1. Iron deprivation-induced reactive oxygen species generation leads to non-autolytic PCD in Brassica napus leaves
Stefan Sassmann, Franz Hadacek, Ingeborg Lang, Rajesh Kumar Tewari Environ Exp Bot . 2013 Jul;91(100):74-83. doi: 10.1016/j.envexpbot.2013.03.006.
Using iron-deprived (-Fe) chlorotic as well as green iron-deficient (5 μM Fe) and iron-sufficient supplied (50 μM Fe) leaves of young hydroponically rearedBrassica napusplants, we explored iron deficiency effects on triggering programmed cell death (PCD) phenomena. Iron deficiency increased superoxide anion but decreased hydroxyl radical (·OH) formation (TBARS levels). Impaired photosystem II efficiency led to hydrogen peroxide accumulation in chloroplasts; NADPH oxidase activity, however, remained on the same level in all treatments. Non-autolytic PCD was observed especially in the chlorotic leaf of iron-deprived plants, to a lesser extent in iron-deficient plants. It correlated with higher DNAse-, alkaline protease- and caspase-3-like activities, DNA fragmentation and chromatin condensation, hydrogen peroxide accumulation and higher superoxide dismutase activity. A significant decrease in catalase activity together with rising levels of dehydroascorbic acid indicated a strong disturbance of the redox homeostasis, which, however, was not caused by ·OH formation in concordance with the fact that iron is required to catalyse the Fenton reaction leading to ·OH generation. This study documents the chain of events that contributes to the development of non-autolytic PCD in advanced stages of iron deficiency inB. napusleaves.
2. Development of an LC-MS/MS method for determination of 2-oxo-clopidogrel in human plasma
Tai-Jun Hang, Min Song, Yu-Han Li J Pharm Anal . 2015 Feb;5(1):12-17. doi: 10.1016/j.jpha.2014.07.004.
A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was established to determine 2-oxo-clopidogrel, a crucial intermediate metabolite in human plasma. A chromatographic separation was performed on a Sapphire C18column following a liquid-liquid extraction sample preparation with methyl t-butyl ether. Detection was carried out on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) with an electrospray ionization (ESI) mode. The method was validated in terms of specificity, accuracy, precision and limit of quantification. The calibration curves ranged from 0.50 to 50.0 ng/mL with good linearity. The stability was fully validated with addition of 1,4-dithio-DL-threitol (DTT) into the plasma sample prior to and in the preparation procedure. The validated method was proved to be suitable for use in pharmacokinetic study after single oral administration of 75 mg clopidogrel tablets in human subjects, which could make contribution to intensive study of the clinical drug-drug interactions of clopidogrel and individual treatment.
3. Development and validation of a chiral UHPLC-MS method for the analysis of cysteine enantiomers in biological samples
Víctor González-Ruiz, Roccaldo Sardella, Serge Rudaz, Joséphine Zangari, Benedetto Natalini, Lucia Pucciarini, Jean-Claude Martinou J Pharm Biomed Anal . 2020 Jan 5;177:112841. doi: 10.1016/j.jpba.2019.112841.
For years, d-amino acids were thought to have a minor function in biological processes compared to that of l-enantiomers. Recently, many studies have shown that d-amino acids are present in high concentrations in microorganisms, plants, mammals and humans and execute specific biological functions. One relevant example is that of d-cysteine, whose hydrogen sulfide-producing properties have been found to protect neurons against oxidative stress and to promote dendritic development. Herein, we introduce a chiral LCMS method for the rapid determination of cysteine enantiomers under polar ionic elution conditions (MeOH/MeCN/H2O 49/49/2 v/v/v, containing 50 mM formic acid and 50 mM ammonium formate) developed on a Chiralpak® ZWIX(+) chiral stationary phase. Cysteine enantiomers were analysed in biological samples after efficient reduction of the disulfide bond in cystine; the latter was achieved with the use of 1,4-dithio-dl-threitol as a reducing agent. A baseline resolution (RS= 2.7) was obtained, and the d-enantiomer eluted before the l-enantiomer. For the enantioselective analysis, cysteine was labelled with AccQ-Tag reagent, resulting in improved chromatographic behaviour and MS detection sensitivity. The method was validated according to the Food and Drug Administration guidelines. Good linearity was determined in the ranges of 0.05-0.50 mg/L for d-cysteine and 0.11-0.56 mg/L for l-cysteine. The repeatability and intermediate precision were found to be lower than 4.0%, with trueness ranging from 95.6 to 100.2% for both enantiomers. The LOD and LOQ values were 0.02 and 0.05 mg/L for d-cysteine and 0.04 and 0.11 mg/L for l-cysteine, respectively. The method was successfully applied to cell culture samples treated with d-cysteine.
