pmedpharmФармация и фармакологияPharmacy & Pharmacology2307-92662413-2241Pyatigorsk Medical and Pharmaceutical Institute - branch of Volgograd State Medical Univer10.19163/2307-9266-2018-6-6-535-547pmedpharm-341Research ArticleФАРМАЦЕВТИЧЕСКАЯ И ТОКСИКОЛОГИЧЕСКАЯ ХИМИЯPHARMACEUTICAL AND TOXICOLOGICAL CHEMISTRYРАЗРАБОТКА МЕТОДИК ОПРЕДЕЛЕНИЯ СПЕЦИФИЧЕСКИХ ПРИМЕСЕЙ В СУБСТАНЦИИ ГЛУТАТИОНА ВОССТАНОВЛЕННОГОDEVELOPMENT OF METHODS FOR DETERMINATION OF SPECIFIC IMPURITIES IN THE GLUTATIONION RESTORED SUBSTANCEhttps://orcid.org/0000-0002-0711-3505АлексееваК. А.AlexeevaK. A.

Алексеева Ксения Александровна – аспирант 3 года обучения кафедры общей химии.

308015, Белгород, ул. Победы, 85

Alekseeva Kseniya Aleksandrovna – a post-graduate student of Pharmaceutical Chemistry and Pharmacognosy Department.

85, Pobedа St., Belgorod, 308015

740890@bsu.edu.ruhttps://orcid.org/0000-0002-2996-7712ПисаревД. И.PisarevD. I.

Писарев Дмитрий Иванович – доктор фармацевтических наук, доцент, профессор кафедры общей химии.

308015, Белгород, ул. Победы, 85

Pisarev Dmitriy Ivanovich – PhD (Pharmacy), Associate Professor, Professor of Pharmaceutical Chemistry and Pharmacognosy Department.

85, Pobedа St., Belgorod, 308015

pisarev@bsu.edu.ruhttps://orcid.org/0000-0001-6170-2151МалютинаА. Ю.MalyutinaA. Yu.

Малютина Анастасия Юрьевна – кандидат фармацевтических наук, доцент кафедры общей химии.

308015, Белгород, ул. Победы, 85

Malyutina Anastasiya Yurevna – PhD (Pharmacy), Associate Professor of Pharmaceutical Chemistry and Pharmacognosy Department.

85, Pobedа St., Belgorod, 308015

malyutina_a@bsu.edu.ruhttps://orcid.org/0000-0001-9222-2935БойкоН. Н.BoykoN. N.

Бойко Николай Николаевич – кандидат фармацевтических наук, младший научный сотрудник научно-образовательного центра «Фармация».

Boyko Nikolay Nikolaevich – PhD (Pharmacy), junior research scientist of Scientific and Educational Centre “Pharmacy”.

85, Pobedа St., Belgorod, 308015

boykoniknik@gmail.comФГАОУ ВО НИУ «Белгородский государственный университет» Минобрнауки РоссииРоссияBelgorod State Research UniversityRussian Federation20180601201966535547Copyright © Алексеева К.А., Писарев Д.И., Малютина А.Ю., Бойко Н.Н., 20192019Алексеева К.А., Писарев Д.И., Малютина А.Ю., Бойко Н.Н.Alexeeva K.A., Pisarev D.I., Malyutina A.Y., Boyko N.N.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.pharmpharm.ru/jour/article/view/341

Глутатион (γ-L-глутамил-L-цистеинилглицин) является важнейшим низкомолекулярным внутриклеточным тиольным трипептидом, состоящим из трёх аминокислот – глицина, цистеина и кислоты глутаминовой. Поскольку в отечественной фармакопее отсутствует нормативная документация на глутатион, разработка фармакопейной статьи на указанную субстанцию является актуальной проблемой.

Цель

Цель. Разработка методик определения посторонних специфических примесей в глутатионе.

Материалы и методы

Материалы и методы. В качестве объектов исследования использованы субстанция – глутатион восстановленный (CAS №70-18-8, EC №2007254, Applichem, Германия), содержащий примеси и стандартный образец глутатиона восстановленного – Sigma Aldrich (Япония). Анализ проводился с помощью методов высокоэффективной жидкостной хроматографии в обращённо-фазном варианте и тонкослойной хроматографии. Хроматографирование с помощью ОФ ВЭЖХ проводили после предварительной дериватизации глутатиона и его специфических примесей дансилхлоридом. Поскольку специфические примеси в глутатионе представляют собой дипептиды и аминокислоты, следовательно, они также как и сам глутатион способны вступать в реакцию с дансилхлоридом с образованием дансильных производных, которые далее можно определить в ходе хроматографического разделения.

Результаты

Результаты. В результате хроматографирования методом ОФ ВЭЖХ дериватизированного дансилхлоридом глутатиона установлено, что данная реакция позволяет обнаружить примеси в нём. Дериваты глутатиона хорошо разделяются при хроматографировании методом ОФ ВЭЖХ и имеют отличающиеся максимумы поглощения. Дериват глутатиона имел максимум поглощения при λmax = 284 нм. Дериваты, принадлежащие специфическим примесям глутатиона, поглощают при λmax = 288 нм и λmax = 296 нм. Данные полученные с помощью ОФ ВЭЖХ были подтверждены методом ТСХ в системе изо-пропанол – вода 2:1. Обнаружено три компонента, один из которых соответствует глутатиону, два остальных – примеси.

Glutathione (γ-L-glutamyl-L-cysteinylglycine) is the most important low molecular weight intracellular thiol tripeptide consisting of three amino acids – glycine, cysteine and glutamic acid. In Russian pharmacopoeia there is no regulatory documentation for glutathione, therefore, the development of a pharmacopoeial item for the specified substance is a relevant problem.

The aim of the article is the development of methods for determining foreign specific impurities in glutathione.

Materials and methods

Materials and methods. The substance of glutathione reduced (CAS 70-18-8, EC 2007254, Applichem, Germany) containing impurities, and a standard sample of reduced glutathione (Sigma Aldrich, Japan) were used as the objects of the study. The analysis was carried out by using a high-performance liquid chromatography method in the reverse phase version and a thin layer chromatography method. The chromatography using RP HPLC was performed after preliminary derivatization of glutathione and its specific impurities with dancil chloride. Specific impurities in glutathione are dipeptides and amino acids. Therefore, they, like glutathione, can react with dancil chloride. Dancil derivatives are formed, and they can be determined by chromatographic separation.

Results

Results. As a result of chromatography by the method of RP HPLC of derivatized dancil chloride glutathione it has been established that this reaction makes it possible to detect impurities in it. Glutathione derivatives are well separated by chromatography by implementing the method of RP HPLC and have different absorption maxima. The glutathione derivative had an absorption maximum at λmax=284 nm. The derivatives belonging to specific glutathione impurities absorb at λmax=288 nm and λmax=296 nm. The data obtained using RP HPLC were confirmed by TLC in the isopropanol-water (2:1) system. Three components were found out, one of which corresponds to glutathione, while two others are impurities.

Conclusion

Conclusion. Methods for determining impurities in the glutathione substance using RP HPLC methods with preliminary derivatization with dancil chloride and TLC with ninhydrin detection have been worked out. A comparative analysis of the data obtained makes it possible to state that the OF-HPLC method with pre-column derivatization is more reliable, since it is more sensitive to impurities, and also makes it possible to study the UV profiles of impurity components better than the TLC method. Therefore, for the detection of impurities in the substance of glutathione, it is more preferable to use RP-HPLC with pre-column derivatization. The results of this study can be recommended for inclusion in the regulatory documentation on the substance of glutathione in the section “Impurities”.

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