had a good linearity in the range of1—1000 μg·L~(-1). The instrument detection limit, instrument quantification limit and average recovery efficiency were 1.0—2.0 μg·L~(-1),2.0—5.0 μg·L~(-1),and 80.0%—113.4%,respectively.When this method was used to analyze the activated sludge from wastewater treatment plant and labscale sequential batch activated sludge reactor( SBR) system,different types of AHLs were detected with a good peak shapes and separation efficiency. Therefore,this method can satisfy the actual requirement for the measurement of AHLs in activated sludge.
N-homoserine lactones( AHLs) are vital quorum-sensing signal molecules of gram-negative bacteria. A high performance liquid chromatography-tandem mass spectrometry( HPLC-MS/MS)method was established to determine the AHLs in activated sludge. A mixture of acetonitrile and water served as the mobile phase and an electrospray positive ion mode was set. After seperation by mobile phase with gradient elution,the sample was measured in multi-reaction monitoring mode with a detection time of 10 min. The results showed that the method had a good linearity in the range of1—1000 μg·L~(-1). The instrument detection limit, instrument quantification limit and average recovery efficiency were 1.0—2.0 μg·L~(-1),2.0—5.0 μg·L~(-1),and 80.0%—113.4%,respectively.When this method was used to analyze the activated sludge from wastewater treatment plant and labscale sequential batch activated sludge reactor( SBR) system,different types of AHLs were detected with a good peak shapes and separation efficiency. Therefore,this method can satisfy the actual requirement for the measurement of AHLs in activated sludge.
引文
[1] REV G,MA A,LIU W,et al. Bacterial signals N-acyl homoserine lactones induce the changes of morphology and ethanol tolerance inSaccharomyces cerevisiae[J]. AMB Express,2016,6(1):117-125.
[2]陈舒涵,李安婕,王越兴,等.厌氧氨氧化污泥群体感应信号分子检测及影响研究[J].环境科学,2017,38(3):1137-1143.CHEN S H,LI A J,WANG Y X,et al. Identification and influence of quorum sensing on anaerobic ammonium oxidation process[J].Environmental Science,2017,38(3):1137-1143(in Chinese).
[3] WATERS C M,BASSLER B L. Quorum sensing:Cell-to-cell communication in bacteria[J]. Annual Review of Cell&DevelopmentalBiology,2005,21(21):319-346.
[4]孙颉,宋协法,马真.两种AHLs信号分子对生物膜法养殖污水处理条件下水体内环境的影响[J].环境工程学报,2013,7(6):2250-2254.SUN J,SONG X F,MA Z. Impacts of two kinds of AHLs on aquaculture environment with biofilm treatment[J]. Chinese Journal ofEnvironmental Engineering,2013,7(6):2250-2254(in Chinese).
[5] LEIPERT J,TREITZ C,LEIPPE M,et al. Identification and Quantification of N-Acyl homoserine lactones involved in bacterialcommunication by small-scale synthesis of internal standards and matrix-assisted laser desorption/ionization mass spectrometry[J]. Journalof the American Society for Mass Spectrometry,2017,28(12):1-10.
[6] LV J,WANG Y,ZHONG C,et al. The effect of quorum sensing and extracellular proteins on the microbial attachment of aerobic granularactivated sludge[J]. Bioresource Technology,2014,152(1):53-58.
[7] TAN C H,KAI S K,CHAO X,et al. The role of quorum sensing signaling in EPS production and the assembly of a sludge community intoaerobic granules[J]. The ISME Journal,2014,8(6):1186-1197.
[8] MA H,WANG X,ZHANG Y,et al. The diversity,distribution and function of N-acyl-homoserine lactone(AHL)in industrial anaerobicgranular sludge[J]. Bioresource Technology,2017,247:116-124.
[9] KUMARI A,PASINI P,DAUNERT S. Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples[J]. Analytical&Bioanalytical Chemistry,2008,391(5):1619-1627.
[10]马晨晨,李柏林,欧杰,等.高效液相色谱-串联质谱法同时测定细菌群体感应效应的11种AHLs类信号分子[J].分析化学,2010,38(10):1428-1432.MA C C,LI P L,O J,et al. Simultaneous determination of 11 AHLs signaling molecules by high performance liquid chromatography-tandem mass spectrometry[J]. Analytical Chemistry,2010,38(10):1428-1432(in Chinese).
[11]李玖龄,孙凯,孟佳,等.超高效液相色谱-串联质谱法检测微氧生物脱氮菌群酰基高丝氨酸内酯信号分子[J].分析化学,2016(8):1165-1170.LI J L,SUN K,MENG J,et al. Detection of acyl oxygen homoserine lactone signaling molecules in microaero biological denitrificationbacteria group with ultra high performance liquid chromatography-tandem mass spectrometry[J]. Analytical Chemistry,2016(8):1165-1170(in Chinese).
[12]曹广霞,徐素平,彭远义.细菌群体感应信号分子N-酰基高丝氨酸内酯的检测[J].生物技术通讯,2007,21(5):433-437.CAO G X,XU S P,PENG Y Y. Research for detection of quorum sensing signal molecules N-acyl-homoserine Lactones in Bacteria[J].Letters in Biotechnology,2007,21(5):433-437(in Chinese).
[13] WANG J,QUAN C,WANG X,et al. Extraction,purification and identification of bacterial signal molecules based on N-acyl homoserinelactones[J]. Microbial Biotechnology,2011,4(4):479-490.
[14] HUANG J,SHI Y,ZENG G,et al. Acyl-homoserine lactone-based quorum sensing and quorum quenching hold promise to determine theperformance of biological wastewater treatments:An overview[J]. Chemosphere,2016,157:137-151.
[15]李正邦.浅议有关物质分析方法验证的接受标准[J].中国药品标准,2016,17(4):246-251.LI Z B. Discussion on acceptance criteria of analytical method validation of related substances[J]. Drug Standards of China,2016,17(4):246-251(in Chinese).
[2]陈舒涵,李安婕,王越兴,等.厌氧氨氧化污泥群体感应信号分子检测及影响研究[J].环境科学,2017,38(3):1137-1143.CHEN S H,LI A J,WANG Y X,et al. Identification and influence of quorum sensing on anaerobic ammonium oxidation process[J].Environmental Science,2017,38(3):1137-1143(in Chinese).
[3] WATERS C M,BASSLER B L. Quorum sensing:Cell-to-cell communication in bacteria[J]. Annual Review of Cell&DevelopmentalBiology,2005,21(21):319-346.
[4]孙颉,宋协法,马真.两种AHLs信号分子对生物膜法养殖污水处理条件下水体内环境的影响[J].环境工程学报,2013,7(6):2250-2254.SUN J,SONG X F,MA Z. Impacts of two kinds of AHLs on aquaculture environment with biofilm treatment[J]. Chinese Journal ofEnvironmental Engineering,2013,7(6):2250-2254(in Chinese).
[5] LEIPERT J,TREITZ C,LEIPPE M,et al. Identification and Quantification of N-Acyl homoserine lactones involved in bacterialcommunication by small-scale synthesis of internal standards and matrix-assisted laser desorption/ionization mass spectrometry[J]. Journalof the American Society for Mass Spectrometry,2017,28(12):1-10.
[6] LV J,WANG Y,ZHONG C,et al. The effect of quorum sensing and extracellular proteins on the microbial attachment of aerobic granularactivated sludge[J]. Bioresource Technology,2014,152(1):53-58.
[7] TAN C H,KAI S K,CHAO X,et al. The role of quorum sensing signaling in EPS production and the assembly of a sludge community intoaerobic granules[J]. The ISME Journal,2014,8(6):1186-1197.
[8] MA H,WANG X,ZHANG Y,et al. The diversity,distribution and function of N-acyl-homoserine lactone(AHL)in industrial anaerobicgranular sludge[J]. Bioresource Technology,2017,247:116-124.
[9] KUMARI A,PASINI P,DAUNERT S. Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples[J]. Analytical&Bioanalytical Chemistry,2008,391(5):1619-1627.
[10]马晨晨,李柏林,欧杰,等.高效液相色谱-串联质谱法同时测定细菌群体感应效应的11种AHLs类信号分子[J].分析化学,2010,38(10):1428-1432.MA C C,LI P L,O J,et al. Simultaneous determination of 11 AHLs signaling molecules by high performance liquid chromatography-tandem mass spectrometry[J]. Analytical Chemistry,2010,38(10):1428-1432(in Chinese).
[11]李玖龄,孙凯,孟佳,等.超高效液相色谱-串联质谱法检测微氧生物脱氮菌群酰基高丝氨酸内酯信号分子[J].分析化学,2016(8):1165-1170.LI J L,SUN K,MENG J,et al. Detection of acyl oxygen homoserine lactone signaling molecules in microaero biological denitrificationbacteria group with ultra high performance liquid chromatography-tandem mass spectrometry[J]. Analytical Chemistry,2016(8):1165-1170(in Chinese).
[12]曹广霞,徐素平,彭远义.细菌群体感应信号分子N-酰基高丝氨酸内酯的检测[J].生物技术通讯,2007,21(5):433-437.CAO G X,XU S P,PENG Y Y. Research for detection of quorum sensing signal molecules N-acyl-homoserine Lactones in Bacteria[J].Letters in Biotechnology,2007,21(5):433-437(in Chinese).
[13] WANG J,QUAN C,WANG X,et al. Extraction,purification and identification of bacterial signal molecules based on N-acyl homoserinelactones[J]. Microbial Biotechnology,2011,4(4):479-490.
[14] HUANG J,SHI Y,ZENG G,et al. Acyl-homoserine lactone-based quorum sensing and quorum quenching hold promise to determine theperformance of biological wastewater treatments:An overview[J]. Chemosphere,2016,157:137-151.
[15]李正邦.浅议有关物质分析方法验证的接受标准[J].中国药品标准,2016,17(4):246-251.LI Z B. Discussion on acceptance criteria of analytical method validation of related substances[J]. Drug Standards of China,2016,17(4):246-251(in Chinese).