Lipoxygenase (LOX) products (5- and 12- HETE) have been implicated in carcinogenesis, contribution to invasion and cancer progression. Several LOX-inhibitors have been studied as potential cancer preventive agents, but the specificity of these inhibitors has been doubted. It is therefore important to have a highly selective method, as UPLC-MS/MS, for assessment of the mechanism of action for LOX inhibitors in the cellular context. Design of Experiments (DoE) is a chemo metrical approach which allows for optimization of a quantification method using a limited number of experiments and interaction between experimental factors can be observed. This paper presents optimization of an UPLC-MS/MS method for accurate quantification of the LOX products, 5- and 12- HETE, utilizing DoE. Significant interaction effects where seen between amount of organic solvent in the mobile phase at initial condition and gradient slope as well as between mobile phase flow rate and capillary voltage. A UPLC-MS/MS quantification method was developed and validated for 5- and 12- HETE. An intraday validation assessment showed that the quantitative determination was linear for 5- and 12- HETE in the range tested (1.00-100 ng/ml), and accuracy and precision met the acceptance criteria with a coefficient of variations lower than 15%. Auto-sampler stability was established for 12 hours at 4°C. The optimized method was specific for evaluation of LOX activity in cultured AsPC-1 pancreatic cancer cells. Without added Arachidonic Acid (AA) as substrate LOX products were not detected. After addition of AA, 5- and 12- HETE were quantified in both supernatants and cell lysates, demonstrating the usefulness for cell-based studies in the evaluation of LOX inhibitors.

This report describes a specific and precise Gas Chromatography coupled to a flame ionization detector (GC-FID) method for the quantification of orto cresol (o-cresol) in human urine as biomarker of exposure to toluene. The procedure included an acid hydrolysis step, a liquid-liquid extraction and the GC-FID determination. The mean recovery ranged between 95.4% and 110.6%. The detection and quantification limits were 0.03 µg/ mL and 0.20 µg/mL respectively. The method described is a promising alternative tool for monitoring toluene exposed workers. Data of exposed and non-exposed population to toluene is shown.

Extraction of active polyphenol oxidase and peroxidase from a plant rich in phenolics and chlorophylls in the post-harvest browning syndrome is described. Initially, general optimization using conventional enzyme extractions was performed. However, along with membrane-bound proteins, chlorophylls and phenols were also released with Triton X (TTX). With a view to obtaining high enzymatic activity, removal of the released chlorophylls and phenols by formation of TTX-114 micelles in the detergent rich phase after high-temperature induced phase separation was tested