Development and validation of the method for the quantitative determination of methotrexate in a transport medium by HPLC-MS/MS

Relevance. 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.

Download Full-text

INFLUENCE OF POLYSACCHARIDE COMPLEXES OF PLANTS OF CENTRAL RUSSIA ON THE P-GLYCOPROTEIN ACTIVITY IN VITRO

chemistry of plant raw material ◽

10.14258/jcprm.2020047444 ◽

2020 ◽

pp. 73-81

Author(s):

Ivan Vladimirovich Chernykh ◽

Aleksey Vladimirovich Shchul'kin ◽

Yekaterina Yevgen'yevna Kirichenko ◽

Sergey Konstantinovich Pravkin ◽

Yelena Nikolayevna Yakusheva

Keyword(s):

High Performance ◽

Protein Activity ◽

Apparent Permeability ◽

Melilotus Officinalis ◽

Polysaccharide Complex ◽

Transport Medium ◽

Transporter Protein ◽

Central Russia ◽

P Glycoprotein

The purpose of this work was to study the effect of polysaccharide complexes isolated from tansy flowers (Tanacetum vulgare L., fam. Asteraceae) and melilotus herb (Melilotus officinalis L., fam. Fabaceae) on P-glycoprotein (Pgp, ABCB1 protein) activity in vitro. On Caco-2 cell line the effect of polysaccharide complex isolated from tansy flowers and melilotus herb on Pgp activity was studied. In vitro Pgp activity was assessed by the transport of fexofenadine in the transwell system. High performance liquid chromatography with UV detection at wavelength 220 nm was used to determine fexofenadine concentration in the transport medium. It was revealed that when polysaccharide isolated from tansy flowers was added to the transport medium in concentrations 10 and 100 μM the ratio of the apparent permeability coefficients of fexofenadine b-a/a-b decreased by 1.81 and 2.65 times, respectively, compared with the series of isolated transport of fexofenadine, which indicated decreased Pgp functional activity under the polysaccharide action. The polysaccharide complex of the melilotus herb did not change the b-a/a-b ratio in any of the applied concentrations, thus it did not affect the activity of this transporter. It is advisable to continue the study of tansy flower polysaccharide complex as an inhibitor of Pgp transporter protein in order to assess the possibility of its clinical use for the treatment of pharmacoresistant forms of cancer by overcoming the phenomenon of multidrug resistance of cells.

Download Full-text

Pegylated magnetic nanocarriers for doxorubicin delivery: A quantitative determination of stealthiness in vitro and in vivo

European Journal of Pharmaceutics and Biopharmaceutics ◽

10.1016/j.ejpb.2012.04.002 ◽

2012 ◽

Vol 81 (3) ◽

pp. 498-505 ◽

Cited By ~ 46

Author(s):

E. Allard-Vannier ◽

S. Cohen-Jonathan ◽

J. Gautier ◽

K. Hervé-Aubert ◽

E. Munnier ◽

...

Keyword(s):

Quantitative Determination ◽

Doxorubicin Delivery ◽

Magnetic Nanocarriers

Download Full-text

Importance of probe design for bioanalysis of oligonucleotides using hybridization-based LC-fluorescence assays

Bioanalysis ◽

10.4155/bio-2019-0154 ◽

2019 ◽

Vol 11 (21) ◽

pp. 1917-1925 ◽

Cited By ~ 2

Author(s):

Yuhuan Ji ◽

Yijiang Liu ◽

Wanhong Xia ◽

Alexander Behling ◽

Min Meng ◽

...

Keyword(s):

Quantitative Determination ◽

Human Plasma ◽

Peptide Nucleic Acid ◽

Dynamic Range ◽

Sample Volume ◽

Probe Design ◽

Assay Sensitivity ◽

Accuracy And Precision ◽

Fluorescence Assays

Aim: The importance of the length and/or structure of fluorescently labeled PNA (peptide nucleic acid) probes for quantitative determination of oligodeoxynucleotides (ODNs) is demonstrated in human plasma using hybridization-based LC-fluorescence assays. The length of the PNA probes impacts the peak shape and chromatographic separation of the resulting PNA/ODN hybridization complexes and affects assay sensitivity, dynamic range and carryover. Methods: For quantitative determination of an 18-mer phosphodiester ODN (DNL1818) in human plasma, an assay utilizing an Atto dye-labeled 12-mer PNA probe provided a linear quantitation range of 0.1–50 ng/ml with excellent accuracy and precision (within -5.3–7.73%). Conclusion: This method provides a convenient method for sensitive and specific quantification of ODNs in biological matrix with limited sample volume and no special extraction.

Download Full-text

A Method for the Routine Determination of Corn Sirup in Prepared Meat Products

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/47.5.903 ◽

1964 ◽

Vol 47 (5) ◽

pp. 903-909

Author(s):

Lester Hankin ◽

Alphonse F Wickroski

Keyword(s):

Statistical Analysis ◽

Quantitative Determination ◽

Correction Factor ◽

Meat Products ◽

Processed Meat ◽

Average Amount ◽

Wide Range ◽

The Difference

Abstract A method has been devised for the determination of corn sirup added to processed meat products. The method is based on the quantitative determination of dextrin added to corn sirup. The dextrins are enzymatically hydrolyzed by α-amylase and β-amylase, and maltose is calculated as the difference in CuO2 found by copper reduction between a treated and an untreated aliquot. A correction factor was devised to determine the average amount of dextrin in corn sirup by testing a number of commercial sirups for their dextrin content and subjecting the data to statistical analysis. With this equation the method is applicable to a wide range of sirups. The method also permits the estimation of dextrose added to meats in excess of that included as one of the components of corn sirup.

Download Full-text

An in vitro method for the quantitative determination of the antimicrobial efficacy of silver-containing wound dressings

International Journal of Pharmaceutics ◽

10.1016/j.ijpharm.2008.09.005 ◽

2009 ◽

Vol 366 (1-2) ◽

pp. 111-116 ◽

Cited By ~ 30

Author(s):

Simon Gaisford ◽

Anthony E. Beezer ◽

Alistair H. Bishop ◽

Michael Walker ◽

David Parsons

Keyword(s):

Quantitative Determination ◽

Wound Dressings ◽

Antimicrobial Efficacy ◽

In Vitro Method

Download Full-text

Optimized External IR Reflection Spectroscopy for Quantitative Determination of Borophosphosilicate Glass Parameters

Applied Spectroscopy ◽

10.1366/0003702971940161 ◽

1997 ◽

Vol 51 (2) ◽

pp. 259-264 ◽

Cited By ~ 5

Author(s):

Lizhong Zhang ◽

James E. Franke ◽

Thomas M. Niemczyk ◽

David M. Haaland

Keyword(s):

Film Thickness ◽

Quantitative Determination ◽

Least Squares ◽

Partial Least Squares ◽

Reflection Spectroscopy ◽

Ir Radiation ◽

Ir Reflection ◽

Wide Range ◽

Borophosphosilicate Glass

Infrared (IR) external reflection spectroscopy has been optimized for the quantitative determination of composition and film thickness of borophosphosilicate glass (BPSG) deposited on silicon wafer substrates. The precision of the partial least-squares calibrations for boron and phosphorus contents and thin-film thickness were measured as the cross-validated standard error of prediction statistic. The results showed that BPSG IR reflection spectra collected over a wide range of incident IR radiation angles (15°, 25°, 45°, and 60°) can be used for the simultaneous quantification of these three BPSG parameters. When high angles of incidence were employed, the measurement was found to be more sensitive to small errors in the angle of incidence. The polarization state of the incident IR radiation did not noticeably affect the prediction of the three calibrated BPSG parameters. The results achieved in this study provide guidelines for at-line process monitoring and quality control of BPSG thin films used in the fabrication of microelectronic devices. Index Headings: IR reflection; Multivariate calibration; Partial least-squares

Download Full-text

Quantitative determination of in vitro immunoglobulin secretion with protein A from Staphylococcus aureus

Journal of Immunological Methods ◽

10.1016/0022-1759(82)90115-6 ◽

1982 ◽

Vol 54 (1) ◽

pp. 73-80 ◽

Cited By ~ 1

Author(s):

Mioara Manciulea

Keyword(s):

Staphylococcus Aureus ◽

Quantitative Determination ◽

Protein A ◽

Immunoglobulin Secretion

Download Full-text

In vitro evaluation of a manganese chloride phantom-based MRI technique for quantitative determination of lumbar intervertebral disc composition and condition

European Spine Journal ◽

10.1007/s00586-010-1664-7 ◽

2010 ◽

Vol 20 (3) ◽

pp. 434-439 ◽

Cited By ~ 3

Author(s):

Lachlan J. Smith ◽

Andrew P. Kurmis ◽

John P. Slavotinek ◽

Nicola L. Fazzalari

Keyword(s):

Intervertebral Disc ◽

Quantitative Determination ◽

Manganese Chloride ◽

Lumbar Intervertebral Disc ◽

In Vitro Evaluation

Download Full-text

Identification and quantitative determination of pinoresinol in Taxus ×media Rehder needles, cell suspension and shoot cultures

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.2014.038 ◽

2015 ◽

Vol 84 (1) ◽

pp. 125-132 ◽

Cited By ~ 2

Author(s):

Paulina Mistrzak ◽

Hanna Celejewska-Marciniak ◽

Wojciech J. Szypuła ◽

Olga Olszowska ◽

Anna K. Kiss

Keyword(s):

Cell Culture ◽

Suspension Culture ◽

Quantitative Determination ◽

Cell Suspension ◽

Dry Weight ◽

Shoot Cultures ◽

Taxus Media ◽

In Vitro Shoot Cultures

The aim of our study was to investigate the presence and quantitative contents of lignans in the tissues of Taxus ×media. The presence of the lignans: pinoresinol, matairesinol and secoisolariciresinol was assessed in needles, shoots cultures and suspension culture. Pinoresinol was the only lignan found in the tissue of T. ×media. The total pinoresinol content in the needles and in the shoots was 1.24 mg/g dry weight (dw) and 0.69 mg/g dw, respectively. Most of the pinoresinol identified was appeared glycosidically bound. In needles, the amount of glycosidically bound pinoresinol (0.81 mg/g dw) was about twice as high as that of free pinoresinol (0.43 mg/g dw). The content of free and glycosidically bound pinoresinol showed the level of 0.18 mg/g dw and 0.51 mg/g dw, respectively in the in vitro shoot cultures. In the cell culture, no pinoresinol was found.

Download Full-text