Optimization of Extraction of Phlorotannins from the Arctic Fucus vesiculosus Using Natural Deep Eutectic Solvents and Their HPLC Profiling with Tandem High-Resolution Mass Spectrometry

Phlorotannins are secondary metabolites produced mainly by brown seaweeds (Phaeophyceae) and belong to the class of polyphenolic compounds with diverse bioactivities. The key factors in the extraction of polyphenols are the selection of a suitable solvent, method of extraction and selection of optimal conditions. Ultrasonic-assisted extraction (UAE) is one of the advanced energy-saving methods suitable for the extraction of labile compounds. Methanol, acetone, ethanol and ethyl acetate are the most commonly used solvents for polyphenol extraction. As alternatives to toxic organic solvents, a new class of green solvents, natural deep eutectic solvents (NADES), has been proposed for the efficient extraction of a wide range of natural compounds including polyphenols. Several NADES were screened previously for the extraction of phlorotannins; however, the extraction conditions were not optimized and chemical profiling of NADES extract was not performed. The purpose of this work was to study the effect of selected extraction parameters on the phlorotannin content in NADES extract from Fucus vesiculosus, optimization of extraction conditions and chemical profiling of phlorotannins in the NADES extract. A fast and green NADES-UAE procedure was developed for the extraction of phlorotannins. Optimization was performed through an experimental design and showed that NADES (lactic acid:choline chloride; 3:1) provides a high yield (137.3 mg phloroglucinol equivalents per g dry weight of algae) of phlorotannins under the following extraction conditions: extraction time 23 min, 30.0% water concentration and 1:12 sample to solvent ratio. The antioxidant activity of the optimized NADES extract was equal to that of EtOH extract. In total, 32 phlorotannins have been identified (one trimer, two tetramers, six pentamers, four hexamers, six heptamers, six octamers and seven nonamers) in NADES extracts from arctic F. vesiculosus using the HPLC-HRMS and MS/MS technique. It was noted that all the above-mentioned phlorotannins were identified in both EtOH and NADES extracts. Our results suggest that NADES could be considered as an alternative to the conventional techniques for the effective extraction of phlorotannins from F. vesiculosus with high antioxidant potential. © 2023 by the authors.121091600104-7; Ministry of Education and Science of the Russian Federation, Minobrnauka; Russian Science Foundation, RSF: 20-66-47017This study was funded by the Ministry of Science and Higher Education of the Russian Federation within the framework of the Government Assignment to the Murmansk Marine Biological Institute Russian Academy of Sciences (State Reg. No. 121091600104-7). HPLC-HRMS and MS/MS analysis were performed with the financial support from the Russian Science Foundation (Grant 20-66-47017)

Влияние условий пробоподготовки и режима хроматографирования на уровень фонового сигнала при ВЭЖХ-УФ-анализе плазмы крови

The HPLC-UV method is widely used for quantitative analysis of drug substances found in biological samples obtained in pharmacokinetics studies, bioequivalence studies, or during therapeutic drug monitoring. The main limitation is associated with a significant background effect of biological matrixes, limiting the sensitivity of HPLC-UV methods.The aim of the study was to evaluate sample preparation and chromatographic conditions in terms of background signal level during HPLC-UV analysis of blood plasma.Material and methods: three types of blood plasma (rat, rabbit and human), three chromatographic modes, and three detection wavelengths were used to assess the effect of the precipitation agent, centrifuge conditions, and the inclusion of the solid-phase extraction (SPE) step into the sample preparation procedure on the background signal level during HPLC-UV analysis.Results. It was established that the background signal was practically unaffected by the chromatographic mode, centrifugation intensity, or introduction of the SPE step. The background signal levels for human and rabbit blood plasma tended to be lower than that for rat blood plasma. The factors that had the greatest effect on the background signal level were the choice of the precipitation agent during sample preparation, and the detection wavelength. It was shown that acetonitrile is preferable to methanol, and that the near UV region should be avoided.Conclusions. The identified key aspects of sample preparation procedures and chromatographic conditions can be used in the development and validation of bioanalytical methods for preclinical and clinical studies.Метод ВЭЖХ с УФ-детектированием широко используют для количественного анализа лекарственных веществ в биообразцах, полученных при изучении фармакокинетики, биоэквивалентности или лекарственного мониторинга. Основное ограничение связано со значимым фоновым влиянием матриц биообразцов, ограничивающим чувствительность ВЭЖХ-УФ-методик.Цель работы: оценка влияния пробоподготовки и условий хроматографирования на уровень фонового сигнала при ВЭЖХ-УФ-анализе плазмы крови.Материалы и методы: на примере трех типов плазмы крови (крысы, кролика и человека) рассмотрено влияние осадителя, режима центрифугирования, включения стадии твердофазной экстракции (ТФЭ) в процедуру подготовки проб на уровень фонового сигнала при анализе методом ВЭЖХ-УФ в трех хроматографических режимах при трех длинах волн.Результаты: установлено, что уровень фонового сигнала практически не зависит от хроматографического режима, интенсивности центрифугирования и введения стадии ТФЭ;  для плазмы крови человека и кролика уровень фонового сигнала имеет тенденцию к снижению по сравнению с сигналом плазмы крови крыс. Наибольшее значение на уровень фонового сигнала оказывает выбор осадителя при пробоподготовке и длина волны детектирования. Показано, что ацетонитрил предпочтительнее, чем метанол, а при выборе длины волны следует по возможности избегать ближневолновой области.Выводы: выявленные особенности ключевых моментов процедуры подготовки проб и выбора условий хроматографического анализа могут быть использованы при разработке и валидации биоаналитических методик, применяемых в доклинических и клинических исследованиях

Экспериментальное исследование фармакокинетики рифабутина в липосомальной форме

On mongrel male rats studied pharmacokinetics of rifabutin in liposomal form, the relative bioavailability and tissue distribution after intratracheal administration, evaluated pharmacokinetic linearity. To determine rifabutin in plasma and organs were developed and validated HPLC method with UV detection. In the course of the study established a new linear pharmacokinetics of liposomal form of rifabutin dose range 6.5-26 mg/kg, calculate the main pharmacokinetic parameters found that rifabutin intensively distributed in highly vascularized organs, its content in poorly vascularized organs is much lower. After injection the highest concentration of active substance is observed at the injection site, namely the lungs. Relative bioavailability of rifabutin in liposomal form in this experiment was 522%.На беспородных крысах-самцах изучена фармакокинетика препарата рифабутин в липосомальной форме, относительная биодоступность и распределение в тканях после эндотрахеального введения препарата, проведена оценка линейности фармакокинетики. Для определения рифабутина в плазме крови и органах был разработан и валидирован метод ВЭЖХ с УФ-детекцией. В ходе проведенного исследования установлена линейность фармакокинетики новой липосомальной формы рифабутина в диапазоне доз 6.5-26 мг/кг, рассчитаны основные фармакокинетические параметры, установлено, что рифабутин интенсивно распределяется в сильно васкуляризированные органы, содержание его в слабо васкуляризированных органах значительно ниже. После введения препарата наибольшая концентрация действующего вещества наблюдается в месте введения, а именно в легких. Относительная биодоступность препарата Рифабутин в липосомальной форме в проведенном эксперименте составила 522%

Effect of Sample Preparation and Chromatographic Conditions on Background Signal

The HPLC-UV method is widely used for quantitative analysis of drug substances found in biological samples obtained in pharmacokinetics studies, bioequivalence studies, or during therapeutic drug monitoring. The main limitation is associated with a significant background effect of biological matrixes, limiting the sensitivity of HPLC-UV methods.The aim of the study was to evaluate sample preparation and chromatographic conditions in terms of background signal level during HPLC-UV analysis of blood plasma.Material and methods: three types of blood plasma (rat, rabbit and human), three chromatographic modes, and three detection wavelengths were used to assess the effect of the precipitation agent, centrifuge conditions, and the inclusion of the solid-phase extraction (SPE) step into the sample preparation procedure on the background signal level during HPLC-UV analysis.Results. It was established that the background signal was practically unaffected by the chromatographic mode, centrifugation intensity, or introduction of the SPE step. The background signal levels for human and rabbit blood plasma tended to be lower than that for rat blood plasma. The factors that had the greatest effect on the background signal level were the choice of the precipitation agent during sample preparation, and the detection wavelength. It was shown that acetonitrile is preferable to methanol, and that the near UV region should be avoided.Conclusions. The identified key aspects of sample preparation procedures and chromatographic conditions can be used in the development and validation of bioanalytical methods for preclinical and clinical studies

DEVELOPMENT AND VALIDATION OF IMMUNOASSAY FOR IMMUNOGENICITY OF INTERFERON-ALPHA 2A DRUGS

Interferons were one of the first groups of cytokines to be cloned and produced in bacteria. Interferon-based drugs are widely used for treatment of various infections, inflammatory and autoimmune diseases and malignant tumors. Clinical use of interferons can result in wide range of side effects, including unwanted influence on immune system of patients. Therefore it is important to study immunogenicity of interferones. The method of indirect ELISA was adopted and validated for analysis of antibodies to human recombinant interferon in mice blood plasma. The specificity was confirmed for mice antibodies. The linearity was 3.75-60.00 ng/ml, the detection limit was 2.19 ng/ml. Accuracy, interday and intraday precision did not exceed 15% at all concentration levels, that satisfies all validation requirements

DETERMINATION OF DARBEPOETIN ALFA IN RABBIT PLASMA BY ELISA

Method of quantitative determination of darbepoetin alfa in rabbit blood plasma was developed. Quantitative determination was performed by ELISA using commercially available ELISA kits for assay «Erythropoietin ELISA-Best». The method was validated on the following parameters: specificity, linearity, precision, accuracy, and the stability samples. Linear range of methods for determining darbepoetin alfa in the blood plasma of rabbits was 67-2000 pg/mL, the limit of detection - 31 pg/ml. The method can be used for bioequivalence and toxicokinetics studies of drugs of darbepoetin alfa

ABNORMAL TOXICITY TEST VALIDATION FOR INSULIN SUBSTANCE STANDARDISATION

Abnormal Toxicity test is obligatory in the Russian Federation for standardisation of biological origin substances, despite an ambiguous assessment of its expediency. Evaluation of abnormal toxicity of insulin by a standard technique is impossible because its introduction causes death of animals as a result of hypoglycemic shock. A change in pharmacopoeial technique defines need of its validation. On the example of substance of human genetically engineered insulin Rosinsulin (LLC «Zavod Medsintez») with mice males as biological test system the optimum conditions of the test were proposed. They include intraperitoneal introduction of glucose solution in 60 min, prior to intravenous administration of insulin. Indicators of intoxication, body weight and blood glucose level were monitored during the test for three bathes of insulin substance. Abnormal Toxicity test was validated on Robutness (with influence of small changes of volume of the entered solution of insulin and time between introduction of glucose and insulin solutions) and a Precision (intra-day and inter-day). Satisfactory results for all validation parameters were received

DISTRIBUTION OF THE LABELED [¹²³I] PEPTIDE-PROTEIN DRUG CELLEX IN RATS

The aim of this work was to study the distribution of the labeled 123I peptide-protein drug Cellex in organs and tissues of rats after single intravenous injection and accumulation. After a single intravenous injection of radiolabeled [123I]Cellex it was found that the drug is characterized by rapid distribution into tissues and organs. The highest content of the drug in all organs and tissues was observed in 15 minutes after its administration. It was established that the drug penetrates the blood-brain barrier and about 6% of the drug accumulates in the brain, which is 10% of the total amount of the drug found in all organs in 15 minutes after administration

Maitohorsman (Ebilobium angustifolium) fermentoinnin optimointi

Maitohorsman (Epilobium angustifolium L.) fermentoiduista lehdistä valmistettua teetä (Ivan tsaj, Ivan-tee, Koporjen tee) käytetään Venäjälle perinteisesti vatsahaavojen, mahakatarrin ja nukkumisvaikeuksien hoitoon. Ivan-teen aineksen perinteinen valmistusmenetelmä sisältää useita käsityötä vaativia vaiheita. Vuosien 2012–14 aikana SPECICROP-hankkeessa tehtiin useita koesarjoja, joissa fermentoitua teetä valmistettiin maitohorsman versoista. Kokeet tehtiin Maa- ja elintarviketalouden tutkimuskeskuksessa (nykyisin Luonnonvarakeskus) Mikkelissä yhteistyössä Venäjällä toimivan Pietarin Farmasia-Instituutin kanssa. Tuloksien pohjalta todettiin, että Ivan-tee voidaan valmistaa kaupallisessa mittakaavassa maitohorsman 40 cm pitkistä tuoreista versoista. Koetulosten pohjalta tuotantoprosessin eri vaiheet optimoitiin ja ne esitellään ehdotetussa tuotantomenetelmässä. Kuvattu menetelmä sopii pienen yrityksen olosuhteisiin.201