液相色谱质谱法测定萤火虫荧光素
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摘要
[目的]建立快速测定D-虫荧光素的液相色谱-质谱(HPLC-MS/MS)分析方法,并对荧光素的碎裂机理进行解析。[方法]采用Shim-pack VP-ODS(150 L×2.0 mm,5μm)色谱柱,甲醇、水梯度洗脱12min进行色谱分离;在质谱正离子模式下,用6对反应监测离子对(MRM)进行定性、定量分析。[结果]目标化合物在0.625～125.000μg/L线性关系良好(r=0.996 9),检出限为0.250μg/L(S/N为5)。[结论]该方法灵敏度高、选择性好,不仅可用于萤火虫发光机理、发光强弱差异、发光颜色差异方面的研究,还可以用于其他需要定量测定荧光素的研究。
[Objective]A rapid high performance liquid chromatography-mass spectrometry(HPLC-MS/MS)method was developed for the determination of D-Luciferin.The D-Luciferin fragmentation mechanism was analyzed.[Method]The HPLC separation was carried on the shim-pack VP-ODS column(150 L×2.0 mm,5 μm) with methanol and water eluting gradiently in 12 min.The D-Luciferin was detected by using 6 multiple reaction monitoring(MRM) in ESI positive mode of mass spectrometer. [Result]There was a good linear relationship within the range of 0.625-125.000 μg/L(r=0.996 9).The quantitative determination limit was 0.250 μg/L(S/N=5).[Conclusion]The method is sensitive and selective. It can be used to detect D-Luciferin rapidly in the firefly or other research.
[Objective]A rapid high performance liquid chromatography-mass spectrometry(HPLC-MS/MS)method was developed for the determination of D-Luciferin.The D-Luciferin fragmentation mechanism was analyzed.[Method]The HPLC separation was carried on the shim-pack VP-ODS column(150 L×2.0 mm,5 μm) with methanol and water eluting gradiently in 12 min.The D-Luciferin was detected by using 6 multiple reaction monitoring(MRM) in ESI positive mode of mass spectrometer. [Result]There was a good linear relationship within the range of 0.625-125.000 μg/L(r=0.996 9).The quantitative determination limit was 0.250 μg/L(S/N=5).[Conclusion]The method is sensitive and selective. It can be used to detect D-Luciferin rapidly in the firefly or other research.
引文
[1] LOWER S E,STANGER-HALL K F,HAL D W.Molecular variation across populations of a widespread North American firefly,Photinus pyrails,reveals that coding changes do not underlie flash color variation or associated visual sensitivity[J].BMC Evolutionary Biology,2018,18:1-14.
[2] YIU V,JENG M L.Oculogryphus chenghoiyanae sp.n.(Coleoptera,Lampyridae):A new ototretine firefly from Hong Kong with descriptions of its biolu minescent behavior and ultraviolet-induced fluorescence in frmales[J].Zookeys,2018,739:65-78.
[3] EBRAHIMI M,HOSSEINKHANI S,HEYDARI A,et al.Improvement of thermostability and activity of firefly luciferase through [TMG][Ac] ionic liquid mediator [J].Applied biochemistry biotechnology,2012,168(3):604-615 .
[4] LESKINEN P,VIRTA M,KARP M.One-step measurement of firefly luciferase activity in yeast [J].Yeast,2003,20(13):1109-1113.
[5] MAGHAMI P,RANJBAR B,HOSSEINKHANI S,et al.Relationship between stability and biolu minescence color of firefly luciferase [J].Photochemical & photobiological sciences,2010,9(3):376-383.
[6] HIRANO T.Molecular origin of color variation in firefly(beetlle) biolu minescence:A chemical basis for biological imaging[J].Current topics in medicinal chemistry,2016,16(24):2638-2647.
[7] KIYAMA M,SAITO R,IWANO S,et al.Muticolor bioluminescence obtained using firefly luciferin[J].Current topics in medicine chemistry,2016,16(24):2648-2655.
[8] NIWA K,NAKAMURA M,MAKI S,et al.Biosynthesis of firefly D-luciferin [J].Luminscence,2006,21(5):286.
[9] OBA Y,YOSHIDA N,KANIE S,et al.Biosynthesis of firefly luciferin in adult lantern:Decarboxylation of L-cysteine is a key step for benzothiazole ring formation in firefly luciferin synthesis [J].PLoS One,2013,8(12):1-15.
[10] NIWA K,NAKAMUA M,OHMIYA Y.Stereoisomeric bio-inversion key to biosynthesis of firefly D-luciferin [J].FEBS Letters,2006,580(22):5283-5287.
[11] 何雄,周静峰,孙金才.银纳米粒子基底制备及表面增强拉曼光谱法检测荧光素钠[J].食品安全质量检测学报,2018,9(5):1084-1088.
[12] 李伯平,郭冬发.分光光度法测定钻井液中示踪剂荧光素钠[J].中国无机分析化学,2013,3(1):38-40.
[13] 蒋俊,王祖翔,任连兵,等.HPLC 法同时测定果汁饮料中曙红、荧光桃红、荧光素钠[J].分析实验室,2011,30(12):34-37.
[14] 奚星林,邵仕萍,徐娟,等.固相萃取-高效液相色谱法同时测定食品中的12种合成色素[J].中国食品卫生杂志,2012,24(3):217-222.
[15] KANIE S,NAKAI R,OJIKA M,et al.2-S-cysteinylhydroquinone is an intermediate for the firefly lusiferin biosynthesis that occurs in the pupal stage of the Japanese firefly,Luciola lateralis[J].Bioorganic chemistry,2018,80:223-229.
[2] YIU V,JENG M L.Oculogryphus chenghoiyanae sp.n.(Coleoptera,Lampyridae):A new ototretine firefly from Hong Kong with descriptions of its biolu minescent behavior and ultraviolet-induced fluorescence in frmales[J].Zookeys,2018,739:65-78.
[3] EBRAHIMI M,HOSSEINKHANI S,HEYDARI A,et al.Improvement of thermostability and activity of firefly luciferase through [TMG][Ac] ionic liquid mediator [J].Applied biochemistry biotechnology,2012,168(3):604-615 .
[4] LESKINEN P,VIRTA M,KARP M.One-step measurement of firefly luciferase activity in yeast [J].Yeast,2003,20(13):1109-1113.
[5] MAGHAMI P,RANJBAR B,HOSSEINKHANI S,et al.Relationship between stability and biolu minescence color of firefly luciferase [J].Photochemical & photobiological sciences,2010,9(3):376-383.
[6] HIRANO T.Molecular origin of color variation in firefly(beetlle) biolu minescence:A chemical basis for biological imaging[J].Current topics in medicinal chemistry,2016,16(24):2638-2647.
[7] KIYAMA M,SAITO R,IWANO S,et al.Muticolor bioluminescence obtained using firefly luciferin[J].Current topics in medicine chemistry,2016,16(24):2648-2655.
[8] NIWA K,NAKAMURA M,MAKI S,et al.Biosynthesis of firefly D-luciferin [J].Luminscence,2006,21(5):286.
[9] OBA Y,YOSHIDA N,KANIE S,et al.Biosynthesis of firefly luciferin in adult lantern:Decarboxylation of L-cysteine is a key step for benzothiazole ring formation in firefly luciferin synthesis [J].PLoS One,2013,8(12):1-15.
[10] NIWA K,NAKAMUA M,OHMIYA Y.Stereoisomeric bio-inversion key to biosynthesis of firefly D-luciferin [J].FEBS Letters,2006,580(22):5283-5287.
[11] 何雄,周静峰,孙金才.银纳米粒子基底制备及表面增强拉曼光谱法检测荧光素钠[J].食品安全质量检测学报,2018,9(5):1084-1088.
[12] 李伯平,郭冬发.分光光度法测定钻井液中示踪剂荧光素钠[J].中国无机分析化学,2013,3(1):38-40.
[13] 蒋俊,王祖翔,任连兵,等.HPLC 法同时测定果汁饮料中曙红、荧光桃红、荧光素钠[J].分析实验室,2011,30(12):34-37.
[14] 奚星林,邵仕萍,徐娟,等.固相萃取-高效液相色谱法同时测定食品中的12种合成色素[J].中国食品卫生杂志,2012,24(3):217-222.
[15] KANIE S,NAKAI R,OJIKA M,et al.2-S-cysteinylhydroquinone is an intermediate for the firefly lusiferin biosynthesis that occurs in the pupal stage of the Japanese firefly,Luciola lateralis[J].Bioorganic chemistry,2018,80:223-229.