This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lipid Extraction The Enoxaparin sodium solution (150 mg/mL) was divided into four 400-uL portions. Three of the portions were spiked with nonadecanoic acid (1, 10 and 100 uL, respectively, of a 0.1 mg/mL solution). 1.85 mL chloroform-methanol (1:2) was added, and the mixtures were vortexed for 15 min. Then, 0.62 mL chloroform was added, and after vortexing for another 5 min, 0.62 mL water. The mixtures were vortexed again for 5 min and centrifuged for 5 min. The organic layers of each sample were separated and dried down under a stream of nitrogen. Lipid Saponification and Derivatization with 1-Pyrenyldiazomethane (PDAM) Each sample was treated with 500 uL 1 M KOH in 50% methanol for 30 min at 95 degree C. After cooling, the samples were acidified with 2 M HCI and extracted with 1 mL chloroform-hexane (1:1). After vortexing 5 min, the organic layers were separated and dried down with a stream of nitrogen. The samples were dissolved in 100 uL methanol and treated with 100 uL 1 mg/mL PDAM in ethyl acetate for 90 min at room temperature. HPLC with Fluorescence Detection HPLC was carried out on an Agilent system using a 4.6x250 mm Phenomenex Prodigy C18 analytical column with 5 um particle size. Analytes were eluted by a linear gradient between water (A) and acetonitrile (B), starting at 90% B (flow rate 1.0 mL/min) increasing to 100 % B (1.2 mL/min) over 40 min, then isocratic at 100 % B (1.2 mL/min) for 50 min. Analytes were detected by a Waters 470 Scanning Fluorescence Detector (ex. 340 nm;em 395 nm). The injection volume was 5 uL.
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