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Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are considered as viable targets in the treatment of Alzheimer's disease and other dementias. However, since clinically used AChE or dual AChE/BuChE inhibitors only provide some symptomatic treatment and lose their therapeutic effectiveness over time, in the last years an intensive search for multi-target compounds has started, which address additional targets besides AChE and BuChE, like endocannabinoid degrading enzymes. For the evaluation of the AChE and BuChE inhibitory potency of the test compounds in such drug development projects, usually the traditional Ellman's method is used. Since this method has some analytical drawbacks, we have developed alternative robust HPLC/UV based assays using pyridin-2-ylmethyl acetate as substrate for AChE and benzoylcholine for BuChE. The enzyme products pyridin-2-ylmethanol and benzoic acid, respectively, were determined by reversed phase HPLC adding the ion pair reagent sodium octane-1-sulfonate to the mobile phase in case to the polar pyridine derivative. AChE from electric eel, human recombinant AChE and BuChE from equine serum were employed as enzymes. For method validation, the IC50-values of the known AChE and BuChE inhibitors physostigmine, rivastigmine and tacrine were measured. The obtained data were within the very wide range of the IC50-values given in the literature for these compounds. [ABSTRACT FROM AUTHOR]

A new method for the determination of trace bisphenol A (BPA) and diethylstilbestrol (DES) in water samples has been developed by using three phase hollow fiber liquid-phase microextraction (HF-LPME) and an ionic liquid (IL, 1-Octyl-3-methylimidazolium hexafluorophosphate, [C8MIM][PF6]) as supported phase (IL-HF-LPME) combined with high performance liquid chromatography/ultraviolet spectrophotometry (HPLC/UV). The optimized microextraction conditions were as follows: [C8MIM][PF6] was used as the extraction solvent, pH 10 NaOH solution, 5 µL, as acceptor phase; 20 mL water sample with NaCl (5%, w/v) as donor phase and the extraction time was 30 min (stirring rate 200 rpm, 25°C). Under the optimal extraction conditions, the proposed method offered: good linearity range, 0.30–100 µg L−1, with correlation coefficients (r2) of 0.9996 and 0.9988; low detection limit, 0.20 and 0.05 µg L−1(S/N = 3) for BPA and DES, respectively; good reproducibility (relative standard deviation, RSD, 7.28% and 8.45%, respectively,n = 5); high enrichment factor, 850 and 624, respectively. The method was used to analyze real water samples, and satisfactory recoveries (75.4–99.2%) were obtained, which confirmed that the developed method was simple and environmentally friendly for the determination of BPA and DES in aquatic systems. Meanwhile, it was found that humic acid had no obvious influence on enrichment factor. [ABSTRACT FROM PUBLISHER]

The chemical compound thymoquinone (THQ) isolated fromNigella sativaseed oil has multiple therapeutic potentials. A simple and rapid reversed-phase high-performance liquid chromatography assay was developed to determine the concentration of THQ in rabbit plasma. The procedure involved extraction of the compound and thymol (internal standard) from the plasma using acetonitrile and methanol. The extract was injected onto Waters 5 µm Atlantis dC-18 Bond pack column (10 μ, 150 × 3.9 mm 1.d.). The mobile phase consisted of methanol and 10 mM KH2PO4buffer in the ratio of 90:10 (v/v, pH = 4.6), which was delivered using isocratic flow at a rate of 0.9 mL/min. THQ and the internal standard were monitored using UV detection at 254 nm with a total run time of 7 min. The method was found to be linear (r2 > 0.997), precise (CVs = 3.69–12.63%) and accurate (recovery = 88–107.77%) in the concentration range of 0.5–50 µg/mL. The limits of detection and quantitation of the method were 0.134 and 0.408 µg/mL, respectively. The within and between-day coefficients of variation were less than 7%. The method has been successfully used to measure THQ plasma concentrations in a rabbit model following an intravenous administration of the drug. [ABSTRACT FROM PUBLISHER]

Determination of malondialdehyde (MDA) is commonly used for the evaluation of lipid peroxidation in biological human samples, especially in plasma, serum, and urine, as a non-invasive biomarker of some oxidative-stress-related diseases. The objective of the present study was to implement a HPLC methodology for the quantification of MDA in cell suspensions and liver, after complexing this compound with thiobarbituric acid (TBA), giving rise to the formation of the complex MDA–(TBA)2. The implemented methodology was validated and allowed a reliable separation and quantification of MDA, with high precision and recovery, and a low detection limit. The stability of MDA in the biological samples, as well as of the MDA–(TBA)2 complex, was also evaluated during storage at different temperatures and for different time periods. The data obtained from the stability studies concerning the MDA–(TBA)2 complex prepared from hepatocyte suspensions and liver samples, indicated that the chromatographic analysis should be performed within the period of 4 hr when it is maintained at ambient temperature or at 40°C, and within the period of 48 hr if the temperatures of the storage are 4°C, −20°C, or −80°C. When the biological samples are conserved for posterior preparation of the complex, MDA can be measured in the cell suspensions conserved at any of these temperatures in the subsequent 24 hr, but liver samples must be conserved at 4°C or −20°C in order to obtain reliable results of MDA. [ABSTRACT FROM AUTHOR]

A precise, sensitive, and reliable RP-HPLC method was developed and validated to enable the separation and quantification of caseinomacro peptide. The optimized method used a polystyrene-divinylbenzene column and gradient elution with two solvents. Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 95% acetonitrile-5% water-0.1% trifluoroacetic acid. The flow rate used was 1 mL/min. The effluent was monitored by a UV detector at 214 nm and enabled the separation of two peaks corresponding to the aglyco components of the two principal genetic variants (A and B), eluted after the less well resolved glyco caseinomacropeptide components. The determinations were performed in the linear range of 15–200 µg/mL. Detection limit was 2 µg/mL. The validity of the method was verified. The recoveries were 93 and 98%. The precision of the method was also evaluated being the %CV less than 4.87%. The method was applied to the analysis of rennet and acid wheys and whey protein concentrates produced by the dairy industry. It was also applied to the detection of rennet whey in powdered milks. [ABSTRACT FROM AUTHOR]