FATTY ACID ASSAY OF ONE SAMPLE BY HPLC WITH FLUORESCENCE DETECTION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 脂质提取 将依诺肝素钠溶液（150 mg/mL）分成4份400 μ L。 向其中三个部分加标十九烷酸（分别为1、10和100 uL，0.1 mg/mL溶液）。 1.85加入10 mL氯仿-甲醇（1：2），涡旋混合物15 min。然后加入0.62 mL氯仿，再涡旋5 min后，加入0.62 mL水。 将混合物再次涡旋5 min并离心5 min。分离各样品的有机层并在氮气流下干燥。 用1-芘基重氮甲烷（PDAM）进行脂质皂化和衍生化 将每个样品用500 μ L IM KOH的50%甲醇溶液在95 ℃下处理30分钟。 冷却后，将样品用2 M HCl酸化并用ImL氯仿-己烷（1：1）萃取。 涡旋5分钟后，分离有机层并用氮气流干燥。 将样品溶于100 uL甲醇中，并在室温下用100 uL lmg/mL PDAM的乙酸乙酯溶液处理90 min。 HPLC-荧光检测 HPLC在Agilent系统上使用4.6x250 mm Phenomenex Prodigy C18分析柱（具有5 μ m粒度）进行。 通过水（A）和乙腈（B）之间的线性梯度洗脱分析物，从90% B（流速1.0 mL/min）开始，经40 min增加至100% B（1.2 mL/min），然后以100% B（1.2 mL/min）等度洗脱50 min。 分析物通过沃茨470扫描荧光检测器（来自340 nm; em 395 nm）。 注射体积为5 uL。

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.