Matthew Joseph Doyle

Determination of menthol and menthol glucuronide in human urine by gas chromatography using an enzyme-sensitive internal standard and flame ionization detection

The rate of peppermint oil absorption and excretion, following peroral administration, was determined by measuring urinary levels of menthol glucuronide. Menthol, a major component of peppermint oil, was liberated from its glucuronide metabolite by treating raw urine with beta-D-glucuronidase (Patella vulgata). Phenyl glucuronide was employed as an enzyme-sensitive internal standard. Menthol and phenol were recovered by ethyl acetate extraction and quantitated by capillary gas chromatography using flame ionization detection. Standard curves were linear between 25 and 250 micrograms/ml with a detection limit (signal-to-noise ratio = 2) of 0.25 microgram/ml. Assay precision was shown to be +/- 1.2% relative standard deviation.

Determination of leukotriene B4 in human plasma by gas chromatography using a mass selective detector and a stable isotope labelled internal standard. Effect of NE-11740 on arachidonic acid metabolism

A highly selective gas chromatographic method, coupled with selected ion monitoring using a mass selective detector and positive electron ionization, was developed for the determination of leukotriene B4 (LTB4) in human plasma. Plasma was separated from whole human blood via centrifugation, proteins precipitated with acetonitrile and LTB4 recovered (approximately 82.0%) by ethyl acetate extraction. The methyl ester, bis-t-butyldimethylsilyl ether derivative of LTB4 was formed prior to analysis and determined quantitatively using [18O]2-LTB4 as an internal standard. The limit of detection (S/N = 2) was 425 pg on column (m/z 335/339) using a 1-microliter injection. Standard curves were linear over two orders of magnitude with an RSD of less than 5.0% (n = 10). NE-11740, a new anti-inflammatory drug, was shown to inhibit, in a dose-dependent manner (ED50 = 22 microM) ionophore-stimulated LTB4 biosynthesis by human whole blood in vitro.

Effects of Tebufelone (NE-11740), a new anti-inflammatory drug, on arachidonic acid metabolism

Tebufelone is a novel nonsteroidal anti-inflammatory drug (NSAID), of the di-tert-butylphenol (DTBP) class, which displays potent anti-inflammatory, analgesic and anti-pyretic properties in a variety of animal models. In this report, the effects of Tebufelone on arachidonic acid (AA) metabolism are reviewed. Tebufelone potently inhibits the formation of prostaglandins (PGE2) a key mediator of pain and inflammation, in isolated enzyme preparations (IC50=1.5 μM,KI=0.35 μM), twoin vitro cellular systems: rat peritoneal macrophages (IC50=0.02 μM) and human whole blood (IC50=0.08 μM), andex vivo in man. In addition to PGE2 inhibition, which is common to all NSAIDs, higher concentrations of Tebufelone block thein vitro formation of products of the lipoxygenase pathway [leukotrienes (LTB4)] in rat macrophages (IC50=20 μM) and human whole blood (IC50=22 μM). Substrate incorporation studies (14C-AA) indicate that Tebufelone reversibly inhibits cyclooxygenase (CO) and 5-lipoxygenase (5-LO) enzymes rather than regulating the release of AA. Tebufelone was shown to be a more potent CO inhibitor than indomethacin and a less potent 5-LO inhibitor than RG-5901. Comparisons to structurally related compounds under development (E-5110, Esai; KME-4, Kanagafuchi), found Tebufelone to be the most potent CO inhibitorin vitro. All three DTBP compounds were equipotent 5-LO inhibitors. It is likely that Tebufelones inhibitory effects on AA metabolism are, in part, responsible for itsin vivo efficacy and enhanced safety profile.

Determination of tebufelone (a new anti-inflammatory drug) strength and stability in bulk drug, dosage formations and feed admixtures by reversed-phase high-performance liquid chromatography

A rugged reversed-phase high-performance liquid chromatographic method suitable for the quantitative determination of tebufelone, a new anti-inflammatory drug, in bulk drug, various pharmaceutical formulations and animal feed admixtures is described. Tebufelone was easily separated from synthetic by-products and detected by ultraviolet absorption (280 nm). Standard curves were linear (r2 greater than 0.999) over 2 orders of magnitude with a detection limit of 0.1 microgram/ml at a signal-to-noise ratio of 2 (0.05 ml injected). Recovery of tebufelone from bulk drug and dosage formulations was greater than 99% with a coefficient of variation of 1.8% throughout the range of the standard curve. Recovery of tebufelone from feed admixtures was 96-102% with a less than 5% relative standard deviation at the levels assayed.