Ed in sterile 1 ml tipcap amber oral syringes (Becton Dickinson, Oxford
Ed in sterile 1 ml tipcap amber oral syringes (Becton Dickinson, Oxford, UK) and made use of inside 1 week of preparation. Fasted subjects had been cannulated through the antecubital vein and blood was drawn into 10 ml EDTA Vacutainer tubes (Becton Dickinson). Subjects then received the dual isotopic oral dose of 2 mg [13C10] -carotene and 1 mg [13C10]retinylFig. 1. -carotene and retinyl acetate metabolism. Position of [13C] labels are shown for [13C10] -carotene and [13C10]retinyl acetate, and derived 13 13 metabolites. Inserts show the [ C20] -carotene and d4-retinyl palmitate employed for system validation. Asterisks () denote position of [ C] labels.Journal of Lipid Investigation Volume 55,acetate in conjunction with a standardized breakfast meal consisting of a muffin and yogurt smoothie. The meal was created to reflect the same nutrient content as described by Borel et al. (5) containing 46.3 g of fat (55.five of total power intake). Blood was subsequently collected at 2, four, six, 8, 10, and 12 h postdose through cannulation, and at 24, 48, 168, and 336 h by very simple venipuncture. Each and every blood sample was right away centrifuged at four upon collection and also the plasma stored at 80 until evaluation.Plasma extraction and analyte recoveryAn ethanolethyl acetate (1:1) solvent extraction was applied to plasma samples to ensure sufficient recovery of all analytes with no coextraction of lipids recognized to interfere with LCMS analyses. All extraction procedures were performed below yellow lighting. To 1 ml of plasma, ten l (50 pmol) each and every of the [13C10]retinyl acetate and [13C20] -carotene internal standards had been added ahead of denaturing with 5 ml of ethanol and 5 ml of ethyl acetate. The sample was then shaken on an orbital shaker for 10 min and centrifuged at 10,000 rpm for 30 min at four . The supernatant was transferred to a clean glass tube plus the solvent evaporated to dryness below a stream of nitrogen. The residue was resuspended in 100 l of ethyl acetate, by vortexing briefly, and transferred to amber glass vials ready for LCMSMS injection. Resulting from endogenous levels of [12C] -carotene, retinol, and retinyl palmitate generally being present in “control” plasma, recovery of target analytes from the plasma matrix was assessed applying the following stable isotopes: [13C10] -carotene, [13C5]retinol, and d4-retinyl palmitate. Blank plasma was generously supplied by the Blood Transfusion Service, Newcastle upon Tyne Hospitals (UK). For extraction efficiency experiments, ten l of [13C10] carotene, [13C5]retinol, and d4-retinyl palmitate in ethanol had been spiked into 1 ml of handle plasma at a final concentration of 5 M. Plasma was then extracted as described above.returned to 80 B for 3 min to re-equilibrate. Flow rate was 1.0 ml min 1 with an injection volume of 10 l. An API4000 triple quadrupole LCMSMS (Applied Biosystems, Carlsbad, CA) was utilised for evaluation with atmospheric stress chemical ionization (APCI) performed in optimistic ion mode working with cIAP-2 Molecular Weight nitrogen gas with all the following optimum settings: collision gas, 7; curtain gas, 10; ion IL-1 list source gas 1, 60; ion source gas 2, 15. Temperature on the heated nebulizer was 400 with an ionspray voltage of five,500. Optimization of MSMS parameters for all analytes was performed by picking precursor ions of [MH] for -carotene, [MH-18] for retinol, [MH-256] for retinyl palmitate, and [MH-60] for retinyl acetate to acquire solution ion spectra. Quantitation of analytes was performed in selected reaction monitoring (SRM) mode; mass transitions and optimized MSMS parame.
