phkineticaФармакокинетика и ФармакодинамикаPharmacokinetics and Pharmacodynamics2587-78362686-8830ООО «Издательство ОКИ»10.37489/2587-7836-2022-4-33-42phkinetica-341Research ArticleМЕТОДЫ ОПРЕДЕЛЕНИЯ ЛЕКАРСТВЕННЫХ СРЕДСТВDRUGS DETERMINATION METHODSКоличественное определение моноаминовых нейротрансмиттеров в гомогенатах головного мозга крыс с помощью ВЭЖХ-МС/МСQuantitative determination of monoamine neurotransmitters in rat brain homogenates using HPLC-MS/MShttps://orcid.org/0000-0002-1792-7414ПоповН. С.PopovN. S.

Попов Никита Сергеевич, к. фарм. н., зав. научно-исследовательской лабораторией, доцент кафедры фармакологии и клинической фармакологии

Тверь

Popov Nikita S., PhD, Cand. Pharm Sci., Head of Research Laboratory, Associate Professor, Department of Pharmacology and Clinical Pharmacology

SPIN code: 1974-7300

Tver

ns.popov@mail.ruГавриленкоД. А.GavrilenkoD. A.

Гавриленко Дмитрий Антонович, асс. кафедры химии

Тверь

Gavrilenko Dmitry A., Assistant of the Department of Chemistry

SPIN code: 5573-2662

Tver

niruef@yandex.ruhttps://orcid.org/0000-0002-5744-7060БалабаньянВ. Ю.BalabanyanV. Yu.

Балабаньян Вадим Юрьевич, д. фарм. н., доцент, в. н. с., лаборатория трансляционной медицины, факультет фундаментальной медицины

Москва

Balabanyan Vadim Yu., Dr. Sci. (Pharm), Associate Professor, Leading Researcher, Laboratory of Translational Medicine, Faculty of Fundamental Medicine

SPIN code: 7351-7328

Moscow

bal.pharm@mail.ruПетроваМ. Б.PetrovaM. B.

Петрова Маргарита Борисовна, д. б. н., профессор, зав. кафедрой биологии

Тверь

Petrova Margarita B., Dr. Sc. (Biol.), Professor, Head of Department of Biology

SPIN code: 4310-3839

Tver

education@tvgmu.ruhttps://orcid.org/0000-0003-3780-5534ДонсковС. А.DonskovS. A.

Донсков Сергей Александрович, к. с.-х. н., доцент кафедры гистологии, эмбриологии и цитологии

Тверь

Donskov Sergey A., PhD, Cand. Agricult Sci., Associate Professor, Department of Histology, Embryology and Cytology

SPIN code: 1026-7460

Tver

donskov_s@mail.ruhttps://orcid.org/0000-0003-1995-2661АтаджановИ. Б.AtadzhanovI. B.

Атаджанов Ильяс Борисович, препаратор кафедры гистологии, эмбриологии и цитологии

Тверь

Atadzhanov Ilyas B., preparator of the Department of Histology, Embryology and Cytology

Tver

atadzanov.ilyas@yandex.ruШатохинаН. А.ShatokhinaN. A.

Шатохина Наталья Александровна, к. м. н., доцент кафедры фармакологии и клинической фармакологии

Тверь

 

Shatokhina Natalya A., PhD, Cand. Med Sci., Associate Professor, Department of Pharmacology and Clinical Pharmacology

SPIN code: 8171-5540

Tver

chatokhina@mail.ruФГБОУ ВО Тверской государственный медицинский университет» Министерства здравоохранения Российской Федерации (ФГБОУ ВО Тверской ГМУ Минздрава России)РоссияFSBEI HE TVER SMU MOH RussiaRussian FederationФГБОУ ВО «Московский государственный университет им. М.В. Ломоносова»РоссияFSEI HPE Lomonosov Moscow State UniversityRussian Federation202218012023043342Copyright © Попов Н.С., Гавриленко Д.А., Балабаньян В.Ю., Петрова М.Б., Донсков С.А., Атаджанов И.Б., Шатохина Н.А., 20232023Попов Н.С., Гавриленко Д.А., Балабаньян В.Ю., Петрова М.Б., Донсков С.А., Атаджанов И.Б., Шатохина Н.А.Popov N.S., Gavrilenko D.A., Balabanyan V.Y., Petrova M.B., Donskov S.A., Atadzhanov I.B., Shatokhina N.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.pharmacokinetica.ru/jour/article/view/341Актуальность

Актуальность. Оценка влияния лекарственных средств на нейромедиаторные процессы является важной составляющей фармакодинамических исследований. Количественное определение моноаминовых нейротрансмиттеров в структурах головного мозга лабораторных животных является актуальной задачей фармакологии и физиологии.

Цель – разработка методики количественного определения серотонина, дофамина, норэпинефрина, гистамина и эпинефрина в гомогенатах головного мозга крыс с помощью ВЭЖХ-МС/МС.

Методы

Методы. Выделение нейромедиаторов из мозга крыс осуществляли путём гомогенизации биоматериала с ацетонитрилом и хлористоводородной кислотой. Очистку извлечения проводили с помощью жидкость-жидкостной экстракции с хлороформом и изопропанолом. Детектирование моноаминов осуществляли с помощью масс-спектрометра AB Sciex QTrap 3200MD, хроматографирование проводили с использованием ВЭЖХ Agilent Technologies 1260 Infinity II. В качестве элюента использовали метанол и деионизированную воду.

Результаты

Результаты. Пробоподготовка представляла собой центрифугирование полученного гомогената, высушивание супернатанта в токе азота, растворение осадка в подвижной фазе, очистку раствора с помощью смеси хлороформа и изопропанола. Для хроматографического разделения моноаминовых нейромедиаторов использовали аналитическую колонку Agilent InfinityLab Poroshell 120 EC-C18 4,6 × 100 мм, 2,7 мкм. Общее время хроматографического анализа составило 12 минут, время удерживания норэпинефрина, эпинефрина, дофамина, серотонина, гистамина составило 2,8; 3,2; 5,4; 7,9; и 2,2 минут, соответственно. Аналитический диапазон методики составил 25,0–5000,0 нг/г для эпинефрина, гистамина и дофамина; 5,0–5000,0 нг/г для серотонина и 50,0–5000,0 для норэпинефрина. Для апробации методики был проведён анализ моноаминовых нейромедиаторов в стриатуме интактных крыс Wistar. Заключение. Разработанная биоаналитическая ВЭЖХ-МС/МС-методика количественного определения моноаминовых нейромедиаторов в головном мозге крыс полностью соответствует валидационным требованиям. Метрологические характеристики методики позволяют с высокой точностью оценить содержание норэпинефрина, эпинефрина, дофамина, серотонина и гистамина в структурах головного мозга крыс.

Relevance

Relevance. Evaluation of the effect of drugs on neurotransmitter processes is an important component of pharmacodynamic studies. The quantitative determination of monoamine neurotransmitters in the brain structures of laboratory animals is an urgent task of pharmacology and physiology.

Purpose of the study

Purpose of the study. Development of a method for the quantitative determination of serotonin, dopamine, norepinephrine, histamine and epinephrine in rat brain homogenates using HPLC-MS/MS.

Methods

Methods. The isolation of neurotransmitters from the brain of rats was carried out by homogenizing the biomaterial with acetonitrile and hydrochloric acid. The extraction was purified by liquid-liquid extraction with chloroform and isopropanol. Monoamines were detected using an AB Sciex QTrap 3200MD mass spectrometer, chromatography was performed using an Agilent Technologies 1260 Infinity II HPLC. Methanol and deionized water were used as eluent.

Results

Results. Sample preparation consisted of centrifugation of the resulting homogenate, drying of the supernatant in a stream of nitrogen, dissolution of the precipitate in the mobile phase, and purification of the solution using a mixture of chloroform and isopropanol. An Agilent InfinityLab Poroshell 120 EC-C18 4.6×100 mm, 2.7 μm analytical column was used to separate monoamine neurotransmitters. The total time of the chromatographic analysis was 12 minutes, the retention time of norepinephrine, epinephrine, dopamine, serotonin, histamine was 2.8; 3.2; 5.4; 7.9; and 2.2 minutes, respectively. The analytical range of the technique was 25.0–5000.0 ng/g for epinephrine, histamine, and dopamine; 5.0–5000.0 ng/g for serotonin and 50.0–5000.0 for norepinephrine. To test the technique, we analyzed monoamine neurotransmitters in the striatum of intact Wistar rats.

Conclusion

Conclusion. The developed bioanalytical HPLC-MS/MS method for the quantitative determination of monoamine neurotransmitters in the rat brain fully complies with the validation requirements. The metrological characteristics of the technique make it possible to estimate the content of norepinephrine, epinephrine, dopamine, serotonin, and histamine in the brain structures of rats with high accuracy.

ВЭЖХ-МС/МСхроматографиямасс-спектрометриядофаминнорэпинефринэпинефринсеротонингистаминHPLC-MS/MSchromatographymass spectrometrydopaminenorepinephrineepinephrineserotoninhistamineReferencesBarandouzi ZA, Lee J, del Carmen Rosas M, et al. Associations of neurotransmitters and the gut microbiome with emotional distress in mixed type of irritable bowel syndrome. Sci Rep. 2022;12(1):1648. DOI: 10.1038/s41598-022-05756-0.Barandouzi ZA, Lee J, del Carmen Rosas M, et al. Associations of neurotransmitters and the gut microbiome with emotional distress in mixed type of irritable bowel syndrome. Sci Rep. 2022;12(1):1648. DOI: 10.1038/s41598-022-05756-0.Федеральный закон Российской Федерации № 61 от 12 апреля 2010 г. «Об обращении лекарственных средств».Federal Law of Russian Federation №61 of 12 April 2010 «Ob obrashchenii lekarstvennykh sredstv». (In Russ). URL: https://base.garant.ru/12174909/ Ссылка активна на: 17.10.2022.Руководство по проведению доклинических исследований лекарственных средств. Часть первая. М.: Гриф и К; 2012. 17–24 с.Rukovodstvo po provedeniyu doklinicheskih issledovanij lekarstvennyh sredstv Part 1. Moscow: Grif i K; 2012. (In Russ).Fei YY, Johnson PA, Omran NAL, et al. Maladaptive or misunderstood? Dopamine fasting as a potential intervention for behavioral addiction. Lifestyle Med. 2021;3(1):e54. DOI: 10.1002/lim2.54.Fei YY, Johnson PA, Omran NAL, et al. Maladaptive or misunderstood? Dopamine fasting as a potential intervention for behavioral addiction. Lifestyle Med. 2021;3(1):e54. DOI: 10.1002/lim2.54.Jung-Klawitter S, Kuseyri Hübschmann O. Analysis of catecholamines and Pterins in inborn errors of monoamine neurotransmitter metabolism—from past to future. Cells. 2019;8(8):867. DOI: 10.3390/cells8080867.Jung-Klawitter S, Kuseyri Hübschmann O. Analysis of catecholamines and Pterins in inborn errors of monoamine neurotransmitter metabolism—from past to future. Cells. 2019;8(8):867. DOI: 10.3390/cells8080867.Kontur PJ, Fechter LD. Brain regional manganese levels and monoamine metabolism in manganese-treated neonatal rats. Neurotoxicol Teratol. 1988;10(4):295–303. DOI: 10.1016/0892-0362(88)90031-1.Kontur PJ, Fechter LD. Brain regional manganese levels and monoamine metabolism in manganese-treated neonatal rats. Neurotoxicol Teratol. 1988;10(4):295–303. DOI: 10.1016/0892-0362(88)90031-1.Seyfried CA, Adam G, Greve T. An automated direct-injection HPLCmethod for the electrochemical/fluorimetric quantitation of monoamines and related compounds optimized for the screening of large numbers of animals. Biomed Chromatogr. 1986;1(2):78–88. DOI: 10.1002/bmc.1130010206.Seyfried CA, Adam G, Greve T. An automated direct-injection HPLCmethod for the electrochemical/fluorimetric quantitation of monoamines and related compounds optimized for the screening of large numbers of animals. Biomed Chromatogr. 1986;1(2):78–88. DOI: 10.1002/bmc.1130010206.Wang W, Wu X, Yang CS, et al. An unrecognized fundamental relationship between neurotransmitters: Glutamate protects against catecholamine oxidation. Antioxidants (Basel). 2021;10(10):1564. DOI: 10.3390/antiox10101564.Wang W, Wu X, Yang CS, et al. An unrecognized fundamental relationship between neurotransmitters: Glutamate protects against catecholamine oxidation. Antioxidants (Basel). 2021;10(10):1564. DOI: 10.3390/antiox10101564.Cannazza G, Carrozzo MM, Cazzato AS, et al. Simultaneous measurement of adenosine, dopamine, acetylcholine and 5-hydroxytryptamine in cerebral mice microdialysis samples by LC–ESI-MS/MS. J Pharm Biome Anal. 2012;71:183–186. DOI: 10.1016/j.jpba.2012.08.004.Cannazza G, Carrozzo MM, Cazzato AS, et al. Simultaneous measurement of adenosine, dopamine, acetylcholine and 5-hydroxytryptamine in cerebral mice microdialysis samples by LC–ESI-MS/MS. J Pharm Biome Anal. 2012;71:183–186. DOI: 10.1016/j.jpba.2012.08.004.Greco S, Danysz W, Zivkovic A, et al. Microdialysate analysis of monoamine neurotransmitters—a versatile and sensitive LC–MS/MS method. Anal Chim Acta. 2013;771:65–72. DOI: 10.1016/j.aca.2013.02.004.Greco S, Danysz W, Zivkovic A, et al. Microdialysate analysis of monoamine neurotransmitters—a versatile and sensitive LC–MS/MS method. Anal Chim Acta. 2013;771:65–72. DOI: 10.1016/j.aca.2013.02.004.Uutela P, Reinilä R, Piepponen P, et al. Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(20):2950–2956. DOI: 10.1002/rcm.2160.Uutela P, Reinilä R, Piepponen P, et al. Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(20):2950–2956. DOI: 10.1002/rcm.2160.Xu N, Qiu C, Wang W, et al. HPLC/MS/MS for quantification of two types of neurotransmitters in rat brain and application: Myocardial ischemia and protection of Sheng-Mai-san. J Pharm Biomed Anal. 2011;55(1):101–108. DOI: 10.1016/j.jpba.2010.12.015.Xu N, Qiu C, Wang W, et al. HPLC/MS/MS for quantification of two types of neurotransmitters in rat brain and application: Myocardial ischemia and protection of Sheng-Mai-san. J Pharm Biomed Anal. 2011;55(1):101–108. DOI: 10.1016/j.jpba.2010.12.015.Kovac A, Somikova Z, Zilka N, et al. Liquid chromatography–tandem mass spectrometry method for determination of panel of neurotransmitters in cerebrospinal fluid from the rat model for tauopathy. Talanta. 2014;119:284–290. DOI: 10.1016/j.talanta.2013.10.027.Kovac A, Somikova Z, Zilka N, et al. Liquid chromatography–tandem mass spectrometry method for determination of panel of neurotransmitters in cerebrospinal fluid from the rat model for tauopathy. Talanta. 2014;119:284–290. DOI: 10.1016/j.talanta.2013.10.027.Syslová K, Rambousek L, Kuzma M, et al. Monitoring of dopamine and its metabolites in brain microdialysates: Method combining freeze-drying with liquid chromatography–tandem mass spectrometry. J Chromatogr A. 2011;1218(21):3382–3391. DOI: 10.1016/j.chroma.2011.02.006.Syslová K, Rambousek L, Kuzma M, et al. Monitoring of dopamine and its metabolites in brain microdialysates: Method combining freeze-drying with liquid chromatography–tandem mass spectrometry. J Chromatogr A. 2011;1218(21):3382–3391. DOI: 10.1016/j.chroma.2011.02.006.Chen D, Zhang J-X, Cui W-Q, et al. A simultaneous extraction/derivatization strategy coupled with liquid chromatography–tandem mass spectrometry for the determination of free catecholamines in biological fluids. J Chromatogr A. 2021;1654:462474. DOI: 10.1016/j.chroma.2021.462474.Chen D, Zhang J-X, Cui W-Q, et al. A simultaneous extraction/derivatization strategy coupled with liquid chromatography–tandem mass spectrometry for the determination of free catecholamines in biological fluids. J Chromatogr A. 2021;1654:462474. DOI: 10.1016/j.chroma.2021.462474.Thomas J, Khanam R, Vohora D. A validated HPLC-UV method and optimization of sample preparation technique for norepinephrine and serotonin in mouse brain. Pharm Biol. 2015;53(10):1539–1544. DOI: 10.3109/13880209.2014.991837.Thomas J, Khanam R, Vohora D. A validated HPLC-UV method and optimization of sample preparation technique for norepinephrine and serotonin in mouse brain. Pharm Biol. 2015;53(10):1539–1544. DOI: 10.3109/13880209.2014.991837.Ma S-R, Yu J-B, Fu J, et al. Determination and application of nineteen monoamines in the gut microbiota targeting phenylalanine, tryptophan, and glutamic acid metabolic pathways. Molecules. 2021;26(5):1377. DOI: 10.3390/molecules26051377.Ma S-R, Yu J-B, Fu J, et al. Determination and application of nineteen monoamines in the gut microbiota targeting phenylalanine, tryptophan, and glutamic acid metabolic pathways. Molecules. 2021;26(5):1377. DOI: 10.3390/molecules26051377.Xie Z, Lorkiewicz P, Riggs DW, et al. Comprehensive, robust, and sensitive UPLC-MS/MS analysis of free biogenic monoamines and their metabolites in urine. J Chromatogr B Analyt Technol Biomed Life Sci. 2018;1099:83–91. DOI: 10.1016/j.jchromb.2018.09.012.Xie Z, Lorkiewicz P, Riggs DW, et al. Comprehensive, robust, and sensitive UPLC-MS/MS analysis of free biogenic monoamines and their metabolites in urine. J Chromatogr B Analyt Technol Biomed Life Sci. 2018;1099:83–91. DOI: 10.1016/j.jchromb.2018.09.012.Minkler P, Stoll M, Ingalls S et al. Quantification of Carnitine and Acylcarnitines in Biological Matrices by HPLC Electrospray Ionization–Mass Spectrometry. Clin Chem. 2008;54(9):1451–1462. DOI:10.1373/clinchem.2007.099226.Minkler P, Stoll M, Ingalls S et al. Quantification of Carnitine and Acylcarnitines in Biological Matrices by HPLC Electrospray Ionization–Mass Spectrometry. Clin Chem. 2008;54(9):1451–1462. DOI:10.1373/clinchem.2007.099226.González-Pardo H, Arias J, Gómez-Lázaro E, et al. Sex-Specific Effects of Early Life Stress on Brain Mitochondrial Function, Monoamine Levels and Neuroinflammation. Brain Sci. 2020;10(7):447. DOI: 10.3390/brainsci10070447.González-Pardo H, Arias J, Gómez-Lázaro E, et al. Sex-Specific Effects of Early Life Stress on Brain Mitochondrial Function, Monoamine Levels and Neuroinflammation. Brain Sci. 2020;10(7):447. DOI: 10.3390/brainsci10070447.Doroshenko Y, Lelevich V. Biogenic Monoamines, Their Precursors, and Metabolites in the Brain of Rats under Experimental Circulatory Failure. Neurochemical Journal. 2020;14(3):295–302. DOI: 10.1134/S1819712420030034.Doroshenko Y, Lelevich V. Biogenic Monoamines, Their Precursors, and Metabolites in the Brain of Rats under Experimental Circulatory Failure. Neurochemical Journal. 2020;14(3):295–302. DOI: 10.1134/S1819712420030034.Mahmood D, Akhtar M, Jahan K, et al. Histamine H3 receptor antagonists display antischizophrenic activities in rats treated with MK-801. J Basic Clin Physiol Pharmacol. 2016;27(5):463–471. DOI: 10.1515/jbcpp-2015-0045.Mahmood D, Akhtar M, Jahan K, et al. Histamine H3 receptor antagonists display antischizophrenic activities in rats treated with MK-801. J Basic Clin Physiol Pharmacol. 2016;27(5):463–471. DOI: 10.1515/jbcpp-2015-0045.Krupina N, Khlebnikova N, Narkevich V, et al. The Levels of Monoamines and Their Metabolites in the Brain Structures of Rats Subjected to Two- and Three-Month-Long Social Isolation. Bull Exp Biol Med. 2020;168(5):605–609. DOI: 10.1007/s10517-020-04761-5.Krupina N, Khlebnikova N, Narkevich V, et al. The Levels of Monoamines and Their Metabolites in the Brain Structures of Rats Subjected to Two- and Three-Month-Long Social Isolation. Bull Exp Biol Med. 2020;168(5):605–609. DOI: 10.1007/s10517-020-04761-5.

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