2020-4-40-54

УДК 616.5:615.07

И. В. Семак¹, В. Б. Климашевич², Е. В. Малюшкова¹, Е. О. Корик¹, О. А. Казючиц³, А. И. Жебентяев⁴

РАЗРАБОТКА И ВАЛИДАЦИЯ МЕТОДИКИ КОЛИЧЕСТВЕННОГО ОПРЕДЕЛЕНИЯ РАНОЛАЗИНА В СЫВОРОТКЕ КРОВИ ЧЕЛОВЕКА ДЛЯ ФАРМАКОКИНЕТИЧЕСКИХ ИССЛЕДОВАНИЙ

¹Белорусский государственный университет,

г. Минск, Республика Беларусь

²Государственное предприятие «АКАДЕМФАРМ»,

г. Минск, Республика Беларусь

³СООО«НАТИВИТА»,

г. Витебск, Республика Беларусь

⁴Витебский государственный ордена Дружбы народов медицинский университет,

г. Витебск, Республика Беларусь

Разработана методика хромато-масс-спектрометрического определения ранолазина в сыворотке крови с использованием для пробоподготовки твердофазной экстракции на гидрофобном сополимере полистирола и дивинилбензола (Chromabond HR-X) с последующим хроматографическим разделением экстрагированных аналитов на колонке со среднеполярной фазой на основе химически модифицированного кремнезема с привитыми цианоалкильными группами (Nucleodur-100-3 CN-RP). Благодаря предложенной схеме твердофазной экстракции достигалась селективность и эффективность выделения ранолазина и минимизировался эффект матрицы при последующем его масс-спектрометрическом детектировании. Сочетание в методике различных механизмов сорбции аналитов при пробоподготовке и при хроматографии обеспечило высокую селективность и чувствительность анализа. В качестве внутреннего стандарта впервые предложено использовать карведилол, который, благодаря наличию ряда общих структурных элементов и сходству физико-химических свойств, обеспечивает высокую воспроизводимость и надежность разработанной методики при анализе большого количества биообразцов. Применение карведилола в качестве альтернативы дорогостоящим и малодоступным меченым внутренним стандартам позволяет снизить себестоимость количественного определения ранолазина при проведении масштабных биоэквивалентных испытаний. Методика валидирована в соответствии с международными требованиями и успешно апробирована при исследовании сравнительной биодоступности лекарственных средств, содержащих ранолазин.

Ключевые слова:

ранолазин, твердофазная экстракция, хромато-масс-спектрометрическое определение, валидация.

SUMMARY

V. Semak, V. B. Klimashevich, E. V. Malyushkova, E. O. Korik, O. A. Kazyuchits, A. I. Zhebentyaev

DEVELOPMENT AND VALIDATION OF THE ASSAY METHOD FOR RANOLASIN IN HUMAN BLOOD SERUM FOR PHARMACOKINETIC STUDIES

1, 2 Minsk, The Republic of Belarus

3, 4 Vitebsk, The Republic of Belarus

A method for chromatography-mass spectrometric determination of ranolazine in blood serum has been developed using solid-phase extraction for sample preparation on hydrophobic polystyrene and divinylbenzene copolymer (Chromabond HR-X) followed by chromatographic separation of the extracted analytes on the column with a mid-polar phase based on chemically modified cyanoalkyl-embedded silica (Nucleodur-100-3 CN-RP). Due to the proposed solid phase extraction scheme, selectivity and efficiency of ranolazine isolation were achieved and matrix effect was minimized during its subsequent mass spectrometric detection. Various mechanisms combination of analytes sorption in the technique during sample preparation and chromatography performance provided high selectivity and sensitivity of analysis. For the first time carvedilol was proposed to be used as an internal standard which, due to the presence of a number of common structural elements and similarity of physicochemical properties, ensures high reproducibility and reliability of the developed technique when analyzing a large number of biosamples. The use of carvedilol as an alternative to expensive and inaccessible labeled internal standards allows to reduce the cost of ranolazine assay during large-scale bioequivalent studies. The methodology is validated in accordance with international requirements and has been successfully tested in the study of comparative bioavailability of the drugs containing ranolazine.

Keywords:

ranolazine, solid phase extraction, chromatography-mass spectrometric determination, validation.

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