7 research outputs found
Development and validation of the method for the quantitative determination of methotrexate in a transport medium by HPLC-MS/MS
No full textRelevance. BCRP is an efflux transporter protein that plays an important role in the pharmacokinetics of a wide range of drugs. The BCRP activity in vitro experiments is assessed by the transport of transporter protein substrates (methotrexate, etc.) across the bilipid membrane of cells overexpressingBCRP, for example, Caco-2 cells. The aim is to develop and validate a method for the quantitative determination of the BCRP substrate, methotrexate, in the transport medium of Caco-2 cells by HPLC-MS/MS. Methods. The work was performed on an Ultimate 3000 HPLC chromatograph (ThermoFisher, USA) with a TSQ Fortis tandem mass-selective detector (ThermoFisher, USA). The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm * 100 mm 5um, 100A, Selectra C18 Guard Cartridges SLC-18GDC46-5UM, separation temperature 35 °С, flow rate 0.3 ml/min, injected sample volume - 2 μl, analysis time - 10 min. Used a gradient elution: the ratio of the solution of 0.1 % formic acid and acetonitrile was at 0 min 75 and 25 %; 0.4 min 60 and 40 %; 6 minutes 20 and 80 %; 8 minutes 75 and 25 %. Under these conditions, the retention time of methotrexate is 3.11 minutes. Detection conditions: methotrexate - positive ionization mode, 455.15 m / z → 308.125 m / z, collision energy 22.99 V, source fragmentation 5, CID gas pressure 2 mTorr. The extraction of methotrexate from the transport medium (Hanks solution with 25 mM Hepes and 1% dimethyl sulfoxide) after incubation with Caco-2 cells for 3 h was carried out with a mixture of methanol + water in a ratio of 1: 1. Results. The developed method was validated according to the following parameters: selectivity, linearity, accuracy, precision, limit of quantitative determination, sample transfer, sample stability. The confirmed analytical range of the method was 60 -10,000 nmol / L in the transport medium. Conclusions: a method for the quantitative determination of methotrexate in the transport medium of Caco-2 cells by HPLC-MS / MS was developed and validated
Mitoxantrone Quantification by HPLC-MS/MS in Caco-2 Culture Media
No full textMitoxantrone is a marker substrate of breast cancer resistance protein (BCRP). BCRP is involved in a number of pharmacokinetic drug-drug interactions. The transporter’s possible saturability makes it advisable to use low concentrations of mitoxantrone for in vitro studies. Consequently, mitoxantrone quantification requires a method with high sensitivity.The aim of the study was to develop and validate a procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.Materials and methods. The authors used an Ultimate 3000 HPLC system and a TSQ Fortis triple quadrupole mass spectrometer by Thermo Fisher Scientific and a Selectra C18 column (4.6×100 mm, 5 μm, 100 Å) by United Chemical Technologies. The elution ran in a gradient mode with a mobile phase of 1% formic acid solution and methanol. Experimental parameters were as follows: eluent flow rate, 0.3 mL/min; separation column temperature, 35 °C; injection volume, 5 μL; ana lysis time, 10 min; approximate mitoxantrone retention time, 5.51 min. The sample preparation involved protein precipitation from the culture medium with methanol, followed by centrifugation at 13,000 g for 10 min. The detection was performed using electrospray ionisation in the positive ion mode. Detection parameters were as follows: electrospray voltage, 3700 V; sheath gas flow rate, 50 L/min; auxiliary gas flow rate, 10 L/min; sweep gas flow rate, 1 L/min; ion-transfer tube temperature, 300 °C; and evaporator temperature, 350 °C. The detection was set at mass transitions of m/z 455 to 88.2 and m/z 455 to 358.1, with the collision energy for these transitions amounting to 25 V and 18 V, respectively. The source fragmentation was at 0, and the CID gas pressure was at 2 mTorr.Results. The analytical procedure showed selectivity, high sensitivity (limit of detection, 10 nmol/L; lower limit of quantification, 50 nmol/L), accuracy, precision, and linearity in the concentration range of 50–1000 nmol/L. The authors observed no carryover or matrix effects. A simulation of real-life storage conditions demonstrated high stability of mitoxantrone samples. Thus, the analytical procedure enables preclinical evaluation of medicinal product effects on the functional activity of BCRP, based on assessing the transcellular mitoxantrone transport in the presence of a test product.Conclusion. The authors developed and validated the analytical procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS
Method for quantitative analysis of the marker substrate ABCB1-protein fexofenadine in Caco-2 cell lysate
No full textOne way to analyze the activity of the ABCB1 protein is to assess the accumulation of its substrate fexofenadine (F.) inside the test cells. The goal is to develop and validate a method for the quantitative analysis of F. in Caco-2 cell lysate using HPLC-MS/MS. Materials and methods. Caco-2 cell lysate was used as a matrix. The analysis was performed on an "Ultimate 3000" chromatograph with a TSQ Fortis triple quadrupole mass detector, a UCT Selectra C18 4.6 mm*100 mm 5 µm column in a gradient elution mode. The mobile phase rate was 0.3 ml/min, the sample volume was 20 µl, the ionization mode was positive, and the internal standard was amantadine (ng/ml). Sample preparation — precipitation of cell lysate protein with acetonitrile. The method was validated for the following parameters: selectivity, linearity, lower limit of quantitation (LLOQ), correctness, precision, sample transfer and sample stability. Results. Chromatograms of the blank lysate of Caco-2 cells showed no peaks with retention times characteristic of F. (5.70 min) and amantadine (3.58 min). NPKO F. was 0.5 ng/ml. F.'s transfer did not exceed 20% of NPKO, and amantadine — 5%. Based on the results of the analysis of three series of calibration standards (0.5; 1; 1.5; 5; 10; 25; 40; 50 ng/ml), linear regression equations were obtained, the correlation coefficients exceeded 0.99. Accuracy and precision were assessed within and between cycles by analyzing F. solutions in the matrix (0.5; 1.5; 25 and 40 ng/ml) within three cycles. The parameters did not exceed 20% for LLPO and 15% for other points. The stability of F. solutions (1.5 and 40 ng/ml) in the lysate was analyzed during storage at room temperature, after 3-fold freezing-thawing, storage at -80 °C for 60 days, after sample preparation and being in the autosampler for 24 hours. The accuracy was within 15% of the nominal values. Conclusions. A method for the quantitative determination of F. in Caco-2 cell lysate using HPLC-MS/MS has been developed and validated
Разработка и валидация методики количественного определения метотрексата в транспортной среде методом ВЭЖХ-МC/МС
No full textRelevance. BCRP is an efflux transporter protein that plays an important role in the pharmacokinetics of a wide range of drugs. The BCRP activity in vitro experiments is assessed by the transport of transporter protein substrates (methotrexate, etc.) across the bilipid membrane of cells overexpressingBCRP, for example, Caco-2 cells. The aim is to develop and validate a method for the quantitative determination of the BCRP substrate, methotrexate, in the transport medium of Caco-2 cells by HPLC-MS/MS. Methods. The work was performed on an Ultimate 3000 HPLC chromatograph (ThermoFisher, USA) with a TSQ Fortis tandem mass-selective detector (ThermoFisher, USA). The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm * 100 mm 5um, 100A, Selectra C18 Guard Cartridges SLC-18GDC46-5UM, separation temperature 35 °С, flow rate 0.3 ml/min, injected sample volume - 2 μl, analysis time - 10 min. Used a gradient elution: the ratio of the solution of 0.1 % formic acid and acetonitrile was at 0 min 75 and 25 %; 0.4 min 60 and 40 %; 6 minutes 20 and 80 %; 8 minutes 75 and 25 %. Under these conditions, the retention time of methotrexate is 3.11 minutes. Detection conditions: methotrexate - positive ionization mode, 455.15 m / z → 308.125 m / z, collision energy 22.99 V, source fragmentation 5, CID gas pressure 2 mTorr. The extraction of methotrexate from the transport medium (Hanks solution with 25 mM Hepes and 1% dimethyl sulfoxide) after incubation with Caco-2 cells for 3 h was carried out with a mixture of methanol + water in a ratio of 1: 1. Results. The developed method was validated according to the following parameters: selectivity, linearity, accuracy, precision, limit of quantitative determination, sample transfer, sample stability. The confirmed analytical range of the method was 60 -10,000 nmol / L in the transport medium. Conclusions: a method for the quantitative determination of methotrexate in the transport medium of Caco-2 cells by HPLC-MS / MS was developed and validated.Актуальность. BCRP – эффлюксный белок-транспортёр, играющий важную роль в фармакокинетике широкого спектра лекарственных веществ. Активность BCRP в опытах in vitro оценивается по транспорту субстратов белка-транспортёра (метотрексата и др.) через билипидную мембрану клеток, гиперэкспрессирующих BCRP, например, клетках линии Caco-2. Цель: разработать и валидировать методику количественного определения субстрата BCRP – метотрексата в транспортной среде клеток линии Caco-2 методом ВЭЖХ-МС/МС. Методы исследования. Работа выполнена на ВЭЖХ-хроматографе «Ultimate 3000» («ThermoFisher», США) с тандемным масс-селективным детектором TSQ Fortis («ThermoFisher», США). Условия хроматографического анализа были следующими: колонка UCT Selectra C18 4,6 mm ×100 mm 5um, 100A, предколонка Selectra C18 Guard Cartridges SLC-18GDC46-5UM, температура разделения – 35 °С, скорость потока – 0,3 мл/мин, объём вводимой пробы – 2 мкл, время анализа – 10 мин. Использовали градиентный режим элюирования: соотношение раствора 0,1 % муравьиной кислоты и ацетонитрила составило на 0 мин 75 и 25 %; 0,4 мин – 60 и 40 %; 6 мин – 20 и 80 %; 8 мин – 75 и 25 %. В данных условиях время удерживания метотрексата – 3,11 мин. Условия детектирования: метотрексат – положительный режим ионизации, 455,15 m/z → 308,125 m/z, энергия столкновения – 22,99 В, фрагментация источника – 5, давление CID-газа – 2 мТорр. Извлечение метотрексата из транспортной среды (раствор Хэнкса с 25 мМ Хепес и 1 % диметилсульфоксида) после инкубирования с клетками линии Сaco-2 в течение 3 ч осуществляли смесью метанол+вода в соотношении 1:1. Результаты. Разработанная методика была валидирована по следующим параметрам: селективность, линейность, точность, прецизионность, предел количественного определения, перенос пробы, стабильность образцов. Подтверждённый аналитический диапазон методики составил 60–10 000 нмоль/л в транспортной среде. Выводы: разработана и валидирована методика количественного определения метотрексата в транспортной среде клеток линии Caco-2 методом ВЭЖХ-МС/МС
Method for Testing of Drugs Belonging to Substrates and Inhibitors of the Transporter Protein BCRP on Caco-2 Cells
No full textIntroduction. Breast cancer resistance protein (BCRP) is an efflux membrane transporter that controls the pharmacokinetics of a large number of drugs. Its activity may change when taking some endo- and exogenous substances, thus making it a link in drug interactions.Aim. The aim of the study was to develop a method for testing of drugs for belonging to BCRP substrates and inhibitors in vitro.Materials and methods. The work was performed on Caco-2 cells overexpressing BCRP, the cultivation was performed in a transwell-system consisting of the apical and basolateral chambers. Cells were seeded at the bottom of the apical chamber, which is a semipermeable membrane. Primarily, the transport of BCRP substrates: methotrexate, mitoxantrone and quercetin was evaluated in the concentration range of 1, 5, 10, and 50 μM in the direction from the basal chamber to the apical one (Papp b-a) and in the opposite direction (Papp a-b). The ratio Papp b-a / Papp a-b more than «2» characterizes the participation of transporter proteins in the transcellular transport of substances. To confirm the participation of BCRP in their transport the experiment was carried out with the addition of a transporter inhibitor, reserpine, to the transport medium at a concentration of 50 μM. The concentration of substrates in the chambers was analyzed by HPLC-MS/MS.Results and their discussion. The addition of methotrexate (1 μM), mitoxantrone (1 μM), and quercetin (1–10 μM) to both the apical or basolateral chambers of the transwell-system, their content in the recipient chamber was not detected. When methotrexate concentration became 5 μM the Papp b-a / Papp a-b ratio was 3.38 ± 0.08, which indicates the involvement of transporters in its transfer. The addition of methotrexate to the donor chamber at concentrations of 10 and 50 μM, Papp b-a / Papp a-b decreased to values below «2». At mitoxantrone concentration of 5 μM Papp b-a / Papp a-b was 2.72 ± 0.16. An increase in the concentration to 10 μM led to an increase in Papp b-a / Papp a-b to 6.18 ± 0.08. With a substance content of 50 μM the indicator decreased but remained above the value «2». In the quercetin concentration of 50 microns, Papp b-a / Papp was below "2". Reserpine reduced Papp b-a / Papp a-b of methotrexate by 3.31 times (p = 0.0002), which indicates the elimination of asymmetry in the transport of the substance. At a mitoxantrone concentration of 10 microns, reserpine reduced its Papp b-a / Papp a-b by 3.36 times (p < 0.0001). The results indicate the participation of BCRP in the control of the transfer of both substances through the cellular monolayer.Conclusion. A method of testing drugs belonging to BCRP substrates and inhibitors using methotrexate (5 μM) and mitoxantrone (10 μM) as marker substrates and reserpine (50 μM) as inhibitor was developed and tested on Caco-2 cells
Количественный анализ митоксантрона методом ВЭЖХ-МС/МС в среде для культивирования клеток Caco-2
No full textMitoxantrone is a marker substrate of breast cancer resistance protein (BCRP). BCRP is involved in a number of pharmacokinetic drug-drug interactions. The transporter’s possible saturability makes it advisable to use low concentrations of mitoxantrone for in vitro studies. Consequently, mitoxantrone quantification requires a method with high sensitivity.The aim of the study was to develop and validate a procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.Materials and methods. The authors used an Ultimate 3000 HPLC system and a TSQ Fortis triple quadrupole mass spectrometer by Thermo Fisher Scientific and a Selectra C18 column (4.6×100 mm, 5 μm, 100 Å) by United Chemical Technologies. The elution ran in a gradient mode with a mobile phase of 1% formic acid solution and methanol. Experimental parameters were as follows: eluent flow rate, 0.3 mL/min; separation column temperature, 35 °C; injection volume, 5 μL; ana lysis time, 10 min; approximate mitoxantrone retention time, 5.51 min. The sample preparation involved protein precipitation from the culture medium with methanol, followed by centrifugation at 13,000 g for 10 min. The detection was performed using electrospray ionisation in the positive ion mode. Detection parameters were as follows: electrospray voltage, 3700 V; sheath gas flow rate, 50 L/min; auxiliary gas flow rate, 10 L/min; sweep gas flow rate, 1 L/min; ion-transfer tube temperature, 300 °C; and evaporator temperature, 350 °C. The detection was set at mass transitions of m/z 455 to 88.2 and m/z 455 to 358.1, with the collision energy for these transitions amounting to 25 V and 18 V, respectively. The source fragmentation was at 0, and the CID gas pressure was at 2 mTorr.Results. The analytical procedure showed selectivity, high sensitivity (limit of detection, 10 nmol/L; lower limit of quantification, 50 nmol/L), accuracy, precision, and linearity in the concentration range of 50–1000 nmol/L. The authors observed no carryover or matrix effects. A simulation of real-life storage conditions demonstrated high stability of mitoxantrone samples. Thus, the analytical procedure enables preclinical evaluation of medicinal product effects on the functional activity of BCRP, based on assessing the transcellular mitoxantrone transport in the presence of a test product.Conclusion. The authors developed and validated the analytical procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.Митоксантрон — маркерный субстрат белка устойчивости рака молочной железы (BCRP), участвующего в ряде фармакокинетических межлекарственных взаимодействий. При проведении исследований in vitro в связи с возможной насыщаемостью транспортера BCRP целесообразно применять невысокие концентрации митоксантрона, поэтому необходим высокочувствительный метод его количественного определения.Цель работы: разработка и валидация методики количественного определения митоксантрона в среде для культивирования клеток Caco-2 методом ВЭЖХ-МС/МС.Материалы и методы: анализ выполняли на высокоэффективном жидкостном хроматографе «Ultimate 3000» (Thermo Fisher Scientific) с тройным квадрупольным масс-спектрометрическим детектором TSQ Fortis и колонкой UCT Selectra C18 4,6×100 мм, 5 мкм, 100 Å. Использовали градиентный режим элюирования, подвижная фаза — смесь раствора муравьиной кислоты 1% и метанола. Скорость подвижной фазы — 0,3 мл/мин, температура разделения — 35 °С, объем пробы — 5 мкл. Длительность анализа — 10 мин, время удерживания митоксантрона — 5,51 мин. Пробоподготовка заключалась в осаждении белка среды для культивирования метанолом и центрифугировании (13000 g, 10 мин). Детектирование проводили в режиме регистрации положительных ионов, метод ионизации — электрораспыление (3700 В), оболочечный газ 50 л/мин, вспомогательный газ 10 л/мин, продувочный газ 1 л/мин, температура трубки для переноса ионов 300 °С, температура испарителя 350 °С. Переходы масс: 455 m/z → 88,2 m/z при энергии столкновения 25 В, 455 m/z → 358,1 m/z при 18 В, фрагментация источника 0, CID gas 2 мТорр.Результаты: методика характеризовалась селективностью, чувствительностью (предел обнаружения — 10 нмоль/л, нижний предел количественного определения — 50 нмоль/л), правильностью, прецизионностью и линейностью (50−1000 нмоль/л). Отсутствовал перенос пробы и матричный эффект, образцы проб обладали стабильностью при моделировании реальных условий хранения. Методика позволяет проводить доклинические исследования влияния лекарственных веществ на активность BCRP путем оценки трансцеллюлярного переноса митоксантрона в присутствии тестируемого вещества.Выводы: разработана и валидирована ВЭЖХ-МС/МС методика количественного анализа митоксантрона в среде для культивирования клеток Caco-2
Методика количественного анализа маркерного субстрата ABCB1-белка фексофенадина в лизате клеток Caco-2
No full textOne way to analyze the activity of the ABCB1 protein is to assess the accumulation of its substrate fexofenadine (F.) inside the test cells. The goal is to develop and validate a method for the quantitative analysis of F. in Caco-2 cell lysate using HPLC-MS/MS. Materials and methods. Caco-2 cell lysate was used as a matrix. The analysis was performed on an "Ultimate 3000" chromatograph with a TSQ Fortis triple quadrupole mass detector, a UCT Selectra C18 4.6 mm*100 mm 5 µm column in a gradient elution mode. The mobile phase rate was 0.3 ml/min, the sample volume was 20 µl, the ionization mode was positive, and the internal standard was amantadine (ng/ml). Sample preparation — precipitation of cell lysate protein with acetonitrile. The method was validated for the following parameters: selectivity, linearity, lower limit of quantitation (LLOQ), correctness, precision, sample transfer and sample stability. Results. Chromatograms of the blank lysate of Caco-2 cells showed no peaks with retention times characteristic of F. (5.70 min) and amantadine (3.58 min). NPKO F. was 0.5 ng/ml. F.'s transfer did not exceed 20% of NPKO, and amantadine — 5%. Based on the results of the analysis of three series of calibration standards (0.5; 1; 1.5; 5; 10; 25; 40; 50 ng/ml), linear regression equations were obtained, the correlation coefficients exceeded 0.99. Accuracy and precision were assessed within and between cycles by analyzing F. solutions in the matrix (0.5; 1.5; 25 and 40 ng/ml) within three cycles. The parameters did not exceed 20% for LLPO and 15% for other points. The stability of F. solutions (1.5 and 40 ng/ml) in the lysate was analyzed during storage at room temperature, after 3-fold freezing-thawing, storage at -80 °C for 60 days, after sample preparation and being in the autosampler for 24 hours. The accuracy was within 15% of the nominal values. Conclusions. A method for the quantitative determination of F. in Caco-2 cell lysate using HPLC-MS/MS has been developed and validated.Один из способов анализа активности ABCB1-белка — это оценка накопления его субстрата фексофенадина (Ф.) внутри тест-клеток. Цель — разработка и валидация методики количественного анализа Ф. в лизате клеток Caco-2 с помощью ВЭЖХ-МС/МС. Материалы и методы. В качестве матрицы использовался лизат клеток Caco-2. Анализ выполняли на хроматографе «Ultimate 3000» с тройным квадрупольным масс-детектором TSQ Fortis, колонкой UCT Selectra C18 4,6 мм×100 мм 5 мкм в градиентном режиме элюирования. Скорость подвижной фазы — 0,3 мл/мин, объём пробы — 20 мкл, режим ионизации — положительный, внутренний стандарт — амантадин (нг/мл). Пробоподготовка — осаждение белка лизата клеток ацетонитрилом. Методику валидировали по параметрам: селективность, линейность, нижний предел количественного определения (НПКО), правильность, прецизионность, перенос пробы и стабильность образцов. Результаты. На хроматограммах холостого лизата клеток Caco-2 не было пиков со временем удерживания, характерным для Ф. (5,70 мин) и амантадина (3,58 мин). НПКО Ф. составил 0,5 нг/мл. Перенос Ф. не превышал 20 % НПКО, а амантадина — 5 %. По результатам анализа трёх серий градуировочных стандартов (0,5; 1; 1,5; 5; 10; 25; 40; 50 нг/мл) получены уравнения линейной регрессии, коэффициенты корреляции превышали 0,99. Правильность и прецизионность оценивали внутри и между циклами, выполняя анализ растворов Ф. в матрице (0,5; 1,5; 25 и 40 нг/мл) в рамках трёх циклов. Параметры не превышали 20 % для НПКО и 15 % — для остальных точек. Стабильность растворов Ф. (1,5 и 40 нг/мл) в лизате анализировали при хранении при комнатной температуре, после 3-кратной заморозки–разморозки, хранении при -80 °С 60 сут., после пробоподготовки и нахождения в автосемплере 24 ч. Правильность находилась в пределах 15 % от номинальных значений. Выводы. Разработана и валидирована методика количественного определения Ф. в лизате клеток Caco-2 с помощью ВЭЖХ-МС/МС
