High-Throughput Analysis of Amino Acids for Protein Quantification in Plant and Animal-Derived Samples Using High Resolution Mass Spectrometry

Current methods for measuring the abundance of proteogenic amino acids in plants require derivatisation, extended run times, very sensitive pH adjustments of the protein hydrolysates, and the use of buffers in the chromatographic phases. Here, we describe a fast liquid chromatography–mass spectrometry (LC–MS) method for the determination of amino acids that requires only three steps: hydrolysis, neutralisation, and sample dilution with a borate buffer solution for pH and retention time stability. The method shows excellent repeatability (repeated consecutive injections) and reproducibility (repeated hydrolysis) in the amino acid content, peak area, and retention time for all the standard amino acids. The chromatographic run time is 20 min with a reproducibility and repeatability of <1% for the retention time and <11% for the peak area of the BSA and quality control (QC) lentil samples. The reproducibility of the total protein levels in the hydrolysis batches 1–4 was <12% for the BSA and the lentil samples. The level of detection on column was below 0.1 µM for most amino acids (mean 0.017 µM).

Effect of Different Austempering Heat Treatments on Corrosion Properties of High Silicon Steel

A novel high silicon austempered (AHS) steel has been studied in this work. The effect of different austenitizing temperatures, in full austenitic and biphasic regime, on the final microstructure was investigated. Specimens were austenitized at 780 °C, 830 °C, 850 °C and 900 °C for 30 min and held isothermally at 350 °C for 30 min. A second heat treatment route was performed which consisted of austenitizing at 900 °C for 30 min and austempering at 300 °C, 350 °C and 400 °C for 30 min. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) have been used to evaluate the microstructural evolution. These techniques revealed that the microstructures were composed of carbide-free bainite, ferrite, martensite and retained austenite (RA) in different volume fractions (Vγ). An aqueous borate buffer solution with 0.3 M H3BO3 and 0.075 M Na2B4O7∂10H2O (pH = 8.4) was used for corrosion tests in order to evaluate the influence of the different volume fractions of retained austenite on the corrosion properties of the specimens. The results showed that when increasing the austenitization temperatures, the volume fractions of retained austenite reached a maximum value at 850 °C, and decrease at higher temperatures. The corrosion properties were investigated after 30 min and 24 h immersion by means of potentiodynamic polarization (after 30 min) and electrochemical impedance spectroscopy (after both 30 min and 24 h) tests. The corrosion resistance of the samples increased with increases in the volume fraction of retained austenite due to lower amounts of residual stresses.

Determination of glyphosate in honey by high-performance liquid chromatography

The article presents the results of the developed highly sensitive and easily implemented method of sample preparation and determination of the content of residual amounts of glyphosate – one of the most widely used non-selective herbicides of systemic action for the destruction of weeds, in flower honey samples by high-performance liquid chromatography (HPLC) with a fluorescent detector (FD) in the framework of ensuring the safety of beekeeping products. The HPLC stage with FD was preceded by sample preparation, which included the following steps: extraction of glyphosate from an artificially contaminated honey sample with a mixture of water/methanol (1:1) with subsequent addition of a four-fold excess of acetonitrile to the resulting solution to separate a complex multicomponent matrix of honey by centrifugation; filtration of the supernatant and evaporation of the filtrate on a rotary evaporator; dissolution of the resulting residue in a borate buffer solution in order to achieve an alkaline pH value of the medium equal to 8.5-9.5; pre-column derivatization of glyphosate in an alkaline medium with an acetonitrile solution of FMOC-Cl for 30 minutes to obtain a fluorescent herbicide derivative – N-alpha-(9-fluorenylmethyloxycarbonyl)-N-alpha-(phosphonomethyl)-glycine; adding at the end of the derivatization process to the reaction mixture of formic acid in order to transition from alkaline to acidic conditions and stabilize the glyphosate derivative; adding dichloromethane to remove the unreacted excess of the derivatizing agent. Subsequent chromatographic analysis of the fluorescent derivative glyphosate was performed on a reverse-phase packed column "Luna 100 C18-2.5 µ", characterized by versatility and stability in the range of pH values from 1.5 to 11.0, in the isocratic elution mode of mobile phase composition acetonitrile/an aqueous buffer solution of potassium phosphoric acid with a pH value equal to 4.5-5.5 (30:70). The developed method was successfully tested on real samples of flower honey.