Development and validation studies for determination of phenylpropanoid‐substituted flavan‐3‐ols in semipurified extract of Trichilia catigua by high‐performance liquid chromatography with photodiode array detection

The phenolic compounds are the main phytochemical constituents of the bark of Trichilia catigua and are commonly used for medicinal purposes. An HPLC method for the simultaneous quantification of phenolic compounds (procyanidin B2 (PB2), epicatechin (EPC), chinchonains Ia, Ib, IIa, IIb, catechin, and chrologenic acid) in T. catigua extract was developed and validated. A suitable chromatographic system was selected, which uses a gradient elution with methanol/ACN (75:25), and water both with 0.05% TFA, as mobile phase, column Luna, 280 nm, and flow 0.4 mL/min. Validation of the analytical method was based on the parameters: linearity, precision, LODs and LOQs, accuracy, robustness, and stability. The method showed linearity for PB2 and EPC, in the range 10–120 μg/mL with good correlation coefficients (>0.996). For precision, the repeatability ranged from 1.89 to 3.23%, and the values for accuracy for PB2 and EPC were 95 and 89%, respectively. The LODs and LOQs for PB2 were 1.36 and 4.12 μg/mL, and for EPC were 2.18 and 6.61 μg/mL, respectively. The method was robust under the conditions employed. The proposed method could be employed for quality assessment of T. catigua, as well as pharmaceutical products.     

Critical evaluation of the potential of radiofrequency pulsed glow discharge–time-of-flight mass spectrometry for depth-profile analysis of innovative materials

The combination of radiofrequency pulsed glow discharge (RF-PGD) analytical plasmas with time-of-flight mass spectrometry (TOFMS) has promoted the applicability of this ion source to direct analysis of innovative materials. In this sense, this emerging technique enables multi-elemental depth profiling with high depth resolution and sensitivity, and simultaneous production of elemental, structural, and molecular information. The analytical potential and trends of this technique are critically presented, including comparison with other complementary and well-established techniques (e.g. SIMS, GD–OES, etc.). An overview of recent applications of RF-PGD–TOFMS is given, including analysis of nano-structured materials, coated-glasses, photovoltaic materials, and polymer coatings     

Targeting prohibited substances in doping control blood samples by means of chromatographic–mass spectrometric methods

Urine samples have been the predominant matrix for doping controls for several decades. However, owing to the complementary information provided by blood (as well as serum or plasma and dried blood spots (DBS)), the benefits of its analysis have resulted in continuously increasing appreciation by anti-doping authorities. On the one hand, blood samples allow for the detection of various different methods of blood doping and the abuse of erythropoiesis-stimulating agents (ESAs) via the Athlete Biological Passport; on the other hand, targeted and non-targeted drug detection by means of chromatographic–mass spectrometric methods represents an important tool to increase doping control frequencies out-of-competition and to determine drug concentrations particularly in in-competition scenarios. Moreover, blood analysis seldom requires in-depth knowledge of drug metabolism, and the intact substance rather than potentially unknown or assumed metabolic products can be targeted. In this review, the recent developments in human sports drug testing concerning mass spectrometry-based techniques for qualitative and quantitative analyses of therapeutics and emerging drug candidates are summarized and reviewed. The analytical methods include both low and high molecular mass compounds (e.g., anabolic agents, stimulants, metabolic modulators, peptide hormones, and small interfering RNA (siRNA)) determined from serum, plasma, and DBS using state-of-the-art instrumentation such as liquid chromatography (LC)–high resolution/high accuracy (tandem) mass spectrometry (LC-HRMS), LC–low resolution tandem mass spectrometry (LC-MS/MS), and gas chromatography–mass spectrometry (GC-MS).     

Asymmetrical flow field-flow fractionation as a novel technique for the analysis of PS-b-PI copolymers

Asymmetrical flow field-flow fractionation (AF4) was used as a fractionation technique to investigate the molecular heterogeneity of poly(styrene-b-isoprene) diblock copolymers synthesized by either sequential living anionic polymerization or coupling of living precursor blocks. AF4 coupled to multi-angle laser light scattering (MALLS), refractive index (RI), and ultraviolet (UV) detectors was used to separate the diblock copolymers from the homopolymers and coupling products, and the molar masses of the different components were analyzed. In order to get more information about the separated block copolymers, homopolymers, and coupling products, fractions were collected directly after the AF4 channel. The collected fractions were analyzed offline by ¹H NMR to provide identification of the different species and additional information on the true chemical composition, and the microstructure of the diblock copolymer was obtained.

Calorimetric Estimation Employing the Unscented Kalman Filter for a Batch Emulsion Polymerization Reactor

Reaction calorimetry is a very useful tool to monitor exothermic polymerization reactions as it is based on the estimation of the heat generated by the reaction. The objective of this work is to analyze the performance of an unscented Kalman filter (UKF) for online monitoring of batch vinyl acetate emulsion polymerization reactions. Reactions are performed in isoperibolic and isothermal conditions. The UKF is compared to an extended Kalman filter that has a very poor performance. The results show that the UKF is able to provide good estimates for the conversion, for the reactor and jacket temperatures, for the overall heat transfer coefficient between the reaction medium and the jacket, and for the heat loss from the jacket to the surroundings.

     

Molecular Recognition of Carbonyl Compounds by Uranyl-salophen Based Neutral Receptors Driven by Van Der Waals Forces

The complexation of the salophen-uranyl metallocleft 2 and of its half-cleft analogue 3 with enones and other carbonyl compounds was assessed in chloroform by UV-Vis titration and, occasionally, by FT-IR measurements. Complexes with receptors 2 and 3 are in all cases more stable than those with the control unsubstituted uranyl-salophen 1 , showing that in addition to the primary binding force provided by coordination of the carbonyl oxygen to the uranium, a significant driving force for complexation, typically in the range of 2-3 kcal/mol, results from van der Waals interactions of the guest with the aromatic walls. Replacement of the phenyl group in 3 with larger aromatic residues to give 4 and 5 , led to enhanced complex stabilities, due to more extended contact surfaces between host and guest.     

Structures and properties of solvated and unsolvated isopropyl functionalised calix[4]arenes

The tetra-isopropyl ethers of calix[4]arene and p-t-butylcalix[4]arene have been isolated in the cone conformation, and structurally characterised as chloroform solvates. Thermogravimetric analysis demonstrated that the parent isopropylcalix[4]arene solvate is significantly more stable than the p-t-butylcalix[4]arene analogue, retaining the solvent up to a temperature of 125°C. It was found that the calix[4]arene ether sublimes at atmospheric pressure, and solvent-free crystals appropriate for structure determination were produced at reduced pressure. The p-t-butylcalix[4]arene ether was also isolated without solvent in the lattice, but in this case the calixarene was crystallised from acetone, as sublimation did not produce crystals of sufficient quality.     

Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K _M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol?L ^?1 and 164 ± 13.4 μmol?L ^?1 , respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for P_i-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.

Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5–100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1–1,000 nM for all MTXPGn (R ²?>?0.99). The coefficient of variation ranged from 1–4% for intraday precision and 6–15% for interday precision. Samples were stable for at least 1 month at ?80 °C. Recovery ranged from 98–100%, and the relative matrix-effect varied from 95–99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.