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1.
An UHPLC-MS/MS Method for Target Profiling of Stress-Related Phytohormones.
Novák, O, Floková, K
Methods in molecular biology (Clifton, N.J.). 2018;:183-192
Abstract
The methodology described here represents an improved strategy for analysis of a broad range of stress-related plant hormones including jasmonates, salicylic acid, abscisic acid, and auxin metabolites. The method conditions are optimized in order to reduce the background effect of complicated plant matrix, allow effective preconcentration and thus perform highly sensitive profiling of multiple plant hormones by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
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Propafenone quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a bioequivalence study
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Severino, B, Luisi, G, Donomae Iwamoto, R, Moreno, RA, Perez Teixeira, V, Di Vaio, P, Saccone, I, Magli, E, Santagada, V, Caliendo, G, et al
International journal of clinical pharmacology and therapeutics. 2018;(6):280-291
Abstract
Propafenone is an antiarrhythmic drug applied to ventricular arrhythmias, initially recognized as a sodium channel blocker. This study aims to evaluate the bioequivalence of two propafenone formulations (300 mg tablet) in healthy subjects under non-fasting conditions. The study was conducted as an open, randomized, 2-period design with a 2-sequence (RT, TR) with a 1-week washout interval. The subjects were selected for the study after having their health status previously assessed by a clinical evaluation and laboratory tests (biochemical and hematological parameters, and urinalysis). Debrisoquine phenotype of healthy subjects was determined by analysis of urinary excretion of debrisoquine and its major metabolite, 4-hydroxydebrisoquine. A single propafenone tablet (300 mg) was given in each occasion. Plasma propafenone concentrations were analyzed by liquid chromatography coupled to tandem mass spectrometry (HPLC/MS/MS) with positive ion electrospray ionization using multiple reactions monitoring (MRM). The geometric mean and 90% confidence intervals (CI) of propafenone/Ritmonorm® (T/R) percent ratio were 100.44% (88.39 - 114.13%) for AUClast, 99.84% (90.31 - 110.36%) for AUCinf, and 99.30% (90.08 - 109.47%) for Cmax. Since the 90% CI for Cmax, AUClast, and AUCinf ratios were all inside the 80 - 125% interval proposed by the US Food and Drug Administration Agency, it was concluded that the propafenone formulation elaborated by Biolab Sanus Farmacêutica Ltda. is bioequivalent to Ritmonorm® formulation for both the rate and the extent of absorption. The drug was well tolerated by the subjects, indicating that it is safe to perform propafenone bioequivalence studies in healthy subjects with intermediate/extensive metabolism.
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UHPLC-MS/MS method for determination of atorvastatin calcium in human plasma: Application to a pharmacokinetic study based on healthy volunteers with specific genotype.
Xia, B, Li, Y, Zhang, Y, Xue, M, Li, X, Xu, P, Xia, T, Chen, S
Journal of pharmaceutical and biomedical analysis. 2018;:428-435
Abstract
A rapid, selective and sensitive ultra high performance liquid chromatography coupled with tandem triple quaternary mass spectrometry (UHPLC-MS/MS) method was developed and validated for the quantitative determination of atorvastatin calcium (AC) in human plasma. Separation of AC and rosuvastatin calcium (internal standard, IS) were achieved on a Dikma Leapsil C18 reversed phase column (100 × 2.1 mm, 2.7 μm) with gradient elution using 0.2% (v/v) formic acid in water and acetonitrile as mobile phases, at the flow rate of 0.3 mL/min. AC and IS were detected using MS/MS with turbo ion pray source in negative mode by monitoring the precursor-to-product ion transitions m/z 557.0→453.0 for AC and m/z 480.0→418.0 for IS. The calibration curves were linear from 0.05 to 50 ng/mL with a correlation coefficient ( r2) of 0.9992 or better. Thereafter, 187 healthy candidates were checked to the genetic polymorphism analysis of SLCO1B1 521T>C(rs4149056), SLCO1B1 388A>G(rs2306283), CYP3A4 1*B(rs2740574), CYP3A4 1*G(rs2242480) and CYP3A5*3(rs776746) using fluorescence in situ hybridization technology. The genotype frequencies of wild-type homozygote, mutant heterozygote and mutant homozygote were 62.57%(TT), 34.22%(TC) and 3.21%(CC) for SLCO1B1 521T>C, and 8.56%(AA), 33.69%(AG) and 57.75%(GG) for SLCO1B1 388A>G, and 62.57%(CC), 34.22%(CT) and 3.21%(TT) for CYP3A4 1 G, and 58.29%(GG), 34.76%(GA) and 6.95%(AA) for CYP3A5*3, respectively. Furthermore, each tested genotype of CYP3A4 1B was wild type. Finally, 5 candidates with specific genotype described above were recruited to carry out the clinical pharmacokinetics of AC (n = 5). The validated UHPLC-MS/MS method was implemented in a high-throughput setting, capable of analyzing up to 288 samples per day, and was successfully applied to the pharmacokinetic study of AC based on healthy volunteers with specific genotype. The Cmax of AC in human volunteers with the specific genotype was nearly 10 times higher than that previous reported, indicating that genetic polymorphisms of these specific genotypes have significant influence on pharmacokinetics of atorvastatin.
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4.
Simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram in plasma using liquid chromatography-tandem mass spectrometry.
Flint, RB, Bahmany, S, van der Nagel, BCH, Koch, BCP
Biomedical chromatography : BMC. 2018;(10):e4290
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Abstract
A simple and specific UPLC-MS/MS method was developed and validated for simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and its active metabolite keto-doxapram. The internal standard was fentanyl-d5 for all analytes. Chromatographic separation was achieved with a reversed-phase Acquity UPLC HSS T3 column with a run-time of only 5.0 min per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate or formic acid in Milli-Q ultrapure water or in methanol with a total flow rate of 0.4 mL min-1 . A plasma volume of only 50 μL was required to achieve adequate accuracy and precision. Calibration curves of all five analytes were linear. All analytes were stable for at least 48 h in the autosampler. The method was validated according to US Food and Drug Administration guidelines. This method allows quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram, which is useful for research as well as therapeutic drug monitoring, if applicable. The strength of this method is the combination of a small sample volume, a short run-time, a deuterated internal standard, an easy sample preparation method and the ability to simultaneously quantify all analytes in one run.
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Development of a rapid and sensitive HPLC method for the identification and quantification of cavoxin and cavoxone in Phoma cava culture filtrates.
Masi, M, Moeini, SA, Boari, A, Cimmino, A, Vurro, M, Evidente, A
Natural product research. 2018;(13):1611-1615
Abstract
Cavoxin is a tetrasubstituted phytotoxic chalcone and cavoxone is the corresponding chroman-4-one, both produced in vitro by Phoma cava, a fungus isolated from chestnut. Cavoxin showed biofungicide potential against fungal species responsible for food moulding. Therefore, cavoxin has potential to be incorporated into biopolymer to generate 'intelligent food packaging'. To reach this objective, large-scale production of cavoxin by P. cava fermentation needs to be optimized. A rapid and efficient method for cavoxin analysis, as well as of cavoxone, in the fungal culture filtrates and the corresponding organic extracts is the first experimental step. Thus, a HPLC method was developed and applied to quantify cavoxin and cavoxone production in two different fungal culture conditions. The analysis proved that cavoxin production in stirred culture filtrates is significantly higher than in static ones.
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Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies.
Jetha, A, Thorsteinson, N, Jmeian, Y, Jeganathan, A, Giblin, P, Fransson, J
mAbs. 2018;(6):890-900
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Abstract
Monoclonal antibody (mAb) candidates from high-throughput screening or binding affinity optimization often contain mutations leading to liabilities for further development of the antibody, such as aggregation-prone regions and lack of solubility. In this work, we optimized a candidate integrin α11-binding mAb for developability using molecular modeling, rational design, and hydrophobic interaction chromatography (HIC). A homology model of the parental mAb Fv region was built, and this revealed hydrophobic patches on the surface of the complementarity-determining region loops. A series of 97 variants of the residues primarily responsible for the hydrophobic patches were expressed and their HIC retention times (RT) were measured. As intended, many of the computationally designed variants reduced the HIC RT compared to the parental mAb, and mutating residues that contributed most to hydrophobic patches had the greatest effect on HIC RT. A retrospective analysis was then performed where 3-dimentional protein property descriptors were evaluated for their ability to predict HIC RT using the current series of mAbs. The same descriptors were used to train a simple multi-parameter protein quantitative structure-property relationship model on this data, producing an improved correlation. We also extended this analysis to recently published HIC data for 137 clinical mAb candidates as well as 31 adnectin variants, and found that the surface area of hydrophobic patches averaged over a molecular dynamics sample consistently correlated to the experimental data across a diverse set of biotherapeutics.
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The validated and sensitive HPLC-DAD method for determination of patulin in strawberries.
Sadok, I, Szmagara, A, Staniszewska, MM
Food chemistry. 2018;:364-370
Abstract
This work outlines HPLC coupled with DAD detection for accurate quantification of patulin (mycotoxin) in strawberries. The optimized extraction of fruit samples through the modified QuEChERS technique in acetonitrile acidified with acetic acid (1%, v/v) and citrate-buffered salts followed by dispersive solid phase extraction using a primary secondary amine and graphitized carbon is described. The method presents LOD and LOQ of 1.5 and 5µg/kg, respectively and has been validated in strawberry matrix at three concentration levels (5, 10 and 50µg/kg), according to SANTE/11945/2015 and the 2002/657/EC Decision requirements. All validated patulin levels show high percentage of patulin recovery. The validation procedure also includes the intermediate precision performed within three successive days in samples spiked with patulin. The developed method was applied for analysis of fresh and frozen strawberries purchased at local markets. Patulin level in analyzed samples was below the level of detection.
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Semi-preparative HPLC purification of δ-tocotrienol (δ-T3) from Elaeis guineensis Jacq. and Bixa orellana L. and evaluation of its in vitro anticancer activity in human A375 melanoma cells.
Beretta, G, Gelmini, F, Fontana, F, Moretti, RM, Montagnani Marelli, M, Limonta, P
Natural product research. 2018;(10):1130-1135
Abstract
In this work, we report a rapid and convenient HPLC-UV-DAD method for the isolation of δ-T3 on semi-preparative scale from two different vitamin E rich processed, commercially available products obtained from the fruits of Elaeis guineensis Jacq. (oil palm) and from the seeds of Bixa orellana L. (achiote tree). Chromatography was run using reverse phase (RP) C-18 columns and HPLC-grade acetonitrile as mobile phase. The purity of the isolated δ-T3, assessed by GC-MS and 1H NMR was above 98%. The δ-T3 cytotoxic activity found in vitro against the proliferation of human A375 melanoma cells compared to that of the other δ-T3 free tocols mixture suggest its primary role in the experimental anticancer activity observed for palm oil derived products. Taken altogether, the results of this study highlight the importance of the application of suitable purification systems for the preparations of tocotrienols prior to their experimental or clinical testing.
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Method Development and Validation of Indaziflam and Its Five Metabolites in Soil, Water, and Fruits by Modified QuEChERS and UHPLC-MS/MS.
Hu, M, Qiu, J, Zhang, H, Fan, X, Liu, K, Zeng, D, Tan, H
Journal of agricultural and food chemistry. 2018;(39):10300-10308
Abstract
A method for simultaneously determining indaziflam and its five metabolites in soil, water, and fruits using ultraperformance liquid chromatography/tandem mass spectrometry was established. The analytes were eluted in <4.5 min. Positive electrospray ionization mode was used. The analytes were extracted using acetonitrile containing 1% ammonium hydroxide, and then the extracts were purified using octadecylsilane and PRiME HLB cartridges. The quantification limits were 0.01-1.01 μg kg-1. The linearities of the calibrations for all analytes were excellent ( R2 > 0.9952). The recoveries at spike concentrations of 0.01, 0.1, and 1 mg kg-1 were 81.3-112.1%. The intraday and interday relative standard deviations were <13.5% and <12.3%, respectively. The method was successfully used to determine indaziflam and its five metabolites in samples from markets and fields. The results confirmed that the method is an effective and robust procedure for routinely determining indaziflam and its metabolites in soil, water, and fruit samples.
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Application of Targeted Metabolomics to Investigate Optimum Growing Conditions to Enhance Bioactive Content of Strawberry.
Akhatou, I, Sayago, A, González-Domínguez, R, Fernández-Recamales, Á
Journal of agricultural and food chemistry. 2017;(43):9559-9567
Abstract
A simple, sensitive, and rapid assay based on liquid chromatography coupled to tandem mass spectrometry was designed for simultaneous quantitation of secondary metabolites in order to investigate the influence of variety and agronomic conditions on the biosynthesis of bioactive compounds in strawberry. For this purpose, strawberries belonging to three varieties with different sensitivity to environmental conditions ('Camarosa', 'Festival', 'Palomar') were grown in a soilless system under multiple agronomic conditions (electrical conductivity, substrate type, and coverage). Targeted metabolomic analysis of polyphenolic compounds, combined with advanced chemometric methods based on learning machines, revealed significant differences in multiple bioactives, such as chlorogenic acid, ellagic acid rhamnoside, sanguiin H10, quercetin 3-O-glucuronide, catechin, procyanidin B2, pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside, and pelargonidin 3-O-rutinoside, which play a pivotal role in organoleptic properties and beneficial healthy effects of these polyphenol-rich foods.