vuzbiochemiИзвестия вузов. Прикладная химия и биотехнологияProceedings of Universities. Applied Chemistry and Biotechnology2227-29252500-1558ИРНИТУ.org/10.21285/2227-2925-2018-8-3-25-32vuzbiochemi-127Research ArticleФИЗИКО-ХИМИЧЕСКАЯ И ОБЩАЯ БИОЛОГИЯPHYSICOCHEMICAL AND GENERAL BIOLOGYОПРЕДЕЛЕНИЕ САКСИТОКСИНА МЕТОДОМ ВЭЖХ-МС С ПРЕДКОЛОНОЧНОЙ ДЕРИВАТИЗАЦИЕЙ 2,4-ДИНИТРОФЕНИЛГИДРАЗИНОMA METHOD FOR DETERMINATION OF SAXITOXINS USING HPLC-MS WITH 2,4-DINITROPHENYLHYDRAZINE PRECOLUMN DERIVATIZATIONЗубковИ. Н.ZubkovI. N.ilyagosldstein@gmail.comКузьминА. В.KuzminA. V.kuzmin@lin.irk.ruТихоноваИ. В.TikhonovaI. V.iren@lin.irk.ruБелыхО. И.BelykhO. I.belykh@lin.irk.ruСмирновВ. И.SmirnovV. I.smirnov@irioch.irk.ruИвановА. В.IvanovA. V.ivanov@irioch.irk.ruШагунВ. А.ShagunV. A.shagun@irioch.irk.ruГрачевМ. А.GrachevM. A.grachev@lin.irk.ruФедороваГ. А.FedorovaG. A.fgalina@mail.ruЛимнологический институт, Сибирское отделение Российской академии наукРоссияLimnological Institute, Siberian Branch of the Russian Academy of SciencesRussian FederationЛимнологический институт, Сибирское отделение Российской академии наук; Иркутский институт химии им. А.Е. Фаворского, Сибирское отделение Российской академии наукРоссияLimnological Institute, Siberian Branch of the Russian Academy of Sciences; A.E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of SciencesRussian FederationИркутский институт химии им. А.Е. Фаворского, Сибирское отделение Российской академии наукРоссияA.E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of SciencesRussian FederationЛимнологический институт, Сибирское отделение Российской академии наук; Восточно-Сибирский филиал Российского государственного университета правосудияРоссияLimnological Institute, Siberian Branch of the Russian Academy of Sciences; Russian State University of JusticeRussian Federation201823092019832532Copyright © Зубков И.Н., Кузьмин А.В., Тихонова И.В., Белых О.И., Смирнов В.И., Иванов А.В., Шагун В.А., Грачев М.А., Федорова Г.А., 20192019Зубков И.Н., Кузьмин А.В., Тихонова И.В., Белых О.И., Смирнов В.И., Иванов А.В., Шагун В.А., Грачев М.А., Федорова Г.А.Zubkov I.N., Kuzmin A.V., Tikhonova I.V., Belykh O.I., Smirnov V.I., Ivanov A.V., Shagun V.A., Grachev M.A., Fedorova G.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vuzbiochemi.elpub.ru/jour/article/view/127

В настоящей работе предложен оригинальный метод определения сакситоксина (STX) основанный на его выделении из цианобактериальной биомассы путем экстракции 0,05 М уксусной кислотой, дериватизации 2,4-динитрофенилгидразином (DNPH) и последующем анализе производного методом ВЭЖХ-МС. Дериватизация проводилась в смеси ацетонитрил-трифторуксусная кислота (98,5:1,5 об./об.) при 65 °С; период полупревращения составляет ~ 3,6 ч. Разработанный метод был применен для анализа природных цианобактериальных образцов, а также культивированного штамма Nostoc Pruniforme. Детектирование гидразона сакситоксина проводилось путем измерения полного ионного тока и последующего экстрагирования ионного тока при m/z 462,15 ± 0,1. Дериватизация 2,4-динитро-фенигидразином осложнена образованием двух стереоизомеров, E и Z, которые дают два отдельных пика при ВЭЖХ. Выполненные нами квантово-химические расчеты показывают, что в случае сакситоксина образуется только один Z-изомер вследствие стерического фактора и, следовательно, только один хроматографический пик гидразона сакситоксина наблюдается на хроматограмме. Для предложенного метода получены следующие метрологические характеристики: предел определения - 20 нг/г (сухая биомасса), повторяемость - 4%, внутрилабораторная прецезионность - ±7%.

A method for saxitoxin (STX) determination is proposed. It consists of target compound extraction with 0,05 M acetic acid, derivatization with 2,4-dinitrophenylhydrazine (DNPH), and subsequent HPLC electrospray ionization MS analysis. The reaction was performed in acetonitrile-trifluoroacetic acid (98,5:1,5 v/v) at 65 °C; the half-life of STX was ca. 3,6 hours. The method was applied to analysis of natural cyanobacteria samples and a cultivated Nostoc Pruniforme strain. Detection was performed by measuring total ion current, and chromatograms for STX were obtained using an extracted ion procedure at m/z 462,15 (STX hydrazone). A frequent complication of the derivatization with DNPH is the formation of two stereoisomers, E and Z, giving two separate peaks under HPLC. Quantum-chemical calculation predicted that, due to steric hindrance, only one isomer (Z) of STX adduct is formed, and hence only one peak of the analyte is present in the chromatogram. The following metrological parameters were achieved: limits of determination (20 ng/g of dry cyanobacterial biomass), reproducibility (4%), and medium-term precision (± 7%).

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The authors declare that there are no conflicts of interest present.