Optimized one-pot derivatization and enantioseparation of cysteine: Application to the study of a dietary supplement.

Cysteine is a sulfur-containing amino acid which plays an outstanding role in many biological pathways in mammals. The analysis and quantification of native cysteine remains a critical issue due to its highly reactive thiol group evolving to the disulfide cystine derivative through oxidation reaction. Aimed at improving the derivative stability, cysteine was labelled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), which reacts with both amino and thiol groups. The derivatization was optimized and the chemical identity of the reaction product was assessed via high-resolution mass spectrometry. The NBD-cysteine derivative resulted stable for 10 days. This derivative was enantioresolved (α and RS equal to 1.25 and 2.70, respectively) thanks to a (R,R)-Whelk-O1 phase with the following chromatographic setting: eluent, MeOH/water-90/10 (v/v) with 15 mM ammonium formate (pwsH 6.0); column temperature, 35 °C; flow rate, 1.0 mL/min. The developed method was validated following the ICH guidelines and applied for the quality control of a L-cysteine containing dietary supplement.

Impurity profiling of N,N'-ethylenebis-l-cysteine diethyl ester (Bicisate).

A HPLC-UV-CAD method with a HILIC column for impurity profiling of the 99mTc chelating agent bicisate has been developed and evaluated. Bicisate and its impurities were separated by means of isocratic elution on a zwitterionic stationary phase using a mixture of 7.5mmol/L trifluoroacetic acid and acetonitrile (47.5:52.5 V/V) as the mobile phase. Five different bicisate batches of a manufacturer were tested using the method. In addition LC-MS experiments were conducted in order to identify the impurities. The predominant impurities found were the oxidation product (disulfide), the monoester of ethylene dicysteine and an unknown compound with an m/z of 293 in ESI positive mode. A new degradation product of bicisate, bicisate lactam, was identified during sample solution stability assessment.

Direct determination of ECD in ECD Kit: a solid sample quantitation method for active pharmaceutical ingredient in drug product.

Technetium-99m ethyl cysteinate dimer (Tc-99m-ECD) is an essential imaging agent used in evaluating the regional cerebral blood flow in patients with cerebrovascular diseases. Determination of active pharmaceutical ingredient, that is, L-Cysteine, N, N'-1,2-ethanediylbis-, diethyl ester, dihydrochloride (ECD) in ECD Kit is a relevant requirement for the pharmaceutical quality control in processes of mass fabrication. We here presented a direct solid sample determination method of ECD in ECD Kit without sample dissolution to avoid the rapid degradation of ECD. An elemental analyzer equipped with a nondispersive infrared detector and a calibration curve of coal standard was used for the quantitation of sulfur in ECD Kit. No significant matrix effect was found. The peak area of coal standard against the amount of sulfur was linear over the range of 0.03-0.10 mg, with a correlation coefficient (r) of 0.9993. Method validation parameters were achieved to demonstrate the potential of this method.

Database of normal human cerebral blood flow measured by SPECT: I. Comparison between I-123-IMP, Tc-99m-HMPAO, and Tc-99m-ECD as referred with O-15 labeled water PET and voxel-based morphometry.

OBJECTIVES: Three accumulative tracers, iodine-123-labeled N-isopropyl-p-iodoamphetamine (I-123-IMP), technetium-99m-labeled hexamethylpropyleneamineoxime (Tc-99m-HMPAO), and technetium-99m-labeled ethyl cysteinate dimer (Tc-99m-ECD) are widely used to measure cerebral blood flow (CBF) in single-photon emission computed tomography (SPECT). In the present study, normal regional distribution of CBF measured with three different SPECT tracers was entered into a database and compared with regional distribution of CBF measured by positron emission tomography (PET) with H2(15)O. The regional distribution of tissue fractions of gray matter determined by voxel-based morphometry was also compared with SPECT and PET CBF distributions. METHODS: SPECT studies with I-123-IMP, Tc-99m-HMPAO, and Tc-99m-ECD were performed on 11, 20, and 17 healthy subjects, respectively. PET studies were performed on 11 healthy subjects. Magnetic resonance (MR) imaging studies for voxel-based morphometry were performed on 43 of the 48 subjects who underwent SPECT study. All SPECT, PET, and MR images were transformed into the standard brain format with the SPM2 system. The voxel values of each SPECT and PET image were globally normalized to 50 ml/100 ml/min. Gray matter, white matter, and cerebrospinal fluid images were segmented and extracted from all transformed MR images by applying voxel-based morphometry methods with the SPM2 system. RESULTS: Regional distribution of all three SPECT tracers differed from that of H2150 in the pons, midbrain, thalamus, putamen, parahippocampal gyrus, posterior cingulate gyrus, temporal cortex, and occipital cortex. No significant correlations were observed between the tissue fraction of gray matter and CBF with any tracer. CONCLUSION: Differences in regional distribution of SPECT tracers were considered to be caused mainly by differences in the mechanism of retention of tracers in the brain. Regional distribution of CBF was independent of regional distribution of gray matter fractions, and consequently the blood flow per gray matter volume differed for each brain region.

Characterization and quality control analysis of 99mTc-bicisate.

The aim of this study was to investigate the mini cartridge versus paper chromatography quality control methods for determining the radiochemical purity (RCP) of (99m)Tc-bicisate. The 4 methods that were compared with the manufacturer's method included Whatman 17 paper/ethyl acetate solvent, instant thin-layer chromatography (ITLC) silica gel paper/saline solvent, reverse-phase C18 mini cartridge/saline solvent, and strong anion exchange mini cartridge/water solvent. At 30 min after reconstitution, (99m)Tc-bicisate was formed at 97%-98% RCP as assayed by the paper and cartridge methods, and the strong anion exchange/water for injection (WFI) system slightly underestimated the percentage at 96%. A significantly lower RCP was obtained for the C18/saline method when a faster flow rate was used. The lipophilic complex moved with ethyl acetate on Whatman 17, was separated from origin impurities on ITLC silica gel/saline, and remained on the column with C18/saline. For strong anion exchange/WFI, components in the radioactive formulation are likely to have influenced the percentage of (99m)Tc-bicisate. The time disadvantage for ITLC silica gel/saline analysis made the method less than ideal. The C18 mini cartridge/saline method was found to be the simplest and fastest; a result was obtained in 2 min with use of a safe solvent of elution.

Comparison of anion-exchange and ion-modified reversed-phase liquid chromatography for the determination of S-sulfocysteine.

A dual Hg-Au amalgam electrode is used to detect S-sulfocysteine (SSC) in this study. There exist two main components in the acetonitrile (ACN) rat brain extracts, namely, Cl- and GSSG (oxidized glutathione), that are active in our detection system (GSH is not extracted in ACN). Two strong anion-exchange columns from different companies were used to separate the samples under different conditions, but SSC and Cl- were not separated at the optimum detection pH of 5.2. The signal from Cl- was greatly decreased by lowering the potential at the downstream electrode, though it cannot be completely eliminated. While a silver cartridge removed Cl- from micromoles to several millimoles without any negative effect on the SSC signal in aqueous standards, a large negative peak which interferes with SSC detection was unfortunately introduced when a silver cartridge was applied to brain tissue samples. However, SSC and Cl- in the samples are successfully separated by ion-modified reversed-phase LC in acetate buffer at the optimum detection pH (5.2). The separation conditions are 20 mM acetic acid, 2% methanol, 0.5 mM cetyltrimethylammonium p-toluene sulfonate (CTMA) (pH 5.2). Most importantly, the sensitivity of SSC under the optimum separation conditions is not sacrificed. The detection limit is 8 nM (20 microl injected).

Toxicity and growth-promoting potential of spermine when fed to chicks.

Previous studies have shown that the feeding of putrescine, a biogenic amine and the precursor of the mammalian polyamines, can promote whole-body growth of chicks. The current study was undertaken to determine the effect of spermine, also a biogenic amine and the most cationic of the polyamines, under similar conditions. In Exp. 1, 120 week-old chicks were fed purified crystalline amino acid-based diets containing 0, .2, .4, .6, .8, or 1.0% spermine for 14 d. Spermine proved highly toxic and growth rates were reduced compared with controls when even .2% was fed. In Exp. 2, chicks were fed 0, .0375, .0750, or .1000% spermine. These concentrations proved less toxic than those used in Exp. 1. Supplemental dietary cysteine was then provided at 0, .3, .6, and .9% together with 0, .025, .050, or .400% spermine (Exp. 3) because depletion of cellular glutathione has been suggested as contributing to spermine's toxicity. Even high levels of cysteine supplementation did not overcome spermine's toxicity. Subsequent dietary provision of L-2-oxothiazolidine-4-carboxylic acid (OTC, Exp. 4), a cysteine prodrug, showed that depletion of cellular glutathione was not likely a cause of spermine toxicosis. A trend toward increased weight gain and feed efficiency was observed when low concentrations of spermine were fed. It was concluded, however, that dietary spermine was more toxic to chicks than was previously seen for putrescine, that any growth-promoting effects of dietary spermine are small, and that supplements of dietary cysteine or OTC are unlikely to increase these effects by overcoming spermine toxicosis.