Accurate determination of amino acids by quadruple isotope dilution-reverse phase liquid Chromatography-Tandem mass spectrometry after derivatization with 2-Naphthoyl chloride.

The determination of amino acids in biological samples is central to the diagnosis of inherited metabolic disorders and also gives significant information about the metabolisms in the cells and living body. The development of analytical method for reliable quantification of amino acids in biological samples is still challenging because of the polar nature of amino acids and complex nature of biological samples causing a high degree of interferences during analysis. In the present study, a pre-column derivatization method using 2-naphtoyl chloride combined with liquid chromatography-tandem mass spectrometry method was developed for the determination of 17 amino acids in human serum and urine matrices. Low detection limits were obtained in the range of 0.015 - 0.266 µmol kg-1 and acceptable recovery results were obtained in human serum and urine samples. Isotopically labelled (15N labelled) amino acids were spiked to standards and samples before derivatization to compensate for the analytical errors in the whole procedure. The combination of quadrupole isotope dilution strategy with the derivatization based reversed phase chromatography allowed to improve method accuracy and precision.

Determination of seventeen free amino acids in human urine and plasma samples using quadruple isotope dilution mass spectrometry combined with hydrophilic interaction liquid chromatography - Tandem mass spectrometry.

Taking into account the growing demand for new analytical procedures that are appropriate for analysis of complex biological samples with increased sensitivity, accuracy and precision, a novel analytical method was described for the determination of underivatized amino acids in human plasma and urine samples. The presented analytical procedure involved the direct analysis of urine samples and the analysis of plasma samples followed by a simple protein precipitation protocol. Samples were analyzed using a simple and fast chromatographic method developed for the determination of 17 different amino acids by liquid chromatography - tandem mass spectrometry. The limit of detection and quantification values for amino acids were ranged between 0.03-2.26 µmol kg-1 and 0.09-7.54 µmol kg-1. Matrix effects of plasma and urine on the quantification of analytes were determined by spiking experiments. The accuracy of method was evaluated by matrix matching and quadruple isotope dilution strategies. Excellent accuracy and precision were obtained with the use isotope labeled amino acids demonstrating the high reliability and reproducibility of the proposed method. The percent recovery values were found to be between 98.70 - 101.68% with%RSD below than 1.62% for human plasma and 99.14 - 101.78% with%RSD below than 2.44% for urine samples.

DNA-damage and cell cycle arrest initiated anti-cancer potency of super tiny carbon dots on MCF7 cell line.

While carbon-based materials have spearheaded numerous breakthroughs in biomedicine, they also have procreated many logical concerns on their overall toxicity. Carbon dots (CDs) as a respectively new member have been extensively explored in nucleus directed delivery and bioimaging due to their intrinsic fluorescence properties coupled with their small size and surface properties. Although various in vitro/in vivo studies have shown that CDs are mostly biocompatible, sufficient information is lacking regarding genotoxicity of them and underlying mechanisms. This study aims to analyze the real-time cytotoxicity of super tiny CDs (2.05 ± 0.22 nm) on human breast cancer cells (MCF7) and human primary dermal fibroblast cell cultures (HDFa) by xCELLigence analysis system for further evaluating their genotoxicity and clastogenicity to evaluate the anti-tumor potential of CDs on breast adenocarcinoma. As combined with flow cytometry studies, comet assay and cytokinesis-block micronucleus assay suggest that the CDs can penetrate to the cell nuclei, interact with the genetic material, and explode DNA damage and G0/G1 phase arrest in cancer cells even at very low concentrations (0.025 ppm) which provide a strong foundation for the design of potentially promising CD-based functional nanomaterials for DNA-damage induced treatment in cancer therapy.

Fluorescent Carbon Dots from Nerium oleander: Effects of Physical Conditions and the Extract Types.

In this original research, the synthesis of carbon nanodots (CDs) from two different solvent extracts of Nerium oleander by the thermal method was investigated under various physical conditions such as pH, reaction temperature, ionic strength, and surface passivation agent (polyethylene glycol, PEG) presence in the reaction media. The effects of extract types and physical conditions on CDs formation were characterized by UV-Visible spectrophotometry, fluorescence spectrophotometry, Fourier transform infrared spectroscopy and dynamic light scattering analysis. Fluorescent CDs were obtained from PEG included reaction media. Additionally, the enhanced fluorescence intensity correlated with ascending reaction temperature was reported. The hydrodynamic particle size of CDs in aqueous solution was determined between ~1 and 235 nm with negative surface potential in the range of -6 mV and -28 mV. Moreover, CDs synthesized from aqueous extract mostly resulted in smaller size than that of ethanol extract based ones. The impact of surface passivation with PEG on the fluorescence feature of CDs was verified. For the relevant extracts of Oleander, CDs synthesized from PEG included formulations at pH 5 and NaCl free reaction media found as better alternatives than CDs synthesized under other conditions taking account their effect on fluorescence feature, hydrodynamic size and etc. Graphical Abstract.

Accurate and sensitive determination of sildenafil, tadalafil, vardenafil, and avanafil in illicit erectile dysfunction medications and human urine by LC with quadrupole-TOF-MS/MS and their behaviors in simulated gastric conditions.

The widespread use of phosphodiesterase-5 inhibitors has attracted broad attention of counterfeiters to develop illicit erectile products with inaccurate amounts, unknown toxicity, and purity of active ingredients. Correspondingly, intake of these products endangers consumer health and needs to be screened for precautionary actions to reduce this risk. Therefore, in this study, a sensitive and rapid analytical method has been developed for simultaneous determination of selected phosphodiesterase-5 inhibitors present in illicit erectile medications and human urine. Quantification of the analytes was performed by liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry system. The chromatographic separation was successfully achieved with a run period of 8 min. Low detection limits were obtained in the range of 1.63-9.81 ng/g with relative standard deviations below 7.72% obtained using the replicate measurements of lowest concentration in calibration plots. The analytical performance of the proposed method proved good linearity, low detection limits, good accuracy and precision with high percent recoveries for human urine samples. Developed method was successfully applied to real samples including four different brands of illicit erectile medications. The results obtained revealed the presence of high levels of sildenafil in analyzed samples. The behaviors of selected phosphodiesterase-5 inhibitors were also studied in simulated gastric conditions.

A kinetic study on sesame cake protein hydrolysis by Alcalase.

In the present study, the hydrolysis of sesame cake protein was performed by Alcalase, a bacterial protease produced by Bacillus licheniformis, to investigate the reaction kinetics of sesame cake hydrolysis and to determine decay and product inhibition effects for Alcalase. The reactions were carried out for 10 min in 0.1 L of aqueous solutions containing 10, 15, 20, 25, and 30 g protein/L at various temperature and pH values. To determine decay and product inhibition effects for Alcalase, a series of inhibition experiments were conducted with the addition of various amounts of hydrolysate. The reaction kinetics was investigated by initial rate approach. The initial reaction rates were determined from the slopes of the linear models that fitted to the experimental data. The kinetic parameters, K(m) and V(max), were estimated as 41.17 g/L and 9.24 meqv/L x min. The Lineweaver-Burk plots showed that the type of inhibition for Alcalase determined as uncompetitive, and the inhibition constant, K(i), was estimated as 38.24% (hydrolysate/substrate mixture). Practical Application: Plant proteins are increasingly being used as an alternative to proteins from animal sources to perform functional roles in food formulation. Knowledge of the kinetics of the hydrolysis reaction is essential for the optimization of enzymatic protein hydrolysis and for increasing the utilization of plant proteins in food products. Therefore, in the present study, the hydrolysis of sesame cake protein was performed by Alcalase, a bacterial protease produced by B. licheniformis, to investigate the reaction kinetics of sesame cake hydrolysis and to determine decay and product inhibition effects for Alcalase.

Kinetic thermal degradation of vitamin C during microwave drying of okra and spinach.

In this present study, the effect of microwave output power and sample amount on vitamin C loss in okra (Hibiscus esculenta L.) and spinach (Spinacia oleracea L.) were investigated using the microwave drying technique. The procedure is based on the reaction between l-ascorbic acid (vitamin C) and 2,6-dichloroindophenol. The proposed method was applied successfully to both okra and spinach for the determination of ascorbic acid (vitamin C) content. It was observed that as the microwave output power increased or as the sample amount decreased, the vitamin C in okra and spinach decreased as well. The activation energy for degradation of vitamin C for both okra and spinach was calculated using an exponential expression based on the Arrhenius equation.