A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

High Resolution Mass Spectrometry Elucidation of Captopril’s Ozonation and Chlorination By-Products

The article evaluated the degradation of the captopril in aqueous solution after ozonation and chlorination. The process was continuously monitored focusing on the identification, mass spectrometry and elucidation of its by-products by applying direct infusion and high performance liquid chromatography, electrospray ionization high resolution mass spectrometry, in the negative ion mode. The cytotoxicity of its by-products solutions were evaluated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. It was observed through that after 30 min of ozonation and chlorination, there was complete oxidation of captopril, i.e., 100% removal efficiency. At these conditions, the rate of mineralization, by total organic carbon, was only 7.63% for ozonation and 6.40% for chlorination, evidencing the formation of degradation by-products. Ten captopril by-products were identified and their respective chemical structures elucidations are proposed. The treated samples and their by-products were nontoxic to HepG2 cells by MTT assay.

Profiling of circulating tumor cells in liquid biopsies from metastatic cancer patients

Aim:Circulating tumor cells(CTCs)are crucial to tumor metastasis and valuable for prediction of clinical outcome in patients with solid tumors.Here,the authors aimed to establish a method for enumeration and characterization of CTCs from liquid biopsies.Methods:Peripheral blood mononuclear cells(PBMCs)were separated from blood samples from patients with metastatic cancer using Ficoll-Hypaque gradients and cultured to isolate and enumerate CTCs.Cultured CTCs were morphologically characterized by light and phase contrast microscopy.The tumorigenicity of Ficoll-Hypaque-separated PBMCs was examined,in addition to their expression of mRNA metastasis markers.Results:CTCs were isolated in culture and enumerated by counting under phase contrast microscopy,demonstrating that 0.01-0.04%of total PBMCs were CTCs.CTCs were dormant,with large,oval-shaped,spiky morphology.PBMCs obtained from liquid biopsies exhibited anchorage-independent growth,forming numerous colonies in soft agar assays.Molecular profiling demonstrated expression of several metastatic genes,but not of cadherin 1(encoding the adhesion protein),in all patients.Conclusion:The authors successfully isolated,enumerated,and characterized CTCs from liquid biopsies of metastatic cancer patients.This study has potential to facilitate the development of new diagnostic and therapeutic methods using liquid biopsies,for application in metastatic cancers.

A Method for Determination of Hexokinase Activity by RP-HPLC

A method was established to determine the activity of hexokinase by reverse-phase high-performance liquid chromatography （RP-HPLC）. The activity of hexokinase was determined by detecting the amount of ADP in the reaction that converts glucose into glucose-6-phosphate. The separation degree of ATP and ADP was as good as 3.46 （separation degree^l.5）. In the range of 0.01-0.40 mmol/L, there was a good linear relationship between concentration of ADP and its peak areas （the correlation coefficient was 0.999）. The relative standard deviation（RSD） of intraday was 1.43%-1.46%, that of interday was 2.12%-2.15%. At 30℃ （optimal temperature） and pH7.0 （optimal pH）, the recovery rate of hexokinase was 95.3%-97.6%. The results showed that the method had good precision and high recovery rate, and the enzyme activity was determined through only one-step reaction. To some extent, this method can reduce the cost.

A novel efficient medium for chromogenic catalysis of tetramethylbenzidine with horseradish peroxidase

The traditional surfactant sodium dodecyl sulfate（SDS） and ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate（[Emim][BF4]） have been combined to create a novel efficient medium for chromogenic catalysis of 3,30,5,50-tetramethylbenzidine with horseradish peroxidase in presence of H2O2. The results have shown the [Emim][BF4] in the mediums can promote the rate of formation of the blue chromogen,the SDS is responsible for the stabilization of the blue chromogen due to the electrostatic attraction between positively charged blue chromogen and the negatively charged surfactant. The SDS/[Emim][BF4]combination not only enhance catalytic activity of HRP remarkably but also stabilize the blue chromogen formed in the HRP oxidation of the substrate TMB compared to the conventional medium. Based on the superior combination of SDS and [Emim][BF4], the colorimetric assay for detecting HRP activity and H2O2 concentration was established. This work demonstrates a novel efficient medium for chromogenic catalysis with potential applications in biosensors and clinical diagnosis.